The FASEB Journal

Published by Federation of American Society of Experimental Biology
Online ISSN: 1530-6860
Print ISSN: 0892-6638
Purpose: Studies have shown that aldosterone would have angiogenic effects, and therefore would be beneficial in the context of cardiovascular diseases. We thus investigated the potential involvement of aldosterone in triggering a cardiac angiogenic response in the context of type-2 diabetes, and the molecular pathways involved. Procedure: 3 week-old male mice, overexpressing aldosterone-synthase (AS), and their controls littermates (WT) were fed with a standard or high fat, high sucrose (HFHS) diet. After 6 months of diet, mice were sacrificed and cardiac samples were assayed by RT-PCR, immuno-blotting and -histology. Findings: HFHS-diet induced type-2 diabetes (D) in WT and AS mice. VEGFa mRNAs were decreased in WT-D (-43%, P<0.05 vs. WT) while increased in AS-D mice (+236%, P<0.01 vs. WT-D). In WT-D hearts, the proapoptotic p38-MAPK was activated (P<0.05 vs. WT and AS-D) whereas Akt activity decreased. The AS-mice, that exhibited a cardiac upregulation of IGF1-R, showed an increase in Akt phosphorylation when diabetic (P<0.05 vs. WT and AS-D). Contrary to WT-D, AS-D hearts did not express inflammatory markers and exhibited a normal capillary density (P<0.05 vs. WT-D) Conclusions: To our knowledge, this is the first study providing new insights into the mechanisms whereby aldosterone prevents diabetes-induced cardiac disorders.
Aging is strongly correlated with the accumulation of oxidative damage in DNA, particularly in mitochondria. Oxidative damage to both mitochondrial and nuclear DNA is repaired by the base excision repair (BER) pathway. The "mitochondrial theory of aging" suggests that aging results from declining mitochondrial function, due to high loads of damage and mutation in mitochondrial DNA (mtDNA). Restriction of caloric intake is the only intervention so far proven to slow the aging rate. However, the molecular mechanisms underlying such effects are still unclear. We used caloric-restricted (CR) mice to investigate whether lifespan extension is associated with changes in mitochondrial BER activities. Mice were divided into two groups, receiving 100% (PF) or 60% (CR) of normal caloric intake, a regime that extends mean lifespan by approximately 40% in CR mice. Mitochondria isolated from CR mice had slightly higher uracil (UDG) and oxoguanine DNA glycosylase (OGG1) activities but marginally lower abasic endonuclease and polymerase gamma gap-filling activities, although these differences were tissue-specific. Uracil-initiated BER synthesis incorporation activities were significantly lower in brain and kidney from CR mice but marginally enhanced in liver. However, nuclear repair synthesis activities were increased by CR, indicating differential regulation of BER in the two compartments. The results indicate that a general up-regulation of mitochondrial BER does not occur in CR.
Human telomerase reverse transcriptase (hTERT) is central to maintain telomeres for continuous cell proliferation, but it remains unknown how extracellular cues regulate telomerase maintenance of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces Smad3 phosphorylation, nuclear translocation, and hTERT gene repression. BMP7 induces Smad3-dependent telomerase inhibition in a time- and concentration-dependent manner in breast cancer cells. Chronic exposure of breast cancer cells to BMP7 results in short telomeres, cell senescence, and apoptosis. Mutation of BMPRII receptor, but not TGFbetaRII, ACTRIIA, or ACTRIIB, blocked BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres, and continued cell proliferation. Expression of hTERT inhibits BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging and death by a mechanism involving inhibition of telomerase activity and telomere maintenance via BMPRII receptor- and Smad3-mediated repression of the hTERT gene.
In pathological conditions, F(0)F(1)-ATPase hydrolyzes ATP in an attempt to maintain mitochondrial membrane potential. Using thermodynamic assumptions and computer modeling, we established that mitochondrial membrane potential can be more negative than the reversal potential of the adenine nucleotide translocase (ANT) but more positive than that of the F(0)F(1)-ATPase. Experiments on isolated mitochondria demonstrated that, when the electron transport chain is compromised, the F(0)F(1)-ATPase reverses, and the membrane potential is maintained as long as matrix substrate-level phosphorylation is functional, without a concomitant reversal of the ANT. Consistently, no cytosolic ATP consumption was observed using plasmalemmal K(ATP) channels as cytosolic ATP biosensors in cultured neurons, in which their in situ mitochondria were compromised by respiratory chain inhibitors. This finding was further corroborated by quantitative measurements of mitochondrial membrane potential, oxygen consumption, and extracellular acidification rates, indicating nonreversal of ANT of compromised in situ neuronal and astrocytic mitochondria; and by bioluminescence ATP measurements in COS-7 cells transfected with cytosolic- or nuclear-targeted luciferases and treated with mitochondrial respiratory chain inhibitors in the presence of glycolytic plus mitochondrial vs. only mitochondrial substrates. Our findings imply the possibility of a rescue mechanism that is protecting against cytosolic/nuclear ATP depletion under pathological conditions involving impaired respiration. This mechanism comes into play when mitochondria respire on substrates that support matrix substrate-level phosphorylation.
The use of xenogeneic cells or tissues for tissue engineering applications may lead to advances in biomedical research. Hyperacute and delayed rejection are immunologic hurdles that must be addressed to achieve xenograft survival in the pig-to-primate setting. Expression of human alpha1,2-fucosyltransferase (HT) in the donor cell or tissue protects from hyperacute rejection (HAR) by reducing expression of Galalpha1,3-Gal epitope, the major xenoantigen recognized by human natural antibodies. We hypothesized that Galalpha1,3-Gal antigen contributes to delayed tissue rejection. To test this hypothesis, we transplanted control or HT-transgenic engineered porcine cartilage s.c. into alpha1,3-galactosyltransferase knockout (Gal KO) mice. Control porcine cartilage grafted in Gal KO mice was not susceptible to HAR but was rejected in several wk by a prominent cellular immune infiltrate and elevated antibody titers. In contrast, Gal KO mice receiving the HT engineered cartilage showed a markedly reduced anti-pig antibody response and no anti-Galalpha1,3-Gal-elicited antibody response. The HT implants had a mild cellular infiltrate that was confined to the graft periphery. Our study demonstrates that a marked reduction of Galalpha1,3-Gal antigen in HT-transgenic porcine cartilage confers resistance to a process of delayed rejection. Further development of tissue engineering applications that use genetically modified porcine tissues is encouraged.
Microsomal 4-hydroxylase of 1,2,3,4-tetrahydroisoquinoline (TIQ), a possible candidate for causing Parkinson disease, was characterized by using rat hepatic microsomes and purified P450 isozymes. Kinetic analysis revealed that Km and Vmax values (mean +/- SE) for hepatic microsomal TIQ 4-hydroxylase of male Wistar rats were 319.6 +/- 26.8 microM and 12.13 +/- 1.43 protein, respectively. When TIQ 4-hydroxylase activity was compared in Wistar (an animal model of extensive debrisoquine metabolizers) and Dark Agouti (an animal model of poor debrisoquine metabolizers) rats, significant strain (Wistar greater than Dark Agouti) and sex (male greater than female) differences were observed. The microsomal activity toward TIQ 4-hydroxylation was increased by pretreatment of male Wistar rats with P448 inducers (beta-naphthoflavone and sudan I), but not with phenobarbital. Pretreatment with propranolol, an inhibitor of P450 isozymes belonging to the P450 IID gene subfamily, decreased TIQ 4-hydroxylase activity. P450 BTL, a P450 isozyme belonging to the IID subfamily, showed TIQ 4-hydroxylase activity of 64.1 pmol.min-1.nmol P450(-1), which was 3.2-fold that of microsomes (20.9 pmol.min-1.nmol P450(-1)). Antibody (IgG) against this isozyme suppressed microsomal TIQ 4-hydroxylase activity concentration-dependently. A male-specific P450 ml (P450IIC11) catalyzed this reaction to a much lesser extent (10.0 pmol.min-1.nmol P450(-1)), and its antibody did not affect the microsomal activity. These results suggest that TIQ 4-hydroxylation in hepatic microsomes are catalyzed predominantly by a P450 isozyme (or isozymes) belonging to the IID gene subfamily in non-treated rats and its immunochemically related P450 isozyme (or isozymes), and that a P450 isozyme (or isozymes) belonging to the IA subfamily also participates in TIQ 4-hydroxylation in rats pretreated with P448-inducers.
Mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 30 mg/kg i.p. twice, 16 h apart). This resulted in changes in motor performance and toxic insult of nigral neurons as evidenced by dopamine depletion in nucleus caudatus putamen. In vitro and in vivo treatment of MPTP caused the generation of hydroxyl radicals (.OH) as measured by a sensitive salicylate hydroxylation procedure. A dopamine agonist, bromocriptine (10 microM and 10 mg/kg i.p.), blocked .OH formation caused by MPTP in vitro (20 microM) and in vivo (30 mg/kg i.p.). An MPTP-induced increase in the activity of catalase and superoxide dismutase in substantia nigra on the seventh day was reduced by bromocriptine pretreatment. Bromocriptine blocked MPTP-induced behavioral dysfunction as well as glutathione and dopamine depletion, indicating its potent neuroprotective action. This study suggests that bromocriptine stimulates antioxidant mechanisms in the brain and acts as a free radical scavenger in addition to its action at dopamine receptors, thus indicating its strength as a valuable neuroprotectant.
Pseudovitamin D-deficiency rickets (PDDR) is an autosomal disease characterized by hyperparathyroidism, rickets, and undetectable levels of 1,25-dihydroxyvitaminD3 (1,25(OH)2D3). Mice in which the 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) gene was inactivated presented the same clinical phenotype as patients with PDDR and were used to study renal expression of the epithelial Ca2+ channel (ECaC1), the calbindins, Na+/Ca2+ exchanger (NCX1), and Ca2+-ATPase (PMCA1b). Serum Ca2+ (1.20+/-0.05 mM) and mRNA/protein expression of ECaC1 (41+/-3%), calbindin-D28K (31+/-2%), calbindin-D9K (58+/-7%), NCX1 (10+/-2%), PMCA1b (96+/-4%) were decreased in 1alpha-OHase-/- mice compared with 1alpha-OHase+/- littermates. Feeding these mice a Ca2+-enriched diet normalized serum Ca2+ levels and expression of Ca2+ proteins except for calbindin-D9K expression. 1,25(OH)2D3 repletion resulted in increased expression of Ca2+ transport proteins and normalization of serum Ca2+ levels. Localization of Ca2+ transport proteins was clearly polarized in which ECaC1 was localized along the apical membrane, calbindin-D28K in the cytoplasm, and calbindin-D9K along the apical and basolateral membranes, resulting in a comprehensive mechanism facilitating renal transcellular Ca2+ transport. This study demonstrated that high dietary Ca2+ intake is an important regulator of the renal Ca2+ transport proteins in 1,25(OH)2D3-deficient status and thus contributes to the normalization of blood Ca2+ levels.
The innate immune system of mammals provides a rapid response to repel assaults from numerous infectious agents including bacteria, viruses, fungi, and parasites. A major component of this system is a diverse combination of cationic antimicrobial peptides that include the alpha- and beta-defensins and cathelicidins. In this study, we show that 1,25-dihydroxyvitamin D3 and three of its analogs induced expression of the human cathelicidin antimicrobial peptide (CAMP) gene. This induction was observed in acute myeloid leukemia (AML), immortalized keratinocyte, and colon cancer cell lines, as well as normal human bone marrow (BM) -derived macrophages and fresh BM cells from two normal individuals and one AML patient. The induction occurred via a consensus vitamin D response element (VDRE) in the CAMP promoter that was bound by the vitamin D receptor (VDR). Induction of CAMP in murine cells was not observed and expression of CAMP mRNA in murine VDR-deficient bone marrow was similar to wild-type levels. Comparison of mammalian genomes revealed evolutionary conservation of the VDRE in a short interspersed nuclear element or SINE in the CAMP promoter of primates that was absent in the mouse, rat, and canine genomes. Our findings reveal a novel activity of 1,25-dihydroxyvitamin D3 and the VDR in regulation of primate innate immunity.
We recently showed that excessive fructose consumption, already associated with numerous metabolic abnormalities, reduces rates of intestinal Ca(2+) transport. Using a rat lactation model with increased Ca(2+) requirements, we tested the hypothesis that mechanisms underlying these inhibitory effects of fructose involve reductions in renal synthesis of 1,25-(OH)(2)D(3). Pregnant and virgin (control) rats were fed isocaloric fructose or, as controls, glucose, and starch diets from d 2 of gestation to the end of lactation. Compared to virgins, lactating dams fed glucose or starch had higher rates of intestinal transcellular Ca(2+) transport, elevated intestinal and renal expression of Ca(2+) channels, Ca(2+)-binding proteins, and CaATPases, as well as increased levels of 25-(OH)D(3) and 1,25-(OH)(2)D(3). Fructose consumption prevented almost all of these lactation-induced increases, and reduced vitamin D receptor binding to promoter regions of Ca(2+) channels and binding proteins. Changes in 1,25-(OH)(2)D(3) level were tightly correlated with alterations in expression of 1α-hydroxylase but not with levels of parathyroid hormone and of 24-hydroxylase. Bone mineral density, content, and mechanical strength each decreased with lactation, but then fructose exacerbated these effects. When Ca(2+) requirements increase during lactation or similar physiologically challenging conditions, excessive fructose consumption may perturb Ca(2+) homeostasis because of fructose-induced reductions in synthesis of 1,25-(OH)(2)D(3).
Our understanding of how vitamin D mediates biological responses has entered a new era. It is now clear that the bulk of the biological responses supported by vitamin D occur as a consequence of its metabolism to its daughter metabolite 1 alpha,25-dihydroxyvitamin D3 (a steroid hormone). The fact that 1,25(OH)2D3 receptors are ubiquitous in tissue distribution opens the possibility for unforeseen biological functions of the vitamin D endocrine system. For example, 1,25(OH)2D3 serves as an immunoregulatory hormone and a differentiation hormone besides its classical role in mineral homeostasis. The avian 1,25)OH)2D3 receptor has recently been cloned and shown to be a member of the nuclear transacting receptor family that includes estrogen, progesterone, glucocorticoid, thyroxine (T3), aldosterone, and retinoic acid receptors. We have compiled an extensive number of RNA polymerase II-transcribed genes that are regulated by 1,25(OH)2D3. Classification of these genes on functional grounds identifies and formulates the several genetic circuits or biochemical systems in which 1,25(OH)2D3 plays an essential regulatory role. These systems include genes that govern oncogene and lymphokine expression as well as those involved in mineral homeostasis, vitamin D metabolism, and regulation of a set of replication-linked genes (c-myc, c-myb, and histone H4), which are critical for rapid cellular proliferation. An integrated analysis of the combinations of genetic circuits regulated by 1,25(OH)2D3 suggests that they may be collectively tied to a DNA replication-differentiation switch.
Platelets are activated by increased cytosolic Ca2+ concentration ([Ca2+]i) following store-operated calcium entry (SOCE) accomplished by calciumrelease-activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca2+ transport is regulated by 1,25(OH)2 vitamin D3 [1,25(OH) 2D3]. 1,25(OH)2D3 formation is inhibited by klotho and excessive in klotho-deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca2+ signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl-mice were analyzed by RT-PCR, Western blotting, confocal microscopy, Fura-2-fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist-induced [Ca2+]i increase, activation-dependent degranulation, integrin αIIbβ3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27-90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38-73%) and 1,25(OH) 2D3-treated WT megakaryocytes. Nuclear NF-kB subunit p50/p65 abundance was significantly reduced in kl/kl-megakaryocytes (by 51-76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG-01 cells (by 46-97%). Low-vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca2+ signaling and activation, an effect at least partially due to 1,25(OH)2D3-dependent down-regulation of NF-kB activity and STIM1/Orai1 expression in megakaryocytes.
Adipose tissue inflammation is an important pathological process in obese people, associated with diabetes and cardiovascular disease. We hypothesized that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] inhibits cytokine secretion from adipocytes via direct inhibition of transcription factor nuclear factor-κB (NF-κB). We utilized two different human models. Bone marrow-derived human mesenchymal stromal cells (hMSCs) differentiated into adipocytes, and adipocytes isolated from biopsies stimulated with lipopolysaccharide (LPS) were treated with or without 1,25(OH)(2)D(3). Expression and secretion of interleukin-6 (IL-6) were measured by quantitative RT-PCR analysis and ELISA. Assessment of NF-κB nuclear translocation, DNA binding activity was performed by immunofluorescence (IF) and electrophoretic mobility assay (EMSA). Inhibitor κB (IκB) and its phosphorylation were detected by Western blot (WB) analysis. Simultaneous 1,25(OH)(2)D(3) cotreatment significantly reduced LPS-stimulated (10 ng/ml) IL-6 secretion dose dependently by 15% at 10(-10) M and 26% at 10(-7) M (P<0.05) in hMSCs, while preincubation with 1,25(OH)(2)D(3) (10(-7) M) for 24 h reduced IL-6 secretion by 24 and 35% (P<0.001) and mRNA levels by 34 and 30% (P<0.05) in hMSCs and isolated adipocytes, respectively. 1,25(OH)(2)D(3) suppressed LPS-stimulated IκB phosphorylation-mediated NF-κB translocation into the nucleus were evident from WB, IF, and EMSA. 1,25(OH)(2)D(3) inhibits LPS-stimulated IL-6 secretion in two human adipocyte models via interference with NF-κB signaling.-Mutt, S. J., Karhu, T., Lehtonen, S., Lehenkari, P., Carlberg, C., Saarnio, J., Sebert, S., Hyppönen, E., Järvelin, M.-R., Herzig, K.-H. Inhibition of cytokine secretion from adipocytes by 1,25-dihydroxyvitamin D(3) via the NF-κB pathway.
Dendritic cells (DCs) are antigen-presenting cells that provide a link between innate and adaptive immunity. Ca(2+)-dependent signaling plays a central regulatory role in DC responses to diverse antigens. DCs are a primary target of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a secosteroid hormone, that, in addition to its well-established action on Ca(2+) homeostasis, possesses immunomodulatory properties. Surprisingly, nothing is known about its effects on DC cytosolic Ca(2+) activity. The present study explored whether 1,25(OH)(2)D(3) modifies the intracellular Ca(2+) concentration ([Ca(2+)](i)) in DCs. Here we show that mouse DCs expressed K(+)-independent (NCX1-3) and K(+)-dependent (NCKX1, 3, 4, and 5) Na(+)/Ca(2+) exchangers. Acute application of LPS (100 ng/ml) to DCs increased [Ca(2+)](i), an effect significantly blunted by prior incubation with 1,25(OH)(2)D(3). 1,25(OH)(2)D(3) increased the membrane abundance of the NCKX1 protein, up-regulated the K(+)- and Na(+)-dependent Ca(2+) entry and enhanced the K(+)-dependent Na(+)/Ca(2+) exchanger currents. The NCKX blocker 3',4'-dichlorobenzamyl (DBZ) reversed the inhibitory effect of 1,25(OH)(2)D(3) on the LPS-induced increase of [Ca(2+)](i). Expression of the costimulatory molecule CD86 was down-regulated by 1,25(OH)(2)D(3), an effect reversed by DBZ. In summary, 1,25(OH)(2)D(3) blunts the LPS-induced increase in [Ca(2+)](i) by stimulation of Na(+)/Ca(2+) exchanger-dependent Ca(2+) extrusion, an effect that contributes to 1,25(OH)(2)D(3)-mediated immunosuppression. The results disclose completely novel mechanisms in the regulation of DC maturation and function.
Recent work has demonstrated that inhibition of nitric oxide production with various nitric oxide synthesis inhibitors (L-NAME, L-NMMA) initiate leukocyte adhesion to postcapillary venules. The objective of this study was to elucidate the mechanism (or mechanisms) that promote the L-NAME-induced leukocyte response. Intravital microscopy was used to examine 25-40 microns venules in the rat mesentery. Nitric oxide synthesis was inhibited with L-NAME and leukocyte adhesion was observed over the first 60 min. The fourfold increase in leukocyte adhesion was independent of alterations in venular red blood cell velocity. The adhesion was superoxide-mediated inasmuch as superoxide dismutase (SOD) abolished the rise in leukocyte adhesion associated with nitric oxide synthesis inhibition. Ketotifen, a mast cell stabilizer, also abolished the rise in leukocyte adhesion induced by L-NAME. Histology revealed that mast cell degranulation occurred only in animals treated with L-NAME but not in animals pretreated with SOD or ketotifen. This observation suggests that mast cells become activated in the absence of nitric oxide production and superoxide contributes to the mast cell activation. The L-NAME-induced leukocyte adhesion could be reproduced by infusing hypoxanthine/xanthine oxidase (a superoxide generating system) or compound 48/80 (an activator of mast cells) and both responses were attenuated by ketotifen. These data suggest that inhibition of nitric oxide synthesis results in a superoxide and mast cell-dependent leukocyte adhesion.
Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) drive calcium signals involved in skeletal muscle excitation-transcription coupling and plasticity; IP(3)R subtype distribution and downstream events evoked by their activation have not been studied in human muscle nor has their possible alteration in Duchenne muscular dystrophy (DMD). We studied the expression and localization of IP(3)R subtypes in normal and DMD human muscle and in normal (RCMH) and dystrophic (RCDMD) human muscle cell lines. In normal muscle, both type 1 IP(3)Rs (IP(3)R1) and type 2 IP(3)Rs (IP(3)R2) show a higher expression in type II fibers, whereas type 3 IP(3)Rs (IP(3)R3) show uniform distribution. In DMD biopsies, all fibers display a homogeneous IP(3)R2 label, whereas 24 +/- 7% of type II fibers have lost the IP(3)R1 label. RCDMD cells show 5-fold overexpression of IP(3)R2 and down-regulation of IP(3)R3 compared with RCMH cells. A tetanic stimulus induces IP(3)-dependent slow Ca(2+) transients significantly larger and faster in RCDMD cells than in RCMH cells as well as significant ERK1/2 phosphorylation in normal but not in dystrophic cells. Excitation-driven gene expression was different among cell lines; 44 common genes were repressed in RCMH cells and expressed in RCDMD cells or vice versa. IP(3)-dependent Ca(2+) release may play a significant role in DMD pathophysiology.
Increases of IP 3 R3 in rapidly renewing tissues. A) IP 3 R3 staining in E20 small intestine is localized to villar cells. 
Increases of IP 3 R3 in developmental neuronal 
Increases of IP 3 R3 in excitotoxic neuronal death. Sagittal sections of 3-month-old rat hippocampus show negli- 
IP 3 R3 antisense oligonucleotides block NGF deprivation-induced neuronal death. A) (1) High-power magnification of IP 3 R3 expression in developing dorsal root ganglion at E15. (2) Protein immunoblot detection of IP 3 R with affinity-purified, subtype-specific rabbit anti-IP 3 R1 and IP 3 R3 antibodies. Lane NGF ϩ : control NGF-treated DRG homogenates. Lane NGF Ϫ : 
Mechanisms accounting for the cellular entry of calcium that mediates cellular proliferation and apoptosis have been obscure. Previously we reported selective augmentation of type 3 inositol (1,4,5) trisphosphate receptors (IP(3)R3) in lymphocytes undergoing programmed cell death, which was prevented by antisense constructs to IP(3)R3. We now report increases in mRNA and protein levels for IP(3)R3 associated with cell death in several apoptotic paradigms in diverse tissues. Elevations of IP(3)R3 occur during developmental apoptosis in early postnatal cerebellar granule cells, dorsal root ganglia, embryonic hair follicles, and intestinal villi. Neurotoxic damage elicited by the glutamate agonist kainate is also associated with IP(3)R3 augmentation. In chick dorsal root ganglia neurons undergoing apoptosis due to deprivation of nerve growth factor, levels of IP(3)R3 are selectively increased and cell death is selectively prevented by antisense oligonucleotides to IP(3)R3. Thus, IP(3)R3 appears to participate actively in cell death in a diversity of tissues.
Of all the intracellular organelles, secretory granules contain by far the highest calcium concentration; secretory granules of typical neuroendocrine chromaffin cells contain approximately 40 mM Ca(2+) and occupy approximately 20% cell volume, accounting for >60% of total cellular calcium. They also contain the majority of cellular inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in addition to the presence of >2 mM of chromogranins A and B that function as high-capacity, low-affinity Ca(2+) storage proteins. Chromogranins A and B also interact with the IP(3)Rs and activate the IP(3)R/Ca(2+) channels. In experiments with both neuroendocrine PC12 and nonneuroendocrine NIH3T3 cells, in which the number of secretory granules present was changed by either suppression or induction of secretory granule formation, secretory granules were demonstrated to account for >70% of the IP(3)-induced Ca(2+) releases in the cytoplasm. Moreover, the IP(3) sensitivity of secretory granule IP(3)R/Ca(2+) channels is at least approximately 6- to 7-fold more sensitive than those of the endoplasmic reticulum, thus enabling secretory granules to release Ca(2+) ahead of the endoplasmic reticulum. Further, there is a direct correlation between the number of secretory granules and the IP(3) sensitivity of cytoplasmic IP(3)R/Ca(2+) channels and the increased ratio of IP(3)-induced cytoplasmic Ca(2+) release, highlighting the importance of secretory granules in the IP(3)-dependent Ca(2+) signaling. Given that secretory granules are present in all secretory cells, these results presage critical roles of secretory granules in the control of cytoplasmic Ca(2+) concentrations in other secretory cells.-Yoo, S. H. Secretory granules in inositol 1,4,5-trisphosphate-dependent Ca(2+) signaling in the cytoplasm of neuroendocrine cells.
FAD-linked presenilin-1 transfection increased the secretion of endogenous GnT-V. A) GnT-V activity in cell lysates and conditioned medium was assayed in specific SK-N-SH cells transfected with control vector (mock), WT presenilin-1 (wild-type), and FAD-linked presenilin-1 (A246E, ⌬ E9), respectively. Values represent the means of 2 or 3 independent experiments; bars, sd . B) Expression and molecular size of endogenous GnT-V were characterized by Western blot of GnT-V in each SK-N-SH transfectant (upper panel). After the same membrane was deprobed, the amount of ␤ 1– 6GlycNAc branching (the product of GnT-V) was assessed by L 4 -PHA lectin blot analysis (L.B) (lower panel). Asterisk indicates several ␤ 1– 6GlcNAc oligosaccharide-con- 
Glycosyltransferases are present in the Golgi apparatus in a membrane-bound form and are released from cells after cleavage by certain proteases. Beta1,6-N-acetylglucosaminyltransferase V (GnT-V), which is cleaved and secreted from the cells, is involved in the biosynthesis of beta1-6GlcNAc branching on N-glycans and has been implicated in tumor progression and metastasis. We recently reported that a secreted type of GnT-V (soluble GnT-V) itself could promote angiogenesis, which is completely different from its original function as a glycosyltransferase, and this might play a role in tumor invasion. In this study, to explore the molecular basis for this functional glycosyltransferase secretion, its cleavage site was examined and the protease(s) involved in that cleavage were identified. The NH2-terminal protein sequence of purified soluble GnT-V (approximately 100 kDa) from GnT-V-overexpressed cells revealed that its terminus started at His31, located at the boundary position between the transmembrane and stem regions. This secretion was not inhibited by a single amino acid mutation at the cleavage site (Leu29, Leu30 to Asp, His31 to Ala), but specifically inhibited by addition of DFK-167, a gamma-secretase inhibitor, suggesting that gamma-secretase is a plausible protease for secretion processing. In addition, transfection of the gene of familial Alzheimer's disease (FAD)[corrected]-linked presenilin-1, a component of gamma-secretase, increased the secretion rate of endogenous GnT-V; the secretion of soluble GnT-V (approximately 100 kDa) was completely inhibited in presenilin-1/2 double-deficient cells, which have no gamma-secretase activity. Collectively, these results demonstrate that Golgi-resident GnT-V is cleaved at the transmembrane region by gamma-secretase, and this might control tumor angiogenesis through a novel pathway.
Up-regulation of core fucosylation catalyzed by α1,6-fucosyltransferase (Fut8) has been observed in hepatocellular carcinoma (HCC). Here, to explore the role of Fut8 expression in hepatocarcinogensis, we established the chemical-induced HCC models in the male wild-type (WT; Fut8(+/+)), hetero (Fut8(+/-)), and knockout (KO; Fut8(-/-)) mice by use of diethylnitrosamine (DEN) and pentobarbital (PB). In the Fut8(+/+) and Fut8(+/-) mice, multiple large and vascularized nodules were induced with an increased expression of Fut8 after DEN and PB treatment. However, the formation of HCC in Fut8(-/-) mice was suppressed almost completely. This potent inhibitory effect of Fut8 deficiency on tumorigenesis was also confirmed by the abolished tumor formation of Fut8 KO human hepatoma cell line cells by use of a xenograft tumor model. Furthermore, loss of the Fut8 gene resulted in attenuated responses to epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in the HepG2 cell line, which provides the possible mechanisms for the contribution of Fut8 to hepatocarcinogensis. Taken together, our study clearly demonstrated that core fucosylation acts as a critical functional modulator in the liver and implicated Fut8 as a prognostic marker, as well as a novel, therapeutic target for HCC.-Wang, Y., Fukuda, T., Isaji, T., Lu, J., Im, S., Hang, Q., Gu, W., Hou, S., Ohtsubo, K., Gu, J. Loss of α1,6-fucosyltransferase inhibits chemical induced hepatocellular carcinoma and tumorigenesis by down-regulating several cell signaling pathways. © FASEB.
Here we present functional and biochemical evidence for a Ca(2+) channel (Ca(V)1.2)/protein kinase C (PKC) signaling complex being a key player in muscarinic regulation of urinary bladder smooth muscle. Muscarinic stimulation induced Ca(2+) signals and concomitant contractions in detrusor muscle from mice that were dependent on functional Ca(2+) channels. These signals were still present in muscles being depolarized by 85 mM extracellular K(+). Muscarinic-induced contractions were reduced by a PKC inhibitor [bisindolylmaleimide I (BIM-I)] and a phospholipase D (PLD) inhibitor (1-butanol). A phorbol ester (PDBu) enlarged muscarinic-induced Ca(2+) signals and contractions. The effects of BIM-I and PDBu were inhibited by isradipine and/or absent in muscles from Ca(V)1.2-deficient mice. Both carbachol and PDBu increased Ca(V)1.2 channel currents in isolated bladder myocytes. Blue native-PAGE electrophoresis revealed that Ca(V)1.2, PKC, and PLD are closely associated in muscles being previously stimulated by carbachol. Immunoprecipitation using anti-Ca(V)1.2 followed by Western blotting demonstrated that Ca(V)1.2 and PKC are coupled in stimulated muscles from wild-type mice. Autoradiography on immunoprecipitates showed that Ca(V)1.2 is a substrate for PKC-mediated phosphorylation. These findings suggest that a signaling complex consisting of Ca(V)1.2, PKC, and, probably, PLD controls muscarinic-mediated phasic contraction of urinary bladder smooth muscle.
The Ca(v)1.2 L-type Ca2+ channel is the dominant voltage-activated Ca2+ channel in heart and smooth muscle. The functional significance of this channel was studied in intestinal smooth muscle from mice carrying a smooth muscle-specific, conditional inactivation of the Ca(v)1.2 gene (Ca(v)1.2SMACKO mice). Inactivation was complete within 4 wk after tamoxifen treatment and confirmed by RT-PCR, Western blot and functional analysis. Ca(v)1.2SMACKO mice show reduced feces excretion, absence of rhythmic contractions in small and large intestinal muscle and signs of paralytic ileus. Extracellular field stimulation evoked smaller contractions in jejunum muscles from Ca(v)1.2SMACKO than from CTR mice, whereas carbachol-induced contractions of similar magnitude in both muscles. The Ca2+ needed for contraction in jejunum was provided mainly by Ca(v)1.2 channels and by store-operated channels in muscles from CTR and Ca(v)1.2SMACKO mice, respectively. In conclusion, the Ca(v)1.2 channel is essential for electromechanical coupling and important for pharmaco-mechanical coupling in intestinal smooth muscle and cannot be substituted functionally by other Ca2+ entry pathways.
Ahnak missense mutation Ile5236Thr. A) Chromato- gram demonstrating the mutation in female mutation carrier. Heterozygous mutation T2611C (GenBank TM accession num- 
Effects of ahnak-derived peptides on Ca 2 ϩ inward 
Ahnak Ile5236Thr affects the ␤ 2 subunit binding. A) The ␤ 2 subunit and ahnak-C1 were expressed in E. coli as GST fusion proteins. Decreasing amounts of ␤ 2 subunit (10, 5, 2.5 ␮ g at lanes 1–3, respectively) as well as ahnak-C1 (10, 5, 2.5 ␮ g at lanes 4 – 6, respectively) were subjected to SDS-PAGE and stained with Coomassie blue (upper panel). Proteins from identical gels were blotted to nitrocellulose and overlaid with biotinylated wild-type ahnak peptide (middle panel) or mutated ahnak peptide (lower panel). B) Equilibrium sedimentation analysis demonstrating the ahnak-C1/ ␤ 2-complex formation. The ␤ 2 subunit (0.16 ␮ M) and ahnak-C1 (0.185 ␮ M) dissolved in 50 mM Tris-HCl (pH 7.4), 500 mM NaCl were centrifuged at 14,000 rpm and 10°C. Radial concentration distribution curves are recorded at 270 nm ( E ), 275 nm ( • ), or 280 nm ( ᮀ ) were globally fitted using the POLYMOLE program (26). The solid curves below represent the free reactants and a 1:1 complex resulting in a dissociation constant (K d ) of 152 Ϯ 17 nM. As an indicator of the fit quality, residuals are given (upper part) that are statistically distributed around the mean values. C) Different mixtures consisting of 0.16 ␮ M ␤ 2 and variable amounts of ahnak-C1 with either isoleucine (wild-type, circle) or threonine (mutated; triangle) at amino acid position 5236 were centrifuged to sedimentation equilibrium and analyzed for complex formation. The K d values were 158 Ϯ 26 nM ( n ϭ 20) and 96 Ϯ 17 nM ( n ϭ 9) for wild-type ahnak-C1 and Ile5236Thr mutated ahnak-C1, respectively. The values are means Ϯ sd with the number of independent experiments in parenthesis. D) Bar graphs demonstrating the effects of synthetic ahnak peptides (wild-type, Ile5236Thr mutated; as in legend to Fig. 2) on ahnak-C1/ ␤ 2 subunit binding expressed as K d values of ahnak-C1/ ␤ 2-complexes (means Ϯ sd for at least 3 independent experiments). K d was solely increased by the mutated ahnak peptide demonstrating its specific uncoupling effect. 
Mutated ahnak fragment, Ile5236Thr ahnak-C1/C mimics ␤ -adrenergic stimulation of I CaL . Time course of peak I CaL (in pA/pF) in a rat cardiomyocyte intracellularly per- 
Proposed model for sympathetic control of I CaL by ahnak/Ca 2 ϩ channel binding. Under basal conditions, I CaL carried by the ␣ 1C subunit is reprimed by strong ahnak- C1/ ␤ 2 subunit binding (left panel). Upon sympathetic stimulation, PKA sites in ahnak (12) and Ser-478 and Ser-479 in ␤ 2 (41) are phosphorylated. This attenuates ahnak-C1/ ␤ 2 subunit binding, resulting in increased I CaL since ␤ 2 subunit is more available for ␣ 1C (right panel). Hence, we propose ahnak-C1/ ␤ 2 subunit binding serves as physiological brake of ␣ 1C conductance. Relief from this inhibition is proposed as a common pathway used by the sympathetic signal cascade and Ile5236Thr mutated ahnak fragments to increase I CaL . Mutated ahnak, like chronic ␤ -adrenergic stimulation, might lead to cardiotoxicity. 
Defective L-type Ca2+ channel (I(CaL)) regulation is one major cause for contractile dysfunction in the heart. The I(CaL) is enhanced by sympathetic nervous stimulation: via the activation of beta-adrenergic receptors, PKA phosphorylates the alpha1C(Ca(V)1.2)- and beta2-channel subunits and ahnak, an associated 5643-amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr-ahnak on I(CaL). Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both beta2 subunit interaction and I(CaL) regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and beta2 subunit decreased by approximately 50% after PKA phosphorylation or in the presence of Ile5236Thr-ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA-effects on I(CaL) increasing the amplitude by approximately 60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr-peptide and Ile5236Thr-fragment (aa 5215-5288) prevented specifically the further up-regulation of I(CaL) by isoprenaline. Hence, we suggest the ahnak-C1 domain serves as physiological brake on I(CaL). Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr-ahnak fragments to increase I(CaL). This genetic ahnak variant might cause individual differences in I(CaL) regulation upon physiological challenges or therapeutic interventions.
Depolarization-and carbachol-induced contraction. Original recordings of tension of CTR (a) and SMACKO (b) detrusor muscle response to depolarization by 85 mM K + (A) or 10 µM CCh (B, C) are shown. Bars indicate the presence of 85 mM K + or 10 µM CCh. Traces after 5 min preincubation with 1 µM isradipine (ISR; A, B), 20 sec after bath application of 10 mM BAPTA (B), 30 min after addition of 1 µM thapsigargin (TG; C) and 15 min after incubation in Ca 2+ free solution containing 1 mM EGTA (C) are graphically superimposed. Statistics of the peak (c) and tonic (d) contractile responses in control conditions (Control) and in the presence of ISR, BAPTA, TG, and EGTA are shown. Tonic responses were obtained 5 min after addition of K + or CCh. Open and filled bars indicate experiments with muscles from CTR and SMACKO mice, respectively. Data represent means ± SEM. Numbers indicate number of experiments. *P<0.05; **P<0.01; ***P<0.001; n.s., non-significant. 
Mice deficient in the smooth muscle Cav1.2 calcium channel (SMACKO, smooth muscle alpha1c-subunit calcium channel knockout) have a severely reduced micturition and an increased bladder mass. L-type calcium current, protein, and spontaneous contractile activity were absent in the bladder of SMACKO mice. K+ and carbachol (CCh)-induced contractions were reduced to 10-fold in detrusor muscles from SMACKO mice. The dihydropyridine isradipine inhibited K+- and CCh-induced contractions of muscles from CTR but had no effect in muscles from SMACKO mice. CCh-induced contraction was blocked by removing extracellular Ca2+ but was unaffected by the PLC inhibitor U73122 or depletion of intracellular Ca2+ stores by thapsigargin. In muscles from CTR and SMACKO mice, CCh-induced contraction was partially inhibited by the Rho-kinase inhibitor Y27632. These results show that the Cav1.2 Ca2+ channel is essential for normal bladder function. The Rho-kinase and Ca2+-release pathways cannot compensate the lack of the L-type Ca2+ channel.
Expression studies of multigene families, such as the immunoglobulin (Ig) loci, are difficult because of their large size and the necessity to introduce germline configured regions into an animal. Antibody diversity from Ig gene miniloci is limited by the number of variable (V) region genes and the need for distal regulatory elements to control expression. Here, we show germline transfer into mice of a 1300 kb human Ig kappa light chain locus on a yeast artificial chromosome that resulted in early DNA rearrangement and highly efficient human light chain expression. The human locus was assembled from a 300 kb authentic region using contig extension by addition of cosmid multimers to supplement the variable gene cluster. This resulted in the addition of about 100 V region genes in germline configuration from different families. In transgenic animals with Ig kappa disruption, this large human kappa locus replaced the endogenous locus, and subsequent down-regulation of Ig lambda light chain contribution led to a dominant expression of the rearranged human genes. Contrary to expectation, rather than providing a solely selective advantage for ensuring repertoire formation controlled by the sheer number of introduced genes, the lambda/kappa ratio in serum appears to be the result of competition for early surface Ig expression maintained in the developing B cell.-Zou, X., Xian, J., Davies, N. P., Popov, A. V., Brüggemann, M. Dominant expression of a 1.3 Mb human Ig kappa locus replacing mouse light chain production.
Analysis of ␣ 1D subunit-specific membrane proteins from RPE cells and rat brain. A) Western blot analysis of membrane proteins. Left panels: Staining with anti- ␣ 1D subunit antibodies reveals the presence of ␣ subunits from L-type channels of the neuroendocrine subtype in membrane proteins from both RPE cells (from serum-containing cultures) and rat brain tissue. Right panel: Staining of the same Western blot using antiphosphotyrosine antibodies (anti-p-tyr) shows the tyrosine phosphorylation of ␣ 1D subunit-specific bands. B) Western blot analysis of immunoprecipitates obtained using anti- ␣ 1D subunit antibodies (RPE cells from serum-containing cultures). Left panels: Staining with anti- ␣ 1D subunit antibodies shows the enrichment of proteins specific for ␣ subunits of neuroendocrine L-type channels (arrows indicate the bands that disappear using the corresponding blocking peptide). Middle panels: Staining of the same precipitates using anti-FGFR2 antibodies reveals the coprecipitation of FGFR2 (arrows indicate the bands that disappear using the corresponding blocking peptide). Right panel: Staining with anti-FGFR1 antibodies did not result in the detection of FGFR1-specific proteins among these precipitates. C) Western blot analysis of immunoprecipitates obtained with anti- ␣ 1D subunit antibodies pretreated with the corresponding blocking peptide. Under these conditions no coprecipitation of FGFR2 with L-type channel ␣ subunits occurred. 
Immunoprecipitation of bFGF receptors. A) Immunoprecipitation of membrane proteins from RPE cells and rat brain using antibodies against FGFR2 (bek). Left panels: Western blot analysis of precipitates stained with anti-FGFR2 antibodies shows the presence of FGFR2 (arrows indicate the bands that disappear using the corresponding blocking peptide). Right panels: Western blot analysis of the precipitates stained with anti- ␣ 1D subunit antibodies (arrows indicate the bands that disappear in the presence of the corresponding blocking peptide). The proteins were isolated from (left to right): rat brain, RPE cells maintained in serum-containing cultures, RPE cells maintained for 24 h in serum-free cultures prior immunoprecipitation experiments, RPE cells maintained in serum-free cultures incubated in bFGF (10 ng/ml; 1 h). Coprecipitation was not observed in precipitates obtained from RPE cells maintained in serum-free cultures. B) Immunoprecipitation with antibodies against FGFR2, where the antibodies were pretreated with the corresponding blocking peptide. Western blot analysis of the precipitates stained with anti- ␣ 1D subunit antibodies shows no coprecipitation with ␣ subunits of L-type channels. C) Immunoprecipitation of membrane proteins from RPE cells and rat brain using antibodies against FGFR1 (flg). Left panels: Western blot analysis of the precipitates indicates the presence of FGFR1 (anti-FGFR1 staining was compared with the corresponding positive control). Right panels: Western blot analysis of the precipitates stained with anti- ␣ 1D subunit antibodies indicate no coprecipitation with ␣ subunits from L-type channels. 
In contrast to the fibroblast growth factor receptor 1 (FGFR1), little is known about intracellular signaling of FGFR2. The signaling cascade of FGFR2 was studied using the perforated patch configuration of the patch-clamp technique in cultured rat retinal pigment epithelial (RPE) cells that express both FGFR1 and FGFR2. Interaction of signaling proteins was studied using immunoprecipitation techniques with membrane proteins from RPE cells and freshly isolated rat brain. When Ba(2+) currents through L-type channels were studied, extracellular application of bFGF (10 ng/ml) led to a shift of the steady-state activation to more negative values. In 50% of cells, an additional increase in maximal current amplitude was observed. This effect was blocked by the tyrosine kinase inhibitor lavendustin A (10(-5) M) but was not influenced by the FGFR1 blocker SU5402 (2 x 10(-5) M) or by the blocker for src-kinase herbimycin A (10(-5) M). Immunoprecipitation of FGFR2 led to coprecipitation of alpha 1D Ca(2+) channel subunits and precipitation of alpha 1D subunits led to coprecipitation of FGFR2. Immunoprecipitation of FGFR1 did not result in the coprecipitation with alpha 1D Ca(2+) channel subunits. The coprecipitation results were comparable when using brain tissue and RPE cells. The alpha 1D subunit-specific band were stained with antiphosphotyrosine antibodies. We conclude that FGFR2 acts via a different signaling cascade than FGFR1. This cascade involves an src-kinase-independent, close functional interaction of FGFR2 and the alpha subunit of neuroendocrine L-type channels.
SDS-PAGE of wild-type, Thr-88, Leu-88, and Leu-90 1.3S proteins after purification by HPLC. Acrylamide concentration was 15% for the running gel. Lane a, wild-type 1.3S; lane b, Thr-88; lane c, standards: myoglobin, 17,201; myoglobin I + II, 14,632; myoglobin I, 8235; myoglobin II, 6383; lane d, Leu-88; lane e, Leu-90; and lane f, P shermanii 1.3S subunit. Protein applied was 
The apo 1.3S subunit of transcarboxylase contains the sequence Ala-87-Met-88-Lys-89-Met-90, and it is Lys-89 that is biotinated. This sequence is highly conserved in all the biotin enzymes that have been sequenced (with the exception of acetyl-CoA carboxylase from chicken liver, which has Val in place of Ala). The role of Met-88 and Met-90 in specifying Lys-89 for biotination by synthetase was examined by site-directed mutagenesis. Genes of the 1.3S subunit coding for Thr-88, Leu-88, or Leu-90 were generated by oligonucleotide-directed in vitro mutagenesis and expressed in Escherichia coli. The mutated apo 1.3S subunits were isolated and the biotination by homogeneous synthetase from Propionibacterium shermanii was compared with that of the apo wild-type subunit. The Vmax for the apo mutants was the same as that for the apo wild type, but when Leu was substituted for Met-88 or Met-90, the Km for the mutant was lower than that of the wild-type or mutant Thr-88. The activity of the synthetase of E. coli was determined by an in vivo assay. During the early log phase of growth, a smaller portion of mutants Thr-88 and Leu-90 was biotinated than with the wild-type or mutant Leu-88. When the cultures progressed to stationary phase, mutants and the wild type were biotinated to the same extent. The overall results show that Met-88 and Met-90 are not required for biotination of the apo 1.3S subunit by the synthetases.
The somatostatin analogs octreotide and lanreotide, selective to receptor subtypes 2 and 5, failed clinical efficacy for the prevention of restenosis after percutaneous transluminal angioplasty. These findings might have been the result of targeting a wrong subset of receptors. In rat arteries, subtypes 1 and 4 are expressed 3-4 times more prominently than 2 and 5, and subtype 1 is the nearly exclusive subtype in atherosclerotic human vessels. Here, we demonstrate that daily s.c. injections (50-500 microg/kg/d) of CH275 (DesAA1,2,5(D-W8,IAmp9)Somatostatine-14), selective to subtypes 1 and 4, dose-dependently inhibited intimal hyperplasia 14 days after rat carotid denudation injury (for intimal area P=0.0002 across the dose range). CH275 was more effective than somatostatin-14 (equal affinity to all five subtypes, P=0.03), or octreotide (selective to subtypes 2 and 5, P=0.098). When rats were given the peptides for 14 days with end-point at 28 days, CH275 still significantly inhibited intimal area expansion. Both CH275 and octreotide inhibited the outgrowth of cells from postinjury aortic tissue punch-explants and the distance migrated in vitro, but not cell replication, which indicated that the effects of somatostatin analogs were directed on the migration of intimal cell progenitors rather than on their proliferation.
The efficiency of ATP synthesis coupled to cell respiration, commonly referred to as the P/O ratio, has been the subject of extensive studies for more that 50 years. The general conclusion from these studies is that respiring mitochondria can convert external ADP to ATP at a maximal P/O ratio of 3 for NAD-linked substrates and 2 for succinate. However, in recent years the validity of these "integral" values has been questioned on both mechanistic and thermodynamic grounds, and a mechanistic P/O ratio of 2.5 for NAD-linked substrates and 1.5 for succinate have been concluded on the basis of experiments with isolated mitochondria. These values have been widely adopted in the scientific literature, including several recent textbooks. In this paper we report that under optimal conditions with respect to preparation and assay procedures, the P/O ratios obtained with isolated rat liver mitochondria consistently exceed 2.5 with NAD-linked substrates and 1.5 with succinate. These results, although not excluding "nonintegral" P/O ratios due to various energy-dissipating side reactions, warrant caution in accepting the reported lower values and, in general, in referring to mechanistic considerations unless the underlying molecular mechanisms are understood.
I Na measurement. A) Total I Na at HP 120 mV, [Na] o 5mM. B) I Na at HP 70 mV, [Na] o 5mM. C) I Na at HP 70 mV with 1 mM Cd 2 , and TTX in WT cells, [Na] o 50 mM. D) I Na at HP 70 mV with 1 mM Cd 2 , and TTX in null cells, [Na] o 50 mM. E) Steady-state inactivation and activation curves of the sodium current at HP 70 mV, with 1 mM Cd 2. F) TTX (100 nM)-sensitive current obtained from panels C, D. Data are averages sd, ANOVA. 
ECG recording. A) Representative recordings of a single sinus beat in a WT mouse (left panel) and in a null mouse (right panel). B) Superposition of QRS-T complexes from mice in panel A. C) Na V 1.6 mutant mice exhibit prolonged PR and QRS intervals. Data are averages sd, ANOVA. 
CV in hyperkalemia. A) Activation maps of WT and null hearts paced at 140 ms from the apex in 12 mM [K ] o. B, C) Plots of data in 9 WT and 9 null mice at different [K ] o , with pacing at 140 (B) and 80 ms (C). Data are averages sd, Mixed. 
APD 80 in hyperkalemia. A) Anterior surface APD 80 maps of WT and null hearts paced at 140 ms from the apex in 12 mM [K ] o. B, C) Plot of data in 9 WT and 9 null mice at different [K ] o , with pacing at 140 (B) and 80 ms (C). Data are averages sd, Mixed. 
Na V 1.6-null mice exhibit a prolonged Ca 2 transient. A) Left panel: time-space plot line. Center panel: representative time-space plot of Rhod-2 fluorescence in WT (top) and null mouse myocytes (bottom). Right panel: superimposed single-pixel recordings. B) Composite plot of Ca 2 transients. Data are averages sd, ANOVA. 
Evidence supports the expression of brain-type sodium channels in the heart. Their functional role, however, remains controversial. We used global Na(V)1.6-null mice to test the hypothesis that Na(V)1.6 contributes to the maintenance of propagation in the myocardium and to excitation-contraction (EC) coupling. We demonstrated expression of transcripts encoding full-length Na(V)1.6 in isolated ventricular myocytes and confirmed the striated pattern of Na(V)1.6 fluorescence in myocytes. On the ECG, the PR and QRS intervals were prolonged in the null mice, and the Ca(2+) transients were longer in the null cells. Under patch clamping, at holding potential (HP) = -120 mV, the peak I(Na) was similar in both phenotypes. However, at HP = -70 mV, the peak I(Na) was smaller in the nulls. In optical mapping, at 4 mM [K(+)](o), 17 null hearts showed slight (7%) reduction of ventricular conduction velocity (CV) compared to 16 wild-type hearts. At 12 mM [K(+)](o), CV was 25% slower in a subset of 9 null vs. 9 wild-type hearts. These results highlight the importance of neuronal sodium channels in the heart, whereby Na(V)1.6 participates in EC coupling, and represents an intrinsic depolarizing reserve that contributes to excitation.
Histatin does not promote cell proliferation. A) Representative micrographs illustrating the number of cells per well after incubation in the presence or absence of Hst1(12-38) at concentrations ranging from 0.1-100 M or rhEGF at 10 ng/ml. HO-1-N-1 cells were seeded (110 4 cells/well); 48 h later, cells were fixed and permeabilized, and subsequently DNA was stained with PI, a red dye that becomes fluorescent on binding to DNA. Scale bar 100 m. B) Total cell numbers were quantified by measuring PI fluorescence of DNA. Hst1(12-38) does not enhance proliferation, whereas rhEGF does. AU, arbitrary units. Data represent means sd; n 12. *P 0.01 vs. control. 
Wounds in the mouth heal faster and with less scarification and inflammation than those in the skin. Saliva is thought to be essential for the superior oral wound healing, but the involved mechanism is still unclear. We have previously discovered that a human-specific peptide, histatin, might be implicated in the wound-healing properties of saliva. Here we report that histatin enhances reepithelialization in a human full-skin wound model closely resembling normal skin. The peptide does not stimulate proliferation but induces cell spreading and migration, two key initiating steps in reepithelialization. Activation of cells by histatin requires a G-protein-coupled receptor that activates the ERK1/2 pathway. Using a stepwise-truncation method, we determined the minimal domain (SHREFPFYGDYGS) of the 38-mer-parent peptide that is required for activity. Strikingly, N- to C-terminal cyclization of histatin-1 potentiates the molar activity approximately 1000-fold, indicating that the recognition of histatin by its cognate receptor requires a specific spatial conformation of the peptide. Our results emphasize the importance of histatin in human saliva for tissue protection and recovery and establish the experimental basis for the development of synthetic histatins as novel skin wound-healing agents.
S-100a(o), the alpha alpha isoform of a subfamily of Ca(2+)-binding proteins of the EF-hand type expressed in cardiac and skeletal muscle cells, is reported to inhibit the assembly of the intermediate filament subunit desmin and to stimulate the disassembly of desmin intermediate filaments in the presence of micromolar levels of free Ca(2+). These effects are dose-dependent with respect to the S-100a(o) concentration and maximal at a desmin/S-100a(o) (dimer) molar ratio of approximately 2. Other members of the S-100 subfamily [S-100a (alpha beta) and S-100b (beta beta) and the unfractionated mixture of S-100a plus S-100b produce qualitatively similar effects on desmin assembly, with a potency that depends on the fraction of S-100alpha subunit (the most potent) or S-100beta subunit (the least potent) present in the S-100 isoforms tested. A binding stoichiometry of 2 mol of desmin/mol of S-100a(o) (dimer) and an affinity in the submicromolar range are calculated. The S-100beta subunit also interacts with desmin, but with a lower affinity compared with S-100alpha. By contrast, the S-100-like proteins calcyclin and p11 neither interact with desmin nor affect desmin assembly. The present data suggest that S-100a(o) might play a role in the regulation of the state of assembly of desmin intermediate filaments.
Screen for l-DOPA-modifying effect of MDMA and -substituted analogues in reserpine-treated rats. MDMA, MBDB, and 5 novel -substituted analogues (all 3 mg/kg, i.p.) were coadministered with l-DOPA (125 mg/kg, i.p.) in reserpine (4 mg/kg, s.c.)-treated rats. Vertical (A) and horizontal (B) locomotor activity was cumulated for 1 h during times of peak l-DOPA action (0 –1 h). Data are means se; n 10 –20. Compounds eliciting a significant reduction in vertical locomotor activity (MDMA, UWA-101, UWA-104, and UWA-001, displayed in color) were selected for further assessment. *P 0.05, ***P 0.001 vs. vehicle-treated animals.  
Effect of MDMA and -substituted analogues on duration and quality of antiparkinsonian action of l-DOPA in MPTP-treated marmosets. MPTP-lesioned marmosets received MDMA (A), UWA-101 (B), UWA-104 (C), or UWA-001 (D) (all vehicle, 1 and 3 mg/kg, s.c.) coadministered with l-DOPA (25 mg/kg, s.c.). Duration of antiparkinsonian action (ON-time; time for which disability scores were mild or less) that was of good (without disabling dyskinesia) or bad (with disabling dyskinesia) quality was assessed for the entire 4-h observation period. Data are means se; n 5 for all treatment groups. *P 0.05 for increase in total ON-time vs. control; # P 0.05, ## P 0.01 for increase in good ON-time vs. control; ANOVA.  
Treatment of Parkinson's disease with dopaminergic agents, such as l-DOPA, is frequently compromised by disabling side effects, particularly dyskinesia and a shortening in duration of antiparkinsonian action. Studies in animal models and anecdotal evidence from a patient with Parkinson's disease show that the illicit drug ecstasy (MDMA) can alleviate these side effects, though with many drawbacks (e.g., psychoactivity). MDMA itself thus has little therapeutic potential. On the basis of known structure-psychoactivity relationships, we designed a series of α-substituted MDMA analogues, one of which, bearing an α-cyclopropyl substituent (UWA-101), enhanced the quality of l-DOPA actions in animal models. Indeed, UWA-101 was more effective than MDMA. Unlike MDMA, UWA-101 did not reduce viability of serotonergic cells, exhibit psychoactive properties, or reduce food intake, and did not substitute for MDMA in drug discrimination assays. UWA-101 displayed a unique receptor/transporter binding profile relative to MDMA, with a >5-fold decrease in affinity for NET and 5-HT(2A) receptors and a 10-fold increase in affinity for DAT. Furthermore, in a functional reuptake assay, UWA-101 inhibited both 5-HT and dopamine reuptake, while having no effect on the reuptake of noradrenaline. UWA-101 is the first selective DAT/SERT inhibitor described with comparable affinities for these two sites. These data identify a new class of therapeutic in Parkinson's disease and highlight the potential benefits of studying illicit drugs that in themselves would never be considered safe for long-term therapy.
Myofibroblasts are present at the invasion front in colon cancer. In an attempt to understand their putative proinvasive activity, we have developed an in vitro model. Myofibroblasts isolated from colon cancer tissue or obtained through transdifferentiation of colon fibroblasts by transforming growth factor (TGF)-beta stimulate invasion of colon cancer cells into collagen type I and Matrigel. We identified two convergent proinvasive agents secreted by myofibroblasts: namely scatter factor/hepatocyte growth factor (SF/HGF) and the TGF-beta-upregulated extracellular matrix glycoprotein tenascin-C (TNC), each of which is necessary though not sufficient for invasion. Myofibroblast-stimulated invasion into collagen type I is characterized by a change from a round, nonmigratory morphotype with high RhoA and low Rac activity to an elongated, migratory morphotype with low RhoA and high Rac activity. RhoA inactivation is determined by the epidermal growth factor (EGF)-like repeats of TNC through EGF-receptor signaling that confers a permissive and priming signal for the proinvasive activity of SF/HGF that activates Rac via c-Met. We confirmed the validity of this mechanism by using pharmacological modulators and dominant negative or constitutive active mutants that interfere with RhoA-Rho kinase and Rac signaling. Our in vitro results point to a new putative proinvasive signal for colon cancer cells provided by myofibroblasts in the tumor stroma.
A common phenotype in breast cancer is the expansion of the estrogen receptor-alpha (ER+) cell population and an inappropriate elevation of ERalpha protein, the latter predisposing patients for a poorer prognosis than those with lower levels of the receptor. A tetracycline-inducible ERalpha overexpression model was developed in the MCF-7 cell line to assess induction of endogenous gene activation and growth in response to elevations in ERalpha protein. Heightened levels of ERalpha resulted in aberrant promoter occupancy and gene activation in the absence of hormone, which was independent of ligand and AF-2 function. This increased receptor activity required the amino-terminal A/B domain and was not inhibited by tamoxifen, which supports an enhancement of AF-1 function, yet was independent of serine-104, 106, and 118 phosphorylation. Ligand-independent transcription was accompanied by an increase in growth in the absence of hormonal stimulation. The results suggest that elevated levels of ERalpha in breast cancer cells can result in activation of receptor transcriptional function in a manner distinct from classical mechanisms that involve ligand binding or growth factor-induced phosphorylation. Further, they describe a potential mechanism whereby increases in ERalpha concentration may provide a proliferative advantage by augmenting ERalpha function regardless of ligand status.
A comprehensive sequence alignment of 64 members of the family of matrix metalloproteinases (MMPs) for the entire sequences, and subsequently the catalytic and the hemopexin-like domains, have been performed. The 64 MMPs were selected from plants, invertebrates, and vertebrates. The analyses disclosed that as many as 23 distinct subfamilies of these proteins are known to exist. Information from the sequence alignments was correlated with structures, both crystallographic as well as computational, of the catalytic domains for the 23 representative members of the MMP family. A survey of the metal binding sites and two loops containing variable sequences of amino acids, which are important for substrate interactions, are discussed. The collective data support the proposal that the assembly of the domains into multidomain enzymes was likely to be an early evolutionary event. This was followed by diversification, perhaps in parallel among the MMPs, in a subsequent evolutionary time scale. Analysis indicates that a retrograde structure simplification may have accounted for the evolution of MMPs with simple domain constituents, such as matrilysin, from the larger and more elaborate enzymes.
Airway smooth muscle (ASM) cells play important physiological roles in the lung, and abnormal proliferation of ASM directly contributes to the airway remodeling during development of lung diseases such as asthma. MicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation; however, little is known about the precise role of microRNAs in the proliferation of the ASM. Here we report that a specific microRNA (miR-10a) controls ASM proliferation through directly inhibiting the phosphoinositide 3-kinase (PI3K) pathway. Next-generation sequencing identified miR-10a as the most abundant microRNA expressed in primary human airway smooth muscle (HASM) cells, accounting for > 20% of all small RNA reads. Overexpression of miR-10a reduced mitogen-induced HASM proliferation by ∼50%, whereas inhibition of miR-10a increased HASM proliferation by ∼40%. Microarray profiling of HASM cells expressing miR-10a mimics identified 52 significantly down-regulated genes as potential targets of miR-10a, including the catalytic subunit α of PI3K (PIK3CA), the central component of the PI3K pathway. MiR-10a directly suppresses PIK3CA expression by targeting the 3'-untranslated region (3'-UTR) of the gene. Inhibition of PIK3CA by miR-10a reduced V-akt murine thymoma viral oncogene homolog 1 (AKT) phosphorylation and blunted the expression of cyclins and cyclin-dependent kinases that are required for HASM proliferation. Together, our study identifies a novel microRNA-mediated regulatory mechanism for PI3K signaling and ASM proliferation and further suggests miR-10a as a potential therapeutic target for lung diseases whose etiology resides in abnormal ASM proliferation.-Hu, R., Pan, W., Fedulov, A. V., Jester, W., Jones, M. R., Weiss, S. T., Panettieri, R. A., Jr., Tantisira, K., Lu, Q. MicroRNA-10a controls airway smooth muscle cell proliferation via direct targeting of the PI3 kinase pathway.
A) Normal cell-cell apposition in a confluent monolayer of RAEC grown on type IV collagen (x 150. B) Subconfluent RAEC positively labeled with pAbvWF (x 690). C) Phase contrast micrograph of cells labeled with pAbvWF
Confluent monolayers of RAEC pretreated (5 mm) with 0.1 M 5(S)-HETE (A) (x 230), 15(S)-HETE (B) (x 230), or 12(R)HETE (C) (x 230) were indistinguishable from controls after 1 h of incubation. In contrast, after 1 h incubation, cells pretreated (5 mm) with 0.1 M I2(S)-HETE (D) (x 230) demonstrated
RAEC monolayers treated with 5(5)-, 15(5)-, 12(R)-, or 12(S)-HETE (0.1 tiM; 5 mm) were incubated for 1 or 36 h before use in an adhesion assay with W256 tumor cells. One h after exposure to each of the above HETEs, only monolayers exposed to 12(S)-HETE demonstrated increased tumor cell adhesion compared with the control (P < 0.01). In contrast, assays performed 36 h after exposure  
A 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), which is produced by platelets and tumor cells, was tested for its ability to induce retraction of endothelial cell monolayers. The induction of endothelial cell retraction is a critical step in tumor cell metastasis. Endothelial cells demonstrated reversible retraction in response to 12(S)-HETE, but did not respond to the stereoisomer 12(R)-HETE or to unrelated 5-lipoxygenase (i.e., 5[S]-HETE) or 15-lipoxygenase (i.e., 15[S]-HETE) metabolites. Endothelial cells did not demonstrate loss of viability in response to 12(S)-HETE. The induction of retraction was both dose and time dependent. Scanning electron microscopy confirmed that 12(S)-HETE induced endothelial cell retraction and revealed collapsed filopodia on their surface, the appearance of spaces between endothelial cells and the underlying subendothelial matrix, in addition to large gaps between adjacent endothelial cells. Tumor cell adhesion to endothelial cell monolayers was enhanced 1 h after pretreatment of monolayers with 12(S)-HETE but not after pretreatment with other lipoxygenase metabolites. Tumor cell adhesion to endothelial cell monolayers 36 h after pretreatment with 12(S)-HETE was not different from adhesion to untreated monolayers. Therefore we suggest that 12(S)-HETE generated during tumor cell-platelet-endothelial cell interactions may induce reversible endothelial cell retraction, allowing tumor cell access to the subendothelial matrix, which is a critical step in their eventual extravasation from the microvasculature during hematogenous metastasis.
Family 19 of the P450 super family is responsible for the conversion of C19 androgenic steroids to the corresponding estrogens, a reaction known as aromatization because it involves conversion of the delta4-3-one A-ring of the androgens to the corresponding phenolic A-ring characteristic of estrogens. The gene encoding human aromatase has been cloned and characterized and shown to be unusual compared to genes encoding other P450 enzymes, because there are numerous untranslated first exons that occur in aromatase transcripts in a tissue-specific fashion due to differential splicing as a consequence of the use of tissue-specific promoters. Thus, expression in the ovary uses a proximal promoter that is regulated primarily by cAMP. On the other hand, expression in the placenta uses a distal promoter located at least 40 kb upstream of the start of transcription that is regulated by retinoids. Other promoters are used in brain and adipose tissue. In the latter case, class I cytokines such as IL-6 and IL-11, as well as TNF-alpha, are important regulatory factors. A common 3'-splice junction located upstream of the start of translation is used in all of the splicing events involved in the use of these various promoters. Thus, the coding region of the transcripts, and hence the protein, are identical regardless of the tissue site of expression; what differs in a tissue-specific fashion is the 5'-end of the transcripts. This pattern of expression has great significance both from a phylogenetic and ontogenetic standpoint, as well as for the physiology and pathophysiology of estrogen formation, as will be discussed in this review.
11β-Hydroxysteroid dehydrogenase type-1 (11β-HSD1) converts inert cortisone into active cortisol, amplifying intracellular glucocorticoid action. 11β-HSD1 deficiency improves cardiovascular risk factors in obesity but exacerbates acute inflammation. To determine the effects of 11β-HSD1 deficiency on atherosclerosis and its inflammation, atherosclerosis-prone apolipoprotein E-knockout (ApoE-KO) mice were treated with a selective 11β-HSD1 inhibitor or crossed with 11β-HSD1-KO mice to generate double knockouts (DKOs) and challenged with an atherogenic Western diet. 11β-HSD1 inhibition or deficiency attenuated atherosclerosis (74-76%) without deleterious effects on plaque structure. This occurred without affecting plasma lipids or glucose, suggesting independence from classical metabolic risk factors. KO plaques were not more inflamed and indeed had 36% less T-cell infiltration, associated with 38% reduced circulating monocyte chemoattractant protein-1 (MCP-1) and 36% lower lesional vascular cell adhesion molecule-1 (VCAM-1). Bone marrow (BM) cells are key to the atheroprotection, since transplantation of DKO BM to irradiated ApoE-KO mice reduced atherosclerosis by 51%. 11β-HSD1-null macrophages show 76% enhanced cholesterol ester export. Thus, 11β-HSD1 deficiency reduces atherosclerosis without exaggerated lesional inflammation independent of metabolic risk factors. Selective 11β-HSD1 inhibitors promise novel antiatherosclerosis effects over and above their benefits for metabolic risk factors via effects on BM cells, plausibly macrophages.-Kipari, T., Hadoke, P. W. F., Iqbal, J., Man, T. Y., Miller, E., Coutinho, A. E., Zhang, Z., Sullivan, K. M., Mitic, T., Livingstone, D. E. W., Schrecker, C., Samuel, K., White, C. I., Bouhlel, M. A., Chinetti-Gbaguidi, G., Staels, B., Andrew, R., Walker, B. R., Savill, J. S., Chapman, K. E., Seckl, J. R. 11β-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis.
Puri fi cation, active site titration, ATPase, and motile activity of NtMD-2R. ( A ) Coomassie gel showing the elution pro fi le of His-tagged NtMD-2R construct by Ni 2+ -NTA chromatography puri fi cation. M, marker; FT, fl ow-through; W1, W2, washing steps; F1 – F8, eluted fractions. ( B ) Plot of the relative amplitude change of mant- fl uorescence as a function of mantATP concentration. From the section of the 2 lines the fraction of active protein was determined. Inset: Stopped- fl ow records of 16, 26, and 52 nM mantATP binding to 150 nM NtMD-2R. ( C ) Steady-state ATP turnover rates of NtMD-2R plotted as a function of F-actin concentration. Inset: dependence of the ATPase on the ATP concentration in the presence of 2 m M actin. ( D ) Gaussian distribution of actin fi lament sliding velocities of NtMD-2R at 30°C and 0.05 mM [Mg 2+ ] free . ( E ) Actin fi lament sliding velocities of NtMD-2R at 24°C in the presence of 4.5 mM (light gray bars) and 0.05 mM (dark gray bars) [Mg 2+ ] free . 
Transient kinetic parameters Construct Type of myosin Nt 175kDa myosin-11 Cc myosin-11 CCMD (19) At myosin-11 MYA1-2R (23) NtMD-2R Full-length (17)
Assignment of myosin mechanical activities to kinetic parameters
Structural modeling of the acto-myosin complex. (A) Predicted 3-dimensional structure of Nt 175kDa myosin-11 motor domain docked to an actin filament. The motor domain is depicted as cartoon representation; the actin filament is shown according to its electrostatic potential as colored surface. Positively and negatively charged regions in actin are blue and red, respectively, and uncharged areas are shown in white (from 21 kT/e to +1 kT/e).
Myosin actin binding interface. Comparison of the electrostatic potentials (A) and hydrophobic surfaces (B) of the motor domains of Nt 175kD myosin-11, Ddmyosin-2, and Ggmyosin-5 as obtained by computational modeling. The actin binding interface is indicated by a dashed line. Act1 and Act2 are 2 actin monomers involved in actin binding (shown as gray loop representations). Myosin binding to actin involves elements in the upper 50K and lower 50K domain, including loop 2, CM loop, loop 3, helix-loop-helix, and the activation loop. Clear differences in the charge density distribution of the actin binding area are found among the 3 myosins. The electrostatic surface potential is shown from 21 kT/e (red) to +1 kT/e (blue). Hydrophobic regions are represented in green. Higher values correlate with higher intensities.
The 175-kDa myosin-11 from Nicotiana tabacum (Nt(175kDa)myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt(175kDa)myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt(175kDa)myosin-11 is a highduty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt(175kDa)myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins.-Diensthuber, R. P., Tominaga, M., Preller, M., Hartmann, F. K., Orii, H., Chizhov, I., Oiwa, K., Tsiavaliaris, G. Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor.
A cycle of cis-to-trans isomerization of the chromophore is intrinsic to vertebrate vision where rod and cone photoreceptors mediate dim- and bright-light vision, respectively. Daylight illumination can greatly exceed the rate at which the photoproduct can be recycled back to the chromophore by the canonical visual cycle. Thus, an additional supply pathway(s) must exist to sustain cone-dependent vision. Two-photon microscopy revealed that the eyes of the zebrafish (Danio rerio) contain high levels of 11-cis-retinyl esters (11-REs) within the retinal pigment epithelium. HPLC analyses demonstrate that 11-REs are bleached by bright light and regenerated in the dark. Pharmacologic treatment with all-trans-retinylamine (Ret-NH2), a potent and specific inhibitor of the trans-to-cis reisomerization reaction of the canonical visual cycle, impeded the regeneration of 11-REs. Intervention with 11-cis-retinol restored the regeneration of 11-REs in the presence of all-trans-Ret-NH2. We used the XOPS:mCFP transgenic zebrafish line with a functional cone-only retina to directly demonstrate that this 11-RE cycle is critical to maintain vision under bright-light conditions. Thus, our analyses reveal that a dark-generated pool of 11-REs helps to supply photoreceptors with the chromophore under the varying light conditions present in natural environments.-Babino, D., Perkins, B. D., Kindermann, A., Oberhauser, V., von Lintig, J. The role of 11-cis-retinyl esters in vertebrate cone vision.
Loss of E-cadherin and up-regulation of mesenchymal cadherins, a hallmark of the epithelial-mesenchymal transition, contributes to migration and dissemination of cancer cells. Expression of human cadherin-11 (Cad11), also known as osteoblast cadherin, in prostate cancer increases the migration of prostate cancer cells. How Cad11 mediates cell migration is unknown. Using the human Cad11 cytoplasmic domain in pulldown assays, we identified human angiomotin (Amot), known to be involved in cell polarity, migration, and Hippo pathway, as a component of the Cad11 protein complex. Deletion analysis showed that the last C-terminal 10 amino acids in Cad11 cytoplasmic domain are required for Amot binding. Further, Cad11 preferentially interacts with Amot-p80 than Amot-p130 isoform and binds directly to the middle domain of Amot-p80. Cad11-Amot interaction affects Cad11-mediated cell migration, but not homophilic adhesion, as deletion of Amot binding motif of Cad11 (Cad11-ΔAmot) did not abolish Cad11-mediated cell-cell adhesion of mouse L cells, but significantly reduced Cad11-mediated cell migration of human C4-2B4 and PC3-mm2 prostate cancer cells and human HEK293T cells. Together, our studies identified Amot-p80 as a novel component of the Cad11 complex and demonstrated that Amot-p80 is critical for Cad11-mediated cell migration.-Ortiz, A., Lee, Y.-C., Yu, G., Liu, H.-C., Lin, S.-C., Bilen, M. A., Cho, H., Yu-Lee, L.-Y., and Lin, S.-H. Angiomotin is a novel component of cadherin-11/β-catenin/p120 complex and is critical for cadherin-11-mediated cell migration. © FASEB.
The effect of medroxyprogesterone 
Angiogenesis is the growth of new blood vessels from existing ones. It is an important aspect of new tissue development, growth, and tissue repair. It is also a component of many diseases including cancer, blindness, and chronic inflammation such as rheumatoid arthritis (RA) and psoriasis. There is considerable evidence to suggest that angiogenesis and chronic inflammation are codependent; recent studies have begun to reveal the nature of this link, which involves both augmentation of cellular infiltration and proliferation and overlapping roles of regulatory growth factors and cytokines. Through these studies, we have begun to understand the codependence of chronic inflammation and angiogenesis, the potential benefits of targeting angiogenesis in the treatment of chronic inflammation, and of targeting chronic inflammation to affect angiogenesis.
Sulfation is a common final step in the biotransformation of xenobiotics and is traditionally associated with inactivation. However, the sulfate group is electron-withdrawing and may be cleaved off heterolytically in some molecules, leading to an electrophilic cation. The stable heterologous expression of sulfotransferases in indicator cells of standard mutagenicity tests has substantially improved the accessibility of this activation pathway. Sulfotransferase-mediated genotoxic effects have been demonstrated for numerous benzylic alcohols derived from polycyclic aromatic hydrocarbons and various aromatic hydroxylamines. Also, hycanthone (a benzylic alcohol), alpha-hydroxytamoxifen (an allylic alcohol), 1'-hydroxysafrole (an allylic/benzylic alcohol), and 2-nitropropane are activated to genotoxicants by sulfotransferases. Various reactive sulfate conjugates show strong mutagenic effects only when they are generated directly within the indicator cell, due to their inefficient penetration of cell membranes. In other cases, secondary membrane-penetrating reactive species are formed from sulfuric acid esters by displacement reactions with medium components, such as chloride or amino acids. Reaction with water regenerates the alcohol, which becomes available for a new cycle of activation. Different sulfotransferases from the same species as well as related forms from rat and human differ in their substrate specificities and tissue distributions. These characteristics and reactivities of the sulfate conjugates formed may explain organotropic effects of the compounds activated via sulfotransferases.
Top-cited authors
Nihal Ahmad
  • University of Wisconsin–Madison
Charles N Serhan
  • Brigham and Women's Hospital
Mauro Perretti
  • Queen Mary, University of London
Miral Dizdaroglu
  • National Institute of Standards and Technology
Mark Evans
  • De Montfort University