Biochemical Journal

Published by Portland Press
Online ISSN: 1470-8728
Print ISSN: 0264-6021
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The question of a role for water in biochemical and cellular events is ignored by most workers (apart from its obvious role in hydrolysis reactions, which is not under discussion here). But much recent research has pointed to the importance of physical, as well as biochemical, processes of the cell, which focus attention on such straightforward elementary questions as position and relationship in space of cell components. In this communication these questions are examined in terms of a new model of water structure. A radically new feature of this model is that water clusters have long-term rather than flickering existence and are as large as the macromolecular components of the cell. These properties allow the clusters and other components to pack together spatially so giving rise to integrated, large-scale, subcellular structures.
 
A method is described where leaching of the column resin by pyridine-acetate buffers is minimal and excessive rise of the base-line of the chromatogram is avoided. The method can be used to fractionate peptide mixtures at a concentration of 2-20nmol/component.
 
Under defined conditions liver mitochondria from hypothyroid rats show an apparent lowering of the ADP/O ratio, which can be corrected by addition in vitro of 0.1 nM-tri-iodothyronine (T3). Nicotinamide prevents this restoration by hormone, lowers the ADP/O ratio of euthyroid-rat mitochondria to hypothyroid-rat values and induces T3-sensitivity in euthyroid-rat mitoplasts indistinguishable from that found with hypothyroid-rat preparations. Incorporation into the trichloroacetic-acid insoluble fraction of mitoplasts and hypothyroid-rat mitochondria of radiolabel from [adenine-14C]-NAD+ was stimulated by T3: this stimulation was abolished by nicotinamide. The findings strongly suggest that this incorporation occurs external to the matrix. Confirming the work of others, PAGE of radiolabelled mitoplasts shows alkali-labile modification of a major species of approx. 30 kDa: both nicotinamide and T3 abolish this modification. By contrast, T3 promotes incorporation of label into a single major 11 kDa species: this incorporated label is somewhat acid-labile, and the incorporation is abolished by nicotinamide. Comparative electrophoresis of purified sub-mitoplast fractions show that the 11 kDa species is in the inner membrane and absent from the matrix. The findings are consistent with a receptor-mediated ADP-ribosylation mechanism for the rapid action of T3 on mitochondria.
 
The crystal structure of the beta-lactamase of Streptomyces albus G has been solved at 0.3 nm resolution by X-ray-diffraction methods. The enzyme is a typical two-domain protein. One domain consists of five alpha-helices, and the other is five-stranded beta-sheet with alpha-helices on both sides of the sheet. The active-site serine residue (Ser-48) is within a cleft located between the two domains.
 
Region surrounding the metal ion in the Cd(II)enzyme electron-density map Six sections perpendicular to b are shown, each separated by 0.1 nm and contoured in multiples of the r.m.s. error in electron density (broken contour level = r.m.s. error). The scale bar represents 0.2 nm. Numbers indicate the a-carbon atom positions as located in the map-stack, and the letters A, B and C indicate electron-density features that correspond to the side chains of histidine residues 88, 210 and 86 respectively (see the text). A region of solvent surrounding one of the crystallographic 2-fold axes may clearly be seen (lower right).  
Crystals of beta-lactamase II (EC 3.5.2.6., 'penicillinase') from Bacillus cereus were grown with Cd(II) in place of the natural Zn(II) cofactor and stabilized by cross-linking with glutaraldehyde. Their space group is C2, the cell dimensions are a = 5.44 nm, b = 6.38 nm, c = 7.09 nm and beta = 93.6 degrees, and there is one molecule in the asymmetric unit. Diffraction data were collected from cross-linked crystals of the Cd(II)-enzyme, the apoenzyme and six heavy-atom derivatives. The electron-density map calculated at 0.35 nm resolution reveals the essential Cd(II) ion surrounded by three histidine residues and one cysteine residue. The position of a glutamic acid residue, modification of which destroys activity [Little, Emanuel, Gagnon & Waley (1986) Biochem. J. 233, 465-469], suggests the probable location of the active site of the enzyme. Two minor Cd(II) sites not essential for activity were also located. The structure of the apoenzyme at this resolution appears to differ from that of the Cd(II)-enzyme only in the orientation of two of the histidine residues and the cysteine residue that surround the metal ion.
 
Three regions of the fi-lactamase electron-density map Twofold axes are shown, and the molecular boundary is outlined in (b) and (c). (a) Superposition of three sections around x = 0. This is the region around the three intersecting twofold axes where the dimers above and below these sections end, creating a large solvent channel. Density in the comers of the Figure belongs to the molecules related to the central dimer by body centring, and is shown more fully in (c). (b) Superposition of thirteen sections from x = 0.341 nm to x = 2.39nm, showing the bulk of the density for a pair of molecules forming a tight dimer. (c) Three sections around x = i, showing the packing between a dimer, the bulk of the density of which is between x = 0 and this region (outlined), and a dimer related to it by the twofold axes in this plane along y = J and z = i. The crosses mark the binding sites for the active-site-directed compounds on the two molecules of the dimer. 
The preparation, crystallization and low-resolution structure determination of beta-lactamase (EC 3.5.2.6, 'penicillinase') from Staphylococcus aureus is described. The enzyme crystallizes in space group I222 with 1 molecule per asymmetric unit and cell dimensions a = 5.45(1), b = 9.39(1) and c = 13.87(2) nm. The structure was determined at 0.5 nm resolution by using phases calculated from (NH4)2Pt(CN)4 and KAu(CN)2 derivatives. The mean figure of merit mean value of m, for the 1106 reflexions used was 0.70. Difference Fourier syntheses for data collected from crystals soaked in platinum D-methionine and in 6-(4-hydroxy-3,5-di-iodobenzamido)penicilloic acid revealed the likely position of the active site of the enzyme.
 
Portion of the electron-density map for GDH The phases derived from the combination of isomorphous replacement and molecular averaging were used to compute a Fourier map at 0.6 nm resolution. This map was averaged about the non-crystallographic threefold axis and plotted in the molecular frame. Sections 15/54 to 19/54 of this map are shown. The long rod of density dominating the view is a segment of helix approx. 4.0 nm in length. 
Quaternary structure of GDH 
Location of the GDH active site A view of the cleft between the two domains in GDH is shown including the bound NAD moiety (darker colour). The probable location of the active site deduced from the fitted position of the nicotinamide ring and a knowledge of the stereospecificity of the hydrogen-transfer reaction is shown as a white dot. 
The structure of glutamate dehydrogenase from Clostridium symbiosum has been solved by single-crystal X-ray-diffraction studies at 0.6 nm resolution by using a combination of isomorphous replacement and molecular averaging. The electron-density map reveals that this glutamate dehydrogenase is a hexameric oligomer, arranged in 32 symmetry, of cylindrical appearance and dimensions, of length 10.8 nm and radius 4.4 nm. From an analysis of this map each subunit appears to contain some 55% alpha-helix and is organized into two distinct globular domains separated by a deep cleft. The subunits associate using the domain closest to the 32-symmetry point, making intimate contacts around the threefold and twofold interfaces. The second domain shows structural homology to the NAD-binding domain of other dehydrogenases, and difference Fourier analysis has shown that the NAD is bound in both a structurally equivalent position and a similar conformation to that observed for those related enzymes.
 
Endoglycosidase H treatment of insulin-receptor components labelled with 13Hlmannose The insulin receptors were pulse-chase labelled with {3H]mannose as described in the legend to Fig. 1. Chase time points were 1 h for the high-and low-density microsomal fractions and 4 h for the plasma-membrane samples. The enzymic digestion (Endo H; 500 munits/mi for 4 h at 37 °C) was performed in the presence of 0.25% SDS after release of the receptors from the immunoadsorbent. The reaction was terminated by precipitation of the proteins with 10% trichloroacetic acid. The precipitates were dissolved in electrophoresis buffer containing 2 % SDS and analysed by electrophoresis and fluorography. 
We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.
 
Hepatic microsomal glucose-6-phosphatase activity was rendered extremely unstable by a variety of techniques: (a) incubation at pH 5.0; (b) extraction of the microsomal fraction in the presence of 1% Lubrol; (c) various purification procedures. These techniques all result in the removal of a 21 kDa polypeptide from the fraction containing glucose-6-phosphatase activity. The 21 kDa protein was purified to apparent homogeneity by solubilization in the detergent Lubrol 12A-9 and chromatography on Fractogel TSK DEAE-650(S) and centrifugation at 105 000 g. The 21 kDa protein stabilizes glucose-6-phosphatase activity, whereas other purified hepatic microsomal proteins do not. The 21 kDa protein appears to be a potential regulator of glucose-6-phosphatase activity.
 
Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.
 
The physicochemical properties of a novel Mr-21 000 Ca2+-binding protein isolated from bovine brain were investigated. The protein exhibited a partial specific volume of 0.724 ml/g, a degree of hydration of 0.47 g of water/g of protein and a mean residue weight of 119. Sedimentation equilibrium analysis revealed Mr = 22 600 in the absence of Ca2+; Ca2+ binding appeared to induce dimerization of the molecule. Size-exclusion chromatography indicated a compacting of the molecule on binding of Ca2+: the Stokes radius decreased from 2.75 nm in the absence of Ca2+ to 2.56 nm in its presence. Far-u.v.c.d. spectroscopy showed the apoprotein to be composed of 44% alpha-helix, 18% beta-pleated sheet and 38% random coil. Addition of either KCl (0.1 M) plus Mg2+ (1 mM), or Ca2+ (2 mM), changed the conformation to 49% alpha-helix, 18% beta-pleated sheet and 33% random coil. Near-u.v.c.d. and u.v. difference spectroscopy both indicated perturbations in the environments of all three types of aromatic amino acids on binding of Ca2+. Ca2+ binding also resulted in a 30% enhancement in the tryptophan fluorescence emission intensity. Ca2+ titration of the far-u.v.c.d. and fluorescence enhancement provided KD values of 9.91 microM and 4.68 microM respectively. Finally, the protein was shown to bind Zn2+ with KD = 1.44 microM (no Mg2+) and 1.82 microM (+ Mg2+). These observations strongly support the possibility that this novel Ca2+-binding protein resembles calmodulin and related Ca2+-binding proteins and undergoes a conformational change on binding of Ca2+ which reflects a physiological role in Ca2+-mediated regulation of brain function.
 
Addition of lutropin (luteinizing hormone, 'LH') and 3-isobutyl-1-methylxanthine to tumour Leydig cells stimulated phosphorylation of five proteins, of 17 000, 22 000, 24 000, 33 000 and 57 000 Da. Phosphorylation of these proteins coincided with increased pregnenolone production. Phosphorylation of a 33 000-Da protein was lutropin-dependent in Leydig cells isolated from a Leydig-cell tumour, from immature testes or from mature testes. In tumour Leydig cells this protein was present in the small ribosomal subunit. Incubation of tumour Leydig cells with either cycloheximide or puromycin inhibited both basal and lutropin-dependent pregnenolone production, by approx. 90% and 98% respectively. In contrast, basal pregnenolone production in Leydig cells from immature and mature testes was insensitive to cycloheximide or puromycin. Cycloheximide or puromycin increased phosphorylation of the 33 000-Da phosphoprotein by approx. 130% and 80% respectively (effect of lutropin/3-isobutyl-1-methylxanthine on phosphorylation: 100%). The molecular mass, the subcellular localization and the sensitivity to phosphorylation in the presence of inhibitors of protein synthesis indicate that the 33 000-Da protein could be similar to ribosomal protein S6.
 
A gelatin-binding 95000-Mr protein was detected in human serum and plasma by immunoblotting using antibodies against the 95000-Mr gelatin-binding protein, a major secretory component of cultured adherent human monocyte/macrophages. Serum and plasma were prepared by incubating blood at 4, 22 or 37 degrees C for different periods of time, and gelatin-binding proteins were isolated from 200 microliter portions by gelatin-Sepharose affinity chromatography. The bound material was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In protein-stained gels, fibronectin and some minor polypeptides were seen, but not the 95000-Mr protein. In immunoblotting of identical serum samples the antibodies detected apparently two closely spaced polypeptide bands at Mr95000, and in plasma samples a single band at the position of the faster-migrating one of the two above-mentioned bands. The immunoperoxidase reaction was stronger when serum and plasma were prepared by incubating for longer periods of time (up to 8 h) or at higher temperatures (up to 37 degrees C). In samples made from plasma, the immunoperoxidase reactions were weaker than in those from serum, indicating a lower quantity of the protein. The results suggest that the 95000-Mr protein is released from monocytes and granulocytes during the incubation of blood and, more likely, when they possibly interact with the blood clot and may become adherent.
 
Cytochrome c3 (M(r) 26000) isolated from Desulfovibrio gigas is a dimeric cytochrome consisting of two identical subunits of 109 amino acids, each of which contains four haem groups. On the basis of its amino acid sequence, this cytochrome clearly belongs to the cytochrome c3 superfamily, and will be classified in class III of the c-type cytochromes as defined by Ambler [(1980) in From Cyclotrons to Cytochromes (Robinson, A. B. and Kaplan, N. O., eds.), pp. 263-279, Academic Press, London]. It contains ten cysteine and nine histidine residues in each subunit, and eight cysteines and eight histidines linked to the four haem groups were found to be invariant on alignment of all known cytochrome c3 sequences. Two intermolecular disulphide bridges have been determined between cysteine residues 5 and 46 of the two monomers. Cytochrome c3 (M(r) 26,000) from D gigas is clearly different from cytochrome c3 (M(r) 13,000) from the same strain, with which it shows only 27% sequence identity. Compared with cytochrome c3 (M(r) 26,000) from D. desulfuricans Norway, the three-dimensional structure of which has been determined, 26.95% of the residues have been conserved. In the enzyme from D. desulfuricans Norway, hydrophobic interactions have been described across the dimer interface. Residues involved in similar interactions seem to be well conserved in the equivalent D. gigas cytochrome. This sequence provides structural data to allow specification of this new subclass of polyhaem cytochromes. Furthermore, D. gigas cytochrome c3 (M(r) 26,000) is the first polyhaem cytochrome shown to contain two disulphide bridges linking two identical subunits, which could induce more rigid folding. The folding and the evolution of this family of polyhaem cytochromes are discussed.
 
Identification ofa J1OkDa endogenous phosphoprotein in cell extracts derivedfrom rat hepatocytes and rabbit brown adipose tissue Samples of wheat-germ-lectin-purified glycoproteins obtained from rat hepatocytes (lanes a-d) or rabbit brown adipose tissue (lanes e andj) were incubated for 90min at 20°C without or with insulin (1 uM), and used in a cell-free phosphorylation assay (15min at 20°C). The reaction was stopped by addition of boiling SDS solution, and the samples were analysed by SDS/polyacrylamide-gel electrophoresis under reducing conditions (lanes a, b, e,J) or nonreducing conditions (lanes c and d) followed by autoradiography. Key, OR, origin; pp I 10, 1 IO kDa phosphoprotein; f,, receptor fl-subunit. Numbers to the left of gels are Mr values (x 10-3) of standards.  
The JJOkDa phosphoprotein derivedfrom rat liver is not immunoprecipitated by antibodies to insulin receptor Samples of wheat-germ-purified glycoproteins obtained from rat hepatocytes were incubated for 90 min at 20°C with insulin (I pM) and used in a cellfree phosphorylation assay (15min at 20°C). The reaction was stopped by adding an ice-cold stopping solution (NaF, 100mM; EDTA, 20mM), and thereafter the samples were exposed to normal serum (lanes a, c) or to serum containing autoantibodies to the insulin receptor (patient B-9; Anti-R) at a 1: 300 dilution (lanes b, d). Immunoprecipitation was achieved by addition of protein A. The immunoprecipitates (lanes a, b) and their supernatants (lanes c, d) were analysed by SDS/polyacrylamidegel electrophoresis under reducing conditions, followed by autoradiography. For key see Fig. I legend.
Addition of insulin to wheat-germ-lectin-purified glycoproteins derived from rat hepatocytes or rabbit brown adipose tissue results in the increased phosphorylation of a Mr-110 000 protein. This naturally occurring glycoprotein appears as a monomeric structure and is not part of the insulin receptor itself, since it is not immunoprecipitated by highly specific antibodies to insulin receptor. Phosphorylation of the Mr-110 000 protein and autophosphorylation of the receptor beta-subunit (Mr 95 000) are stimulated by insulin in a remarkably similar dose-dependent fasion, with half-maximal stimulation at 1 nM-insulin. Further, kinetic studies suggest that the phosphorylation of the Mr-110 000 protein occurs after autophosphorylation of the insulin-receptor kinase. In conclusion, the present identification of an endogenous substrate for the insulin-receptor kinase could suggest that some, if not all, effects of insulin may be mediated through activation of this kinase.
 
mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of approximately 10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.
 
The human MICA gene, encoding a ligand for the NKG2D receptor, is highly polymorphic. A group of MICA alleles, named MICA 5.1 (prototype, MICA*008), produce a truncated protein due to a nucleotide insertion in the transmembrane domain. These alleles are very frequent in all the human populations studied and they have different biological properties, compared to full-length alleles, eg. recruitment into exosomes, which makes them very potent for downmodulating the NKG2D receptor in effector immune cells. Moreover, MICA*008 is not affected by viral immune evasion mechanisms that target other MICA alleles. Here we demonstrate that MICA*008 acquires a glycosyl-phosphatidyl-inositol (GPI) anchor and that this modification is responsible for many of the distinct biological features of the truncated MICA alleles, including recruitment of the protein to exosomes. MICA*008 processing is also unusual as it is observed in the Endoplasmic Reticulum as a Triton X-114 soluble protein, partially undergoing GPI modification while the rest is exocytosed, suggesting a new model for MICA*008 release. This is the first report of a GPI-anchored MICA allele. The finding that this modification occurs in both families of human of NKG2D-ligands, as well as in the murine system, suggests positive pressure to maintain this biochemical feature.
 
The NUAK1 and NUAK2 are members of the AMPK family of protein kinase that are activated by the LKB1 tumour suppressor kinase. Recent work suggests they play roles in regulating key biological processes including Myc driven tumourigenesis, senescence, cell adhesion and neuronal polarity. We describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms, whereas HTH-01-015 inhibits only NUAK1. These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases tested including 10 AMPK family members. WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterised substrate namely MYPT1 that is phosphorylated by NUAK1 at Ser445. We identify a mutation that does not affect basal NUAK1 activity but renders it resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating phosphorylation of MYPT1 we find that in cells overexpressing drug resistant NUAK1, but not wild type NUAK1, phosphorylation of MYPT1 at Ser445 is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to mouse embryonic fibroblasts (MEFs) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1 knock-out. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knock-down. WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knock-down. Our data indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAKs .
 
Overview of PDK1-kinase domain bound to staurosporine  
PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors.
 
Distribution of 35S-labelled macromolecules in the cell layer of confluent UMR 106-01 cells after 24 h labelling 
Disaccharide analysis of CS PGs Distribution of 3H radioactivity is expressed as a percentage of total radioactivity co-eluting with disaccharide standards. Samples are representative of many similar analyses. 
The proteoglycans synthesized by an osteoblast-like cell line of rat origin (UMR 106-01) were defined after biosynthetic labelling with [35S]sulphate and [3H]glucosamine. Newly synthesized labelled proteoglycans were characterized by differential enzymic digestion in combination with analytical gel filtration and SDS/PAGE. UMR 106-01 cells were found to synthesize three major species of proteoglycan: a large chondroitin sulphate proteoglycan of Mr approximately 1 x 10(6), with a core protein of Mr approximately 350,000-400,000; a small chondroitin sulphate-containing species of Mr approximately 120,000 with a core protein of Mr 43,000; and a heparan sulphate proteoglycan of Mr approximately 150,000, with a core protein of Mr approximately 80,000. Over 70% of the newly synthesized intact proteoglycan species are associated with the cell layer of near-confluent cells; however, accessibility to trypsin digestion suggests an extracellular location. Chemical characteristics of the proteoglycans and preliminary mRNA hybridization indicate that the small chondroitin sulphate proteoglycan is probably PG II (decorin). The large chondroitin sulphate proteoglycan is most likely related to a hyaluronate-aggregating species from fibroblasts (versican), and the heparan sulphate proteoglycan bears striking similarities to cell-membrane-intercalated species described for a number of cell types.
 
Superose 6 chromatography of trypsin-released heparan sulphate proteoglycan (a) Before heparitinase digestion; (b) after heparitinase digestion; (c) heparan sulphate proteoglycan isolated as in (a) without prior treatment with trypsin and chromatographed on Superose 6 after heparitinase digestion. The arrow indicates heparan sulphate core protein. Symbols are as for Fig. 2. 
The heparan sulphate (HS) proteoglycans associated with the cell layer of a rat osteosarcoma cell line [UMR 106-01 (BSP)] were compared with similar cell-associated proteoglycans from other cells, and their interaction with the plasma membrane was studied. HS proteoglycans were metabolically labelled by incubation of cell cultures with [3H]glucosamine or [3H]leucine and [35S]sulphate. HS proteoglycan core protein preparation generated by heparitinase digestion of the major species from UMR 106-01 (BSP) cells co-migrated on PAGE with identical preparations from ovarian granulosa cells and parathyroid cells (at approximately 70 kDa). The hydrophobic nature of the major HS proteoglycans from these diverse cell lines, based on elution position from octyl-Sepharose, were also comparable. Linkages of the HS proteoglycan to the cell membrane were investigated by labelling plasma-membrane preparations with a lipid soluble photoactivatable reagent, 3-(trifluoromethyl)-3- (m-[125I]iodophenyl)diazirine (TID), which selectively labels plasma-membrane-spanning peptide domains. Purified HS proteoglycan from UMR 106-01 (BSP) cells was shown to be accessible to the [125I]TID, and the core protein portion of the molecule was labelled, confirming its close association with the plasma membrane. Approx. 36% of 35S-labelled HS proteoglycans were released from the cell surface by phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol-linked proteins. In the presence of insulin, the metabolism of the phospholipase C-sensitive population was unaltered; however, release of the phospholipase C-insensitive population into the medium was increased. These data indicate that a subpopulation of HS proteoglycans are covalently bound to the plasma membrane by a glycosylphosphatidylinositol structure, with the remainder representing those species directly inserted into the plasma membrane via a hydrophobic peptide domain. These observations are similar to those reported for ovarian granulosa cells [Yanagishita & McQuillan (1989) J. Biol. Chem. 264 17551-17558], and thus may represent a general phenomenon for many cell types.
 
A structural study was performed by 13C-n.m.r. spectroscopy and methylation analysis of the O-chain of lipopolysaccharide (LPS) from Vibrio bioserogroup 1875 possessing antigenic factor(s) in common with O1 Vibrio cholerae. It was demonstrated to contain a linear homopolymer of (1-->2)-linked N-3-hydroxypropionyl-alpha-D-perosamine [4-(3-hydroxypropanamido)-4,6-dideoxy-alpha-D-mannopyranose], which is very similar to, but not identical with, both (1-->2)-linked linear N-3-deoxy-L-glycero-tetronyl(S-2,4-dihydroxybutyryl)-alpha-D - perosamine homopolymer and (1-->2)-linked linear N-acetyl-alpha-D-perosamine homopolymer which constitute the O-chains of O1 V. cholerae and non-O1 V. cholerae bioserogroup Hakata LPS respectively.
 
l(2)01810 causes glutamine-dependent megamitochondrial formation when it is overexpressed in Drosophila cells. In the present study, we elucidated the function of l(2)01810 during megamitochondrial formation. The overexpression of l(2)01810 and the inhibition of glutamine synthesis showed that l(2)01810 is involved in the accumulation of glutamate. l(2)01810 was predicted to contain transmembrane domains and was found to be localized to the plasma membrane. By using (14)C-labelled glutamate, l(2)01810 was confirmed to uptake glutamate into Drosophila cells with high affinity (K(m)=69.4 μM). Also, l(2)01810 uptakes glutamate in a Na(+)-independent manner. Interestingly, however, this uptake was not inhibited by cystine, which is a competitive inhibitor of Na(+)-independent glutamate transporters, but by aspartate. A signal peptide consisting of 34 amino acid residues targeting to endoplasmic reticulum was predicted at the N-terminus of l(2)01810 and this signal peptide is essential for the protein's localization to the plasma membrane. In addition, l(2)01810 has a conserved functional domain of a vesicular-type glutamate transporter, and Arg(146) in this domain was found to play a key role in glutamate transport and megamitochondrial formation. These results indicate that l(2)01810 is a novel type of glutamate transporter and that glutamate uptake is a rate-limiting step for megamitochondrial formation.
 
In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium mutant M-0198, isopenicillin N was converted into a penicillinase-resistant material that behaved like deacetoxycephalosporin C on high-pressure liquid chromatography analysis. This activity was found to be unstable to storage at -80 degrees C; 70-80% of the activity was lost after 1 day.
 
Properties of trappin family members and SLPI Abbreviation : HUSI, human seminal fluid inhibitor. 
Interaction between elastase and the inhibitory domain of trappin-2 (SKALP/elafin) (a) Backbone drawing of the inhibitory domain of human trappin-2 (SKALP/elafin). The domain indicated (Thr 11-Gln 57 ) corresponds to residues 71-117 of the trappin-2 precursor. Residues involved in the interaction with elastase are labelled. Ala 24 , Met 25 and Leu 26 represent the variable region indicated in Figure 1(e). (b) Backbone drawing of pig pancreatic elastase (residues Val 16-Asn 245 ). The catalytic triad, comprising Asp 102 , His 56 and Ser 195 , in the active centre is highlighted. (c) Structure of the trappin-2-elastase complex determined by X-ray crystallography [56]. 
Structure and evolution of trappin genes (a) Structure of the gene and biosynthetic processes of human trappin-2. The untranslated regions are depicted in white, the presequence in grey, the transglutaminase (TGase) substrate domain in pink and the WAP motif in black. (b) Evolutionary history of trappin genes. 
Recently, several new genes have been discovered in various species which are homologous to the well-characterized human epithelial proteinase inhibitor elafin/SKALP (skin-derived anti-leukoproteinase). Because of the high degree of conservation and the similarities in genomic organization, we propose that these genes belong to a novel gene family. At the protein level, the family members are defined by: (1) an N-terminal domain consisting of a variable number of repeats with the consensus sequence Gly-Gln-Asp-Pro-Val-Lys that can act as an anchoring motif by transglutaminase cross-linking, and (2) a C-terminal four-disulphide core or whey acidic protein (WAP) domain, which harbours a functional motif involved in binding of proteinases and possibly other proteins. We have proposed the name trappin gene family as a unifying nomenclature for this group of proteins (trappin is an acronym for TRansglutaminase substrate and wAP domain containing ProteIN, and refers to its functional property of 'getting trapped' in tissues by covalent cross-linking). Analysis of the trappin family members shows extensive diversification in bovidae and suidae, whereas the number of primate trappins is probably limited. Recent biochemical and cell biological data on the human trappin family member elafin/SKALP suggest that this molecule is induced in epidermis by cellular stress. We hypothesize that trappins play an important role in the regulation of inflammation and in protection against tissue damage in stratified epithelia.
 
FVII activation by FXa 
FX activation by soluble TF-FVIIa complex FXa generation was assayed in reaction buffer using 100 nM FX, 10 nM soluble TF, 100 µM PCPS and 1 nM FVIIa. The FVIIa proteins included wild-type FVIIa ($), Novoseven TM ( ), FVII(a) A294V (>). Aliquots of the reaction were quenched at time points between 0 and 3.5 min using 20 mM Hepes/150 mM NaCl/50 mM EDTA/0.1 % PEG 8000, pH 7.4. Spectrozyme fXa (100 µM) was used to determine FXa generation.
FIX activation by soluble TF–FVIIa complex 
Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa-TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa-TF complex, but also in the interaction of FVII with both its activators and cofactor TF.
 
Molecular modelling of the 4-52 amino acid sequence of the Ntermlnal lobe of hLf (a) and the corresponding regions of the N-terminal lobe of bLf (b), the N-terminal lobe of rabbit serum transterrin (c) and the Cterminal lobe of hit (d) Arrowheads in (a) indicate the 28-34 loop region of hLf. 
The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties. A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence. Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM). Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS. Like hLf, bLf also contains a low- and a high-affinity LPS-binding site. The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf. Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction. Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe. The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding. Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.
 
Parathyroid hormone (PTH) and PTH-related protein (PTHrP) act via PTH receptors in bone to stimulate bone resorption. Bone resorption is also stimulated by certain cytokines, which are produced in bone and bone marrow. The effects of such cytokines on the PTH-receptor system were studied in the osteoblast-like osteosarcoma cell line UMR 106-06. 125I-labelled PTHrP-(1-84)-peptide bound specifically to the cells, and PTHrP-(1-34) and -(1-84) competed with equimolar affinity for binding to UMR 106-06 cells. The specific binding of 125I-PTHrP-(1-84) could be completely blocked by PTH. Therefore 125I-PTHrP-(1-84) bound to a classical receptor in UMR 106-06 cells. Preincubation for 3 days with either tumour necrosis factor alpha (TNF alpha) or retinoic acid (RA) both decreased the specific binding of 125I-PTHrP-(1-84) to about 40% of control levels. These effects were specific for PTH binding, since there was little effect on 125I-salmon-calcitonin binding. Both TNF alpha and RA required 24 h exposure to cells to produce a measurable effect. The decrease in 125I-PTHrP-(1-84) binding was due to a reduced number of binding sites, with little apparent change in affinity. Half-maximal effects were seen with 1 ng of TNF alpha/ml, whereas 1 microM-RA was needed to observe the loss of PTH receptors. Combinations of RA and TNF alpha produced a greater effect than that of either agonist alone. The loss of PTH receptors was accompanied by a specific loss of PTH-stimulated cyclic AMP production. Preincubation with TNF alpha increased the basal plasminogen activator (PA) activity in the cells and decreased the amplitude of the response of PA activity to PTH compared with control cells. Furthermore TNF alpha decreased sensitivity to PTH (50% stimulation of PA activity with 0.1 nM-PTH in control cells versus 50% stimulation with 0.3 nM-PTH in TNF alpha-treated cells). In contrast, TNF alpha pretreatment increased the amplitude of the response of PA activity to calcitonin, whereas sensitivity to calcitonin was not altered. These data are consistent with a specific down-regulation of PTH receptors in osteoblast-like UMR 106-06 cells after exposure to TNF alpha or RA. The loss of PTH receptors is accompanied by a decreased responsiveness to PTH, as measured with the PA system in these cells. A loss of PTH receptors could modulate PTH responses in osteoblasts, either in the local control of bone formation and resorption, or in pathological conditions such as humoral hypercalcaemia of malignancy.
 
1. Gliorosein was obtained in excellent yield (150mg./200ml. of Raulin-Thom medium) from surface cultures of Gliocladium roseum. Its nuclear-magnetic-resonance spectrum showed conclusively that it is 1,6-dihydro-3,4-dimethoxy-6-methyltoluquinone. 2. Sodium [2-(14)C]acetate was incorporated into gliorosein and the related products (3.3% conversion). The specific activities of these substances increased in the order gliorosein, 3,4-dimethoxy-6-methyltoluquinol, the related quinhydrone and quinone, indicating that gliorosein was the actual metabolite that was secreted and that the other compounds were derived from it in the medium. 3. 6-Methylsalicylic acid was not taken up by the mycelium and could be recovered unchanged. Orsellinic acid was decarboxylated by G. roseum and an equivalent amount of orcinol was secreted into the medium. The methyl esters of 6-methylsalicylic acid and orsellinic acid were both hydrolysed by an esterase present in the mycelium. Some of the 6-methylsalicylic acid thus produced was secreted into the medium and the orsellinic acid was decarboxylated. 4. Washed mycelium of G. roseum converted aurantiogliocladin and 3,4-dimethoxy-6-methyltoluquinol quantitatively into gliorosein within 18hr. More critical experiments with (14)C-labelled substrates demonstrated that 3-hydroxy-4-methoxy-6-methyltoluquinol and 3,4-dimethoxy-6-methyltoluquinol, and their respective quinones, were effectively incorporated into gliorosein and related products (49, 68, 30 and 57% respectively). 5. The following sequence of reactions is proposed for the biosynthesis of gliorosein: acetyl-CoA+3 malonyl-CoA+S-adenosyl-methionine --> 5-methylorsellinic acid --> 3-hydroxy-4-methoxy-6-methyltoluquinol --> 3,4-dimethoxy-6-methyltoluquinol --> gliorosein. 6. Since gliorosein is optically active (dextrorotatory), the final tautomerization reaction leading to its formation must be enzyme-catalysed.
 
The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Here we examine the possibility that ubiquitin-proteasome is involved in regulating the levels of Bcl-2, which is abundantly expressed in M-07e cells, a granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent human leukaemic cell line. Apoptosis in M-07e cells, induced by GM-CSF withdrawal, was associated with a gradual cleavage of Bcl-2 into a 22 kDa fragment. Treatment of M-07e cells with benzyloxycarbonyl-Leu-Leu-l-leucinal (Z-LLL-CHO; MG-132), a reversible ubiquitin-proteasome inhibitor, markedly accelerated the cleavage of Bcl-2 and promoted cell death through the apoptotic pathway. The cleavage of Bcl-2 was inhibited by a caspase-3 (CPP32)-specific inhibitor [acetyl-Asp-Glu-Val-Asp-CHO (DEVD-CHO)] but not caspase 1 inhibitor (acetyl-Tyr-Val-Ala-Asp-CHO), suggesting that Bcl-2 is a proteolytic substrate of a caspase-3-like protease activated during apoptosis. The simultaneous addition of recombinant human GM-CSF (rhGM-CSF) to M-07e cultures delayed the activation of caspase 3 and Bcl-2 cleavage triggered by Z-LLL-CHO, suggesting that the activation of the GM-CSF signalling pathway can partly overcome the apoptotic effect induced by Z-LLL-CHO. Apoptosis induced by inhibition of the proteasome pathway was verified in studies with lactacystin, a highly specific and irreversible proteasome inhibitor. Lactacystin-induced apoptosis in M-07e cells was remarkably similar to that induced by Z-LLL-CHO, which included caspase 3 activation, cleavage of Bcl-2 into a 22 kDa fragment and, ultimately, cell death. These results showed that inhibition of the ubiquitin-proteasome pathways can lead to the activation of a DEVD-CHO-sensitive caspase and induces Bcl-2 cleavage, which might have a role in mediating apoptosis in M-07e cells.
 
Transgenic mouse models of mammary tumorigenesis and analyses of human breast tumour samples have indicated a role for Src proteins in the tumorigenic process. The downstream effectors of Src function in mammary epithelial cells are less well understood. STAT proteins constitute a family of transcription factors whose activation by cytokine and non-cytokine receptors leads to tyrosine phosphorylation, dimerization and translocation from the cytoplasm to the nucleus. In the nucleus they activate the transcription of specific genes by binding to consensus DNA elements. STATs 1 and 3 can be activated by both cytokine and non-cytokine receptors, and bind as homodimers or heterodimers to viral simian sarcoma virus (sis)-inducible elements such as that found in the c-fos promoter. Here we report that one of the downstream effectors of Src function in mammary epithelial cells is STAT3. We demonstrate that v-src expression in mammary epithelial cells induces Tyr-705 phosphorylation, nuclear translocation and DNA binding of STAT3. Furthermore, we demonstrate that v-src can induce STAT3-dependent transcription. These observations are the first direct evidence that v-src can regulate transcription through the activation of STAT proteins, and add a further level of complexity to the understanding of the mode of action of v-src.
 
The effects in vivo of dichloromethanediphosphonate and 1-hydroxyethane 1,1-diphosphonate on collagen solubility, hydroxylation of lysine and proline and on the formation of collagen intermolecular cross-links were studied by using rat bone, cartilage and skin tissues. Dichloromethanediphosphonate decreased bone collagen solubility both in acetic acid and after pepsin treatment. Although none of the diphosphonates had any effect on the hydroxylation of proline, dichloromethane-diphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, increased the number of hydroxylysine residues in the alpha-chains of bone, skin and cartilage collagen. The stimulatory effect was dose-dependent. The dichloromethanediphosphonate-mediated increase in hydroxylysine residues in bone and cartilage was manifested in an increase of dihydroxylysinonorleucine, the cross-link that is formed by the condensation of two hydroxylysine residues. The cross-link hydroxylysinonorleucine, a condensation product of hydroxylysine and lysine, on the other hand, was decreased. The total number of intermolecular cross-links was not changed by the diphosphonate.
 
Investigations were carried out to assess the effects of disodium 1-hydroxyethane-1,1-diphosphonate and disodium dichloromethanediphosphonate (compounds containing a P-C-P bond) on isolated rabbit articular chondrocytes in culture. Studies on growth behaviour showed that both diphosphonates displayed inhibitory actions, dichloromethanediphosphonate producing the larger effect. Both compounds inhibited the uptake of 2-deoxy-d-glucose, dichloromethanediphosphonate once more being the more potent of the two. The uptake of alpha-aminoisobutyrate was considerably increased by chondrocytes treated with dichloromethanediphosphonate, whereas 1-hydroxyethane-1,1-diphosphonate showed no effects. The biosynthesis of sulphated extracellular macromolecules secreted by the cells into the pericellular space as well as into the growth medium was greatly increased by dichloromethanediphosphonate but not by 1-hydroxyethane-1,1-diphosphonate. The stimulatory effect was dose-dependent. Short-term exposure of already confluent cells to dichloromethanediphosphonate as opposed to growing the cells to confluence in the presence of the diphosphonate revealed that the stimulatory effects were already evident after 24h, indicating that cell division is not necessarily required to produce the observed effects. The increment in proteoglycan synthesis was still evident with cells that were exposed continuously to the diphosphonate in primary as well as secondary culture. Pulse-chase experiments together with studies on the enzyme arylsulphatase revealed that the appearance of increased amounts of proteoglycans was the result of a stimulation in synthesis and not due to an inhibition in turnover.
 
Investigations were performed to assess the effects of dichloromethanediphosphonate on the synthesis of collagen by (1) isolated rabbit articular chondrocytes, (2) isolated rat calvaria bone cells and (3) bone explants from rats treated with the diphosphonates. The studies showed that dichloromethanediphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, causes articular chondrocytes to increase net collagen biosynthesis, both when measured as 3H-labelled or as non-radioactive material, in a dose-related fashion. The increment in collagen synthesis was still evident with cells that were exposed continuously to the diphosphonate in primary as well as secondary culture; however, it declined with cells in tertiary culture and was absent after the fourth subculture. The type of collagen was not affected by the diphosphonate. The synthesis of collagen by bone cells was likewise increased with dichloromethanediphosphonate. No effects were detected with 1-hydroxyethane-1,1-diphosphonate was tested. Finally, when calvaria and tibiae from diphosphonate-treated rats were cultured in vitro, the positive effect of dichloromethanediphosphonate on collagen synthesis was also evident. 1-Hydroxyethane-1,1-diphosphonate, on the other hand, decreased the incorporation of [3H]proline into the collagen of calvaria and osseous tibial shafts and showed no effect on the collagen synthesis of the cartilaginous tibial heads.
 
Rats were given [1,1-2H2]ethanol in a single dose, and the 2H content was determined in testicular steroids and in organic acids of low molecular mass in the testis, liver and blood. The acids were quantified by capillary gas chromatography/mass spectrometry of t-butyldimethylsilyl derivatives with [2H4]lactate as internal standard. In addition to lactate, pyruvate, 3-hydroxybutyrate and acids of the tricarboxylic acid cycle, the testis was shown to contain 2-hydroxybutyrate, 2-hydroxy-2-methylbutyrate, 2-hydroxyisohexanoate and glycerate. No 2H was found in pregnenolone, 5-androstene-3 beta,17 beta-diol or testosterone, whereas the abundance of monodeuterated molecules of 5 alpha-androstane-3 alpha,17 beta-diol and its 3 beta-isomer were 7.6% and 11.2% respectively. The abundance of monodeuterated lactate was 7.0% in the testis and 5.3% in the blood. The other acids were less labelled but 3-hydroxybutyrate had a higher 2H content in the testis (3.1%) than in the liver. These results support the contention that ethanol is oxidized in an alcohol dehydrogenase-catalysed reaction in testis in vivo and that the acute inhibition of the testosterone production is due at least partly to a redox effect. The labelling and increased concentration of 3-hydroxybutyrate in the testis indicate that a change in the mitochondrial redox state might be involved.
 
Decreased intestinal absorption of Ca2+ occurs in response to treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP). The effect is due to decreased 1-hydroxylation of calcidiol (25-hydroxycholecalciferol) in the kidney. In an attempt to establish whether impairment of vitamin D metabolism at steps beyond kidney hydroxylation occurs due to treatment with EHDP, chicks were depleted of vitamin D and were treated with calcitriol (1,25-dihydroxycholecalciferol) as their sole source of the vitamin. The chicks were then divided into two groups, one being treated with EHDP while the second group served as control. Intestinal absorption of Ca2+ in the EHDP-treated group was found to be impaired, along with decreases in concentrations of calbindin D28K (the 28,000-Mr vitamin D-dependent Ca2+-binding protein). When the chicks were dosed with [3H]calcitriol, significantly lower concentrations of the sterol were detected in the duodena of EHDP-treated birds. Measurement of levels of receptors for calcitriol in duodena showed no difference between groups, but levels of calcitriol in sera were considerably lower in the EHDP-treated group along with the elevated biliary and urinary excretion of glucuronidated conjugates. It is therefore concluded that treatment with EHDP results in increased catabolism of calcitriol in addition to the known suppression of the renal production of the hormone.
 
Top-cited authors
Philip Cohen
  • University of Dundee
Martin D Brand
  • Buck Institute for Research on Aging
Maria Deak
  • Nestlé Institute of Health Sciences S.A.
David G Campbell
  • University of Dundee
Nick Morrice
  • AB-Sciex