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ABSTRACT: Overexpression of the zinc enzyme carbonic anhydrase (CA; EC ) XII is observed in certain human cancers. This bitopic membrane protein contains an N-terminal extracellular catalytic domain, a membrane-spanning alpha-helix, and a small intracellular C-terminal domain. We have determined the three-dimensional structure of the extracellular catalytic domain of human CA XII by x-ray crystallographic methods at 1.55-A resolution. The structure reveals a prototypical CA fold; however, two CA XII domains associate to form an isologous dimer, an observation that is confirmed by studies of the enzyme in solution. The identification of signature GXXXG and GXXXS motifs in the transmembrane sequence that facilitate helix-helix association is additionally consistent with dimeric architecture. The dimer interface is situated so that the active site clefts of each monomer are clearly exposed on one face of the dimer, and the C termini are located together on the opposite face of the dimer to facilitate membrane interaction. The amino acid composition of the active-site cleft closely resembles that of the other CA isozymes in the immediate vicinity of the catalytic zinc ion, but differs in the region of the nearby alpha-helical "130's segment." The structure of the CA XII-acetazolamide complex is also reported at 1.50-A resolution, and prospects for the design of CA XII-specific inhibitors of possible chemotherapeutic value are discussed.
Proceedings of the National Academy of Sciences 09/2001; 98(17):9545-50. · 9.68 Impact Factor
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ABSTRACT: Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.
Proceedings of the National Academy of Sciences 03/2001; 98(4):1918-23. · 9.68 Impact Factor
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ABSTRACT: Carbonic anhydrase XII (CA XII) is a transmembrane glycoprotein with an active extracellular CA domain that is overexpressed on cell surfaces of certain cancers. Its expression has been linked to tumor invasiveness. To characterize its catalytic properties, we purified recombinant secretory forms of wild-type and mutant CA XIIs. The catalytic properties of these enzymes in the hydration of CO(2) were measured at steady state by stopped-flow spectrophotometry and at chemical equilibrium by the exchange of (18)O between CO(2) and water determined by mass spectrometry. The catalysis of CO(2) hydration by soluble CA XII has a maximal value of k(cat)/K(m) at 34 microM(-1) small middle dots(-1), which is similar to those of the membrane-associated CA IV and to soluble CA I. The pH profiles of this catalysis and the catalyzed hydrolysis of 4-nitrophenylacetate indicate that the pK(a) of the zinc-bound water in CA XII is 7.1. His64 in CA XII acts as a proton shuttle residue, as evidenced by the reduced rate constant for proton transfer in the mutants containing the replacements His64 --> Ala and His64 --> Arg, as well as by the selective inhibition of the proton transfer step by cupric ions in wild-type CA XII. The catalytic rate of CO(2) hydration by the soluble form of CA XII is identical with that of the membrane-bound enzyme. These observations suggest a role for CA XII in CO(2)/HCO(3)(-) homeostasis in cells in which it is normally expressed. They are also compatible with a role for CA XII in acidifying the microenvironment of cancer cells in which CA XII is overexpressed, providing a mechanism for CA XII to augment tumor invasiveness and suggesting CA XII as a potential target for chemotherapeutic agents.
Proceedings of the National Academy of Sciences 12/2000; 97(26):14212-7. · 9.68 Impact Factor
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ABSTRACT: A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitochondria isolated from transfected COS cells. Northern blot analyses of total RNAs from mouse tissues and Western blot analyses of mouse tissue homogenates showed differences in tissue-specific expression between CA VB and CA VA. CA VB was readily detected in most tissues, while CA VA expression was limited to liver, skeletal muscle, and kidney. The human orthologue of murine CA VB was recently reported also. Comparison of the CA domain sequence of human CA VB with that reported here shows that the CA domains of CA VB are much more highly conserved between mouse and human (95% identity) than the CA domains of mouse and human CA VAs (78% identity). Analysis of phylogenetic relationships between these and other available human and mouse CA isozyme sequences revealed that mammalian CA VB evolved much more slowly than CA VA, accepting amino acid substitutions at least 4.5 times more slowly since each evolved from its respective human-mouse ancestral gene around 90 million years ago. Both the differences in tissue distribution and the much greater evolutionary constraints on CA VB sequences suggest that CA VB and CA VA have evolved to assume different physiological roles.
Proceedings of the National Academy of Sciences 03/2000; 97(4):1677-82. · 9.68 Impact Factor
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ABSTRACT: Lysosomal beta-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine beta-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human beta-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg-GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that (i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg.
Archives of Biochemistry and Biophysics 01/2000; 372(1):53-61. · 2.93 Impact Factor
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ABSTRACT: Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose that residues Glu(451), Glu(540), and Tyr(504) in hGUSB are involved in catalysis, Glu(451) being the acid-base residue and Glu(540) the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu(451), Glu(540), and Tyr(504) hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. The k(cat)/K(m) values (mM(-1).s(-1)) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu(451) as the acid-base catalyst. We conclude that, like their homologues in E. coli beta-galactosidase, Glu(540) is the nucleophilic residue, Glu(451) the acid-base catalyst, and Tyr(504) is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB.
Journal of Biological Chemistry 09/1999; 274(33):23451-5. · 4.77 Impact Factor
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ABSTRACT: The technique of reverse transcriptase-polymerase chain reaction differential display was used to identify thyroid hormone (TH) responsive mRNAs in the adult rat cerebral tissue. A partial cDNA (0.76 kb) was cloned and sequenced. Comparison of the sequence to the GenBank data base showed almost 100% homology to mouse translational repressor (NAT-1) mRNA 3'-end. In a northern blot analysis this cDNA hybridized with a mRNA whose expression in hyperthyroid rat cerebral tissue was approximately 6-fold higher than in euthyroid rats. The time course studies showed a rapid induction of this mRNA within 3 h following thyroxine administration. This mRNA is widely expressed in various tissues, and in hepatic tissue it is also TH responsive. To determine if TH responsiveness of this mRNA persists during aging, 25-month-old aged rats were studied and the results were compared with those of 4-month-old rats. Unlike young mature rats, the TH responsiveness of NAT-1 mRNA in both the cerebral and hepatic tissue of aged rats was blunted. It is concluded that cerebral tissue in aging rats beyond the developmental stages, like the hepatic tissue, is associated with altered TH responsiveness.
European Journal of Endocrinology 01/1999; 139(6):649-53. · 3.42 Impact Factor
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O Türeci,
U Sahin,
E Vollmar,
S Siemer,
E Göttert,
G Seitz,
A K Parkkila, G N Shah,
J H Grubb,
M Pfreundschuh,
W S Sly
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ABSTRACT: We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30-42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.
Proceedings of the National Academy of Sciences 07/1998; 95(13):7608-13. · 9.68 Impact Factor
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ABSTRACT: To determine if aging in rats is associated with insensitivity of cerebral tissue to thyroid hormones (TH), the expression of a TH responsive protein or (THRP) in cerebral tissue was studied in male Fischer rats at 4, 12 and 24 months of age during euthyroid, hypothyroid and hyperthyroid states. The basal levels of THRP mRNA was significantly increased in 24-month-old and in 12-month-old rats while THRP mass measured by Western blots was decreased compared to 4-month-old rats. Compared to euthyroid rats, hyperthyroidism in 4-month-old rats was associated with 5.1-fold increase in THRP mRNA and 3.7-fold increase in protein content while in hyperthyroid aged rats, the increase of THRP mRNA was only 1.6-fold and the increase in the protein was 2.4-fold. Hypothyroidism did not significantly alter THRP or its mRNA in either young or aged rats. It is concluded that aging in rats is associated with reduced cerebral tissue responsiveness to thyroid hormones.
Brain Research 06/1998; 793(1-2):302-4. · 2.73 Impact Factor
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ABSTRACT: To determine the molecular mechanisms of age-related changes in the expression of glucose transporter 1 (GLUT-1) mRNA in cerebral tissue, male Fischer 344 rats at 4, 12, and 24 months of age were studied. The GLUT-1 mRNA in cerebral tissue was not significantly different among the various age groups. The in vitro translatability of GLUT-1 mRNA of 24-month-old rats (0.867 +/- 0.066 arbitrary units) was significantly lower than that in 4-month-old (1.403 +/- 0.153) P < 0.01 or 12-month-old rats (1.387 +/- 0.122) P < 0.01. The poly (A) tail length of GLUT-1 mRNA decreased from 200-350 nt in 4-month-old rats to only 50-100 nt in 24-month-old rats. Twelve-month-old rats also showed reduced poly (A) tail lengths. The poly (A) tail of G3PDH mRNA was not altered with age. The changes in GLUT-1 mRNA translatability did not correlate with GLUT-1 content in total cerebral tissue homogenate or in isolated cerebral microvessels, suggesting that GLUT-1 protein turnover is altered with age. It is concluded that aging is associated with significant changes in the structure and function of GLUT-1 mRNA.
Proceedings of The Society for Experimental Biology and Medicine 12/1997; 216(3):380-5.
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ABSTRACT: A gene responsive to thyroid hormone (TH) has been identified in the adult rat brain cerebral tissue. A cDNA probe differentially expressed in euthyroid, hypothyroid and hyperthyroid rat cerebral tissue, generated by reverse transcriptase-PCR differential display of mRNA, was used to screen the rat brain cDNA library. A 3.4 kb positive clone hybridized in Northern blots with a 3.8 kb mRNA that proved to be TH responsive (THR). The remaining coding sequence and a part of the 5' untranslated region of this cDNA were obtained by 5' rapid amplification of cDNA ends. The deduced amino acid sequence revealed that THR protein (THRP), a 68 kDa moiety, has 83% sequence similarity with c-Abl interactor protein (Abi-2), which is a substrate for tyrosine kinase activity of c-Abl. The extensive similarity between the two proteins suggests a potential role for THRP as a substrate for c-Abl. Northern analysis showed that the expression of THR mRNA in hyperthyroid rats is 6-fold that in euthyroid rats. There is also a 4-6-fold increase in the concentration of THRP, as analysed by Western analysis. Owing to the extensive similarity between rat THRP and human Abi-2, a polyclonal anti- (human Abi-2) antibody was successfully used for Western analysis of proteins from the rat tissues. The observed increase in both the mRNA and the protein did not decline after beta-adrenergic system blockade with propranolol, suggesting that the action of TH on the expression of this gene is not mediated through the beta-adrenergic system. Immunohistochemical studies revealed that neuronal cells were particularly rich in THRP. Both THR mRNA and THRP are rapidly induced in vivo after intravenous administration of thyroxine. Tissue distribution studies indicated that the cerebral tissue was particularly enriched with THR mRNA and 68 kDa THRP. A cDNA clone for a THR gene could provide a useful tool to study the molecular mechanisms of TH effects on cerebral tissue in adult animals.
Biochemical Journal 11/1997; 327 ( Pt 2):617-23. · 4.90 Impact Factor
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ABSTRACT: To determine if dietary carbohydrates modulate apolipoprotein A1 (ApoA1) expression, plasma ApoA1 protein and hepatic ApoA1 mRNA levels were measured in young and aged rats maintained on a high-fructose (60% of diet weight consisting of fructose), or high-glucose (60% glucose) diet or fed regular rat chow for 10 days. Aged rats on regular chow had significantly higher plasma ApoA1 concentrations and hepatic ApoA1 mRNA than young rats maintained on this diet. Plasma ApoA1 and hepatic ApoA1 mRNA levels in young rats or aged rats maintained on the 60% fructose diet were significantly higher than in rats within the same age group maintained on regular rat chow (P < .01). Similar induction of ApoA1 protein and mRNA was found in rats maintained on the 60% glucose diet (P < .01). It is concluded that ApoA1 expression in rats is modulated by factors related to the nature of dietary carbohydrates rather than insulin resistance associated with high-fructose feeding.
Metabolism 10/1997; 46(10):1132-6. · 2.66 Impact Factor
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ABSTRACT: To determine the molecular mechanisms of diabetes-related changes in the expression of GLUT-1 in cerebral tissue, streptozotocin-induced diabetic rats and vehicle injected controls were studied after 4 weeks of diabetes. The GLUT-1 mass in cerebral microvessels was reduced in diabetic rats by approximately 38% (P < 0.01). The GLUT-1 concentration in insulin-treated diabetic group was not significantly different from controls. The GLUT-1 mRNA content of cerebral tissue in diabetic rats (0.064 +/- 0.007) was significantly reduced compared to control rats (0.122 +/- 0.011) or insulin-treated diabetic rats (0.122 +/- 0.015) P < 0.01. The in vitro translation of GLUT-1 mRNA of diabetic rats (0.793 +/- 0.047 arbitrary units) was also significantly lower than that in control rats (1.403 +/- 0.153) P < 0.01 or insulin-treated diabetic rats. (1.124 +/- 0.083) P < 0.01. These changes occurred in asssociation with a reduction in poly (A) tail length of GLUT-1 mRNA which decreased from a control value of 200-350 nt to only 50-100 nt in diabetic rats. Shortening of poly (A) tail of mRNAs is a novel mechanism of diabetes-related changes in the expression of specific genes which are regulated at a translational level.
Brain Research 04/1997; 754(1-2):213-20. · 2.73 Impact Factor
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ABSTRACT: To determine the age-related changes in the effect of thyroid hormone on apolipoprotein A1 (ApoA1) gene expression, male Fischer 344 rats at 4 (young) and 26 (aged) mo of age were studied. Hyperthyroidism was induced with daily intraperitoneal injections of 3,5,3'-triiodothyronine (15 micrograms/100 g body wt) for 10 days. Hypothyroidism was induced with 0.025% methimazole in drinking water for 4 wk. Hyperthyroidism was associated with increased serum ApoA1 levels in young rats [7.52 +/- 0.41 vs. 3.67 +/- 0.30 optical density (OD); P < 0.01]. The increase in aged rats (6.5 +/- 0.87 vs. 5.14 +/- 0.09 OD) did not reach statistical significance. Hypothyroidism was not associated with significant changes in serum ApoA1 levels in either young or aged rats. Hyperthyroidism was associated with a 2.5-fold increase in ApoA1 mRNA in young rats and a 1.7-fold increase in aged rats. Hypothyroidism was associated with a 3.6-fold reduction in ApoA1 mRNA in young rats, but there was no significant change in aged hypothyroid rats. Mobility shift assays indicated that the binding of transacting factors to ApoA1 promoter increased in hyperthyroid young rats but not in hyperthyroid aged rats. It is concluded that aging in rats is associated with reduced ApoA1 responsiveness to thyroid hormones. This altered responsiveness could partly be the result of changes in the binding activity of nuclear factors to the ApoA1 promoter.
The American journal of physiology 01/1997; 271(6 Pt 2):R1602-7.
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ABSTRACT: To determine if thyroid hormone (TH) modulates the expression of cerebral glucose transporter one (GLUT-1) and whether there are age-related differences in TH effect, young male Fischer 344 rats (6 months old) and aged rats (26 months old) were studied at euthyroid, hyperthyroid and hypothyroid conditions. Immunoblot analysis indicated that 55 kDa GLUT-1 mass was decreased in hypothyroid young rats (174 +/- 15 arbitrary units) and hyperthyroid rats (144 +/- 22) compared to euthyroid young rats (395 +/- 57) P < 0.01. Jr. aged rats the 55 kDa GLUT-1 mass was significantly increased in hyperthyroidism (392 +/- 49) compared to euthyroid aged rats (237 +/- 27) P < 0.05. The 45 kDa isoform of GLUT-1 in rat cerebral tissue did not significantly change with age or thyroidal state. The changes in 55 kDa GLUT-1 mass did not correlate with the changes of GLUT-1 mRNA content. It is concluded that alterations in cerebral GLUT-1 content in response to altered thyroid state are age-specific.
Brain Research 01/1997; 747(1):144-6. · 2.73 Impact Factor
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ABSTRACT: When cDNAs for human and rodent beta-glucuronidases were expressed in COS-7 cells using several different promoters, rodent beta-glucuronidases were produced three times more than human beta-glucuronidase, although their transcriptional levels were similar. Similar observations were also recorded in LMTK- cells using SV40 early or chicken beta-actin promoters. In hopes of enhancing yields of recombinant human beta-glucuronidase for enzyme replacement therapy, we sought to determine the region within the linear sequences responsible for the higher levels of expression of the rodent cDNAs. To do so, we made various rat-human chimeric cDNAs utilizing conserved restriction enzyme sites. The levels of products expressed from these chimeric cDNAs in COS cells were assessed by activity assay and by metabolic labeling of the proteins followed by immunoprecipitation and SDS-PAGE. From the results of these expression studies, we identified a 155-bp ClaI (643)-AflII (797) fragment in the rat open reading frame responsible for the increased rate of translation of the rat beta-glucuronidase (RBG) cDNA. Replacement of the homologous ClaI (683)-AflII (838) fragment in human beta-glucuronidase (HBG) with this 155-bp fragment from RBG increased the translation level of the resulting chimeric HRaH. Conversely, substitution of the 155-bp human fragment for that of rat in RBG cDNA reduced the total synthesis of the resulting chimeric HHaR. Placement of the 155-bp segment between the initiation ATG and the promoter has only negative effects on the expression of either cDNA. A more stable secondary structure of the human cDNA in this region might explain a reduced rate of translation. However, secondary structure analysis of mRNAs from the 155-bp fragment of rat and human cDNAs predicted that, while both can form stem-loops, the rat fragment (delta Gzero = -42.1 kcal/ mol) is actually more stable than the human fragment (delta Gzero = -32.1 kcal/mol). In fact, the free energy of stability of the first 50 bp within this ClaI-AflII fragment from rat (-10.3 kcal/mol) indicates that the secondary structure is considerably more stable than the corresponding 50 bp from human (-2.1 kcal/mol). This segment of the rat sequence also contains a tar-like sequence in a stem-loop. Although tar-like sequences can enhance rates of translation, altering this sequence by mutagenesis had no effect on the rate of synthesis of rat beta-glucuronidase. Thus, although the region conferring enhanced rate of synthesis from rat cDNA has been identified, the mechanism by which it does so is not yet clear.
Archives of Biochemistry and Biophysics 10/1996; 333(2):385-93. · 2.93 Impact Factor
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ABSTRACT: To determine the molecular mechanisms of age-related changes in hepatic albumin gene expression, male Fisher 344 rats at 4, 12, and 24 months of age were studied. The hepatic albumin content was modestly increased by 20% and plasma albumin concentration was not altered with age. There were no age-related changes in albumin mRNA content of the liver. The in vivo synthetic rate of albumin as measured with [14C]-leucin incorporation in 4-month-old rats (350 +/- 24.5 cpm) was not significantly different from that in 24-month-old rats (393 +/- 30.3 cpm). However, in vitro translational efficiency of albumin mRNA from 24-month-old rats was only 49% of that in 4-month-old rats. This was correlated with shortening of the poly(A) tail of albumin mRNA from approximately 300-450 bases in 4-month-old rats to only 50-100 bases in 24-month-old rats. The reduced albumin mRNA translational efficiency in vitro along with shortening of the poly(A) tail with age is probably an adaptive response since hepatic albumin content was increased and plasma albumin concentrations were not altered with age. The age-related shortening of the poly(A) tail is yet another mechanism of age-related alterations in the expression of specific genes where the changes seen with age are mostly translational.
Archives of Biochemistry and Biophysics 01/1996; 324(1):105-10. · 2.93 Impact Factor
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ABSTRACT: To determine the age-related changes in apolipoprotein A-I (ApoA1) expression, male Fischer 344 rats at 4 (young), 12 (intermediate age), and 24-26 (aged) months of age were studied. Immunoblot analysis of plasma proteins indicated that 26-month-old rats (1.79 +/- 0.16 mg/ml) and 12-month-old rats (2.23 +/- 0.11 mg/ml) have significantly higher plasma ApoA1 concentrations compared to 4-month-old rats (1.14 +/- 0.15 mg/ml) P < 0.001. Hepatic ApoA1 mRNA was approx. 2-fold higher in aged rats compared to 12-month-old and 4-month-old rats. This increase in hepatic ApoA1 mRNA in aged rats was also reflected in the increased translation of ApoA1 mRNA in vitro. Reduced mRNA turnover may account for the increased hepatic ApoA1 mRNA content in 26-month-old rats, since the rate of ApoA1 gene transcription as measured with nuclear run off assays was significantly reduced with age. The ApoA1 synthesis in vivo, as measured by [14C]leucine incorporation at 30 min, was reduced in aged rats compared to young rats (170.5 +/- 10.2 vs. 253.9 +/- 7.7 cpm per liver) P < 0.001 probably as a result of changes related to cellular metabolism rather than an alteration inherent to the ApoA1 mRNA translatability. The age-related increase in plasma ApoA1 protein is probably secondary to reduced metabolic clearance rate of ApoA1 protein or is the result of increased intestinal synthesis of ApoA1.
Biochimica et Biophysica Acta 01/1996; 1259(3):277-82. · 4.66 Impact Factor
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ABSTRACT: We used rat pancreatic acini as well as COS-7 cells transfected with the cloned pancreatic cholecystokinin (CCK) receptor and measured the abilities of CCK octapeptide (CCK-8) and L-364,718 (a CCK receptor antagonist) to inhibit binding of 125I-labeled CCK-8 (125I-CCK-8) and [3H]L-364,718. With pancreatic acini 125I-CCK-8 bound to two different states of the CCK receptor. The high-affinity state (1% of the receptors) had a Kd for CCK-8 of 985 pM and the low-affinity state (19% of the receptors) had a Kd for CCK-8 of 30 nM. [3H]L-364,718 bound to low-affinity receptors and to a previously unrecognized very-low-affinity state (80% of the receptors) having a Kd for CCK-8 of 13 microM. L-364,718 had the same affinity (Kd 3 nM) for each of the three different states of the CCK receptor. Similar measurements using transfected COS cells also identified three different states of the CCK receptor, with the very-low-affinity state being the most abundant. Thus, the ability of the CCK receptor to exist in three different states is an intrinsic property of the CCK receptor molecule itself.
Proceedings of the National Academy of Sciences 04/1994; 91(5):1868-72. · 9.68 Impact Factor
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ABSTRACT: Aging of the cerebral microcirculation results in significant alteration in the blood-brain barrier (BBB). The barrier function appears to remain intact in older animals, although it may be more susceptible to disruption by external factors (hypertension) and drugs (haloperidol). While overall transport processes do not change with age, aging animals and humans have altered BBB function of select carrier mediated transport systems including the transport of choline, glucose, butyrate and triiodothyronine. These age-related changes are the result of either alteration in the carrier molecules or the physiochemical properties of the cerebral microvessels. At the present time, it is not known whether changes in the BBB contribute to the age-related neurodegenerative diseases or are merely epiphenomena of aging.
Experimental Gerontology 32(4-5):501-19. · 3.74 Impact Factor
