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M Sternberg

Publications and source records attributed to M Sternberg.

At least 37 records · Page 2Linked to original sources
PubMed1996

Molecular docking programs successfully predict the binding of a beta-lactamase inhibitory protein to TEM-1 beta-lactamase.

Crystallization of the 1:1 molecular complex between the beta-lactamase TEM-1 and the beta-lactamase inhibitory protein BLIP has provided an opportunity to put a stringent test on current protein-docking algorithms. Prior to the successful determination of the structure of the complex, nine laboratory groups were given the refined atomic coordinates of each of the native molecules. Other than the fact that BLIP is an effective inhibitor of a number of beta-lactamase enzymes (KI for TEM-1 approximately 100 pM) no other biochemical or structural data were available to assist the practitioners in their molecular docking. In addition, it was not known whether the molecules underwent conformational changes upon association or whether the inhibition was competitive or non-competitive. All six of the groups that accepted the challenge correctly predicted the general mode of association of BLIP and TEM-1.

Amino Acid Sequence↗
PubMed1996

Production of type IV collagen and 72-kDa gelatinase by human endothelial cells cultured in high glucose. Effects of a protein kinase C inhibitor, GF 109203X.

Since diabetic microangiopathy and macroangiopathy are characterized by type IV collagen accumulation in vascular basement membranes, it was of interest to study type IV collagen production and type IV collagenase secretion by endothelial cells (EC) cultured in high glucose and to evaluate the role of protein kinase C (PKC) activation in the alterations induced by high glucose. Primary cultures of human umbilical vein EC were exposed to high glucose concentration for 3 days at the beginning of confluence. The number of EC decreased with glucose concentration from 5 to 50 mM. At 16.7 mM glucose concentration, the amount of type IV collagen, determined by a two-step ELISA, increased in the culture supernatant and in the insoluble fraction associated with the extracellular matrix and cells; proline incorporation was more markedly elevated in the collagenous than in the total proteins of the culture supernatant and of the extracellular matrix and cell extracts. Gelatin zymography of the culture supernatant showed that EC mainly produce a 72-kDa gelatinase known to degrade type IV collagen. At 16.7 mM glucose concentration, total gelatinase activity per millilitre of culture supernatant was reduced and the 72-kDa gelatinase activity measured on the zymogram scan was lowered. When EC were exposed to 16.7 mM glucose, the specific PKC inhibitor GF 109203X corrected the increases in type IV collagen concentration and in proline incorporation into the collagenous or total proteins present in he culture supernatant or in the extract of the insoluble fraction, including the extracellular matrix and cells. Our results show that soluble and insoluble type IV collagen accumulation by EC cultured at high glucose concentration is not only associated with increased synthesis of the collagenous and total proteins but also with decreased total 72-kDa gelatinase activity in the extracellular fluid. The observed effects of GF 109203X are in favor of the involvement of PKC activation in the type IV collagen accumulation.

Cell-Free System↗
PubMed1995

[Effects of glycation process on the macromolecular structure of the glomerular basement membranes and on the glomerular functions in aging and diabetes mellitus].

Three stages can be distinguished during the glycation process: initiation with the formation of Amadori product; spreading with glyco-oxidation reactions; terminal formation of advanced glycation end products (AGEs). Some AGEs have been isolated and characterized: pyrraline linked to one aminoacid, pentosidine linked to two aminoacids and forming a cross-link between peptidic chains. The AGE-induced cross-links alter the biophysical properties of the proteins with increased stiffness of the fibrous proteins and resistance to proteases. Glycation of the glomerular basement membrane (GBM) macromolecules modifies the architecture of the glomerular filtration barrier. Type IV collagen is the major constituent of the GBM and the mesangial matrix and is a substrate for prolonged glycation, due to its long half-life. In the GBM, AGE level (particularly pentosidine level per mg collagen) increases with age; it is higher in diabetic or uremic patients than in age-matched controls. In insulin-dependent diabetes mellitus, a correlation has been shown between the pentosidine level of skin collagen and the severity of vascular complications. Glycation inhibits the homotypic polymerization interactions between two type IV collagen molecules through their NC1 ends. Glycation also affects the heterotypic interactions between different GBM macromolecules: the affinity of glycated fibronectin for type IV collagen is diminished. Besides, glycation modifies the interactions between type IV collagen and adjacent cells: mesangial and endothelial cells are less adherent on a glycated type IV collagen matrix and their morphology modified. GBM treated with dimethylmalonimidate, which induces cross-links between amines as does advanced glycation, are more permeable to proteins.

Aging↗
PubMed1994

Renal and microvascular effects of an aldose reductase inhibitor in experimental diabetes. Biochemical, functional and ultrastructural studies.

Aldose reductase inhibitors, and particularly sorbinil, have been reported to prevent glomerular basement membrane thickening (GBMT) and albuminuria development in diabetic rats, but contradictory observations have been published. The aim of this study was to answer the following questions (i) is the corrective effect of sorbinil on GBMT, if confirmed, associated with an effect on collagen metabolism alterations? (ii) Is it associated with an effect on microvascular functional alterations? We therefore studied the influence of sorbinil on glucosyl-galactosyl-hydroxylysyl-glucohydrolase activity (GGHG; EC 3.2.1.107 which is involved in the catabolism of collagen disaccharide units), 3- and 4-hydroxyproline content and GBMT by ultrastructural morphometry in the kidney cortex of streptozotocin-diabetic rats after 5 months of disease. In parallel, the effects on albumin renal clearance and another functional alteration, the microvascular response to norepinephrine, were evaluated. We confirmed a corrective effect of sorbinil on both renal albumin clearance and GBMT. In the diabetic rats, sorbinil diminished the 3-hydroxyproline (but not the 4-hydroxyproline) content, whether expressed per mg protein or per total kidney cortex relative to body weight. Sorbinil reduced GGHG activity measured in the dialysed 10,000 g supernatant whether expressed per mg protein or per total kidney cortex; this activity has been shown to be increased in diabetes. Sorbinil also corrected the microvascular response to norepinephrine which is altered in diabetes.

Albuminuria↗
PubMed1994

Relation between pancreatic islet cellular infiltration and plasma fibrinogen or alpha 1-acid glycoprotein levels in spontaneously and streptozotocin-diabetic rats: an increase in these protein levels is not necessary for inducing microcirculatory erythrocyte velocity alteration.

Plasma levels of fibrinogen, alpha 1-acid glycoprotein (AG) and albumin, pancreatic insulitis quantitative scores, and erythrocyte velocity in the mesoappendix microvessels were measured in BB diabetic (BBD) and streptozotocin-diabetic rats (WSTZ) in order to answer the following questions: (a) Does hyperfibrinogenemia or increase in AG plasma level occur in BBD and WSTZ rats, and if so, are these alterations secondary to the hyperglycemia or to an inflammatory process such as insulitis? (b) Is there a decrease in microcirculatory flow in the BBD and WSTZ rats, and if so, is it secondary to the hyperfibrinogenemia and/or the hyperglycemia? Insulitis was present in the BBD rats after 5 weeks of disease (with a score of 2.9 +/- 0.1 vs. 1.4 +/- 0.6 in the normoglycemic controls), but absent in WSTZ rats after 5 months of disease (1.2 +/- 0.06 vs. 1.1 +/- 0.06). Increase in fibrinogen and AG plasma levels was observed in the BBD rats only and appears linked to the insulitis. The major acute phase protein AG level is increased in BBD rats already on the first day of appearance of glycosuria. In the WSTZ rats, without insulitis, chronic hyperglycemia alone did not induce an increase in fibrinogen and AG plasma levels. A decreased microcirculatory erythrocyte velocity has been found in both BBD and WSTZ rats. Thus an increase in fibrinogen or AG plasma levels is not necessary for inducing a decrease in erythrocyte velocity. Hyperglycemia is probably the main factor responsible for the decrease in microcirculatory flow in the BBD and WSTZ rats.

Animals↗
PubMed1993

[Changes in collagen type IV metabolism in diabetes].

In Diabetes Mellitus, type IV collagen biosynthesis is increased: the alpha 1(IV) procollagen specific mRNA concentration is elevated, particularly in the kidney, and the type IV collagen protein is accumulating is the thickened basement membranes. Aldose reductase inhibitors like sorbinil do prevent basement membrane thickening and type IV collagen overproduction. The latter seems related to intracellular sorbitol accumulation and also to protein kinase C activation. Autocrine or paracrine TGF beta may be involved in the type IV collagen oversecretion. The secreted type IV collagen is subject to posttranslational alterations, especially glycation which leads to advanced glycation end-products and covalent crosslinks. This decreases collagen extractability and susceptibility to collagenases and favours basement membrane thickening. Disaccharide unit-specific alpha-glucosidase activity is inhibited by glucose (Kp = 7.5 mM). Type IV collagenase activity secreted by endothelial cells cultured at high glucose concentrations appears to be diminished. Therefore type IV collagen catabolism may be decreased in Diabetes Mellitus.

Animals↗
PubMed1992

Dementia and the nursing home: association with care needs.

OBJECTIVE: To determine whether RUG reimbursement categories accurately predict requirements for care in nursing homes. DESIGN: Prospective descriptive study of residents in lower reimbursement categories according to RUG. SETTING: Three nursing homes in New York City. PARTICIPANTS: Convenience sample of 173 residents who agreed to participate, not significantly different from 201 who did not agree to participate. MAIN MEASURES: Chart review; assessment of residents' cognitive and functional abilities; nursing assistants' ratings of residents' functional abilities, behavioral problems, the amount of effort required in care; and time-motion studies of staff-resident interactions. RESULTS: Both the residents' RUG classification (P less than 0.01) and the level of ADL independence (P less than 0.001) had significant impacts on the staff effort required in their care, with more dependent residents requiring greater effort. The residents' level of cognitive impairment also had a significant impact on the staff effort, with the severely impaired requiring greater effort (P less than 0.05). The time-motion analysis indicated that residents within the same RUG category differed in the number of staff-resident interactions based on their level of cognitive impairment. CONCLUSIONS: Cognitive impairment is a significant morbidity (or co-morbidity) in determining the quantity of staff effort required by the resident, and behavioral interventions are an important care component. There is marked heterogeneity within lower (RUG) reimbursement categories which translates into strikingly different care requirements.

Activities of Daily Living↗
PubMed1992

Sex and age dependence of rat kidney sialidase.

Rat kidney sialidase levels have been reported to be markedly altered in pathological states such as diabetes. This was associated with a modification of sialic acid levels. Therefore, it was interesting to study the variations of kidney sialidase and sialyltransferase activities and sialic acid content according to sex and age. This was carried out from birth to 210 days of age. The substrates used were sialyl alpha(2-3)[3H]-lactitol for sialidase activity, asialofetuin and [14C]-CMPNeu5Ac for sialyltransferase activity. In males sialidase activity increased until 32 days then slightly declined. In females, the activity increased and leveled off at 135 days of age. Higher sialidase activity was observed in females than in males from 56 days of age. Gonadectomy had no effect on this activity. In both sexes, sialyltransferase activity decreased markedly with age. This activity was higher in females than in males, whereas sialic acid levels varied only moderately with age and were slightly higher in females.

Aging↗
PubMed1990

Kidney sialidase and sialyltransferase activities in spontaneously and experimentally diabetic rats. Influence of insulin and sorbinil treatments.

Kidney cortex sialic acid level, sialidase and sialyltransferase activities have been measured in spontaneously diabetic BB rats and in streptozotocin-diabetic rats (STZ). In untreated diabetic BB rats, at the onset of the disease, sialidase specific activity was found to be increased by 21% when compared with diabetes-resistant BB controls (P less than 0.05) whereas sialyltransferase activity was not significantly modified and bound sialic acid concentration was diminished (P less than 0.05). In diabetic BB rats submitted to a minimal insulin therapy, during 3 months of disease, sialidase activity and sialic acid concentration were similar to those of Wistar age-matched controls. In STZ-diabetic Wistar rats, sialidase specific activity was increased by 76% after 5 months of disease when compared to age-matched Wistar controls (P less than 0.01); in contrast, specific sialyltransferase activity was decreased by 21% (P less than 0.05); these enzymatic alterations were associated with a decrease in bound sialic acid concentration (P less than 0.01); 1 month's insulin therapy, started 4 months after onset of the disease, normalized sialidase activity but had no effect on sialyltransferase activity and sialic acid concentration; treatment with sorbinil prevented cataract development but had no effect on sialidase activity whereas it emphasized the decrease in sialyltransferase activity and sialic acid concentration. The disturbances in the enzyme activities concerned with sialoglycoconjugate metabolism observed in experimental and spontaneous diabetes may be responsible for the decreased bound sialic acid content observed in the rat kidney cortex.

Animals↗
PubMed1989

Modification of enzyme activities concerned with collagen metabolism in the heart of spontaneously hypertensive and aortic-constricted rats.

The activities of three enzymes concerned with collagen metabolism 4-prolyl hydroxylase, UDP-glucose: collagen glucosyltransferase and glucosyl-galactosyl-hydroxylysine glucohydrolase and 4-hydroxyproline content have been studied in the cardiac ventricles of spontaneously hypertensive rats (SHR) during prehypertensive, hypertensive and sustained hypertensive stages (respectively 4.5, 12 and 19 weeks of age). They were compared with values observed in age-matched normotensive Wistar Kyoto rats (WKY). The same studies have been performed in parallel on aortic-constricted rats (ACR) 8 days after suprarenal constriction of the abdominal aorta. The most striking finding was a significant increase in cardiac prolyl hydroxylase specific activity in the ACR but not in the SHR. No variation in 4-hydroxyproline concentration was found in the hearts of ACR. In contrast, a decrease in 4-hydroxyproline concentration was found in the hearts of SHR at 19 weeks. Cardiac glucosyltransferase specific activity was significantly elevated only in the SHR at 12 weeks. No variation in glucohydrolase specific activity was detected in the hearts of either SHR or ACR. The cardiac enzyme activities all decreased with age. These data show that the alterations in cardiac collagen metabolism are different in SHR and ACR. The patterns of the alterations found in the heart mirror those observed in the aorta in both models under the same experimental conditions.

Age Factors↗
PubMed1988

Aging and diabetes increase the aggregating potency of rat skin collagen towards normal platelets.

Acid-soluble collagen samples were prepared from individual skins of 24 month old rats (n = 8), 2 month old young controls (n = 8) and from 6 month old streptozotocin-diabetic rats (n = 5) and their age-matched controls (n = 10). Less collagen was obtained by acid extraction and salt precipitations from skins of diabetic and aged rats than from those of their respective controls. The collagen preparations from diabetic and aged rats showed an increased ratio of beta/alpha components. The rate of "in vitro" fibrillogenesis was less for collagen from diabetic rats than from controls. It was not modified for collagens from aged rats. The aggregating potency towards normal human platelets was markedly increased for collagens from aged and diabetic rats: reduced latency time (p less than 0.01) and increased velocity (p less than 0.01) were observed for collagens from aged rats when compared with young rats (16.5 micrograms/ml). Increased velocity (p less than 0.01) was also observed for collagens from diabetic rats (8.25, 11 and 16.5 micrograms/ml), without modification of latency time.

Aging↗
PubMed1988

Alteration in sialidase and other glycosidase activities in the kidney of spontaneously hypertensive rats: persistence after preventive treatment with hydralazine.

Because kidney microangiopathy with capillary basement membrane thickening has been reported in spontaneous hypertension, we have studied the activities of three lysosomal glycosidases able to degrade the carbohydrate moieties of basement membrane constituents in the kidney cortex of 12-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar Kyoto rats (WKY). These activities were also determined in SHR and WKY treated from 6 to 12 weeks of age with hydralazine (mean dose, 18 mg/kg per day in drinking water). Sialidase specific activity on sialyl-alpha 2-3-[3H]lactitol was markedly decreased in the kidney of untreated SHR, 40% activity remaining relative to that found in untreated age-matched WKY (p less than 0.001). beta-Galactosidase specific activity on p-nitrophenyl-beta-D-galactoside was also decreased, 86% activity remaining relative to that found in untreated WKY (p less than 0.001). Glucosyl-galactosyl-hydroxylysyl glucohydrolase specific activity on glucosyl-galactosyl-hydroxylysine was equally diminished, 74% activity remaining relative to that found in untreated age-matched WKY (p less than 0.001). In contrast, the activities of two control glycosidases inactive on the carbohydrate moieties of basement membrane constituents, alpha-glucosidase assayed with p-nitrophenyl-alpha-D-glucoside as substrate and beta-glucosidase assayed with p-nitrophenyl-beta-D-glucoside as substrate, were significantly increased. All the alterations in enzyme activities observed in the kidney of SHR were also present in the long-term treated normotensive SHR. No effect of the hydralazine treatment on the three enzyme activities investigated could be demonstrated in the WKY. Thus the alterations observed in the kidneys of SHR appear to be independent of blood pressure level.

Animals↗
PubMed1985

Properties of a microsomal enzyme system from Linum usitatissimum (linen flax) which oxidizes valine to acetone cyanohydrin and isoleucine to 2-methylbutanone cyanohydrin.

Microsomal preparations from flax seedlings have recently been shown to convert L-valine to acetone cyanohydrin, the precursor of the cyanogenic glucoside linamarin [A. J. Cutler and E. E. Conn (1981) Arch. Biochem. Biophys. 212, 468-474]. Further details of this four-step biosynthetic sequence and also details of the analogous reactions in lotaustralin biosynthesis have been obtained. The lotaustralin precursor, 2-methylbutyraldoxime, is the best substrate for cyanide production (Vmax = 413 nmol h-1 g fresh wt-1) and inhibits the conversion of valine and isoleucine into products. Similarly, the linamarin precursor isobutyraldoxime is an excellent substrate (Vmax = 400 nmol h-1 g fresh wt-1) and also inhibits oxidation of the amino acids. The substrate specificity of the oxime-metabolizing step is low and a variety of aliphatic oximes are converted to cyanide. On the other hand, the activity of the microsomal extract is highly selective with regard to the amino acid substrate since, of the aliphatic amino acids tested, only valine and isoleucine are metabolized. We were unable to demonstrate product formation from isobutyronitrile (a linamarin precursor) but did observe detectable cyanide formation from 2-methylcyanobutane, the corresponding precursor of lotaustralin. Competition experiments showed that the biosynthesis of linamarin and lotaustralin is not likely to be catalyzed by separate enzyme systems.

Chromatography, Gas↗
PubMed1985

Aorta collagen metabolism in spontaneously hypertensive and aortic-constricted rats: variations in enzyme activities concerned with disaccharide unit synthesis and degradation according to blood pressure and age.

Two enzyme activities concerned with collagen disaccharide unit metabolism (UDP-glucose: collagen glucosyltransferase and glucosyl-galactosyl-hydroxylysine glucohydrolase) have been studied in the thoracic aortic wall together with 4-prolyl hydroxylase activity and 4-hydroxyproline content in spontaneously hypertensive rats (SHR) at the prehypertensive, hypertensive and sustained hypertensive stages (respectively 32 days, 12 weeks and 19 weeks of age). They were compared with values observed in age-matched normotensive Wistar Kyoto rats (WKY). The same studies have been performed in parallel on aortic-constricted rats (ACR) 8 days after suprarenal constriction of the abdominal aorta. Negative regressions of all three specific activities as function of age were observed. The most striking difference observed between the SHR and the WKY was the increase of glucosyltransferase specific activity, already found at the prehypertensive stage and continuing thereafter; the glucohydrolase specific activity was increased only during the establishment of hypertension whereas no modification was found with prolyl hydroxylase at any stage. However, a diminution of hydroxyproline concentration was seen at all ages while total hydroxyproline mass remained unaffected. The alterations of the aortic collagen metabolism observed in the ACR recall those seen in the SHR at the prehypertensive stage: the only significant modification was that of glucosyltransferase activity. Correlation was found between glucosyltransferase activity and blood pressure level in the two animal models.

Age Factors↗
PubMed1985

Connective tissue in diabetes mellitus: biochemical alterations of the intercellular matrix with special reference to proteoglycans, collagens and basement membranes.

Diabetes mellitus induces alterations in the metabolism of the macromolecules present in the intercellular matrices and particularly in the basement membranes. These contribute to the morphological changes characteristic of the disease : basement membrane thickening, skin thickening and induration. Accumulation of overglycosylated collagens and diminution of sulfated proteoglycan concentrations are the most generally reported biochemical modifications in human or animal diabetic states. More limited data are available concerning elastin, fibronectin and laminin in diabetes.

Animals↗
PubMed1984

Studies on kidney sialidase in normal and diabetic rats.

Rat kidney cortex sialidase was studied using alpha-sialyl-(2----3)-[3H]lactitol and alpha-sialyl-(2----6)-[3H]lactitol as substrates. The enzyme was found mainly in the lysosomal fraction. Only 23% of the sialidase activity of this fraction could be solubilized by a combination of freezing-thawing, sonication and Triton X-100 treatment. The optimal pH for the lysosomal enzyme activity was 4.2 and the enzyme's Km values for alpha-sialyl-(2----3)-lactitol and alpha-sialyl-(2----6)-lactitol were 0.28 and 0.41 mM, respectively. The specific activity was twice as high with the former substrate than with the latter. Sialidase activities in dialyzed kidney cortex homogenates of streptozotocin-diabetic rats and of age-matched control rats were compared. The specific activity was found to be significantly increased in the diabetic animals when using both substrates 5950 +/- 720 (S.E.) dpm/h per mg protein (n = 7) vs. 3970 +/- 370 in the controls (n = 8) with alpha-sialyl-(2----3)-lactitol (P less than 0.025) and 2870 +/- 300 vs. 1820 +/- 170 with alpha-sialyl-(2----6)-lactitol (P less than 0.02). The activities were also found to be increased when expressed per whole kidney cortex (P less than 0.005 and P less than 0.001, respectively). The elevated sialidase activity in diabetic kidney cortex may be related to the reported decrease in sialic acid content of the glomerular basement membrane, which lowers its negative charges and which may contribute to an increased permeability to proteins.

Animals↗