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Biomedical subjects

F Ahmad

Publications and source records attributed to F Ahmad.

At least 73 records · Page 4Linked to original sources
PubMed1997

Effect of tumor necrosis factor-alpha on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases.

Tumor necrosis factor-alpha (TNF-alpha) can modulate the signalling capacity of tyrosine kinase receptors; in particular, TNF-alpha has been shown to mediate the insulin resistance associated with animal models of obesity and noninsulin-dependent diabetes mellitus. In order to determine whether the effects of TNF-alpha might involve alterations in the expression of specific protein-tyrosine phosphatases (PTPases) that have been implicated in the regulation of growth factor receptor signalling, KRC-7 rat hepatoma cells were treated with TNF-alpha, and changes in overall tissue PTPase activity and the abundance of three major hepatic PTPases (LAR, PTP1B, and SH-PTP2) were measured in addition to effects of TNF-alpha on ligand-stimulated autophosphorylation of insulin and epidermal growth factor (EGF) receptors and insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation. TNF-alpha caused a dose-dependent decrease in insulin-stimulated IRS-1 phosphorylation and EGF-stimulated receptor autophosphorylation to 47-50% of control. Overall PTPase activity in the cytosol fraction did not change with TNF-alpha treatment, and PTPase activity in the particulate fraction was decreased by 55-66%, demonstrating that increases in total cellular PTPase activity did not account for the observed alterations in receptor signalling. However, immunoblot analysis showed that TNF-alpha treatment resulted in a 2.5-fold increase in the abundance of SH-PTP2, a 49% decrease in the transmembrane PTPase LAR, and no evident change in the expression of PTP1B. These data suggest that at least part of the TNF-alpha effect on pathways of reversible tyrosine phosphorylation may be exerted through the dynamic modulation of the expression of specific PTPases. Since SH-PTP2 has been shown to interact directly with both the EGF receptor and IRS-1, increased abundance of this PTPase, may mediate the TNF-alpha effect to inhibit signalling through these proteins. Furthermore, decreased abundance of the LAR PTPase, which has been implicated in the regulation of insulin receptor phosphorylation, may account for the less marked effect of TNF-alpha on the autophosphorylation state of the insulin receptor while postreceptor actions of insulin are inhibited.

Animals↗
PubMed1996

Protein stability: urea-induced versus guanidine-induced unfolding of metmyoglobin.

We have studied the denaturation of metmyoglobin at pH 6.0 and 25 degrees C by urea and guanidine hydrochloride (GdnHCl) which are known to unfold the protein to the same extent. It has been observed that estimates of protein stability (delta G0N-U) from urea-induced and GdnHCl-induced denaturations do not agree with one another; the linear extrapolation method gave delta G0N-U values of 7.59 +/- 0.33 and 5.35 +/- 0.10 kcal mol-1 for urea and GdnHCl denaturations, respectively. Measurements of the effect of the addition of KCl in the concentration range 0.1-1.0 M to urea denaturation have suggested that this disagreement is not due to the nonionic and ionic characters of urea and GdnHCl, respectively. The functional dependence of the free energy change of unfolding (delta GN-U) on [denaturant], the molar concentration of the denaturant, has been investigated for understanding the cause(s) of the disagreement between the two estimates of delta G0N-U of metmyoglobin. For this purpose, we have studied the GdnHCl-induced denaturation of the protein in the presence of different urea concentrations at pH 6.0 and 25 degrees C and vice versa. These measurements yield delta GN-U values in the full concentration range [Ahmad et al. (1994) J. Biochem. 115, 322-327], and these results provide strong evidence that the delta GN-U dependence on [urea] is linear (linear free energy model of denaturation) and the relation between delta GN-U and [GdnHCl] is curved (binding model of denaturation). It has been observed that the extrapolated value of delta GN-U in urea using the linear free energy model becomes identical to the extrapolated value of delta GN-U in GdnHCl using the binding model.

Animals↗
PubMed1996

Effects of salts of alkali earth metals and calcium chloride on the stability of cytochrome c and myoglobin.

This study suggests a procedure by which binding of denaturants could be detected without any additional information other than that provided in the denaturation profiles of proteins. Two predominantly alpha-proteins, namely ferricytochrome c and metmyoglobin were denatured by guanidine hydrochloride (GdnHCl) in the presence of low fixed concentrations of salts at 25 degrees C and transition between native and denatured states was followed by absorbance measurements in the visible region (500-350 nm). The raw data were converted into transition curves from which Cm, the midpoint of GdnHCl-induced transition, and deltaGapp, the free energy changes on denaturation, were calculated assuming a two-state mechanism, and values of deltaGapp at zero concentration of the denaturant were estimated. It has been observed (1) that chlorides of Na, K, Cs, and Rb do not affect the native conformation of proteins, (2) that GdnHCl-induced denaturations of proteins in presence and absence of sodium bromide, sodium perchlorate and salts of lithium and calcium are reversible, (3) that optical properties of the GdnHCl-denatured state of proteins remain unchanged in presence of the second denaturant, (4) Cm decreases with an increase in the denaturant concentration, and (5) that except for GdnHCl there exist one or more binding sites on the native proteins for the denaturants.

Animals↗
PubMed1996

Preferential oxidation of glycogen in isolated working rat heart.

We tested the hypothesis that glycogen is preferentially oxidized in isolated working rat heart. This was accomplished by measuring the proportion of glycolytic flux (oxidation plus lactate production) specifically from glycogen which is metabolized to lactate, and comparing it to the same proportion determined concurrently from exogenous glucose during stimulation with epinephrine. After prelabeling of glycogen with either 14C or 3H, a dual isotope technique was used to simultaneously trace the disposition of glycogen and exogenous glucose between oxidative and non-oxidative pathways. Immediately after the addition of epinephrine (1 microM), 40-50% of flux from glucose was directed towards lactate. Glycogen, however, did not contribute to lactate, being almost entirely oxidized. Further, glycogen utilization responded promptly to the abrupt increase in contractile performance with epinephrine, during the lag in stimulation of utilization of exogenous glucose, suggesting that glycogen serves as substrate reservoir to buffer rapid increases in demand. Preferential oxidation of glycogen may serve to ensure efficient generation of ATP from a limited supply of endogenous substrate, or as a mechanism to limit lactate accumulation during rapid glycogenolysis.

Animals↗
PubMed1996

Ultrasound-assisted synthesis of santalbic acid and a study of triacylglycerol species in Santalum album (Linn.) seed oil.

Methyl ricinoleate (1) was treated with bromine and the dibromo derivative (2) was reacted with ethanolic KOH under ultrasonic irradiation to give 12-hydroxy-octadec-9-ynoic acid upon acidification with dil. HCI. The latter compound was methylated with BF3/methanol to give methyl 12-hydroxy-octadec-9-ynoate (3). Compound 3 was treated with methanesulfonyl chloride in the presence of triethylamine in CH2Cl2 to give methyl 12-mesyloxy-octadec-9-ynoate (4). Reaction of methyl 12-mesyloxy-octadec-9-ynoate with aqueous KOH under ultrasonic irradiation (20 kHz) gave (11E)-octadecen-9-ynoic acid (5, santalbic acid, 40%) and (11Z)-octadecen-9-ynoic acid (6, 60%) on acidification with dil. HCI. These isomers were separated by urea fractionation. The 13C nuclear magnetic resonance (NMR) spectroscopic properties of the methyl ester and the triacylglycerol (TAG) esters of these enynoic fatty acid isomers were studied. The carbon shifts of the unsaturated carbon nuclei of the methyl ester of the E-isomer were unambiguously assigned as 88.547 (C-9), 79.287 (C-10), 109.760 (C-11), and 143.450 (C-12) ppm, while the unsaturated carbon shifts of the (Z)-enynoate isomer appeared at 94.277 (C-9), 77.561 (C-10), 109.297 (C-11), and 142.668 (C-12) ppm. In the 13C NMR spectral analysis of the TAG molecules of type AAA containing either the (Z)- or (E)-enyne fatty acid, the C-1 to C-6 carbon atoms on the alpha- and beta-acyl positions were differentiated. The unsaturated carbon atoms in the alpha- and beta-acyl chains were also resolved into two signals except that of the C-11 olefinic carbon. Sandal (Santalum album) wood seed oil (a source of santalbic acid) was separated by silica chromatography into three fractions. The least polar fraction (7.2 wt%) contained TAG which had a random distribution of saturated and unsaturated fatty acids, of which oleic acid (69%) was the predominant component. The second fraction (3.8 wt%) contained santalbic acid (58%) and oleic acid (28%) together with some other normal fatty acids. Santalbic acid in this fraction was found in both the alpha- and beta-acyl positions of the glycerol "backbone." The most polar fraction (89 wt%) consisted of TAG containing santalbic acid only. The distribution of the various fatty acids on the glycerol "backbone" was supported by the results from the 13C NMR spectroscopic analysis.

Carbon Isotopes↗
PubMed1996

Characterization of an acquired IgG inhibitor of coagulation factor XIII in a patient with systemic lupus erythematosus.

An acquired IgG inhibitor to factor XIII was identified in a 30-year-old Vietnamese woman with systemic lupus erythematosus. The IgG fraction was isolated from plasma by chromatography on an agarose gel column and by protein G adsorption. The anti-factor XIII activity, identified within the IgG fraction, inhibited factor XIII from both normal plasma (a2b2) and platelets (a2). A normal level of the b subunit was measured by immunoelectrophoresis of the patient's plasma, but the a subunit was not detected. These results suggested the presence of an IgG immunoglobulin which recognized the a subunit and interfered with its activity.

Adult↗
PubMed1996

Delineation of a 1-cM region on distal 5q containing the locus for corneal dystrophies Groenouw type I and lattice type I and exclusion of the candidate genes SPARC and LOX.

Granular Groenouw type I (CDGG1) and lattice type 1 (CDL1) corneal dystrophies are two distinct potentially blinding conditions. These two entities were recently mapped to a region on chromosome 5q. We have investigated 2 families of Swiss origin with CDGG1 and CDL1 by linkage analysis. Our data show a maximum lod score of 5.38 at theta = 0.00 for marker D5S393 in CDL1 and 4.17 at theta = 0.00 for D5S658 in CDGG1. When combined, these families show a maximum low score of 9.22 for D5S393 at theta = 0.00. This confirms previous reports. Furthermore, we describe a recombination centromeric to D5S399 in a member of the CDL1 family. Haplotype analysis in the 4 branches of the CDGG1 family demonstrated a common chromosomal region including D5S393 and D5S399 in all the affected members. By combining our data with previously reported mapping information and assuming that CDGG1 and CDL1 are allelic manifestations of the same gene, we can refine the location of the CDGG1/CDL1 gene to a 1-cM region on chromosome 5q. Using candidate genes in the 5q22-q32 interval, we investigated the possibility that mutations in the SPARC or LOX genes cause these corneal diseases. Several recombinations occurred between these two genes and CDGG1/CDL1 in our 2 families, thus excluding this hypothesis.

Alleles↗
PubMed1996

Use of laparoscopy in the treatment of acute and chronic right lower quadrant pain.

Laparoscopy and the availability of procedures which can be easily performed through the laparoscope, including appendectomy, alter the surgeon's approach towards the management of right lower quadrant pain, both acute and chronic. Laparoscopy can be utilized in a variety of clinical settings, a number far greater than those patients which would otherwise be considered proper subjects for open appendectomy. Laparoscopy can be diagnostic, therapeutic, and also prophylactic. A two year experience of managing right lower quadrant pain in a rural hospital setting was reviewed; the procedure naturally lent itself to a variety of patients because of its ease, safety, and diagnostic accuracy. Chart review indicates an extraordinary degree of success in alleviating both acute and chronic symptoms; only one patient appears to have recurrent right lower quadrant pain out of our entire group. The use of laparoscopy in this manner creates a paradigm for a type of medical practice that is problem-oriented instead of specialty-oriented. The pathological status of the appendix was not the only factor that predicted patients' clinical response to the procedure.

Abdomen, Acute↗
PubMed1996

Effect of alloxan induced mild insulin dependent diabetes mellitus on rat erythrocyte cytosolic dehydrogenases.

In order to understand how the red cell of mild insulin dependent diabetes mellitus rat perform the normal physiological function and maintain integrity cytosolic dehydrogenases were assayed. Lactate dehydrogenase produces the cofactor for glycolytic enzymes while glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase produces the coenzymes for oxygen radical scavanging enzymes. Decrease in activity of cytosolic dehydrogenase renders diabetic erythrocyte population more susceptible to oxidant stress.

Animals↗
PubMed1996

Stability of D-amino acid oxidase: denaturation by guanidine hydrochloride and urea.

The guanidine hydrochloride-induced and urea-induced denaturations of swine D-amino acid oxidase (EC 1.4.3.3) dimer were studied by the measurements of the difference absorption spectra in the near-ultra violet region and specific activity. Spectral measurements were made at different pH values (8.3 and 3.2) and temperatures (27, 37 and 47 degrees C). It has been observed that (1), enzyme looses all its activity in concentrated solutions of the denaturants, and (2), the product of urea and guanidine hydrochloride denaturation is only partially unfolded as judged by the measurements of the intrinsic viscosity and exposures of the tyrosyl, tryptophyl and sulphhydryl residues.

Animals↗
PubMed1995

Osmotic loading of neutralizing antibodies demonstrates a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway.

Protein-tyrosine phosphatases (PTPases) have been postulated to balance the steady-state phosphorylation and the activation state of the insulin receptor and its substrate proteins. To explore whether PTP1B, a widely expressed, non-receptor-type PTPase, regulates insulin signaling, we used osmotic shock to load rat KRC-7 hepatoma cells with affinity-purified neutralizing antibodies that immunoprecipitate and inactivate the enzymatic activity of recombinant rat PTP1B in vitro. In cells loaded with PTP1B antibody, insulin-stimulated DNA synthesis and phosphatidylinositol 3'-kinase activity were increased by 42% and 38%, respectively, compared with control cells loaded with preimmune IgG (p < 0.005). In order to characterize the potential site(s) of action of PTP1B in insulin signaling, we also determined that insulin-stimulated receptor autophosphorylation and insulin receptor substrate 1 tyrosine phosphorylation were increased 2.2- and 2.0-fold, respectively, and that insulin-stimulated receptor kinase activity toward an exogenous peptide substrate was increased by 57% in the PTP1B antibody-loaded cells. Osmotic loading did not alter the cellular content of PTP1B protein, suggesting that the antibody acts in the cell by sterically blocking catalytic interactions between PTP1B and its physiological substrates. These studies demonstrate that PTP1B has a role in the negative regulation of insulin signaling and acts, at least in part, directly at the level of the insulin receptor. These results also show that insulin signaling can be enhanced by the inhibition of specific PTPases, a maneuver that has potential clinical relevance in the treatment of insulin resistance and Type II diabetes mellitus.

Animals↗
PubMed1995

Purification, identification and subcellular distribution of three predominant protein-tyrosine phosphatase enzymes in skeletal muscle tissue.

Protein-tyrosine phosphatases (PTPases) play a key role in the regulation of insulin action. In order to identify PTPases in skeletal muscle, the major site of insulin-mediated glucose disposal in vivo, we purified PTPases from rat muscle tissue fractions by a series of column chromatographic techniques. PTPase activities were assayed by measuring the dephosphorylation of a rat insulin receptor kinase domain, derivatized lysozyme and p-nitrophenylphosphate, and the enzymes were further characterized by immunoblotting. Of the total PTPase activity in muscle homogenates, 51-64% was localized to the solubilized particulate fraction, with the specific PTPase activity 3.3-fold and 5.6-fold higher in the particulate fraction towards RCM-lysozyme or the insulin receptor, respectively. The major peak (> 75%) of PTPase activity in the particulate fraction was purified further to 700-fold; 75% of this activity passed through a Blue-3GA column and revealed immunoreactivity for both LAR and SH-PTP2. PTPase activity retained on the Blue-3GA column contained PTPase1B. The major peak (> 70%) from muscle cytosol was further purified to 1500-fold. After the Blue-3GA step, immunoblotting revealed both SH-PTP2 and PTPase1B in the cytosol fraction, but LAR was absent from this fraction. LRP (RPTP-alpha) was not detected by blotting the PTPase activities from the purified particulate or cytosol fractions. Immunodepletion studies demonstrated that LAR, SH-PTP2 and PTPase1B were quantitatively major PTPase activities in the initial muscle homogenate, together accounting for over 70% of the total activity towards RCM-lysozyme. These studies provide insight into the relative abundance and subcellular distribution of specific PTPases in muscle tissue that are involved in the regulation of reversible tyrosine phosphorylation in this tissue.

Animals↗
PubMed1995

Use of narrow-bore high-performance liquid chromatography-diode array detection for the analysis of intermediates of the biological degradation of 2,4,6-trinitrotoluene.

A single method was developed for the separation and quantitation of hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene (TNT), and most of the known and suspected biodegradation intermediates of TNT by RP-HPLC and diode array detection. The known biodegradation intermediates of TNT analyzed were 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,6-diamino-4-nitrotoluene, 2,4-diamino-6-nitrotoluene, 2,4,6-triaminotoluene, 2,2',6,6'-tetranitro-4,4'-azoxytoluene, and 4,4',6,6'-tetranitro-2,2'-azoxytoluene. The suspected biodegradation intermediates of TNT included 1,2,3-benzenetriol (pyrogallol), 1,3,5-benzenetriol (phloroglucinol), 2-methyl-1,3,5-benzenetriol (methyl phloroglucinol) and 4-methylphenol (p-cresol). Mobile phases consisting of aqueous buffers adjusted to three different pH values in a gradient with acetonitrile were examined for their efficiency in separating the intermediate compounds and for the minimization of speciation of the ionizable intermediates (e.g. 2,4,6-triaminotoluene). A final aqueous buffer pH of 3.2 was selected to minimize the interference to the separation caused by 2,4,6-triaminotoluene speciation. Solvent consumption was minimized by the use of a narrow-bore column. All of the known reduction products as well as p-cresol and methyl phloroglucinol were identified in culture supernatants from TNT-degrading cultures while pyrogallol and phloroglucinol were not.

Biodegradation, Environmental↗
PubMed1995

Regulation of pyruvate carboxylase in 3T3-L1 cells.

When 3T3-L1 fibroblasts differentiate to adipocytes, the specific activity of pyruvate carboxylase (PC) increases about 25-fold in parallel with its intracellular protein concentration. The increase in PC protein concentration is accompanied by a 9-10-fold increase in the relative abundance of 4.2 kb PC mRNA measured by Northern-blot analysis using a cDNA probe encoding a segment of the PC gene of 3T3-L1 adipocytes. The effects of cyclic AMP (cAMP) alone and together with insulin on levels of cellular protein, PC activity, PC protein and on the relative abundance of PC mRNA were examined in mature 3T3-L1 adipocytes. Adipocytes exposed to cAMP for 24 h exhibited a 25% decrease in cellular protein and marked decreases in enzyme activity (88%) and PC mRNA abundance (98%) compared with untreated adipocyte controls. After 48 h of exposure to cAMP, PC activity and PC mRNA diminished to levels approaching their detection limits. When exposed to medium containing cAMP plus insulin, adipocyte enzyme activity and PC mRNA declined more slowly during the first 24 h exposure (about 20% decrease) but after 48 h fell to values comparable with those of adipocytes exposed to cAMP alone. Despite these decreases in enzyme activity, the PC protein content of adipocytes treated with cAMP alone or cAMP plus insulin are nearly identical with that of control adipocytes. The inactivation of PC in cAMP-treated adipocytes does not involve loss of the prosthetic group from the holoenzyme. Cross-linking experiments suggest that the spatial arrangement of protomers in inactive PC may differ from that in the active tetrameric enzyme. Data presented suggest that, in addition to inducing inactivation, cAMP may also regulate adipocyte PC by decreasing transcription of the PC gene and/or enhancing the rate of degradation of PC mRNA.

3T3 Cells↗
PubMed1995

Increased abundance of specific skeletal muscle protein-tyrosine phosphatases in a genetic model of insulin-resistant obesity and diabetes mellitus.

Resistance to the biological action of insulin in its target tissues is a cardinal feature of non-insulin-dependent diabetes mellitus. Protein-tyrosine phosphatases (PTPases) have been postulated to play a key role in the regulation of the insulin action pathway, especially in skeletal muscle, the major site of insulin-mediated glucose disposal in vivo. To evaluate whether changes in the activity and/or abundance of candidate skeletal muscle PTPases is associated with severe resistance to insulin in an animal model, we measured PTPase enzyme activity and PTPase protein level by immunoblotting in subcellular fractions of skeletal muscle in lean (+/?), insulin-resistant obese (fa/fa), and diabetic (ZDF/Drt-fa/fa) Zucker rats. Using a phosphotyrosylmyelin basic protein substrate, the solubilized-particulate fraction PTPase activity was increased by 65% and 74% (P < .05) and in vitro dephosphorylation of a recombinant rat insulin receptor kinase domain was increased by 104% and 114% in obese and diabetic animals, respectively (P < .01). These changes in PTPase activity were associated with an increase in specific immunoreactivity of leukocyte common antigen-related PTPase ([LAR] by 42% and 50%), PTPase 1B (by 61% and 69%), and the SHZ domain containing PTPase (SH-PTP2) (by 44% and 48%) in the solubilized-particulate fraction of obese and diabetic animals, respectively (P < .05). In diabetic muscle, increased SH-PTP2 abundance was also associated with a shift of SH-PTP2 to a plasma membrane component, which may have important consequences for the activation of this enzyme in the insulin-resistant state. These results provide evidence that specific PTPases play a role in the insulin resistance of this genetic model of obesity and non-insulin-dependent diabetes.

Animals↗
PubMed1995

Alterations in specific protein-tyrosine phosphatases accompany insulin resistance of streptozotocin diabetes.

To test whether protein tyrosine phosphatases (PTPases) may play a role in the insulin resistance of insulinopenic diabetes, we assessed PTPase activity as well as the protein and mRNA abundance of three major candidate PTPases in subcellular fractions of liver and skeletal muscle of streptozotocin-diabetic rats before and after insulin treatment. PTPase activity against the insulin receptor in liver and muscle cytosol increased to 120-125% of control in the diabetic animals and by an additional 5-10% after insulin treatment. In the particulate fraction, PTPase activity decreased to 65-70% of control in diabetic liver and muscle and increased to 115-120% of control after insulin treatment. Protein for the leukocyte common antigen-related PTPase paralleled the changes in the PTPase activity in the particulate fraction. SH-PTP2/syp and PTPase 1B were both significantly increased in diabetes. SH-PTP2/syp also exhibited an increased ratio of particulate to cytosol distribution in diabetic tissues (1.8-1.9) that was reversed after insulin treatment (0.79-0.95). Northern analysis suggested that the PTPases were regulated at a pretranslational level. These changes in the abundance and distribution of specific PTPases may be involved in the pathogenesis of insulin resistance in insulinopenic diabetes.

Animals↗