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J J Herbst

Publications and source records attributed to J J Herbst.

At least 19 recordsLinked to original sources
PubMed2002

High-throughput fluorescence polarization method for identification of FKBP12 ligands.

FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His(6)-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K(i) values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.

Drug Evaluation, Preclinical↗
PubMed1997

Trafficking kinetics of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes.

In fat and muscle cells insulin causes the marked translocation of the glucose transporter GLUT4 from its intracellular location to the plasma membrane. We and others have discovered an insulin-regulated membrane aminopeptidase (designated IRAP) that colocalizes with intracellular GLUT4 and also translocates markedly in response to insulin. This study describes the trafficking kinetics of IRAP in 3T3-L1 adipocytes. By means of a surface biotinylation method, the half-time for the increase in IRAP at the plasma membrane in response to insulin was found to be 2 min. The increase was completely blocked by the phosphatidylinositol 3-kinase inhibitor, wortmannin. In insulin-treated cells, biotinylated IRAP, initially at the plasma membrane, equilibrated with the intracellular pool with a half-time of 2 min. Thus, IRAP continuously recycles. Finally, vesicles isolated from the intracellular membranes with antibodies against IRAP and GLUT4 showed the same protein composition. In conjunction with results in the literature, these findings indicate that IRAP and GLUT4 traffic through the same intracellular compartments.

3T3 Cells↗
PubMed1997

Distribution and regulation of ornithine decarboxylase activity along the villus-crypt axis in the small intestine.

Distribution of ornithine decarboxylase activity in rat intestinal villi and crypts was determined by serially sectioning frozen mucosa and measuring enzyme activity in pools of sections composed of villi or crypts. Contents of the pools was determined by histological examination of representative sections, and simultaneous measurement of sucrase as a marker of villus samples demonstrated excellent separation of villi and crypts. In fasted and ad lib fed rats, enzyme activity was highest in the villus-crypt junctional area and in crypts (P < 0.05). Refeeding after a fast increased enzyme activity 15-fold, with greatest activity in villus tips and the villus-crypt junctional area. Luminal 0.4 M glycine stimulated enzyme activity only in villus and villus-crypt junctional samples, while luminal 10 mM putrescine stimulated activity only in crypts. Parenteral epidermal growth factor caused increased enzyme activity in all mucosal areas, but the 18-28-fold increase in the three villus samples (top, middle and bottom) was significantly greater (P < 0.05) than the 7-9-fold increase in crypt and junctional samples. In rats refed after a fast, parenteral putrescine (2 mmol/kg) depressed enzyme activity in all mucosal areas. Ornithine decarboxylase activity is usually greatest in junctional and crypt cells, and villus and crypt cells respond differently to luminal and systemic stimuli.

Animals↗
PubMed1997

Insulin stimulates cell surface aminopeptidase activity toward vasopressin in adipocytes.

We previously discovered that insulin stimulates the marked translocation of a novel membrane aminopeptidase, designated vp165 for vesicle protein of 165 kDa, to the cell surface in adipocytes. To examine the hypothesis that this enzyme acts on peptide hormones, we assessed the relative affinity of the enzyme for 22 peptide hormones by measuring the inhibitory effect of each on the hydrolysis of a fluorogenic substrate, and we directly assayed the cleavage of four of these. Angiotensin III, angiotensin IV, and Lys-bradykinin bound to the enzyme with half-saturation constants between 20 and 600 nM and were cleaved by vp165. Vasopressin bound with lower affinity but at saturation was cleaved more rapidly. Subsequently, the effect of insulin on the rates of cleavage of 125I-labeled vasopressin by intact 3T3-L1 and rat adipocytes was determined. With both cell types, vasopressin cleavage was stimulated approximately threefold. These findings indicate that a physiological role for vp165 may be the processing of peptide hormones and that insulin could enhance the cleavage of extracellular substrates by eliciting the translocation of vp165 to the cell surface.

3T3 Cells↗
PubMed1996

The antihyperglycemic agent englitazone prevents the defect in glucose transport in rats fed a high-fat diet.

The effects of englitazone in male Wistar rats fed a high-fat diet (59% of calories as fat) were compared with control rats fed a high-carbohydrate diet (69% of calories as carbohydrate) (5-15 animals per group). Insulin-stimulated (17 nmol/l) 2-deoxy-D-glucose (2-DG) uptake was inhibited 31% in adipocytes isolated from rats on the high-fat diet for 3 weeks, but englitazone (50 mg/kg for the last 7 days) normalized the response. There was a selective decrease in GLUT4 (54 +/- 5% of high-carbohydrate) in epididymal fat from rats on the high-fat diet for 3 weeks, but englitazone treatment did not reverse the defect in GLUT4 (43 +/- 8% of high-carbohydrate) or increase GLUT1 (81 +/- 12% of high-carbohydrate). Englitazone normalized oral glucose (1 g/kg body wt) intolerance and excessive (210% of high-carbohydrate) liver glycogen deposition (from [14C]glucose) caused by the high-fat diet. The high-fat diet tended to decrease insulin receptor substrate-1 (IRS-1) and phosphatidylinositol-3'-kinase (PI-3-kinase) expression in epididymal fat (26% decrease; P < 0.1). Englitazone did not reverse this decrease in IRS-1 and PI-3-kinase levels in fat from high-fat-fed rats (there was a further 25-30% decrease, P < 0.05), nor did it increase PI-3-kinase activity in 3T3-L1 adipocytes under conditions (48 h incubation) where it stimulated 2-DG uptake sixfold or enhanced insulin-stimulated 2-DG uptake. In summary, englitazone prevented the insulin resistance associated with a high-fat diet, but the mechanism of action does not involve changes in fat or muscle glucose transporter content and may not involve activation of the insulin signaling pathway via PI-3-kinase.

Adipocytes↗
PubMed1995

Effect of the activation of phosphatidylinositol 3-kinase by a thiophosphotyrosine peptide on glucose transport in 3T3-L1 adipocytes.

Insulin causes the activation of phosphatidylinositol 3-kinase (PI 3-kinase) through complexation of tyrosine-phosphorylated YMXM motifs on insulin receptor substrate 1 with the Src homology 2 domains of PI 3-kinase. Previous studies with inhibitors have indicated that activation of PI 3-kinase is necessary for the stimulation of glucose transport in adipocytes. Here, we investigate whether this activation is sufficient for this effect. Short peptides containing two tyrosine-phosphorylated or thiophosphorylated YMXM motifs potently activated PI 3-kinase in the cytosol from 3T3-L1 adipocytes. Introduction of the phosphatase-resistant thiophosphorylated peptide into 3T3-L1 adipocytes through permeabilization with Staphylococcus aureus alpha-toxin stimulated PI 3-kinase as strongly as insulin. However, under the same conditions the peptide increased glucose transport into the permeabilized cells only 20% as well as insulin. Determination of the distribution of the glucose transporter isotype GLUT4 by confocal immunofluorescence showed that GLUT4 translocation to the plasma membrane can account for the effect of the peptide. These results suggest that one or more other insulin-triggered signaling pathways, besides the PI 3-kinase one, participate in the stimulation of glucose transport.

3T3 Cells↗
PubMed1994

Evidence for a role of phosphatidylinositol 3-kinase in the regulation of glucose transport in Xenopus oocytes.

We have used two experimental approaches to examine the possible role of phosphatidylinositol 3-kinase (PI 3-kinase) in the regulation of glucose transport in oocytes isolated from Xenopus laevis. Incubation of oocytes with the PI 3-kinase inhibitor wortmannin inhibited insulin-like growth factor-1-stimulated deoxyglucose uptake. Half-maximal inhibition was observed at concentrations approximately 20 nM. Conversely, we also examined the effects of microinjection of synthetic peptides designed to interact with Src homology 2 domains of the regulatory subunit of PI 3-kinase on deoxyglucose transport in oocytes. We show that a bifunctional synthetic peptide containing two YMXM consensus sequences for binding to SH2 domains stimulated both PI 3-kinase activity and deoxyglucose transport when both tyrosine residues were phosphorylated. However, non-phosphorylated or bisphosphonotyrosine peptides with the identical amino acid sequence failed to stimulate transport or PI 3-kinase activity. Taken together, these data argue strongly for a role for PI 3-kinase in the regulation of glucose transport in oocytes.

Amino Acid Sequence↗
PubMed1994

Potent activation of phosphatidylinositol 3'-kinase by simple phosphotyrosine peptides derived from insulin receptor substrate 1 containing two YMXM motifs for binding SH2 domains.

The phosphotyrosine form of the major substrate for the insulin receptor tyrosine kinase, insulin receptor substrate 1 (IRS-1), associates with and activates the enzyme phosphatidylinositol 3'-kinase (PtdIns 3'-kinase). IRS-1 contains nine potential tyrosine phosphorylation sites within YMXM or YXXM sequences known to bind to the two SH2 domains on the 85-kDa regulatory subunit of PtdIns 3'-kinase. We used sequences within IRS-1 as a model for synthesizing phosphotyrosine and nonhydrolyzable phosphonotyrosine peptides containing two YMXM motifs and tested them for their ability to bind to the SH2 domains of PtdIns 3'-kinase and stimulate its activity. We demonstrated for the first time that IRS-1-derived peptides containing two tyrosine phosphorylated YMXM motifs are capable of stimulating PtdIns 3'-kinase activity in the cytosol of 3T3-L1 adipocytes at nanomolar concentrations, similar to that required by purified phosphoryl-IRS-1 [Lamphere, M., Carpenter, C. L., Sheng, Z., Kallen, R. G., & Lienhard, G. E. (1994) Am. J. Physiol. 266 (Endocrinol. Metab. 29), E486-E489] and the extent of activation by these peptides was similar to that seen by maximal stimulation of cells with insulin. In contrast, those phosphotyrosine peptides containing only a single YMXM motif were able to stimulate PtdIns 3'-kinase activity only at concentrations over 10 microM. We conclude from these results that the high-affinity activation of PtdIns 3'-kinase requires the simultaneous binding of two phosphorylated YMXM motifs on IRS-1 to the two SH2 domains of PtdIns 3'-kinase.

Amino Acid Sequence↗
PubMed1994

Regulation of postendocytic trafficking of the epidermal growth factor receptor through endosomal retention.

Little is known about the regulation of EGF receptor (EGF-R) trafficking following endocytosis. We investigated this by using a series of EGF-R with altered cytoplasmic tails and comparing their ability to undergo recycling and lysosomal targeting in both the occupied and empty state. We found that 2-3% of empty EGF-R are internalized each minute, but rapidly recycle (t1/2 approximately 5 min). This constitutive internalization and recycling of empty receptors was independent of cytoplasmic receptor sequences. Occupied EGF-R, in contrast, displayed a much slower rate of recycling (t1/2 between 10-23 min) due to retention within recycling endosomes. Endosomal retention of different EGF-R correlated with lysosomal targeting of EGF. Intrinsic receptor tyrosine kinase activity had no discernible effect on postendocytic trafficking of EGF. Although sequences within the cytoplasmic tail of the EGF-R appear to be required for occupancy-dependent endosomal retention, they are distinct from those required for ligand-induced endocytosis. Our studies indicate that intracellular trafficking of the EGF-R is regulated by endosomal components that preferentially recognize occupied receptors. Down-regulation of the EGF-R thus involves two distinct regulatory processes: one at the level of internalization and one at the level of recycling.

Animals↗
PubMed1993

The effect of glycemic control on plasma prealbumin levels in type-1 diabetic children.

Plasma prealbumin levels have been used as a sensitive biochemical index in the assessment of nutritional status. The present study was undertaken to determine if diabetic children with uncontrolled diabetes are at greater nutritional risk than those with controlled glycemia. Plasma prealbumin was determined using a radial immunoassay in 42 diabetics and 20 age-matched normal volunteers. Results (mean +/- SE) show that prealbumin levels are significantly (p < 0.02) lower in diabetics (21.5 +/- 1.0) than in normals (25.8 +/- 1.3). Compared with normals, prealbumin levels in controlled diabetics are similar; whereas uncontrolled diabetics have significantly lower (p < 0.05) prealbumin levels. Prealbumin levels were 20.4 +/- 1.7 in uncontrolled diabetics (GHb > 9.00), 21.8 +/- 2.3 in intermediately controlled diabetics (GHb 7.0-9.0), and 22.2 +/- 1.5 in controlled diabetics (GHb < 7.0). The data suggest that diabetics in good control are nutritionally similar to the normal population and that patients in poor control have significantly lower prealbumin than the normal population indicating that metabolic derangement may result in malnutrition.

Adolescent↗
PubMed1992

The effect of glycemic control and duration of diabetes on cholesterol and phospholipid classes in erythrocytes of type I diabetes.

Abnormalities in the lipid composition of erythrocytes can alter blood rheology and viscosity. These alterations have been implicated in the pathogenesis of microvascular disease in diabetic patients. The present study was undertaken to examine whether or not long-term glycemic control or duration of diabetes has any role in the altered membrane cholesterol and phospholipid composition of erythrocytes in type I diabetes. Long-term glycemic control was assessed by measuring glycosylated hemoglobin (GHb) from diabetic patients and age-matched normal volunteers. There was no significant correlation between GHb or duration of diabetes with total cholesterol, phospholipid, and cholesterol to phospholipid molar ratios in erythrocytes of these patients. Among phospholipid classes, GHb showed a significantly negative relationship with sphingomyelin (SM) (r = .55, P less than .01) levels, but was not related to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels of erythrocytes. Duration of diabetes had no effect on SM, PC, or PE levels of erythrocytes.

Adolescent↗
PubMed1991

The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor.

Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent down-regulation, indicating that the proximal cause of down-regulation is occupancy-induced endocytosis. Tyrosine kinase activity greatly enhances this process by stabilizing receptor association with the endocytic apparatus.

Animals↗
PubMed1990

Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation.

To assess the functional significance of phosphorylation of the epidermal growth factor (EGF) receptor at Thr654, we compared the effects of 12-O-tetradecanoyl-13-acetate (TPA) on ligand-induced internalization and down-regulation between wild-type and mutant receptors that contain an alanine substitution at position 654. Activation of protein kinase C with TPA blocked EGF-induced internalization and down-regulation of Thr654 receptors and inhibited in vivo tyrosine kinase activity by 80%. TPA did not inhibit transferrin receptor internalization or constitutive EGF receptor internalization, suggesting that protein kinase C activation inhibits only the ligand-induced process. Inhibition by TPA of induced internalization, down-regulation, and kinase activity required threonine at position 654 since full-length Ala654 EGF receptors were significantly resistant to TPA inhibition of these ligand-induced activities. However, C'-terminal truncation further enhanced this resistance to TPA inhibition. The EGF-dependent internalization of kinase-inactive receptors truncated at residue 1022 was also impaired by TPA in Thr654 receptors, but not in Ala654 receptors, indicating that phosphorylation at Thr654 also interferes with tyrosine kinase-independent receptor activities. We conclude that the dominant regulatory effect of protein kinase C on the EGF receptor is mediated through phosphorylation at Thr654 which effectively inactivates the receptor. The submembrane region of the EGF receptor appears to regulate transmission of conformational information from the extracellular ligand-binding site to the cytoplasmic kinase and regulatory domains.

Alanine↗
PubMed1990

Low plasma prealbumin and carotenoid levels in sickle cell disease patients.

This study was undertaken to discover whether reduced levels of micronutrients in patients with sickle cell disease are due to the generalized undernutrition. Protein undernutrition was determined by measuring plasma levels of prealbumin. One micronutrient, carotene, was also simultaneously assayed in the plasma. Eighteen sickle cell disease patients were enrolled at random. Controls were 19 normal volunteers and seven family members of the patients. The patients showed significantly reduced levels of prealbumin and carotenoids compared with either normals or family members. Levels of carotenoids did not significantly correlate with level of prealbumin, which suggests that the undernutrition was not the only factor contributing to lower levels of carotenoids in these patients.

Adolescent↗
PubMed1989

Electron microscopic evaluation of bacterial adherence to polyvinyl chloride endotracheal tubes used in neonates.

Studies in adults suggest a possible association between pulmonary nosocomial infection and bacterial adherence to endotracheal tubes (ETT). Some strains of coagulase-negative Staphylococcus produce a mucoid extracellular material known as slime or glycocalyx that helps bacteria adhere to the surface of biomaterials. We examined by scanning and transmission electron microscope the surface of 29 polyvinyl ETT removed from neonatal patients, and ten unused ETT. Multiple surface irregularities were found in the unused tubes. These irregularities were most prominent in the tip and around the side hole. Amorphous material containing slime-producing bacteria was found on 29 ETT removed from patients. Tubes in place for longer times had greater areas of surface covered; the amorphous material was more concentrated in the areas where surface irregularities were most prominent. No clinical correlation with pulmonary nosocomial infection was established.

Bacteria↗