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Biomedical subjects

S A Berger

Publications and source records attributed to S A Berger.

At least 19 recordsLinked to original sources
PubMed2000

Differential stimulation of c-Kit mutants by membrane-bound and soluble Steel Factor correlates with leukemic potential.

The authors investigated the roles of PI3-kinase and PLC-gamma in stimulation by Steel Factor (SLF) through c-Kit. c-Kit mutants YF719, YF728, and a YF719/YF728 double mutant were expressed in 32D myelomonocytic cells. KitYF719 fails to recruit PI3-kinase after stimulation with SLF, whereas KitYF728 fails to stimulate PLC-gamma phosphorylation or mobilize Ca(++). Both single mutants responded mitogenically to soluble SLF (sSLF) in a manner indistinguishable from wild type (WT), although sSLF failed to stimulate or promote the survival of cells expressing the double mutant. In contrast, although cells expressing WT or YF719 were mitogenically stimulated by membrane-bound SLF (mSLF), stimulation of cells expressing KitYF728 was impaired. Similarly, cells expressing WT or YF719 receptors were stimulated by plate-bound anti-Kit antibodies, whereas cells expressing the YF728 receptor were not stimulated. Neomycin sulfate, a PLC antagonist, inhibited cells expressing YF719 receptors stimulated by sSLF. Neomycin also inhibited cells expressing the WT receptor that were stimulated by mSLF or immobilized anti-Kit antibodies but did not inhibit stimulation of cells expressing WT or YF719 receptors by sSLF. 32D cells expressing KitWT, KitYF719, or KitYF728 were injected into mice and the presence of cells was evaluated by colony assays 6 to 7 weeks later. Although both KitWT and KitYF719 expressing cells could be recovered from the spleen and bone marrow, recovery of KitYF728 cells from these organs was severely reduced. These results indicate that Kit tyrosine 728 is of particular importance for mitogenic stimulation by mSLF or immobilized ligand and is required for full maintenance of cells in vivo, likely through activation of PLC-gamma. (Blood. 2000;96:3734-3742)

Animals↗
PubMed2000

Epo regulates erythroid proliferation and differentiation through distinct signaling pathways: implication for erythropoiesis and Friend virus-induced erythroleukemia.

We have recently isolated the erythroleukemic cell line, HB60-5, that proliferates in the presence of erythropoietin (Epo) and stem cell factor (SCF), but undergoes terminal differentiation in the presence of Epo alone. Ectopic expression of the ets related transcription factor Fli-1 in these cells resulted in the establishment of the Epo-dependent cell line HB60-ED that proliferates in the presence of Epo. In this study, we utilized these two cell lines to examine the signal transduction pathways that are activated in response to Epo and SCF stimulation. We demonstrate that Epo, but not SCF, phosphorylates STAT-5 in both HB60-5 and HB60-ED cells. Interestingly, SCF activates the Shc/ras pathway in HB60-5 cells while Epo does not. However, both Epo and SCF are capable of activating the Shc/ras pathway in HB60ED cells. Furthermore, enforced expression of gp55 in HB60-5 cells by means of infection with the Spleen Focus Forming virus-P (SFFV-P), confers Epo independent growth, which is associated with the up-regulation of Fli-1. Activation of the Shc/ras pathway is readily detected in gp55 expressing cells in response to both Epo and SCF, and is associated with a block in STAT-5B tyrosine phosphorylation. These results suggest that STAT-5 activation, in the absence of Shc/ras activation, plays a role in erythroid differentiation. Moreover, Fli-1 is capable of switching Epo-induced differentiation to Epo-induced proliferation, suggesting that this ets factor regulated genes whose products modulate the Epo-Epo-R signal transduction pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Influence of stenosis morphology on flow through severely stenotic vessels: implications for plaque rupture.

Flow patterns and flow-related stresses contribute to the characterization of health risks, particularly the risk of plaque rupture, posed by a particular atherosclerotic stenosis. Blood flow in the presence of significant plaque deposits is investigated, and the influence of factors such as stenosis morphology and surface irregularity is evaluated. Solutions for three-dimensional, unsteady flow in these stenotic vessels are obtained for an incompressible, Newtonian fluid. The equations of motion are solved numerically using a finite volume formulation. The resulting flow patterns and shear and normal stresses are interpreted with respect to diagnostic implications, including the possibility of plaque rupture. The inadequacy of "percent stenosis" to characterize the risks posed by a particular plaque is demonstrated. Surface irregularity, stenosis aspect ratio, and the shape of the pulsatile waveform all have considerable influence on the flow field and on the stresses on the plaque. A measure of surface irregularity or plaque symmetry, in particular, may complement percent stenosis in diagnosing the risk of plaque rupture.

Arteriosclerosis↗
PubMed2000

Clinical significance and antibiotic resistance patterns of Leminorella spp., an emerging nosocomial pathogen.

Although Leminorella spp., members of the family Enterobacteriaceae, were previously isolated from feces and urine specimens, clinical correlates have not been studied. We conducted a retrospective study to investigate the clinical significance and disease spectrum of these organisms, as well as their antibiotic susceptibility patterns. Identification and susceptibility testing were performed by an automated system. Eighteen cases were identified retrospectively during a 28-month period (1/97 to 4/99), representing an incidence of 11 cases per 100,000 patient admissions. The medical records of 14 patients were reviewed. The average patient age was 67 years, and 78% were males. Patients had multiple and diverse underlying conditions which might have predisposed them to infection. Leminorella spp. were classified as definite pathogens in 43% of the cases, probable pathogens in 29%, and possible pathogens in 21%. In one case of asymptomatic bacteriuria, the isolate had no clinical significance. All infections but one were nosocomial. Clinical syndromes included urinary tract infection in six patients, surgical site infection in three patients, and primary bacteremia, peritonitis, respiratory tract infection, and soft tissue infection in one patient each. Isolates were uniformly susceptible to imipenem. Other beta-lactam agents had poor activity against the isolates. We conclude that Leminorella spp. are significant nosocomial pathogens that are capable of causing a variety of clinical syndromes and are resistant to multiple antibiotic agents.

Adult↗
PubMed1999

A transforming mutation enhances the activity of the c-Kit soluble tyrosine kinase domain.

An activating mutation (DY814) located in the catalytic domain of the c-Kit receptor has been found in mastocytomas from human, mouse and rat. We evaluated the enzymic properties of purified wild-type (WT) and DY814 tyrosine kinase domains expressed in Pichia pastoris. A linker encoding the Flag epitope was fused to c-Kit cDNA species, enabling affinity purification of the proteins with anti-Flag antibodies. Yeast lysates expressing DY814 contained multiple tyrosine-phosphorylated proteins, whereas WT lysates had no detectable tyrosine phosphorylation. Purification of the WT and mutant kinases in the presence of vanadate demonstrated that both enzymes undergo autophosphorylation. Kinetic analyses of WT and DY814 kinases indicated that at 20 nM enzyme concentration the mutation increases the specific activity 10-fold and decreases the apparent Km for ATP 9-fold. WT activity displayed a hyperbolic dependence on enzyme concentration, consistent with a requirement for dimerization or aggregation for activity. This activity was also enhanced by anti-Flag antibodies. In contrast, the dependence of DY814 activity on enzyme concentration was primarily linear and only marginally enhanced by anti-Flag antibodies. Gel-filtration analysis showed that the WT kinase migrated as a monomer, whereas the DY814 mutant migrated as a dimer. These results indicate that this point mutation promotes dimerization of the c-Kit kinase, potentially contributing to its transforming potential in mast cells.

Animals↗
PubMed1998

Protection from apoptosis by steel factor but not interleukin-3 is reversed through blockade of calcium influx.

Steel factor (SLF), the ligand for the c-Kit receptor, protects hemopoietic progenitors and mast cells from apoptosis. We show here that protection of 32D-Kit cells or mast cells from apoptosis by SLF is abrogated through concurrent inhibition of Ca2+ influx. In contrast, cell survival promoted by interleukin-3 is not affected by Ca2+ influx blockers. In the presence of blockers, increasing stimulation by SLF leads to greater levels of cell death in the population, indicating that it is the combination of activation by SLF with concurrent blockade of Ca2+ influx that results in apoptosis. The p815 mastocytoma, which expresses a mutated, constitutively active c-kit receptor, dies apoptotically in the presence of Ca2+ influx blockers alone. Ionomycin protects cells from SLF plus blocker-induced apoptosis, confirming specificity for Ca2+ ion blockade in cell death induction. Overexpression of bcl-2, which protects 32D-Kit cells from factor withdrawal, does not protect cells from apoptosis by SLF plus blocker. In contrast, caspase inhibitors YVAD-CHO, DEVD-FMK, and Boc-Asp-FMK protect cells from SLF plus blocker-induced death. These observations highlight the importance of SLF-stimulated Ca2+ influx in the protection of cells from apoptosis and demonstrate a new mechanism for inducing bcl-2 insensitive, caspase-dependent apoptosis through the combination of SLF stimulation with Ca2+ influx blockade.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Phosphatidylinositol 3-kinase and Ca2+ influx dependence for ligand-stimulated internalization of the c-Kit receptor.

We have evaluated the role of phosphatidylinositol 3-kinase (PI3-kinase) and Ca2+ influx in ligand-stimulated internalization of the c-Kit receptor. The wild type (wt) c-Kit receptor and YF719, a mutant receptor in which the SH2-mediated binding site for the p85 subunit of PI3-kinase is disrupted, were expressed in DA-1 cells. YF719 internalized with similar kinetics as wt c-Kit although the receptor remained localized close to the plasma membrane. However, in the absence of extracellular Ca2+, or in the presence of the competitive Ca2+ influx blocker Ni2+, the YF719 mutant failed to internalize. Failure to internalize in the absence of Ca2+ was also observed for the wt c-Kit receptor in cells that were pretreated with the PI3-kinase inhibitor, wortmannin. Following stimulation with ligand, clathrin heavy chains were found to co-immunoprecipitate with c-Kit. However, under conditions in which PI3-kinase activity is inhibited and Ca2+ influx is blocked, clathrin failed to co-immunoprecipitate with c-Kit. Our results demonstrate that both Ca2+ influx and PI3-kinase activity influence c-Kit endocytosis, and inhibition of these two signals disrupts the earliest stages of ligand-mediated internalization.

3T3 Cells↗
PubMed1996

Identification of a bacterial inhibitor of protein kinases. Mechanism and role in host cell invasion.

We show that Escherichia coli produce a factor that inhibits the activity of tyrosine and serine/threonine protein kinases. The factor is a protein found in the periplasmic compartment and is also secreted into the culture medium. Using a particle concentration fluorescence immunoassay specific for tyrosine kinase activity and inhibition of the tyrosine kinase p56(lck), we purified this factor to apparent homogeneity. Analysis of trypsin-digested fragments by mass spectrometry identified the inhibitor as the bacterial periplasmic protein UDP-sugar hydrolase, an enzyme with potent and nonspecific 5'-nucleotidase activity. Overexpression of the enzyme in bacteria leads to coordinate increases in both 5'-nucleotidase and p56(lck) inhibitory activity, confirming the identity of the inhibitor. The kinase inhibitory activity appears to be due to the formation of adenosine, which we show is inhibitory for p56(lck), cAMP-dependent protein kinase, and casein kinase. Overexpression of UDP-sugar hydrolase leads to an increase in the recovery of enteropathogenic E. coli following infection of HeLa cell monolayers and corresponding alterations in tyrosine-phosphorylated host proteins. These results suggest that UDP-sugar hydrolase may be an important factor affecting host cell function following intracellular bacterial infection.

Adenosine↗
PubMed1996

Calculation of the magnetization distribution for fluid flow in curved vessels.

The signal intensity in magnetic resonance angiography (MRA) images reflects both morphological and flow-related features of vascular anatomy. A thorough understanding of MRA, therefore, demands a careful analysis of flow-related effects. Computational fluid dynamics (CFD) methods are very powerful in determining flow patterns in 3D tortuous vessels for both steady and unsteady flow. Previous simulations of MRA images calculated the magnetization of flowing blood by tracking particles as they moved along flow streamlines that had been determined by a CFD calculation. This manuscript describes MRA simulations that use CFD calculations to determine magnetization variation at a fixed point and, therefore, do not require streamline tracking to calculate the distribution of magnetization in flowing fluids. This method inherently accounts for uniform particle density, avoids problems associated with tracking particles close to the wall, and is well-suited to modeling pulsatile flow.

Blood Flow Velocity↗
PubMed1996

Molecular genetic analysis of glucocorticoid and mineralocorticoid signaling in development and physiological processes.

To understand the role of glucocorticoid and mineralocorticoid signalling during development and in whole animal physiology, we have disrupted the mouse glucocorticoid and mineralocorticoid receptor gene by gene targeting. Most of the mice with a disrupted glucocorticoid receptor gene die within the first hours after birth due to severe lung atelectasis. Perinatal induction of gluconeogenic enzymes in the liver is impaired. Regulation of the glucocorticoid synthesis via the hypothalamic-pituitary-adrenal axis is perturbed, leading to increased plasma levels of corticosterone and adrenocorticotrophic hormone. Activation of the hypothalamic-pituitary-adrenal axis results in extensive hypertrophy and hyperplasia of the cortical zones of the adrenal and induction of genes involved in steroid biosynthesis. The adrenal medulla is disorganized and severely reduced in size; no cells capable of adrenaline synthesis can be detected. Mineralocorticoid receptor deficient mice die mainly at day 9/10 after birth. Weightloss precedes death of homozygous mutant mice and is correlated with an increase in the haematocrit. As a consequence of this mutation, plasma levels of renin and aldosterone are high elevated.

Adrenal Medulla↗
PubMed1996

Central intraluminal saturation stripe on MR angiograms of curved vessels: simulation, phantom, and clinical analysis.

PURPOSE: To investigate the appearance of reduced signal intensity in the center of blood vessels on magnetic resonance (MR) angiograms that can mimic intraluminal thrombus. MATERIALS AND METHODS: Simulations and phantom studies were performed to analyze MR angiogram appearance distal to a pronounced curve. RESULTS: Saturation effects substantially lower the signal strength in the center of the vessel relative to that at the vessel periphery. These effects appeared even though the flow was well ordered and laminar. In curved geometries, secondary flow patterns produced counter-rotating vortices, which moved the fastest-moving particles to the outside of the curve and folded the slow-moving particles to the center of the vessel. CONCLUSION: Imaging parameter choices that reduce saturation, such as acquisition of a two-dimensional section transverse to the vessel and through the questionable region, effectively eliminate the central hypointensity effect in vivo.

Adult↗