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N Sherman

Publications and source records attributed to N Sherman.

At least 19 recordsLinked to original sources
PubMed2000

Mass spectrophotometric evidence for P-III/P-IV metalloproteinases in the venom of the Boomslang (Dispholidus typus).

The Boomslang, Dispholidus typus, is a mid- to rear-fanged arboreal colubrid widely distributed throughout much of the African continent. Envenoming by this species is rare although deaths have been recorded. Typical symptoms associated with envenoming include diffuse intravascular coagulation (DIC) caused by fibrinogen consumption and consequent incoagulable blood together with haemorrhage into tissues such as muscle and brain; together, these procoagulant and haemorrhagic effects of the venom result in a very poor prognosis in patients who receive a large dose of venom and who are not treated with antivenom. Renal failure may also result from acute tubular necrosis resulting from pigment nephropathy. Little is known about the toxic components present in the venom; however, proteolytic activity has been reported although the proteinases involved have not been identified. In this study we provide LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) data supporting the presence of class P-III/P-IV snake venom metalloproteinases (SVMPs) in Boomslang venom. Using a polyclonal antibody raised against the P-III haemorrhagic toxin (Jararhagin) obtained from the venom of the Brazilian pit viper, Bothrops jararaca, we identified by western blot a 65 kDa protein from Boomslang venom which cross-reacted with the jararhagin antibody. A corresponding band from SDS-PAGE was subjected to tryptic digestion followed by LC/MS/MS sequence analysis of the digestion mixture. A variety of peptide sequences were identified in the digest, one of which was clearly homologous with a highly conserved region of the disintegrin-like domains of P-III/P-IV SVMPs. These data provide the first structural evidence for the presence of SVMPs in Boomslang venom; it is possible that SVMPs may also be present in the venoms of other colubrids, which cause similar symptoms in envenomed humans. In other snake venoms, most notably those of the Viperinae and Crotalinae subfamilies, many of the coagulopathic and haemorrhagic syndromes associated with systemic and local envenoming are attributed to SVMPs. The identification of a P-III/P-IV SVMP sequence in D. typus venom suggests that many of the pathological signs resulting from envenoming by this species may also be due to the presence of SVMPs in the venom. It is hoped that these results may accelerate research into colubrid venoms and may provide new insights into novel and more efficacious treatments for colubrid envenoming.

Amino Acid Sequence↗
PubMed1999

Cell adhesion to a population of laminin isoforms isolated from normal renal tissue.

To assess whether cells react differently towards a population of several laminin isoforms, as found in vivo, vs. a single isoform, we have compared the biological activity of kidney laminins to that of pure laminin 1. The kidney laminin preparation contained laminin 1 and further isoforms. Both substrates induced adhesion of a large spectrum of cell types, with kidney laminins being the most active. Unfolding of the coil-coiled conformation of the kidney isoforms negatively affected cell adhesion-promoting activity, which indicated that conformation-dependent cell binding is a characteristic feature of many or all laminins. Cellular interactions with kidney laminins were mediated by alpha3beta1 and alpha6beta1 integrins, with the contribution of alpha3beta1 being apparently lower than that of alpha6beta1 integrins. Immunofluorescence staining of vinculin and integrin subunits decorated focal adhesions on kidney laminins which differed in morphology from those formed on laminin 1 alone, in spite of the presence of the latter in the kidney preparation. These observations collectively indicate that tissue specific but often overlapping expression of laminin isoforms might modulate cell behavior by the activation of distinct sets of integrins and by the induction of distinct molecular assemblies within the cell adhesion signaling complexes.

Amino Acid Sequence↗
PubMed1999

FSP95, a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa.

Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of approximately 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.

Amino Acid Sequence↗
PubMed1998

Phosphotyrosine (p-Tyr)-dependent and -independent mechanisms of p190 RhoGAP-p120 RasGAP interaction: Tyr 1105 of p190, a substrate for c-Src, is the sole p-Tyr mediator of complex formation.

p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.

Amino Acid Sequence↗
PubMed1997

Mental health home care aide program. Enhancing services all around.

Home care aides are a vital link in the home care continuum. One agency realized that specialty training for home care aides would not only make them even more valuable to the agency but also provide a career ladder for aides. One of the agency's first areas of specialty training was mental health.

Career Mobility↗
PubMed1996

Conservation of minor histocompatibility antigens between human and non-human primates.

It is well accepted that minor histocompatibility antigens (mHag) can function as transplantation barriers between HLA-matched individuals. Little is known about the molecular nature and evolutionary conservation of mHag. It is only very recently that the first human mHag were identified. The HLA-A2.1-restricted mHag HA-2 and the HLA-B7-restricted mHag H-Y appeared to be peptides derived from polymorphic self proteins. Here we show that the HLA-A2.1-restricted mHag HA-1, HA-2, and the H-Y peptides are conserved between man, chimpanzees and rhesus macaques. Human cytotoxic T cell clones specific for the HLA-A2.1-restricted mHag HA-1, HA-2, and H-Y recognized HLA-A2.1 gene-transfected chimpanzee and rhesus macaque cells. High-pressure liquid chromatography fractionation of HLA-A2.1-bound peptides isolated from the HLA-A2.1-transfected chimpanzee cells revealed that the chimpanzee HA-1 and HA-2 co-eluted with the human HA-1 and HA-2. Subsequent amino acid sequencing showed that the chimpanzee HA-2 peptide is identical to the human HA-2 peptide. Our functional and biochemical results demonstrate that mHag peptides are conserved for over 35 million years.

Amino Acid Sequence↗
PubMed1996

Providing social work without insurance.

Home care agencies, particularly nonprofit ones, often face financial dilemmas when patients who do not qualify for insurance coverage desperately need social work services. One visiting nurse association found a way to provide social services to these patients through innovative funding sources.

Aged↗
PubMed1994

Definition of specific peptide motifs for four major HLA-A alleles.

Allele-specific motifs for the human MHC class I molecules, HLA-A1, A3, A11, and A24 were characterized by three complementary approaches. First, amino acid sequence analysis of acid eluted peptide pools from affinity purified class I molecules defined putative motifs 9 or 10 amino acids in length and bearing critical anchor residues at position 2 and at the COOH-terminal. These motifs were distinct, with the exception of the HLA-A3 and A11 motifs that were very similar to each other. Second, the correctness of these putative motifs was verified by analyzing the binding capacity of polyalanine peptide analogues to purified HLA-A molecules. Several alternative anchor residues that were not obvious from the pooled peptide sequencing analysis were identified. Third, sequences of individual peptides eluted from HLA-A1, A11, and A24 were determined by tandem mass spectrometry. Nonamers were the predominant species, although peptides of 8, 10, 11, and 12 amino acids in length were also identified. These peptides displayed anchor residues predicted by the specific motifs at position 2 and at the COOH-terminal, regardless of peptide length. Synthetic versions of the naturally processed peptides were shown to bind to the appropriate HLA-A alleles with IC50 values in the 0.3- to 200-nM range. A rational approach to search Ags with known amino acid sequences for epitopes restricted by some of the most common HLA-A types and of potential clinical importance is now feasible.

Alleles↗
PubMed1994

Influence of surgical margins on outcome in patients with preoperatively irradiated extremity soft tissue sarcomas.

BACKGROUND: Limb-sparing surgery for soft tissue sarcomas of the extremities may result in microscopically positive surgical margins. The consequences of these microscopically positive margins are unknown. We have analyzed the influence of surgical margins on local disease control and overall survival in patients with extremity soft tissue sarcomas who received preoperative radiation therapy followed by limb-sparing surgery. METHODS: Ninety-five consecutive patients with intermediate and high grade extremity sarcomas who received preoperative radiation therapy and limb-sparing surgery were identified from a soft tissue sarcoma data-base. The clinical outcome of 24 patients who had microscopically positive surgical margins was compared with that of 71 patients who had clear surgical margins. RESULTS: Multivariate statistical analysis revealed that patients with microscopically positive surgical margins or intraoperative tumor violation had an increased risk for local failure. High grade, large size, and intraoperative violation of the tumors were associated with decreased overall survival. However, neither the presence of a positive surgical margin nor the occurrence of a local failure adversely affected overall survival. CONCLUSIONS: Achieving negative surgical margins in patients with intermediate and high grade extremity sarcomas enhances local disease control but does not measurably improve overall survival. These data should be factored into patient management decisions in cases where the goal of achieving clear surgical margins requires amputation or the significant functional compromise of the extremity.

Actuarial Analysis↗
PubMed1994

Radiation therapy for alveolar soft-part sarcoma.

A clinical experience with radiotherapy in 18 patients with alveolar soft-part sarcoma is presented. Adjuvant radiotherapy was associated with prolonged local control in six of six patients without metastatic disease at diagnosis; later one patient relapsed systematically. Meaningful palliation was achieved in all patients with extra-skeletal (and possibly skeletal) metastatic disease. Radiation therapy may be beneficial for patients with alveolar soft-part sarcoma by enhancing local control achieved with limited surgery, by retarding progression of metastatic deposits, and by providing meaningful palliation.

Adult↗
PubMed1993

Coexistence of deep venous thrombosis and heterotopic ossification in the pediatric patient.

A 14-year-old girl and a 15-year-old boy with severe traumatic brain injuries were noted to have marked asymmetry in the lower extremities. Clinical and radiologic evaluation demonstrated the coexistence of deep venous thrombosis (DVT) and heterotopic ossification (HO). These cases demonstrate that although DVT are rare in children, they can be present and may coexist with the more common presentation of a swollen lower extremity secondary to HO.

Adolescent↗
PubMed1992

Na+/K(+)-ATPase activity and its alpha II subunit gene expression in rat skeletal muscle: influence of diabetes, fasting, and refeeding.

We have examined the effects of diabetes, fasting, and refeeding on Na+/K(+)-adenosine triphosphatase (ATPase) activity and its catalytic alpha II subunit gene expression in skeletal muscle. Two hypoinsulinemic states, streptozotocin-induced diabetes and 48-hour fasting caused a significant decrease (P less than .05) in skeletal muscle Na+/K(+)-ATPase activity and a marked increase (P less than .01) in the levels of alpha II subunit mRNA. A decrease in enzyme activity was observed on the 2nd and the 14th day of diabetes, whereas an increase in alpha II mRNA levels was found only on the 14th day. The levels of alpha I mRNA were not affected, while the levels of mRNA of the structural beta subunit were decreased on the 14th day of diabetes. Correction of hyperglycemia with insulin restored enzyme activity and alpha II isoform mRNA levels toward normal in diabetic animals. Refeeding for 48 or 72 hours restored these parameters to normal in skeletal muscle of previously fasting rats. These observations suggest that a decrease in muscle Na+/K(+)-ATPase activity may lead to a compensatory increase in its alpha II subunit gene expression. The levels of insulin and not of glycemia appear to be critical in modulating Na+/K(+)-ATPase activity and gene expression.

Animals↗
PubMed1990

Postprandial changes in cytosolic free calcium and glucose uptake in adipocytes in obesity and non-insulin-dependent diabetes mellitus.

We evaluated the possible relationship between [Ca2+]i and glucose uptake in the postabsorptive state and postprandially in adipocytes obtained from normal and obese subjects, as well as from patients with non-insulin-dependent diabetes mellitus (NIDDM). Adipocytes isolated from overnight-fasted obese and NIDDM patients revealed high levels of [Ca2+]i (p less than 0.05 vs. control) in association with a decreased insulin-stimulated glucose uptake (p less than 0.05 vs. controls). In obese and NIDDM patients treated with oral hypoglycemic agents, the overnight fasting levels of [Ca2+]i were increased postprandially (p less than 0.05), concomitantly with a further decrease in insulin-stimulated 2-deoxyglucose uptake. Although the precise nature of the relationship between [Ca2+]i in specific insulin target tissues and diminished insulin action remains unknown, it is clear that high levels of [Ca2+]i may contribute to the development of insulin resistance.

Adipose Tissue↗
PubMed1989

Mechanism of insulin resistance induced by sustained levels of cytosolic free calcium in rat adipocytes.

We have recently provided evidence that elevated levels of cytosolic free Ca2+ ([Ca2+]i) decreased insulin-stimulated glucose uptake in isolated rat adipocytes. To investigate the mechanism of Ca2+ action, we examined the effects of elevated levels of [Ca2+]i on insulin binding, autophosphorylation, and tyrosine kinase activity (TKA) of insulin receptors as well as basal and insulin-stimulated cellular distribution of glucose transporters. The latter was assessed by cytochalasin-B binding to plasma membrane and cytosolic fractions. Elevated concentrations of [Ca2+]i were maintained by incubating adipocytes with a depolarizing concentration of K+ (40 mM). Basal nonstimulated glucose uptake was not altered by increased levels of [Ca2+]i. Adipocytes with higher [Ca2+]i (220 +/- 15 nM) showed 30% reduction in insulin-stimulated 2-deoxyglucose uptake compared with control cells ([Ca2+]i, 140 +/- 18 nM). Moreover, adipocytes with higher levels of [Ca2+]i demonstrated an approximately 10% reduction in autophosphorylation and TKA of insulin receptors without a change in insulin binding. Both basal and insulin-stimulated distributions of glucose transporters were unaffected by sustained levels of [Ca2+]i. The effects of elevated [Ca2+]i were not mimicked by protein kinase-C activation. These observations suggest that 1) elevated or sustained levels of [Ca2+]i impair insulin-stimulated glucose uptake; and 2) Ca2+-induced impairment appears to reside at the postbinding steps of insulin action and probably interferes with the TKA of insulin receptors and the intrinsic activity of glucose transporters.

Adipose Tissue↗
PubMed1988

Exocytosis in normal anterior pituitary cells. Quantitative correlation between growth hormone release and the morphological features of exocytosis.

We have used high-pressure freezing techniques to study exocytosis in rat anterior pituitary cells. The cells were either unstimulated or exposed to 1 nM growth hormone releasing factor (GRF) for 10 min before ultrarapid freezing. The magnitude of growth hormone (GH) release was then correlated with the number of exocytotic events observed with freeze-fracture electron microscopy. High-pressure freezing of unfixed and uncryoprotected specimens permits cryofixation of samples up to 1 mm diam (0.5 mm thick) without ice crystal damage, and arrests exocytotic events within 10 ms. Our studies comparing conventionally fixed specimens with those prepared by high-pressure freezing confirm that areas of intramembrane particle clearing at potential exocytotic sites are an artifact of conventional fixation and/or cryoprotection techniques. The cells exposed to 1 nM GRF released approximately fivefold more GH than did unstimulated cells. Morphologically, we have observed a 3.3-fold increase in the number of exocytotic events in GRF-stimulated cells, 33.7 events/100 micron2 compared with 10.4 events/100 micron2 for unstimulated cells. In additional experiments, we studied the effects of two inhibitors of GRF-induced exocytosis, somatostatin and sodium isethionate. Both compounds elicit the same response, a parallel decrease in exocytotic events and in secreted product. We conclude that high-pressure freezing, combined with freeze-fracture and freeze-substitution processing techniques, is an excellent tool for studying the morphological aspects of exocytosis. In the present investigation, it has allowed us to quantitatively relate the biochemistry and morphology of exocytosis in anterior pituitary cells.

Animals↗
PubMed1988

Relationship between cytosolic free calcium concentration and 2-deoxyglucose uptake in adipocytes isolated from 2- and 12-month-old rats.

We have examined the relationship between insulin-stimulated 2-deoxyglucose uptake and cytosolic free calcium concentrations, [( Ca2+]i), in adipocytes isolated from 2- and 12-month-old rats. The basal rates of glucose uptake and the levels of cytosolic Ca2+ were only minimally reduced in 12-month-old animals. In contrast, insulin-stimulated glucose up-take and [Ca2+]i were significantly decreased in older adipocytes at all insulin concentrations (P less than 0.01). When the rate of glucose uptake was plotted as a function of [Ca2+]i, insulin-stimulated glucose uptake was almost identical in older and younger animals at any given level of [Ca2+]i. Similar to insulin, glyburide and K+ increased [Ca2+]i in both younger and older adipocytes. However, glyburide- and K+-elicited responses were lower in older rats (P less than 0.01). The effects of insulin, glyburide, and K+ on [Ca2+]i are mediated via voltage-dependent Ca2+ channels. Thus, the present observations suggest an impairment in either function and/or availability of the voltage-dependent Ca2+ channels in older animals. This was supported by the finding of reduced [3H]nitrendipine binding in adipocytes isolated from older animals (6.5% vs. 3.3% in 2- and 12-month-old rats, respectively; P less than 0.01). The results of these experiments indicate that the postreceptor changes in adipocyte responsiveness to insulin in aging may involve inadequate increases in [Ca2+]i. The latter probably occurs as a result of decreased availability and/or function of the voltage-dependent calcium channels.

Adipose Tissue↗
PubMed1987

The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes.

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.

Adipose Tissue↗