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Biomedical subjects

L D Adams

Publications and source records attributed to L D Adams.

18 recordsLinked to original sources
PubMed1992

Tissue distribution and cellular localization of hsp56, an FK506-binding protein. Characterization using a highly specific polyclonal antibody.

Heat shock protein 56 (hsp56) has been shown to be involved in two cellular pathways, as an immunophilin for FK506 and as a component of steroid receptor complexes. To help define its role in these cellular pathways, we have developed UPJ56, a polyclonal antibody raised against hsp56 purified from Jurkat cells. In Western blot experiments, hsp56 was highly expressed in rat thymus, liver, and spleen, with low levels in lung and muscle. In immunofluorescence experiments using untreated LLC-PK1 cells, fibrillar staining was seen in the cytoplasm, suggesting a cytoskeletal localization of hsp56. The nuclei were brightly stained, except for the nucleoli. Confocal microscopy demonstrated that the staining was present in all planes of the nucleus. These results suggest that hsp56 is expressed in tissues enriched in steroid receptors and is highly expressed in tissues involved in T cell function. Furthermore, the localization of hsp56 with the cytoskeleton and throughout the nucleus is consistent with its association with steroid receptor complexes.

Animals↗
PubMed1992

Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.

The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.

Animals↗
PubMed1992

HIV-1 protease cleaves actin during acute infection of human T-lymphocytes.

Actin, one of the most abundant proteins of the cell, is hydrolyzed by the human immunodeficiency virus type 1 (HIV-1) protease during acute infection of cultured human T lymphocytes. The actin fragments produced during the course of infection are identical to those obtained by recombinant HIV-1 protease digests of (1) a lysate from uninfected T lymphocytes and (2) globular actin itself. Hydrolysis by the HIV-1 protease of physiologically important host cellular proteins during infection may have important consequences relative to viral pathogenesis.

Actins↗
PubMed1990

Effects of trospectomycin on serum sensitivity of Escherichia coli UC 9451.

Trospectomycin sulfate, a chemically synthesized analog of spectinomycin, exhibits a broad range of activity against both aerobes and anaerobes, including the etiological agents of sexually transmitted diseases. Its activity in vitro against Escherichia coli is considered only moderate. At subinhibitory levels, however, trospectomycin induced changes in a pathogenic strain of E. coli, UC 9451, which significantly increased its sensitivity to serum lysis. This strain of E. coli shows high-level resistance to serum in vitro, typically growing twofold within a 45-min incubation period. Following exposure to one-fifth the MIC of trospectomycin, greater than 99% of the bacteria were killed in 25% serum within 15 min. Surviving bacteria were static in this level of serum for over 3 h. Killing was due to lysis mediated by both the classical and alternative complement pathways. The bacteria exposed to trospectomycin were enlarged in both diameter and length, but they still grew at rates comparable to those of untreated bacteria. No other visible morphological changes could be directly related to the increase in serum sensitivity. The profile of outer membrane proteins obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical for trospectomycin-treated or untreated bacteria. However, the relative proportion of four major outer membrane proteins varied considerably.

Anti-Bacterial Agents↗
PubMed1990

Detection and recovery of proteins from gels following zinc chloride staining.

This report describes a method for detecting proteins in SDS polyacrylamide gels using ZnCl2 and their recovery using passive elution. Washing unfixed gels in a dilute solution of ZnCl2 produces two desirable effects. First, it makes the proteins easily visible as clear zones in a white background, and second, it prevents loss of proteins due to diffusion into the wash solution. Compared to other commonly used methods of protein detection such as Coomassie, KCl, copper or silver staining, the zinc stain offers some distinct advantages. Zinc staining can be completed in 15 min for most applications, making it much faster than Coomassie or most silver stains. The zinc stain is more sensitive than Coomassie, KCl or copper stain. Since no harsh chemical conditions are used in the zinc staining procedure, recovery of proteins from the gel is facilitated. More than 90% of selected proteins were recovered from 2-D gels by simple elution from the gel pieces with a buffer containing 10 mM EDTA.

Animals↗
PubMed1989

Rapid modulation of a 64 K dalton fibroblast protein: a PDGF mediated early cellular event.

The results presented here reveal a novel platelet derived growth factor (PDGF) mediated early cellular event. Treatment of growth arrested Balb/c3T3 fibroblasts with PDGF induces a specific and rapid modulation of a 64,000 Dalton (64 KD) protein preexisting in quiescent cells. The kinetics of 64 KD protein modulation indicate that, temporally, this PDGF mediated step lies between the membrane associated immediate events such as receptor autophosphorylation or ion mobilization and the earliest known transcriptional event, the activation of the proto-oncogene c-fos.

Animals↗
PubMed1989

Dietary management of canine and feline chronic renal failure.

Nutritional therapy is the mainstay of management of chronic renal failure in dogs and cats. Diets designed for use in renal failure are typically reduced in protein, phosphorus, and sodium content. These and other dietary modifications are designed to prevent or ameliorate clinical signs of uremia, minimize disturbances associated with excesses or losses of electrolytes and minerals, arrest or retard progression of renal failure, and maintain adequate nutrition.

Animal Nutritional Physiological Phenomena↗
PubMed1988

Mode of fibroblast growth enhancement by human interleukin-1.

Previous studies have demonstrated that interleukin-1 (IL-1) stimulates fibroblast growth (Schmidt, J. A., S. B. Mizel, D. Cohn, and I. Green. 1982. J. Immunol. 128:2177-2182) and binds to specific, high affinity receptors of BALB/c3T3 cells (Bird, T. A., and J. Saklatval. 1986. Nature (Lond.). 324:263-265, 266-268). We have investigated the mechanism of fibroblast growth stimulation by IL-1. Addition of fibroblast growth factor derived from platelets (PDGF) to a quiescent culture of BALB/c3T3 cells produced 8-10-fold increase in DNA synthesis during 24-h incubation. The cellular action of PDGF was mediated through competence induction and required synergistic action of plasma-derived factors for full mitogenic activity. When tested at a wide range of concentrations (0.1-100 pM), natural IL-1 or recombinant IL-1 produced only a maximum of 5-10% of DNA synthesis elicited in response to PDGF or serum. Induction of DNA synthesis required continuous presence of IL-1 and did not exhibit synergism with plasma. Competence induction and mitogenic stimulation by PDGF was associated with early induction of proteins P32, P38, P46-48, P75, and changes in cytoskeletal organization. Examination of these early cellular changes showed that IL-1 did not produce similar induction of cellular proteins and the morphological changes associated with growth stimulation. These results suggest that the mode of IL-1 action on BALB/c3T3 was not through competence induction. When IL-1 was added to cells rendered competent by brief exposure to PDGF, 10-15% additional DNA synthesis occurred during the first 24 h. Extended incubation of PDGF-treated cells in the presence of IL-1 revealed that the stimulation by IL-1 occurred predominantly during the subsequent cycle of DNA replication, wherein DNA synthesis reached three- to fivefold higher than the untreated cultures. We conclude (a) IL-1 alone is not a potent mitogen for BALB/c3T3 cells, and does not bring cells out of the growth arrest Go phase, (b) treatment with PDGF renders the cells more responsive to IL-1, (c) part of the IL-1 action on competent cells may be characterized as progression inducing activity, further, (d) our results indicate that action of IL-1 on PDGF-treated cells produces sustained DNA synthesis for an extended period, perhaps by preventing the entry of cells into growth arrest Go phase.

Animals↗
PubMed1988

Increased amount of a 25-kilodalton phosphoprotein after v-mos transfection of CHO cells.

We transfected Chinese hamster ovary (CHO) cells with a cloned v-mos gene (pHT25). The mos family of oncogenes has previously been shown to have serine-threonine kinase activity. This kinase activity may be required for oncogenic transformation, although its exact biological role is unknown. We found that the transfected cells had an altered morphology, a slower doubling time, and an apparent increase in the amount of a 25-kilodalton (kDa) phosphoprotein that appeared to be of low abundance. Transfection of CHO cells with a cloned temperature-sensitive mos gene (ts159) led to isolation of a cell line that showed the presence of the 25-kDa phosphoprotein at the permissive but not at the nonpermissive temperature, suggesting a direct relationship between mos activity and the presence of this phosphoprotein. The characteristics of altered morphology and depressed growth rate were reminiscent of changes seen after the activation of the cyclic AMP-dependent protein kinase (PKA) in CHO cells. However, PKA activation did not stimulate phosphorylation of this 25-kDa protein, nor was there a change in total PKA activity in these cells. We suggest that the increased presence of the 25-kDa phosphoprotein is a consequence of the v-mos transfection and that it may be involved in the change of morphology and growth rate seen in the CHO cells. Phosphorylation of this protein may be a useful marker of mos and have some functional importance in the transformation of cells by the v-mos oncogene.

Animals↗
PubMed1986

A nonurea electrophoretic gel system for resolution of polypeptides of Mr 2000 to Mr 200,000.

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system which resolves proteins and peptides from Mr 2000 to Mr 200,000 is described. Gradients of polyacrylamide, crosslinker, and glycerol buffered in Tris-phosphate (pH 6.8) are employed. Neither urea nor a stacking gel is required. This system has been used to separate molecules below Mr 3000 which differed by only seven amino acid residues, yet has the capacity to survey masses up to Mr 200,000 on the same gel. Examples are given for separations of myoglobin cyanogen bromide fragments and adrenocorticotropin peptides. Utilizing the same gradient slab gel system in tandem with isoelectric focusing, a two-dimensional separation pattern of mammalian liver cell lysate is shown. A comparison of two different silver stain methods with this system is also given.

Buffers↗
PubMed1985

The antigenic relatedness of proteins from human and simian prostate fluid.

Two-dimensional electrophoretic analysis of human prostate fluid reveals an abundant protein migrating to a molecular weight of 15 kD and an isoelectric point of 5.5. Polyclonal antibodies were raised specifically to microgram quantities of electrophoresed, excised, and eluted PSP15 (prostate secretory protein). Western immunoblot analysis using these antibodies showed they not only react to PSP15, but cross-react with simian prostate and human seminal fluid proteins of similar molecular weights. Two-dimensional gel immunoblots strongly suggest that the seminal protein and PSP15 are the same, thereby providing a more accessible source of the protein. The antibody to the human PSP15 cross-reacted with neither prostate fluid from the ventral lobe of the rat prostate nor the prostate fluid from the beagle dog.

Animals↗
PubMed1984

Insulin-induced rapid decrease of a major protein in fat cell plasma membranes.

In order to increase our understanding of the mode of action of insulin in rat fat cells, we investigated the effect of insulin on protein concentrations in purified fat cell fractions using two-dimensional electrophoresis in combination with an ultrasensitive color silver stain technique. Incubation of fat cells with insulin caused a 90% decrease in the plasma membrane concentration of a major plasma membrane protein with a molecular mass of 90 kDa. The insulin effect was dose-dependent with a half-maximal effect at 9.5 microunits/ml, and time-dependent with a t 1/2 of less than 20 s. Insulin-like growth factor I, orthovanadate, and lanthanum mimicked the effect of insulin. Likewise, fractionation of adipocytes in the presence of divalent cation chelating agents caused a similar reduction in the concentration of the 90 kDa protein, and it was possible to overcome the effects of the chelating agents by adding equivalent amounts of calcium. This suggests the involvement of calcium. The 90 kDa protein was also found in low and high density microsomes, but it was not affected in those fractions by either insulin or chelators. It is suggested from the study that the movement of a 90 kDa protein in fat cell plasma membranes probably represents part of the transmission system in the mechanism of insulin action in rat adipocytes.

Adipose Tissue↗
PubMed1983

Alloxan-induced hyperglycemia in rats is reduced by 16,16-dimethyl-PGE2.

Female rats were treated with subcutaneous 16,16-dimethyl-PGE2 (1-75 micrograms/kg) for 24, 18, and 0.5 h prior to and 6, 24, and 48 h after intravenous beta cell destruction. Protection was assessed by morphological examination of beta cells and the level of fasting hyperglycemia seen 72 h after alloxan treatment. Prostaglandin reduced the degree of alloxan-induced hyperglycemia in a dose-dependent fashion but had no demonstrable effect on morphological assessment of beta cell destruction. However, prostaglandin treatment by itself induced transient (0-2 h) hyperglycemia that could be correlated inversely with the level of fasting blood glucose observed 72 h after alloxan treatment. Administration of oral glucose, which mimics the prostaglandin-induced hyperglycemia, afforded protection against alloxan challenge comparable to that produced by the prostaglandin. Thus, it appears that reduction of alloxan-induced hyperglycemia by 16,16-dimethyl-PGE2 may be linked to the transient hyperglycemia produced prior to alloxan administration.

16,16-Dimethylprostaglandin E2↗
PubMed1977

Measuring various drug use dimensions with a calendar method.

This note presents a technique used to acquire a variety of time-specific data on drug use. The calendar technique involves self-reported patterns of consumption and any changes therein, including cessation. Advantages of this technique over other approaches are discussed, and examples of the variety of use measures that can be derived from the calendar method are illustrated.

Humans↗
PubMedDate not supplied

Tumor progression- and metastasis-associated proteins identified using a model of locally recurrent rat mammary adenocarcinomas.

A recently established model for local breast cancer recurrence using the 13762NF rat mammary adenocarcinoma was used to evaluate biologic and biochemical properties related to clinical outcome for this class of tumors. Sublines isolated from local tumor regrowths following surgical resection differed from each other and from the 'parental' cell lines for multiple phenotypes, including metastatic propensity. Local recurrence- and primary tumor-derived sublines were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), lectin binding to electrophoretically separated proteins, and lactoperoxidase-catalyzed cell surface iodination; and differential protein patterns were compared to tumor progression and metastatic potential. 2D-PAGE revealed several quantitatively different spots which correlated with lung colonization potential. In particular, quantities of an apparently unique, non-cell-surface protein, P50.9 (Mr approximately 50,900, pI approximately 7.3) correlated inversely with metastatic propensity, suggesting that it may be associated with, among other possibilities, the negative regulation of the metastatic phenotype. P50.9 was unrelated to four similarly sized metastasis-associated proteins--tumor autocrine motility factor; the rat analog of tumor suppressor, p53; rat cytokeratin 14 or procathepsin D--as determined by amino acid analysis. A major wheat germ agglutinin binding sialoglycoprotein, gp93 (Mr approximately 93,000), was present in smaller amounts as cells were passaged in vivo and re-established as in vitro cultures [MTF7 greater than 'primary' tumor-derived lines (sc1, sc3) much greater than local recurrence-derived lines (LR1, LR1a, LR3, LR4, LR5, LR6)]. Besides cell surface glycoprotein losses, two of six local recurrence-derived sublines expressed a wheat germ agglutinin-binding sialoglycoprotein, gp110 (Mr approximately 110,000), previously undetected on any of the other cell lines including the parental populations. gp110 was found in LR3 and LR6 which were relatively highly metastatic; however, correlation with metastatic potential failed because gp110 was not present on the metastatic parental cell line, MTF7. These results demonstrate specific quantitative and qualitative protein differences associated with the selection of locally recurrent mammary tumors.

Adenocarcinoma↗