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Biomedical subjects

L M Graves

Publications and source records attributed to L M Graves.

At least 55 records · Page 3Linked to original sources
PubMed1994

Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation.

Directed migration or chemotaxis of arterial smooth muscle cells (SMC) contributes to intimal SMC accumulation, a key event in the development of atherosclerotic lesions and in restenosis after angioplasty. The present study compares and contrasts insulin-like growth factor I (IGF-I) and platelet-derived growth factor (PDGF-BB) as chemoattractants and mitogens for human arterial SMC. Compared with PDGF-BB, IGF-I is a weaker SMC mitogen. Thus, PDGF-BB, but not IGF-I, evokes a strong and rapid activation of mitogen-activated protein (MAP) kinase kinase and MAP kinase. However, IGF-I is a potent stimulator of directed migration of human arterial SMC, as measured in a Boyden chamber assay. The half-maximal concentration for migration is similar to the Kd for IGF-I receptor interaction. An IGF-I receptor-blocking antibody blocks the effects of IGF-I, IGF-II, and insulin, indicating that the effects are indeed mediated through the IGF-I receptor. The maximal effect of IGF-I on directed migration ranges between 50% and 100% of the effect of PDGF-BB, the strongest known chemoattractant for SMC. The ability of IGF-I and PDGF-BB to induce chemotaxis coincides with their ability to stimulate phosphatidylinositol turnover, diacylglycerol formation, and intracellular Ca2+ flux and suggests that these signaling pathways, but not activation of the MAP kinase cascade, are required for chemotaxis of human arterial SMC.

Amino Acid Sequence↗
PubMed1993

Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells.

Stimulation of aortic smooth muscle cells with platelet-derived growth factor BB homodimer (PDGF-BB) leads to the rapid activation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MAPKK). Compounds that increase cAMP and activate protein kinase A (PKA)--prostaglandin E2, isoproterenol, cholera toxin, and forskolin--were found to inhibit the PDGF-BB-induced activation of MAPKK and MAPK. Forskolin, but not the inactive analogue 1,9-dideoxyforskolin, inhibited PDGF-BB-stimulated MAPKK and MAPK activation in a dose-dependent manner. PKA antagonism of MAPK signaling was observed at all doses of PDGF-BB or PDGF-AA. PKA did not inhibit MAPKK and MAPK activity in vitro, and MAPKK and MAPK from extracts of forskolin-treated cells could be activated normally with purified Raf-1 and MAPKK, respectively, suggesting that PKA blocked signaling upstream of MAPKK. Neither PDGF-BB-stimulated tyrosine autophosphorylation of the PDGF receptor beta subunit nor inositol monophosphate accumulation was affected by increased PKA activity, suggesting that PKA inhibits events downstream of the PDGF receptor. This study provides an example of cross talk between two important signaling systems activated by physiological stimuli in smooth muscle cells--namely, the PKA pathway and the growth factor-activated MAPK cascade.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Metabolic labeling of mitogen-activated protein kinase kinase in A431 cells demonstrates phosphorylation on serine and threonine residues.

Mitogen-activated protein (MAP) kinase kinase is an enzyme that activates the growth factor-regulated MAP kinase in vitro by a mechanism that involves direct phosphorylation of MAP kinase on tyrosine and threonine residues. MAP kinase kinase is stimulated by growth factor treatment of cells and has been shown to be inactivated with protein phosphatases, suggesting that it is regulated by protein phosphorylation. Analysis of two epidermal growth factor-stimulated forms of MAP kinase kinase, purified from 32P-labeled A431 cells, shows that the kinase is phosphorylated on serine and threonine residues and that treatment with protein phosphatases leads to serine dephosphorylation. Under conditions that lead to complete inactivation, only partial dephosphorylation of MAP kinase kinase is observed. Consistent with this finding, inactive forms of MAP kinase kinase, which separate from active forms during the course of purification, are also observed to be phosphorylated in intact cells.

Cell Line↗
PubMed1992

Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases.

Mitogen-activated protein (MAP) kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that specifically phosphorylate and activate MAP kinases in response to extracellular signals. Here, we report the cloning of two forms of cDNA that encode this protein from human T-cells. MKK1a encodes a protein with predicted molecular size of 43,439 Da. Overexpression of this clone in COS cells led to elevated levels of protein and phorbol ester-stimulated MAP kinase kinase activity, confirming that MKK1a encodes the predicted protein. MKK1b, which appears to be an alternatively spliced form of the MKK1a gene, encodes a protein with predicted molecular size of 40,745 Da. Northern analysis revealed that the MKK1 cDNA hybridizes with a single 2.6-kilobase mRNA species in all human tissues examined. Sequence comparison shows homology to a group of yeast kinases that participate in signal transduction and to subdomain XI of other dual specificity kinase.

Amino Acid Sequence↗
PubMed1991

Ribosomal DNA fingerprinting of Listeria monocytogenes using a digoxigenin-labeled DNA probe.

A nonisotopic ribosomal DNA fingerprinting technique was developed for the characterization of Listeria monocytogenes. Plasmid pKK3535 (a pBR322-derived plasmid containing rrnB ribosomal RNA operon of Escherichia coli) was labeled with digoxigenin-11-dUTP by random priming and used to probe EcoRI fragments of L. monocytogenes chromosomal DNA on nylon filters. The method was successfully applied to the characterization of two sets of patient and food isolates of L. monocytogenes.

DNA Fingerprinting↗
PubMed1991

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay.

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.

DNA Probes↗
PubMed1991

Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group.

We compared the cold enrichment (CE) and U.S. Department of Agriculture (USDA) methods for isolating Listeria monocytogenes by examining 402 food samples. The food samples were collected from refrigerators of listeriosis patients as part of a multistate active surveillance project to determine the role of foods in sporadic listeriosis in the United States. L. monocytogenes was isolated from 51 food samples (13%). The USDA method was significantly better (P less than 0.001) than the CE method. The isolation efficiencies of the USDA and CE methods were 96 and 59%, respectively. Quantitation of L. monocytogenes in the food samples revealed that many food samples containing less than 0.3 CFU/g were negative as determined by the CE method but positive as determined by the USDA method.

Cold Temperature↗
PubMed1990

Aspartokinase II from Bacillus subtilis is degraded in response to nutrient limitation.

Aspartokinase II from Bacillus subtilis was shown by immunochemical methods to be regulated by degradation in response to starvation of cells for various nutrients. Ammonium starvation induced the fastest aspartokinase II decline (t1/2 = 65 min), followed by amino acid starvation (t1/2 = 80 min) and glucose limitation (t1/2 = 120 min). Loss of enzyme activity was closely correlated with the disappearance of the alpha subunit; degradation of the beta subunit was somewhat delayed or slower under some conditions. Pulse-chase experiments demonstrated that aspartokinase II was stable during exponential growth; the synthesis of the enzyme rapidly declined in response to nutrient exhaustion. The degradation of aspartokinase II was interrupted by inhibitors of energy production and protein synthesis but was not changed in a mutant lacking a major intracellular protease. Mutants lacking a normal stringent response displayed only a slight decrease in the rate of aspartokinase II degradation, even though aspartate transcarbamylase was degraded more slowly in the same mutant cells. These results indicate that although energy-dependent degradation of biosynthetic enzymes is a general phenomenon in nutrient-starved B. subtilis cells, the degradation of specific enzymes probably involves different pathways.

Amino Acid Sequence↗
PubMed1990

Replica plating of colonies from Listeria-selective agars to blood agar to improve the isolation of Listeria monocytogenes from foods.

Bacterial colonies from Listeria-selective agars were replica plated to sheep blood agar to screen for beta-hemolysis. By using the replica plating method to test for the beta-hemolytic characteristic of all the colonies growing on Listeria-selective agars instead of picking 3 to 10 suspected colonies for further testing, we recovered Listeria monocytogenes from 59 of 142 Listeria-selective agar plates which contained colonies of hemolytic and nonhemolytic Listeria species and were negative when tested by conventional colony picks.

Agar↗
PubMed1990

Aspartokinase III, a new isozyme in Bacillus subtilis 168.

A previously undetected Bacillus subtilis aspartokinase isozyme, which we have called aspartokinase III, has been characterized. The new isozyme was most readily detected in extracts of cells grown with lysine, which repressed aspartokinase II and induced aspartokinase III, or in extracts of strain VS11, a mutant lacking aspartokinase II. Antibodies against aspartokinase II did not cross-react with aspartokinase III. Aspartokinases II and III coeluted on gel filtration chromatography at Mr 120,000, which accounts for the previous inability to detect it. Aspartokinase III was induced by lysine and repressed by threonine. It was synergistically inhibited by lysine and threonine. Aspartokinase III activity, like aspartokinase II activity, declined rapidly in B. subtilis cells that were starved for glucose. In contrast, the specific activity of aspartokinase I, the diaminopimelic acid-inhibitable isozyme, was constant under all growth conditions examined.

Amino Acids↗
PubMed1989

A cluster of blister-associated toxic shock syndrome in male military trainees and a study of staphylococcal carriage patterns.

In March-April 1985, toxic shock syndrome (TSS) developed in three male military trainees at one base secondary to infected blisters acquired while marching during basic combat training. One trainee died. Staphylococcus aureus was isolated from the blisters in all three patients, and the two strains available for testing both produced toxic shock syndrome toxin-1 (TSST-1). A culture survey identified carriage of S. aureus in 44% (145/333) of the trainees; carriage rates did not differ between the company with two ill trainees and a control company. Carriage was more frequent in the nose than foot (41% versus 9%, p less than 0.0001). White trainees were more likely to carry S. aureus than black trainees (47% versus 28%, p = 0.013), although the same proportion of strains was positive for TSST-1 in both races. The two strains obtained from the patients had similar but not identical phage typing patterns, but had identical antibiograms, and neither strain carried any plasmids. Based upon phage typing of the TSST-1 positive S. aureus strains isolated in the study, widespread carriage of the case strain(s) in other trainees was not found nor was clustering noted by barrack room. It is possible that differences in carriage rates may partially explain the low rate of TSS in blacks.

Adult↗
PubMed1988

Characterization of the interaction of ophiobolin A and calmodulin.

1. The fungal toxin ophiobolin A reacts with the epsilon-amino group of lysine to give a conjugated enamine produce with lambda max at 272 nm and a molar extinction of 19,200 per M/cm. 2. Bovine brain calmodulin reacts with ophiobolin A to give a lambda max at 272 nm. 3. One mol of calmodulin reacts with two moles of ophiobolin A. Reaction of 1 mol of ophiobolin A inactivates 1 mol of calmodulin. 4. Ophiobolin A-treated calmodulin is resistant to tryptic cleavage at lysine 77. 5. Ophiobolin A also inhibits Dictyostelium calmodulin which has glutamine instead of lysine at residue 77.

Animals↗
PubMed1987

Production and characterization of monoclonal antibodies to toxic shock syndrome toxin 1 and use of a monoclonal antibody in a rapid, one-step enzyme-linked immunosorbent assay for detection of picogram quantities of toxic shock syndrome toxin 1.

Twenty-six hybridoma cell lines that produced monoclonal antibodies to toxic shock syndrome toxin 1 (TSST-1) were generated by immunizing mice with a highly purified preparation of TSST-1 and fusing their splenic lymphocytes with SP2/0-Ag-14 cells. One monoclonal antibody of the immunoglobulin G1 isotype, designated as PEC-1 10-2SCH, was selected for extensive study. The specificity of this antibody was determined by testing spent culture fluid filtrates of TSST-1- and non-TSST-1-producing strains of Staphylococcus aureus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunoelectrotransfer blot techniques. Monoclonal antibody PEC-1-10-2SCH was specific for TSST-1-producing strains of S. aureus, reacting with TSST-1 and two other proteins which appear to be unique to S. aureus strains that produce TSST-1. Monoclonal antibody PEC-1-10-2SCH was used in conjunction with polyclonal rabbit antibodies to TSST-1 in a rapid, one-step, sensitive, specific, and quantitative enzyme-linked immunosorbent assay. This assay was shown to be more sensitive, faster, and simpler to perform than previously described isoelectric focusing, immunodiffusion, and solid-phase radioimmunoassays for TSST-1. Monoclonal antibody PEC-1-10-2SCH was not reactive with Staphylococcus protein A under the conditions of the test.

Antibodies, Monoclonal↗
PubMed1986

Isolation of Listeria monocytogenes from raw milk.

During a recent outbreak of listeriosis, we examined 121 raw milk samples and 14 milk socks (filters). Listeria monocytogenes was recovered from 15 (12%) of 121 milk specimens and 2 (14%) of 14 milk socks. The optimal processing method consisted of cold enriching diluted milk for 1 month with culture to selective broth, followed by plating.

Agar↗
PubMed1985

Staphylococcus aureus isolates from patients with nonmenstrual toxic shock syndrome. Evidence for additional toxins.

Although toxic shock syndrome toxin-1 (TSST-1) has been proposed as the toxin responsible for toxic shock syndrome, its role in this disease has not been proved. To study this question, we examined Staphylococcus aureus strains isolated from normally sterile sites in patients with nonmenstrual toxic shock syndrome for the presence of TSST-1 production. Only 20 (62.5%) of 32 produced TSST-1, compared with 41 (93%) of 44 vaginal isolates from patients with menstrual toxic shock syndrome. Of strains of S aureus from patients with nonmenstrual toxic shock syndrome, TSST-1-negative isolates were more likely to be associated with a fatal outcome and to not be phage group 1 than TSST-1-positive isolates. Seven of the TSST-1-negative strains were evaluated in a rabbit subcutaneous chamber model of toxic shock syndrome. Fifteen (60%) of 25 rabbits developed a toxic shock syndrome-like illness and nine died. Clinical signs and histopathologic findings in the rabbits were similar to those seen in rabbits inoculated with TSST-1-positive S aureus isolates. These results suggest that other, as yet unrecognized, toxins play a role in toxic shock syndrome, and that TSST-1 production may not be essential to the pathogenesis of toxic shock syndrome.

Adolescent↗
PubMed1984

Production of toxic-shock-associated protein(s) in Staphylococcus aureus strains isolated from 1956 through 1982.

A total of 281 Staphylococcus aureus strains selected from those submitted to the Centers for Disease Control for phage typing between 1956 and 1982 were tested for the production of toxic-shock-associated protein (TSAP) by isoelectric focusing (IEF) and solid-phase radioimmunoassay. The results suggest that the observed temporal trends in the incidence of toxic-shock syndrome were not primarily due to changes in the distribution of TSAP-positive strains of S. aureus. Overall, 39 (14%) were TSAP positive by both methods. The earliest positive strain was an isolate submitted in 1957. TSAP-positive strains were most prevalent in the group of isolates submitted in 1976 for which 29% reacted, but the percent positive subsequently declined for isolates submitted in later years. TSAP production was more frequent among strains of phage types 29, 29/52, and 52 than among other strains. The use of IEF to identify TSAP detected false-positive proteins. Seven strains were positive by IEF and negative by solid-phase radioimmunoassay, whereas only one was positive by solid-phase radioimmunoassay and negative by IEF.

Bacterial Proteins↗
PubMed1983

Toxic shock syndrome: modification and comparison of methods for detecting marker proteins in Staphylococcus aureus.

Development of a new medium and modification of incubation conditions increased production of toxic shock syndrome marker proteins and enabled detection of small volumes of pyrogenic exotoxin C (PEC) by isoelectric focusing and staphylococcal enterotoxin F (SEF) by a newly developed solid-phase radioimmunoassay. The results were compared with those obtained with previously described methods. The results were identical, and all PEC-positive isolates were SEF positive. In a second study of 262 randomly selected Staphylococcus aureus isolates examined by isoelectric focusing and solid-phase radioimmunoassay but grown in fresh beef heart medium, 47 (17.9%) isolates were PEC and SEF positive; however, 9 (3.4%) were PEC positive and SEF negative, and 3 (1.1%) were SEF positive and PEC negative. When grown in buffered beef heart yeast extract medium, six of the previously PEC-positive and SEF-negative isolates were PEC negative. Autoradiographic analysis of selected isolates demonstrated that PEC- and SEF-positive strains bound SEF antitoxin to the protein at isoelectric point 7.2, suggesting that in staphylococci from patients with toxic shock syndrome, PEC and SEF are the same protein. In screening staphylococci for toxic shock syndrome marker proteins, isoelectric focusing to identify PEC may detect false-positive proteins and may be more susceptible to technical variation than immunological methods to detect SEF.

Bacterial Proteins↗