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Biomedical subjects

J L Martin

Publications and source records attributed to J L Martin.

At least 19 recordsLinked to original sources
PubMed1992

The topography of trifluoroacetylated protein antigens in liver microsomal fractions from halothane treated rats.

Sera from patients with halothane hepatitis contain immunoglobulin G (IgG) antibodies to trifluoroacetylated liver microsomal proteins of 100, 76, 59, 57 and 54 kDa, which are produced as a consequence of metabolism of halothane to trifluoroacetyl halide by cytochrome(s) P450. In the present study, the membrane topographies of the various antigens in rat liver microsomal fractions were investigated. Liver microsomal fractions from rats treated with halothane in vivo, and rat liver microsomal fractions which had been incubated with halothane in vitro, were used as the source of trifluoroacetyl antigens. The antigens were detected by immunoblotting. Whereas the 100, 76, 59 and 57 kDa antigens were solubilized from the microsomal membrane by either 0.1 M sodium carbonate or 0.1% (w/v) sodium deoxycholate, the 54 kDa antigen was not solubilized by 0.1% (w/v) sodium deoxycholate. In intact microsomal fractions, the 100, 76, 59 and 57 kDa antigens were not degraded appreciably by trypsin unless detergent was added to permeabilize the microsomal membrane. These results indicate that the 54 kDa antigen is an integral membrane protein, whereas the 100, 76, 59 and 57 kDa antigens are peripheral membrane proteins situated within the lumen of microsomal vesicles, and hence presumably located within the lumen of the endoplasmic reticulum in vivo.

Animals↗
PubMed1992

VIP receptor subtypes in mouse cerebral cortex: evidence for a differential localization in astrocytes, microvessels and synaptosomal membranes.

The binding characteristics of a monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) to the membranes of astrocytes, intraparenchymal microvessels and synaptosomes were analyzed in mouse cerebral cortex. Binding to astrocytes, studied in primary cultures, indicates the presence of a single class of high affinity binding sites with a Kd of 3.3 nM and a Bmax of 565 fmol/mg protein. The structurally related peptide secretin does not compete for sites labeled by M-[125I]VIP. In cultured astrocytes, VIP has been previously shown to promote glycogenolysis. Secretin, despite its lack of interaction with sites labeled by M-[125I]VIP, stimulates glycogenolysis with an EC50 of 0.5 nM, thus demonstrating the presence in astrocytes of functional secretin receptors independent from those for VIP. Trypsinization of the primary astrocyte cultures followed by replating as secondary cultures, reveals a second class of low affinity binding sites, with a Kd of 41.3 nM and a Bmax of 881 fmol/mg protein. Secretin does not compete for this class of low affinity binding sites either. Binding of M-[125I]VIP to intraparenchymal microvessels reveals the presence of two classes of binding sites with Kd of 1.4 and 30.3 nM, and Bmax of 7.1 and 73.8 pmol/mg protein, respectively. Similar to what is observed in primary or secondary astrocyte cultures, secretin does not interact with these sites. In this cell type VIP stimulates cAMP formation with an EC50 of 18 nM, while secretin is ineffective. Finally, in agreement with previous reports in rat and guinea pig cerebral cortex, two classes of binding sites are observed in synaptosomal membranes: a high affinity class with a Kd of 4.9 nM and a Bmax of 316 fmol/mg protein, and a low affinity class with a Kd of 42.8 nM and a Bmax of 1578 fmol/mg protein. In contrast to what is observed in non-neuronal membranes, in synaptosomal membranes, secretin effectively competes for sites labeled by M-[125I]VIP with an EC50 of approximately 150 nM. These results indicate that secretin may represent a useful tool to discriminate between neuronal and non-neuronal VIP binding sites, since it competes with M-[125I]VIP exclusively for the neuronal class of binding sites.

Animals↗
PubMed1992

Femtosecond spectral evolution of the excited state of bacterial reaction centers at 10 K.

The femtosecond spectral evolution of reaction centers of Rhodobacter sphaeroides R-26 was studied at 10 K. Transient spectra in the near infrared region, obtained with 45-fs pulses (pump pulses centered at 870 nm and continuum probe pulses), were analyzed with associated kinetics at specific wavelengths. The t = 0-fs transient spectrum is very rich in structure; it contains separate induced bands at 807 and 796 nm and a bleaching near 760 nm, reflecting strong changes in interaction between all pigments upon formation of the excited state. A complex spectral evolution in the 800-nm region, most notably the bleaching of the 796-nm band, takes place within a few hundred femtosecond--i.e., on a time scale much faster than electron transfer from the primary donor P to the bacteriopheophytin acceptor HL. The remarkable initial spectral features and their evolution are presumably related to the presence of HL, as they were not observed in the DLL mutant of Rhodobacter capsulatus, which lacks this pigment. A simple linear reaction scheme with an intermediate state cannot account for our data; the initial spectral evolution must reflect relaxation processes within the excited state. The importance for primary photochemistry of long distance interactions in the reaction center is discussed.

Cold Temperature↗
PubMed1992

Alterations in tumor angiogenesis associated with stable expression of the HIV tat gene.

Recent evidence suggests that the human immunodeficiency virus type 1 (HIV) trans-activator gene (tat) has transforming properties and may be a causative factor in the development of certain types of cancers, in particular Kaposi's sarcoma (i.e., Vogel J. et al. Nature 335:606-611, 1988). To help elucidate the potential role or roles of the HIV tat gene in neoplastic transformation, cell lines were constructed that constitutively express a functional tat gene product. HeLa cells were coelectroporated with two plasmids, one containing the HIV tat gene in an expression cassette and another containing the dominant selectable marker gene xanthine guanine phosphoribosyltransferase (XGPRT). After XGPRT selection, single-cell clones that expressed a functional tat protein were identified by measuring chloramphenicol acetyltransferase (CAT) activity after electroporating a plasmid containing the CAT gene transcriptionally controlled by HIV trans-activation-responsive region (tar). Phenotypic alterations resulting from the expression of tat were then determined. Control cells and tat-expressing cells grew at similar rates in culture. However, when grown as tumors in nude mice, tat-expressing cells produced a lower percentage of tumors, and the tumors that were produced either regressed, stopped growing, or grew at a very reduced rate compared with cells not expressing tat. These differences may have resulted from a tat-associated reduction in neovascularization in the tumors. A comparison of total cellular proteins by two-dimensional polyacrylamide gel electrophoresis indicated only one reproducible alteration in a polypeptide of approximately 44 kDa and pl of approximately 6.2 associated with tat expression. These cells may be very useful in future in vitro and in vivo studies designed to examine the effects of HIV tat on endothelial and vascular smooth-muscle cells and the role of tat in the etiology of Kaposi's sarcoma.

Animals↗
PubMed1992

Inhibition of human fibroblast insulin-like growth factors binding protein (IGFBP) production by IGFBP-3.

Human neonatal fibroblasts in monolayer culture secrete a number of insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, which may alter paracrine or autocrine IGF activity. Studies in vitro have demonstrated that exogenous IGFBP-3 can both inhibit and potentiate IGF action in these cells; however, it is not known to what extent there is regulatory interaction between the IGFBPs. In this study we report that exogenous and endogenous IGFBP-3 inhibit production of an IGF inducible IGFBP. When analyzed by SDS-PAGE and [125I]IGF-II ligand blotting, human neonatal fibroblasts secrete IGFBP-3, an IGFBP of 29-31 kDa, and a 22-24 kDa IGFBP after treatment with 50 ng/ml IGF-I. When IGF-I treatment was carried out in the presence of increasing concentrations (50-1000 ng/ml) of pure human serum-derived IGFBP-3, there was a dose-dependent decrease in the 29-31 kDa protein. In the presence of excess (250 ng/ml) IGF-I, IGFBP-3 had approximately 20-fold reduced potency in inhibiting 29-31 kDa IGFBP. When endogenous production of IGFBP-3 was increased by treatment with transforming growth factor-beta 1 (TGF beta 1), there was complete inhibition of 29-31 kDa IGFBP, while at high IGF-I concentrations TGF beta 1 had 2 to 3-fold reduced potency. These results demonstrate that fibroblast IGFBP production can be altered by exogenous and endogenous IGFBP-3, and suggest the existence of regulatory interactions between fibroblast IGFBPs.

Carrier Proteins↗
PubMed1992

Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 release IGF-binding protein-3 from human fibroblasts by different mechanisms.

Human neonatal fibroblasts in monolayer culture secrete insulin-like growth factor-binding proteins (IGFBPs), which may modulate IGF action. To examine whether an increase in extracellular concentrations of IGFBPs in response to IGF-I is due to the release of cell-associated IGFBPs, we measured secreted and cell-associated IGFBP-3 immunologically in fibroblast monolayers treated with IGF-I and IGF analogs with altered affinities for the IGF receptors and IGFBPs. IGFBP-3 in medium conditioned by fibroblasts treated with IGF-I was significantly increased (P < 0.05) compared with that in medium from untreated cultures; concomitantly, cell-associated IGFBP-3 was significantly decreased (P < 0.05). [Ser24]IGF-I (reduced affinity for IGF receptors) also increased secreted IGFBP-3 and decreased cell-associated IGFBP-3. In contrast, IGFBP-3 concentrations in medium conditioned by fibroblasts treated with B-chain IGF-I (reduced affinity for IGFBPs) were not significantly increased, and cell-associated IGFBP-3 was unchanged. Heparin, which releases proteins attached to cell surface proteoglycans, increased medium concentrations of IGFBP-3 and decreased IGFBP-3 binding to fibroblasts. An IGFBP of 29-31 kilodaltons (kDa) showed a pattern of regulation similar to that of IGFBP-3, while a third IGFBP, of 24 kDa, was decreased in IGF-I- and [Ser24]IGF-I-conditioned medium and unchanged by B-chain IGF-I and heparin. Preincubation with transforming growth factor-beta 1 (TGF beta 1), which stimulates fibroblast IGFBP-3 production, or human serum-derived IGFBP-3 did not increase cell-associated IGFBP-3. Analysis of total RNA isolated from fibroblasts revealed that IGFBP-3 mRNA was increased by TGF beta 1, but not by IGF-I. These data suggest that IGFs and TGF beta 1 release fibroblast IGFBPs by distinct mechanisms: IGFs by binding and subsequent release of cell-associated IGFBP-3 and 29- to 31-kDa IGFBP, and TGF beta 1 by increased de novo synthesis of IGFBP-3.

Carrier Proteins↗
PubMed1992

Experimental aspects of cerebrospinal hemodynamics: the relationship between blood flow velocity waveform and cerebral autoregulation.

The dynamic relationships among mean flow velocity, its pulsatile amplitude (FVa), cortical cerebral blood flow (CBF), and cerebral perfusion pressure (CPP) were studied in normal rabbits and rabbits with subarachnoid hemorrhage using 8-MHz pulsed transcranial Doppler ultrasound and hydrogen clearance under conditions of systemic hypotension and intracranial hypertension. A two-slope relationship was observed between FVa and CPP with a break point that correlated closely with the lower limit of CBF autoregulation in each animal. Below this CPP break point, FVa varied directly with CPP, and above the break point FVa varied inversely with CPP. In this experimental model, an inverse correlation between FVa and CPP indicates intact CBF autoregulation, whereas loss of that correlation implies exhaustion of autoregulatory reserve. Simultaneous recording and computation of FVa, CPP, and the correlation coefficient between FVa and CPP may be a means of monitoring CBF autoregulation in clinical practice.

Animals↗
PubMed1992

Comparison of extraction methods for insulin-like growth factor-I in rat serum.

This study was undertaken to compare various extraction methods for insulin-like growth factor-binding proteins (IGFBPs) from insulin-like growth factor-I (IGF-I) in rat serum systematically, before measurement of IGF-I by radioimmunoassay (RIA). The values obtained in the IGF-I RIA following acid-ethanol (AE), acid-ethanol cryoprecipitation (AEC) and formic acid-acetone (FA) extraction methods were compared with the IGF-I values obtained following high-performance liquid chromatography (HPLC), which was the reference method. Radioligand blots were used to determine the pattern and degree of IGFBP removal by these methods. Over a wide range of circulating IGF-I levels, AE and AEC extraction gave IGF-I levels comparable with those obtained following HPLC. FA extraction resulted in IGF-I levels that were consistently higher (P < 0.01) than those obtained following HPLC and gave non-parallel displacement curves in comparison with recombinant IGF-I standards (P <0.01). Ligand blots demonstrated a similar pattern of IGFBP removal among the three methods with almost complete removal of IGFBP-3 but only 30-40% removal of the lower molecular weight IGFBPs. These lower molecular weight IGFBPs did not interfere with the RIA measurements of IGF-I from AE and AEC extracts. Therefore the AE extraction method of Daughaday, originally validated for use in human serum, is also satisfactory for use in rat serum. The complete removal of IGF-binding activity does not appear essential for accurate measurement of IGF-I by RIA, although this may depend on the specific binding characteristics of the IGF-I antiserum.

Animals↗
PubMed1992

High pressure liquid chromatographic determination of the new non-steroidal anti-inflammatory agent butibufen.

High performance liquid chromatography (HPLC) method for the determination and validation of a new non-steroidal anti-inflammatory agent, butibufen (CAS 55837-18-8), microencapsulated butibufen and its pharmaceutical forms--tablets, sachets (microencapsulated butibufen with excipients), microemulsion and cream--is described. Acetonitrile proved to be the best solvent for the extraction of butibufen from its pharmaceutical forms. The results submitted in this paper suggest that the HPLC method for the quantitative determination of butibufen is linear, accurate, precise and sensitive.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1992

Leukoedema: a review of the literature.

Leukoedema, a grayish-white lesion of the oral mucosa in humans, was once thought to be a probable precursor to leukoplakia. Clinical examination differentiates leukoedema from leukoplakia, lichen planus, white sponge nevus, and pathomimia morsicatio buccarum. Prevalence rates vary greatly in different countries and in different ethnic groups. For many years, leukoedema was alleged to occur only in adult populations until Martin and Crump found this lesion in children and youth. There is a definite predilection for this lesion in black Americans. Although the etiology is unknown, it has been suggested that leukoedema develops in areas of local irritation. This article reviews the literature related to the prevalence of leukoedema and correlates the independent findings, which might pertain to its etiology.

Black or African American↗
PubMed1991

Glucose analogue inhibitors of glycogen phosphorylase: the design of potential drugs for diabetes.

The T-state crystal structure of the glucose-phosphorylase b complex has been used as a model for the design of glucose analogue inhibitors that may be effective in the regulation of blood glucose levels. Modeling studies indicated room for additional atoms attached at the C1-beta position of glucose and some scope for additional atoms at the C1-alpha position. Kinetic parameters were determined for alpha-D-glucose: Ki = 1.7 mM, Hill coefficient n = 1.5, and alpha (synergism with caffeine) = 0.2. For beta-D-glucose, Ki = 7.4 mM, n = 1.5, and alpha = 0.4. More than 20 glucose analogues have been synthesized and tested in kinetic experiments. Most were less effective inhibitors than glucose itself and the best inhibitor was alpha-hydroxymethyl-1-deoxy-D-glucose (Ki = 1.5 mM, n = 1.3, alpha = 0.4). The binding of 14 glucose analogues to glycogen phosphorylase b in the crystal has been studied at 2.4-A resolution and the structure have been refined to crystallographic R values of less than 0.20. The kinetic and crystallographic studies have been combined to provide rationalizations for the apparent affinities of glucose and the analogues. The results show the discrimination against beta-D-glucose in favor of alpha-D-glucose is achieved by an additional hydrogen bond made in the alpha-glucose complex through water to a protein group and an unfavorable environment for a polar group in the beta pocket. The compound alpha-hydroxymethyl-1-deoxy-D-glucose has an affinity similar to that of glucose and makes a direct hydrogen bond to a protein group. Comparison of analogues with substituent atoms that have flexible geometry (e.g., 1-hydroxyethyl beta-D-glucoside) with those whose substituent atoms are more rigid (e.g., beta-azidomethyl-1-deoxyglucose or beta-cyanomethyl-1-deoxyglucose) indicates that although all three compounds make similar polar interactions with the enzyme, those with more rigid substituent groups are better inhibitors. In another example, alpha-azidomethyl-1-deoxyglucose was a poor inhibitor. In the crystal structure the compound made several favorable interactions with the enzyme but bound in an unfavorable conformation, thus providing an explanation for its poor inhibition. Attempts to utilize a contact to a buried aspartate group were partially successful for a number of compounds (beta-aminoethyl, beta-mesylate, and beta-azidomethyl analogues). The beta pocket was shown to bind gentiobiose (6-O-beta-D-glucopyranosyl-D-glucose), indicating scope for binding of larger side groups for future studies.

Deoxyglucose↗
PubMed1991

Direct observation of vibrational coherence in bacterial reaction centers using femtosecond absorption spectroscopy.

It is shown that vibrational coherence modulates the femtosecond kinetics of stimulated emission and absorption of reaction centers of purple bacteria. In the DLL mutant of Rhodobacter capsulatus, which lacks the bacteriopheophytin electron acceptor, oscillations with periods of approximately 500 fs and possibly also of approximately 2 ps were observed, which are associated with formation of the excited state. The kinetics, which reflect primary processes in Rhodobacter sphaeroides R-26, were modulated by oscillations with a period of approximately 700 fs at 796 nm and approximately 2 ps at 930 nm. In the latter case, at 930 nm, where the stimulated emission of the excited state, P*, is probed, oscillations could only be resolved when a sufficiently narrow (10 nm) and concomitantly long pump pulse was used. This may indicate that the potential energy surface of the excited state is anharmonic or that low-frequency oscillations are masked when higher frequency modes are also coherently excited, or both. The possibility is discussed that the primary charge separation may be a coherent and adiabatic process coupled to low-frequency vibrational modes.

Bacterial Chromatophores↗
PubMed1991

Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase.

Serial human immunodeficiency virus type-1 (HIV-1) isolates were obtained from five individuals with acquired immunodeficiency syndrome (AIDS) who changed therapy to 2',3'-dideoxyinosine (ddI) after at least 12 months of treatment with 3'-azido-3'-deoxythymidine (zidovudine, AZT). The in vitro sensitivity to ddI decreased during the 12 months following ddI initiation, whereas AZT sensitivity increased. Analysis of the reverse transcriptase coding region revealed a mutation associated with reduced sensitivity to ddI. When this mutation was present in the same genome as a mutation known to confer AZT resistance, the isolates showed increased sensitivity to AZT. Analysis of HIV-1 variants confirmed that the ddI resistance mutation alone conferred ddI and 2',3'-dideoxycytidine resistance, and suppressed the effect of the AZT resistance mutation. The use of combination therapy for HIV-1 disease may prevent drug-resistant isolates from emerging.

Acquired Immunodeficiency Syndrome↗
PubMed1991

A metabolite of halothane covalently binds to an endoplasmic reticulum protein that is highly homologous to phosphatidylinositol-specific phospholipase C-alpha but has no activity.

When the inhalation anesthetic halothane was administered to rats, a 58 kDa protein in the liver became covalently labeled by the trifluoroacetyl chloride metabolite of halothane. The amino acid sequences of the N-terminal and of several internal peptide fragments of the protein were 99% homologous to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha. The purified trifluoroacetylated 58 kDa protein or native 58 kDa protein, however, did not have phosphatidylinositol-specific phospholipase C activity. We conclude that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha may encode for a microsomal protein of unknown function.

Amino Acid Sequence↗
PubMed1991

Ligand binding and protein relaxation in heme proteins: a room temperature analysis of NO geminate recombination.

Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well.

Binding Sites↗