Search PubMed⌕ Search

Biomedical subjects

J Moss

Publications and source records attributed to J Moss.

At least 163 records · Page 9Linked to original sources
PubMed1997

The alpha 7 integrin as a target protein for cell surface mono-ADP-ribosylation in muscle cells.

A membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified 215,000-fold from rabbit skeletal muscle and its gene was isolated from a skeletal muscle cDNA library. The enzyme was a glycosylphosphatidyl-inositol-linked protein, present on the surface of differentiated skeletal muscle myoblasts (myotubes). Following incubation of cultured, intact myotubes with [adenylate-32P]NAD and analysis by SDS-PAGE, a major radiolabeled protein of 97/140 kDa (reduced/nonreduced conditions) was observed. It was identified as integrin alpha 7 based on its size, binding to a laminin affinity column, immunoprecipitation with a monoclonal antibody, and partial amino acid sequencing. Since ADP-ribosylarginine hydrolase, the enzyme responsible for cleavage of the ADP-ribosylarginine bond and a component with the transferase of a putative ADP-ribosylation cycle, is cytosolic, whereas the transferase is attached via a GPI-anchor to the cell surface, the processing of ADP-ribosylated integrin alpha 7 was investigated. 32P label was rapidly removed from [32P]ADP-ribosylated integrin alpha 7, a process inhibited by free ADP-ribose or p-nitrophenylthymidine-5'-monophosphate, alternative substrates for 5'-nucleotide phosphodiesterase. The processed integrin alpha 7 was not susceptible to subsequent ADP-ribosylation, although the amount of surface integrin alpha 7 remained constant. During the processing, no loss of label was observed from integrin alpha 7 radiolabeled with [14C]NAD, containing 14C in the nicotinamide-proximal ribose, consistent with a degradation of the ADP-ribose moiety by a cell surface 5'-nucleotide phosphodiesterase. Thus, cell surface ADP-ribosylation, in contrast to intracellular ADP-ribosylation, is not readily reversed by the presently known ADP-ribosylarginine hydrolase and seems to operate outside the postulated ADP-ribosylation cycle.

ADP Ribose Transferases↗
PubMed1997

Activation of toxin ADP-ribosyltransferases by the family of ADP-ribosylation factors.

ADP-ribosylation factors or ARFs are 20-kDa guanine nucleotide-binding proteins, initially identified as stimulators of cholera toxin-catalyzed ADP-ribosylation of Gs alpha. We now know that ARFs play a critical role in many vesicular trafficking events and ARF activation of a membrane-associated phospholipase D (PLD) has been recognized. ARF is active and associates with membranes when GTP is bound. The active state is terminated by hydrolysis of bound GTP, producing inactive ARF-GDP. The nucleotide effect on ARF association with membranes is related to alteration in orientation of the N-terminal myristoyl moiety that is important for ARF function. Cycling of ARF between active and inactive states involves guanine nucleotide-exchange proteins (GEPs) that accelerate replacement of bound GDP with GTP and GTPase-activating proteins (GAPS) that are responsible for ARF inactivation. Six mammalian ARFs have been identified by cDNA cloning. Class I ARFs 1 and 3 have been studied most extensively. Their activation (GTP binding) is catalyzed by a GEP now purified from spleen cytosol. In crude preparations, GEP was inhibited by brefeldin A (BFA), which in cells causes apparent disintegration of Golgi. Demonstration that the approximately 60 kDa purified GEP was not inhibited by BFA means that contrary to earlier belief, there must be another protein to mediate BFA inhibition. GEP activity was greatly enhanced by phosphatidyl serine. The purified GEP, equally active with ARFs 1 and 3, was inactive with ARFs 5 and 6 (Classes II and III); myristoylated ARFs were better substrates than were their non-myristoylated counterparts. ARF GAP purified from bovine spleen cytosol in our laboratory had much broader substrate specificity than the GEP. It used both ARFs 5 and 6 at least as well as ARFs 1 and 3; myristoylation was without effect. It also accelerated GTP hydrolysis by certain ARF mutants and an ARF-like protein (ARL1) that does not have ARF activity. The purified GAP also differed from the GEP in its rather specific requirement for phosphatidylinositol bisphosphate. This was also observed with a seemingly different ARF GAP that was purified and subsequently cloned in Cassel's laboratory. Activation and inactivation of ARFs present many potential sites for physiological regulation and, therefore, for pathological disruption of ARF function.

ADP-Ribosylation Factors↗
PubMed1997

Natural inhibitors of cholinesterases: implications for adverse drug reactions.

PURPOSE: Acetylcholinesterase and butyrylcholinesterase are two closely related enzymes important in the metabolism of acetylcholine and anaesthetic drugs, including succinylcholine, mivacurium, and cocaine. The solanaceous glycoalkaloids (SGAs) are naturally occurring steroids in potatoes and related plants that inhibit both acetylcholinesterase and butyrylcholinesterase. There are many clinical examples of direct SGA toxicity due to cholinesterase inhibition. The aim of this study was to review the hypotheses that (1) SGAs may be the evolutionary driving force for atypical butyrylcholinesterase alleles and that (2) SGAs may adversely influence the actions of anaesthetic drugs that are metabolized by acetylcholinesterase and butyrylcholinesterase. SOURCE: The information was obtained by Medline search and consultation with experts in the study of SGAs and cholinesterases. PRINCIPAL FINDINGS: The SGAs inhibit both acetylcholinesterase and butyrylcholinesterase in numerous in vitro and in vivo experiments. Although accurate assays of SGA levels are difficult, published data indicate human serum SGA concentrations at least ten-fold lower than required to inhibit acetylcholinesterase and butyrylcholinesterase in vitro. However, we review evidence that suggests the dietary ingestion of SGAs can initiate a cholinergic syndrome in humans. This syndrome appears to occur at SGA levels lower than those which interfere with anaesthetic drug catabolism. The world distribution of solanaceous plants parallels the distribution of atypical alleles of butyrylcholinesterase and may explain the genetic diversity of the butyrylcholinesterase gene. CONCLUSION: Correlative evidence suggests that dietary SGAs may be the driving force for atypical butyrylcholinesterase alleles. In addition, SGAs may influence the metabolism of anaesthetic drugs and this hypothesis warrants experimental investigation.

Animals↗
PubMed1997

The safety and efficacy of oral methylnaltrexone in preventing morphine-induced delay in oral-cecal transit time.

Methylnaltrexone is a quaternary opioid antagonist with limited ability to cross the blood-brain barrier that has the potential to antagonize the peripherally mediated gastrointestinal effects of opioids. In recent trials in human volunteers, we demonstrated that intravenous methylnaltrexone prevented morphine-induced changes in gastrointestinal motility and transit, without affecting analgesia. In this study, 14 healthy volunteers were first given three ascending oral doses of methylnaltrexone to obtain safety and tolerance data (phase A study). In phase B, these subjects were then given single-blind oral placebo and intravenous placebo, followed by randomized, double-blind oral placebo and intravenous morphine (0.05 mg/kg) or oral methylnaltrexone (19.2 mg/kg, an established highest and safe dose based on previous administrations of two smaller doses of 0.64 mg/kg and 6.4 mg/kg in phase A) and intravenous morphine (0.05 mg/kg). Oral-cecal transit time was assessed by the pulmonary hydrogen measurement technique after lactulose ingestion. Morphine significantly increased oral-cecal transit time from 114.6 +/- 37.0 minutes (mean +/- SD) to 158.6 +/- 50.2 minutes (p < 0.001). Oral methylnaltrexone (19.2 mg/kg) completely prevented morphine-induced increase in oral-cecal transit time (110.4 +/- 45.0 minutes; not significant compared with baseline; p < 0.005 compared with morphine alone). These sessions were then followed by single-blind evaluations of descending doses of methylnaltrexone. We observed that 6.4 mg/kg oral methylnaltrexone significantly attenuated the morphine-induced delay in oral-cecal transit time (p < 0.005 compared with morphine alone), and a dose-dependent response was obtained. There was no correlation between oral methylnaltrexone effects on the transit time and the drug plasma concentration, suggesting direct preferential luminal effects of oral methylnaltrexone. Oral methylnaltrexone may have a clinical value in the prevention and treatment of constipation induced by long-term opioid use.

Administration, Oral↗
PubMed1997

Immunohistochemical study of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in pulmonary lymphangioleiomyomatosis (LAM).

To evaluate the role of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) in the pathogenesis of the structural damage and cystic lesions found in pulmonary lymphangioleiomyomatosis (LAM), immunohistochemical studies were made of the localization of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, HMB-45, and type IV collagen in sections of lung biopsy specimens from four patients with this disorder. These studies showed increased immunoreactivity compared with that in normal bronchiolar and vascular smooth muscle cells, of MMP-2 and, to a lesser extent, MMP-9 and MMP-1 in the LAM cells. MMP-2 was also localized in some elastic fibers and in the basement membranes of LAM cells and overlying epithelial cells. The basement membranes in both of these sites often showed colocalization of MMP-2 and type IV collagen. Some epithelial basement membranes showing this colocalization were disrupted. These changes were not accompanied by increased immunoreactivity for TIMPs. Taken together with previous observations showing structural damage to elastic fibers and collagen fibrils, and with the absence of demonstrable neutrophil or pancreatic types of elastase, these findings suggest that MMP-2 and MMP-9 (both of which can degrade elastin as well as collagens) are responsible for the connective tissue destruction and cyst formation in LAM.

Adult↗
PubMed1997

In vitro synergy of paclitaxel (Taxol) and vinorelbine (navelbine) against human melanoma cell lines.

Paclitaxel (PTXL) (Taxol), a taxane, and vinorelbine (VRB), a semisynthetic vinca alkaloid drug, have tubulin as their common intracellular target, but inhibit growth by binding to different sites. We evaluated in vitro the antiproliferative activity of these two drugs as single agents and in combination, against two human melanoma cell lines, G361 and StM111a. The SRB (sulphorhodamine B) assay was used to determine growth inhibition. Possible drug-drug interaction at the cellular level was assessed by constructing Isoboles (Isobologram analysis) and applying the concept of an 'envelope of additivity'. Both agents were active in the nanomolar range at clinically achievable concentrations. The mean IC50 for G361 was 46.6 nM (PTXL) and 19.9 nM (VRB) after a 1 h drug exposure. Mean IC50 (1 h) for StM111a was 9.7 nM (PTXL) and 26.9 nM (VRB). Isobole analysis at the isoeffect levels of 25%, 50% and 75% indicated that drug interaction was predominantly synergistic (supra-additive) when paclitaxel and VRB were added concurrently for 1 h to cultures of StM11 1a or G361. In some experiments, this synergy was observed with particularly low concentrations of paclitaxel (3 nM) and VRB (0.01 nM). A new points were located within the envelope of additivity or in the subadditive (antagonism) region of the isobole. An overall synergy was also found if the data were analysed by the median effect analysis. The effect of these agents on the cytoskeleton and ultrastructure were studied with immunofluorescence and electron microscopy, respectively. These results confirm the in vitro inhibitory activity of paclitaxel and VRB against malignant melanoma, but more importantly the two drugs appear to act synergistically at relatively low concentrations.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1997

Research: doing it right the first time.

This manuscript presents the beginning portions of a research project as well as a critique of the design and methods. Although it is not a complete proposal, this article may given those readers who are thinking of doing research some idea of how easy it is to make fatal flaws in the design of a project. Suggestions for how a research novice might avoid these classic mistakes are made.

Humans↗
PubMed1997

The lack of histamine release with cisatracurium: a double-blind comparison with vecuronium.

A prospective, randomized, double-blind study was performed in 62 patients (ASA Classes I and II) treated with either 0.15 or 0.25 mg/kg cisatracurium or 0.15 mg/kg vecuronium administered as a rapid bolus. We wished to determine whether the muscle relaxants caused cutaneous, systemic, or chemical evidence of histamine release. Six minutes after induction of anesthesia with thiopental, patients received one of the muscle relaxants over 5 s. Plasma histamine levels were measured by radioimmunoassay after thiopental administration and 3 and 5 min after the administration of the relaxant. Additionally, plasma was assayed for tryptase, a marker of mast cell release. Cutaneous manifestations to both thiopental and the muscle relaxant were graded by an independent observer. Arterial blood pressure and heart rate were measured every minute. Although systolic and diastolic blood pressure decreased and heart rate increased significantly after thiopental administration (P < 0.0001), there were no further hemodynamic changes after either cisatracurium or vecuronium. One patient who received 0.25 mg/kg cisatracurium exhibited a slight elevation in plasma histamine level 5 min after hemodynamic changes. Cutaneous signs of histamine release were noted in five patients after thiopental administration (flush in four, erythema in one), but no further cutaneous reactions were observed after administration of either cisatracurium or vecuronium. We conclude that cisatracurium and vecuronium do not cause systemic or cutaneous histamine release. Tryptase levels showed no evidence of mast cell degranulation.

Atracurium↗
PubMed1997

A new biological assay for measuring cyanide in blood.

UNLABELLED: Clinical diagnosis of cyanide poisoning is complicated by the lack of an easy, convenient assay for cyanide concentration in blood. Therapy may be delayed with unconfirmed diagnosis because the conventional antidote to cyanide poisoning exposes patients to substantial risks. We developed a new spectrophotometric assay to measure cyanide by extraction into a sodium hydroxide trap, followed by the addition of exogenous methemoglobin as a colormetric indicator. Samples of blood from 15 healthy subjects and 5 patients who had received prolonged nitroprusside infusions were assayed. To optimize assay characteristics, methemoglobin concentrations, pH, temperature, incubation time, and buffer strengths were varied. Duplicate samples were assayed by using the polarographic method for assay validation. Over a range from 300 ng/mL to 7 microg/mL, the correlation between methods was r = 0.983. Interassay and intraassay variability were 5% and 2%, respectively. Samples drawn from the five patients and tested by using both methods yielded a correlation of r = 0.978. This new assay for cyanide in blood may greatly facilitate the diagnosis and treatment of cyanide ingestion. The use of methemoglobin as the colorimetric indicator in the assay contributes to its low cost and ease of use. IMPLICATIONS: Cyanide, an important factor in death from burn-related inhalation injury, is difficult and time-consuming to measure. We developed a new, rapid blood test for cyanide using methemoglobin as a colormetric indicator. A rapid, accessible test for cyanide may speed the diagnosis and treatment of cyanide poisoning.

Adult↗
PubMed1997

Borrelia recurrentis characterization and comparison with relapsing-fever, Lyme-associated, and other Borrelia spp.

Borrelia recurrentis, the cause of louse-borne relapsing fever, has until recently been considered noncultivable, which has prevented characterization of this spirochete. We successfully cultivated 18 strains from patients with louse-borne relapsing fever and present the initial characterization of these isolates. Electron microscopy revealed spirochetal cells with pointed ends, an average wavelength of 1.8 microns, an amplitude of 0.8 micron, and 8 to 10 periplasmic flagella. The G+C ratio was 28.4 mol%. Whole DNA-DNA hybridizations showed similarity between the isolates of B. recurrentis but not with Borrelia hermsii, Borrelia parkeri, Borrelia turicatae, or the Lyme-associated borreliae. Sequencing studies of both the flagellin and 16S RNA genes revealed that the greatest similarity was between B. recurrentis and Borrelia duttonii. Analysis of the sodium dodecyl sulfate-polycarylamide gel electrophoresis profiles of strains revealed four groups based on the position of a major protein band (one of the groups showed some heterogeneity and was subdivided into four subgroups). Pulsed-field gel electrophoresis revealed five distinct patterns.

Borrelia↗
PubMed1997

Safety and tolerance of methylnaltrexone in healthy humans: a randomized, placebo-controlled, intravenous, ascending-dose, pharmacokinetic study.

N-methylnaltrexone bromide (methylnaltrexone) is a quaternary opioid antagonist with a limited ability to cross the blood-brain barrier. In animal models it reverses at peripheral receptors such side effects of opioids as decreased gastrointestinal motility, emesis, and cough suppression without affecting the desired analgesic effect mediated by central nervous system receptors. Methylnaltrexone thus may be a clinically useful compound for the prevention and treatment of opioid-induced side effects. This study was designed to examine the safety and tolerance of methylnaltrexone in healthy human participants over a range of doses and to identify any adverse effects or toxicity associated with methylnaltrexone and the doses at which these adverse effects occur. Healthy male volunteers received intravenous methylnaltrexone in six ascending doses with a placebo randomly inserted into the sequence. Each participant was observed for subjective and hemodynamic changes. Electrocardiogram and laboratory studies were also performed. The dose-limiting adverse effect of methylnaltrexone was orthostatic hypotension at 0.64 mg/kg (n = 3) or 1.25 mg/kg (n = 5), which was transient and self-limiting. Plasma levels of methylnaltrexone in excess of 1,400 ng/mL were observed to be associated with orthostatic hypotension. There were no significant subjective changes, no release of histamine, and no changes in physical examination or laboratory studies during the course of the study. Pharmacokinetic analysis revealed an elimination half-life of 117.5 minutes (+/-53.2), and a clearance of 38.8 L/hr (+/-17.4) with a methylnaltrexone dose of 0.64 mg/kg. Our results indicate that methylnaltrexone is well tolerated at doses of 0.32 mg/kg in healthy humans.

Adult↗
PubMed1997

The influence of diabetes on the vasomotor responses of saphenous vein and the development of infra-inguinal vein graft stenosis.

There has been a prejudice that diabetes modulates the function of saphenous vein in a manner that predisposes to bypass graft failure, although most of the evidence accrues from animal studies. We have investigated the effect of diabetes on the vasodilator responses and ultrastructure of saphenous vein harvested from patients undergoing infrainguinal bypass surgery for limb salvage and the development of stenoses within the vein grafts. Of 55 consecutive patients undergoing vein bypass surgery for critical ischemia, 16 (29%) were diabetic: diabetes was not a risk factor for graft stenosis, which occurred in 17 of 56 (30%) grafts. Endothelium-dependent relaxation by nitric oxide pathways stimulated after receptor activation (bradykinin and thrombin) was not different in vein rings from diabetic (n = 12) and nondiabetic patients (n = 12). Prostarioid-mediated vasorelaxation was absent in vein rings from diabetic patients, and the production of 6-keto prostaglandin F(1alpha) (PGF(1alpha)) from diabetic vein was only 66 +/- 27 pg x cm-2 x min-1 compared with 112 +/- 20 pg x cm-2 x min-1 from control vein (P = 0.011). Fibrinogen-mediated vasorelaxation, normally inhibited by K+ channel blockers, was negligible in vein from diabetic patients. No ultrastructural differences were observed between the endothelium of saphenous vein harvested from diabetic and nondiabetic patients. However, diabetes was associated significantly with the presence of spiraled collagen in media. The maintenance of receptor-activated stimulation of nitric oxide pathways and the damping of the response to fibrinogen in saphenous vein endothelium may provide, in part, for the good prognosis of vein graft surgery in diabetic patients: diabetes is not a risk factor for early (12 months) infrainguinal vein graft stenosis.

Aged↗
PubMed1997

Mouse RT6 locus 1 and rat RT6.2 are NAD+. Arginine ADP-ribosyltransferases with auto-ADP-ribosylation activity.

We report that rat RT6.2 and recombinant mouse Rt6 locus 1 proteins possess auto-ADP-ribosyltransferase activity and that Rt6, but not RT6, catalyzes the ADP-ribosylation of exogenous histones. Based on NH2OH sensitivity, it appeared that the ADP-ribose was attached to arginine residues on proteins. We also observed that the NAD+ concentration in culture medium correlates inversely with the proliferation of rat RT6+ T cells. The data suggest that lymphocyte surface ADP-ribosyltransferases could be involved in signaling and immunoregulatory processes.

ADP Ribose Transferases↗
PubMed1996

A steady-state labelling approach to the measurement of proteoglycan turnover in vivo and its application to glomerular proteoglycans.

Rats were implanted with mini osmotic pumps delivering sodium [35S]sulphate and their newly synthesized proteoglycans were labelled over a 146 h period (steady-state labelling). Proteoglycan turnover was measured in vivo using a chase protocol. Glomerular proteoglycans were recovered quantitatively and the perlecan present was isolated by immunoprecipitation. The procedure allows newly synthesized proteoglycans to be quantified in mass units (pmol of glycossminoglycan sulphate) after labelling and during the chase. Ultrastructural-immunogold experiments identified the location of perlecan as the glomerular basement membrane and mesangial matrix. Perlecan in the basement membrane was quantified using the ultrastructural-immunogold technique. Perlecan comprises about 10% of the total glomerular proteoglycans, which are otherwise associated with glomerular cells and the mesangium. Both the total glomerular heparan sulphate proteoglycans and perlecan turn over rapidly (t1/2 approximately 3-4 h and < 3 h respectively). In contrast, turnover of proteoglycans in other tissues was slow, except in the liver where the heparan sulphate and chondroitin sulphate t1/2 values were 16 h and 9 h respectively. Microalbuminuria was induced with a low-dose regimen of puromycin aminonucleoside. At the onset of microalbuminuria (5 days) there was no change in the level of newly synthesized perlecan, or in perlecan in the glomerular basement membrane detected by immunogold labelling. Newly synthesized perlecan had undergone a minimal change in turnover rate by day 5 in puromycin aminonucleoside-treated rats. In contrast, the total glomerular proteoglycan population showed a dramatic decrease in turnover by day 5. Since there was no evidence of accumulation of glomerular proteoglycans on either day 5 or day 6, it is likely that decreased turnover of cell-associated proteoglycans is accompanied by an equivalent decrease in their synthesis.

Albuminuria↗
PubMed1996

Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain.

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J.B. & Rothman, J.E. (1992) Nature (London) 360, 352-354; Donaldson, J.G., Finazzi, D. & Klausner, R.D. (1992) Nature (London) 360, 350-352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305-309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE-Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (approximately 670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of approximately 190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.

ADP-Ribosylation Factors↗