Biomedical subjects

G M Jenkins

Publications and source records attributed to G M Jenkins.

15 recordsLinked to original sources

PubMed1997

Involvement of yeast sphingolipids in the heat stress response of Saccharomyces cerevisiae.

A role for sphingolipids in the yeast heat stress response has been suggested by the isolation of suppressors of mutants lacking these lipids, which are unable to grow at elevated temperatures. The current study examines the possible role of sphingolipids in the heat adaptation of yeast cells as monitored by growth and viability studies. The suppressor of long chain base auxotrophy (SLC, strain 7R4) showed a heat-sensitive phenotype that was corrected by transformation with serine palmitoyltransferase. Thus, the deficiency in sphingolipids and not the suppressor mutation was the cause of the heat-sensitive phenotype of the SLC strain 7R4. The ability of sphingolipids to rescue the heat-sensitive phenotype was examined, and two endogenous yeast sphingoid backbones, phytosphingosine and dihydrosphingosine, were found to be most potent in this effect. Next, the effect of heat stress on the levels of the three major classes of sphingolipids was determined. The inositol phosphoceramides showed no change over a 1.5-h time course. However, the four detected species of sphingoid bases increased after 15 min of heat stress from 1.4- to 10.8-fold. The largest increases were seen in two sphingoid bases, C20 phytosphingosine and C20 dihydrosphingosine, which increased 6.4- and 10.8-fold over baseline, respectively. At 60 min of heat stress two species of yeast ceramide increased by 9.2- and 10.6-fold over baseline. The increase seen in the ceramides was partially decreased by Fumonisin B1, a ceramide synthase inhibitor. Therefore, heat stress induces accumulation of sphingoid bases and of ceramides, probably through de novo synthesis. Taken together, these results demonstrate that sphingolipids are involved in the yeast heat stress adaptation.

Chromatography, High Pressure Liquid↗

PubMed1997

Identification and characterization of Saccharomyces cerevisiae dihydrosphingosine-1-phosphate phosphatase.

We have identified the yeast sphingosine resistance gene (YSR2) of Saccharomyces cerevisiae as encoding a protein that specifically dephosphorylates dihydrosphingosine 1-phosphate (DHS-1-P), and we refer to this protein as dihydrosphingosine-1-phosphate phosphatase. Overexpression of YSR2 conferred sphingosine resistance to the dihydrosphingosine-1-P lyase-defective mutant (JS16) of S. cerevisiae, which is hypersensitive to sphingosine. The ysr2Delta deletion mutant of S. cerevisiae accumulated DHS-1-P compared with its wild type strain upon labeling with D-erythro-[4, 5-3H]dihydrosphingosine, whereas overexpression of YSR2 increased dephosphorylation of DHS-1-P. An epitope-tagged fusion protein (YSR2-Flag) was partially purified and found to specifically dephosphorylate DHS-1-P to yield dihydrosphingosine. YSR2 failed to dephosphorylate ceramide 1-phosphate or phosphatidic acid. Functionally, the mutant bearing the ysr2Delta deletion decreased labeling of sphingolipids and increased labeling of glycerolipids dramatically following in vivo labeling with D-erythro-[3H]dihydrosphingosine, but it slightly affected labeling of sphingolipids with inositol. Taken together, these results identify YSR2 as dihydrosphingosine-1-phosphate phosphatase. They also raise the intriguing possibility that phosphorylation followed by dephosphorylation is required for incorporation of exogenous long chain sphingoid bases into sphingolipids.

Drug Resistance, Microbial↗

PubMed1997

Expression of neutral sphingomyelinase identifies a distinct pool of sphingomyelin involved in apoptosis.

The activation of sphingomyelinase and generation of ceramide have been implicated as important regulatory pathways in cell growth and apoptosis. Bacterial sphingomyelinase has been used in many cell systems to mimic the activation of endogenous sphingomyelinase. These studies, however, have been complicated by the inability of exogenously applied bacterial sphingomyelinase to perform many of the effects of short chain cell permeable ceramides, indicating that there may be a distinct signal transducing pool of sphingomyelin not accessible to exogenous sphingomyelinase or that endogenous ceramide is not sufficient to induce these changes. We cloned the Bacillus cereus sphingomyelinase gene by polymerase chain reaction and subcloned it into a mammalian expression vector under the control of an inducible promoter. Upon stable transfection and induction of B. cereus sphingomyelinase, there were increases in neutral sphingomyelinase activity, cellular ceramide levels, cleavage of the death substrate poly(ADP-ribosyl)polymerase, and cell death. In contrast, exogenously applied B. cereus sphingomyelinase, despite causing higher elevations in ceramide levels, was unable to induce poly(ADP-ribosyl)polymerase cleavage or cell death. These results support the existence of a signal transducing pool of sphingomyelin that is distinct from the pool accessible to exogenous sphingomyelinase.

Animals↗

PubMed1995

Neurotrophins induce sphingomyelin hydrolysis. Modulation by co-expression of p75NTR with Trk receptors.

We examined neurotrophin-induced sphingomyelin hydrolysis in cells expressing solely the low affinity neurotrophin receptor, p75NTR, and in PC12 cells that co-express p75NTR and Trk receptors. Nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and NT-5 stimulated sphinomyelin hydrolysis with similar kinetics in p75NTR-NIH-3T3 cells. Although brain-derived neurotrophic factor (10 ng/ml) was slightly more potent than NGF at inducing sphingomyelin hydrolysis, NT-3 and NT-5 induced significant hydrolysis (30-35%) at 0.1 to 1 ng/ml in p75NTR-NIH-3T3 cells. NT-3 did not induce sphingomyelin hydrolysis in Trk C-NIH-3T3 cells nor in cells expressing a mutated p75NTR containing a 57-amino acid cytoplasmic deletion, thus demonstrating the role of p75NTR in this signal transduction pathway. In p75NTR-NIH-3T3 cells, neurotrophin-induced sphingomyelin hydrolysis 1) localized to an internal pool of sphingomyelin, 2) was not a consequence of receptor internalization, and 3) showed no specificity with respect to the molecular species of sphingomyelin hydrolyzed. In contrast to cells expressing solely p75NTR, NGF (100 ng/ml) did not induce sphingomyelin hydrolysis in PC12 cells. Interestingly, NT-3 (10 ng/ml) induced the same extent of sphingomyelin hydrolysis in PC12 cells as was apparent in p75NTR-NIH-3T3 cells. However, in the presence of NGF, NT-3 was unable to induce sphingomyelin hydrolysis, raising the possibility that Trk was modulating p75NTR-dependent sphingomyelin hydrolysis. Inhibition of Trk tyrosine kinase activity with 200 nM K252a enabled both NGF and NT-3 in the presence of NGF to induce sphingomyelin hydrolysis. These data support that p75NTR serves as a common signaling receptor for neurotrophins through induction of sphingomyelin hydrolysis and that crosstalk pathways exist between Trk and p75NTR-dependent signaling pathways.

3T3 Cells↗

PubMed1995

Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat.

Despite significant improvements in the primary success rate of the medical and surgical treatments for atherosclerotic disease, including angioplasty, bypass grafting, and endarterectomy, secondary failure due to late restenosis continues to occur in 30-50% of individuals. Restenosis and the later stages in atherosclerotic lesions are due to a complex series of fibroproliferative responses to vascular injury involving potent growth-regulatory molecules (such as platelet-derived growth factor and basic fibroblast growth factor) and resulting in vascular smooth muscle cell (VSMC) proliferation, migration, and neointimal accumulation. We show here, based on experiments with both taxol and deuterium oxide, that microtubules are necessary for VSMCs to undergo the multiple transformations contributing to the development of the neointimal fibroproliferative lesion. Taxol was found to interfere both with platelet-derived growth factor-stimulated VSMC migration and with VSMC migration and with VSMC proliferation, at nanomolar levels in vitro. In vivo, taxol prevented medial VSMC proliferation and the neointimal VSMC accumulation in the rat carotid artery after balloon dilatation and endothelial denudation injury. This effect occurred at plasma levels approximately two orders of magnitude lower than that used clinically to treat human malignancy (peak levels achieved in this model were approximately 50-60 nM). Taxol may therefore be of therapeutic value in preventing human restenosis with minimal toxicity.

Angioplasty, Balloon↗

PubMed1994

Modulation of cardiac sarcoplasmic reticulum ryanodine receptor by sphingosine.

Excitation contraction (EC) coupling in muscle cells involves the movement of calcium through the calcium release channel of the sarcoplasmic reticulum (SR) membrane known as the ryanodine receptor. We have recently shown that the novel second messenger, sphingosine, can block calcium release from skinned skeletal muscle fibers and from isolated skeletal muscle SR membranes (Sabbadini et al., J Biol Chem 267: 15475-15484, 1992). In this report, we demonstrate that sphingosine also inhibits calcium release from isolated canine cardiac SR membranes containing the ryanodine receptor when release is induced by caffeine, doxorubicin or by calcium. Sphingosine also prevents the augmentation of [3H]-ryanodine binding normally produced by caffeine and doxorubicin and exerts noncompetitive inhibition with regard to both releasing agents. Sphingosine significantly reduces in a dose-dependent manner [3H]-ryanodine binding to the high affinity site of the receptor and increases by several-fold the Kd for binding, which is consistent with a blocking action of sphingosine on the ryanodine receptor calcium channel. Sphingosine inhibits the extent of calcium-induced calcium release (CICR) and significantly shifts the threshold for CICR so that a higher level of trigger calcium is required to initiate CICR. The sphingosine inhibition of CICR is consistent with the near abolition of calcium dependent [3H]-ryanodine binding. HPLC analysis of cardiac sphingosine content indicates that sphingosine is present in the cardiac cell at moderately high levels (29.4 nmol/g wet wt for the entire cell and approximately 0.4 microM for the cytosol) which are sufficient to produce significant inhibition by sphingosine on calcium release and ryanodine binding. The data suggest that sphingosine acts on the cardiac ryanodine receptor by opposing the physiological stimulus (e.g. trigger calcium entering via the dihydropyridine receptor). We propose that sphingosine is produced by the T-tubule membranes and that sphingosine is released into the protected intracellular environment of the T-tubule/SR junction to negatively modulate calcium release. Consequently, it is possible that sphingosine is a physiologically relevant regulator of calcium levels in the heart.

Animals↗

PubMed1991

Prevention of coronary heart disease in black adults.

Development of strategies to prevent CHD in blacks is impeded by the virtual absence of clinical trials demonstrating the feasibility and effectiveness of interventions in blacks. The wholesale generalization that interventions effective (or ineffective) in whites are similarly effective in blacks may risk the employment of worthless or even dangerous interventions in blacks. Using available epidemiologic data, a number of risk factors may be more important in blacks than whites by virtue of higher prevalence, increased relative risk, or both. These may include hypertension, lipoprotein (a), smoking, diabetes, and obesity. Thus, health agencies might emphasize these risk factors when developing preventive programs targeted at black populations. Prevention programs may best seek to prevent the onset of risk factors found highly prevalent in black communities, rather than the costly and side-effect-prone interventions to treat risk factors once established. Thus, there is a role for community-based as well as a high-risk approaches. The community-based approaches should seek to work with organizations such as churches, which traditionally play strong roles in the black community. Physicians treating black patients should be aware of the potentially different roles played by risk factors, and treat aggressively those individuals identified to be at high risk. Risk factor management should be emphasized, rather than reduced, in patients with already established CHD. CHD has been clearly shown to be preventable; both blacks and whites should benefit from specific interventions aimed toward this worthy goal.

Adult↗

PubMed1990

Electrochemistry of galvanic couples between carbon and common metallic biomaterials in the presence of crevices.

In vitro experiments were conducted upon some common metallic biomaterials and carbons, both isolated or forming galvanic couples, in a cell specially designed for crevice corrosion studies. The alloys examined were AISI 316L stainless steel, Ti6AI4V and Co-Cr-Mo. The types of carbon were glassy carbon and carbon fibre-reinforced carbon. The surface modifications were evaluated by SEM, AES and ESCA-XPS analyses. AISI 316L stainless steel suffered localized corrosion in open-circuit experiments whilst the other materials remained unattacked. Galvanic currents between metal-carbon couples were measured by zero resistance ammetry. The carbon-metal area ratio was 1:1. The results showed that 316L stainless steel and the Co-Cr-Mo alloy were prone to accelerated corrosion, whilst the Ti6AI4V alloy remained unattacked. The galvanic corrosion currents were also predicted using mixed potential theory from polarization curves obtained for each material. The experimental and theoretical values showed good agreement for the stainless steel and Co-Cr-Mo alloy. Long-term immersion tests with the same couples showed that the only metal not to suffer degradation was the Ti6AI4V alloy.

Alloys↗

PubMed1988

Mineral structure and preferred orientation in the fin bones of the plaice, Pleuronectes platessa.

The technique of collagenase etching of a polished surface of plaice fin bone has been used to reveal the structural detail of the mineral component and hence to explain the preferred orientation previously deduced from X-ray diffraction experiments. High resolution electron micrographs reveal units approximately 20 nm across, which aggregate to form units approximately 100 nm across, which in turn coalesce to produce rods up to approximately 1 micron diameter and of substantial length. These rods show a preferred orientation with their axes parallel to the long axis of the bone, thus demonstrating a preferred orientation of the mineral component. Additionally, collagenase-etched transverse surfaces reveal numbers holes from which collagen fibrils parallel to the long axis of the bone have been removed, whereas similarly treated longitudinal surfaces do not show such holes. This is consistent with a predominance of collagen fibrils running along the bone axis and explains the previously observed preferred orientation of the collagen component of fish fin bones.

Animals↗

PubMed1987

Collagen fibre orientation in bovine secondary osteons by collagenase etching.

The orientation of collagen fibres in bovine secondary osteons has been investigated in the scanning electron microscope (SEM) by removal independently of firstly the mineral component and secondly the collagen fibres. Demineralization of polished transverse sections reveals a lamellar structure for the collagen component but the precise orientation of the collagen in each ring is not unequivocably determined. However, by using a collagenase solution to etch away the collagen component of a polished surface, holes are produced in the mineral revealing the former position of the fibres. The greater rigidity of the mineral component ensures that the structure does not collapse and produce artifacts. A specimen cut so that transverse and longitudinal sections are simultaneously observed allows the relationship between the structural features on each surface to be revealed. Analysis of such micrographs indicates a model for the collagen component of osteons in which the lamellar structure contains fibres with orientations alternately parallel to and circumferential to the long axis of the osteon. Tilting the samples to look directly down the holes shows that the fibres are not precisely longitudinal and circumferential but are tilted from these ideals by a variable angle (typically 20 degrees) the precise angle probably being an important factor related to the in vivo mechanical property requirements.

Animals↗

PubMed1985

Bone microstructure by collagenase etching.

A novel technique has been developed for microstructural studies of bone. The spatial organizations of the mineral and collagen fibres in bone have been a matter of discussion for some time, with numerous diverse observations arising from various preparative techniques. In this latest investigation details of the mineral structure are clearly revealed in the SEM by treating a cut and polished surface of bone with collagenase to remove the major organic component. This new procedure has minimal effect on the mineral and hence reveals microstructural detail which is far closer to that in vivo than in previous investigations. This paper concentrates on two aspects of the studies, namely the detailed morphology of the mineral component and the arrangement of the collagen fibres in the osteons of compact bone. Firstly, the mineral component is revealed as comprising 'crystallites' (approximately 20 nm diam.) which aggregate to form larger contiguous 'spheroidal particles' (approximately 100 nm diam.), which in turn form 'granules' (approximately 500 nm diam.). Secondly, the regions from which collagen fibres have been removed are clearly revealed, showing that within an osteon, alternating lamellae have collagen fibres oriented approximately parallel to and circumferential to the Haversian canal respectively.

Animals↗

PubMed1983

Developmental changes of mouse red cell pyruvate kinase.

The increase in red cell 2,3-diphosphoglycerate (2,3-DPG) concentration during mouse postnatal development is highly correlated with the decrease in red cell pyruvate kinase (PK) activity. Immunoneutralization curves for newborn and adult red cell PK are parallel, which suggests that the higher PK activity in newborn mouse red cells is due primarily to a greater quantity of PK protein. Mouse newborn and adult red cell PK also differ in mobility on cellulose acetate electrophoresis (pH 7.5). This is true for two strains of mice (C57BL/6 and IS/Cam) whose adult red cell PK electrophoretic patterns differ (single vs. double bands, respectively). Trypsinization does not alter the electrophoretic mobilities of C57BL/6 newborn and adult red cell PK, but it does convert the IS/Cam adult PK to a single band of greater mobility than the single band of C57BL/6 adult PK. These data are consistent with the hypothesis that mice have structurally different forms of PK for newborn and adult red cells, analogous to the fetal gamma- and adult beta-globins of humans.

Aging↗

PubMed1981

The spatial arrangement of bone mineral as revealed by ion bombardment.

Ion etching and thinning using ionized argon beams have been used to prepare surfaces and thin sections of human and animal compact cortical bone for examination in both scanning and transmission modes of the electron microscope. Using these techniques the mineral component in bone is revealed as a continuous, vermiform microskeleton composed of spheroidal segments approximately 0.1 micrometer in diameter. Electron diffraction and micro-analysis have confirmed that the mineral segments contain calcium and phosphate ions arranged in a poorly crystallised form of hydroxyapatite.

Animals↗

PubMed1979

The fabrication of artifacts out of glassy carbon and carbon-fiber-reinforced carbon for biomedical applications.

Polymeric carbons are produced by the carbonization of a wide range of organic polymeric systems. We have concentrated on the fabrication of two types of polymeric carbons, glassy carbon and carbon-fiber-reinforced carbon (CFRC), both involving phenolic resin precursors. We describe herein the technology which enables us to make dental implants and heart valves out of glassy carbon. We also show how carbon-fiber-reinforced carbon can be made in the form of rods and plates for orthopedic use and molded before firing to produce complex, rigid, individually sculptured shapes suitable for maxillofacial bone replacement. The mechanical properties will be discussed in relation to the structure of these various forms of polymeric carbon. The main purpose of the work is to show that the technology of polymeric-carbon manufacture is essentially simple and the manufacturing process is readily carried out in laboratories which have already been equipped to fabricate standard dental prostheses.

Acrylonitrile↗

PubMedDate not supplied

Increased expression of membrane-type matrix metalloproteinase and preferential localization of matrix metalloproteinase-2 to the neointima of balloon-injured rat carotid arteries.

BACKGROUND: Remodeling of the injured vascular wall is dependent on the action of several extracellular proteases. Previous studies have shown that expression of matrix metalloproteinases (MMP-2 and MMP-9) is upregulated after vascular injury and that MMP-2 is required for the migration of cultured vascular smooth muscle cells across complex extracellular matrix barriers. The present study examined changes in the expression of membrane-type metalloproteinase (MT-MMP-1), a putative regulator of MMP-2, in the tissue localization of MMP-2, and in the expression of activated and latent forms of MMP-2 and the tissue inhibitor of metalloproteinases, TIMP-2, in rat carotid arteries subjected to balloon catheter injury. METHODS AND RESULTS: MT-MMP-1 mRNA levels increased sixfold after 3 days of injury, coinciding with an increase in MMP-2 activation assessed by gelatin zymography. Western blotting and gelatin zymography showed an increase in MMP-2 protein levels beginning 5 to 7 days after injury; immunocytochemistry and Western blotting showed that the increase occurred preferentially in the developing neointima. CONCLUSIONS: These results show that increased expression of MT-MMP-1 and activation of MMP-2 occurs early after injury to the rat carotid artery and that at later times MMP-2 is preferentially localized to the developing neointima.

Angioplasty, Balloon↗