Prediction of effects of dairy selection indexes on methane emissions.

Global warming caused by greenhouse gas emissions is a threat to the survival of humans and other organisms living on Earth. The greenhouse gases released from the dairy sector of New Zealand accounted for 18.2 Mt of carbon dioxide equivalent (CO2-eq) in 2016, mainly from methane generated by enteric fermentation in the rumen of milking cows and their replacement stock. A productivity commission established by the New Zealand government in 2018 estimated that methane emissions from livestock needed to be reduced from 2016 levels by 10 to 22% by 2050 (i.e., 2.8 to 6.1 million t lower), so as to restrict future increases in global temperature to less than 2°C. In this study, we evaluated genetic effects of 8 traits included in the New Zealand national dairy breeding objective, on 3 types of methane emissions metrics: gross methane emissions per dairy cow per year (E), methane emissions per hectare (EH), and methane emissions intensity per milk protein equivalents (EI), as carbon dioxide equivalents. These effects were then aligned with recent genetic changes in these traits brought about by breeding schemes, so that the overall genetic trend for each metric into the future was estimated. The results showed that EH and EI are currently being reduced at rates of -2.31 kg of CO2-eq per hectare per cow per year (current average is 6,915 kg of CO2-eq/ha per cow per year) and -0.04 kg of CO2-eq per kg of milk protein equivalents per cow per year, respectively (current average is 9.7 kg of CO2-eq/milk protein-eq per cow per year). These improvements directly reflect increased production efficiency through selection for farm profitability. If the pastureland area in New Zealand remains the same, at the same productivity and with no increase in supplementation rates from external land sources, in 20 years gross emissions would be reduced by only 0.6%, or 89 Mt. Increased production efficiency will likely result in corresponding changes to the stocking rate, to fully utilize the pasture resource available, and might further encourage a greater rate of intensification via supplementary feeding. Both consequences of current genetic selection could negate any benefits for the national greenhouse gas inventory. New selection criteria for reduced methane production are needed to help achieve New Zealand's national methane reduction targets.

Short communication: Estimates of genetic parameters for dairy fertility in New Zealand.

Reproductive performance of dairy cows in a seasonal calving system is especially important as cows are required to achieve a 365-d calving interval. Prior research with a small data set has identified that the genetic evaluation model for fertility could be enhanced by replacing the binary calving rate trait (CR42), which gives the probability of a cow calving within the first 42d since the planned start of calving at second, third, and fourth calving, with a continuous version, calving season day (CSD), including a heifer calving season day trait expressed at first calving, removing milk yield, retaining a probability of mating trait (PM21) which gives the probability of a cow being mated within the first 21d from the planned start of mating, and first lactation body condition score (BCS), and including gestation length (GL). The aim of this study was to estimate genetic parameters for the proposed new model using a larger data set and compare these with parameters used in the current system. Heritability estimates for CSD and PM21 ranged from 0.013 to 0.019 and from 0.031 to 0.058, respectively. For the 2 traits that correspond with the ones used in the current genetic evaluation system (mating trait, PM21 and BCS) genetic correlations were lower in this study compared with previous estimates. Genetic correlations between CSD and PM21 across different parities were also lower than the correlations between CR42 and PM21 reported previously. The genetic correlation between heifer CSD and CSD in first parity was 0.66. Estimates of genetic correlations of BCS with CSD were higher than those with PM21. For GL, direct heritability was estimated to be 0.67, maternal heritability was 0.11, and maternal repeatability was 0.22. Direct GL had moderate to high and favorable genetic correlations with evaluated fertility traits, whereas corresponding residual correlations remain low, which makes GL a useful candidate predictor trait for fertility in a multiple trait evaluation. The superiority of direct GL genetic component over the maternal GL component for predicting fertility was demonstrated. Future work planned in this area includes the implementation and testing of this new model on national fertility data.

Phenotypic associations between gestation length and production, fertility, survival, and calf traits.

Gestation length may be a useful selection criterion in the genetic evaluation of fertility for New Zealand's predominantly seasonally calving dairy herd. However, it is unknown if calves born following shorter gestation lengths have lower survival or are compromised in their subsequent performance as a milking cow. In this study, data from a large number (~38,000) of cows were first analyzed to determine if those animals born following a short (shortest 5%) or a long (longest 5%) gestation length differed in their subsequent fertility, milk production, and survival compared with intermediate-gestation-length animals. To determine the effect of gestation length on calving difficulty and perinatal mortality, the gestation records of the calves born to these cows (from their heifer and subsequent 6 parities) were also analyzed. Animals born following short gestation lengths had improved fertility (specifically, their probability of being presented for mating in the first 21 d of the mating season was increased by 4 to 5 percentage points and the day of the calving season at which they calved was 2 to 5d earlier), whereas those born following long gestation lengths had decreased fertility (3 to 4% less likely to be presented for mating in the first 21 d of the calving season and calved 3 to 5d later) compared with animals with average gestation lengths. Both short- and long-gestation-length animals produced significantly less milk and solids (e.g., 1.3 to 1.4 kg of protein over a standardized 270-d lactation) relative to intermediate-gestation-length cows, after adjusting for the day of the year they were born. However, for short-gestation-length cows, this effect disappeared when the earlier birth advantage was retained. Short-gestation-length cows did not exhibit a significant reduction in survival compared with intermediate-gestation-length cows. Short gestation length did not affect calving difficulty but long gestation length was negatively associated with this trait (i.e., about 2% higher incidence). Calves gestated for shorter or longer periods were more likely to die in the perinatal period than other calves (3 and 7% higher incidence of mortality, respectively). Overall, the net effects of shortened gestation lengths are likely to be economically positive.

Cardiolipin deficiency releases cytochrome c from the inner mitochondrial membrane and accelerates stimuli-elicited apoptosis.

Cardiolipin (CL) is a mitochondria-specific phospholipid synthesized by CL synthase (CLS). We describe here a human gene for CLS and its analysis via RNAi knockdown on apoptotic progression. Although mitochondrial membrane potential is unchanged in cells containing only 25% of the normal amount of CL, free cytochrome c (cyt. c) is detected in the intermembrane space and the mitochondria exhibit signs of reorganized cristae. However, the release of cyt. c from the mitochondria still requires apoptotic stimulation. Increased sensitivity to apoptotic signals and accelerated rates of apoptosis are observed in CL-deficient cells, followed by elevated levels of secondary necrosis. Apoptosis is thought to progress via binding of truncated Bid (tBid) to mitochondrial CL, followed by CL oxidation which results in cyt. c release. The exaggerated and accelerated apoptosis observed in CL-deficient cells is matched by an accelerated reduction in membrane potential and increased cyt. c release, but not by decreased tBid binding. This study suggests that the CL/cyt. c relationship is important in apoptotic progression and that regulating CL oxidation or/and deacylation could represent a possible therapeutic target.

Phospholipase D: a lipid centric review.

Phospholipase D (PLD) hydrolyzes the phosphodiester bond of the glycerolipid phosphatidylcholine, resulting in the production of phosphatidic acid and free choline. Phosphatidic acid is widely considered to be the intracellular lipid mediator of many of the biological functions attributed to PLD. However, phosphatidic acid is a tightly regulated lipid in cells and can be converted to other potentially bioactive lipids, including diacylglycerol and lysophosphatidic acid. PLD activities have been described in multiple organisms, including plants, mammals, bacteria and yeast. In mammalian systems, PLD activity regulates the actin cytoskeleton, vesicle trafficking for secretion and endocytosis, and receptor signaling. PLD is in turn regulated by phosphatidylinositol-4,5-bisphosphate, protein kinase C and ADP Ribosylation Factor and Rho family GTPases. This review focuses on the lipid precursors and products of mammalian PLD metabolism, especially phosphatidic acid and the roles this lipid performs in the mediation of the functions of PLD.

The emerging role for sphingolipids in the eukaryotic heat shock response.

Eukaryotic cells have a highly conserved response to an increase in temperature, termed the heat shock response. Recent research has revealed multiple roles for various sphingolipids in the heat shock responses of both yeast and mammalian cells. Heat stressed or shocked yeast and mammalian cells have an acute activation of serine palmitoyltransferase, resulting in the de novo biosynthesis of sphingolipids. Also, both mammalian and yeast cells were shown to increase ceramide levels upon heat stress or shock. In yeast cells, several functions have emerged for the de novo produced sphingoid bases in terms of the heat stress response. These functions include a role in accumulation of trehalose, a role in the heat-induced transient G0/G1 cell cycle arrest and phytosphingosine activation of a ubiquitin protein degradation pathway. However, in mammalian systems, ceramides have been demonstrated as bioactive lipids. Ceramides produced in response to heat shock were demonstrated to induce the production of c-jun, leading to apoptosis, and to be upstream of dephosphorylation of serine-rich proteins. Increasingly, sphingolipids are emerging as bioactive signaling molecules involved in numerous aspects of the eukaryotic heat shock response.

Evolution of base composition and codon usage bias in the genus Flavivirus.

The extent to which base composition and codon usage vary among RNA viruses, and the possible causes of this bias, is undetermined in most cases. A maximum-likelihood statistical method was used to test whether base composition and codon usage bias covary with arthropod association in the genus Flavivirus, a major source of disease in humans and animals. Flaviviruses are transmitted by mosquitoes, by ticks, or directly between vertebrate hosts. Those viruses associated with ticks were found to have a significantly lower G+C content than non-vector-borne flaviviruses and this difference was present throughout the genome at all amino acids and codon positions. In contrast, mosquito-borne viruses had an intermediate G+C content which was not significantly different from those of the other two groups. In addition, biases in dinucleotide and codon usage that were independent of base composition were detected in all flaviviruses, but these did not covary with arthropod association. However, the overall effect of these biases was slight, suggesting only weak selection at synonymous sites. A preliminary analysis of base composition, codon usage, and vector specificity in other RNA virus families also revealed a possible association between base composition and vector specificity, although with biases different from those seen in the Flavivirus genus.

Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosis.

BACKGROUND: Despite limiting elastic recoil and late vascular remodeling after angioplasty, coronary stents remain vulnerable to restenosis, caused primarily by neointimal hyperplasia. Paclitaxel, a microtubule-stabilizing drug, has been shown to inhibit vascular smooth muscle cell migration and proliferation contributing to neointimal hyperplasia. We tested whether paclitaxel-coated coronary stents are effective at preventing neointimal proliferation in a porcine model of restenosis. METHODS AND RESULTS: Palmaz-Schatz stents were dip-coated with paclitaxel (0, 0.2, 15, or 187 microgram/stent) by immersion in ethanolic paclitaxel and evaporation of the solvent. Stents were deployed with mild oversizing in the left anterior descending coronary artery (LAD) of 41 minipigs. The treatment effect was assessed 4 weeks after stent implantation. The angiographic late loss index (mean luminal diameter) decreased with increasing paclitaxel dose (P<0.0028 by ANOVA), declining by 84.3% (from 0.352 to 0.055, P<0.05) at the highest level tested (187 microgram/stent versus control). Accompanying this change, the neointimal area decreased (by 39.5%, high-dose versus control; P<0.05) with increasing dose (P<0.040 by ANOVA), whereas the luminal area increased (by 90.4%, high-dose versus control; P<0.05) with escalating dose (P<0.0004 by ANOVA). Inflammatory cells were seen infrequently, and there were no cases of aneurysm or thrombosis. CONCLUSIONS: Paclitaxel-coated coronary stents produced a significant dose-dependent inhibition of neointimal hyperplasia and luminal encroachment in the pig LAD 28 days after implantation; later effects require further study. These results demonstrate the potential therapeutic benefit of paclitaxel-coated coronary stents in the prevention and treatment of human coronary restenosis.

Evidence for the non-quasispecies evolution of RNA viruses [corrected].

The quasispecies model of RNA virus evolution differs from those formulated in conventional population genetics in that neutral mutations do not lead to genetic drift of the population, and natural selection acts on the mutant distribution as a whole rather than on individual variants. By computer simulation, we show that this model could be inappropriate for many RNA viruses because the neutral sequence space may be too large to allow the formation of a quasispecies distribution. This view is supported by our analysis of gene sequences from vesicular stomatitis virus, which is considered a prototype RNA virus quasispecies. Our results are relevant to the evolution of RNA systems in general.

Role for de novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae.

The recent findings of sphingolipids as potential mediators of yeast heat stress responses led us to investigate their possible role in the heat-induced cell cycle arrest and subsequent recovery. The sphingolipid-deficient yeast strain 7R4 was found to lack the cell cycle arrest seen in the isogenic wild type. Furthermore, strain lcb1-100, which harbors a temperature-sensitive serine palmitoyltransferase, lacked increased de novo generated sphingoid bases upon heat stress. Importantly, this strain was found to lack the transient heat-induced G0/G1 arrest. These results indicate a role for sphingolipids and specifically those generated in the de novo pathway in the cell cycle arrest response to heat. To determine the bioactive sphingolipid regulating this response, an analysis of key mutants in the sphingolipid biosynthetic and degradation pathways was performed. Strains deleted in sphingoid base kinases, sphingoid phosphate phosphatase, lyase, or dihydrosphingosine hydroxylase were found to display the cell cycle arrest. Also, the knockout of a fatty acyl elongation enzyme, which severely attenuates ceramide production, displayed the arrest. These experiments suggested that the active species for cell cycle arrest were the sphingoid bases. In further support of these findings, exogenous phytosphingosine (10 microM) was found to induce transient arrest. Stearylamine did not induce an arrest, demonstrating chemical specificity, and L-erythro- was not as potent as D-erythro-dihydrosphingosine showing stereospecificity. To investigate a possible arrest mechanism, we studied the hyperstable Cln3 (Cln3-1) strain LDW6A that has been previously shown to be resistant to heat stress-induced cell cycle arrest. The strain containing Cln3-1 was found to be resistant to cell cycle arrest induced by exogenous phytosphingosine, indicating that Cln3 acts downstream of the sphingoid bases in this response. Interestingly, cell cycle recovery from the transient arrest was found to be dependent upon the sphingoid base kinases (LCB4, LCB5). Overall, this combination of genetic and pharmacologic results demonstrates a role for de novo sphingoid base biosynthesis by serine palmitoyltransferase in the transient G0/G1 arrest mediated through Cln3 via a novel mechanism.

Testing the extent of sequence similarity among viroids, satellite RNAs, and hepatitis delta virus.

A Monte Carlo method was used to test the extent of sequence similarity among viroids, satellite RNAs, and hepatitis delta virus. This analysis revealed that there is insufficient sequence similarity among these pathogens to support the hypothesis that they have a common evolutionary origin. Furthermore, while definite patterns of sequence similarity were observed among some viroids, there was a clear lack of overall similarity, indicating that a monophyletic origin for even this group cannot be reliably supported from sequence data alone.

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.

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.

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.

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.

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.

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.

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.

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.

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.