The role of hepatic arterial flow on portal venous and hepatic venous wedged pressure in the isolated perfused CCl4-cirrhotic liver

In cirrhosis, hepatic venous pressure gradient is used to measure portal venous and sinusoidal pressures, as well as drug-induced decreases of elevated pressures. The aim of this study was to investigate the influence of hepatic arterial flow (HAF) changes on portal venous perfusion (PVPP) and wedged hepatic venous pressure (WHVP). Normal and CCl4-cirrhotic rats were subjected to a bivascular liver perfusion with continuous measurements of PVPP, WHVP, and hepatic arterial perfusion pressure. Flow-pressure curves were performed with the use of different flows either through the portal vein (PVF: 20-32 ml/min) or HAF (5-15 ml/min). Increases in HAF lead to significant absolute and relative increases in PVPP (P = 0.002) and WHVP (P < 0.001). Absolute changes in HAF correlated to absolute changes in PVPP (cirrhosis: r = 0.64, P < 0.001; control: r = 0.67, P < 0.001) and WHVP (cirrhosis: r = 0.71, P < 0.001; control: r = 0.82, P < 0.001). Changes in PVPP correlated to changes in WHVP due to changes in PVF only in cirrhosis (r = 0.75, P < 0.001), whereas changes in HAF correlated in both cirrhosis (r = 0.92, P < 0.001) and control (r = 0.77, P < 0.001). In conclusion, increases and decreases in HAF lead to respective changes in PVPP and WHVP. This suggests a direct influence of HAF on PVPP and WHVP most likely due to changes in sinusoidal perfusion.     

Effects of changes in circulating volume and in arterial pressure on plasma renin activity in the intact rat

The effects of changes in arterial pressure and in circulating volume on Plasma Renin Activity (PRA) in the intact rat were compared by two experimental procedures. Gradual volume depletion was induced by intraperitoneal injection of a hyperoncotic polyethyleneglycol solution (PEG) in absence of acute changes in Systolic Arterial Pressure (SAP). SAP was measured in the conscious state by the tail cuff technique. Plasma Protein Concentration (PPC) and Hematocrit (Hct) increases after PEG injection were compared as the index for measuring the Plasma Volume Reduction (PVR). PRA showed a significant (p less than 0.001) linear relationship with PPC, suggesting a direct dependence of renin secretion on volume depletion. Acute changes in the circulating volume were induced by controlled hemorrhages of 5.0, 10.0, 15.0 and 20.0 ml of blood/kg body weight. The increase in PRA showed a significant relationship with the changes in circulating volume, but it did not show any dependence on the changes in Mean Arterial Pressure (MAP). Our results suggest that, in the intact and conscious rat, renin secretion responds to the information from the cardiopulmonary volume receptors rather than to that from the high pressure receptors.     

PARTICLE-INDUCED ERYTHROPOIETIN-INDEPENDENT EFFECTS ON ERYTHROID PRECURSOR CELLS IN MURINE BONE MARROW

A possible regulatory action of phagocytic cells on erythropoiesis was investigated by infusion of inert polystyrene latex particles (LAT). LAT appeared to induce changes in the femoral content of erythroid progenitor cells. These changes were most pronounced in primitive erythroid progenitor cells (BFUe) and appeared to be gradually damped in more differentiated populations (CFUe and erythroblasts). LAT did not influence granulocyte/macrophage progenitor cells (CFUc). The effects of LAT could not be attributed to changes in the systemic erythropoietin (EP) concentration. Administration of dexamethason nullified the effect of low doses of LAT, suggesting that phagocytosis of the particles is essential to the observed effects. Erythroid burst formation was previously found to be dependent on a bone marrow associated activity, termed BFA (burst feeder activity). BFA acts as an in vitro inducer of EP-responsiveness in BFUe. In this study it was found that LAT-induced changes in femoral erythroid progenitor cell content were characteristically preceded by corresponding changes in BFA. It was concluded that BFA-associated cells probably play a role in vivo in the early differentiation of erythroid progenitor cells. The present data are interpreted as direct in vivo evidence supporting a two-step regulatory model operating in erythropoiesis and provide evidence that phagocytic cells are a component of the erythroid haemopoietic inductive micro-environment.     

Free radicals in chronic fatigue syndrome: cause or effect?

We have demonstrated that certain morphological and biochemical changes occur in chronic fatigue syndrome (CFS) and in rheumatoid arthritis (RA). These changes in RA can be explained by the well-established inappropriate increase in free radical generation. The similar changes in CFS suggest a similar explanation and a possible role for free radicals in the aetiology of this condition.     

Changes in tissue water content measured with multiple-frequency bioimpedance and metabolism measured with 31P-MRS during progressive forearm exercise.

Multiple-frequency bioimpedance analysis (MFBIA) has been used to determine the cellular water composition in the human body. It is noninvasive and has demonstrated good correlations with other invasive measures of tissue water. However, the ability of this method to study transient changes in tissue water in specific muscle groups has not been explored. In this study, MFBIA was used to assess changes in forearm intracellular water (ICW), extracellular water (ECW), and total water (TW) in seven healthy volunteers during and after a progressive wrist flexion exercise protocol. In an identical trial, (31)P magnetic resonance spectroscopy ((31)P-MRS) was used to assess changes in intracellular pH and phosphocreatine (PCr). At the completion of exercise, forearm ICW increased 12.6% (SD 0.07, P = 0.003), TW increased 10.1% (SD 0.06, P = 0.005), and no significant changes were recorded for ECW. A significant correlation was found between the changes in intracellular pH and changes in ICW during exercise (r = -0.84, P = 0.018). With the use of regression analysis, average changes in P(i), PCr, and pH were found to predict changes in ICW (R(2) = 0.98, P = 0.005). In conclusion, MFBIA was sensitive enough to measure transient changes in the exercising forearm muscle. The changes seen were consistent with the hypothesis that intracellular acidification and PCr hydrolysis are important mediators of cellular osmolality and therefore may be responsible for the increased volume of water in the intracellular space that is often recorded after short-term high-intensity exercise.     

Two processes lead to a stable all-trans and 13-cis isomer equilibrium in dark-adapted bacteriorhodopsin; effect of high pressure on bacteriorhodopsin,bacteriorhodopsin mutant D96N and fluoro-bacteriorhodopsin analogues

The combination of absorption spectroscopy and extraction techniques was applied to study the effect of high pressure on the dark-adapted state of bacteriorhodopsin, 14-(12-,10-)fluoro-bacteriorhodopsin, a D96N bacteriorhodopsin mutant, and 14-(12-,10-)fluoro-D96N. Evidence is presented that, at high pressure, the isomers' equilibrium is shifted from all- trans isomers towards the 13-cis isomers. Two groups of values for calculated molar volume changes indicate that there are at least two different processes leading to a stable all-trans and 13-cis isomers' equilibrium called the dark-adapted bacteriorhodopsin. The first process may be attributed to changes in the distances and rearrangement of functionally important residues and a retinal Schiff base. It is suggested that the moved residues (probably Asp-212 with the contribution of Tyr-185 and/or Asp-85) closer to the chromophore could catalyse its trans-cis isomerization. These changes require smaller pressure changes and induce larger volume changes (large-volume-change process). The second process may be attributed to the formation of the three hydrogen bonds that additionally decrease the volume and strengthen further stabilization of the 13-cis isomer. To induce these changes, larger changes of pressure are required and the final molar volume changes are smaller (small-volume-change process). The total molar volume change between all-trans bacteriorhodopsin and 13-cis bacteriorhodopsin in the dark-adapted state of native bacteriorhodopsin was found to be about -28 mL/mol, which is much higher than the value of about -7 mL/mol obtained previously (Tsuda and Ebrey 1980, Schulte and Bradley 1995). The data provide a novel insight into factors leading to stable isomer equilibrium in dark-adapted bacteriorhodopsin.     

Accuracy of measurements of small changes in soft tissue mass by use of dual-photon absorptiometry

Dual-photon absorptiometry (DPA) has recently been applied to the assessment of body composition. To evaluate the accuracy of DPA in detecting small changes in the lean soft tissue mass, we performed DPA with the use of the Norland 2600 Dichromatic densitometer on six healthy adult males before and after a 30-ml/kg transfusion of saline and before and after exercise in a warm environment, resulting in a greater than or equal to 1-kg weight loss. Absolute weight [baseline pretransfusion r2 = 0.999, standard error of estimate (SEE) = 590 g; posttransfusion r2 = 0.999, SEE = 300 g; baseline pretranspiration r2 = 0.999, SEE = 230 g; posttranspiration r2 = 0.999, SEE = 240 g] was accurately reflected in DPA total mass. Weight changes due to transfusion were poorly reflected by changes in DPA total mass (r2 = 0.417, SEE = 404 g). However, changes posttranspiration were accurately reflected in the DPA total mass (r2 = 0.886, SEE = 106 g posttranspiration). Similarly, weight changes due to transfusion were poorly measured by changes in DPA soft mass (r2 = 0.478, SEE = 365 g), but changes posttranspiration were highly correlated with DPA soft mass changes (r2 = 0.909, SEE = 92 g). Weight changes were not reflected by changes in the DPA lean soft tissue mass (r2 = 0.006, SEE = 1,737 posttransfusion, r2 = 0.094, SEE = 1,038 g posttranspiration). DPA-derived nonfat mass was highly correlated with skinfold-derived nonfat mass (r2 = 0.96, SEE = 2,400 g). Accuracy of total and soft tissue measurements implied correct mineral mass assessment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological characteristics of motor units in the brachioradialis muscle across fatiguing low-level isometric contractions.

The purpose of this study was to determine (i) if decomposition-based quantitative electromyography (DQEMG) could detect changes in motor unit potential (MUP) morphology and motor unit (MU) firing pattern statistics associated with muscle fatigue, (ii) if any detected changes are correlated with surface electromyographic (SEMG) signs of fatigue, and (iii) if significant fatigue-dependent changes are repeatable within individuals. Mean MU firing rates and the morphology of MUPs detected using needle and surface electrodes during constant-torque isometric contractions held until exhaustion were investigated in the brachioradialis (BR) muscle in 10 healthy volunteers (mean age=28.6 yr, SD+/-3.9). Time dependant changes were investigated using an analysis of variance with normalized time as a main effect. Partial correlation coefficients were computed using a repeated measures analysis of covariance to determine if changes in MU firing rates, needle-detected MUPs and surface-detected MUPs (SMUPs) were related to changes in SEMG signal amplitude and frequency parameters. Intraclass correlation coefficients (ICCs) were used to determine the within-subject repeatability of changes in MU firing rates, and MUP and SMUP parameters. Significant decreases in mean MU firing rates were found along with significant increases in various duration and area related parameters in both MUPs and SMUPs across the fatiguing contraction. The SEMG signal demonstrated the expected changes with fatigue: an increase in amplitude and a decrease in frequency content. SEMG amplitude was significantly positively correlated with SMUP peak-to-peak voltage (r=0.85, p<0.05), and SMUP area (r=0.86, p<0.05). Mean power frequency was significantly negatively correlated with SMUP negative peak duration (r=-0.74, p<0.05). The significant time-dependent changes were reliably observed (ICCs were 0.94 for MUP peak to peak amplitude, 0.97 for MUP area and 0.95 for MUP area to amplitude ratio, 0.95 for SMUP peak-to-peak voltage, 0.83 for SMUP area, 0.99 for SMUP negative peak amplitude and 0.88 for SMUP negative peak area). The decreases in mean MU firing rates measured along with the increases in amplitude, duration and area parameters of MUPs and SMUPs and their partial correlation with SEMG amplitude during submaximal fatiguing contractions of the BR, suggest that recruitment is a main cause of increased SEMG amplitude parameters with fatigue. We conclude that DQEMG can be effectively and reliably used to detect changes in physiological characteristics of MUs that accompany fatigue.     

Association between cutaneous occlusive vascular disease, cigarette smoking, and skin slough after rhytidectomy

This prospective study attempted to determine if nonreversible occlusive vascular changes in the skin contribute to skin slough after rhytidectomy. The dermal microvasculature from 83 consecutive rhytidectomies was evaluated for intimal proliferation and/or hyalin sclerosis. Occlusive vascular disease increased progressively with age in all patients, but smokers and ex-smokers had significantly greater involvement than nonsmokers at any given age (p = 0.03). Severe occlusive vascular disease and skin slough were associated (p = 0.02), and there was a strong trend toward an association between active smoking and skin slough (p = 0.06). Among smokers, there was a significant relationship between skin slough and failure to abstain from smoking postoperatively (p = 0.006). We conclude that with aging, nonreversible occlusive changes develop in the dermal microvasculature. These changes appear to be accelerated by cigarette smoking. Our data, however, show that these nonreversible occlusive vascular changes by themselves do not completely account for the occurrence of skin slough after rhytidectomy.     

Histochemical and histoenzymatic liver changes in Guérin tumour-grafted rats

In order to follow up the morphological changes of the rat liver as a reaction to the tumour graft, three groups of Guérin tumour-grafted animals were used: a first control group, a second one treated with leucotrophine (LT) and a third one treated with LT and thiamine diphosphate (TDP). The tumour-grafted rats showed hepatic changes affecting mainly the organelles involved in cellular respiration and synthesis, as well as some morphological changes of Kupffer cells expressing an increased endocytosis. The protection by immunostimuli determined the diminution of histochemical and histoenzymatic changes both in hepatocytes and Kupffer cells.     

Selected genetic polymorphisms and plasma coagulation factor VII changes with exercise training.

We assessed the effects of coagulation factor VII (FVII) gene polymorphisms, lipid-related polymorphisms, and exercise training-induced plasma lipoprotein lipid changes on FVII level changes with exercise training in middle- to older-aged men and women. Forty-six healthy sedentary men and women were stabilized on a low-fat diet and then underwent baseline testing, 6 mo of endurance exercise training, and final testing. Plasma FVII-Ag levels decreased with exercise training (106.7 +/- 1.4 vs. 104.2 +/- 1.6%, P = 0.005). There were no significant differences in FVII-Ag changes with exercise training between -323 (0/10 bp)/-401 (G/T) haplotype or -402 (G/A) genotype groups. FVII-Ag changes with training were not correlated with changes in plasma lipoprotein lipids. In linear regression analyses, FVII-Ag changes with training remained significant after adjusting for training-induced plasma lipoprotein lipid changes (P = 0.01). FVII changes with training were associated with apolipoprotein E genotype (P = 0.012); this relationship was still evident after adjusting for training-induced plasma lipoprotein lipid changes (P = 0.047). FVII changes with training also were significantly associated with human ATPase binding cassette-1 genotype (P = 0.018); this relationship persisted after accounting for the effect of the training-induced plasma lipoprotein lipid changes (P = 0.045). We conclude that plasma FVII-Ag changes with exercise training are more closely related to selected lipid-related genotypes than FVII gene promoter variants.     

STUDIES ON THE BIOCHEMISTRY OF MITOCHONDRIA AND CELL MORPHOLOGY IN THE NEONATAL SWINE HEPATOCYTE

Mitochondrial preparations isolated from neonatal swine hepatocytes show a marked increase in oxidative and concomitant phosphorylative capacity between birth and 2 days postpartum. There are no changes in the coupling parameters (respiratory control ratio and adenosine diphosphate/O ratio) with age. Changes in sedimentation properties in a sucrose gradient suggest qualitative changes in the mitochondria. Some of the lipid measurements (increased phospholipid) might be interpreted as supportive of this suggestion, although most could also be regarded as indicative of quantitative changes (increased number of mitochondria). Electron microscopy of isolated mitochondria and of the hepatocyte demonstrated an increased number of mitochondria but no change in shape, size, or structure as the pig developed. An increase in a number of cytoplasmic components (Golgi apparatus and endoplasmic reticulum) and a decrease in glycogen were also observed. The functional changes in mitochondria seem to occur within a short period of time (6–12 hr postpartum).     

Light-induced unfolding of photoactive yellow protein mutant M100L

Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila. To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYP(M) by 3 orders of magnitude, as previously reported for the M100A mutant [Devanathan, S., Genick, U. K., Canestrelli, I. L., Meyer, T. E., Cusanovich, M. A., Getzoff, E. D., and Tollin, G. Biochemistry (1998) 37, 11563-11568]. The FTIR difference spectrum of PYP(M) and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYP(M). The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the alpha-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure. The optical activity of the p-coumaric acid chromophore completely vanished upon PYP(M) in contrast to the dark state, indicating deformation of the binding pocket structure in PYP(M). The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYP(M). These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery. The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYP(M) to PYP(dark), which can be regarded as a process of folding of the secondary and the tertiary structures of the "PAS" domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization.     

Influence of dietary protein level on growth: effect of clenbuterol.

1. The influence has been studied of a beta-agonist, clenbuterol, on body weight increase, feed efficiency and muscular tissue growth in Wistar rats of two different weights (70 and 200 g) fed with a diet of differing protein levels (4, 10 and 15%). 2. Treatment with clenbuterol had a negative influence on body growth and feed efficiency when the animals were fed with low protein diets (4%). 3. With a higher protein content (10 and 15%), the situation changed completely, and these indices increased, with a relationship appearing between these changes, the agonist dose and the animal's age. 4. In all the experiments clenbuterol improved muscle/body ratio significantly (P less than 0.001). The favourable effect of clenbuterol on muscle growth is due to a specific increase in protein deposition and not to changes in the water content of muscular tissue, even though these changes may be produced.     

斑翅蝗科四种蝗虫卵子发生阶段及种间差异的统计分析

通过组织学观察与测量对斑翅蝗科(直翅目,蝗总科)中4种蝗虫卵子发生各阶段进行测量和分析比较,4种蝗虫分别为:大垫尖翅蝗Epacromius coerulipes(Ivan.)、黄胫小车蝗Oedaleus infernalis Sauss.、花胫绿纹蝗Aiolopus tamulus(Fabricius)和疣蝗Trilophidia annulata(Thunberg).结果显示,在蝗虫卵子发生过程中,卵母细胞体积和形态变化显著,呈现指数增长模式;细胞核体积增长在发育初期增长显著,后期变化不显著;滤泡细胞体积和形态及数量呈现周期性变化规律;进而比较种间差异,可见不同种蝗虫的卵子发生存在不同的发育进程.     

Free radicals in chronic fatigue syndrome: cause or effect?

Abstract

We have demonstrated that certain morphological and biochemical changes occur in chronic fatigue syndrome (CFS) and in rheumatoid arthritis (RA). These changes in RA can be explained by the well-established inappropriate increase in free radical generation. The similar changes in CFS suggest a similar explanation and a possible role for free radicals in the aetiology of this condition.     

Activation of command fibres to the stomatogastric ganglion by input from a gastric mill proprioceptor in the crab,Cancer pagurus †

In semi‐intact preparations of the crab Cancer pagurus the normal output from the stomatogastric ganglion (StG) was a regular pyloric cycle (Figure 4). Repeated stimulation of the posterior stomach nerve (psn) of the posterior gastric mill proprioceptor (PSR) often induced series of spontaneous gastric cycles. We were therefore able to describe the normal gastric cycle as recorded in the output nerves from StG and to identify most of the relevant motor neurones by reference to the muscles that they innervate (Figure 10). The gastric cycle output was variable (Figures 5, 6), although in many preparations one complex type of output predominated (Figure 7). The basic feature of the gastric cycle was an alternation of activity between the single cardio‐pyloric neurone (CP) and a complex variable burst in the lateral cardiac (LC), the gastro‐pyloric (GP), the gastric (GM), and other associated neurones. During this normally occurring complex gastric burst significant changes occurred in the pyloric cycle, notably an increase in activity of the pacemaker pyloric dilator (PD) group and an inhibition of the lateral pyloric (LP), inferior cardiac (IC) and ventricular dilator (VD) neurones (Figures 6, 7, 8, 9). These changes are probably associated with an opening of the cardio‐pyloric valve and food passage into the pyloric filter. The gastric output was related to the normally observed movements of the dorsal ossicles of the gastric mill and thus to the operation of the teeth of the mill (Figure 11). Increased input from the PSR is associated with the grinding action of the teeth which is caused by the complex gastric burst (Figure 12).

Stimulation of the psn during an ongoing regular pyloric output caused changes in the cycle which mimicked those occurring during the spontaneous gastric cycle (Figure 13; Table 1). Stimulation of the psn during ongoing gastric activity also affected the gastric units (Figure 14). The input pathway from the PSR is shown to be through the stomatogastric nerve (sgn), the connection between the commissural ganglia and the stomatogastric ganglion (Figure 15). The commissural ganglia are known to receive most of the sensory input from the foregut and PSR input is probably processed there. Recordings from the sgn show that psn stimulation activates a small number of centrally originating units, and that the activity of these units coincides with the pyloric output changes (Figures 15, 16). We therefore label the units command interneurones. Their effects could be mediated by direct connections to only the PD pacemaker neurones of the pyloric cycle. Control experiments showed that PSR input is not necessary for the pyloric output changes to occur during gastric output but that similar output changes can be evoked by input resulting from induced gastric movements (Figure 15(E)). We think that the pyloric cycle output changes are normally controlled by a number of mechanisms at different levels (Figure 17). We cannot easily explain the effects of PSR input on the gastric cycle neurones.

These findings are important because they allow us to study a specific input to the StG without disrupting its normal input‐output pathways to the central nervous system. Further experiments on the system designed to test the assumption that the sgn units are in fact responsible for the pyloric output changes, and to investigate the processing of the PSR input are outlined.     

Altered cholesterol ester proportions in embryonic tissues of dystrophic chicken

The concentrations of cholesterol esters in tissues of dystrophic chicken embryos are altered from normal. These changes are accompanied by significant changes in the proportions of the esterified fatty acids (the fatty acid profile). In serum and pectoral muscles there is a shift to a higher proportion of unsaturated fatty acids (in particular 18:1). Thigh muscle esters are little changed and in liver and brain the proportion of unsaturated fatty acids decreases.     

Sensing minute changes in biological cell monolayers with THz differential time-domain spectroscopy

We used terahertz differential time-domain spectroscopy (THz-DTDS) to measure minute changes of bovine lung microvessel endothelial cells (BLMVEC) in response to vascular endothelial growth factor (VEGF). These changes were reflected by alterations in THz wave attenuations and THz dielectric properties of the treated cells. The VEGF-induced THz attenuations of cell monolayers correlated well with changes in transendothelial resistance, as measured using electric cell-substrate impedance sensing (ECIS). However, the morphological differences that gave rise to these changes were not observed with standard optical phase contrast microscopy. We conclude that THz-DTDS is a highly sensitive, non-invasive, powerful new tool to measure minute changes in the morphology of live, cultured cell monolayers. This method enables spectroscopic investigations of cells in the THz band, providing information unavailable through other conventional methods such as optical phase contrast microscopy and ECIS.     

Sib-pair linkage analysis of longitudinal changes in lipoprotein risk factors and lipase genes in women twins

Based on longitudinal twin data in women, we have previously demonstrated a genetic influence on changes in lipoprotein risk factors, blood pressure measurements, and body mass index over a decade. The present study examined the linkage between changes in lipoprotein variables and candidate genes encoding the hormone-sensitive lipase (HSL), hepatic lipase (HL), and lipoprotein lipase (LPL). The sample consisted of 126 dizygotic (DZ) pairs of women twins who participated in the two examinations of the Kaiser Permanente Women Twins Study, performed a decade apart. Using quantitative sib-pair linkage analysis, a linkage was demonstrated between the locus for hormone-sensitive lipase and age-adjusted changes in plasma triglyceride (P = 0.015), which became more significant after adjustment for environmental factors and the exam-1 level (P = 0.005). There was also evidence suggesting linkage between the locus for hepatic lipase and changes in triglyceride (P = 0.023), but no linkage was detected for lipoprotein lipase and changes of lipid levels with time. These findings suggest that variation at these candidate gene loci may underlie a portion of the intraindividual variations in these coronary heart disease (CHD) risk factors, and that studies to identify the functional variants could provide new insights into genetic susceptibility to cardiovascular disease.