Recovery of prostacyclin capacity of irradiated endothelial cells and the protective effect of vitamin C

Ionizing irradiation has been reported to affect prostacyclin (PGI2) production by intact blood vessels and cultured endothelial cells (EC) due to damage of enzymes of the arachidonate cascade. In the present study, we investigated whether EC can recover from radiation injury and regain their capacity to produce PGI2. Bovine aortic EC were exposed to radiation doses of 3 and 6 Gy and their capacity to produce PGI2 in response to stimulation with arachidonic acid was tested, at various times after irradiation. The results of these experiments showed clearly that EC exposed to single or fractionated irradiation could recover their capacity to produce PGI2 depending on the radiation dose and the time period following radiation. Radiation damage is associated with oxidant stress and the production of free radicals. We therefore tested the ability of an oxygen radical scavenger, vitamin C, to protect the capacity of irradiated EC to produce PGI2. Pretreatment of EC with low concentrations of vitamin C inhibited the radiation induced release of PGI2 to the culture medium. Vitamin C also enhanced the capacity of irradiated EC to produce PGI2 following short stimulation with arachidonic acid. Treatment with this scavenger however, did not protect the cells against the cytopathic effects of radiation.     

Growth measurements of terrestrial microbial species by a continuous-flow technique

A continuous nutrient flow system has been developed to measure microbial activity in soil with various concentrations of added substrate. The system consists of a thin soil layer through which substrate was added continuously over periods up to 4.5 days. Substrate utilization was determined by effluent analysis. Respiration was measured manually by injecting a sample into a gas chromatograph or automatically by coupling the growth chamber to a computer-controlled gas sampling valve. This permitted respiratory CO₂ to be measured by the gas chromatograph at intervals selected by the investigator. Software controlling the valve and gas chromatograph not only automated gas phase sampling, but also provided a scan of CO₂ evolution and a preliminary data summary. This included the date and time of sample, peak height, and percent CO₂ in the gas phase. Data for growth on glucose using a microbial population native to a California annual grassland soil demonstrated that the direct cell count and respiratory techniques for biomass estimation give comparable results. This procedure provides the potential for detailed analyses of substrate utilization in studies of the growth and maintenance of soil microorganisms.     

Cultured endothelial cells increase their capacity to synthesize prostacyclin following the formation of a contact inhibited cell monolayer

The synthesis of the prostaglandins (PG), prostacyclin (PGI2), PGE2, and thromboxane A2 (TXA2), has been investigated in actively growing and contact-inhibited bovine aortic endothelial cell cultures. Cells were stimulated to synthesize prostaglandins by exposure to exogenous arachidonic acid or to the endoperoxide PGH2 and by the liberation of endogenous arachidonic acid from cellular lipids with melittin or ionophore A23187. Increased capacity of the cells to synthesize PGI2 and PGE2 was observed as a function of time in culture, regardless of the type of stimulation. TXA2 production increased with time only upon stimulation of the cells with ionophore A23187. This increased PG synthetic capacity was independent of cell density since it was mainly observed in confluent, nondividing endothelial cell cultures. The fact that increased PGI2 production in confluent cells was also observed with PGH2, a direct stimulator of PGI2 synthetase, implies that this process is independent of the arachidonate concentration within the cells or in the culture medium. This increased capacity is likely to reflect an increased activity of the PG synthetase system associated with the formation of a contact inhibited endothelial cell monolayer. A similar time-dependent increase in the PGI2 production capacity was also observed during growth of cultured bovine corneal endothelial cells.     

Envelope glycoprotein of avian hemangioma retrovirus induces a thrombogenic surface on human and bovine endothelial cells.

Vascular endothelial cells are a target for blood-borne pathogens which may affect their integrity and thromboresistant properties. Here, we report that cultured bovine and human endothelial cells lose their thromboresistance following interaction with the avian hemangioma-inducing retrovirus. We show that the envelope (env) gene product, glycoprotein 85, is responsible for this effect, which appears soon after infection without viral replication or cell transformation. Induction of thrombogenicity is associated with a reduction in prostacyclin release and increased expression of tissue factor. These observations may explain the occurrence of thrombosis frequently observed in association with the hemangiosarcomas induced by avian hemangioma-inducing retrovirus. These unique endothelial cell-virus interactions may also be a model for the pathogenesis of various vascular diseases.     

Conversion of Biovolume Measurements of Soil Organisms, Grown Under Various Moisture Tensions, to Biomass and Their Nutrient Content

Direct microscopic measurements of biomass in soil require conversion factors for calculation of the mass of microorganisms from the measured volumes. These factors were determined for two bacteria, five fungi, and a yeast isolated from soil. Moisture stress conditions occurring in nature were simulated by growth in two media using shake cultures, on agar plates, and on membranes held at 34, 330, and 1,390 kPa of suction. The observed conversion factors, i.e., the ratio between dry weight and wet volume, generally increased with increasing moisture stress. The ratios for fungi ranged from 0.11 to 0.41 g/cm³ with an average of 0.33 g/cm³. Correction of earlier data assuming 80% water and a wet-weight specific gravity of 1.1 would require a conversion factor of 1.44. The dry-weight specific gravity of bacteria and yeasts ranged from 0.38 to 1.4 g/cm³ with an average of 0.8 g/cm³. These high values can only occur at 10% ash if no more than 50% of the cell is water, and a specific conversion factor to correct past data for bacterial biomass has not yet been suggested. The high conversion factors for bacteria and yeast could not be explained by shrinkage of cells due to heat fixing, but shrinkage during preparation could not be completely discounted. Moisture stress affected the C, N, and P content of the various organisms, with the ash contents increasing with increasing moisture stress. Although further work is necessary to obtain accurate conversion factors between biovolume and biomass, especially for bacteria, this study clearly indicates that existing data on the specific gravity and the water and nutrient content of microorganisms grown in shake cultures cannot be applied when quantifying the soil microbial biomass.     

Cooperativity of lectin binding to lymphocytes,and its relevance to mitogenic stimulation

The relationship between the binding patterns of soybean agglutinin, peanut agglutinin (both in their native (unaggregated) form and in their polymerized form), and of Phaseolus vulgaris leucoagglutinin, to neuraminidase-treated lymphocytes from different sources, and the mitogenic activity of these lectins, was studied. In all cases investigated, binding of a lectin to lymphocytes which resulted in stimulation was a positive cooperative process. Our findings support the assumption that clustering of receptors and conformational changes in membrane structure are prerequisites for mitogenic stimulation.     

A systematic review of the literature examining the diagnostic efficacy of measurement of fractionated plasma free metanephrines in the biochemical diagnosis of pheochromocytoma

BACKGROUND: Fractionated plasma metanephrine measurements are commonly used in biochemical testing in search of pheochromocytoma. METHODS: We aimed to critically appraise the diagnostic efficacy of fractionated plasma free metanephrine measurements in detecting pheochromocytoma. Nine electronic databases, meeting abstracts, and the Science Citation Index were searched and supplemented with previously unpublished data. Methodologic and reporting quality was independently assessed by two endocrinologists using a checklist developed by the Standards for Reporting of Diagnostic Studies Accuracy Group and data were independently abstracted. RESULTS: Limitations in methodologic quality were noted in all studies. In all subjects (including those with genetic predisposition): the sensitivities for detection of pheochromocytoma were 96%-100% (95% CI ranged from 82% to 100%), whereas the specificities were 85%-100% (95% CI ranged from 78% to 100%). Statistical heterogeneity was noted upon pooling positive likelihood ratios when those with predisposition to disease were included (p < 0.001). However, upon pooling the positive or negative likelihood ratios for patients with sporadic pheochromocytoma (n = 191) or those at risk for sporadic pheochromocytoma (n = 718), no statistical heterogeneity was noted (p = 0.4). For sporadic subjects, the pooled positive likelihood ratio was 5.77 (95% CI = 4.90, 6.81) and the pooled negative likelihood ratio was 0.02 (95% CI = 0.01, 0.07). CONCLUSION: Negative plasma fractionated free metanephrine measurements are effective in ruling out pheochromocytoma. However, a positive test result only moderately increases suspicion of disease, particularly when screening for sporadic pheochromocytoma.     

Neuronal assemblies: single cortical neurons are obedient members of a huge orchestra

Spontaneous cortical activity of single neurons is often either dismissed as noise, or is regarded as carrying no functional significance and hence is ignored. Our findings suggest that such concepts should be revised. We explored the coherent population activity of neuronal assemblies in primary sensory area in the absence of a sensory input. Recent advances in real-time optical imaging based on voltage-sensitive dyes (VSDI) have facilitated exploration of population activity and its intimate relationship to the activity of individual cortical neurons. It has been shown by in vivo intracellular recordings that the dye signal measures the sum of the membrane potential changes in all the neuronal elements in the imaged area, emphasizing subthreshold synaptic potentials and dendritic action potentials in neuronal arborizations originating from neurons in all cortical layers whose dendrites reach the superficial cortical layers. Thus, the VSDI has allowed us to image the rather illusive activity in neuronal dendrites that cannot be readily explored by single unit recordings. Surprisingly, we found that the amplitude of this type of ongoing subthreshold activity is of the same order of magnitude as evoked activity. We also found that this ongoing activity exhibited high synchronization over many millimeters of cortex. We then investigated the influence of ongoing activity on the evoked response, and showed that the two interact strongly. Furthermore, we found that cortical states that were previously associated only with evoked activity can actually be observed also in the absence of stimulation, for example, the cortical representation of a given orientation may appear without any visual input. This demonstration suggests that ongoing activity may also play a major role in other cortical function by providing a neuronal substrate for the dependence of sensory information processing on context, behavior, memory and other aspects of cognitive function.     

Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy

Twenty flavonoid compounds of five different subclasses were selected, and the relationship of their structure to the inhibition of low-density lipoprotein (LDL) oxidation in vitro was investigated. The most effective inhibitors, by either copper ion or 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) induction, were flavonols and/or flavonoids with two adjacent hydroxyl groups at ring B. In the presence of the later catechol group, the contribution of the double bond and the carbonyl group at ring C was negligible. Isoflavonoids were more effective inhibitors than other flavonoid subclasses with similar structure. Substituting ring B with hydroxyl group(s) at 2' position resulted in a significantly higher inhibitory effect than by substituting ring A or ring B at other positions. The type of LDL inducer had no effect in flavonoids with catechol structure. Calculated heat of formation data (deltadeltaH(f)) revealed that the donation of a hydrogen atom from position 3 was the most likely result, followed by that of a hydroxyl from ring B. Position 3 was favored only in the presence of conjugated double bonds between ring A to ring B. This study makes it possible to assign the contribution of different functional groups among the flavonoid subclasses to in vitro inhibition of LDL oxidation.     

Vitamin D sensitizes breast cancer cells to the action of H2O2: mitochondria as a convergence point in the death pathway

Calcitriol, the hormonal form of vitamin D3, sensitizes breast cancer cells to reactive oxygen species (ROS)-dependent cytotoxicity induced by various anticancer modalities. This effect could be due to increased generation of ROS and/ or to increased sensitivity of the target cells to ROS. This work examined the effect of calcitriol on the damage inflicted on breast cancer cells by the direct action of ROS represented by H2O2. Treatment of MCF-7 cells with H2O2 resulted in activation of caspase 7 as well as induction of caspase-independent cell death. Both were enhanced by 48-72 h of pretreatment with calcitriol. This effect was not due to modulation of H2O2 degradation or to a specific effect on *OH-mediated cytotoxicity. The H2O2-induced drop in mitochondrial membrane potential and release of cytochrome c were enhanced by calcitriol. These findings indicate that calcitriol sensitizes breast cancer cells to ROS-induced death by affecting event(s) common to both caspase-dependent and -independent modes of cell death upstream to mitochondrial damage.