Endothelium specific Weibel-Palade bodies in a continuous human cell line,EA.hy926

Summary Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

Thiol-induced oligomerization of α-lactalbumin at high pressure

Denaturation and aggregation of-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding of-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation of-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield of-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers of-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecular-sheets.     

Induction of leaf senescence in Arabidopsis thaliana by long days through a light-dosage effect

Given the influence of photoperiod on reproductive development and whole-plant senescence in monocarpic plants, one would suspect that leaf senescence in these plants might be under photoperiodic control. In Arabidopsis thaliana , which is monocarpic and also a nonobligate long-day (LD) plant, LDs (16 h, 300 μmol m⁻² s⁻¹) caused leaves to die earlier than did short days (SDs, 10 h). Since leaf longevity was not paralleled by the reproductive development in the present study, the reproductive structures did not seem to be the primary controls of leaf senescence. The LD effect appeared to depend on the amount of light rather than on day length, for leaves given LDs at reduced light intensity (180 μmol m⁻² s⁻¹) lived longer than those in LDs with full light. In addition, the higher light intensity promoted chlorophyll loss and anthocyanin accumulation in LDs. Thus, senescence of these leaves seems to be governed by light dosage rather than photoperiod. Light may play a natural role in promoting the senescence of A. thaliana leaves.     

Biology of glomerular cells in culture

The glomerulus is a complex structure including four cell types, namely mesangial, visceral epithelial, parietal epithelial and endothelial cells. Mesangial cells resemble smooth muscle cells and play a major role in the synthesis of the components of the glomerular basement membrane and in the vasoreactivity of the glomerular tuft. In particular, they express receptors for angiotensin II which mediate mesangial cell contraction, this effect resulting in the decrease of the filtration area. They are also the site of synthesis of a variety of inflammatory agents which are involved in the development of glomerular injury in glomerulonephritis. Visceral epithelial cells, also referred to a podocytes, also participate in the synthesis of the normal constituents of the glomerular basement membrane. They express receptors for atrial natriuretic factor and possess on their surface a number of ectoenzymes. They also, in concert with mesangial cells, release metalloproteases which contribute to the degradation of the extracellular matrix. Parietal epithelial cells have been little studied. They represent the main constituent of the crescents observed in extracapillary proliferative glomerulonephritis. Endothelial cells secrete vasodilatory agents such as nitric oxide and prostacyclin and vasoconstrictor agents such as endothelin which act on the adjacent mesangial cells. New methods of culture of glomerular cells are in progress. Their aim is to keep as long as possible the physiological phenotype of these cells. Another progress is the availability of stable transformed cell lines which represent an abundant source of material for biochemical studies.     

Vascular endothelial cells: Targets for studying the activity of hair follicle cell-produced VEGF

Fetal bovine aortic endothelial cells (FBAEC) were exposed to purified fractions of conditioned medium from cultures of hair dermal papilla cells (DPC) to determine the existence of any vascular endothelial growth factor (VEGF)-like paracrine activity of the latter. Such fractions were tested for stimulation of growth and migration of cultured FBAEC. In addition, VEGF secretion by DPC was measured by radioassay of VEGF receptors using FBAEC as target cells. The results showed that stimulation of FBAEC proliferation and migration following exposure to purified conditioned medium was dose-dependent. Radioreceptor assays of recombinant VEGF and purified DPC-conditioned medium showed competitive VEGF binding in FBAEC.Abbreviations CM conditioned medium - DMEM Dulbecco's modified eagle's medium - DPC dermal papilla cells - EDTA ethylenediaminetetra-acetic acid - FBAEC fetal bovine aortic endothelial cells - FCS fetal calf serum - VEGF vascular endothelial growth factor     

Production of fuel alcohol from oats by fermentation

Very high gravity (>30 g dissolved solids per 100 ml) mashes were prepared from hulled and hulless oats and fermented at 20° C with active dry yeast to produce ethanol. Excessive viscosity development during mashing was prevented by hydrolyzing -glucan with crude preparations of -glucanase or Biocellulase. Both these preparations possessed endo--glucanase activity. By using these enzymes and by decreasing the water to grain ratio, very high gravity mashes with low viscosity were prepared. Unlike wheat and barley mashes, oat mashes contained sufficient amounts of assimilable nitrogen to promote a fast rate of fermentation. The free amino nitrogen (FAN) content of oat mash could be predicted by the equation, mg FAN L^–1=8.9n wheren is the number of grams of dissolved solids in 100 ml of mash supernatant fluid. Ethanol yields of 353.2±3.7 L and 317.6±1.3 L were obtained per tonne (dry weight basis) of hulless (59.8% starch) and hulled (50.8% starch) oats respectively. The efficiency of conversion of starch to ethanol was the same in normal and very high gravity mashes.     

锰对作业工人血液流变学的影响及清栓酶的疗效观察

本文对１０３例锰作业工人测定了血液流变学中的七项指标,结果表明：全血粘度（高切、低切）、血浆粘度、红细胞压积、纤维蛋白原、红细胞电泳、血小板粘附、血沉等项指标,均明显高于正常对照组（Ｐ＜０．０１）,证实锰作业工人普遍存在高粘滞血症,为临床治疗锰中毒开辟新的途径提供了依据。对其中的５２例锰中毒（包括症状较重的观察对象）,用清栓酶治疗后,复查其七项指标与治疗前比较,均有明显下降（Ｐ＜０．０１）,其症状与体征得以改善,进一步说明清栓酶是治疗锰中毒的理想药物之一。     

Characteristics of microwave evoked body movements in mice

Microwave evoked body movements were studied in mice. A resonant cavity was used to provide head and neck exposure of the mouse to pulsed and gated continuous wave (CW) 1.25 GHz microwaves. No difference in response to pulsed and gated CW stimuli of equal average power was found. The incidence of the microwave evoked body movements increased proportionally with specific absorption (dose) when the whole-body average specific absorption rate was at a constant level (7300 W/kg). Under a constant average specific absorption rate, the response incidence reached a plateau at 0.9 kJ/kg. For doses higher than 0.9 kJ/kg, response incidence was proportional to the specific absorption rate and reached a plateau at 900 W/kg. Body movements could be evoked by a single microwave pulse. The lowest whole-body specific absorption (SA) tested was 0.18 kJ/kg, and the corresponding brain SA was 0.29 kJ/kg. Bulk heating potentials of these SAs were less than 0.1 °C. For doses higher than 0.9 kJ/kg, the response incidence was also proportional to subcutaneous temperature increment and subcutaneous heating rate. The extrapolated absolute thresholds (0% incidence) were 1.21 °C temperature increment and 0.24 °C/s heating rate. Due to high subcutaneous heating rates, these microwaves must be perceived by the mouse as an intense thermal sensation but not a pain sensation because the temperature increment was well below the threshold for thermal pain. Results of the present study should be considered in promulgation of personnel protection guideline against high peak power but low average power microwaves. © 1994 Wiley-Liss, Inc.