Evolution of several cytoskeletal proteins of astrocytes in primary culture: Effect of prenatal alcohol exposure

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein -tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of -tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti - and anti--tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin.In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed in vivo.Special issue dedicated to Dr. Santiago Grisolia     

1H-NMR heme resonance assignments by selective deuteration in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl alpha-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole pi bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.     

Pregnancy diagnosis by the ultrasonographical device and observation of fetal growth in the squirrel monkey (Saimiri sciureus)

This paper describes usefulness of the ultrasonographical device (USD) for the diagnosis of pregnancy and the observation of fetal growth in the squirrel monkeys (Saimiri sciureus) conceived under group breeding conditions. Pregnancy was diagnosed on the basis of the detection of gestational sac (GS) in the uterus. The GS was first detected 127 +/- 10 days before delivery. The heart beat of embryo was detected around 114 days before delivery. It was able to judge conditions of fetal growth by measuring the size of GS and the biparietal diameter. No difference in uterine size between nonpregnant and pregnant animals was observed 135 days before delivery.     

Internal structure of the postcapillary high-endothelial venules of rodent lymph nodes and Peyer's patches and the transendothelial lymphocyte passage

Scanning electron microscopy (SEM) shows that the postcapillary high-endothelial venules of lymph nodes and Peyer's patches consist of two segments each with a different surface relief: a proximal segment with a cobblestone surface pattern and a distal segment of interlacing cytoplasmic plates. Both segments have deep adluminal crevices in which lymphocytes are lodged. The internal structural configuration of this endothelium has been examined by transmission electron microscopy (TEM) of serial sections of lymph nodes and Peyer's patches of mice, rats, and guinea pigs. The serial sections revealed that the endothelial cell bodies and their cytoplasmic extensions were disposed in a direction generally lateral to the luminal surface and intruded into the intercellular spaces of similarly disposed neighboring endothelial cells, resulting in a complex interlacing cellular pattern. Lymphocytes penetrated the endothelial cell body and secondarily followed an intracellular pathway through which they entered the extravascular compartment. At the exposed surfaces of the adluminal venule wall, recirculating lymphocytes were seen in SEM images to enter the endothelium by penetrating the endothelial cell body. The mode of migration of lymphocytes lodged in the endothelial crevices could be determined by SEM and has been examined by TEM of serial sections. At these locations as at the exposed surfaces, lymphocytes also entered the venule by penetrating the endothelial cell body. At both sites this transcellular pathway was followed by lymphocyte entry into the intercellular spaces from which they migrated into the extravascular compartment.     

In vivo exocytosis of lysosomes by the endothelium of the venous sinuses of bone marrow and liver: visualization at normal and low body temperature

We have visualized the exocytosis of lysosomes into the peripheral circulation by the phagocytic endothelia of the venous sinuses of liver and bone marrow of rats. Perfusion fixation at normal body temperature produced images of the earliest stages of lysosomal exocytosis. After fixation at low body temperatures (7-12 degrees C), advanced stages of this process became evident, showing extrusion of lysosomes and their contents into the circulation. It is postulated that this form of exocytosis has escaped structural detection because of its rapidity and relative infrequency as compared to merocrine secretory exocytosis, and that fixation at low body temperatures arrests or slows down these exocytic events in sufficient measure for ultrastructural visualization. The possibility that this lysosomal exocytosis contributes to the presence of lysosomal enzymes detected in the peripheral blood should be considered. In addition, it is likely that lysosomal degradation products may be discharged by exocytosis into the circulation.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement and prediction of flow through a replica segment of a mildly atherosclerotic coronary artery of man

Pressure distributions were measured along a hollow vascular axisymmetric replica of a segment of the left circumflex coronary artery of man with mildly atherosclerotic diffuse disease. A large range of physiological Reynolds numbers from about 60 to 500, including hyperemic response, was spanned in the flow investigation using a fluid simulating blood kinematic viscosity. Predicted pressure distributions from the numerical solution of the Navier-Stokes equations were similar in trend and magnitude to the measurements. Large variations in the predicted velocity profiles occurred along the lumen. The influence of the smaller scale multiple flow obstacles along the wall (lesion variations) led to sharp spikes in the predicted wall shear stresses. Reynolds number similarity was discussed, and estimates of what time averaged in vivo pressure drop and shear stress might be were given for a vessel segment.     

Multifaceted freezing injury in human polymorphonuclear cells at high subfreezing temperatures

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.     

Effect of Ca2+ on the ouabain-insensitive, active Na+ uptake in inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells

The ouabain-insensitive, active Na+ uptake of inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can be increased by the presence of micromolar concentrations of Ca2+ in the assay medium. The concomitant ATP hydrolysis associated with the Na+ uptake is also increased by the presence of Ca2+. The Na+ uptake and the concomitant ATP hydrolysis are inhibited by 2 mM furosemide. The effect of Ca2+ is not due to the activity of an Na+-Ca2+ exchanger. The present results are in accordance with our previous model (Proverbio, F., Proverbio, T. and Marín, R. (1982) Biochim. Biophys. Acta 688, 757-763) in which we proposed that Ca2+ seems to modulate the activity of the ouabain-insensitive Na+ pump, in two different ways: (1) in a strong association with the membranes in which Ca2+ (stable component) is essential for the pump activity and (2) in a weak association with the membranes in which Ca2+ (labile component) can be quickly and easily removed by reducing the free Ca2+ concentration of the assay medium to values lower than 1 microM. The Ka for Ca2+ (for the labile component) is around 5 microM. The Ca2+ modulation of the ouabain-insensitive Na+ pump is an indication that Ca2+ could regulate the magnitude of the Na+ extrusion accompanied by Cl- and water present in rat kidney proximal tubular cells.