A predicted three-dimensional structure for the human immunodeficiency virus binding domains of CD4 antigen

A predicted three-dimensional structure of the two N-terminal extracellular domains of human CD4 antigen, a cell surface glycoprotein, is reported. This region of CD4, particularly the first domain, has been identified as containing the binding region for the envelope gp120 protein of the human immunodeficiency virus. The model was predicted based on the sequence homology of each domain with the variable light chain of immunoglobulins. The framework beta-sheet regions were taken from the crystal coordinates of REI. For one region in the first domain of CD4 there was an ambiguity in the alignment with REI and two alternate models are presented. Loops connecting the framework were modelled from fragments selected from a database of main chain coordinates from all known protein structures. Residues identified as involved in binding gp120 have been located in several other studies within the first domain of CD4. Epitopes from eight monoclonal antibodies have been mapped onto residues in both domains. Competition of these antibodies with each other and with gp120 can be interpreted from the structural model.     

The second stiffest axis of a beam-column: implications for cervical spine trauma

Based on an idealized model of a homogeneous, isotropic beam-column, the second stiffest axis under static loading was derived. The maximum allowable force for the second stiffest axis was then examined. The ratio of the maximum allowable forces of the second stiffest axis to the stiffest axis was established. The stiffness ratio of the second stiffest axis to the stiffest axis was also found. Taking buckling into consideration, the safe load region for all possible acting directions was derived. The implications of the idealized model for cervical spine trauma are discussed.     

Microcalorimetric measurements of tissue cells in vitro

A culture flask was designed for the microcalorimetric measurements of tissue cells by an MS 80 standard calvet microcalorimeter. Tissue cells cultured in this flask behaved in the same manner as in the common culture flask used in cytobiological studies. The thermograms of human adenocarcinoma gastric cells (SGc 7901) and HeLa cells were obtained. The heat output power of SGc 7901 cells continuously increased for 70 h with an initial cell number of 3.0 X 10(5). The thermogram was reproducible under strictly controlled conditions. The relationship between the heat output power and the number of SGc 7901 cells within 48 h was obtained. The heat output power was 40 pW/cell to 49 pW/cell when the cell number was in the range 4.5 X 10(5) to 10.4 X 10(5). It was 62.3 +/- 2.9 pW/cell for HeLa cells when the cell number was 6 X 10(5).     

Plant regeneration from callus of Puccinellia distans (L.) Parl

Callus was induced from seeds of Puccinellia distans (L.) Parl. on MS medium supplemented with 2 mgl^-1 2,4-dichlorophenoxyacetic acid and 0.5 mgl^-1 kinetin. Morphogenesis initiation was achieved during subculture on medium containing 0.1 mgl^-1 2,4-D. From the point of morphogenetic capacity, 3 types of callus were selected. High frequency of plant regeneration was obtained by selection of embryogenic type of callus, and culture on N₆ medium and N₆ medium supplemented with kinetin (5–10 mgl^-1), or kinetin (2 mgl^-1) and IAA (0.5 mgl^-1). A high ratio of albinos among regenerants was observed.     

Calcitonin gene-related peptide (CGRP) in normotensive and spontaneously hypertensive rats

With the techniques of specific radioimmunoassay and gel filtration it was found that CGRP was distributed in various tissues of normotensive (WKY) and spontaneously hypertensive rats (SHR) with the highest concentration in the lumbar spinal cord (1197 +/- 94.8 pg/mg tissue) and the lowest in the auricle (15.0 +/- 2.1 pg/mg tissue). In comparison with WKY, CGRP concentration in the plasma was decreased and in the abdominal aorta and hypothalamus was increased in SHR. Gel filtration revealed only one major CGRP molecular form in the tissues. In addition, CGRP reduced the mean arterial pressure (MAP) in SHR in a dose-dependent manner. These data suggest that CGRP may play an important role in the pathogenesis of hypertension and its possible therapy.     

Measurement of extracellular 5-HT and 5-HIAA in hippocampus of freely moving rats using microdialysis: long-term applications

Extracellular levels of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in the ventral hippocampus of the awake rat using microdialysis. The basal level of 5-HT in hippocampal dialysates was very close to the detection limit of our assay. However, addition of a 5-HT re-uptake blocker, citalopram, to the perfusion medium resulted in a 3-fold rise of 5-HT levels in dialysates and provided a stable baseline. Under these conditions, extracellular levels of 5-HT and 5-HIAA could be measured up to 11 days after dialysis probe implantation. 5-HT measured in dialysate was Ca²⁺-dependent both 24 h and 7 days after surgery. In comparison K⁺-induced depolarization caused a 9-fold increase in 5-HT output 24 h after probe implantation and this effect had disappeared 10 days later, although behavioral activation following K⁺ application was similar 24 h and 11 days after surgery. Systematic administration of -trytophan had no significant effect on 5-HT but increased 5-HIAA levels by 90%. These results suggest that in the presence of citalopram, 5-HT in hippocampal dialysates is derived from serotonergic neurones. The microdialysis method appears most useful for measurement of extracellular 5-HT 24–72 h after probe implantation. Finally, our data indicate that extracellular 5-HIAA mainly reflects intraneuronal metabolism of 5-HT prior to release.     

A method of isolation of apical membranous sheets from frog urinary bladder epithelium by stripping with gelatin

We have developed a technique for recovering apical membranous sheets from amphibian urinary bladders by gelatin stripping. The tissue is mounted on a lucite support and the apical surface is first stuck onto a gelatin-coated glass slide at 30 degrees C. This sandwich is then chilled on ice and the bladder is pulled away from the slide. Preliminary results indicate that this simple technique could be used to remove membranous apical sheets of various sizes, almost devoid of cytoplasmic contamination and without significant damage to the underlying cell structures. The method could also be adapted to prepare perforated cells and to study the cohesive forces between the different layers of the tissue.     

Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridine antagonists and activators

The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct detection of beta-1,3-glucanase isozymes on polyacrylamide electrophoresis and isoelectrofocusing gels

A procedure to assay isozymes of beta-1,3-glucanase directly on polyacrylamide gel electrophoresis (PAGE) and isoelectrofocusing (IEF) gels by using 2,3,5-triphenyltetrazolium chloride is described. The reagent reacts with reducing sugars released by beta-1,3-glucanases from the substrate laminarin. Acidic and neutral isozymes of beta-1,3-glucanase were detected and quantified on 17.5% native PAGE gels run with an anodic buffer system. A significant linear relationship (alpha = less than 0.01, R = 0.991) was observed between amounts of beta-1,3-glucanase loaded and intensity of bands stained with the reagent on native PAGE gels. A full isozyme pattern was obtained on 7.5% IEF gels with a pH range of 3.5-9.5. The IEF gels were heated in a microwave oven during the staining process to minimize diffusion.     

Low nanogram detection of nucleotides using fast atom bombardment-mass spectrometry

The effect of trimethylsilyl (TMS) derivatization on detection limits of mononucleotides in fast atom bombardment-mass spectrometry (FAB-MS) was examined. FAB-MS methods were developed to optimize sensitivity using adenosine 5'-monophosphate as a model compound and then applied to reference standards of two clinically important nucleotides: tricyclic nucleoside-5'-monophosphate (TCNMP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). The detection limit for the TMS derivative of TCNMP was 2.5-5 ng/microliters and less than 2.5 ng/microliters for FdUMP as its TMS derivative. This is greater than two orders of magnitude more sensitive than the FAB-MS analysis of the corresponding free compounds. These low detection limits for the TMS derivatives were obtained using a narrow scan range, signal averaging, detection in the negative ion mode, and 3-nitrobenzyl alcohol as the matrix. Hydrolysis of one or more of the labile TMS groups did occur, with the extent of hydrolysis being greatest in the more protic matrices.