Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Ultrasonographic assessment of the endometrium in rhesus monkeys during the normal menstrual cycle

This study was undertaken to determine whether cyclical changes in the endometrium of the rhesus monkey could be observed by using ultrasound. Three indices of endometrial size were examined: the antero-posterior (or ventro-dorsal), longitudinal, and transverse diameters. Changes in the ultrasonic reflectivity of the endometrium were also assessed. We have attempted to correlate these endometrial parameters with the hormonal status of the animal. Ultrasonography was performed for an average of 12 consecutive days during 19 menstrual cycles. All ultrasonic recordings were normalized to the day of the estradiol (E2) peak (Day 0). We found that the reflectivity of the endometrium was dependent on the stage of the cycle: during the follicular phase, the endometrium appeared less echogenic (darker) compared to the myometrium; in the luteal phase, the endometrium was more echogenic (lighter). During the follicular phase (Days -9 to 0), there was a linear increase in the antero-posterior (p less than 0.001), longitudinal (p less than 0.05), and transverse (p less than 0.001) diameters. In the luteal phase (Days 1-15), no significant changes were observed in these diameters. An estimated endometrial volume (EEV) was obtained by the product of the antero-posterior, longitudinal, and transverse diameters. Each animal observed during the follicular phase (n = 14) exhibited a peak in the EEV, which correlated with the day of the E2 peak (p less than 0.01). From this study, we conclude that the sonographic appearance of the endometrium of the rhesus monkey reflects the cyclical changes that occur during the menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Particle-bound glutathione-S-transferases.

The incubation of liver microsomes or mitochondria with glutathione, in the presence of electrophilic compounds, decreased the glutathione concentration in the incubation medium. Product analysis revealed that glutathione conjugates were formed.     

Water-soluble polysaccharides from Ginkgo biloba leaves.

The water-soluble polysaccharides from dried Ginkgo biloba leaves were isolated after exhaustive extraction with organic solvents. The polysaccharide mixture could be separated into a neutral (GF1) and two acidic (GF2 and GF3) polysaccharide fractions by ion exchange chromatography. According to the Mr distribution GF1 and GF3 seemed to be homogenous, whereas GF2 could be further fractionated into two subfractions (GF2a and GF2b) by gel permeation chromatography. GF1 (Mr 23,000) showed the structural features of a branched arabinan. The main chain was composed of 1,5-linked arabinose residues and three in 12 arabinose molecules were branched via C-2 or C-3. GF2a (Mr 500,000) consisted mainly of 1,2,4-branched mannose (29%), 1,4-linked glucuronic (32%) and galacturonic (8%) acid as well as terminal rhamnose (25%). After removal of ca 70% of the terminal rhamnose the remaining polysaccharide showed a decrease in 1,2,4-branched mannose and an increase in 1,2-linked mannose indicating that at least half of the rhamnose residues were linked to mannose via C-4. GF3 (Mr 40,000) consisted of 1,4-linked galacturonic (30%) and glucuronic (16) acid, 1,3,6-branched galactose (15%), 1,2-linked (5%) and 1,2,4-branched (3.5%) rhamnose as well as 1,5-linked arabinose (11%). Rhamnose (5%) and arabinose (10%) were present as terminal groups. Mild acid hydrolysis selectively cleaved arabinose and the remaining polysaccharide showed an increased amount of 1,6-linked and terminal galactose and a decreased quantity of 1,3,6-branched galactose. These results indicated that the terminal as well as the 1,5-linked arabinose were mainly connected to galactose via C-3. The GF3 polysaccharide appeared to be a rhamnogalacturonan with arabinogalactan side chains.     

Identification of hydrogen selenide and other volatile selenols by derivatization with 1-fluoro-2,4-dinitrobenzene

A procedure is described for the trapping and identification of hydrogen selenide and methyl selenol ( CH3SeH ). The volatile selenols were generated by reducing selenious acid or dimethyldiselenide with Zn dust and hydrochloric acid under a stream of nitrogen and passing into a trapping solution composed of 50 mM 1-fluoro-2,4-dinitrobenzene plus 83 mM sodium bicarbonate in 67% dimethylformamide:33% water. The selenols react rapidly to form stable dinitrophenyl (DNP) selenoethers that can be extracted into benzene; these are easily identified by TLC, HPLC, or mass spectrometry. Hydrogen selenide is trapped in 90-99% yield, primarily as the di-DNP- monoselenide with a trace of di-DNP- diselenide .     

Identification of selenocysteine in glutathione peroxidase by mass spectroscopy

A convenient procedure was developed for identifying selenocysteine in selenoproteins by mass spectroscopy, based on formation of the 2,4-dinitrophenyl (DNP) derivative. Pure ovine erythrocyte glutathione peroxidase was reduced with sodium borohydride and reacted with 1-fluoro-2,4-dinitrobenzene at neutral pH under anaerobic conditions in 4 M guanidine. The inactivated enzyme was hydrolyzed with 6 N HCl for 20 h at 110 degrees C under anaerobic conditions. Following extraction of the hydrolysate with benzene, Se-(2,4-dinitrophenyl)selenocysteine in the aqueous phase was separated from non-DNP-amino acids by gel-filtration chromatography and then separated from other water-soluble DNP-amino acids by reversed-phase high-performance liquid chromatography. The Se-(2,4-dinitrophenyl)selenocysteine was converted to Se-methyl-N-(2,4-dinitrophenyl)selenocysteine by the addition of sodium barbital to induce an intramolecular Se leads to N shift (Smiles rearrangement) under anaerobic conditions, in the presence of methyl iodide to trap the liberated selenol group. Following esterification of the product's carboxyl group with methanol and hydrochloric acid, it was subjected to direct probe mass spectroscopy and identified as the methyl ester of Se-methyl-N-(2,4-dinitrophenyl)selenocysteine. This procedure allows selenocysteine to be isolated quite easily as a readily identifiable derivative and has permitted the first identification of a seleno amino acid in a protein by mass spectroscopy.     

Sequence and type-specific immunogenicity of the amino-terminal region of type 1 streptococcal M protein

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.     

Is male back space limiting? An investigation into the reproductive demography of the giant water bug,Abedus indentatus (Heteroptera: Belostomatidae)

Because male giant water bugs in the subfamily Belostomatinae provide parental care by brooding eggs on their back, an accurate assessment can be made of both the actual and potential reproductive capacity of males. Two operational sex-ratio (OSR) indices were developed and empirically measured for a population of giant water bug, Abedus indentatus,in California. One index was based on reproductive rates measured in the laboratory; the other index was based on reproductive resources observed in the field. Both OSR indices suggest that the operational sex ratio fluctuates between maleskewed ratios in the summer and femaleskewed ratios in the winter. This pattern appears to be the consequence of two factors. First, the adult sex ratio is significantly female biased. Second, although males can outreproduce females at high ambient temperatures, the reverse is true at low temperatures. Possible reasons for the female-skewed adult sex ratio are examined, including differential recruitment, differential mortality, and sampling bias.