The phylogenic expression of plasmolipin in the vertebrate nervous system

Plasmolipin is a plasma membrane proteolipid is a major myelin membrane component (Cochary et al., 1990). In this study we report the phylogenic expression of plasmolipin in the vertebrate nervous system. Using Western blot analysis with polyclonal antibodies, we have analyzed membrane fractions, including myelin, from elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. On the basis of immune detection, plasmolipin appears to be restricted to the mammalian nervous system. Comparison of the central and peripheral nervous systems of mammals showed only minor differences in the level of plasmolipin in these two regions. Within mammals, little quantitative differences were observed when rat, human and bovine membrane fractions were compared. The late evolutionary expression of plasmolipin which results in its restriction to mammals makes it unique among the (major) myelin proteins. The potential physiologic significance of these data are discussed.Abbreviations EDTA Ethylene diamine N.,NN tetracetic acid - EGTA Ethylene glycol bis-(B-Aminoethyl Ether) N,,NN tetracetic acid - MES ([N-Morpholino] ethane sulfonic acid) DCCD, N, Dicyclohexyl carbodiimide     

Optimum growth conditions and light utilization efficiency of Spirulina platensis (= Arthrospira fusiformis) (Cyanophyta) from Lake Chitu, Ethiopia

Spirulina platensis (= Arthrospira fusiformis) was isolated from Lake Chitu, a saline, alkaline lake in Ethiopia, where it forms an almost unialgal population. Optimum growth conditions were studied in a turbidostat. Cultures grown in modified Zarrouk's medium and exposed to a range of light intensities (20–500 µmol photons m^–2s^–1) showed a maximum specific growth rate (µ_max) of 1.78 d^–1. Quantum yield for growth (µ) was 3.8% at the optimum light for growth of 330 µmol photons m^–2s^–1, and ranged from 2.8 to 9.4%. With increase in irradiance, the chlorophyll a concentration decreased, and the carotenoids/chlorophyll a ratio increased by a factor of 2.4. The phosphorus to carbon ratio (P/C) showed some variation, while the nitrogen to carbon ratio (N/C) remained relatively constant, thus causing fluctuations in the N:P ratio (7–11) of cells. An optimum N:P ratio of about 7 was attained in cells growing at the optimum light for growth. Results from the continuous culture experiments agreed well with maximum values of photosynthetic efficiency given in the literature for natural populations of S. platensis in the soda lakes of East Africa, Lake Arenguade (Ethiopia), and Lake Simbi (Kenya).     

Application of high-performance liquid chromatography to competitive labeling studies: The chemical properties of functional groups of glucaton

A competitive-labeling study of glucagon was carried out using [³H]- and [¹⁴C]-1-fluoro-2,4-dinitrobenzene to determine simultaneously the chemical properties of the α-amino and imidazole groups of the N-terminal histidine residue, and the lysine and tyrosine residues, under conditions where glucagon is in its physiologically active monomer form. The dinitrophenyl derivatives of these groups were purified by high-performance liquid chromatography which greatly simplified the separation steps of the procedure. The results showed the α-amino and tyrosine groups to have relatively normal behavior, with pK values of 7.98 and 10.22, respectively, while the lysine had a low pK of 8.46. The imidazole function had an apparent pK of 7.84, substantially higher than previous estimates. This difference may be accounted for by the effect of the charged form of the adjacent α-amino group on the nucleophilicity of the imidazole group.     

Sexual differentiation in quail: Conversion of androgen to estrogen mediates testosterone-induced demasculinization of copulation but not other male characteristics

Previous research has shown that administration of either testosterone or estradiol to male quail embryos will demasculinize behavior and morphology. Six experiments in which embryos were treated were conducted to test the hypothesis that this testosterone-induced demasculinization is due to conversion of testosterone to estrogen (aromatization). In Experiment 1, dihydrotestosterone propionate, a nonaromatizable androgen, failed to demasculinize copulatory behavior, but did demasculinize crowing, strutting, and proctodeal glands. In Experiment 2, injection of the aromatizable androgens testosterone propionate (TP), testosterone, or androstenedione demasculinized copulatory behavior, the nonaromatizable androgen androsterone failed to have such an effect, and all androgens demasculinized proctodeal glands. In Experiment 3, Silastic implants of testosterone demasculinized all male characteristics, whereas implants of androsterone demasculinized only proctodeal glands. In Experiment 4, the antiestrogen tamoxifen prevented TP from demasculinizing copulatory behavior, but had no such effect with respect to crowing and strutting. In Experiments 5 and 6, the aromatization inhibitor 1,4,6-androstatrien-3,17-dione (ATD) prevented TP but not estradiol benzoate from demasculinizing copulatory behavior. Thus (1) in quail, testosterone-induced demasculinization of copulatory behavior is due to androgen aromatization, whereas testosterone-induced demasculinization of crowing, strutting, and proctodeal glands is not; (2) the distinct components of normal male reproductive behavior exhibit different patterns of steroid specificity during the organizational period, as was previously shown for the activational period; (3) the steroid specificity of crowing, strutting, and proctodeal glands changes between the organizational and activational periods. During organization, there is little specificity, whereas during activation, these characteristics respond only to androgens, never to estrogens. This difference suggests that developmental changes have occurred in the underlying biochemical substrates.     

A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana

Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues.     

Characterization of cpsF and its product CMP-N-acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli

Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N -acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP-Neu5Ac-synthetase activity (Wessels et al ., 1992). In this paper, we report the identification and characterization of cpsF , a gene interrupted by transposon insertion in the previously described Neu5Ac-deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP-Neu5Ac synthetase encoded by the Escherichia coli K1 gene, neuA . The enzymatic function of the protein encoded by cpsF was established by cloning the gene in E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP-Neu5Ac. In addition, when a CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed with cpsF , K1 antigen expression was restored. We conclude that cpsF encodes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule-synthesis of a heterologous bacterial species.     

Determination of a HIV protease inhibitor (DMP 450) in animal and human plasma by solid-phase extraction and high-performance liquid chromatography

Extraction of DMP 450 from plasma was performed with C₂ solid-phase extraction columns, using 0.1 M ammonium acetate in 90% methanol to elute DMP 450. The extraction recovery over the range of 10 to 10 000 ng/ml averaged 81.0, 96.2, 77.4, 95.2 and 68.0% from rat, dog, monkey, chimpanzee (25–10 000 ng/ml) and human plasma, respectively. HPLC analysis was carried out with a C₁₈ column and a mobile phase of acetonitrile, methanol and 30 mM potassium phosphate (pH 3), the composition dependent on the type of plasma being analyzed, and monitored at a wavelength of 229 nm. Intra-day and inter-day coefficients of variation were less than 9.9 and 12.9%, respectively. Absolute differences were less than 11.5%.     

Measurement of the novel antitumor agent 17-(allylamino)-17-demethoxygeldanamycin in human plasma by high-performance liquid chromatography

A sensitive HPLC assay has been developed to determine the concentration of 17-(allylamino)-17-demethoxygeldanamycin (AAG) in human plasma over the concentration range of 12.5 to 2500 nM (7.33 to 1465 ng/mL). After the addition of 1000 nM geldanamycin as the internal standard, 1 mL samples of human plasma were subjected to solid-phase extraction, via Bond-Elut C₁₈ cartridges, followed by analysis using an isocratic reversed-phase HPLC assay with UV detection. A Phenomenex Kingsorb, 3 micron, C18, 150×4.60 mm column and a Phenomenex Security Guard pre-column, C₁₈ (ODS, Octadecyl), were used to achieve separation. AAG and GM were monitored at 334 and 308 nm, respectively, on a Hewlett-Packard 1050 Diode-Array Detector. The mobile phase, run at a flow-rate of 1 mL/min, was composed of 50% (v/v) 25 mM sodium phosphate (pH 3.00) with 10 mM triethylamine and 50% acetonitrile. HPLC effectively resolved AAG with retention times of 14.60 0.54 min and the internal standard geldanamycin at 10.72±0.38 min (n=15). This assay was able to measure plasma concentrations of AAG, the lower limit of quantitation being 12.5 nM, at a starting dose of 10 mg/m² infused intravenously over 1 h in a Phase I clinical trial in adult patients with solid tumors.