Location and dynamics of alpha-tocopherol in model phospholipid membranes with different charges.

Studies were made on the position and dynamics of the OH-group of alpha-tocopherol in phospholipid membranes. There was no difference in the spin-lattice (T1) relaxation times at the 5a-position of alpha-tocopherol labeled with 13C- or C19F3-determined from the nuclear magnetic resonance (NMR) spectra of liposomes positively charged with stearylamine (SA) and negatively charged with dicetylphosphate (DCP). The zeta-potentials of egg yolk phosphatidylcholine (EYPC) liposomes with and without SA or DCP were not affected by incorporation of 20 mol% alpha-tocopherol, though incorporation of 10 mol% ascorbyl-palmitate decreased the zeta-potentials of EYPC and EYPC-SA liposomes. The P==O stretching band (1235 cm-1) of the phosphate group and C==O stretching band (1734 cm-1) of the acyl ester linkage in dimyristoylphosphatidylcholine (DMPC) liposomes, measured by Fourier transform-infrared (FT-IR) spectroscopy, were not changed by incorporation of alpha-tocopherol. These results suggest that no specific interaction occurred between the OH-group of alpha-tocopherol and the polar interfacial region of the bilayer. The dynamic quenching effects of n-(N-oxy-4,4'-dimethyloxazolidine-2-yl)stearic acids (n-NSs) on the intrinsic fluorescence of alpha-tocopherol were in the order 5-NS > 7-NS = 12-NS > 16-NS. Acrylamide, a water-soluble fluorescence quencher with a very low capacity to penetrate through phospholipid bilayers, had very low quenching efficiency. These results indicate that the bulk of the chromanol moiety of alpha-tocopherol is located in a position close to that occupied by the nitroxide group of 5-NS in the membranes and is poorly exposed at the membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and sequence analyses of cDNAs encoding vasotocin and isotocin precursors of chum salmon,Oncorhynchus keta: evolutionary relationships of neurohypophysial hormone precursors

Summary The nucleotide sequences of cloned cDNAs were used to determine the primary structures of the precursors of vasotocin (sVT) and isotocin (sIT) from the hypothalamus of the chum salmon,Oncorhynchus keta. Two different cDNAs were obtained for each of sVT and sIT precursors (sVT-I and sVT-II; sIT-I and sIT-II). Both sVT and sIT precursors were found to contain a signal peptide and hormone that is connected to a neurophysin by a Gly-Lys-Arg sequence. Northern and Southern blot analyses showed that the sVT and sIT genes are expressed by the same chum salmon hypothalamus, but not by the liver and kidney. Microheterogeneity was found in the nucleotide and amino acid sequences of sVT precursors between our results and the previously reported data (Heierhorst et al. 1990). The conspicuous difference is the occurrence of a stop codon in the middle of sVT-II cDNA. The carboxyl termini of both sVT and sIT neurophysins are about 30 amino acids longer than neurophysins of toad and mammalian neurohypophysial hormone precursors. Although these extended regions do not contain a glycosylation site, they show striking similarity with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. The central portion of the neurophysins shows highest homology among corresponding regions of sVT and sIT precursors. Moreover, calculation of nucleotide substitution rates suggests that a recent gene conversion may have occurred which encompasses the exon that encodes the central segment of the sVT and sIT precursors. A possible pathway for the evolution of precursor molecules of neurohypophysial hormones is discussed.Abbreviations AVP vasopressin - C carboxyl - h human - IT isotocin - MT mesotocin - N amino - OXT oxytocin - S chum salmon - SDS sodium dodecyl sulfate - t toad - VT vasotocin     

Membrane-stabilizing effect of vitamin E: effect of alpha-tocopherol and its model compounds on fluidity of lecithin liposomes

The effects of vitamin E (alpha-tocopherol) and its model compounds on the fluidity of liposomes composed of dipalmitoylphosphatidylcholin (DPPC) and fatty acids were investigated by the measurement of the fluorescent polarization (P) using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a plobe. Although all tocopherols decreased the fluidity of liposomes which was perturbed by the inclusion of an unsaturated fatty acid having more than one double bond, alpha-tocopherol was more effective than the others. The fluidity in arachidonic acid-containing liposomes was decreased most in the presence of alpha-tocopherol and was decreased considerably by the inclusion of model compounds having a side chain at least one isoprene unit or a long straight chain instead of isoprenoid side chain. However, the chromanol with methyl group instead of the above side chain, and phytol, having no chromanol moiety, had no effect. These results show that a structural requirement for a membrane stabilization is to be either the chromanol moiety with methyl groups born on its aromatic ring or a side chain of appropriate length; an isoprenoid side chain of full length or one containing 4'a- and 8'a-methyl groups is not necessarily needed.     

The human alpha-fetoprotein gene. Sequence organization and the 5' flanking region

The human alpha-fetoprotein (AFP) gene was isolated into three overlapping clones in bacteriophage lambda vectors and its sequence organization analyzed by restriction endonuclease mapping and nucleotide sequencing. The human AFP gene is about 20 kilobase pairs long and contains 15 exons and 14 introns. The overall organization of the human AFP gene is similar to that of the mouse AFP gene, with all but two exons showing identical sizes. Nucleotide sequences at all exon/intron junctions display similarity to the consensus boundary sequence (Breathnach, R., and Chambon, P. (1981) Annu. Rev. Biochem. 50, 349-383), with the GT-AG rule applied to the splicing point. The cap site maps 44 nucleotides upstream from the translation initiation site. The "TATA box" is located 27 nucleotides upstream from the putative cap site and is flanked by sequences with dyad symmetry. The TATA box can thus be placed in the loop portion of a possible stem-loop structure formed by intrastrand base-pairing. Other characteristic nucleotide sequences in the 5' flanking region include a CCAAC pentamer, a 14-base pair (bp) enhancer-like sequence, and a 9-bp sequence homologous to the glucocorticoid responsive element. A long (90 bp) direct repeat and several alternating purine/pyrimidine sequences are also present in the 5' flanking region. A 736-bp sequence of the 5' flanking region adjacent to the cap site of the human AFP gene shows a 61% similarity with the corresponding region of the mouse AFP gene. There are two Alu family sequences and two poly(dT-dG) repeats in the human AFP gene that show different distribution patterns from those in the mouse AFP gene.     

Androgen Regulates Gene Expression of Cytoskeletal Proteins in Adult Rat Motoneurons

Expression of β-actin and β-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding chick β-actin and mouse β-tubulin, respectively. Both hybridizable β-actin and βtubulin mRNAs were localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both β-actin and β-tubulin mRNAs in the SNB motoneurons, whereas the changes were prevented by testosterone treatment. In contrast, castration or testosterone treatment induced little or no change in the expression levels of these mRNAs in the much less androgen-sensitive motoneurons of the retrodorsolateral nucleus (RDLN). These results suggest that androgen regulates the expression of β-actin and β-tubulin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.     

Changes in growth hormone and prolactin messenger ribonucleic acid levels during seawater adaptation of amago salmon (Oncorhynchus rhodurus).

To examine the changes in secretion of growth hormone (GH) and prolactin (PRL) with reference to their osmoregulatory roles, changes in pituitary mRNA levels and plasma concentrations of these hormones were examined during seawater adaptation in silvery juveniles (smolts) and precociously mature males (dark parr) of amago salmon (Oncorhynchus rhodurus). Transfer to seawater increased plasma sodium levels in both smolts and dark parr. Smolts adjusted their plasma sodium to the level associated with seawater-adaptation (165 mEq/liter) within 3 days, whereas no adjustment was seen in dark parr; the latter failed to survive in seawater for more than 3 days. In smolts, plasma GH levels increased significantly 1 day after transfer, whereas there was no significant change in dark parr. An increase in GH mRNA levels was observed in smolts in association with increased plasma GH, whereas there was no change in dark parr. In contrast, a reduction in plasma PRL levels was consistently observed in both smolts and dark parr after transfer to seawater. However, there was no significant change in PRL mRNA levels in either smolts or dark parr. These results suggest that both gene expression and release of GH are activated by seawater transfer only in smolts with adequate seawater adaptability, whereas PRL gene expression is decreased after seawater transfer regardless of seawater adaptability.     

Characteristics of the core protein of the aggregating proteoglycan from the Swarm rat chondrosarcoma

A ternary complex of hyaluronic acid-binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid-binding region has a molecular weight of ? 65,000 (HA-BR₆₅). N-terminal amino acids in the complex were selectively ^l4C-carbamylated. The resulting derivatized HA-BR₆₅ was isolated, and tryptic peptide maps were prepared and developed on two-dimensional TLC sheets. A single, labeled peptide was obtained which gave a M_r by ? 8,000 by SDS-PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA-BR₆₅ to a polypeptide of ? 55,000 (HA-BR₅₅) which still retains the same N-terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid-binding region. Direct SDS-PAGE analysis revealed one major intermediate with M_r ? 109,000 (HA-BR₁₀₉) as well as HA-BR₆₅. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS-PAGE gel at M_r ? 120,000 (HA-BR₁₂₀) and ? 140,000 (HA-BR₁₄₀). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid-binding region, and all of them contained the same N-terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid-binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus.     

Prevalence of feline viral antibodies in random-source laboratory cats

Over a period of 1973 to 1979, a serologic survey of virus infections was conducted on feline sera collected in four universities which located in different prefectures; Obihiro, Saitama, Kanagawa and Tokyo. A significant hemagglutination-inhibition (HI) antibody titer of 1 : 8 or higher to feline panleukopenia virus (FPLV) was detected in 130 (58%) of the 226 sera used. No remarkable difference in the HI antibody prevalence in cats to FPLV was recognized by years or localities. Of a total of 188 cats tested, 99 (53%) presented positive serum neutralizing (SN) antibody titers to the No. 1 strain of feline calicivirus (FCV). Especially in Kanagawa, 17 (77%) of the 22 cats had positive SN titers. However, only 42 (22%) of the 188 sera showed positive SN titers to the Kyoritsu strain of FCV. Such lower positivity in the cats was observed with 13% in the SN test to human reovirus type 3 (Reo-3). The incidence of positive SN antibodies to feline rhinotracheitis virus (FRV) also remained in low values of 20 to 27% with the exception of high percentage of 86 in Tokyo. The dissemination of FPLV, FRV, FCV and Reo-3 was briefly discussed in relation with the age distribution of viral antibodies in cats.