The Photoperiodic Entrainment and Induction of Reproductive Rhythms in Male Blackheaded Buntings (Emberiza Melanocephala)

Groups of photosensitive, unstimulated or stimulated, male blackheaded buntings were subjected to photoregimes of 15 hr of green light of three intensities and 9 hr of dark per day. In some groups green light was interrupted with 90 min of bright fluorescent light at different times in the subjective day. While gonads did not develop or regressed in some groups, birds in others behaved as if exposed to long daylengths. The results besides suggesting the involvement of endogenous circadian rhythm during initiation and maintenance of gonadal growth indicate that the reproductive rhythms are entrained and induced by environmental photoperiod.     

Microbial production of d-amino acids

The production of d-aminoacylase by Alcaligenes denitrificans and Alcaligenes faecalis has been studied. The enzyme was inducibly produced and N-acetyl-d-leucine and N-acetyl-d-valine were the most effective inducers. d-methionine, d-valine, d-phenylalamine and d-leucine were produced by the enzymic hydrolysis of the appropriate N-acetyl-d-amino-acids with whole cell biomass. The hydrolysis of N-acetyl-d-methionine by A. denitrificans and N-acetyl-d-valine by A. faecalis was preferential. Maximum yields of d-methionine and d-valine were 94.3 and 84.7% at a specific product formation rate of 20.10 and 19.19 μmol min⁻¹ mg⁻¹ of wet cells at 20 mM substrate concentration and 5 mg ml⁻¹ of cell density.     

Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus)

Summary Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities. These results are consistent with the hypothesis that peptides having significant sequence homology with human neuropeptide Y and porcine neuropeptide Y are present in the nerve fibers that permeate the islet.     

Synthesis and biological properties of 4-norleucine-neuropeptide Y; secondary structure of neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acid peptide amide isolated from porcine brain. The NPY analog, 4-norleucine-NPY was synthesized by a solid-phase method and purified to homogeneity in 20% yield by reverse-phase chromatography. Investigation of the biological properties indicated that the analog is an agonist of NPY. Secondary structural analyses revealed that NPY and the analog exhibited predominantly alpha-helical and beta-sheet structures, respectively; however, experiments in trifluoroethanol indicated that the analog has the potential of assuming an alpha-helical structure. Based on circular dichroism (CD), Raman spectroscopy and Chou-Fasman analyses, a model has been proposed for the secondary structure of NPY.     

Design of liposome to improve encapsulation efficiency of gelonin and its effect on immunoreactivity and ribosome inactivating property

Gelonin, purified from the seeds of Gelonium multiflorum, using cation-exchange and gel-filtration chromatography was characterised for its purity, homogeneity and molecular weight by reverse-phase HPLC (RP-HPLC) and SDS-PAGE analysis. The HPLC purified gelonin was used for entrapment studies in the liposomes. Liposomes were prepared by reverse phase evaporation (REV) technique using three different types of lipid composition in the same molar ratio. The method resulted in 75–80% entrapment efficiency of gelonin in the liposomes. Entrapped and unentrapped gelonin was characterized for physico-chemical, immunochemical and biological properties. The immunoreactivity of entrapped gelonin was fully preserved but the ribosome-inactivating property was slightly inhibited. The method involved mild conditions, highly reproducible and the liposomes produced appeared to be stable for several months. It has important implications in the development of cell type specific cytotoxic agents where a chemical cross-linking is involved which significantly inhibits both immunoreactivity and ribosome-inactivating ability of the toxin.     

Substitution of Ser61----Gly61 in human apolipoprotein C-II does not alter its activation of lipoprotein lipase

Lipoprotein lipase (LpL) activity is enhanced by apolipoprotein C-II (apoC-II), a 79 amino acid residue peptide. The minimal apoC-II sequence required for activation of LpL resides between residues 56-79. To determine the possible role of an acyl-apoC-II intermediate involving Ser61 in enzyme catalysis, a synthetic peptide of apoC-II containing residues 56-79 was synthesized and compared to the corresponding peptide with serine at position 61 being substituted with glycine. With two different LpL assay systems, both peptides enhanced enzyme activity. Since glycine does not contain a hydroxyl group, these results rule out the possibility that an acyl-apoC-II intermediate with Ser61 is required for enzyme activation.     

Selection and characterization of an L-thiazolidine-4-carboxylic acid resistant callus culture of Vigna radiata (L.) Wilczek var. radiata

Callus cultures were established from seedling root tips of mungbean (Vigna radiata (L.) Wilczek var. radiata) cv. K 851. The growing calli were exposed to increasing concentrations of thioproline — an analog of proline, in the medium. A concentration of 3.0 mM thioproline completely inhibited the growth of the cells. However, after 25 days incubation 5 cell clones were obtained which could grow on this concentration of thioproline. Out of them one vigorously growing cell clone was further characterized. This selected clone contained higher endogenous levels of free proline (5 fold) and K⁺ (1.5 fold) and exhibited elevated tolerance, not only to thioproline but also to exogenously applied NaCl in the growth medium, as compared to the normal sensitive callus cells. Higher endogenous levels of free proline and K⁺ appear to impart dual resistance to thioproline and NaCl to the selected cell strain.     

N-α-biotinylated-neuropeptide Y analogs: Syntheses, cardiovascular properties, and application to cardiac NPY receptor visualization

Two monobiotinylated analogs of neuropeptide Y (NPY) were synthesized by coupling the N-hydroxysuccinimidyl esters of biotin and (6-biotinylamido)-hexanoic acid, respectively, to the free alpha-NH2 group of the side chain protected NPY peptide resin. Crude peptides obtained by HF cleavage were purified by RPLC and their integrities were confirmed by amino acid and mass spectral analysis. As with NPY, both biotinylated analogs inhibited 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes in a dose-dependent manner. N-alpha-[(6-biotinylamido)-hexanoyl]-NPY exhibited potencies comparable to that of NPY whereas N-alpha-biotinyl-NPY was slightly less potent. In the in vivo experiments, however, both the biotinylated analogs exhibited responses comparable to NPY in increasing arterial blood pressure and decreasing heart rate in anesthetized rats. The responses of the biotinyl analogs were longer lasting than those of NPY. Histochemical studies revealed that N-alpha-[(6-biotinylamido)-hexanoyl]-NPY could label the NPY receptors in rat cardiac ventricular tissues. This labeling was specific since intact NPY inhibited the staining. These studies show that biotinyl-NPY analogs exhibit biological potencies comparable to intact NPY and can therefore be used to further probe the NPY-receptor interaction.     

Hormonotoxins: the role of positive charge of lysine residue on the immunological,biological and cytotoxic properties of ovine lutropin-S-S-gelonin conjugates

Since the positive charge on the lysine residues plays an important role in the receptor recognition ability of oLH, the hormonotoxin has been synthesised with the use of 2-iminothiolane HC1 (2IT) and N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The oLH activated with 2IT (oLH-10) was then mixed with SPDP activated gelonin (gelonin-30) in order to obtain a oLH-S-S-gelonin hormonotoxin. The conjugation mixture containing hormonotoxin was purified by gel-filtration chromatography according to the molecular weight and a complete physico-chemical, immunochemical and biochemical analysis were performed. The linkage occured through the -NH₂ groups of -subunit of oLH as judged from RP-HPLC analysis. A 11 (oLH:gelonin) molar ratio was obtained when determined with the use of several techniques. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity. The competitive displacement analysis indicate that the binding occurs via the hormone part leaving the gelonin free which was probed with the gelonin antibodies. The presently described (C150A-02, C160A-02 and C170A-02) hormonotoxins exhibited higher receptor binding and toxicity to the target cells than the hormonotoxins prepared with the use of SPDP only. Therefore it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH which was preserved during the conjugation process.Abbreviations BSA Bovine Serum Albumin - CMC Carboxy methyl Cellulose - DTT Dithiothreitol - DMEM Dulbeco's Modified Eagle's Medium - DTNB Ellman's reagent [5,5-dithio-bis-(2-nitrobenzoic acid)] - EDTA Ethylenediaminetetraacetic acid - FPLC Fast Protein Liquid Chromatography - FCA Freund's Complete Adjuvant - FCS Fetal Calf Serum - Gelonin-30 Gelonin modified by SPDP - GnRH Gonadotropin-Releasing Hormone - Gelonin-SPDP SPDP modified derivative of gelonin - HEPES (N-[2-hydroxyethyl] piperazine-N-[-2-ethanesulphonic acid]) - IFA Incomplete Freund's Adjuvant - 2IT 2-Iminothiolane - IODOGEN 1,3,4,6-tetrachloro 3,6-diphenylglycouril - oLH Ovine Luteinizing Hormone - oLH-SPDP SPDP modified derivative of oLH - oLH-10 oLH modified by 2IT - oLH2IT Molar ratio of oLH and 2IT - PDP 2-Pyridyl-dithiopropionate - PAP Pokeweed Antiviral Protein - RIP Ribosome Inactivating Protein - RP-HPLC Reverse-Phase High Performance Liquid Chromatography - RPMI Roswell Park Memorial Institute - RIA Radioimmunoassay - RRA Radioreceptor Assay - SPDP N-Succinimidyl-3(2-pyridyldithio)propionate - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - TCA Trichloroacetic acid - TFA Trifluroacetic acid