Comparison by 1H-nmr spectroscopy of the conformation of the 2600 dalton antifreeze glycopeptide of polar cod with that of the high molecular weight antifreeze glycoprotein

The antifreeze glycopeptide (AFGP-8) from polar cod, B. saida, is a 14-amino acid polypeptide having alternating glycotripeptide sequences of Ala-[Gal(β1 → 3)GalNAc(β1 → O)]-Thr-Pro and Ala-[Gal(β1 → 3)GalNAc(β1 → O)]-Thr-Ala, with alanyl residues at amino and carboxy terminals. Conformational studies of AFGP-8 have been carried out by ¹H-nmr and empirical energy calculations to investigate the difference in its antifreeze behavior from that of the more active high-molecular weight AFGP 1-4 of P. borchgrevinki. The ¹H-nmr spectra, including the resonances of the exchangeable amide protons, were assigned by two-dimensional correlated spectroscopy (COSY), one-dimensional difference decoupling, and nuclear Overhauser effect (NOE) measurements. For the four threonyl residues, the amide proton coupling constants and the small coupling constants between H^α and H^β indicate similar conformations, despite significant chemical shift differences. The strong NOE between the α protons and the amide protons of the residue following together with large temperature coefficients of chemical shifts, indicate an extended conformation not consisting of α-helix, turns or bends. Energy computations indicate several low-energy conformations consistent with the observed coupling constants for ?. Among these, a left-handed helical conformation with three repeating residues per turn has been proposed, which is in accordance with the observed NOE between the methyl group of the α-GalNAc and Ala H^βs. While the observed Overhauser effects in the threonyl side chain suggest a certain amount of conformational averaging, the effect involving the acetmido methyl of α-GalNAc and H^βs of Ala indicate that it as is a major conformer. In view of the close similarity between the conformations of AFGP-8 and the more active antifreeze polymer, AFGP 1-4, we propose that the difference in their activities is due to the length of the regular repeating structure with glycosylation at every third amino acid residue, and not due to any fundamental difference in their conformations.     

p82H identifies sequences at every human centromere

Summary A cloned alphoid sequence, p82H, hybridizes in situ to the centromere of every human chromosome. After washing under stringent conditions, no more than 8% of the grains are located on any specific chromosome. p82H thus differs from other centromeric sequences which are reported to be chromosome specific, because it detects sequences that are conserved among the chromosomes. Two experimental approaches show that the p82H sequences are closely associated with the centromere. First, p82H remains with the relocated centromeres in an inv(19) and an inv(6) chromosome. Second, p82H hybridizes at the centromere but not to the centromeric heterochromatin of chromosomes 1, 9 and 16 that have elongated 1qh, 9qh and 16qh regions produced by short growth in 5-azacytidine. The only noncentromeric site of hybridization is at the distal end of the 9qh region.     

Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy

Peptide binding and lymph node T cell activation studies have been used to characterize T cell recognition of an encephalitogenic T cell autoantigen from myelin basic protein in (PL/J x SJL)F1 mice. Amino acids that determine interactions with either the restriction element of the major histocompatibility complex (MHC) or the encephalitogenic T cell receptor are defined. This information enables the design of peptides that bind MHC yet do not cross-react with the autoantigen. A peptide analog of the encephalitogenic epitope is shown to be "heteroclitic" for MHC binding and activation of encephalitogenic T cells in vitro. This analog is not immunogenic for encephalitogenic T cells in vivo and is shown to inhibit disease that is induced by the autoantigen itself.     

Prostaglandins can modify gamma-radiation and chemical induced cytotoxicity and genetic damage in vitro and in vivo

The effect of prostaglandin E1, E2, and F2 alpha on gamma-radiation, benzo(a)pyrene and diphenylhydantoin-induced cytotoxicity in vivo and genotoxicity in vitro was investigated. Prostaglandin E1 prevented both cytotoxic and genotoxic actions of all the three agents, where as both PGE2 and PGF2 alpha were ineffective. In fact, it was seen that both PGE2 and PGF2 alpha are genotoxic by themselves. Gamma-linolenic acid and dihomogamma-linolenic acid, the precursor of PGE1 were also as protective as that of PGE1, where as arachidonic acid, the precursor of 2 series PGs, has genotoxic actions to human lymphocytes in vitro. These results suggest that prostaglandins and their precursors can determine the susceptibility of cells to cytotoxic and genotoxic actions of chemicals and radiation. This study is particularly interesting since, it is known that some tumor cells contain excess of PGE2 and PGF2 alpha and many carcinogens can augment the synthesis of 2 series of PGs.     

Interleukin-1 stimulates prostaglandin biosynthesis by human amnion

The purpose of these studies was to determine if Interleukin-1 (IL-1) alters the rate of prostaglandin biosynthesis by human amnion. Primary monolayer cultures of amnion cells were established from women undergoing elective cesarean section before the onset of labor. Natural purified and recombinant human IL-1 alpha and IL-1 beta were incubated with amnion cells in culture, and prostaglandin E2 (PGE2) biosynthesis was measured by radioimmunoassay in cell-free media. A concentration-dependent increase in PGE2 production by amnion cells occurred in response to natural purified and recombinant IL-1 preparations. No differences in the parameters of the dose-response curves between the two IL-1 gene products could be determined (p greater than 0.05). Indomethacin blocked the effect of IL-1 in prostaglandin biosynthesis by human amnion. Interleukin-1, a fever mediator, could serve as a signal for the initiation of labor in cases of intrauterine or systemic infection.     

Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.     

Protective role of fructose 1,6-bisphosphate during CCl4 hepatotoxicity in rats.

Rats were injected intraperitoneally with CCl4 (2.5 ml/kg body wt.) and the hepatotoxicity was compared with that of rats receiving the same dose of CCl4 and an intraperitoneal injection of fructose 1,6-bisphosphate (2 g/kg body wt.). A 50-70% decrease in plasma aspartate aminotransferase and alanine aminotransferase activities was observed in the latter treatment, indicating a protective role of the sugar bisphosphate in CCl4 hepatotoxicity. The protection was accompanied by elevated hepatic activities of ornithine decarboxylase at 2, 6 and 24 h, S-adenosylmethionine decarboxylase at 6 h, and spermidine N1-acetyltransferase at 2 h. The increase in the enzymes involved in polyamine metabolism was shown in our previous work [Rao, Young & Mehendale (1989) J. Biochem. Toxicol. 4, 55-63] to correlate with increased polyamine synthesis or interconversion, which was related to the extent of hepatocellular regeneration. The hepatic contents of fructose 1,6-bisphosphate and ATP significantly decreased after CCl4 treatment, and administration of the sugar bisphosphate increased hepatic ATP. Fructose 1,6-bisphosphate, an intermediary metabolite of the glycolytic pathway, may decrease CCl4 toxicity by increasing the ATP in the hepatocytes. The ATP generated is useful for hepatocellular regeneration and tissue repair, events which enable the liver to overcome CCl4 injury.     

A selective differential medium for the isolation of Listeria monocytogenes

A new medium has been developed for the isolation of Listeria monocytogenes from clinical specimens with a mixed flora. Almost complete inhibition of unwanted organisms was achieved and recognition of colonies of Listeria spp. was usually possible after 24 h using the aesculin-ferric ammonium citrate indicator system. Compared to McBride agar the new medium was more inhibitory to representative contaminating species in pure culture and more successful in isolating small numbers of L. monocytogenes from artificially seeded clinical specimens.