Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.     

Effect of Simulated Acid Rain on Bursaphelenchus xylophilus Infection of Pine Seedlings

White, Scots, and Austrian 3-year-old pine seedlings were treated with conditions simulating acid rain and inoculated with the white pine specific pathotype of Bursaphelenchus xylophilus, VPSt-1. Oleoresin concentration increased slightly and carbohydrate concentration decreased in all seedlings treated with simulated acid rain (SAR). The changes were significantly increased after inoculation of SAR-treated white and Scots pine seedlings with VPSt-1. Wilting was delayed and nematode reproduction decreased in SAR-treated white pine seedlings inoculated with VPSt-1. SAR-treated Austrian pine seedlings were resistant to VPSt-1, but SAR-treated Scots pine seedlings lost tolerance to VPSt-1 and wilted 50-60 days after inoculation.     

Humoral Immunity to Commensal Oral Bacteria: Quantitation,Specificity and Avidity of Serum IgG and IgM Antibodies Reactive with Actinobacillus actinomycetemcomitans in Children

The levels, specificity and avidities of serum IgM and IgG antibodies reactive with Actinobacillus actinomycetemcomitans (Aa) serotypes a, b and c were determined in periodontally healthy (PH) children and compared with subjects with localized juvenile periodontitis (LJP). All PH children exhibited IgM and IgG Aa-reactive antibodies whether or not Aa was detected subgingivally but the antibodies were not specific for Aa. In contrast, LJP sera contained high concentrations of IgM and IgG antibodies reactive with Aa that were largely specific for this bacterium. IgM and IgG antibodies in both PH and LJP subjects were of low avidity. With one exception, the avidities of IgG anti-Aa antibodies were significantly greater than those of IgM antibodies in both PH and LJP subjects. However, although the LJP subjects had as much as 115-fold more Aa-reactive IgG antibody than did the PH subjects the avidities of their IgG antibodies were no greater than those of the PH group. The induction by the host of low-avidity antibodies, that are ineffective in immune elimination, may be a reason why commensal bacteria persist at mucosal surfaces and why persons with LJP fail to eliminate Aa from their periodontal pockets.     

Activation of Host Defense Mechanisms by Elevated Production of H2O2 in Transgenic Plants

Active oxygen species have been postulated to perform multiple functions in plant defense, but their exact role in plant resistance to diseases is not fully understood. We have recently demonstrated H2O2-mediated disease resistance in transgenic potato (Solanum tuberosum) plants expressing a foreign gene encoding glucose oxidase. In this study we provide further evidence that the H2O2-mediated disease resistance in potato is effective against a broad range of plant pathogens. We have investigated mechanisms underlying the H2O2-mediated disease resistance in transgenic potato plants. The constitutively elevated levels of H2O2 induced the accumulation of total salicylic acid severalfold in the leaf tissue of transgenic plants, although no significant change was detected in the level of free salicylic acid. The mRNAs of two defense-related genes encoding the anionic peroxidase and acidic chitinase were also induced. In addition, an increased accumulation of several isoforms of extracellular peroxidase, including a newly induced one, was observed. This was accompanied by a significant increase in the lignin content of stem and root tissues of the transgenic plants. The results suggest that constitutively elevated sublethal levels of H2O2 are sufficient to activate an array of host defense mechanisms, and these defense mechanisms may be a major contributing factor to the H2O2-mediated disease resistance in transgenic plants.     

The preparation and characterisation of some complexes of iron(II) with amino acids

The following complexes of iron(II) with the amino acids glycine, alanine, phenylglycine, phenylalanine, leucine, serine, aspartic acid, glutamic acid, glutamine, tryptophan, histidine, methionine, S-methylcysteine, cystine, and glycylglycine have been isolated: Fe(Gly)₂, Fe(Ala)₂, Fe(Phegly)₂, Fe(Phe)₂·2H₂0, Fe(Leu)₂·2H₂0, Fe(Ser)₂, Fe(Asp)·2H₂0, Fe(Glu)·2H₂0, Fe(Gln)₂, Fe(Trp)₂, Fe(His)₂·H₂0 and 2H₂0, Fe(Met)₂, Fe(MeCys)₂, Fe(CysCys) and Fe(GlyGly)₂. Their magnetic behaviour, reflectance spectra, and Mössbauer parameters are consistent with high spin, hexacoordinate iron(II), and imply extended structures involving carboxylate bridges.     

Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate

The pure reticulocyte lipoxygenase converts 15LS-hydroxy-5,8,11,13(Z,Z,Z,E)-icosatetraenoic acid (15LS-HETE) methyl ester to a complex mixture of products containing 5DS,14LR,15LS-trihydro(pero)xy-6E,++ +8Z,10E,12E-icosatetraenoate methyl ester (lipoxin B methyl ester), 5DS,15LS-DiH(P)ETE methyl ester and four 8,15LS-DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy-icosatetraenoic acid]. After a short incubation period (15 min) 5DS,15LS-DiH(P)ETE methyl ester was found to be the main product, whereas after a 3-h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400-MHz 1H-NMR. Atmospheric oxygen was incorporated at carbon-5 and carbon-14 into the major product. 5DS,15LS-DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15LS-HETE and 5DS,15LS-DiHETE. The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.     

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.     

Immunological responses following early embryonic surgical bursectomy

Hormonal and late embryonic surgical bursectomy have been shown to produce chicks deficient in IgM and/or IgG, depending on the time of bursectomy. Some of these hormonally bursectomized birds were reported to have atrophy of the thymic cortex.We have developed a new technique for early embryonic surgical bursectomy (at 70 hr in ovo). Results of stimulation tests with BSA and SRBC indicates a few of the bursectomized birds are capable of producing small amounts of antibody. Early embryonic surgical bursectomy also results in incomplete development of the thymic cortex, indicating a developmental interrelationship exists between the bursa and thymus.     

Biological activities of isomers of 8,15-dihydroxyeicosatetraenoic acid

Chemically synthesized 8(S), 15(S)-dihydroxy-5,11-cis-9, 13-trans-eicosatetraenoic acid and 8(R), 15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid were inactive, in comparison to leukotriene B4, in a human polymorphonuclear leukocyte chemokinetic assay and a rat polymorphonuclear leukocyte aggregation assay.