Some new variants of serum protease inhibitors in Meishan pigs

Serum samples of Meishan (13 animals) and Meishan x Wild Boar crosses (361 animals) were analysed by means of two-dimensional electrophoresis. Some new variants in protease inhibitor systems PO1A, PO1B and PI2 are reported.     

Detection and characterization of mitochondrial DNA rearrangements in Pearson and Kearns-Sayre syndromes by long PCR

We used a strategy based on long PCR (polymerase chain reaction) for detection and characterization of mitochondrial DNA (mtDNA) rearrangements in two patients with clinical signs suggesting Pearson syndrome and Kearns-Sayre syndrome (KSS), respectively, and one patient with myopathic symptoms of unidentified origin. Mitochondrial DNA rearrangements were detected by amplification of the complete mitochondrial genome (16.6 kb) using long PCR with primers located in essential regions of the mitochondrial genome and quantified by three-primer PCR. Long PCR with deletion-specific primers was used for identification and quantitative estimation of the different forms of rearranged molecules, such as deletions and duplications. We detected significant amounts of a common 7.4-kb deletion flanked by a 12-bp direct repeat in all tissues tested from the patient with Pearson syndrome. In skeletal muscle from the patient with clinical signs of KSS we found significant amounts of a novel 3.7-kb rearrangement flanked by a 4-bp inverted repeat that was present in the form of deletions as well as duplications. In the patient suffering from myopathic symptoms of unidentified origin we did not detect rearranged mtDNA in blood but found low levels of two rearranged mtDNA populations in skeletal muscle, a previously described 7-kb deletion flanked by a 7-bp direct repeat and a novel 6.6-kb deletion with no repeat. These two populations, however, were unlikely to be the cause of the myopathic symptoms as they were present at low levels (10–40 ppm). Using a strategy based on screening with long PCR we were able to detect and characterize high as well as low levels of mtDNA rearrangements in three patients. Received: 10 March 1997 / Accepted: 20 May 1997     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

An immuno-dot blot assay for detection of thermostable protease from Pseudomonassp. AFT-36 of dairy origin

A dot-ELISA technique for the detection of Pseudomonas protease was developedusing IgG of anti- Pseudomonas AFT-36 protease as capture antibody. The detection limitof protease in buffer or milk was 1·01 ng ml⁻¹. The procedure was performedat room temperature, took about 2·5 h and was economical. Protease AFT-36 isimmunologically related to five out of seven Pseudomonas spp. The results suggest thatthe assay could be used to detect proteases in dairy products.     

Metmyoglobin promotes arachidonic acid peroxidation at acid pH

The ability of metmyoglobin and other heme proteins to promote peroxidation of arachidonic acid under acidic conditions was investigated. Incubation of metmyoglobin with arachidonic acid resulted in a pH-dependent increase in lipid peroxidation as measured by the formation of thiobarbituric acid reactive products and oxygen consumption. Increased peroxidation was observed at pH levels below 6.0, reaching a plateau between pH 5.5 and 5.0. At comparable heme concentrations, metmyoglobin was more efficient than oxymyoglobin, methemoglobin, or ferricytochrome c in promoting arachidonic acid peroxidation. Metmyoglobin also promoted peroxidation of 1-palmityl-2-arachidonyl phosphatidylcholine and methylarachidonate but at significantly lower rates than arachidonic acid. Addition of fatty acid-free albumin inhibited arachidonic acid peroxidation in a molar ratio of 6 to 1 (arachidonic acid:albumin). Both ionic and non-ionic detergents inhibited metmyoglobin-dependent arachidonic acid peroxidation under acidic conditions. The anti-oxidants butylated hydroxytoluene and nordihydroguaiaretic acid and low molecular weight compounds with reduced sulfhydryl groups inhibited the reaction. However, mannitol, benzoic acid, and deferoxamine were without significant effect. Visible absorption spectra of metmyoglobin following reaction with arachidonic acid showed minimal changes consistent with a low level of degradation of the heme protein during the reaction. These observations support the hypothesis that metmyoglobin and other heme proteins can promote significant peroxidation of unsaturated fatty acids under conditions of mildly acidic pH such as may occur at sites of inflammation and during myocardial ischemia and reperfusion. This may be the result of enhanced aggregation of the fatty acid and/or interaction of the fatty acid with heme under acidic conditions.     

Nigexine, a phospholipase A2 from cobra venom with cytotoxic properties not related to esterase activity. Purification, amino acid sequence, and biological properties

The venoms of the Naja species are known to be cytotoxic. This toxicity has been attributed to the presence of small nonenzymatic polypeptides of 60 amino acid residues, designated as cardiotoxins or cytotoxins. We investigated the cytotoxic potency of Naja nigricollis venom fractions and isolated another type of cytotoxic component which is even more potent than cardiotoxins. This cytotoxic compound, which was designated as nigexine, was purified to homogeneity and its amino acid sequence was determined. Nigexine is a basic phospholipase A2 consisting of a single chain of 118 amino acids. A detailed investigation of the cytotoxic effects on epithelial FL cells, C-13T neuroblastoma cells, and promyelocytic leukemia HL 60 cells revealed that nigexine not only altered cell viability but also prevented cell proliferation. This is a property that was specific to nigexine since other phospholipases A2 from various sources had no detectable cytotoxic activity. The cytotoxic activity of nigexine was not dependent on the presence of divalent cations, unlike its enzymatic activity. In particular, the cytotoxic activity of nigexine was identical in the presence or absence of either 2 mM Ca2+ or Sr2+, or 6 mM EDTA. We also present evidence based on chemical modifications that cytotoxic activity was not correlated with enzymatic activity. Thus, modification with parabromophenacyl bromide totally abolished the enzymatic activity of nigexine, which nevertheless retained 6-20% of the cytotoxicity of native nigexine. Conversely, treatment with cyanogen bromide gave a compound that retained 7% of the enzymatic activity of the parent molecule but was devoid of detectable cytotoxicity.     

Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae

Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF alpha 1 prepro leader peptide/IGF-I gene fusion. Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant. Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively. The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36). No other glycosylation sites were found. Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I. The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be alpha-D-Manp(1----2)alpha-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure. No other post-translational modifications could be identified in the glycosylated IGF-I form. Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay.     

Activation of formylmethanofuran synthesis in cell extracts of Methanobacterium thermoautotrophicum.

In cell extracts of Methanobacterium thermoautotrophicum, formylmethanofuran (formyl-MFR) synthesis (an essential CO2 fixation reaction that is an early step in CO2 reduction to methane) is subject to a complex activation that involves a heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine O3-phosphate (CoM-S-S-HTP). In this paper we report that titanium(III) citrate, a low-potential reducing agent, stimulated CO2 reduction to methane and activated formyl-MFR synthesis in cell extracts. Titanium(III) citrate functioned as the sole source of electrons for formyl-MFR synthesis and enabled this reaction to occur independently of CoM-S-S-HTP. In addition, CoM-S-S-HTP was found to activate an unknown electron carrier that reduced metronidazole. The activation of formyl-MFR synthesis by CoM-S-S-HTP may involve the activation of a low-potential electron carrier.