Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.     

Induction by mycoplasmas of tumor necrosis factor-alpha from macrophages

Several species of mycoplasmas including M. pneumoniae, the causative agent of human respiratory infection, were investigated for tumor necrosis factor-alpha (TNF-alpha) induction. The cytotoxic activity to Meth A cells of peritoneal macrophages purified from BALB/c mice was enhanced markedly when cultured with either viable or nonviable mycoplasmas. The supernatant of macrophage culture mixed with mycoplasmas, M. pneumoniae or A. laidlawii, showed a potent cytotoxic activity to TNF-alpha-sensitive but not to TNA-alpha-insensitive L cells. Addition of anti-TNA-alpha antiserum inhibited completely the cytotoxic activity of the supernatant, indicating that the cytotoxic activity is due mostly to TNF-alpha. These results strongly suggest that mycoplasmas possess an activity to induce TNF-alpha, which enhances the cytotoxic activity of macrophages and prevent infection with mycoplasmas in vivo.     

Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast

A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes.     

Peri-operative immunotherapy with OK-432

Pre- and postoperative intradermal administration of OK-432 enhanced the SU-PS skin reaction in patients with gastric cancer, but failed to prevent a fall in the NK activity induced by the operation.The change in NK activity was not associated with a change in the proportion of Leu 7-positive cells, but was related to Leu 11a-positive cells. Intradermal injection of OK-432 increased the proportion of Leu 7-positive cells in the patients in whom they accounted for less than 20% of lymphocyte population. The case was the same with Leu 11a-positive cells.Intravenous injection of OK-432 tended to increase suppressor-inducer T cells (CD4⁺2HA⁺ cells), B cells and Leu 7-positive cells. Particularly, the proportions of OK-M₁-positive cells and MHC class II antigen-positive cells increased in all patients. Immunotherapy with OK-432 given intravenously at a dose of 0.1 KE appeared to be safe because no side effects were essentially observed.     

One- and two-dimensional NMR studies on the conformation of DNA containing the oligo(dA)oligo(dT) tract.

The resonances of the imino protons and all of the non-exchangeable protons (except for H5'/H5') of d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) have been assigned by means of one- and two-dimensional NMR spectroscopies. Qualitative analyses showed that the overall structure is of the B-form, but local conformational deviations exist. The NOEs between the imino protons of thymines and H2 of adenines suggest that the A-T base pairs are propeller-twisted to almost the same degree as in crystals. A remarkable chemical shift of H1' was observed for the residue located just before the oligo(dA)oligo(dT) tract, suggesting the presence of conformational discontinuity at the junctions between the oligo(dA)oligo(dT) tract and the other portions. Analyses of cross peaks in NOESY spectra between H2 of adenines and H1' of the 3'-neighbouring residues on the complementary strand revealed that the minor groove of the oligo(dA)oligo(dT) tract is narrow and compressed gradually, from 5' to 3', along the tract.     

Overlapping two genes in human DNA: a salivary amylase gene overlaps with a gamma-actin pseudogene that carries an integrated human endogenous retroviral DNA

The human salivary amylase gene (amy1), consisting of eleven exons, is expressed in the salivary gland and in some amylase-producing tumors. Its uppermost exon and the following intron, along with the 5'-flanking region of this gene, are shown to be superimposed with a gamma-actin pseudogene sequence, a portion of which is transcribed into salivary amylase mRNA and another portion of which serves as a promoter for the amy1 gene. In the further upstream region, the gamma-actin pseudogene sequence is interrupted by a human endogenous retroviral nucleotide sequence.     

Clerodane diterpenoids from the roots of Portulaca pilosa

Three trans-clerodane diterpenoids, pilosanol A, B and C, the last compound being a glucoside, have been isolated from the roots of Portulaca pilosa. They show a marked contrast in skeletal type with the constituents of aerial part. Evolutionary changes in the biosynthetic abilities of Portulaca species is discussed.     

Participation of microsomal aldehyde reductase in long-chain fatty alcohol synthesis in the rat brain

The participation of microsomal aldehyde reductase in long-chain fatty alcohol synthesis in the rat brain was examined. A reaction mixture of [1-14C]hexadecanoic acid with brain microsomes and NADPH formed two radioactive products having the same mobilities as pure hexadecanal (RF 0.61) and hexadecanol (RF 0.22), respectively, on TLC plates. The product of the RF 0.61 spot was further identified as hexadecanal using gas-liquid chromatography after methylation and TLC of its reduced product with LiAlH4 and semicarbazide. The ratio of hexadecanal to hexadecanol varied from 0.4 to 1.2 under the present experimental conditions. When solubilized rat brain microsomes were applied to a Sepharose 4B column coupled with the rabbit antibody raised against rat liver microsomal NADPH-cytochrome-c reductase, which reacts with aldehyde reductase from rat brain, the eluted fraction ceased to form [14C]hexadecanol but continued to form [14C]hexadecanal from [14C]hexadecanoic acid. These results strongly indicate that hexadecanal is the intermediate in the synthesis of hexadecanol from hexadecanoic acid in rat brain microsomes with the participation of microsomal aldehyde reductase.     

"Trembler"--a nervous disorder in chickens

The trembler chickens, which spontaneously occurred in the Poultry farm of Animal Genetics Laboratory of Nagoya University, showed the phenotypic symptoms as of the tremor of the head, neck and body. Mild symptoms of shaking were observed in most of the day-old affected chicks, but this sign will be kept calm during 1-4 weeks old. Subsequently, trembling became clearly evident. Progressing with age, the trembling severely increased inducing the chickens to walk with a stumbling gait. In the terminal stages the chickens were hardly to stand and die from inanition. Mating experiments between half-sib and sire-daughter have revealed that the responsible gene (tr) for the trembler chicken is an autosomal recessive gene.     

Characterization of two cysteine residues in cytochrome P-450scc: chemical identification of the heme-binding cysteine residue

It was found that there were only two cysteine residues in highly purified cytochrome P-450scc molecule from bovine adrenocortical mitochondria by titration with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in denatured conditions. Only one cysteine residue at position 303 of cytochrome P-450scc could be specifically modified with DTNB in the native state. The resulting cytochrome P-450scc-5-thio-2-nitrobenzoic acid complex (cytochrome P-450scc-TNB) showed no distinct differences in absorption spectra, cholesterol binding, or electron transferring from adrenodoxin, compared to those of untreated cytochrome P-450scc. These observations indicated that the 303rd cysteine residue does not play a role in heme binding, cholesterol (substrate) binding or adrenodoxin binding. The other cysteine residue at 461 could be modified with DTNB only in a denatured condition. These assignments of cysteine residues were made by the subsequent S-cyanylation with KCN followed by incubation in 6 M guanidine hydrochloride at alkaline pH, which causes enhanced cleavage of peptide bonds adjacent to the cyanylated cysteine residues. Analyses of fragmented polypeptides by SDS-polyacrylamide gel electrophoresis confirmed that there were only two cysteine residues in the molecule and indicated that the cleavage rate of the peptide bond between 460 and 461 becomes high only when both cysteine residues (303 and 461) are cyanylated. These results clearly established that the 461st cysteine residue in cytochrome P-450scc plays a role as the heme fifth ligand on the basis of the general agreement that a thiolated cysteine residue coordinates to the heme iron.