Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.     

Effect of TPA on the mutagenicity of caffeine in the soybean mutation test

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a well-known tumor promoter in mouse-skin carcinogenesis. Its effects on mutagenesis in a soybean test system were examined, and the effects were judged from the appearance of spots of various colors on the leaves. When soybean seeds were treated with TPA plus 0.03% caffeine, the frequency of spots per leaf decreased significantly and in proportion to the concentration of TPA. TPA alone at concentrations of 1–20 μg/ml did not induce any mutations. Mutations induced by γ-rays were not affected by administration of TPA either before or after exposure to γ-rays. The mechanism of suppression by TPA of mutations induced by caffeine is discussed.     

Ultra-fastChara myosin: A test case for the swinging lever arm model for force production by myosin

Recent breakthroughs and technological improvements are rapidly generating evidence supporting the “swinging lever arm model” for force production by myosin. Unlike previous models, this model posits that the globular domain of the myosin motor binds to actin with a constant orientation during force generation. Movement of the neck domain of the motor is hypothesized to occur relative to the globular domain much like a lever arm. This intramolecular conformational change drives the movement of the bound actin. The swinging lever arm model is supported by or consistent with a large number of experimental data obtained with skeletal muscle or slime mold myosins, all of which move actin filaments at rates between 1 and 10 μm/sin vitro. Recently myosin was purified, fromChara internodal cells.In vitro the purifiedChara myosin moves actin filaments at rates one order of magnitude faster than the “fast” skeletal muscle myosin. While this ultra fast movement is not necessarily inconsistent with the swinging lever arm model, one or more specific facets of the motor must be altered in theChara motor in order to accommodate such rapid movement. These characteristics are experimentally testable, thus the ultra fast movement byChara myosin represents a powerful and compelling test of the swinging lever arm model.     

The glycinin box: a soybean embryo factor binding motif within the quantitative regulatory region of the 11S seed storage globulin promoter

The soybean embryo factor binding sequence in the glycinin A₂B_1a gene promoter was delimited to an A/T-rich 9 bp sequence, 5-TAATAATTT-3, designated as the glycinin box, by DNA footprinting and gel mobility shift assay using synthetic oligonucleotides. It was shown that the interaction with the factor takes place at a defined DNA sequence rather than at random A/T-rich sequence blocks in the glycinin 5 flanking region. There are four glycinin boxes in the quantitative regulatory region between positions – 545 and – 378 of the glycinin A₂B_1a promoter. Multiple nonamer motifs similar to the glycinin box were also found in the equivalent regions of other glycinin and legumin promoters, suggesting that they must be conserved as a binding site for the embryo factor that activates the differential and stage-specific expression of seed 11S globulin genes in leguminous plants.     

Glutelin accumulation and changes in the levels of its mRNA in the superior and inferior spikelets of rice ear during ripening

Glutelin accumulation in the apical spikelet of the top primary branch (superior spikelet) and the second spikelet of the lowest secondary branch (inferior spikelet) of the ear of the rice plant (Oryza sativa L.) was characterized during grain filling.In the superior spikelet, the accumulation of dry matter and nitrogen started immediately after flowering and rapidly reached the maturation level by 20 days after heading (DAH). At 7 DAH, total RNA content had already reached its maximum level and glutelin mRNA content 70% of its maximum. The increase in glutelin mRNA was followed by a rapid increase in glutelin between 7 and 16 DAH.In the inferior spikelet dry matter, nitrogen and glutelin accumulation were low immediately after flowering and increased only after grain filling of the superior spikelet was almost complete. Total RNA and glutelin mRNA increased much later at slower rates than in the superior spikelet.It is very likely that the retardation of dry matter, total nitrogen and glutelin accumulation in the inferior spikelet is due to retardation of differentiation and development of endosperm tissue, and to glutelin gene expression in endosperm cells. It is suggested that the delayed development resulted from limited partitioning of nutrients to the inferior spikelet at the early stage of ripening.     

Augmentation of antitumor immunity in regional lymph nodes by local immunotherapy

We have previously reported that the antitumor effect of OK-432, a streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (OK-432/fbg) [1]. In order to elucidate the effects of this immunotherapy on regional lymph nodes (RLN), we carried out both morphological and functional analyses of the RLN from colonic cancer patients treated with OK-432/ fbg. Computer-aided morphometry revealed that the maximal cross-sectional areas and the broadest diameters of the RLN were significantly greater (p<0.01) in patients who had undergone local immunotherapy than in patients who had not. The component structures of RLN, such as sinus, follicle and paracortex, were all enlarged in the OK-432/fbg-treated patients, and necrosis of metastatic tumors was observed. RLN lymphocytes recovered from OK-432/fbg treated patients showed elevated reactivity to phytohemagglutinin (PHA) and the stimulation index was clearly higher than that of control patients. Flow cytometric analysis revealed a predominance of T-cells, especially CD4 subsets, and higher positivity for both CD25 and HLA-DR. Furthermore, RLN lymphocytes killed more effectively K562 and Daudi cells in the patients who had had immunotherapy. These results suggest that the effect of local immunotherapy with OK-432/fbg is not restricted to the site of injection but extends to the lymph nodes, and contributes to tumor regression through the augmentation of cellular immunity.Abbreviations RLN regional lymph node(s) - OK-432/fbg OK-432/fibrinogen solution - PHA phytohemagglutinin - NK natural killer - LAK lymphocyte activated killer     

Accumulation of poly-(hydroxybutyrate) by a non-sulfur photosynthetic bacterium,Rhodobacter sphaeroides RV at different pH

Summary Effect of pH of culture media on intracellular accumulation of poly-(hydroxybutyrate) (PHB) by a non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides strain RV was studied in pH-stat cultures. Sub-optimal pH for growth, 8.0 or 8.5 gave the higher content of PHB rather than optimal pH 7.5 for growth. These results show that growth and PHB accumulation of the bacteria can be controlled by pH of culture media.     

Alcohol and aldehyde dehydrogenase genotypes and drinking behavior of Chinese living in Shanghai

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), the principal enzymes responsible for oxidative metabolism of ethanol, exist in multiple, genetically determined molecular forms. Widely different kinetic properties in some of these isozymes account for the individual differences in alcohol sensitivity. In this study we used the polymerase chain reaction/restriction fragment length polymorphism method to determine the genotypes of the ADH2 and ALDH2 loci of alcoholic and nonalcoholic Chinese living in Shanghai. We also investigated the subjects' drinking patterns by means of semistructured interviews. The alcoholics had significantly lower frequencies of the ADH2² and ALDH2² alleles than did the nonalcoholics, suggesting the inhibitory effects of these alleles for the development of alcoholism. In the nonalcoholic subjects, ADH2² had little, if any, effect, despite the significant effect of the ALDH2² allele in decreasing the alcohol consumption of the individual. Taken together, these results fit the proposed hypothesis for the development of alcoholism, i.e., drinking behavior is greatly influenced by the individual's gentoypes of alcohol-metabolizing enzymes, and the risk of becoming alcoholic is proportionate with the ethanol consumption of the individual.     

Murine defense mechanism against Candida albicans infection. I. Collaboration of cell-mediated and humoral immunities in protection against systemic C. albicans infection

Mice immunized with viable C. albicans cells demonstrated a high incidence of cell-mediated and a low incidence of humoral immune response. There was good agreement between the final survival rate of C. albicans infected mice and the rate of simultaneous cell-mediated and humoral immune response acquisition. Immunized mice with positive delayed hypersensitivity (DTH) against C. albicans crude antigen showed significant protection against intravenous challenge with C. albicans. Furthermore, the transfer of immunoglobulins from rabbit anti-C. albicans serum to DTH-positive mice enhanced protection, while it did not protect control mice against a subsequent challenge with C. albicans. These results suggest that cell-mediated immunity plays a major role and humoral immunity a side role in the defense mechanism(s) of C. albicans infected mice.