DNA hydrolysis by rare-earth metal ions.

Plasmid DNA and poly(dA) are cleaved by rare-earth(III) ions at pH 7-8 and 50 degrees C. The cleavage has been confirmed by prompt conversion of supercoiled pBR 322 plasmid DNA (Form I) to a relaxed Form II. Furthermore, degradation of poly(dA) to shorter oligonucleotides is clearly evidenced by HPLC. A possible application of the metal ions (and their complexes) to artificial nucleases is indicated.     

Transformation of a Methylotrophic Bacterium, Methylobacterium extorquens, with a Broad-Host-Range Plasmid by Electroporation

An efficient method for the transformation of the methylotrophic bacterium Methylobacterium extorquens NR-2 with a broad-host-range plasmid, pLA2917, by electroporation was examined. Transformants of M. extorquens NR-2 expressing resistance to kanamycin were obtained after electric pulse. These transformants were found to harbor a single plasmid which was electrophoretically identical and homologous to pLA2917 obtained from Escherichia coli. Several factors which determined the transformation efficiency were optimized, resulting in a transformation efficiency of up to 8 × 10³ transformants per μg of plasmid DNA by 10 pulses at a field strength of 10 kV/cm and a pulse duration of 300 μs.     

Possible endocrine control by hepatocyte growth factor of liver regeneration after partial hepatectomy.

Hepatocyte Growth Factor (HGF), which is a most potent growth factor for primary cultured hepatocytes, may act as a trigger for liver regeneration. After 70% of the rat liver was removed, HGF activity in the remnant liver began to increase within 24 h. In parallel with the activity, the HGF mRNA level in the remnant liver increased at 12 h after the operation and reached a maximum at 24 h. Increases in HGF activity and in the mRNA level were much lower and later than those in the liver of rats with hepatitis induced with CCl4. However, the first increase in HGF activity in the plasma of hepatectomized rats was noted 3 h after the resection, that is much earlier than the initial DNA synthesis in the remnant liver. Thus, while HGF production was induced in the remnant liver during regeneration after partial hepatectomy, the initial trigger may not be the liver-derived HGF, rather, it may be HGF derived from extrahepatic organs, via blood circulation.     

Possible mechanism of the preventive effect of BCG against diabetes mellitus in NOD mouse. I. Generation of suppressor macrophages in spleen cells of BCG-vaccinated mice.

With the aim of clarifying the mechanism of the suppressive action of BCG against insulitis and overt diabetes in NOD mice, we studied the effects of BCG on spleen cell populations and on the in vitro immune responses of spleen cells. The spleen cells of BCG-vaccinated mice showed much lower responsiveness to various mitogens such as Con A, PHA, PWM, and LPS than those of saline-treated mice. Low responsiveness to alloantigens was also observed. Flow cytometric analysis of the spleen cells revealed that Mac-1+ and Mac-2+ cells had increased while T and B cells had decreased in the BCG-vaccinated mice compared with the saline-treated mice at the time when the maximum level of inhibition of mitogen responses of BCG-vaccinated mice was observed. This suggests that the decreased in vitro immune response was due to the increase in macrophages which suppress lymphocyte functions. Support for this interpretation comes from the following two findings: (1) the restoration of mitogen responses of spleen cells when macrophages were eliminated by plastic adhesion or FACS sorting and (2) resuppression of PHA and Con A responses of plastic-nonadherent spleen cells by addition of adherent cells or flow cytometrically sorted Mac-1+ cells obtained from BCG-vaccinated mice. These results indicate the generation of suppressor macrophages after BCG vaccination and suggest that these macrophages prevent the autoimmune pathogenesis leading to diabetes in NOD mice.     

Structure of the fission yeast centromere cen3: Direct analysis of the reiterated inverted region

We determined the structure of the Schizosaccharomyces pombe centromere cen3 using direct genomic mapping and cosmid walking. The repetitive region of cen3 is approximately 110 kb, much longer than that of the previously determined cen1 and cen2 regions. The 30 kb long left and 60 kb right repetitive sequences are arranged with an inverted symmetry and flank the 1520 kb central domain. The repeat motifs in cen3, although they consist of the common centromeric repeat elements, are slightly different from those in cen1 and cen2. The cen3 repeat motifs appear to be reiterated four times in the left and nine times in the right side repetitive regions. We found that the central domain consists of the common 5 kb core sequence associated with the pair of innermost inverted sequences, most of which are reiterated only twice in the genome. Although their sizes differ significantly, the general features of cen1, cen2 and cen3 are similar, and a prototype, consensus structure for the fission yeast centromere may be deduced.by J.A. Huberman     

Two-step mobilization of arachidonic acid in platelet activation induced by low concentrations of TP 82, a monoclonal antibody against CD9 antigen.

Arachidonic acid mobilization in platelets activated by low concentrations (less than or equal to 1.6 micrograms/ml) of TP 82, a monoclonal antibody against CD9, appears to consist of two distinct phases. In the first phase, limited arachidonic acid release occurs concomitantly with a shape change induced by TP 82. This appears to be dependent upon phospholipase A2 activation, since it is well preserved in the presence of aspirin, which completely blocked both intracellular Ca2+ elevation and phosphatidic acid formation which would indicate phospholipase C activation. The Na+ Exchange was also found to participate in the first phase of arachidonic acid mobilization, since extracellular Na+ depletion and ethylisopropylamiloride, a specific inhibitor of the Na+/H+ exchanger, effectively blocked this limited mobilization of arachidonic acid. The second, much larger, phase of arachidonic acid mobilization occurs with the beginning of platelet aggregation. A limited amount of thromboxane A2 formed during the first phase of arachidonic acid release plays an important role in induction of the massive arachidonic mobilization in the second phase. Factors, as yet unidentified, also appear to work synergistically with thromboxane A2 to induce the full picture of arachidonic acid mobilization.     

Reversible effects of sodium butyrate on the differentiation of F9 embryonal carcinoma cells

We have studied effects of sodium butyrate on embryonal carcinoma F9 cell differentiation. In the presence of sodium butyrate, F9 cells underwent rapid and drastic morphological changes and expressed marked increases in mRNA levels of various differentiation markers. When sodium butyrate was removed from the cultures, all the examined phenotypes of F9 cell differentiation rapidly reverted to the characteristics of undifferentiated stem cells. However, under the same conditions, when cycloheximide or actinomycin D was added to the cultures, such phenotypic reversion was not observed, but high mRNA levels of the differentiation markers as well as altered cell morphology were retained. These results indicated that the effects of sodium butyrate on induction of teratocarcinoma cell differentiation were reversible and that de novo syntheses of some mRNA(s) and protein(s) were necessary for the reversion of differentiated cells to stem cells.     

Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes non-specific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidin-biotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

Age-related changes of glucose metabolism in rat cerebral cortex with reference to glucose-derived amino acids

The parietal cortical slices obtained from 8 week-old (young) and 78 week-old (middle-aged) male Wistar rats were incubated withd-[U-¹⁴C]glucose in oxygensaturated Gey's balanced salt solution. Subsequently, the radioactivities of liberated CO₂ and glucose-derived amino acids (alanine, aspartate, GABA, glutamate and glutamine) obtained from the slices were measured. In the middle-aged rats as compared to the young rats, the amount of radioactivity of CO₂ (P<0.01) and glutamate (P<0.05) showed a significant raduction with glutamine unchanged, while that of alanine (P<0.01), aspartate (P<0.05) and GABA (P<0.05) increased significantly. The results indicate that with advancing age the overall glucose oxidation in the cerebral cortex declines but the metabolic pathway to form amino acids is not uniformly suppressed. Therefore, the above characteristic glucose metabolism could be related to the development of heterogeneous enzyme activities associated with aging in the brain.