Replication initiator protein mRNA of ColE2 plasmid and its antisense regulator RNA are under the control of different degradation pathways

Replication of the ColE2 plasmid requires a plasmid-coded initiator protein (Rep). Rep expression is controlled by antisense RNA (RNAI) against the Rep mRNA at a translational step. In this paper, we examined the effects of host RNA degradation enzymes on the degradation process of the Rep mRNA and its degradation intermediates especially those carrying the 5' untranslated region. We showed that the Rep mRNA is subjected to complex degradation pathways involving at least RNase I, RNase II, RNase III, RNase E, RNase G and PNPase. RNase II acts as a major exoribonuclease and PNPase plays a minor role. We also showed that the PcnB (polyA polymerase I) plays only a minor role in the Rep mRNA degradation process. The RNA degradation pathways of the Rep mRNA and RNAI of the ColE2 plasmid are quite different. Based on these results, we speculate that the ColE2 Rep mRNA and RNAI are endowed with individual RNA half lives required for the efficient copy number control by being subjected to different RNA degradation systems.     

High Cadmium Accumulation Among Humans and Primates: Comparison Across Various Mammalian Species—A Study from Japan

The majority of existing literature reports that cadmium (Cd) is toxic to humans and most living organisms. This paper reports the results of our study that measured Cd levels in the livers and kidneys of humans and other 50 mammalian species under normal conditions in Japan. The study tests the differences in the Cd concentrations across different mammalian species and sexes. Our results revealed that (1) there is a strong correlation between the Cd levels in the livers and kidneys across all examined species, (2) humans exhibit the highest Cd accumulation level in both organs, (3) primates also show a high Cd concentration at a level close to humans, (4) mice and rats show low Cd levels in both organs, indicating that humans accumulate about a few thousand times more Cd than mice and rats, and (5) the Cd concentration of female mammals is more than double of males for both organs. Our results indicate that these cross-sex as well as cross-species discrepancies cannot be explained by the difference in daily Cd intake. While further research is necessary to determine any potential role of Cd accumulation, we speculate that Cd plays some physiological function in the renal cortex of humans and primates.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Dual role of endogenous nitric oxide in development of dextran sodium sulfate-induced colitis in rats.

The role of nitric oxide (NO) in the etiology of ulcerative colitis is controversial with reports of the improvement and aggravation of colonic lesions by inducible NO synthase (iNOS) inhibitors. In the present study, we compared the effect of the selective iNOS inhibitor aminoguanidine and the nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on a dextran sulfate sodium (DSS)-induced model of colitis in rats. Experimental colitis was induced by a 3% DSS-solution added to drinking water for 7 days. Aminoguanidine (5 approximately 20 mg/kg) and L-NAME (10 mg/kg) were administered p.o. twice daily for the first 3 days, the last 3 days or all 6 days of DSS treatment. Body weight and severity of colitis (diarrhea, bloody feces) were observed over a period of 7 days. DSS treatment resulted in severe colonic lesions, accompanied by diarrhea, bloody feces, decrease of body weight and colon shortening. All of the parameters investigated improved significantly with aminoguanidine treatment at 20 mg/kg for 6 days or the last 3 days of DSS-treatment, but L-NAME did not significantly affect the colitis during these periods. When L-NAME or aminoguanidine was given in the first 3 days of DSS treatment, the colonic lesions were slightly aggravated by L-NAME but not affected by aminoguanidine. The expression of iNOS mRNA was observed from the 3(rd) day of DSS treatment. These results suggested that endogenous NO exerts a biphasic influence on DSS-induced colitis, depending on the NOS isoenzyme; a beneficial effect of NO derived from constitutive NOS and a detrimental effect of NO produced by iNOS in the development of colitis.     

Crystal structure determinations of oxidized and reduced pseudoazurins from Achromobacter cycloclastes. Concerted movement of copper site in redox forms with the rearrangement of hydrogen bond at a remote histidine.

The crystal structures of oxidized and reduced pseudoazurins from a denitrifying bacterium, Achromobacter cycloclastes IAM1013, have been determined at 1.35- and 1.6-A resolutions, respectively. The copper site in the oxidized state exhibits a distorted tetrahedral structure like those of other pseudoazurins. However, not only a small change of the copper geometry, but concerted peptide bond flips are identified. The imidazole ring of remote His6 has a hydrogen bonding distance of 2.73 A between N-delta1(His6) and O-gamma1(Thr36) in the oxidized protein. When the protein is reduced at pH 6.0, the imidazole ring rotates by 30.3 degrees and moves 1.00 A away from the position of the oxidized state. A new hydrogen bond between N-epsilon2(His6) and O-epsilon1(Glu4) is formed with a distance of 3.03 A, while the hydrogen bond between N-delta1(His6)-O-gamma1(Thr36) is maintained with an interatomic distance of 2.81 A. A concomitant peptide bond flip of main chain between Ile34 and Thr36 occurs.     

Fluorescence digital image analysis of serotonin-induced calcium oscillations in single blood platelets

The intracellular concentration of Ca2+ [( Ca2+]i) was monitored continuously in single rabbit blood platelets by digital imaging microscopy in conjunction with Fura-2, a specific Ca(2+)-indicator dye. Ionomycin as well as aluminium fluoride caused sustained increase in [Ca2+]i in the platelet, but oscillations of [Ca2+]i were not observed. Serotonin (5-HT) induced oscillatory increases in [Ca2+]i in the presence of 1 mM CaCl2; these had not been detectable in cell populations because the oscillations were not in synchrony. This effect of 5-HT was diminished when CaCl2 was omitted from the medium, and was antagonized by 1 microM ketanserin, a specific 5-HT2 receptor antagonist. Furthermore, DOI, a specific 5-HT2 agonist, had the same effect as 5-HT at lower concentration. A specific effector mechanism, not fully understood at present, therefore appears to mediate 5-HT2 receptors thereby allowing rabbit platelets to generate [Ca2+]i oscillations. It is suggested that protein kinase C in platelets might play a key role in the regulation of [Ca2+]i, and possibly in [Ca2+]i oscillations.     

Production of vitamin B12 in an upflow anaerobic filter continuous reactor using Acetobacterium sp.

The accumulation of biofilm by Acetobacterium sp. during continuous culture in an upflow anaerobic filter (UAF) growing on methanol-formate was the result of space velocity and inlet concentrations of substrate and Co⁺². To achieve good development of biofilm, a space velocity of 0.38 h^–1, inlet substrate concentrations of 125 mM of both methanol and formate, and Co⁺² at 0.16 mM were required. Cell productivities in the effluent of the UAF-reactor were about 6-fold higher than in chemostat cultures (0.20 g l^–1 h^–1 for UAF and 0.035 g l^–1 h^–1 for chemostat) (previous studies), and the maximum vitamin B₁₂ specific concentration was 5.1 mg g cell^–1.