A new method for improving the enantioselectivity of lipase-catalyzed hydrolysis in organic solvent containing a small amount of water in the presence of metal ions

The hydrolysis of racemic butyl 2-(4-substituted phenoxy)propionates having various substituents catalyzed by lipase MY from Candida rugosa was achieved in di-isopropyl ether containing 0.75% (v/v) of 2.4 M LiCl or 1.2 M MgCl₂ aqueous solution. Water molecules hydrated to the metal ion in isopropyl ether acted as a nucleophile to cause the hydrolysis of these esters as with water alone. Metal ions used significantly enhanced their enantioselectivities by 100-fold or above, as compared with the ordinary reaction media.     

Photosynthetic activities of a thermophilic blue-green alga

Photosynthetic activities of a thermophilic blue-green alga,a species of Synechococcus, were studied with special referenceto its growth at high temperatures. A rapid algal growth occurredin the temperature range between 50 and 60?C, showing the maximumrate, six doublings per day, at about 57?C. Photosynthetic oxygenevolution and methyl viologen photoreduction in the cells werealso active at high temperatures and the optimum temperaturesfor these activities agreed with that of the algal growth. Thegrowth and photosynthetic activities were very low at room temperatureand irreversibly inactivated at temperatures above 60?C. The thylakoid membranes isolated from the alga were also photochemicallyactive at high temperatures. The membranes mediated ferricyanidephotoreduction coupled with a stoichiometric oxygen evolutionat a rate comparable to that of photosynthetic oxygen evolutionin the cells. The optimum temperature for the reaction was ashigh as 50?C. The membranes also showed a photosystem I-mediatedreaction at high temperatures. These observations indicate thatthe thylakoid membranes are intrinsically thermophilic in thisorganism. Thus the growth of the alga at high temperatures canbe well correlated to thermophilic properties of the photosyntheticapparatus. (Received February 20, 1978; )     

Molecular typing of Pasteurella pneumotropica isolated from rodents by amplified 16S ribosomal DNA restriction analysis and pulsed-field gel electrophoresis

A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed-field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with Hae III revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty-two percent of the 23 isolates identified as a-1 were derived from mice, whereas all the isolates identified as a-3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a-2 and a-4, respectively. By restriction analysis of genomic DNA, Apa I and Not I digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a-2 and a-4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with Hae III.     

A Basic Study on the Biological Monitoring for Vanadium—Effects of Vanadium on Vero Cells and the Evaluation of Intracellular Vanadium Contents

A high concentration of vanadium (V) has toxic effects on human and animals and is one of environmental pollutants. In the present study, we have conducted a fundamental study using cultured Vero cells from monkey kidney for the future environmental monitoring. Orthovanadate (VAN), one of V compounds, of 10⁻¹⁰ and 10⁻⁸ M did not affect the cell growth although the higher concentration of above 10⁻⁶ M VAN inhibited the cell growth accompanied with the decrease in cell numbers and morphological changes. Given that the washing method with ice-cold Li is also effective for determination of the cellular Na content, we used this method for the determination of the V content of the Vero cells. The V distributions in Vero cell; in the 10⁻³ M VAN solution, extracellular and intracellular were obtained as 1:0.564:0.036 and 1:0.662:0.098 at 60 and 120 min after the treatment of VAN. The intracellular V content was 10% of the applied concentration of VAN. Consequently, it was suggested that V concentration of 10⁻⁷ and 10⁻⁶ M in the tissue and environment, respectively, might become the threshold concentration; a criterion of the environmental contamination when we carry out environmental monitoring.     

A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences

MOTIVATION: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis. RESULTS: Based on the structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3'-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5'-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.     

Human recombinant stem cell factor promotes spermatogonial proliferation,but not meiosis initiation in organ culture of newt testis fragments

We previously showed that mammalian FSH stimulates the proliferation of newt spermatogonia and induces their differentiation into primary spermatocytes in vitro. In the current study, to examine a possibility that stem cell factor (SCF) is involved in the proliferation of newt spermatogonia and/or their differentiation into primary spermatocytes, human recombinant SCF (rhSCF) was added to organ culture of testicular fragments. rhSCF was found to stimulate the spermatogonial proliferation and the spermatogonia progressed to the seventh generation that is the penultimate stage before primary spermatocyte stage. However, the spermatogonia did not differentiate into primary spermatocytes, but instead died of apoptosis. These results indicate that rhSCF promotes the proliferation of newt spermatogonia, but not the initiation of meiosis.     

Time course alterations of myocardial endothelin-1 production during the formation of exercise training-induced cardiac hypertrophy

Endothelin (ET)-1 is produced by endothelial cells and cardiac myocytes. ET-1 has positive inotropic and chronotropic effects on the heart and causes myocardial cell hypertrophy. Exercise training induces a physiologic cardiac hypertrophy. To study whether myocardial ET-1 is involved in the formation of exercise training-induced cardiac hypertrophy, we investigated time-course alterations of myocardial ET-1 gene expression and ET-1 peptide level in the heart of rats during a formative process of exercise training-induced cardiac hypertrophy. We used the hearts of rats that had been exercise-trained for 4 weeks (4WT) or 8 weeks (8WT) and sedentary control rats for 4 weeks (4WC) or 8 weeks (8WC). Exercise-trained rats performed treadmill running for 5 days/week (60 mins/day). Left ventricular mass index and wall thickness and stroke volume index, measured using echocardiography, in the 8WT group were significantly greater than in the 8WC group, although there were no differences between the 4WC and 4WT groups in these parameters. These results indicated that the 8WT rats developed physiologic cardiac hypertrophy, whereas the 4WT rats did not yet have cardiac hypertrophy. Myocardial ET-1 gene expression and tissue ET-1 concentration in the heart were significantly higher in the 8WT group than in the 8WC group, whereas these values did not differ between the 4WC and 4WT groups. The present study suggests that an alternation of myocardial ET-1 production corresponds with the formation of exercise training-induced cardiac hypertrophy. Therefore, the exercise training-induced change in myocardial ET-1 production may participate in a mechanism of exercise training-induced cardiac adaptation (e.g., cardiac hypertrophy).     

Peripheral tolerance and the qualitative characteristics of autoreactive T cell clones in primary biliary cirrhosis

Primary biliary cirrhosis is characterized by autoreactive T cells specific for the mitochondrial Ag PDC-E2(163-176). We studied the ability of eight T cell clones (TCC) specific for PDC-E2(163-176) to proliferate or become anergic in the presence of costimulation signals. TCC were stimulated with either human PDC-E2(163-176), an Escherichia coli 2-oxoglutarate dehydrogenase mimic (OGDC-E2(34-47)), or analogs with amino acid substitutions using HLA-matched allogeneic PBMC or mouse L-DR53 fibroblasts as APC. Based on their differential responses to these peptides (human PDC-E2(163-176), E. coli OGDC-E2(34-47)) in the different APC systems, TCC were classified as costimulation dependent or independent. Only costimulation-dependent TCC could become anergic. TCC with costimulation-dependent responses to OGDC-E2 become anergic to PDC-E2 when preincubated with mimic, even if costimulation is independent for PDC-E2(163-176). Anergic TCC produced IL-10. One selected TCC could not become anergic after preincubation with PDC-E2(163-176)-pulsed L-DR53 but became anergic using L-DR53 pulsed with PDC-E2 peptide analogs with a substitution at a critical TCR binding site. TCC that only respond to peptide-pulsed PBMC, but not L-DR53, proliferate with peptide-pulsed CD80/CD86-transfected L-DR53; however, anergy was not induced with peptide-pulsed L-DR53 transfected with only CD80 or CD86. These data highlight that costimulation plays a dominant role in maintaining peripheral tolerance to PBC-specific Ags. They further suggest that, under specific circumstances, molecular mimicry of an autoantigen may restore rather than break peripheral tolerance.     

Sulfite induces adherence of polymorphonuclear neutrophils to immobilized fibrinogen through activation of Mac-1 beta2-integrin (CD11b/CD18)

Sulfite is a major air pollutant which can cause respiratory tract inflammation characterized by an influx of polymorphonuclear neutrophils (PMN). We have previously shown that human PMN can produce sulfite either spontaneously or in response to stimulation with lipopolysaccharide. We now demonstrate that sulfite activates PMN to adhere to immobilized fibrinogen via the beta2-integrin Mac-1 (CD11b/CD18). Mac-1 expression is not altered by treatment with this agent. Although unaffected by pertussis toxin, sulfite-triggered PMN adhesion was abrogated by pretreating cells with the membrane-impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a modifier of thiol groups on the cell surface. These results suggest that sulfite-induced PMN adhesion is dependent on a modification of thiols at the cell surface. Given its potent antioxidant and antimicrobial activities, sulfite may act as an endogenous mediator in host defense and/or inflammation.