Enzymatic resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase

2-(Substituted phenoxy)-1-propanols, e.g. 2-(4-chlorophenoxy)-1-propanol, belonging to primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, as judged by the value of enantiomeric ratio E (up to 27), through the enantioselective acylation with vinyl butanoate mediated by the little-known lipase from Achromobacter sp. in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors.     

Formaldehyde Fixation Contributes to Detoxification for Growth of a Nonmethylotroph, Burkholderia cepacia TM1, on Vanillic Acid

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [¹⁴C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.     

Functional relationship between bovine brain phosphodiesterase activator protein and bovine heart troponin C

Phosphodiesterase activator protein has been purified from bovine brain and its properties compared with that of bovine heart troponin C. While both proteins activate ‘activator depleted phosphodiesterase’ in the presence of Ca²⁺, a 200-fold greater concentration of troponin C was necessary and the maximal effect was less than that with the activator protein. The activator protein formed a Ca²⁺ -dependent complex with bovine heart troponin I during electrophoresis in 6 M urea-polyacrylamide gel. However, the mobility of this complex was different from that of troponin C · troponin I complex and the affinity between troponin C and troponin I was much stronger than that between the activator protein and troponin I. Ca²⁺ induced changes in the electrophoretic mobility of activator protein and the pattern of its elution during gel filtration which were similar to the Ca²⁺-dependent conformational changes observed with troponin C. Bovine heart troponin I reduced basal, troponin C and the activator protein stimulation of phosphodiesterase activity. These results are compatible with the concept that phosphodiesterase activator protein and troponin C might have a functional relationship.     

Accumulation of Nucleic Acid-related Substances by Microorganisms

The induction of adenosine-producing mutants from an inosine-producing mutant previously derived from a Bacillus strain was attempted, and it was found out that the xanthine-requiring mutants lacking of adenase produce a large amount of adenosine.

The outline of the processes for the derivation of these mutants was described. Main product of these mutants was adenosine, and the culture broth contained a little amount of adenine as a by-product.

The culture conditions optimal for the production of adenosine were investigated, and the yield of adenosine in the culture broth was more than 16 mg/ml.     

Purification and Characteristics of an Autolytic Chitinase of Piromyces communis OTS1 from Culture Medium

An autolysis chitinase was purified from the cultural medium of the anaerobic fungus Piromyces communis OTS1 by ammonium sulfate precipitation, affinity chromatography with regenerated chitin, chromato-focusing, gel filtration, and chromato-focusing again. The optimal pH and temperature were 6.0 and 50°C, respectively, for a 20-min assay. The chitinase was stable from pH 6.0 to 8.0, but was unstable at 70°C for 20 min. The molecular mass of chitinase was estimated by SDS-PAGE to be 44.9 kDa, and its pI was 4.4. The enzyme activity, which was of the ‘endo’ type, was inhibited by Hg²⁺ and allosamidin. The chitinase hydrolyzes chitin powder and fungal cell walls at a higher rate than an artificial chitin substrate. It can be concluded that extracellular chitinase is similar to cytosolic chitinase, but they are not the same protein. Received: 3 December 1996 / Accepted: 28 January 1997     

Identification of intracellular target proteins of the calcium-signaling protein S100A12.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.     

Strain difference in pituitary-adrenal axis between Wistar-Imamichi and Long Evans adult male rats.

In the present study, we used closed colony-Wistar-Imamichi (WI), inbred WI and Long Evans (LE) adult male rats to examine the secretion of ACTH and corticosterone in response to restraint stress. Blood (0.3 ml) was withdrawn through a jugular cannula at 0, 15, 30, 60 and 120 min after the onset of restraint stress. Plasma concentrations of ACTH and corticosterone increased after stress in all groups, but the responses of ACTH and corticosterone secretion were higher in LE rats than in WI rats. Present data suggest that the LE rat might be a good model as a high-response strain and the closed colony or the inbred WI rat might be a good model as a low-response strain in restraint stress experiments.     

Analysis of Genetic Relationships and Antimicrobial Susceptibility of Verotoxin-Producing Escherichia coli Strains Isolated in Nagasaki Prefecture,Japan in 1996

A total of 19 Escherichia coli O157 isolates were obtained in Nagasaki Prefecture, in the southwestern part of Japan, between 1990 and 1996. Pulsed-field gel electrophoresis (PFGE) and computer-assisted analysis were applied to determine genetic relationships among these strains. Fragment patterns of the isolates in Nagasaki, as determined by PFGE, were compared with those of isolates in other areas where large outbreaks and sporadic cases of E. coli O157 infection occurred. Similarity values of all the strains isolated in Nagasaki Prefecture were over 0.65 except for E. coli O26. Some strains were identical to the strains isolated from the areas where large outbreaks occurred. All strains were susceptible to ampicillin, fosfomycin, minocycline, amikacin, ofloxacin and sulfamethoxazole-trimethoprim.     

Biosynthesis of Thiamine Pyrophosphate in Escherichia coli

In Escherichia coli, thiamine pyrophosphate is synthesized from thiamine monophosphate. Free thiamine is not involved as an intermediate in de novo synthesis of thiamine pyrophosphate.