Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin

Class A beta-lactamases are known to hydrolyze substrates through a Ser70-linked acyl-enzyme intermediate, although the detailed mechanism remains unknown. On the basis of the tertiary structure of the active site, the role of Glu166 of class A enzymes was investigated by replacing the residue in RTEM-1 beta-lactamase with Ala, Asp, Gln, or Asn. All the mutants, in contrast to the wild-type, accumulated a covalent complex with benzylpenicillin which corresponds to an acyl-enzyme intermediate. For the Asp mutant, the complex decayed slowly and the hydrolytic activity was slightly retained both in vivo and in vitro. In contrast, the other mutants lost the hydrolytic activity completely and their complexes were stable. These results indicate that the side-chain carboxylate of Glu166 acts as a special catalyst for deacylation. Residues for deacylation have not been identified in other acyl enzymes, such as serine proteases and class C beta-lactamases. Furthermore, the acyl-enzyme intermediates obtained are so stable that they are considered to be ideal materials for crystallographic studies for elucidating the catalytic mechanism in more detail. In addition, the mutants can more easily form inclusion bodies than the wild-type, when they are produced in a large amount, suggesting that the residue also plays an important role in proper folding of the enzyme.     

Phosphorylation and subcellular distribution of calpastatin in human hematopoietic system cells

Calpastatin is an endogenous inhibitor protein acting specifically on calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase). The phosphorylation of calpastatin was investigated in human hematopoietic system cell lines. Microheterogeneity of calpastatin was observed, in which 118- and 116-kDa forms were named calpastatin a and b, respectively. The phosphorylation of both calpastatins was identified in all cell lines examined and occurred mainly at serine residues with trace amounts of phosphothreonine in vivo. The incubation of cells with 12-O-tetradecanoylphorbol-13-acetate increased the incorporation of 32P-orthophosphate into calpastatin a. Two-dimensional maps of 32P-labeled phosphopeptide from both calpastatins were identical except for additional minor spots for calpastatin a. [35S]methionine-labeled calpastatins a and b were localized mainly in the cytosol, and only 6% of cellular calpastatins were detected in the membrane fraction. By contrast, more than 30% of the 32P-labeled calpastatins a and b were distributed in the membrane fraction. Thus, the phosphorylation of calpastatin may be involved in regulating the calpain-calpastatin protein kinase system by its subcellular distribution.     

Study of bilirubin metabolism by high-performance liquid chromatography: stability of bilirubin glucuronides

The stabilities of bilirubin (BR) glucuronide, monoglucuronide (BMG), and diglucuronide (BDG) were studied under various conditions by HPLC. In aqueous media, BMG showed a pronounced lability and was easily transformed into equimolar BDG and BR. It was proved by direct analysis of tetrapyrrole isomers that BDG and BR were formed from dipyrrole exchange of BMG molecules. All reducing agents examined (sodium ascorbate, cysteine, GSH, dithiothreitol, NADH, and NADPH) suppressed the transformation of BMG into BDG and BR. Bovine serum albumin and rat liver cytosol fractions also stabilized BMG strongly. BDG was fairly stable in aqueous media as compared with BMG. When BMG was incubated both with and without liver plasma membranes (N2 fraction) from Wistar rats, the formation rates of BDG and BR in both incubation mixtures were exactly the same. The composition of BDG and BR isomers was the same in both mixtures. Also, heat denaturation of the plasma membranes did not affect formation rates. Moreover, the reaction was completely inhibited by sodium ascorbate. These findings indicate that rat liver plasma membranes have no enzyme activity for BDG formation from BMG.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

How is autonomic nervous system activity in subjects who are sleepy but are unable to sleep in the daytime?

We investigated changes in the activity of the autonomic nervous system (ANS) in the relaxed condition in subjects who felt sleepy, but were unable to sleep. A total of 1021 subjects underwent daytime polysomnography. The sleep latency (SL) and the visual analog scale (VAS) were used to assess “immediate” objective and subjective sleepiness, respectively. The subjects were assigned to an “Alert-Alert” group (VAS ≤ 25 mm, SL ≥ 8 min), a “Sleepy-Alert” group (VAS ≥ 75 mm, SL ≥ 8 min), or a “Sleepy-Sleepy” group (VAS ≥ 75 mm, SL ≤ 4 min). In order to assess the ANS, the spectral analysis and the geometric method were used. The ANS data collected during the relaxed condition (after lights off, post-LO) was compared to that obtained during the control condition (before lights off, pre-LO). From the spectral analysis, a significant decrease of sympathetic function and an increase of parasympathetic function at post-LO in the Sleepy-Sleepy group, a tendency for sympathetic function decrease at post-LO in the Alert-Alert group, and no significant changes to sympathetic and parasympathetic function in the Sleepy-Alert group were observed. The results from the geometric method supported the results of the spectral analysis in the Alert-Alert group and the Sleepy-Sleepy group. The results of this study suggest that the ANS plays a role in individuals who are unable to sleep even though they feel sleepy and are given the opportunity to sleep.

     

Bsr-REMI: An improved method for gene tagging using a new vector inDictyostelium

Using a plasmid pBsr2 which carries a blasticidin S-resistant gene, we have improved the method of REMI (restriction enzyme-mediated integration) provided for insertional mutagenesis inDictyostelium discoideum (bsr-REMI). To confirm usefulness of thebsr-REMI, transformation efficiency, copy number of integrated DNA, and randomness of integration into genome were examined.     

Unprecedented olefin-dependent histidine-kinase inhibitory of zerumbone ring-opening material

Zerumbone ring-opening derivative, 4 (10E/10Z=3/2), inhibited autophosphorylation of the essential histidine-kinase YycG existing in Bacillus subtilis constituting a two-component system (TCS). Generation of 4E-form could be regulated chemically using the difference from the ring-opening reactivity of the precursor forming of 4 and pure 4E was isolated. The stereoisomer, 4E, showed main inhibition activity of autophosphorylation of YycG (IC(50)=63.5 microM).     

Engineered sorbitol accumulation induces dwarfism in Japanese persimmon

A cDNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH), which is a key enzyme in sorbitol biosynthesis in Rosaceae, was introduced into the Japanese persimmon (Diospyros kaki) to increase the environmental stress tolerance. Resultant transformants exhibited salt-tolerance with dwarfing phenotypes. Therefore, we studied two transgenic lines to understand the physiological mechanism of this dwarfism: lines PS1 and PS6 accumulated high and moderate levels of sorbitol, respectively. The average length of shoots was significantly shorter as compared with the wild-type in line PS1, while no such decrease was observed in line PS6. The myo-inositol and glucose 6-phosphate (G6P) contents were measured in the transgenic lines because previous work with tobacco transformed with S6PDH had suggested that growth inhibition was due to depletion of these metabolites. Although the myo-inositol content was decreased in PS1 plants, the decrease was much smaller than that observed in transgenic tobacco that accumulates sorbitol. The G6P contents were the same in PS1 plants and phenotypically normal PS6 plants. The level of indole-3-acetic acid (IAA), which affects stem elongation, in line PS1 was similar to the levels in the other lines. A decrease in gibberellin (GA) content generally induces dwarfism in plants. However, GA was not decreased in PS1 plants compared with wild-type or control plants. Therefore, we focused on sorbitol accumulation as the most remarkable feature of PS1 plants. As one possibility, the observed growth inhibition was likely caused by an osmotic imbalance between the cytosol and vacuole.