Improved efficiency of asymmetric hydrolysis of 3-substituted glutaric acid diamides with an engineered amidase.

AIMS: To improve the efficiency of asymmetric hydrolysis of 3-(4-chlorophenyl) glutaric acid diamide (CGD) using a recombinant Comamonas sp. KNK3-7 amidase (CoAM) produced in Escherichia coli. METHODS AND RESULTS: The CoAM gene was cloned, sequenced and found to comprise 1512 bp, encoding a polypeptide of 54 054 Da. CoAM-transformed E. coli were able to perform R-selective hydrolysis of CGD; however, complete conversion of 166·2 mmol l(-1) CGD in 28 h could not be obtained. We attempted to optimize the reactivity of CoAM by mutating single amino acids in the substrate-binding domain. Notably, the methionine-substituted L146M mutant enzyme showed increased reactivity, completing the conversion of 166·2 mmol l(-1) CGD in just 4 h. The Km value for L146M was lower than that of CoAM. CONCLUSIONS: We succeeded in creating the L146M mutant of CoAM with increased substrate affinity and found that this was the best mutant for the hydrolysis of CGD. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing the efficiency of hydrolysis of 3-substituted glutaric acid diamides is useful to improve the synthesis of optically active 3-substituted gamma-aminobutyric acid. This is the first report of efficient hydrolysis of CGD using amidase mutant-producing E. coli cells.

Improvement of (R)-3-hydroxybutyric acid secretion during Halomonas sp. KM-1 cultivation with saccharified Japanese cedar by the addition of urea.

UNLABELLED: Japanese cedar (Cryptomeria japonica) is a major species in artificial Japanese forests. The Halomonas sp. KM-1 was recently isolated and found to grow effectively on saccharified Japanese cedar wood, resulting in the intracellular storage of poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. Under microaerobic conditions, the extracellular secretion of (R)-3-hydroxybutyric acid ((R)-3-HB) led to the degradation of intracellular PHB. In this study, the production of PHB and the secretion of (R)-3-HB using saccharified Japanese cedar were much improved in cultures that were grown in the presence of urea. The level of intracellular PHB production after 36 h under aerobic cultivation was 23·6 g l(-1) ; after shifting to microaerobic conditions for 24 h, the (R)-3-HB concentration in the medium reached 21·1 g l(-1) . Thus, KM-1 efficiently utilizes saccharified Japanese cedar to produce PHB and secretes (R)-3-HB, making it a practical candidate for use in the industrial production of (R)-3-HB. SIGNIFICANCE AND IMPACT OF THE STUDY: Japanese cedar is a major species grown in artificial Japanese forests, and its thinning is crucial for the health of artificial forests and the Japanese economy. Halomonas sp. KM-1 grew effectively on saccharified Japanese cedar wood, resulting in intracellular storage of poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. Under microaerobic conditions, extracellular secretion of (R)-3-hydroxybutyric acid ((R)-3-HB) caused intracellular PHB degradation. (R)-3-HB is a chiral compound that is useful in the chemical, health food and pharmaceutical industries. The production of PHB and secretion of (R)-3-HB using saccharified wood was dramatically improved, which may positively affect its future industrial production.

Microbial asymmetric hydrolysis of 3-substituted glutaric acid diamides.

AIMS: Micro-organisms were screened for their ability to produce (R)-3-(4-chlorophenyl) glutaric acid monoamide (CGM) from 3-(4-chlorophenyl) glutaric acid diamide (CGD) through stereoselective hydrolysis. (R)-CGM is a useful synthetic intermediate for arbaclofen. METHODS AND RESULTS: Four CGD-assimilating micro-organisms were found to be potential catalysts for (R)-CGM production. Among these micro-organisms, Comamonas sp. KNK3-7 (NITE BP-963) produced (R)-CGM with the highest optical purity [98.7% enantiomeric excess (e.e.)] and was selected as the most promising strain. In addition, Comamonas sp. KNK3-7 could asymmetrically hydrolyse 3-isobutyl glutaric acid diamide (IBD) to produce (R)-3-isobutyl glutaric acid monoamide [(R)-IBM] with high optical purity (>99.0% e.e.). CONCLUSION: The synthesis of a (R)-3-substituted glutaric acid monoamide by desymmetrization of 3-substituted glutaric acid diamide with a micro-organism and an enzyme has not been previously reported. This finding indicates the possibility of the preparation of a variety of optically active 3-substituted glutaric acid monoamides using the amidase from Comamonas sp. KNK3-7. SIGNIFICANCE AND IMPACT OF THE STUDY: The amidase from Comamonas sp. KNK3-7 may be useful for the chemoenzymatic synthesis of various kinds of chiral gamma-aminobutyric acids and may be used in a 'green' process to produce gamma-aminobutyric acids.

Circumvention of vincristine and Adriamycin resistance in vitro and in vivo by calcium influx blockers.

Calcium influx blockers, diltiazem, nicardipine, nifedipine, niludipine, and nimodipine, which possess coronary vasodilator activity, greatly enhanced the cytotoxicity of vincristine (VCR) in tumor cells and especially in VCR-resistant sublines of P388 leukemia (P388/VCR) and human K562 myelogenous leukemia. The extent of enhancement was different among the drugs, and up to a 50- to 70-fold increase in VCR cytotoxicity occurred in P388/VCR cells with nontoxic or marginally toxic concentrations of diltiazem and nicardipine. A 50- to 100-fold enhancement occurred in VCR-resistant human K562 myelogenous leukemia cells with diltiazem, nicardipine, niludipine, and nimodipine. VCR resistance of these cell lines was circumvented completely by these blockers. Calcium influx blockers also enhanced the cytotoxicity of Adriamycin in P388 leukemia cells and especially in its Adriamycin-resistant subline. The extent of enhancement, however, was lower than that which occurred in VCR-resistant tumor lines with VCR. An approximately 10- to 30-fold increase in Adriamycin cytotoxicity occurred in P388 Adriamycin-resistant subline cells with diltiazem, nicardipine, niludipine, and nimodipine. Although VCR alone at 10 to 200 micrograms/kg did not confer a significant therapeutic effect in P388/VCR-bearing mice, calcium influx blockers in doses of 30 to 125 mg/kg administered daily for 10 days with VCR enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. A maximum of approximately a 40 to 50% increase in life span occurred with diltiazem, nicardipine, niludipine, and nimodipine. The calcium influx blockers also enhanced the therapeutic effect of Adriamycin in P388 Adriamycin-resistant subline-bearing mice, although the extent of enhancement was smaller than that observed with VCR in P388/VCR-bearing mice.

Post-translational modification is essential for catalytic activity of nitrile hydratase.

Nitrile hydratase from Rhodococcus sp. N-771 is an alphabeta heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, alphaCys112 and alphaCys114 are modified to a cysteine sulfinic acid (Cys-SO2H) and a cysteine sulfenic acid (Cys-SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The alphabeta complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC/MS analysis showed that the anaerobically reconstituted alphabeta complex did not have the modification of alphaCys112-SO2H and aerobic incubation induced the modification. The activity of the reconstituted alphabeta complex correlated with the amount of alphaCys112-SO2H. Furthermore, ESI-LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that alphaCys114 is modified to Cys-SOH together with the sulfinic acid modification of alphaCys112. These results suggest that alphaCys112 and alphaCys114 are spontaneously oxidized to Cys-SO2H and Cys-SOH, respectively, and alphaCys112-SO2H is responsible for the catalytic activity solely or in combination with alphaCys114-SOH.

Pemphigus IgG induces expression of urokinase plasminogen activator receptor on the cell surface of cultured keratinocytes.

We previously found that the binding of pemphigus IgG to desmogleins caused marked activation of phospholipase C, a transient increase in inositol 1,4,5-trisphosphate production, and a concomitant increase in the intracellular calcium concentration in DJM-1 cells, a squamous cell carcinoma line. The binding of pemphigus IgG to cell membranes increased the activity of urokinase plasminogen activator in culture medium and induced subsequent cell-cell detachment in DJM-1 cells. Because urokinase plasminogen activator activates the conversion of plasminogen to plasmin by binding to urokinase plasminogen activator receptor evading inhibitors in serum, it is likely that plasmin is generated only in microenvironments adjacent to urokinase plasminogen activator receptor on the cell surface. It is not known whether pemphigus IgG causes acantholysis by inducing urokinase plasminogen activator receptor expression on the cell surface and secreting urokinase plasminogen activator in inhibitor-rich environments. We examined the effects of pemphigus IgG on urokinase plasminogen activator receptor expression in DJM-1 cells and normal keratinocytes by immunoblot analysis and immunofluorescence microscopy using antibodies to urokinase plasminogen activator receptor. IgG were obtained from serum samples from eight patients with bullous pemphigoid, five patients with pemphigus vulgaris, seven patients with pemphigus foliaceus, and eight normal subjects. Pemphigus vulgaris and pemphigus foliaceus IgG significantly increased the urokinase plasminogen activator receptor expression on the surface of DJM-1 cells and normal keratinocytes after 3- and 7-d incubation compared with normal IgG. These results suggest that enhanced urokinase plasminogen activator activity and urokinase plasminogen activator receptor expression activates plasmin in the limited cell surface of pemphigus IgG-bound keratinocytes and may contribute to the pathogenesis of differential acantholysis in pemphigus vulgaris and pemphigus foliaceus.

Activin A induces terminal differentiation of cultured human keratinocytes.

Activin A, a member of the TGFbeta-superfamily, is well known to play important roles in the growth and differentiation of various target cells. We have previously demonstrated that activin A is produced at an early stage of cultivation at both the protein and the mRNA levels in cultured human keratinocytes. In this study, the effects of activin A on differentiation and proliferation of human keratinocytes were examined. Activin A (> or =1 nM) induced cornified envelope formation and the synthesis of loricrin, keratin 1, involucrin, and transglutaminase 1. In addition, transglutaminase activity and mRNA level of transglutaminase 1 were increased by activin A. [3H]Thymidine incorporation and cell number were reduced by activin A (> or =1 nM) compared with control, suggesting an inhibitory effect of activin A on cell proliferation. On the basis of these findings, it is likely that activin A contributes to differentiation and suppression of proliferation in human keratinocytes.

Amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum.

The amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum has been determined. It is composed of 260 amino acid residues and its N-terminus is acetylated. The molecular mass is calculated to be 29 851 Da. The presence of six calcium-binding sites (I-VI) has been proposed, two of them (sites II and VI) have lost their calcium-binding function through amino acid replacements, and the other four are able to bind calcium. Six calcium-binding domains are supposed to be derived from two gene duplications of the two ancestral calcium-binding domains. In comparison with the sequence of chick intestinal calcium-binding protein deduced from a cDNA sequence [(1985) Nucleic Acids Res. 13, 8867-8881], the bovine calcium-binding protein is two amino acid residues shorter at the N-terminus and the other parts show 78.5% identity.

Expression of activin A in human keratinocytes at early stages of cultivation.

Activins are members of the TGF-beta superfamily and are classified into 3 types: activin A, which consists of a homodimer of betaA, activin B, which consists of a homodimer of betaB, and activin AB, which consists of a heterodimer of betaAbetaB. We studied the expression of activin mRNAs by RT-PCR in normal human epidermis, cultured keratinocytes, and DJM-1 cells (a squamous cell carcinoma line). We could detect only activin A mRNA (betaA) in normal human epidermis. In cultured keratinocytes and DJM-1 cells, activin betaA mRNA was observed at 4 h but not at 96 h after plating. Activin A activity was detected in the conditioned medium of DJM-1 cells within 48 h. In addition, although follistatin mRNA was not observed in human epidermis in situ, it was transiently expressed in cultured cells at 4 h after plating. These findings suggest that the expression of these molecules in keratinocytes is associated with cell proliferation. In an in vitro tissue injury model, activin A was observed at the wound edge, where cell migration and proliferation may be activated. In DJM-1 cells cultured for 92 h, betaA mRNA was observed 4 h after injury treatment. These findings suggest that activin A acts as a potent inducer of proliferation in vitro, at least in keratinocytes.

Cobalt-substituted Fe-type nitrile hydratase of Rhodococcus sp. N-771.

When the genes encoding alpha and beta subunits of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 were expressed in Escherichia coli in Co-supplemented medium without co-expression of the NHase activator, the NHase specifically incorporated not Fe but Co ion into the catalytic center. The produced Co-substituted enzyme exhibited rather weak NHase activity, initially. However, the activity gradually increased by the incubation with an oxidizing agent, potassium hexacyanoferrate. The oxidizing agent is likely to activate the Co-substituent by oxidizing the Co atom to a low-spin Co(3+) state and/or modification of alphaCys-112 to a cysteine-sulfinic acid. It is suggested that the NHase activator not only supports the insertion of an Fe ion into the NHase protein but also activates the enzyme via the oxidation of its iron center.

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine.

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.

Determination of the active sites serine of the poly (3-hydroxybutyrate) depolymerases of Pseudomonas lemoignei (PhaZ5) and of Alcaligenes faecalis.

Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S138 (P. lemoignei) and S139 (A. faecalis) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase box of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S195 (P. lemoignei) and S196 (A. faecalis), resulted in mutant proteins with almost full activity and proved that S195 and S196 are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.

Behavioral, electroencephalographic and histopathological studies on mongolian gerbils with occluded common carotid arteries.

The effect of brain ischemia on passive avoidance test was investigated in mongolian gerbils with ischemia induced by a 5 min bilateral occlusion of the carotid arteries. Severe impairment of memory was apparent when the training session of the passive avoidance test was carried out 2 or 14 days after the bilateral ischemia. Two days after the occlusion, the amplitude of hippocampal theta waves were slightly decreased and Nissl's degradation was apparent in the CA1 neurons in the hippocampus. The changes in hippocampal neurons become progressively more severe. The amplitude of the hippocampal theta waves diminished considerably and the CA1 neurons in the hippocampus disappeared 14 days after the occlusion. We suggest that the hippocampal damage, especially abnormalities in the CA1 neurons, evidenced by histopathological and electroencephalographic results, may be related to deficits in memory following bilateral common carotid arteries occlusion.

Fe-type nitrile hydratase.

The characteristic features of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 are described. Through the biochemical analyses, we have found that nitric oxide (NO) regulates the photoreactivity of this enzyme by association with the non-heme iron center and photoinduced dissociation from it. The regulation is realized by a unique structure of the catalytic non-heme iron center composed of post-translationally modified cysteine-sulfinic (Cys-SO2H) and -sulfenic acids (Cys-SOH). To understand the biogenic mechanism and the functional role of these modifications, we constructed an over-expression system of whole NHase and individual subunits in Escherichia coli. The results of the studies on several recombinant NHases have shown that the Cys-SO2H oxidation of alphaC112 is indispensable for the catalytic activity of Fe-type NHase.

Arginine 56 mutation in the beta subunit of nitrile hydratase: importance of hydrogen bonding to the non-heme iron center.

Arginine 56 in the beta subunit (betaArg56) of the iron-containing nitrile hydratase (NHase), one of the strongly conserved residues within the NHase family, is known to form hydrogen bonds to the sulfinyl (-SO2H) and sulfenyl (-SOH) groups of the post-translationally modified cysteine residues in the catalytic center. BetaArg56 was substituted by tyrosine, glutamate or lysine, respectively, and the respective mutant enzymes generated by reconstitution were characterized. The betaR56K mutant complex exhibited about 1% of the enzymatic activity of native NHase, while the others were totally inactive. The kinetic analysis of the betaR56K mutant complex exhibited a drastic decrease in turnover number and decreases in kinetic constants for substrate and inhibitors as compared to the native NHase. Changes in UV-visible absorption and light-induced Fourier transform infrared difference spectra suggest that betaArg56 is involved in the positioning of the -SO2H and -SOH groups of the modified Cys residues in the catalytic center so as to fine tune the electronic state of the iron center suitable for catalysis. Thus, betaArg56 is essential for catalysis.

Partial amino acid sequence of an amyloid fibril protein from unusual cutaneous cystic lesions in myeloma-associated amyloidosis.

Although common cutaneous lesions in myeloma-associated systemic amyloidosis are petechiae, purpura, ecchymoses, plaques, waxy, translucent or purpuric papules or nodules, we encountered an unusual case of myeloma-associated amyloidosis with multiple cystic nodules. We isolated amyloid substance from the cutaneous cystic nodules of this patient and characterized it ultrastructurally, immunologically, and biochemically. Electron microscopy demonstrated that amyloid substances isolated by distilled water were principally straight and non-branching fibrils with a diameter of 8 to 10 nm, which was morphologically similar to amyloid fibrils. SDS-PAGE showed that these fibrils consisted of the 20 kDa and 29 kDa peptides, which reacted with the antibody to kappa light chain of immunoglobulin by immunoblot study. Partial amino acid sequence of N-terminal residues of this 20 kDa peptide showed a homology to kappa immunoglobulin light chain of variable subgroup I. These results suggest that amyloid fibrils in this unusual case with cutaneous cystic nodules may be derived from kappa I light chain of immunoglobulin.

Screening of fungal beta-xylanases for production of acidic xylooligosaccharides using in situ reduced 4-O-methylglucuronoxylan as substrate.

Fungal beta xylanases were screened for production of acidic xylooligomers from 4-O-methylglucuronoxylan. In situ reduced hardwood xylan was used as substrate because the products of neutral- and acidic-branched xylooligomers help define substrate specificity of the enzymes. Borohydride reduction in situ transformed 30% of 4-O-methyl-alpha-D-glucopyranosyluronic acid residues into 4-O-methyl-alpha-D-glucopyranosyl residues and reduced C-1 end groups in the xylan backbones. A total of ten beta-xylanase fractions from four fungi were partially purified by chromatography by anion exchange and molecular sieving, and graded qualitatively for enzymatic hydrolysis of the substrate. The yield of acidic xylooligomers was highly affected by whether alpha-glucuronidases were present in the beta-xylanase fractions. Some fractions gave free 4-O-methyl-alpha-D-glucopyranosyluronic acid, but none of the enzyme fractions could release free 4-O-methyl-alpha-D-glucose. Among the beta-xylanase fractions studied, xylanase II of Trichoderma viride was the best producer of aldotetraouronic acid [2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylotriose]. The results obtained suggested that there was a difference in the steric hindrance of the branch point on fungal beta-xylanases between 4-O-methyl-alpha-D-glucopyranosyl and 4-O-alpha-D-glucopyranosyluronic acid residues.

Simple bone cyst: investigation on the presence of gas in the cavity using computed tomography--review of 52 cases.

OBJECTIVE: The purpose of this study was to evaluate whether gas is present in the cavities of simple bone cysts. STUDY DESIGN: Computed tomographic images of 52 simple bone cyst cases were reviewed and compared with the findings at surgery. RESULTS: Whereas gas was confirmed at the time of surgery in 28 cases, no computed tomographic images indicated the presence of gas in the cavity. Bone expansion was noted in 13 of these 28 cases. CONCLUSIONS: From these findings, we concluded that the presence of gas in the simple bone cyst cavity at surgery has been erroneously interpreted.

Effect of various classes of drugs on complete ischemia induced by decapitation and cyanide intoxication in mice.

Effects of various classes of drugs on complete ischemia (gasping) induced by decapitation and cyanide intoxication in mice were investigated. The average gasping duration in complete ischemia and survival time in potassium cyanide injection of control mice were 21.0 +/- 0.1 and 26.1 +/- 0.4 sec (mean +/- SEM), respectively. Some antidepressants, neuroleptics and tranquilizers significantly and dose-dependently prolonged the duration of gasping and survival time, and decreased locomotor activity. Vincamine and its analogues are markedly effective for treating cyanide intoxication. It is conceivable that this is a characteristic phenomenon for vincamine. Xanthine analogues significantly and dose-dependently shortened the duration of gasping and survival time and increased locomotor activity. These results suggest that the cerebral protecting effect primarily relates to the cerebral energy demand.

[Correlation of cerebro-cardiovascular findings to mortality of middle-aged and elderly in a population from a fishing village in Shizuoka Prefecture].

Six hundred and eighteen men and 993 women ranging in age from 30-69, living in Kamo Village, Shizuoka Prefecture, underwent baseline health examinations in 1964-1966. During the follow-up period of 20-years, 175 men and 170 women died. The most frequent cause of death was malignant neoplasms (57 men and 45 women), followed by stroke (47 men and 44 women) and heart disease (29 men and 37 women). The relationship of 22 cerebro-cardiovascular disease variables investigated in the baseline examination to stroke and heart disease mortalities, and, in addition, to cancer and all-cause mortalities were analyzed using Cox's proportional hazard model. In univariate analysis controlled for age and sex, systolic and diastolic blood pressures, albuminuria, hypertensive and sclerotic changes in fundus oculi, body mass index, atrial fibrillation, and use of antihypertensive drugs had significant positive relationships to stroke mortality. As for heart disease mortality, albuminuria, glucosuria, high R wave, and ST and/or T changes in ECG were positively and significantly related. Only Q.QS in ECG significantly correlated with cancer mortality. Variables showing significant relationship to all-cause mortality were systolic and diastolic blood pressures, albuminuria, glucosuria, hypertensive changes in fundus oculi, Q.QS, high R wave, ST and/or T changes, atrial fibrillation, use of antihypertensive drugs, and history of stroke. In multivariate analysis (step-wise) of all examination variables including age and sex, stroke mortality was significantly related to age, atrial fibrillation, use of antihypertensive drugs, and hypertensive changes in fundus oculi. Age, albuminuira, and ST changes in ECG were significantly related to heart disease mortality. Age, sex, and Q.QS in ECG were significantly related to cancer mortality. The relationship of age, sex, albuminuria, Q.QS, and ST changes in ECG to all-cause mortality was significant.