Production of S-lactoylglutathione by glycerol-adapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells

Summary The enzymatic production of S-lactoylglutathione was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli cells dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevisiae cells was markedly induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-fold higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5 mmol/1 (2 g/l) of S-lactoylglutathione was produced from 10 mM methylglyoxal and 50 mM glutathione within 1 h. The extracts of E. coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for production of S-lactoylglutathione and, under optimal conditions, about 40 mmol/l (15 g/l) of S-lactoylglutathione was produced from 50 mM methylglyoxal and 100mM glutathione within 1 h.     

The promoter of Milk vetch dwarf virus component 8 confers effective gene expression in both dicot and monocot plants

The activity of a predicted promoter, PMC8, from Milk vetch dwarf virus was evaluated by comparing it with the cauliflower mosaic virus 35S RNA promoter (P35S) and PNCR, a promoter from Soybean chlorotic mottle virus. When the GUS fusion gene was introduced into tobacco, PMC8 showed a similar expression profile to P35S but with a more intense expression in proliferating tissues. The usefulness of PMC8 was confirmed by driving NPTII for selection of kanamycin-resistant tobacco plants with improved transformation efficiency. PMC8 was also effective in transgenic rice plants. Thus, PMC8 is useful as an alternative to P35S in both dicotyledonous and monocotyledonous plants, especially for gene expression in proliferating tissues.     

Elucidations of the Catalytic Cycle of NADH-Cytochrome b5 Reductase by X-ray Crystallography: New Insights into Regulation of Efficient Electron Transfer

NADH-Cytochrome b₅ reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b₅ (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68 Å resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD⁺. Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78 Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.     

Degradation of corn fiber by Clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein CbpA

Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.     

Gentamicin Components,Produced by a New Isolate of Micromonospora

A novel expression system was developed for the high level production of a labile protein in Escherichia coli. The regulatory signal of bacteriophage T4 uvsY gene was fused in frame with the coding region of human ventricular myosin alkali light chain (VLC1) gene. Expression from the regulatory signal was enhanced and continued in a lysis-inhibition state by infection with a cytosine-substituting T4 phage mutant. VLC1 protein was produced at a low level without infection because of its instability in the cells. Although the productivity was partly improved in a lon-deficient mutant without infection, it was improved about 100-fold with T4 phage infection. T4 phage produces protease inhibitor(s) (pin gene product) against proteases of host cell including the lon gene product (protease La).     

Increased risk of temporomandibular joint closed lock: a case-control study of ANKH polymorphisms

Objectives

This study aimed to carry out a histological examination of the temporomandibular joint (TMJ) in ank mutant mice and to identify polymorphisms of the human ANKH gene in order to establish the relationship between the type of temporomandibular disorders (TMD) and ANKH polymorphisms.

Materials and Methods

Specimens from the TMJ of ank mutant and wild-type mice were inspected with a haematoxylin and eosin staining method. A sample of 55 TMD patients were selected. Each was examined with standard clinical procedures and genotyping techniques.

Results

The major histological finding in ank mutant mice was joint space narrowing. Within TMD patients, closed lock was more prevalent among ANKH-OR homozygotes (p = 0.011, OR = 7.7, 95% CI 1.6–36.5) and the elder (p = 0.005, OR = 2.4, 95% CI 1.3–4.3).

Conclusions

Fibrous ankylosis was identified in the TMJ of ank mutant mice. In the human sample, ANKH-OR polymorphism was found to be a genetic marker associated with TMJ closed lock. Future investigations correlating genetic polymorphism to TMD are indicated.