Efficient Expression of Peroxidatic Activity of Catalase in the Coupled System with Glucose Oxidase—a model of Yeast Peroxisomes

Catalase functioned exclusively to degrade hydrogen peroxide in a reaction mixture containing methanol and hydrogen peroxide, while, when the enzyme was coupled with glucose oxidase, successful conversion of methanol to formaldehyde occurred at the optimized ratio of glucose oxidase to catalase: activity, 1.0 × 10 ^?3; number of molecules, 1.3; protein content, 1. These values in the coupled system were very similar to the ratio of alcohol oxidase to catalase in peroxisomes, one of the subcellular organelles from a methanol-assimilating yeast, Kloeckera sp. 2201, in which these enzymes were coupled to metabolize methanol efficiently. The presence of the optimum ratio in the coupled system in vitro was confirmed by the kinetic analysis of the expression of the peroxidatic activity of catalase coupled with glucose oxidase. Construction of the immobilized system of the coupled enzymes at the optimum ratio demonstrated that the oxidation of methanol through the peroxidatic function of catalase could be continuously and stably operated, the results indicating the usefulness of the system as a model of yeast peroxisomes. Thus, the coupled reaction with glucose oxidase brought out the latent function of catalase, which could not be expected in the system including only catalase.     

Antibacterial Activity of the Lactoperoxidase System Combined with Edible Laminaria Hot-Water Extract as a Source of Halide Ions

Hot-water extracts prepared from nine out of 12 samples of dried edible Laminaria reduced the viable numbers of Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, and Esherichia coli below the detection limit after incubation for 5 min when combined with lactoperoxidase, glucose oxidase, and glucose. Some extracts showed higher bactericidal activity and a higher OI^? concentration in the assay mixture after ultrafiltration.     

The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide—Localization of a putative binding site

Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high‐affinity titanium dioxide (TiO₂)–binding protein (TiBP), purified from Rhodococcus ruber , has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO₂‐binding capabilities with TiBP. Intrigued by the unique TiO₂‐binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti‐based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein‐TiO₂ surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO₂, indicating that neither the enzyme undergoes major conformational changes nor the TiO₂ binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO₂ through similar regions located far from the active site and the dimerization sites. The putative TiO₂‐binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal‐binding sites in proteins.     

Production of Recombinant Der fI with the Native IgE-Binding Activity Using a Baculovirus Expression System

Der fI is a cysteine protease contained in feces of mites and is one of major mite allergens. Recombinant Der fI (reDer fI) that is produced using a baculovirus expression system contains pro-sequences of different lengths. Most of these can be removed by acid treatment. However, IgE-binding activity of acid-treated reDer fI is lower than that of native Der fI at high protein concentrations, and N-terminal amino acids of acid-treated reDer fI are not uniform. Now, a method for processing of the pro-sequence has been developed by producing reDer fI E(?1)K with baculovirus expression system in which the carboxy terminal amino acid of the pro-sequence (glutamate) was replaced by lysine using site directed mutagenesis. No difference in the amount of production was observed upon introducing the mutation into the pro-sequence. Addition of lysylendopeptidase into the culture medium led to processing of the pro-sequence of reDer fI E(?1)K and proceeded the degradation of the other proteins in the medium. Lysylendopeptidase-treated reDer fI E(?1)K was easily purified with an anion exchange column, resulting in 20% increase of the yield. Lysylendopeptidase-treated reDer fI E(?1)K obtained through these processes was compared with the native Der fI. Although some differences were found in protease activity and reactivity with lectins, their N-terminal amino acid and the IgE-binding activity were the same as those of the native one, indicating its usefulness for diagnostic purpose.     

Studies on the Nucleic Acid and Protein Content in the Cotyledon Cells of Lotus Using Fluorescent Imaging System with a Scientific grade Charge Coupled Device

Enzyme dissociated cotyledon cells were obtained from red lotus ( Nelumbo sp. ) on the 5th day and 20th day after fertilization respectively. DNA, RNA and total protein content of individual 5 d and 20 d cell were measured correlatively by microfiuorescent imaging system with a cooled scientific-grade Charge coupled deviced (CCD) camera. The results showed that DNA content of the 20 day cell has become polyploidized from 4C to 8C approximately. The window analysis indicated that relative content of RNA and total protein of the 20 day cell was more than 11 times that of the 5 day cell in the same 4C DNA window respectively. The multiple in-creased of RNA and total protein content was about the same as that of DNA in 2 DNA windows (4C and 8C) of the 20 day cell. These data showed that the enormous accumulation of storage proteins in the cotyledon cells of red lotus depends on both DNA ploidy and selective expression of the subunit genes of the storage proteins during the developmental process.     

Association of the + 45T > G adiponectin gene polymorphism with insulin resistance in non-diabetic Saudi women

Background

The human adiponectin gene variations are associated with obesity, insulin resistance, and diabetes. However, these associations have not been fully examined in a non-diabetic population in Saudi Arabia. We aimed to investigate the association of 45T > G single nucleotide polymorphism (SNP) in the adiponectin gene with total adiponectin levels, insulin resistance (IR), fasting blood glucose (FBG) and other markers of obesity in non-diabetic Saudi females.

Methods

One hundred non diabetic Saudi females were enrolled in this study. They were further divided according to their body mass index (BMI) into two groups. Group I, 46 non diabetic subjects with normal body weight and group II, 54 overweight and obese females. Adiponectin 45T/G polymorphism was detected by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). Serum adiponectin was measured by ELISA.

Results

Obese women exhibited a higher distribution of TG/GG genotype compared with non-obese women. SNP + 45T > G genotypes were associated with higher FBG, insulin levels and HOMA–IR with lower total adiponectin levels in obese Saudi women. Otherwise the all estimated variables revealed non-significant differences among the non-obese genotypes. The observed differences in insulin resistance markers were very significant among women with a higher body weight but not among normal body weight women, thus suggesting that SNP + 45T > G effects on insulin sensitivity may depend upon body weight and body fat status.

Conclusion

SNP + 45T > G of adiponectin gene has a significant role in the development of insulin resistance in Saudi women possibly through an interaction with increase body weight and hypoadiponectinemia.     

Design and synthesis of new antitumor agents with the 1,7-epoxycyclononane framework. Study of their anticancer action mechanism by a model compound

This article describes the design, synthesis and biological evaluation of a new family of antitumor agents having the 1,7-epoxycyclononane framework. We have developed a versatile synthetic methodology that allows the preparation of a chemical library with structural diversity and in good yield. The synthetic methodology has been scaled up to the multigram level and can be developed in an enantioselective fashion. The study in vitro of a model compound, in front of the cancer cell lines HL-60 and MCF-7, showed a growth inhibitory effect better than that of cisplatin. The observation of cancer cells by fluorescence microscopy showed the presence of apoptotic bodies and a degradation of microtubules. The study of cell cycle and mechanism of death of cancer cells by flow cytometry indicates that the cell cycle arrested at the G₀/G₁ phase and that the cells died by apoptosis preferably over necrosis. A high percentage of apoptotic cells at the subG₀/G₁ level was observed. This indicates that our model compound does not behave as an antimitotic agent like nocodazole, used as a reference, which arrests the cell cycle at G₂/M phase. The interaction of anticancer agents with DNA molecules was evaluated by atomic force microscopy, circular dichroism and electrophoresis on agarose gel. The results indicate that the model compound has not DNA as a target molecule. The in silico study of the model compound showed a potential good oral bioavailability.