Characterization of peroxidase in buckwheat seed

A peroxidase (POX)-containing fraction was purified from buckwheat seed. The POX consisted of two isozymes, POX I and POX II, that were purified 6.6- and 67.4-fold, respectively. Their molecular weights were estimated to be 46.1 kDa (POX I) and 58.1 kDa (POX II) by gel filtration. While POX I and II each oxidized quercetin, o-dianisidine, ascorbic acid and guaiacol, only POX II oxidized ABTS. Kinetic studies revealed that POX I and II had lower K(m) values for quercetin (0.071 and 0.028 mM), ABTS (0.016 mM for POX II) and ascorbic acid (0.043 and 0.029 mM) than for o-dianisidine (0.229 and 0.137 mM) and guaiacol (0.288 and 0 ). The optimum pHs of POX I and II for various substrates were almost the same, except for quercetin; pH 8.0 for POX I and pH 4.5 for II. Their optimal temperatures were 30 degrees C (POX I) and 10 degrees C (POX II), and POX I was more stable than POX II above 30 degrees C.     

High‐throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy

Protein aggregation is an essential molecular event in a wide variety of biological situations, and is a causal factor in several degenerative diseases. The aggregation of proteins also frequently hampers structural biological analyses, such as solution NMR studies. Therefore, precise detection and characterization of protein aggregation are of crucial importance for various research fields. In this study, we demonstrate that fluorescence correlation spectroscopy (FCS) using a single‐molecule fluorescence detection system enables the detection of otherwise invisible aggregation of proteins at higher protein concentrations, which are suitable for structural biological experiments, and consumes relatively small amounts of protein over a short measurement time. Furthermore, utilizing FCS, we established a method for high‐throughput screening of protein aggregation and optimal solution conditions for structural biological experiments.     

Selective production of two diastereomers of disaccharide sugar alcohol, mannosylerythritol by Pseudozyma yeasts

Mannosylerythritol (ME) is the hydrophilic backbone of mannosylerythritol lipids as the most promising biosurfactants produced by different Pseudozyma yeasts, and has been receiving attention as a new sugar alcohol. Different Pseudozyma yeasts were examined for the sugar alcohol production using glucose as the sole carbon source. P. hubeiensis KM-59 highly produced a conventional type of ME, i.e., 4-O-β-d-mannopyranosyl-d-erythritol (4-ME). Interestingly, P. tsukubaensis KM-160 produced a diastereomer of 4-ME, i.e., 1-O-β-d-mannopyranosyl-d-erythritol (1-ME). In shake flask culture with 200 g/l of glucose, strain KM-59 produced 4-ME at a yield of 33.2 g/l (2.2 g/l/day of the productivity), while strain KM-160 produced 1-ME at 30.0 g/l (2.0 g/l/day). Moreover, the two strains were found to produce ME from glycerol; the maximum yields of 4-ME and 1-ME from 200 g/l of glycerol were 16.1 g/l (1.1 g/l/day) and 15.8 g/l (1.1 g/l/day), respectively. The production of 1-ME as the new diastereomer was further investigated in fed batch culture using a 5-l jar-fermenter. Compared to the flask culture, strain KM-160 gave three times higher productivity of 1-ME at 38.0 g/l (6.3 g/l/day) from glucose and at 31.1 g/l (3.5 g/l/day) from glycerol, respectively. This is the first report on the selective production of two diastereomers of ME, and should thus facilitate the functional development and application of the disaccharide sugar alcohol in the food and relative industries.     

Genome Sequence of a Novel HIV-1 Circulating Recombinant Form 54_01B from Malaysia

We report here the first novel HIV-1 circulating recombinant form (CRF) 54_01B (CRF54_01B) isolated from three epidemiologically unlinked subjects of different risk groups in Malaysia. These recently sampled recombinants showed a complex genome organization composed of parental subtype B′ and CRF01_AE, with identical recombination breakpoints observed in the gag, pol, and vif genes. Such a discovery highlights the ongoing active generation and spread of intersubtype recombinants involving the subtype B′ and CRF01_AE lineages and indicates the potential of the new CRF in bridging HIV-1 transmission among different risk groups in Southeast Asia.     

Structure and Bitterness in Flavanone Glycosides

Naringenin-7-β-kojibioside, -7-β-sophoroside, -7-[α-d-galactosyl(l→2)β-d-glucoside], -7-[β-d-glucosyl(l→2)β-d-galactoside], and also hesperetin-7-β-kojibioside and -7-β-sophoroside were prepared by the coupling of naringenin or hesperetin with the α-acetobromo derivatives of the appropriate disaccharides, followed by saponification.

Their relative bitterness values were discussed in comparison with naringin and neo-hesperidin.     

Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain

Previous studies indicated that single-headed smooth muscle myosin and S1 (a single head fragment) are not regulated through phosphorylation of the regulatory light chain (RLC). To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of Delta MD lacked the motor domain, that of Delta(MD+ELC) lacked the motor and essential light chain binding domains, and single-headed HMM had one intact head alone. The basal ATPase activities of the three mutants decreased as the KCl concentration became less than 0.1 M. Such a decrease was not observed for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of Delta MD and Delta(MD+ELC), but that of single-headed HMM, were phosphorylated. When their RLCs were unphosphorylated, the three mutants exhibited similar actin-activated ATPase levels. However, when they were phosphorylated, the actin-activated ATPase activities of Delta MD and Delta(MD+ELC) increased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We propose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs. We also propose that a cooperative interaction between two motor domains is required for a higher level of actin activation.     

Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline.

The structural gene (FDH1) coding for NAD(+)-dependent formate dehydrogenase (FDH) was cloned from a genomic library of Candida boidinii, and the FDH1 gene was disrupted in the C. boidinii genome (fdh1 delta) by one-step gene disruption. In a batch culture experiment, although the fdh1 delta strain was still able to grow on methanol, its growth was greatly inhibited and a toxic level of formate was detected in the medium. In a methanol-limited chemostat culture at a low dilution rate (0.03 to 0.05 h[-1]), formate was not detected in the culture medium of the fdh1 delta strain; however, the fdh1 delta strain showed only one-fourth of the growth yield of the wild-type strain. Expression of FDH1 was found to be induced by choline or methylamine (used as a nitrogen source), as well as by methanol (used as a carbon source). Induction of FDH1 was not repressed in the presence of glucose when cells were grown on methylamine, choline, or formate, and expression of FDH1 was shown to be regulated at the mRNA level. Growth on methylamine or choline as a nitrogen source in a batch culture was compared between the wild type and the fdh1 delta mutant. Although the growth of the fdh1 delta mutant was impaired and the level of formate was higher in the fdh1 delta mutant than in the wild-type strain, the growth defect caused by FDH1 gene disruption was small and less severe than that caused by growth on methanol. As judged from these results, the main physiological role of FDH with all of the FDH1-inducing growth substrates seems to be detoxification of formate, and during growth on methanol, FDH seems to contribute significantly to the energy yield.     

Immunohistochemical study on fetal raphe samples transplanted into the leptomeningeal tissue of 5,6-dihydroxytryptamine-treated adult rats

Summary Pieces of fetal midbrain raphe containing serotonergic and dopaminergic neurons were transplanted into the leptomeningeal tissue (see Fig. 3) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. One, 2 and 5 months after transplantation, the rate of neuronal survival in the grafted tissue and the extent of axonal outgrowth into the host brain were studied by use of serotonin and tyrosine hydroxylase (TH) immunohistochemistry. The survival rate of the grafts in the 1-month group was approximately 70%. Neurons containing either serotonin or catecholamine were demonstrated by means of immunocytochemical procedures in the grafts. Two and 5 months after transplantation, serotonin-immunoreactive nerve fibers were densely distributed throughout the graft tissue, while TH-immunoreactive fiber elements were restricted to an area near the somata of TH-positive neurons. Numerous serotonin-immunoreactive fibers derived from the transplant were found in the leptomeningeal tissue surrounding the graft, on the wall of neighboring blood vessels, and also in the adjacent parenchyma of the host brain. Outgrowing TH-immunoreactive nerve fibers were not observed in the host brain, although such elements occurred in the leptomeningeal tissue and the wall of the larger blood vessels. These results suggest that the serotonergic and catecholaminergic (dopaminergic) neurons located in transplants of the raphe nuclei show different patterns when reinnervating the host tissue.     

Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.     

Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.