Metabolic response against sulfur-containing heterocyclic compounds by the lignin-degrading basidiomycete Coriolus versicolor

The fungal conversions of sulfur-containing heterocyclic compounds were investigated using the lignin-degrading basidiomycete Coriolus versicolor. The fungus metabolized a series of sulfur compounds--25 structurally related thiophene derivatives--via several different pathways. Under primary metabolic conditions, C. versicolor utilized thiophenes, such as 2-hydroxymethyl-, 2-formyl-, and 2-carboxyl-thiophenes, as a nutrient sulfur source for growth; thus, the fungus degraded these compounds more effectively in a non-sulfur-containing medium than in conventional medium. The product analysis revealed that several redox reactions, decarboxylation reactions, and C-S cleavage reactions were involved in the fungal conversion of non-aromatic thiophenes. On the other hand, benzothiophene (BT) and dibenzothiophene (DBT) skeletons were converted to water-soluble products. All the products and metabolic intermediates were more hydrophilic than the starting substrates. These metabolic actions seemed to be a chemical stress response against exogenously added xenobiotics. These metabolic reactions were optimized under ligninolytic conditions, also suggesting the occurrence of a fungal xenobiotic response. Furthermore, the fungus converted a series of BTs and DBTs via several different pathways, which seemed to be controlled by the chemical structure of the substrates. DBT, 4-methylDBT, 4, 6-dimethylDBT, 2-methylBT, and 7-methylBT were immediately oxidized to their S-oxides. BTs and DBTs with the hydroxymethyl substituent were converted to their xylosides without S-oxidation. Those with carboxyl and formyl substituents were reduced to form a hydroxymethyl group, then xylosidated. These observations strongly suggested the involvement of a fungal substrate-recognition and metabolic response mechanism in the metabolism of sulfur-containing heterocyclic compounds by C. versicolor.     

Improvement of mass transfer characteristics and productivities of inclined tubular photobioreactors by installation of internal static mixers

The feasibility of improving mass transfer characteristics of inclined tubular photobioreactors by installation of static mixers was investigated. The mass transfer characteristics of the tubular photobioreactor varied depending on the type (shape) and the number of static mixers. The volumetric oxygen transfer coefficient ( k(L)a) and gas hold up of the photobioreactor with internal static mixers were significantly higher than those of the photobioreactor without static mixers. The k(L)a and gas hold up increased with the number of static mixers but the mixing time became longer due to restricted liquid flow through the static mixers. By installing the static mixers, the liquid flow changed from plug flow to turbulent mixing so that cells were moved between the surface and bottom of the photobioreactor. In outdoor culture of Chlorella sorokiniana, the photobioreactor with static mixers gave higher biomass productivities irrespective of the standing biomass concentration and solar radiation. The effectiveness of the static mixers (average percentage increase in the productivities of the photobioreactor with static mixers over the productivities obtained without static mixers) was higher at higher standing biomass concentrations and on cloudy days (solar radiation below 6 MJ m(-2) day(-1)).     

Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain

Rhodococcus sp. KT462, which can grow on either benzothiophene (BT) or dibenzothiophene (DBT) as the sole source of sulfur, was newly isolated and characterized. GC and GC-MS analyses revealed that strain KT462 has the same BT desulfurization pathway as that reported for Paenibacillus sp. A11-2 and Sinorhizobium sp. KT55. The desulfurized product of DBT produced by this strain, as well as other DBT-desulfurizing bacteria such as R. erythropolis KA2-5-1 and R. erythropolis IGTS8, was 2-hydroxybiphenyl. A resting cells study indicated that this strain was also able to degrade various alkyl derivatives of BT and DBT.     

Screening of a molecule endowing Saccharomyces cerevisiae with n-nonane-tolerance from a combinatorial random protein library

A combinatorial random protein library was constructed from random DNA fragments generated by "DNA random priming", an improved method of "random-priming recombination" using random-sequence primers and template cDNA from the yeast Saccharomyces cerevisiae. In order to express this library on the yeast cell surface, a yeast multicopy cassette vector was constructed, in which the random-protein-encoding DNA fragments were fused to a gene encoding the C-terminal 320 amino acids of alpha-agglutinin. Fluorescent labeling of the immuno-reaction of RGS(His)(6) epitope confirmed the surface display of random proteins. The surface display of heterologous random proteins on yeast cells will have a wide application. As an example, an n-nonane-tolerant yeast strain that could grow very well in nonane-overlaid culture medium was screened out from transformants displaying this combinatorial library. n-Nonane tolerance was dependent on the transformed plasmid, and the related protein was confirmed to localize on the cell surface by papain treatment and immunofluorescent labeling. Analysis of this displayed protein was also carried out. This strain is the first one to have been endowed artificially with organic solvent tolerance. This is a good example of creating cells exhibiting new phenotypes using a combinatorial protein library.     

Mechanisms by which endogenous glucocorticoid protects against indomethacin-induced gastric injury in rats

We investigated the mechanisms underlying the protective action of glucocorticoids against indomethacin-induced gastric lesions. One-week adrenalectomized rats with or without corticosterone replacement (4 mg/kg sc) were administered indomethacin (25 mg/kg sc), and gastric secretion (acid, pepsin, and mucus), motility, microvascular permeability, and blood glucose levels were examined. Indomethacin caused gastric lesions in sham-operated rats, with an increase in gastric motility and microvascular permeability as well as a decrease in mucus secretion. Adrenalectomy significantly worsened the lesions and potentiated these functional disorders. Glucose levels were lowered by indomethacin in sham-operated rats, and this response was enhanced by adrenalectomy. The changes observed in adrenalectomized rats were prevented by supplementations of corticosterone at a dose mimicking the indomethacin-induced rise in corticosterone, whereas the protective effect of corticosterone was attenuated by RU-38486, a glucocorticoid receptor antagonist. We conclude that the gastroprotective action of endogenous glucocorticoids may be provided by their support of glucose homeostasis and inhibitory effects on enhanced gastric motility and microvascular permeability as well as maintaining the production of mucus.     

Changes in center-of-pressure dynamics during upright standing related to decreased balance control in young adults: fractional Brownian motion analysis

We investigated the relationships between the ability to maintain balance in an upright stance and center-of-pressure (COP) dynamic properties in young adults. Included in this study were 10 healthy male subjects in each of two groups with respect to balance ability. Balance ability was evaluated according to the length of time a subject stood on one leg with his eyes closed. The means and ranges of this one-leg balancing time were 17.9 s (3-43 s) and 118.3 s (103-120 s) for the off-balance and balance groups, respectively. The time-varying displacements of the COP under a subject's feet during quiet two-leg (normal) standing were measured by an instrumented force platform. Each subject was tested in both the eyes-open and eyes-closed conditions. The COP trajectories were analyzed as fractional Brownian motions according to the procedure of 'stabilogram-diffusion analysis', proposed by Collins and De Luca (1993). The extracted parameters were the effective diffusion coefficients (D) for the short-term (less than about 1.0 s) and long-term intervals, respectively, as well as the Hurst exponents (H) for the short-term and long-term intervals, and some critical-point coordinates (i.e., critical mean square displacements and critical time intervals). The off-balance group showed significantly higher values for short-term D, short-term H, and critical mean square displacements than the balance group. No significant differences between the groups were found in the long-term D and H or in the critical time intervals. That is, for the off-balance subjects, an increase in the stochastic activity and positively correlated (persistent) behavior of the postural sway during shorter timescales may cause postural instability. These results suggest that the difference in balance ability for young adults is related to the open-loop (i.e., short-term) control mechanisms but not to the corrective feedback (i.e., long-term) mechanisms used to maintain balance in an upright stance.     

LET dependence of lethality in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> irradiated by heavy ions

To clarify the effect of heavy ions in plants, dry seeds of Arabidopsis were irradiated with carbon, neon, and argon ions with various linear energy transfer (LET) values. The relative biological effectiveness (RBE) for lethality peaked at LET values over 350 keV/microns for neon and argon ions. This LET giving the peak RBE was higher than the LET of 100-200 keV/microns which was reported to have a maximum RBE for other types of cells, such as mammalian cells. Furthermore, sterility showed a higher RBE at an LET of 354 keV/microns with neon ions than that at an LET of 113 keV/microns with carbon ions. Lethality and sterility are both considered to be caused by damage to DNA. The results indicate that the LET having a maximum of RBE for lethality is higher in Arabidopsis seeds than in other unicellular systems. The most likely explanation for this shift of LET is that the DNA in dry seeds has a different chemical environment and/or hydration state than the DNA in cells in culture.     

Human Rad54B is a double-stranded DNA-dependent ATPase and has biochemical properties different from its structural homolog in yeast, Tid1/Rdh54

The RAD52 epistasis group genes are involved in homologous recombination, and they are conserved from yeast to humans. We have cloned a novel human gene, RAD54B, which is homologous to yeast and human RAD54. Human Rad54B (hRad54B) shares high homology with human Rad54 (hRad54) in the central region containing the helicase motifs characteristic of the SNF2/SWI2 family of proteins, but the N-terminal domain is less conserved. In yeast, another RAD54 homolog, TID1/RDH54, plays a role in recombination. Tid1/Rdh54 interacts with yeast Rad51 and a meiosis-specific Rad51 homolog, Dmc1. The N-terminal domain of hRad54B shares homology with that of Tid1/Rdh54, suggesting that Rad54B may be the human counterpart of Tid1/Rdh54. We purified the hRad54 and hRad54B proteins from baculovirus-infected insect cells and examined their biochemical properties. hRad54B, like hRad54, is a DNA-binding protein and hydrolyzes ATP in the presence of double-stranded DNA, though its rate of ATP hydrolysis is lower than that of hRad54. Human Rad51 interacts with hRad54 and enhances its ATPase activity. In contrast, neither human Rad51 nor Dmc1 directly interacts with hRad54B. Although hRad54B is the putative counterpart of Tid1/Rdh54, our findings suggest that hRad54B behaves differently from Tid1/Rdh54.     

Single nucleotide polymorphisms of estrogen receptor alpha in human renal cell carcinoma

Estrogens and their receptors (ERs) have been shown to play a role in various cancers. We hypothesize that polymorphisms and genotypic changes of the ERalpha gene are involved in renal cell carcinoma. To test this hypothesis, DNA samples from 113 cases of human renal cell cancer were analyzed by sequence-specific polymerase chain reaction to determine the genotypic frequency of six different polymorphic loci on ERalpha gene (codon 10 T-->C, codon 87 G-->C, codon 243 C-->T, codon 325 C-->G, codon 594 G-->A, and intron 1 C-->T). The relative risk of variant genotype was calculated by comparison with 200 healthy controls. The results of this study demonstrate that the distribution of genotypes on codon 10 differs between renal cancer patients and healthy normal controls (p<0.05). The relative risk of the genotype 10C/C was calculated as 2.51. No differences in genotypes were observed at all other loci. We also analyzed DNA from pairs of cancerous and normal tissues from 96 cases of human renal cell cancer to characterize genotypic changes at these loci. Genotypic changes were detected in nine cancer samples on exon 1 (codons 10 and 87) of ERalpha, although none were detected at other regions. The present study demonstrates for the first time that codon 10 polymorphism on exon 1 of ERalpha may be involved in renal cancer risk.     

The specific mitochondrial DNA polymorphism found in Klinefelter's syndrome

Hypervariable segments of mitochondrial DNA (mtDNA) (HV1 and HV2) were analyzed in Klinefelter's syndrome and compared to normal population data. One pair of samples consisting of a Japanese mother and affected son with Klinefelter's syndrome (involved in a criminal case), and seven unrelated DNA samples from Caucasian Klinefelter males (two involved in criminal cases and five diagnosed) were collected in Japan and the United States. The diagnosis of Klinefelter's syndrome was established previously by multiplex XY-STR typing detecting two X alleles and one Y allele in the samples. Haplotype analysis of the mtDNA sequence in Klinefelter males was found to be identical, unique, and specific, as it was not found in the normal population. Astonishingly, family data exhibited that the haplotype of the mtDNA in the son was apparently different from the mother's, suggesting that the mtDNA of Klinefelter male would not be inherited from mother to son. Our data indicate that possible interaction of the sex chromosome and the mtDNA exists, and suggests that the specific mtDNA haplotype could cause the abnormal cell to fertilize and reproduce itself.