Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°C. The pH range for growth is 5.0–9.0, with an optimum around 7.0. Strain RG-20 requires 1%–4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 μg/ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si′cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349). Received: May 11, 1998 / Accepted: July 24, 1998     

Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and beta-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and beta-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.     

Modulation of rat cecal microbiota by administration of raffinose and encapsulated Bifidobacterium breve

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.     

Cell structure degradation in Escherichia coli and Thermococcus sp. strain Tc-1-95 associated with thermal death resulting from brief heat treatment

The thermal death mechanism of microorganisms when heated at lethally high temperatures is still not fully understood. In this study, we examined the relationship between thermal death and degradation of the cell structure in the mesophilic bacterium Escherichia coli strain W3110 and the hyperthermophilic archaeon Thermococcus sp. strain Tc-1-95. By heating the microorganisms at lethally high temperatures only briefly (1.5 s duration) in a flow-type apparatus, we studied the microbial cells at very early and critical stages of the thermal death process. For E. coli, it was found that the loss of viability was not associated with thermal damage to the cell envelope. Deformation of the nucleoid was observed. These results suggest that the thermal death of E. coli is attributed to thermal denaturation or degradation of cytoplasmic molecules. On the other hand, the thermal death of Thermococcus sp. strain Tc-1-95 was strongly associated with rupture of the cell envelope. Furthermore, massive deformation of the S-layer with lethal thermal stress was observed. These results demonstrate that the thermal deaths of the two microorganisms investigated proceed via very different mechanisms. The contrast can be attributed to the difference in their cell envelope structures.     

Involvement of IL-10 and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells

To analyze the possibility that immunological alteration in asbestos-related diseases (ARDs) such as asbestosis (ASB) and malignant mesothelioma (MM) may affect the progression of cancers, a human adult T cell leukemia virus-immortalized T cell line (MT-2Org) was continuously exposed to 10 μg/ml of chrysotile-B (CB), an asbestos. After at least 8 months of exposure, the rate of apoptosis in the cells became very low and the resultant subline was designated MT-2Rst. The MT-2Rst cells were characterized by (i) enhanced expression of bcl-2, with regain of apoptosis-sensitivity by reduction of bcl-2 by siRNA, (ii) excess IL-10 secretion and expression, and (iii) activation of STAT3 that was inhibited by PP2, a specific inhibitor of Src family kinases. These results suggested that the contact between cells and asbestos may affect the human immune system and trigger a cascade of biological events such as activation of Src family kinases, enhancement of IL-10 expression, STAT3 activation and Bcl-2 overexpression. This speculation was partially confirmed by the detection of elevated bcl-2 expression levels in CD4 + peripheral blood T cells from patients with MM compared with those from patients with ASB or healthy donors. Further studies will be required to verify the role of T cells with enhanced bcl-2 expression in tumor progression induced by asbestos exposure.     

Induction by mycoplasmas of tumor necrosis factor-alpha from macrophages

Several species of mycoplasmas including M. pneumoniae, the causative agent of human respiratory infection, were investigated for tumor necrosis factor-alpha (TNF-alpha) induction. The cytotoxic activity to Meth A cells of peritoneal macrophages purified from BALB/c mice was enhanced markedly when cultured with either viable or nonviable mycoplasmas. The supernatant of macrophage culture mixed with mycoplasmas, M. pneumoniae or A. laidlawii, showed a potent cytotoxic activity to TNF-alpha-sensitive but not to TNA-alpha-insensitive L cells. Addition of anti-TNA-alpha antiserum inhibited completely the cytotoxic activity of the supernatant, indicating that the cytotoxic activity is due mostly to TNF-alpha. These results strongly suggest that mycoplasmas possess an activity to induce TNF-alpha, which enhances the cytotoxic activity of macrophages and prevent infection with mycoplasmas in vivo.     

The Influence of 5-HTTLPR Genotype on the Association between the Plasma Concentration and Therapeutic Effect of Paroxetine in Patients with Major Depressive Disorder

Introduction

The efficacy of treatment with selective serotonin reuptake inhibitors in patients with major depressive disorder (MDD) can differ depending on the patient''s serotonin transporter-linked polymorphic region (5-HTTLPR) genotype, and the effects of varying plasma concentrations of drugs can also vary. We investigated the association between the paroxetine plasma concentration and clinical response in patients with different 5-HTTLPR genotypes.

Methods

Fifty-one patients were enrolled in this study. The Montgomery-Asberg Depression Rating Scale (MADRS) was used to evaluate patients at 0, 1, 2, 4, and 6 weeks. The patients'' paroxetine plasma concentrations at week 6 were measured using high-performance liquid chromatography. Additionally, their 5-HTTLPR polymorphisms (alleles S and L) were analyzed using a polymerase chain reaction with specific primers. We divided the participants into two groups based on their L haplotype: the SS group and the SL and LL group. We performed single and multiple regression analyses to investigate the associations between MADRS improvement and paroxetine plasma concentrations or other covariates for each group.

Results

There were no significant differences between the two groups with regard to demographic or clinical data. In the SS group, the paroxetine plasma concentration was significantly negatively correlated with improvement in MADRS at week 6. In the SL and LL group, the paroxetine plasma concentration was significantly positively correlated with improvement in MADRS at week 6 according to the results of the single regression analysis; however, it was not significantly correlated with improvement in MADRS at week 6 according to the results of the multiple regression analysis.

Conclusion

Among patients with MDD who do not respond to paroxetine, a lower plasma concentration or a lower oral dose of paroxetine might be more effective in those with the SS genotype, and a higher plasma concentration might be more effective in those with the SL or LL genotype.     

Thinopyrum 7Ai-1-derived small chromatin with Barley Yellow Dwarf Virus (BYDV) resistance gene integrated into the wheat genome with retrotransposon

Thinopyrum intermedium is a useful source of resistance genes for Barley Yellow Dwarf Virus (BYDV), one of the most damaging wheat diseases. In this study, wheat/Th. intermedium translocation lines with a BYDV resistance gene were developed using the Th. intermedium 7Ai-1 chromosome. Genomic in situ hybridization (GISH), using a Th. intermedium total genomic DNA probe, enabled detection of 7Ai-1-derived small chro-matins containing a BYDV resistance gene, which were translocated onto the end of wheat chromosomes in the lines Y95011 and Y960843. Random amplified polymorphic DNA (RAPD) analyses using 120 random 10-mer primers were conducted to compare the BYDV-resistant translocation lines with susceptible lines. Two primers amplified the DNA fragments specific to the resistant line that would be useful as molecular markers to identify 7Ai-1-derived BYDV resistance chromatin in the wheat genome. Additionally, the isolated Th. intermedium-specific retrotransposon-like sequence pTi28 can be used to identify Th. intermedium chromatin transferred to the wheat genome.     

N-(2-Pyridyl)acetamide complexes of palladium(II), cobalt(II), nickel(II), and copper(II)

N-(2-Pyridyl)acetamide (aapH) complexes of palladium(II), cobalt(II), nickel(II), and copper(II) have been studied by means of magnetic susceptibilities, and infrared, electronic, and PMR spectra. In the octahedral complexes M(aapH)₂X₂(M = Co, Ni, Cu; X = Cl, Br, NCS, NO₃), bidentate aapH is chelated through the pyridine-N and amid-O atomes, whereas in the square-planar Pd(aapH)₂X₂ (X = Cl, Br) unidentate aapH is coordinated through the pyridine-N atom alone. Under alkaline conditions aapH is deprotonated in the presence of palladium(II) to form Pd(aap)₂·4H₂O, aap being an anionic bidentate ligand and chelating through the pyridine-N and amide-O atoms.