Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE

Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinesis, when their extracts were treated with E-64.     

Psychological stress contributed to the development of low-grade fever in a patient with chronic fatigue syndrome: a case report

Background

Low-grade fever is a common symptom in patients with chronic fatigue syndrome (CFS), but the mechanisms responsible for its development are poorly understood. We submit this case report that suggests that psychological stress contributes to low-grade fever in CFS.

Case presentation

A 26-year-old female nurse with CFS was admitted to our hospital. She had been recording her axillary temperature regularly and found that it was especially high when she felt stress at work. To assess how psychological stress affects temperature and to investigate the possible mechanisms for this hyperthermia, we conducted a 60-minute stress interview and observed the changes in the following parameters: axillary temperature, fingertip temperature, systolic blood pressure, diastolic blood pressure, heart rate, plasma catecholamine levels, and serum levels of interleukin (IL)-1β and IL-6 (pyretic cytokines), tumor necrosis factor-α and IL-10 (antipyretic cytokines). The stress interview consisted of recalling and talking about stressful events. Her axillary temperature at baseline was 37.2°C, increasing to 38.2°C by the end of the interview. In contrast, her fingertip temperature decreased during the interview. Her heart rate, systolic and diastolic blood pressures, and plasma levels of noradrenaline and adrenaline increased during the interview; there were no significant changes in either pyretic or antipyretic cytokines during or after the interview.

Conclusions

A stress interview induced a 1.0°C increase in axillary temperature in a CFS patient. Negative emotion-associated sympathetic activation, rather than pyretic cytokine production, contributed to the increase in temperature induced by the stress interview. This suggests that psychological stress may contribute to the development or the exacerbation of low-grade fever in some CFS patients.

     

Stable nuclear transformation of the diatom Chaetoceros sp.

A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5–6.0 transformants per 10⁸ cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture.     

Clinical analysis on steroids. XII. Occurrence of D-homoannulation during the hot acid hydrolysis of 5β-pregnane-3α,20α-diol disulfate

Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III).     

Investigation of the microbial community in a microbiological additive used in a manure composting process

The objectives of this study were to investigate the fate of microorganisms by using cultivation methods as well as DNA analyses in a commercial microbiological additive (MA) in the course of the composting. Almost all the predominant species in the microbial succession during composting process determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were in disagreement with those determined by the clone library method. None of the microbial species in the composting stages corresponded to the microorganisms identified in the MA either by the cultivation method or DNA analysis. The results in regard to predominant microorganisms of the MA detected from the liquid medium by the PCR-DGGE did not correspond with those detected from the MA itself and composting processes. Although no evidence was found that predominant species in the MA itself dominate in the composting process, predominant species diversity in the MA itself was markedly changed after culturing at different thermophilic temperatures. These results suggested that cultivable microorganisms in the MA did not become predominant in the composting process: however, some microorganisms that are detected from the MA itself by the DNA analysis may act effectively in the composting process.     

Induction of SCEs in CHL cells by dichlorobiphenyl derivative water pollutants, 2-phenylbenzotriazole (PBTA) congeners and river water concentrates

We recently identified dichlorobiphenyl (DCB) derivatives and 2-phenylbenzotriazole (PBTA) congeners as major mutagenic constituents of the waters of the Waka River and the Yodo River system in Japan, respectively. In this study we examined sister chromatid exchange (SCE) induction by two dichlorobiphenyl derivatives, 3,3′-dichlorobenzidine (DCB, 4,4′-diamino-3,3′-dichlorobiphenyl) and 4,4′-diamino-3,3′-dichloro-5-nitrobiphenyl (5-nitro-DCB); three PBTA congeners, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1), 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), and 2-[2-(acetylamino)amino]-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6); and water concentrates from the Waka River in Chinese hamster lung (CHL) cells. Concentration-dependent induction of SCE was found for all DCBs and PBTAs examined in the presence of S9 mix, and statistically significant increases of SCEs were detected at 2 μg per ml of medium or higher concentrations. SCE induction of MeIQx was examined to compare genotoxic activities of these water pollutants. According to the results, a ranking of the SCE-inducing potency of these compounds is the following: 5-nitro-DCB ≈ MeIQx > PBTA6 > PBTA-1 ≈ PBTA-2 > DCB.Water samples collected at a site at the Waka River showed concentration-related increases in SCEs at 6.25–18.75 ml-equivalent of river water per ml of medium with S9 mix. The concentrations of 5-nitro-DCB and DCB in the river water samples were from 2.5 to 19.4 ng/l and from 4100 to 18,900 ng/l, respectively. However, these chemicals showed only small contribution to SCE induction by the Waka River water.     

The Role of Individual Domains and the Significance of Shedding of ATP6AP2/(pro)renin Receptor in Vacuolar H+-ATPase Biogenesis

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H⁺-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.     

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes

Phosphoinositides (PI) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples using PI-binding domains labeled with quantum dots (Qdot) as specific probes. This method allowed us to simultaneously quantify three distinct PIs, phosphatidylinositol 3,4,5-triphosphatase [PtdIns(3,4,5)P(3)), PtdIns(3,4)P(2), and PtdIns(4,5)P(2), in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allowed the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P(2) at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P(3) was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events.