Surface plasmon resonance biosensor detects the downstream events of active PKCbeta in antigen-stimulated mast cells

Surface plasmon resonance (SPR) biosensors detect large changes of angle of resonance (AR) when RBL-2H3 mast cells are cultured on a sensor chip and stimulated with antigen. However, the detail of molecular events that are responsible for such large changes of AR remained unknown. In this study, we investigated the relationship between intracellular signaling events induced by antigen and the change of AR, by genetic manipulation of intracellular signaling molecules; spleen tyrosine kinase (Syk), src-like adaptor protein (SLAP), linker for activation of T cells (LAT), growth-factor-receptor-bound protein 2 (Grb2), Grb2-related adaptor protein (Gads), and isotypes of protein kinase C (PKC). RBL-2H3 mast cells overexpressing dominant-negative Syk or SLAP, which both interfere with active Syk, exhibited only minimal increase of AR in response to antigen stimulation. Likewise, the interference of the activation of LAT and Gads, by expressing dominant-negative LAT and Gads, respectively, resulted in nearly complete suppression of the antigen-induced increase of AR. The cells overexpressing PKCs, apart from PKCbeta, showed a reduced extent of increase of AR in response to antigen stimulation. Moreover, the introduction of the small interfering RNA targeted against PKCbeta suppressed the antigen-induced increase of AR. These results indicate that the activation of Syk, LAT, Gads, and subsequent PKCbeta is indispensable for the antigen-induced increase of AR of mast cells detected by SPR biosensors.     

Heat coma and its relationship to ocean dynamics in the oceanic sea skaters of Halobates (Heteroptera: Gerridae) inhabiting Indian and Pacific Oceans

This study was performed to clarify how weather and current dynamics affect the resistance to temperature change in the oceanic sea skaters, Halobates. Heat coma temperature (HCT) was measured for the adults and 5th instar larvae of four Halobates species collected from a fixed sampling location (12°00′N, 135°00′E ) in western tropical Pacific Ocean and from 13 locations in the eastern area of the India Ocean ranging from 08°00′N-06°00′S and 86°00-76°00′E. Both the gap temperature for heat coma (GTHC, mean±SD: 7.83±1.86 °C, n=32) and the heat coma temperature (HCP, 35.03±1.80 °C, n=32) of individuals collected from the Pacific Ocean, during the first half (10 days) of the sampling period at the fixed sampling point, were significantly higher than those during the second half (GTHC: 5.10±2.05 °C, n=63; HCP: 34.03±2.02 °C, n=63). The reduction in heat tolerance shown in the second half of the 20 day period may have been caused by a decrease in air temperature due to rainfall that occurred around the sampling point accompanied with the arrival of Typhoon No. 6.In the study of individuals collected from the Indian Ocean, significantly higher average GTHCs of >8 °C were recorded for the adult H. micans collected at 02°00′S and 06°00′S (89°00′E) than those at 0°00-8°00′N in the eastern Indian Ocean. Dynamic mixture of water from northern and southern currents occurs at 02°00-6°00′S of the Indian Ocean and might relate to such high heat tolerance.Temperature dynamics in the ocean habitat might directly affect the temperature resistance of the oceanic sea skaters.     

Detection of Coxiella burnetii in cow's milk by PCR-enzyme-linked immunosorbent assay combined with a novel sample preparation method.

The use of an adequate concentration of Triton X-100 enhanced immunomagnetic separation of Coxiella burnetii from milk. PCR-enzyme-linked immunosorbent assay (PCR-ELISA) could detect coxiellas more sensitively than could conventional PCR. PCR-ELISA is therefore thought to be suitable for the simultaneous assay of a large number of samples. However, the number of cows from which raw milk tested positive for coxiellas by PCR-ELISA was inconsistent with that found with the antibody to coxiella by indirect immunofluorescence assay. The inconsistency is thought to be associated with the differences in the infectious route, infectious dose, or the timing of yielding the antibody and the period of duration of the antibody.     

Cloning, Overexpression, and Mutagenesis of the Sporobolomyces salmonicolor AKU4429 Gene Encoding a New Aldehyde Reductase, Which Catalyzes the Stereoselective Reduction of Ethyl 4-Chloro-3-Oxobutanoate to Ethyl (S)-4-Chloro-3-Hydroxybutanoate

We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolor AKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3β-hydroxysteroid dehydrogenase–plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303–2310, 1996). The ARII protein was overproduced in Escherichia coli about 2,000-fold compared to the production in the original yeast cells. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif G₁₉-X-X-G₂₂-X-X-A₂₅, which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G₁₉→A and G₂₂→A mutant enzymes by 4-COBE did not occur. The A₂₅→G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.     

Evidence of Horizontal Transfer of the EcoO109I Restriction-Modification Gene to Escherichia coli Chromosomal DNA

A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5'-(A/G)GGNCC(C/T)-3', was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.     

Effects of polyamines on two strains of Trypanosoma brucei in infected rats and in vitro culture

We studied the effects of polyamines, which are necessary for proliferation and antioxidation in Trypanosoma brucei gambiense Wellcome strain (WS) and Trypanosoma brucei brucei ILtat 1.4 strain (IL). No difference was found in activity of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis in trypanosomes, in both strains maintained in vitro; higher (P < 0.05) ODC values were found in IL in vivo. However, WS in vivo exhibited higher proliferation rates with higher spermidine content and decreased host survival times than IL. The in vitro proliferation and polyamine contents of WS increased with the addition of polyamine to the 1-difluoromethylornithine culture medium, but not IL. These results suggested that WS uses extracellular polyamine for proliferation. In the in vitro culture, WS was less tolerant of hydrogen peroxide (oxidative stress) than IL, and malondialdehyde levels in WS were higher than in IL. The expression of trypanothione synthetase mRNA in WS in vitro was higher than in IL. These results suggest that IL is dependent on the synthesis of polyamines for proliferation and reduction of oxidative stress, whereas WS is dependent on the uptake of extracellular polyamines. A thorough understanding of the differences in the metabolic capabilities of various trypanosomes is important for the design of more effective medical treatments.