RPAP3 splicing variant isoform 1 interacts with PIH1D1 to compose R2TP complex for cell survival

We previously characterized RNA polymerase II-associated protein 3 (RPAP3) as a cell death enhancer. Here we report the identification and characterization of splicing isoform of RPAP3, isoform 1 and 2. We investigated the interaction between RPAP3 and PIH1 domain containing protein 1 (PIH1D1), and found that RPAP3 isoform 1, but not isoform 2, interacted with PIH1D1. Furthermore, knockdown of RPAP3 isoform 1 by small interfering RNA down-regulated PIH1D1 protein level without affecting PIH1D1 mRNA. RPAP3 isoform 2 potentiated doxorubicin-induced cell death in human breast cancer T-47 cells although isoform 1 showed no effect. These results suggest that R2TP complex is composed of RPAP3 isoform 1 for its stabilization, and that RPAP3 isoform 2 may have a dominant negative effect on the survival potency of R2TP complex.     

Microbial Mat Boundaries between Chemolithotrophs and Phototrophs in Geothermal Hot Spring Effluents

Among the various microbial mats that develop in geothermal hot springs in solfataric fields, colorless sulfur-turf (ST)—macroscopic white bundles consisting of large sickle-shaped bacteria belonging to Aquificales and elemental sulfur particles–develops in a limited environment of geothermal effluent containing hydrogen sulfide with neutral pH and low in oxygen. Photosynthetic cyanobacterial mat (CY) often grow just downstream of chemolithotrophic ST, or they coexist with ST where the temperature is slightly lower. Knowledge of the environmental regimes of these microbial mats will lead to better understanding of the distribution of thermophilic microorganisms on the Earth and provide clues about evolutionary processes in the microbial ecosystems of the Precambrian era. We studied the environmental parameters of the boundary zone and examined the distribution of these types of mats and measured the in situ growth rates of the microorganisms composing them. In situ examination revealed that temperature and Eh constrain the development of the microbial mats. At the boundary between ST and CY, temperature and Eh ranged between 51.1°C and 63.2°C and between ?112 mV and ?25 mV, respectively. These environmental parameters were not significantly different among Japanese, Yellowstone (North American), and Icelandic hot spring effluents with genetically similar thermal sulfur oxidizers. Sickle-shaped bacteria rarely coexist with cyanobacteria, although they can potentially grow in some CY environments. This suggests that the boundary between ST and CY might be partly determined by exclusive ecological competition.     

Two new β-glucosidases from ethanol-fermenting fungus Mucor circinelloides NBRC 4572: enzyme purification, functional characterization, and molecular cloning of the gene

Two β-glucosidases (BGLs 1 and 2) were purified to homogeneity from the extracellular enzyme preparations of the ethanol-fermenting Mucor circinelloides NBRC 4572 statically grown on rice straw. BGLs 1 and 2 are monomeric glycoproteins whose apparent molecular masses (Ms) are around 78 kDa, which decreased by approximately 10 kDa upon enzymatic deglycosylation. Both BGLs showed similar enzyme characteristics in optimal temperature and pH, stability, and inhibitors. They were active against a wide range of aryl-β-glucosides and β-linked glucose oligosaccharides. Their amino acid sequences shared 81 % identity and exhibited less than 60 % identity with the known family-3 BGLs. Considering properties such as reduced inhibition by ethanol, glucose, and cellobiose, low transglucosylation activity, wider substrate range, less binding affinity to lignocellulosic materials, and abundant expression, BGL1 is likely to be more suitable for bioethanol production than BGL2 via simultaneous saccharification and fermentation of rice straw with M. circinelloides.     

The photosynthetic response of tobacco plants overexpressing ice plant aquaporin McMIPB to a soil water deficit and high vapor pressure deficit

We investigated the photosynthetic capacity and plant growth of tobacco plants overexpressing ice plant (Mesembryanthemum crystallinum L.) aquaporin McMIPB under (1) a well-watered growth condition, (2) a well-watered and temporal higher vapor pressure deficit (VPD) condition, and (3) a soil water deficit growth condition to investigate the effect of McMIPB on photosynthetic responses under moderate soil and atmospheric humidity and water deficit conditions. Transgenic plants showed a significantly higher photosynthesis rate (by 48 %), higher mesophyll conductance (by 52 %), and enhanced growth under the well-watered growth condition than those of control plants. Decreases in the photosynthesis rate and stomatal conductance from ambient to higher VPD were slightly higher in transgenic plants than those in control plants. When plants were grown under the soil water deficit condition, decreases in the photosynthesis rate and stomatal conductance were less significant in transgenic plants than those in control plants. McMIPB is likely to work as a CO₂ transporter, as well as control the regulation of stomata to water deficits.     

Possible ideas on carbon and nitrogen trophic fractionation of food chains: a new aspect of food-chain stable isotope analysis in Lake Biwa, Lake Baikal, and the Mongolian grasslands

Trophic fractionation of carbon and nitrogen isotopes (Δδ¹³C, Δδ¹⁵N) was examined using previously complied databases for food chains in Lake Biwa, Lake Baikal, and Mongolian grassland. The following two features were clarified: (1) For each ecosystem, the ratios of trophic fractionation of carbon and nitrogen isotopes (Δδ¹⁵N/Δδ¹³C) throughout food chain could be obtained as the slope of linear regression line on the δ¹⁵N–δ¹³C plot. (2) Further, analysis of covariance (ANCOVA) revealed the slopes on δ¹⁵N–δ¹³C were not significantly different among these various ecosystems and allowed us to have the regression by setting δ¹⁵N as the response variable: δ¹⁵N = 1.61 δ¹³C + [ecosystem specific constant] with standard errors of [±0.41] and [±9.7] for the slope and the intercept, respectively. It was suggested that the slope of the regression (or the ratio Δδ¹⁵N/Δδ¹³C) could be applicable to more complicated food webs in case nitrogen and carbon isotope ratios of primary producers can be assumed constant in space and time within the ecosystems. The results from simple linear regression analyses coincided well with the ANCOVA results for these ecosystems, although there was some discrepancy between the results of the two statistical analyses. Possible factors that govern the linear relationship between δ¹⁵N and δ¹³C along a food chain are discussed together with a new scope for the stable isotope food chain analyses.     

Synthesis of Rat Casein Antigen in Escherichia coli

Fumes from phospholipids pyrolyzed at 500~700°C did not themselves show any mutagenicity on Salmonella strains, but when the pyrolyzates were treated with a sodium chloride precipitate, active carbon, or an anionic exchange resin, the filtrates were found to be mutagenic on Salmonella TA 100. Tests confirmed that the phospholipid pyrolyzates contained both mutagenic and inactivating substances of this mutagenicity. Low level mutagenicity was produced on Salmonella TA 98, but there was no such activity on the other strains. Preincubation of the pyrolyzates with S-9 mix had no activating effect on mutagenicity. The inactivating substances of the mutagenicity were isolated and identified as long chain fatty acids.     

Ag + -Sensitive NADH Dehydrogenas in the Na+-Motive Respiratory Chain of the Marine Bacterium Vibrio alginolyticus

Components of the Na⁺ -motive NADH : quinone oxi-doreductase segment in the respiratory chain of Vibrio alginolyticus were examined in membranes prepared from wild type, Na⁺ -pump-defective mutants, and a spontaneous revertant. Ag⁺ distinguished two kinds of respiratory NADH dehydrogenases. The Na⁺ -pump-defective mutants lacked Ag⁺ -sensitive NADH dehydrogenase activity. Incubation of the Ag⁺ -sensitive NADH dehydrogenase with solubilized membrane proteins of the mutant led to the reconstitution of Na⁺ -motive NADH : quinone oxidoreductase activity. We think that Ag⁺ -sensitive NADH dehydrogenase is an essential component of the respiratory Na⁺ pump of this organism.     

A single‐molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed

The replisome catalyses DNA synthesis at a DNA replication fork. The molecular behaviour of the individual replisomes, and therefore the dynamics of replication fork movements, in growing Escherichia coli cells remains unknown. DNA combing enables a single‐molecule approach to measuring the speed of replication fork progression in cells pulse‐labelled with thymidine analogues. We constructed a new thymidine‐requiring strain, eCOMB (E. coli for combing), that rapidly and sufficiently incorporates the analogues into newly synthesized DNA chains for the DNA‐combing method. In combing experiments with eCOMB, we found the speed of most replication forks in the cells to be within the narrow range of 550–750 nt s^?1 and the average speed to be 653 ± 9 nt s^?1 (± SEM). We also found the average speed of the replication fork to be only 264 ± 9 nt s^?1 in a dnaE173‐eCOMB strain producing a mutant‐type of the replicative DNA polymerase III (Pol III) with a chain elongation rate (300 nt s^?1) much lower than that of the wild‐type Pol III (900 nt s^?1). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.