Hypertonicity-induced cation channels rescue cells from staurosporine-elicited apoptosis

Cell shrinkage is one of the earliest events during apoptosis. Cell shrinkage also occurs upon hypertonic stress, and previous work has shown that hypertonicity-induced cation channels (HICCs) underlie a highly efficient mechanism of recovery from cell shrinkage, called the regulatory volume increase (RVI), in many cell types. Here, the effects of HICC activation on staurosporine-induced apoptotic volume decrease (AVD) and apoptosis were studied in HeLa cells by means of electronic cell sizing and whole-cell patch-clamp recording. It was found that hypertonic stress reduces staurosporine-induced AVD and cell death (associated with caspase-3/7 activation and DNA fragmentation), and that this effect was actually due to activation of the HICC. On the other hand, staurosporine was found to significantly reduce osmotic HICC activation. It is concluded that AVD and RVI reflect two fundamentally distinct functional modes in terms of the activity and role of the HICC, in a shrunken cell. Our results also demonstrate, for the first time, the ability of the HICC to rescue cells from the process of programmed cell death.     

Phylogenetic and enzymatic diversity of deep subseafloor aerobic microorganisms in organics- and methane-rich sediments off Shimokita Peninsula

"A meta-enzyme approach" is proposed as an ecological enzymatic method to explore the potential functions of microbial communities in extreme environments such as the deep marine subsurface. We evaluated a variety of extra-cellular enzyme activities of sediment slurries and isolates from a deep subseafloor sediment core. Using the new deep-sea drilling vessel "Chikyu", we obtained 365 m of core sediments that contained approximately 2% organic matter and considerable amounts of methane from offshore the Shimokita Peninsula in Japan at a water depth of 1,180 m. In the extra-sediment fraction of the slurry samples, phosphatase, esterase, and catalase activities were detected consistently throughout the core sediments down to the deepest slurry sample from 342.5 m below seafloor (mbsf). Detectable enzyme activities predicted the existence of a sizable population of viable aerobic microorganisms even in deep subseafloor habitats. The subsequent quantitative cultivation using solid media represented remarkably high numbers of aerobic, heterotrophic microbial populations (e.g., maximally 4.4 x 10(7) cells cm(-3) at 342.5 mbsf). Analysis of 16S rRNA gene sequences revealed that the predominant cultivated microbial components were affiliated with the genera Bacillus, Shewanella, Pseudoalteromonas, Halomonas, Pseudomonas, Paracoccus, Rhodococcus, Microbacterium, and Flexibacteracea. Many of the predominant and scarce isolates produced a variety of extra-cellular enzymes such as proteases, amylases, lipases, chitinases, phosphatases, and deoxyribonucleases. Our results indicate that microbes in the deep subseafloor environment off Shimokita are metabolically active and that the cultivable populations may have a great potential in biotechnology.     

Seasonality of herbivory and communication between individuals of sagebrush

Seasonal changes in herbivore numbers and in plant defenses are well known to influence plant–herbivore interactions. Some plant defenses are induced in response to herbivore attack or cues correlated with risk of attack although seasonal variation in these defenses is relatively poorly known. We previously reported that sagebrush becomes more resistant to its herbivores when neighboring plants have been experimentally clipped with scissors. In this study we asked whether herbivory to leaves of sagebrush varied seasonally and whether there was seasonal variation in natural levels of damage when neighbors were clipped. We found that sagebrush accumulated most chewing damage early in the season, soon after the spring flush of new leaves. This damage was caused by generalist grasshoppers, deer, specialist caterpillars, beetles, gall makers, and other less common herbivores. Sagebrush showed no evidence of preferentially abscising leaves that had been experimentally clipped. Experimental clipping by Trirhabda pilosa beetle larvae caused neighbors to accumulate less herbivore damage later that season, similar to results in which clipping was done with scissors. Induced resistance caused by experimentally clipping a neighbor was affected by season; plants with neighbors clipped in May accumulated less damage throughout the season relative to plants with unclipped neighbors or neighbors clipped later in the summer. We found a correlation between seasonal herbivore pressure, damage accumulated by plants, and induced responses to experimentally clipping neighbors. The causal mechanisms responsible for this correlation are unknown although a strong seasonal effect was clear.     

Microwave discharge electrodeless lamps (MDEL). III. A novel tungsten-triggered MDEL device emitting VUV and UVC radiation for use in wastewater treatment.

Exposure to low doses of the xenoestrogen bisphenol A (BPA) and to the hormonal 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide, an environmental endocrine disruptor, can have serious health consequences such as the induction of mammary gland ductal hyperplasias and carcinoma (LaChapelle et al., Reprod. Toxicol., 2007, 23, 20; Murray et al., Reprod. Toxicol., 2007, 23, 383). To the extent that these toxins are present in wastewaters (Donald et al., Sci. Total Environ. 1999, 231, 173; Brotons et al., Environ. Health Perspect. 1994, 103, 608; Olea et al., Environ. Health Perspect. 1996, 104, 298; Biles et al., J. Agric. Food Chem. 1997, 45, 3541; Markey et al., J. Steroid Biochem. Mol. Biol., 2003, 83, 235), we examined their oxidative destruction in aqueous media by a novel light source. A tungsten-triggered microwave discharge electrodeless lamp (W-MDEL) was fabricated for possible use in wastewater treatment using vacuum UV-transparent quartz in which a tungsten trigger, also embedded in quartz, was attached to the MDEL to aid in the self-ignition of the lamp on irradiation at low microwave power levels. The quantity of mercury gas in the W-MDEL was optimized by monitoring the continuous radiation and peak intensities of the emitted light in the vacuum UV (VUV) and UVC regions. The usefulness of the W-MDEL device was assessed through the degradation of 2,4-D and BPA in air-equilibrated aqueous media and in oxygen-saturated aqueous media. Enhanced degradation of these two xenoestrogenic toxins was achieved by increasing the number of W-MDEL devices while keeping constant the microwave radiation feeding each W-MDEL lamp. This novel lamp provides an additional light source in the photooxidation of environmental contaminants without the need for a metal-oxide photocatalyst. Under our conditions, process dynamics using the W-MDEL light source are greater than with the more conventional photochemical methods that employ low-pressure Hg arc electrode lamps in synthetic quartz to degrade these two toxic contaminants.     

Production of Strigolactones by Arabidopsis thaliana responsible for Orobanche aegyptiaca seed germination

The germination stimulants produced by Arabidopsis thaliana, a host of root parasitic plants Orobanche spp. but not of arbuscular mycorrhizal (AM) fungi were examined. Root exudates from the hydroponically grown A. thaliana plants were subjected to reverse phase high performance liquid chromatography (HPLC) and retention times of germination stimulants inducing O. aegyptiaca seed germination were compared with those of strigolactone standards. In addition, the root exudates were analyzed by using HPLC linked with tandem mass spectrometry (LC/MS/MS). A. thaliana was found to exude at least three different germination stimulants of which one was identified as orobanchol. This is the first report of strigolactone production by a non-mycotrophic plant. These results together with recent knowledge imply that strigolactones have other unrevealed functions in plant growth and development.     

Expression of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> butanol synthetic genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), β-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.     

Overproduction of Escherichia coli DNA polymerase DinB (Pol IV) inhibits replication fork progression and is lethal

Escherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We suggest that DinB targets Pol III, thereby acting as a brake on replication fork progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication to safeguard genome stability.     

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.