Identical Hik-Rre systems are involved in perception and transduction of salt signals and hyperosmotic signals but regulate the expression of individual genes to different extents in synechocystis

In previous studies, we characterized five histidine kinases (Hiks) and the cognate response regulators (Rres) that control the expression of approximately 70% of the hyperosmotic stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we screened a gene knock-out library of Rres by RNA slot-blot hybridization and with a genome-wide DNA microarray and identified three Hik-Rre systems, namely, Hik33-Rre31, Hik10-Rre3, and Hik16-Hik41-Rre17, as well as another system that included Rre1, that were involved in perception of salt stress and transduction of the signal. We found that these Hik-Rre systems were identical to those that were involved in perception and transduction of the hyperosmotic stress signal. We compared the induction factors of the salt stress- and hyperosmotic stress-inducible genes that are located downstream of each system and found that these genes responded to the two kinds of stress to different respective extents. In addition, the Hik33-Rre31 system regulated the expression of genes that were specifically induced by hyperosmotic stress, whereas the system that included Rre1 regulated the expression of one or two genes that were specifically induced either by salt stress or by hyperosmotic stress. Our observations suggest that the perception of salt and hyperosmotic stress by the Hik-Rre systems is complex and that salt stress and hyperosmotic stress are perceived as distinct signals by the Hik-Rre systems.     

Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.     

Pharmacokinetics/pharmacodynamics of Y-700, a novel xanthine oxidase inhibitor, in rats and man

The pharmacokinetics and pharmacodynamics of a novel xanthine oxidase (XO) inhibitor, Y-700, were evaluated in rats and healthy male volunteers. In a rat model of hyperuricemia, oral Y-700 (0.3-10 mg/kg) showed a more potent and a longer-lasting hypouricemic action than allopurinol. A single oral dosing of Y-700 (5, 20 or 80 mg) to volunteers caused a dose-dependent reduction of serum uric acid levels indicating close relationship to plasma concentrations of the compound. In addition, Y-700 was hardly excreted in urine but mainly excreted in feces in rats and volunteers. These results suggested that Y-700 is a new effective inhibitor of XO in rats and humans with high oral bioavailability being predominantly eliminated via the liver unlikely to allopurinol.     

Direct inhibition of microtubule-based kinesin motility by local anesthetics

Local anesthetics are known to inhibit neuronal fast anterograde axoplasmic transport (FAAT) in a reversible and dose-dependent manner, but the precise mechanism has not been determined. FAAT is powered by kinesin superfamily proteins, which transport membranous organelles, vesicles, or protein complexes along microtubules. We investigated the direct effect of local anesthetics on kinesin, using both in vitro motility and single-molecule motility assays. In the modified in vitro motility assay, local anesthetics immediately and reversibly stopped the kinesin-based microtubule movement in an all-or-none fashion without lowering kinesin ATPase activity. QX-314, a permanently charged derivative of lidocaine, exerted an effect similar to that of lidocaine, suggesting that the effect of anesthetics is due to the charged form of the anesthetics. In the single-molecule motility assay, the local anesthetic tetracaine inhibited the motility of individual kinesin molecules in a dose-dependent manner. The concentrations of the anesthetics that inhibited the motility of kinesin correlated well with those blocking FAAT. We conclude that the charged form of local anesthetics directly and reversibly inhibits kinesin motility in a dose-dependent manner, and it is the major cause of the inhibition of FAAT by local anesthetics.     

Influence of a 3‐month training program on muscular damage and neutrophil function in male university freshman judoists

We studied the effects of a high intensity and high frequency 3‐month training program on muscle damage and neutrophil function in male judoists. The study included 15 male judoists who started intensive judo training program after a 6‐month break. Creatine kinase (CK), neutrophil counts and reactive oxygen species (ROS) production capability as well as phagocytic activity (PA) of neutrophils were measured at 2 stages; entering university (pre‐training) and after 3‐month training (post‐training). At both points, we investigated parameters three times: just before, immediately after and 24 h after a 2‐h practice session. Practice‐mediated change in CK was lower at post‐training than at pre‐training. Neutrophil count significantly increased after 2‐h practice but recovered 24 h later whereas it showed no subsequent and further increased at 24 h post‐practice. Although neutrophil ROS production capability and PA both decreased (breakdown) after practice session, ROS production capability increased and PA decreased (well‐adapted) at the post‐training. Long‐term training strengthened muscular function and improved neutrophil reaction against practice‐mediated stress. Copyright © 2012 John Wiley & Sons, Ltd.     

The diffusive search mechanism of processive myosin class-V motor involves directional steps along actin subunits

It is widely accepted that the vesicle-transporter myosin-V moves processively along F-actin with large steps of approximately 36 nm using a hand-over-hand mechanism. A key question is how does the rear head of two-headed myosin-V search for the forward actin target in the forward direction. Scanning probe nanometry was used to resolve this underlying search process, which was made possible by attaching the head to a relatively large probe. One-headed myosin-V undergoes directional diffusion with approximately 5.5 nm substeps to develop an average displacement of approximately 20 nm, which was independent of the neck length (2IQ and 6IQ motifs). Two-headed myosin-V showed several approximately 5.5 nm substeps within each processive approximately 36 nm step. These results suggest that the myosin-V head searches in the forward direction for the actin target using directional diffusion on the actin subunits according to a potential slope created along the actin helix.     

Cargo-binding makes a wild-type single-headed myosin-VI move processively

Class VI myosin is an intracellular vesicle and organelle transporter that moves along actin filaments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin filaments with a hand-over-hand mechanism like other myosin organelle transporters. Recently, however, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting a doubt on its processivity. By using single molecule techniques, we show that green-fluorescent-protein-tagged single-headed, wild-type myosin-VI does not move processively. However, when coupled to 200-nm polystyrene beads (comparable to intracellular vesicles in size) at a ratio of one head per bead, single-headed myosin-VI moves processively with large (40-nm) steps. The characteristics of this monomer-driven movement were different to that of artificial dimer-driven movement: Compared to the artificial dimer, the monomer-bead complex had a reduced stall force (1 pN compared to 2 pN), an average run length 2.5-fold shorter (91 nm compared to 220 nm) and load-dependent step size. Furthermore, we found that a monomer-bead complex moved more processively in a high viscous solution (40-fold higher than water) similar to cellular environment. Because the diffusion constant of the bead is 60-fold lower than myosin-VI heads alone in water, we propose a model in which the bead acts as a diffusional anchor for the myosin-VI, enhancing its rebinding following detachment and supporting processive movement of the bead-monomer complexes. Although a single-headed myosin-VI was able to move processively with a large cargo, the travel distance was rather short. Multiple molecules may be involved in the cargo transport for a long travel distance in cells.     

Entrapment of phenylalanine ammonia-lyase in silk fibroin for protection from proteolytic attack

Phenylalanine ammonia-lyase was entrapped in silk fibroin. The entrapped enzyme showed a similar Km for Phe and pH optimum to the free enzyme. It was resistant against chymotrypsin and trypsin in vitro. To assess the activity in vivo, the free or entrapped enzymes and then Phe were injected into rat duodenum, and cinnamate, a product, in plasma was determined as the most direct evidence of the enzyme activity. The entrapped enzyme but not the free form caused a marked raise of plasma cinnamate. It declined with a half life of about 45 min, which was significantly longer than that (10-15 min) observed upon i.v. administration of cinnamate. These results indicated that the entrapped enzyme was actively degrading Phe in the intestinal tract. Entrapment of phenylalanine ammonia-lyase in fibroin thus provides a new prospect for oral enzyme therapy of phenylketonuria.     

Cloning and expression of cDNA encoding human basic fibroblast growth factor

A cDNA encoding human basic fibroblast growth factor (bFGF) was isolated from a human foreskin fibroblast cDNA library. The cDNA, 4 kilobases in size, had a coding sequence, 5' and 3' untranslated regions, and a poly(A) chain. Isolation of additional cDNA clones that had a short 3' untranslated region suggested the presence of multiple mRNA forms. By Northern blot analysis, at least five bFGF mRNA species were detected in cultured fibroblast cells. Transfection of the cDNA to COS cells resulted in the detection of mitogenic activity in the culture medium of the transfected cells, suggesting that a part of the synthesized protein might be secreted from cells despite its unusual short signal sequence.