Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages

Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.     

Phenolics with PPAR-gamma ligand-binding activity obtained from licorice (Glycyrrhiza uralensis roots) and ameliorative effects of glycyrin on genetically diabetic KK-A(y) mice

The EtOAc extract of licorice (Glycyrrhiza uralensis roots) exhibited considerable PPAR-gamma ligand-binding activity. Bioassay-guided fractionation of the extract using a GAL-4-PPAR-gamma chimera assay method resulted in the isolation of two isoflavenes, one of which is a new compound named dehydroglyasperin D, an isoflavan, two 3-arylcoumarins, and an isoflavanone as the PPAR-gamma ligand-binding active ingredients of licorice. The isoprenyl group at C-6 and the C-2' hydroxyl group in the aromatic ring-C part in the isoflavan, isoflavene, or arylcoumarin skeleton were found to be the structural requirements for PPAR-gamma ligand-binding activity. Glycyrin, one of the main PPAR-gamma ligands of licorice, significantly decreased the blood glucose levels of genetically diabetic KK-A(y) mice.     

Physical map of the region surrounding the OTOFERLIN locus on chromosome 2p22-p23

The autosomal recessive form of nonsyndromic deafness DFNB9 has been mapped to a 2-cM region on chromosome 2p22-p23, and the responsible gene, OTOF, has been recently identified by positional cloning combined with a candidate gene approach. In the course of this gene cloning, we established a contig of yeast artificial chromosomes, bacterial artificial chromosomes, and P1 phage artificial chromosomes delimited by polymorphic markers D2S2170 and D2S170, i.e. , extending over approximately 3500 kb. Sixty expressed sequence tags or genes and 14 sequence-tagged sites, 11 of which are polymorphic, were mapped to this contig and assigned to 21 chromosomal intervals.     

ATP-induced transconformation of myosin revealed by determining three-dimensional positions of fluorophores from fluorescence energy transfer measurements

The method of fluorescence resonance energy transfer (FRET) is one of the most important techniques for measuring the distance between two fluorophores and for detecting the changes in protein structure under physiological conditions. The use of green fluorescent protein is also a powerful technology that has been used to elucidate dynamic molecular events. From these we have developed a novel method to determine the three-dimensional positions of fluorophores by combining the FRET data and other structural information available. Using this method, we could determine the ATP-induced changes of three-dimensional structure of truncated Dictyostelium myosin in solution. The myosin structure with ADP in solution was found to be similar to that of the crystal structure of MgADPBeFx-bound truncated Dictyostelium myosin (type I structure), whereas myosin with ATP in solution was similar to the crystal structure of MgAdPVi-bound one (type II structure). However, the crystal structure of MgADP-bound scallop myosin (type III structure) could not be explained by any of our FRET data under various conditions. This indicates that the type III crystal structure might represent a transient intermediate conformation that could not be detected using fluorescence energy transfer.     

Acetylation at the N-terminus of actin strengthens weak interaction between actin and myosin

The N-terminus of all actins so far studied is acetylated. Although the pathways of acetylation have been well studied, its functional importance has been unclear. A negative charge cluster in the actin N-terminal region is shown to be important for the function of actomyosin. Acetylation at the N-terminus removes a positive charge and increases the amount of net negative charges in the N-terminal region. This may augment the role of the negative charge cluster. To examine this possibility, actin with a nonacetylated N-terminus (nonacetylated actin) was produced. The nonacetylated actin polymerized and depolymerized normally. In actin-activated heavy meromyosin ATPase assays, the nonacetylated actin showed higher K(app) without significantly changing V(max), compared with those of wild-type actin. This is in contrast to the effect of the N-terminal negative charge cluster, which increases V(max) without changing K(app). These results indicate that the acetylation at the N-terminus of actin strengthens weak actomyosin interaction.     

Role of residues 230 and 236 of actin in myosin-ATPase activation by actin-tropomyosin

The Dictyostelium/Tetrahymena-chimeric actin (Q228K/T229A/A230Y) showed higher Ca(2+)-activation of myosin S1 ATPase in the presence of tropomyosin-troponin. The crystal structure of the chimeric actin is almost the same as that of wild-type except the conformation of the side chain of Leu236. Here, we introduced an additional mutation (L236A), in which the side chain of Leu236 was truncated, into the chimeric actin (Q228K/T229A/A230Y/L236A). Without regulatory proteins, the new mutant actin showed normal myosin S1 activation and normal sliding velocity. However, in the presence of tropomyosin, the new mutant actin activated myosin S1 ATPase higher than the wild-type actin and showed higher velocities in in vitro motility assay at low HMM concentrations. These results suggest that the mutations of A230Y and L236A in the actin subdomain-4 facilitate the transition of thin filaments from a "closed" state to an "open" state.     

Increased serum oxysterol concentrations in patients with chronic hepatitis C virus infection

Oxidative stress and dysregulated cholesterol metabolism are characteristic features of chronic hepatitis C virus infection (CHC). Therefore, we analyzed serum oxysterol profiles in CHC patients and examined the significance of oxysterols in CHC. The concentrations of 7α-hydroxycholesterol, 4β-hydroxycholesterol and 25-hydroxycholesterol as determined by LC–ESI–MS/MS were significantly elevated by +236%, +29% and +44%, respectively, in CHC patients compared with controls. Moreover, the elevated levels were significantly decreased by anti-viral therapy using PEGylated-interferon and ribavirin for 3 months. In contrast, 24S-hydroxycholesterol, 27-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one concentrations were not affected by CHC or anti-viral treatment. These results suggest that some oxysterols that are elevated in CHC are produced by cholesterol autoxidation due to oxidative stress or inflammation in the liver. Oxysterols may represent novel targets for the inhibition of disease progression and the prevention of hepatocarcinogenesis in CHC patients.     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Expression of semaphorin 3A in the rat corneal epithelium during wound healing

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.