Performance of affinity chromatography columns

Performance of affinity chromatography columns was studied by measuring the rates of adsorption and elution of trypsin in a Sepharose 4B-soybean trypsin inhibitor column and a Sepharose 4B-arginine peptides column. The volumetric coefficient for trypsin transfer was evaluated from the break-through curves of trypsin, and elution profiles bed height of Sepharose 4B-STI column was estimated based on these results.     

TMPRSS13, a type II transmembrane serine protease, is inhibited by hepatocyte growth factor activator inhibitor type 1 and activates pro-hepatocyte growth factor

Type II transmembrane serine proteases (TTSPs) are structurally defined by the presence of a transmembrane domain located near the N-terminus and a C-terminal extracellular serine protease domain. The human TTSP family consists of 17 members. Some members of the family have pivotal functions in development and homeostasis, and are involved in tumorigenesis and viral infections. The activities of TTSPs are regulated by endogenous protease inhibitors. However, protease inhibitors of most TTSPs have not yet been identified. In this study, we investigated the inhibitory effect of hepatocyte growth factor activator inhibitor type 1 (HAI-1), a Kunitz-type serine protease inhibitor, on several members of the TTSP family. We found that the protease activity of a member, TMPRSS13, was inhibited by HAI-1. A detailed analysis revealed that a soluble form of HAI-1 with one Kunitz domain (NK1) more strongly inhibited TMPRSS13 than another soluble form of HAI-1 with two Kunitz domains (NK1LK2). In addition, an in vitro protein binding assay showed that NK1 formed complexes with TMPRSS13, but NK1LK2 did not. TMPRSS13 converted single-chain pro-hepatocyte growth factor (pro-HGF) to a two-chain form in vitro, and the pro-HGF converting activity of TMPRSS13 was inhibited by NK1. The two-chain form of HGF exhibited biological activity, assessed by phosphorylation of the HGF receptor (c-Met) and extracellular signal-regulated kinase, and scattered morphology in human hepatocellular carcinoma cell line HepG2. These results suggest that TMPRSS13 functions as an HGF-converting protease, the activity of which may be regulated by HAI-1.     

Nitrogen fixation throughout growth,and varietal differences in nitrogen fixation by the rhizosphere of rice planted in pots

Summary The nitrogen-fixing activity in the rhizospheres of various rices was measured by the acetylene-reduction method throughout plant growth in green-house pots. The activity began to increase 4 weeks after transplanting, increased until heading stage, then decreased. The concentrations of exchangeable ammonium and sugars in the soil were not related to the variation of nitrogen-fixing activity during rice growth.The nitrogen-fixing activities in the rhizospheres of 41 rice varieties in pots were measured to discover varietal differences. Levels of nitrogen fixation were highly correlated with the rices' dry root weight at heading stage.     

Xylitol Production by an Enterobacter Species

A xylose-utilizing bacterial strain was isolated from soil.

The strain, No. 553, was identified as Enterobacter liquefaciens from the result of the taxonomical studies. This bacterium grew well on D-xylose as a sole carbon source and accumulated pentitol extracellularly in shaking culture.

Pentitol produced was isolated from the culture broth and identified as xylitol.

The xylitol production reached the maximum after the cessation of the cell growth with a yield of 33.3 mg per ml in a medium containing 10% D-xylose as a sole carbon source and no significant decline of the amount of xylitol was observed through the period of the cultivation.     

Isolation and Characterization of PDE-I and II,the Inhibitors of Cyclic Adenosine-3′,5′-monophosphate Phosphodiesterase

In the screening for inhibitors of cyclic adenosine-3′,5′-monophosphate phosphodiesterase, two compounds, PDE-I (C₁₃H₁₃N₃O₅) and PDE-II (C₁₄H₁₄N₂O₅), were isolated from culture filtrates of a Streptomyces. Concentrations for 50% inhibitions of PDE-I and PDE-II against the high Km enzyme were 15 µm and 13 µm, and those against the low Km enzyme were 65 µm and 130 µm, respectively. Production, isolation and characterization of these compounds are described.     

Studies on Glyoxalase Inhibitor Isolation of a New Active Agent,MS-3, from a Mushroom Culture

Cultured broths were screened by measuring the inhibition of glyoxalase, using 105,000 g supernatant of rat liver homogenates as a crude enzyme. An active agent, MS–3 (C₂₁H₂₄O₇), was isolated from a cultured mushroom. ID₅₀ value of MS–3 against the crude glyoxalase was 12 mcg/ml. MS–3 has low toxicity and inhibited the growth of Yoshida rat sarcoma cells in cell culture. The production, isolation, and properties of MS–3 are described.     

The Reaction Mechanism of Rat Liver Glyoxalase I and Its Inhibition by MS-3

Glyoxalase I from rat liver was purified about 25-fold by acetone fractionation and ion-exchange chromatography on CM-Sephadex and DEAE-cellulose columns. The kinetic study of the enzymatic reaction supported the one-substrate mechanism : the hemimercaptal adduct produced nonenzymatically from methylglyoxal and glutathione is the substrate. The Km value determined was 0.1 mm and similar to that of porcine erythrocytes enzyme but differed significantly from that of yeast enzyme. It was inhibited by free glutathione competitively (Ki 1.2 mm). Kinetic studies on inhibition of glyoxalase I by MS–3 which was obtained from a cultured mushroom, Stereum hirsutum, indicated the inhibition type was competitive with the hemimercaptal adduct (Ki 4.6 × 10^?6 m). By the graphical study of the multiple inhibition kinetics free glutathione and MS–3 were shown to bind at the same sites of the enzyme.     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

Natural recombinant between equine herpesviruses 1 and 4 in the ICP4 gene

Equine herpesvirus 1 (EHV-1) is a pathogen causing rhinopneumonia in young horses, abortion in mares, and myeloencephalitis in adult horses. Two types, EHV-1 P and EHV-1 B, have recently been dominant among 16 electropherotypes. EHV-1 P and EHV-1 B viruses were compared by long and accurate polymerase chain reaction (LA-PCR) and restriction fragment length polymorphism (RFLP) analysis. Differences in restriction sites were found to be focused in ORF64, which encodes the infected cell protein 4 (ICP4), and downstream of the ICP4 gene. The 3 ' -end and downstream of ICP4 gene of EHV-1 B were found to be replaced by the corresponding region of EHV-4, indicating that EHV-1 B is a naturally occurring recombinant virus between progenitors of EHV-1 P and EHV-4. This is the first report showing a natural interspecies recombinant in alphaherpesviruses.