Aromatic amino acid transaminase in rat intestine

The transamination of aromatic l-amino acids (5-hydroxytryptophan, tryptophan, tyrosine, phenylalanine and kynurenine) was shown to be catalysed by enzyme preparations from rat small intestine. On the basis of the partial purification and characterization of these aromatic amino acid transaminases, it is suggested that rat small intestine contains several kinds of aromatic amino acid transaminases.     

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.     

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1 fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries.     

Extracellular myocilin affects activity of human trabecular meshwork cells

The trabecular meshwork (TM), a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of this tissue is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. The protein product has been localized to both intra and extracellular sites, but its function still remains unclear. This study was to determine whether extracellular myocilin presented in the matrix affects adhesion, morphology, and migratory and phagocytic activities of human TM cells in culture. Cell adhesion assays indicated that TM cells, while adhering readily on fibronectin, failed to attach on recombinant myocilin purified from bacterial cultures. Adhesion on fibronectin was also compromised by myocilin in a dose dependent manner. Myocilin in addition triggered TM cells to assume a stellate appearance with broad cell bodies and microspikes. Loss of actin stress fibers and focal adhesions was observed. TM cell migration on fibronectin/myocilin to scratched wounds was reduced compared to fibronectin controls. Myocilin, however, had little impact on phagocytic activities of TM cells. Cell attachment on fibronectin and migration of corneal fibroblasts, a control cell type, were not altered by myocilin. These results demonstrate that extracellular myocilin elicits anti-adhesive and counter-migratory effects on TM cells. Myocilin in the matrix of tissues could be exerting a similar influence on TM cells in vivo, impacting the flexibility and resilience required for maintenance of the normal aqueous outflow.     

A retrospective analysis of germline competence in rat embryonic stem cell lines

The factors responsible for conferring germline competence in embryonic stem (ES) cell lines remain unidentified. In the present study, rat ES cell lines (n = 17) were established with 3i medium (SU5402, PD0325901, CHIR99021), 2i medium (PD0325901, CHIR99021) or 2iF medium (PD0325901, CHIR99021, forskolin), and their potential for germline transmission to the G1 generation was examined. Rat strains were divided into an albino group (F344, Wistar or CAG/Venus transgenic rats with the Wistar background) or a colored coat group (Brown-Norway, Dark-Agouti, or BLK rats selected from >F3 generations of Wistar × Dark-Agouti rats based on their black coat color). Successful germline transmission was observed in 57 % (4/7), 40 % (2/5) and 100 % (5/5) of the ES cells established with 3i, 2i and 2iF media, respectively. ES cell lines from the homozygous CAG/Venus transgenic rats were established in all three media, but only the lines established with the 2iF medium were germline-competent. Neither coat-color (albino: 64 %, 7/11; colored: 67 %, 4/6) nor gender of the ES cell lines (XX: 67 %, 2/3; XY: 64 %, 9/14) were likely to affect germline transmission.     

Milk-clotting Enzyme from Microorganisms

The milk-clotting activity of Mucor-rennin obtained from Mucor pusillus Lindt, was not changed by the addition of DFP in the reaction mixture. This finding suggested the probable absence of a serine residue at the active center of the enzyme. Sulfhydryl reagents such as Nekelgon, N-ethyl maleimide, PCMB failed to influence the milk-clotting reaction, indicating that a. reactive sulfhydryl group is not required for the enzymatic activity. The activity was inhibited when Mucor-rennin was treated with iodine at pH higher than 5.0. It was shown that ¹³¹I₂ was incorporated into the enzyme. When Mucor-rennin was photooxidized in the presence of methylene blue, the milk-clotting activity was inactivated. In this case, tyrosine, tryptophan, and histidine residues in the enzyme were oxidized. Among these amino acids, the histidine residue was more rapidly oxidized than other amino acids. A parallel relation was observed between the decrease of the amount of histidine residue and the inactivation of the enzyme. From these results, it is concluded that the histidine residue present in Mucor-rennin has a relation to the active center of this enzyme.     

Microscopical Investigation of the Efifect of Helminthosporol on Rice Seedlings

The elongation of the second leaf sheaths of rice seedlings was promoted strongly by helminthosporol as well as by gibberellic acid. A greater dosage of helminthosporol was required in order to induce the same amount of elongation. Cells of treated plants were somewhat longer than those of controls. The elongation of the second leaf sheath was caused mainly by an increase in cell number. Both helminthosporol and gibberellic acid promoted the elongation of cells in the basal portion of the second leaf sheath.