Integrative taxonomic approach of trypanosomes in the blood of rodents and soricids in Asian countries, with the description of three new species.

Trypanosoma lewisi (Kinetoplastea: Trypanosomatida: Trypanosomatidae) with a cosmopolitan distribution is the type species of the subgenus Herpetosoma, which includes ca. 50 nominal species isolated mainly from rodents. Since members of Herpetosoma in different host species have an almost identical morphology of bloodstream forms, these trypanosomes are referred to as 'T. lewisi-like', and the molecular genetic characterization of each species is necessary to verify their taxonomy. In the present study, we collected blood samples from 89 murid rodents of 15 species and 11 soricids of four species in Indonesia, Philippines, Vietnam, Taiwan, and mainland China for the detection of hemoprotozoan infection. T. lewisi and T. lewisi-like trypanosomes were found in the blood smears of 10 murid animals, which included Bandicota indica (two rats), Rattus argentiventer (one rat), and Rattus tiomanicus (two rats) in Indonesia; Rattus rattus (one rat) in the Philippines; and Niviventer confucianus (four rats) in mainland China. Furthermore, large- or medium-sized non-T. lewisi-like trypanosomes were detected in two soricids, Crocidura dracula in Vietnam and Anourosorex yamashinai in Taiwan, respectively. Molecular genetic characterization of the small subunit (SSU) ribosomal RNA gene (rDNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene indicated that the trypanosomes from all the murid hosts had identical SSU rDNA or gGAPDH gene nucleotide sequences except for those in N. confucianus in mainland China. These N. confucianus-infecting trypanosomes also showed several unique morphological features such as smaller bodies, anteriorly positioned nuclei, and larger rod-shaped kinetoplasts when compared with T. lewisi trypomastigotes. Trypanosoma (Herpetosoma) niviventerae n. sp. is erected for this new species. Similarly, based on morphological and molecular genetic characterization, Trypanosoma sapaensis n. sp. and Trypanosoma anourosoricis n. sp. are proposed for the trypanosomes in C. dracula in Vietnam and A. yamashinai in Taiwan, respectively. More effort directed toward the morphological and molecular genetic characterization of the trypanosomes of rodents and soricids is required to fully understand the real biodiversity of their hemoflagellates.

Mutations in the COPII vesicle component gene SEC24B are associated with human neural tube defects.

Neural tube defects (NTDs) are severe birth malformations that affect one in 1,000 live births. Recently, mutations in the planar cell polarity (PCP) pathway genes had been implicated in the pathogenesis of NTDs in both the mouse model and in human cohorts. Mouse models indicate that the homozygous disruption of Sec24b, which mediates the ER-to-Golgi transportation of the core PCP gene Vangl2 as a component of the COPII vesicle, will result in craniorachischisis. In this study, we found four rare missense heterozygous SEC24B mutations (p.Phe227Ser, p.Phe682Leu, p.Arg1248Gln, and p.Ala1251Gly) in NTDs cases that were absent in all controls. Among them, p.Phe227Ser and p.Phe682Leu affected its protein stability and physical interaction with VANGL2. Three variants (p.Phe227Ser, p.Arg1248Gln, and p.Ala1251Gly) were demonstrated to affect VANGL2 subcellular localization in cultured cells. Further functional analysis in the zebrafish including overexpression and dosage-dependent rescue study suggested that these four mutations all displayed loss-of-function effects compared with wild-type SEC24B. Our study demonstrated that functional mutations in SEC24B might contribute to the etiology of a subset of human NTDs and further expanded our knowledge of the role of PCP pathway-related genes in the pathogenesis of human NTDs.

[Purification and characteristics of creatininase from Arthrobacter sp].

A creatininase produced from a Arthrobacter sp. was purified 145-fold by a series of steps including heat treatment, ammonium sulfate precipitation, DEAE-Cellulose ion-exchange and hydrophobic chromatography. The specific activity of the pure enzyme was 209u/mg. The subunit molecular mass of creatininase was estimated to be 33 700D by SDS-PAGE. The creatininase was stable in the pH range between 6.0 - 9.0 and below 60 degrees C . Its Km value for creatinine was estimated to be 21.14 mmol/L. The enzyme was markedly inactivated by incubation with 1 mmol/L of Hg2+, Ag2+, Li+, Cu2+ and 20 mmol/L of 1, 11-Phananthroline respectively. Activation was observed when the enzyme was incubated with 1 mmol/L of Co2+ and Mn2+.

[Study on the properties of a novel glycine amino peptidase from Actinomucor elegans].

The glycine amino peptidase of Actinomucor elegans was studied in this work. For the enzyme production Actinomucor elegans was cultured with an enzyme producing medium. Then the cells were collected and subjected to enzyme purification. The glycine aminopeptidase was purified 592 times by a DEAE-Toyopearl column, a Toyopearl HW 65-C column and a Superdex 200 column subsequently and the purified enzyme had a specific activity of 14.2 u/mg. The enzyme was estimated to have molecular mass of 320kD by gel filtration and a subunit size of 56.5kD by SDS-PAGE. It hydrolyzes glycine residue containing substrates such as glycine-betanaphthylamine more efficiently than those containing other amino acid residue. Addition to Gly-betaNA, the enzyme could also hydrolyze Ala-betaNA, Met-betaNA, Leu-betaNA, Arg-betaNA and Ser-betaNA but it had no activity on the substrates such as Trp-betaNA, Pyr-betaNA, Pro-betaNA, Asp-betaNA, Lys-betaNA, Val-betaNA. It was also observed when the glycine-betanaphthylamine concentration was higher than 2mmol/L the enzyme showed a substrate inhibition, and at the 20 mmol/L the enzyme only showed about 55% activity as it showed at the 2mmol/L. Whereas no such phenomenon was observed on the other substrate such as alanine-betanaphthylamine. The optimal temperature and pH for the reaction of this enzyme is 30 degrees C and pH 8.0, respectively. The Km and Kcat of the enzyme for glycine-betanaphthylamine is 0.24 mmol/L and 100.8 s(-1), respectively. Zn2+, Cu2+ and Cd2+ suppress almost all activities of the enzyme at the concentration of 1.0 mmol/L. Based on the study of chelating reagents, GAP belongs to the metalloenzyme. When a gelatin solution was hydrolyzed with 0.5% of alkaline proteinase together with glycine aminopeptidase at 50 degrees C for 18 hours, the glycine aminopeptidase could improve the hydrolysis degree of the protease. The total free amino acid was improved about 13% and although the enzyme mainly had the activity to hydrolyze the glycine residue, individual amino acids analysis with an amino acid analyzer showed that the contents of glycine, proline, alanine, arginine and glutamate were considerably increased. The results of this study showed that the glycine aminopeptidase would be useful in the food industry.

Phylogenetic study on Shiraia bambusicola by rDNA sequence analyses.

In this study, 18S rDNA and ITS-5.8S rDNA regions of four Shiraia bambusicola isolates collected from different species of bamboos were amplified by PCR with universal primer pairs NS1/NS8 and ITS5/ITS4, respectively, and sequenced. Phylogenetic analyses were conducted on three selected datasets of rDNA sequences. Maximum parsimony, distance and maximum likelihood criteria were used to infer trees. Morphological characteristics were also observed. The positioning of Shiraia in the order Pleosporales was well supported by bootstrap, which agreed with the placement by Amano (1980) according to their morphology. We did not find significant inter-hostal differences among these four isolates from different species of bamboos. From the results of analyses and comparison of their rDNA sequences, we conclude that Shiraia should be classified into Pleosporales as Amano (1980) proposed and suggest that it might be positioned in the family Phaeosphaeriaceae.