Thelohanellus testudineus n. sp. (Myxosporea: Bivalvulida) infecting the skin of allogynogenetic gibel carp Carassius auratus gibelio (Bloch) in China.

A Thelohanellus species was encountered during a survey on Thelohanellus diversity of Carassius auratus gibelio (Bloch) in China. The infection is characterized by the presence of large cysts of 1.4-3.2 cm in diameter in the skin of host. Mature spores were ampullaceous in frontal view and testudinate in lateral view, measuring 19.7 ± 0.7 (18.6-20.8) µm long, 7.6 ± 0.4 (6.6-8.4) µm wide and 7.3 ± 0.5 (6.6-8.8) µm thick. The single polar capsule was elongated pyriform, with 11.1 ± 0.5 (10.0-11.9) µm long and 5.3 ± 0.3 (4.3-5.8) µm wide. Polar filaments coiled with 7-8 turns. Scanning electron microscopy revealed a smooth spore surface with flat side and convex side. The sutural line was straight or 'S' like, running near the middle of the valves. Histologically, the large cysts consisting of numerous small plasmodia developed in the dermis of the skin. The BLAST search indicated that the newly obtained ssrRNA gene sequences did not match any available sequences in GenBank and phylogenetic analysis placed it in the Thelohanellus clade. Based on morphology and molecular differences with reported Thelohanellus spp., this parasite was described as a new species of genus Thelohanellus.

Isolation and characterization of 32 microsatellite loci for topmouth culter (Culter alburnus Basilewsky).

The topmouth culter (Culter alburnus) is an economically important freshwater fish, which is widely distributed throughout large rivers, reservoirs, and lake areas of China. We report here the isolation and characterization of 32 new polymorphic microsatellite loci isolated from genomic DNA in this species enriched by (CA)12 and (GA)12 probes. The variability of these microsatellites was tested on 30 individuals cultured. The average allele number was 6.6 per locus, ranging from 3 to 12. The observed heterozygosity was from 0.4667 to 0.9000, and the expected heterozygosity was from 0.6163 to 0.9085. After using Bonferroni's correction for multiple tests, there was no evidence of linkage disequilibrium between pairs of loci, but deviations from Hardy-Weinberg equilibrium were found in 3 loci. These microsatellites can be used to study QTL of economic importance, population genetic diversity and the construction of genetic maps for C. alburnus in the future.

Myxobolus musseliusae (Myxozoa: Myxobolidae) from the gills of common carp Cyprinus carpio and revision of Myxobolus dispar recorded in China.

During a survey of myxozoan parasites of common carp Cyprinus carpio in Honghu Lake, Hubei Province, China, a parasite was collected that was identified as Myxobolus dispar based on an earlier description from China. However, the small subunit ribosomal DNA of this species shared only 90 % similarity with M. dispar, instead matching M. musseliusae with 100 % identity. To resolve this apparent taxonomic conflict, the validity of M. dispar reported from China was investigated. The species encountered here and in the earlier report from China both bear spores that are notably smaller than those of M. dispar in Europe. In the present study, a mucous envelope was adhered to the posterior of many fresh spores and was observed to expand and surround the spore. This structure has never been reported from fresh spores of M. dispar. Histology showed extravascular plasmodia in the gill filaments in close contact with the cartilaginous ray of the filament, which contrasts with the plasmodia of M. dispar which develop in the arteries of the gill filaments. Phylogenetically, the current species is distinct from M. dispar, instead forming a sister group with M. musseliusae. The data presented here allow us to conclude that the species isolated is M. musseliusae and that prior reports of M. dispar in China are unsubstantiated.

[Comparison of risk factors for hemorrhagic stroke and ischemic stroke, a prospective long-term follow-up cohort study].

Objective: To analyze and compare the risk factors for hemorrhagic stroke and ischemic stroke and understand the exposure levels in population. Methods: A cohort study of risk factors of stroke was conducted in a rural community in Fengxian District of Shanghai in 2003, and the common risk factors of stroke were investigated at baseline survey, the cerebrovascular hemodynamics indexes were detected, the cerebrovascular function score was calculated according to the unified integral rule, and the incidence of stroke was observed in follow up. The risk factors for hemorrhagic stroke and ischemic stroke were analyzed by cohort study. The risk factors for two subtypes of stroke were compared. Result: A total of 10 565 participants were included in the study, with a mean follow-up period of (11.15±2.26) years, and 103 hemorrhagic stroke cases and 268 ischemic stroke cases were observed during follow-up period. The independent risk factors of hemorrhagic stroke included decreased cerebrovascular function score [hazard ratio (HR)=1.56, 95%CI: 1.23-1.98], history of alcohol consumption (HR=2.46, 95%CI: 1.39-4.34), hypertension (HR=1.75, 95%CI: 1.00-3.07) and older age (HR=1.07, 95%CI: 1.04-1.10). The independent risk factors of ischemic stroke included decreased cerebrovascular function score (HR=1.43, 95%CI: 1.25-1.65), smoking history (HR=1.52, 95%CI: 1.13-2.05), hypertension (HR=1.51, 95%CI: 1.10-2.07), family history of stroke (HR=1.89, 95%CI: 1.13-3.15), left ventricular hypertrophy (HR=1.74, 95%CI: 1.07-2.81) and older age (HR=1.07, 95%CI: 1.05-1.08). Conclusions: Decreased cerebrovascular function score, hypertension, and older age were common independent risk factors of both types of stroke, alcohol consumption history was an independent risk factor of hemorrhagic stroke, and smoking history, and family history of stroke and left ventricular hypertrophy were independent risk factors of ischemic stroke.

Myxobolus honghuensis n. sp. (Myxosporea: Bivalvulida) parasitizing the pharynx of allogynogenetic gibel carp Carassius auratus gibelio (Bloch) from Honghu Lake, China.

Myxobolus honghuensis n. sp. is described from allogynogenetic gibel carp Carassius auratus gibelio (Bloch), during a survey of myxosporean parasites in Honghu Lake, Hubei Province, China. It is characterized by the presence of round plasmodia of 5-12 mm in diameter in the pharynx of host. Mature spores of M. honghuensis were pyriform in frontal view and anterior pointed with bluntly round posterior, they measured 16.9 ± 0.5 (15.1-19.5) µm long, 10.4 ± 0.4 (9.0-11.3) µm wide, and 8.4 ± 0.4 (7.9-9.1) µm thick. Two polar capsules were pyriform and slightly unequal with larger polar capsule 8.4 ± 0.4 (7.6-10.2) µm × 3.9 ± 0.2 (3.0-4.5) µm and smaller capsule 7.9 ± 0.2 (7.0-9.3) µm × 3.7 ± 0.3 (2.8-4.1) µm. Polar filaments coiled with seven to eight turns. Both morphology and DNA sequence data revealed that M. honghuensis n. sp. was distinct from other described Myxobolus species. Phylogenetic analysis placed M. honghuensis n. sp. in a clade of gill-infecting myxobolids.

Some additional data to the occurrence, morphology and validity of Myxobolus turpisrotundus Zhang, 2009 (Myxozoa: Myxosporea).

Myxobolus turpisrotundus Zhang, 2009, infects allogynogenetic gibel carp Carassius auratus gibelio (Bloch) and is always regarded as synonymous with Myxobolus rotundus Nemeczek, 1911, since its first report in goldfish Carassius auratus auratus (L.) in China in 1955. In this study, it was comprehensively examined by morphological and molecular biological methods. The round spores of M. turpisrotundus are similar to those of M. rotundus from common bream Abramis brama (L.) in morphology; however, we detected slight differences in morphometry. The ratios of the length and width of the spore to the length and width of the polar capsule of M. turpisrotundus are usually below 2.0 and 1.9, respectively, however these ratios are always above 2.0 and 1.9 in M. rotundus. The plasmodium size of M. turpisrotundus is 600-6,200 microm in diameter and that of M. rotundus is 60-180 microm in diameter. Scanning observation showed the spore surface of M. turpisrotundus was generally pitted. Yet the surface of M. rotundus is smooth. Sequence comparison revealed the small subunit ribosomal RNA gene sequence of M. turpisrotundus did not match any published sequences of M. rotundus (EU710583, 85% over 742 bp; FJ851447, 85% over 742 bp, FJ851448, 85% over 742 bp; FJ851449, 85% over 742 bp). Moreover, phylogenetic analysis showed M. turpisrotundus clustered with the species from allogynogenetic gibel carp with high bootstrap values (100% neighbor-joining, NJ; 100% maximum parsimony, MP) and M. rotundus from common bream composed a new cluster with high bootstrap values (100% NJ, 100% MP). From the morphological and molecular biological data, we gain enough evidences to support the validity of M. turpisrotundus.

Myxobolus turpisrotundus (Myxosporea: Bivalvulida) spores with caudal appendages: investigating the validity of the genus Henneguya with morphological and molecular evidence.

Spores of the myxozoan parasite Myxobolus turpisrotundus Zhang 2009 were observed for the first time bearing caudal appendages. Most spores had the typical Myxobolus spp. morphology, but approximately 10% of spores possessed a spore body that was slightly elongated with a short tail projecting from the spore valve. In other spores, the tail was much more clearly visible and elongate. The spore body of these unusual spores is consistent in morphology and dimension to the normal spores of M. turpisrotundus. Both spore types were found within individual cysts, and the small subunit ribosomal RNA (ssrRNA) gene sequence from parasite cysts of this type was nearly identical to the previously published sequence of M. turpisrotundus from allogynogenetic gibel carp Carassius auratus gibelio (Bloch). The phenomenon of Myxobolus spores with caudal appendages provides additional evidence that the use of this character to separate Myxobolus and Henneguya into distinct genera is not reflective of an evolutionarily accurate classification scheme. Phylogenetic analysis of ssrDNA sequence from Myxobolus and Henneguya species showed clustering of species in some locations of the tree, but ultimately these genera are intermixed. The use of a single character to delineate species in the two most species-rich myxozoan genera has been consistently challenged where DNA analyses are used. The present finding of a single species bearing both Myxobolus-type and Henneguya-type spores emphasizes the inadequacy of this classification scheme, and highlights the need for careful consideration of these variable characteristics when describing myxozoan species.

Antileukemic roles of human phospholipid scramblase 1 gene, evidence from inducible PLSCR1-expressing leukemic cells.

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated protein which is localized in either the cell membrane or nucleus depending on its palmitoylated state. The increasing evidence showed the biological roles of PLSCR1 in cell signaling, maturation and apoptosis. To investigate the functions of PLSCR1 in leukemic cells, we generated an inducible PLSCR1-expressing cell line using myeloid leukemic U937 cells. In this cell line, PLSCR1 was tightly regulated and induced upon tetracycline withdrawal. Our results showed that inducible PLSCR1 expression arrested the proliferation of U937 cells at G1 phase. Meanwhile, PLSCR1-overexpressing U937 cells also underwent granulocyte-like differentiation with increased sensitivity to etoposide-induced apoptosis. Furthermore, we also found that PLSCR1 induction increased cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1) proteins, together with downregulation of S phase kinase-associated protein 2 (SKP2), an F-box subunit of the ubiquitin-ligase complex that targets proteins for degradation. Additionally, PLSCR1 induction significantly decreased c-Myc protein and antiapoptotic Bcl-2 protein. Although the exact mechanism by which PLSCR1 regulates these cellular events and gene expression remains unresolved, our results suggest that PLSCR1 plays the antagonistic role regarding leukemia development. These data will shed new insights into understanding the biochemical and biological functions of PLSCR1 protein.

Isolation and complete sequence of the purL gene encoding FGAM synthase II in Lactobacillus casei.

The purL gene from Lactobacillus casei, encoding phosphoribosylformylglycinamidine synthase II involved in the de novo synthesis of purines, was cloned and sequenced. The putative purL product of 741 amino acids (M(r) of 79,575) shows 25% and 53% identity to the homologous enzymes from Escherichia coli and Bacillus subtilis, respectively. In addition, partial sequences of two other pur genes (purQ and purF) and a possible third gene (purC) were obtained. All these genes are organized in an operon similar to that of B. subtilis. In contrast, the corresponding genes from E. coli and Salmonella typhimurium are scattered through the genome.

New bioactive adjacent bis-THF annonaceous acetogenins from Annona bullata.

Five new adjacent bis-THF annonaceous acetogenins, 32-hydroxybullatacin, 31-hydroxybullatacin, 30-hydroxybullatacin, and (2,4-cis and trans)-28-hydroxybullatacinones, were isolated from the ethanolic extract of the bark of Annona bullata Rich. (Annonaceae). The absolute configurations of the above five compounds, as well as those of (2,4-cis and trans)-32-, 31-, and 30-hydroxybullatacinones and (2,4-cis and trans)-bulladecinones, previously isolated from the same extract, were defined by the application of the advanced Mosher ester [methoxy(trifluoromethyl)phenyl acetate or MTPA] methodology. The determination of the absolute configuration of C-20 of (2,4-cis and trans)-bulladecinones to be S supports our hypothesis that the cyclization of the THF rings of (2,4-cis and trans)-bulladecinones starts from C-12 (the right side). The first five compounds listed above showed potent bioactivities in the brine shrimp lethality test (BST) and among six human solid tumour cell lines.

Mono-THF ring annonaceous acetogenins from Annona squamosa.

Continuing work on the bark of Annona squamosa Rich. (Annonaceae), directed by the brine shrimp lethality test (BST), has resulted in the isolation of three new Annonaceous acetogenins, 4-deoxyannoreticuin, cis-4-deoxyannoreticuin, and (2,4-cis and trans)-squamoxinone. The first two are additional examples of acetogenins isolated from this plant species which contain the unusual feature of an oxygen functionality at the C-9 position. They have a hydroxylated mono-THF ring with respective threo/trans/threo and threo/cis/threo relative stereochemistries. The latter compound is a ketolactone mixture which has the same relative stereochemistry around the THF ring and the same spatial relationship between the THF ring and the hydroxyl group along the aliphatic chain as 4-deoxyannoreticuin, but is two methylene units longer. Additionally, the isolated hydroxyl group is at C-11, while the THF ring starts at C-17, instead of at C-9 and C-15, respectively, as for the first two compounds. All three compounds showed moderate, but significant, cytotoxicities against a panel of six human tumor cell lines with (2,4 cis and trans)-squamoxinone showing promising selectivity against the pancreatic cell line (PACA-2).

Role of aorto-angioplasty in hypertension caused by Takayasu's arteritis.

Sixteen patients with Takayasu's arteritis and a long-stenotic segment of the descending thoracic aorta and/or proximal and middle portion of the abdominal aorta, with or without renal artery stenosis, were treated by percutaneous transluminal aorto-angioplasty (PTAA). These patients might be subdivided into three groups: A. with normal renal artery, B. with severe renal artery stenosis or occlusion without treatment or with failure of dilatation, and C. with renal artery stenosis but relieved by percutaneous transluminal renal angioplasty (PTRA). It was found that the hypertension may be caused by the suprarenal aortic stenosis itself. PTAA has an excellent effect to normalize the blood pressure in Group A. The elevated blood pressure may also be decreased in various extent in the patient with severe renal artery stenosis after PTAA, but not to normal. An additional PTRA or other renal vesel reconstruction procedure is necessary in this group. No recurrence of the aorta stenosis was found by non-invasive examinations or aortography during the long-term follow-up. PTAA may be considered as the first choice for the aortic stenosis caused by Takayasu's arteritis.

[A study of commonly used substitutes for Chinese drug cortex Eucommiae(Duzhong)].

We have found that 48 species belonging to 10 families and 17 genera are being used as ethnic substitutes for Cortex Eucommiae (Duzhong). A key to all the species has been made to identify them and to distinguish them from Eucommia ulmoides.

Supplemental diagnosis of a myxozoan parasite from common carp Cyprinus carpio: synonymy of Thelohanellus xinyangensis with Thelohanellus kitauei.

Thelohanellus kitauei Egusa et Nakajima, 1981, was described from common carp Cyprinus carpio L. in Japan. In China, a myxosporean infecting the intestinal tissue of the same host species was described as Thelohanellus xinyangensis Xie, Gong, Xiao, Guo, Li et Guo, 2000, despite many similarities to T. kitauei. To examine the potential conspecificity of these species, a morphological and molecular investigation of T. xinyangensis was conducted. Comparing myxospore morphology, the mean spore length and width of each species is not identical between species, but ranges of dimensions overlap. These data are more suggestive of intraspecific variation than distinct species. Comparison of relative ratios of spore length to polar capsule length and spore width to polar capsule width of T. xinyangensis and T. kitauei reveal no differences and scanning electron microscopy reveals a smooth spore surface of T. xinyangensis, which is consistent with that of T. kitauei. Most convincingly, DNA sequences of the small subunit ribosomal RNA (ssrRNA) gene of the two species were identical. From the morphological and molecular biological data, we propose T. xinyangensis from the intestine of common carp is not a distinct species and is synonymous with T. kitauei.

Isolation and complete sequence of the yeast isoleucyl-tRNA synthetase gene (ILS1).

The isoleucyl-tRNA synthetase gene (ILS1) from the yeast Saccharomyces cerevisiae was cloned and sequenced. This gene was initially cloned because it cross-hybridizated to what is now presumed to be the isoleucyl-tRNA synthetase gene (cupC) from the protozoan Tetrahymena thermophila. The ILS1 gene was determined to be 1,072 amino acids in length. A comparison with a recently published sequence of ILS1 from another laboratory (Englisch et al. 1987) was made and differences noted. Two promoter elements were detected, one for general amino acid control and one for constitutive transcription. A heat shock protein (hsp70) gene (probably SSA3) was found 237 bp upstream from the ILS1 translation start site. The ILS1 amino acid sequence was compared to isoleucyl-tRNA synthetases from other organisms, as well as to valyl-, leucyl- and methionyl-tRNA synthetases. Regions of conservation between these enzymes were found.

Two new bioactive monotetrahydrofuran Annonaceous acetogenins from the bark of Xylopia aromatica.

Xylopien [1] and xylomatenin [2], two new bioactive monotetrahydrofuran Annonaceous acetogenins, have been isolated from an EtOH extract of the bark of Xylopia aromatica, using bioactivity-directed fractionation employing lethality to brine shrimp. These new compounds each have a double bond in the hydrocarbon chain and have been identified as C-23, C-24 dehydro analogs of xylopiacin and xylomaticin. Their structures were elucidated by spectral analyses of the parent compounds and/or simple chemical derivatives. Their absolute stereochemistries have been established by 1H- and 2D nmr experiments utilizing the production of Mosher esters. These acetogenins showed cytotoxic potencies superior to adriamycin against three human solid tumor cell lines.