Biochemical properties of purified cathepsin B from Schistosoma mansoni

A previously described “major acidic proteinase” of adult Schistosoma mansoni, believed to play a key role in the parasite's metabolism, has been identified as a cathepsin B (Sm31). Purified Sm cathepsin B was not recognized by anti-Sm32 or anticathepsin L antibodies. The enzyme hydrolyzes the synthetic protease substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC as well as protein substrates. Its pH optimum is 3.0 with serum albumin, 4.0–5.0 with globin and 5.5–6.0 with the synthetic substrates. The enzyme was inactivated by cysteine proteinase inhibitors. Its activity against protein substrates would support the hypothesis that it plays a role in schistosome nutrition.     

Schistosoma mansoni: Permanence of modulation of the granulomatous inflammatory response in mice cured in the chronic phase

Persistence of down regulation of granoloma size was studied in mice chronically infected with Schistosoma mansoni and cured by chemotherapy. The animals were reinfected at 20-, 50-, 110- and 140-day intervals after treatment, and sacrificed 60 days post-reinfection. Reinfected animals were able to modulate the granulomatous inflammatory response, thus preventing a new acute phase. These findings may contribute to the explanation for the decrease of morbidity from human schistosomiasis seen in endemic areas following mass treatment.     

核糖体转录间隔子2应用于鱼类种属的鉴别

为了防止珍稀鱼类的非法捕捞和销售, 鱼类种属的鉴别就成为非常关键的问题, 特别是形态学方法无法区分的样品(如鱼苗、鱼鳞、鱼卵、鱼肉及其加工产品等)。为了帮助珍稀鱼类资源的管理和保护, 文章报道了一种利用核糖体基因的转录间隔子2鉴别鱼类种属的分子遗传学方法: (1) 利用同一目鱼类5.8S rRNA和28S rRNA基因的保守性, 设计出扩增鲤形目鱼类这两个基因间转录间隔子2 DNA片段, 测序获得它们的碱基排列顺序; (2) 再根据不同鱼类转录间隔子2序列的差异, 设计出每种鱼的种属特异引物、种属鉴别标准物, 构建鱼类分子分类图谱, 利用PCR复合扩增技术鉴别鱼类种属。通过对国内不同地方采集的5种鲤形目鱼类的210个单一品种样本和40个混合样本的鉴别检验, 该方法能够准确、灵敏和快速鉴别这5种鱼, 可用于鱼类资源保护和评估、管理和开发, 特别是在渔业管理人员渔业执法、海关打击珍稀鱼类走私、防止商业欺诈和外来有害生物入侵等方面非常有用     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

Chimerical nature of the ribosomal RNA gene of a Nosema species

Taxonomic resolution of the Nosema/Vairimorpha clade has been augmented with DNA sequences of the small subunit (SSU) and large subunit (LSU) ribosomal RNA (rRNA) and the arrangement of SSU and LSU. Based on the two characteristics, the clade is largely divided into two, i.e. ‘true’ Nosema sub-group and non-‘true’ Nosema sub-group within the clade. Our study shows that a novel Nosema species isolated from Pieris rapae has mixed characteristics of the ‘true’ and non-‘true’ Nosema sub-group based on the topology of SSU and LSU sequences. To our knowledge, this may be the first case of the incongruent phylogenetic placement of SSU and LSU in the Nosema/Vairimorpha clade. Additionally, the length of internal transcribed spacer (ITS) can be a diagnostic tool to distinguish ‘true’ Nosema from non-’true’ Nosema in the Nosema/Vairimorpha clade based on its nucleotide length as reported before.     

Effect of F-2 and T-2 fusariotoxins on experimental Cryptosporidium baileyi infection in chickens

The course of Cryptosporidium baileyi infection in chickens fed with different doses of fusariotoxins was compared with that of control groups. F-2 toxin levels of 0.187–1.5 mg kg⁻¹ and T-2 toxin levels of 0.187–6.0 mg kg⁻¹ were investigated. The experimental amimals were orally infected with 6 × 10⁵ C. baileyi oocysts at 1 week of age. Total daily oocyst output was monitored by a quantitative method. Acquired immunity was tested at the age of 4 weeks, by ELISA and by a challenge infection with an equal number of oocysts, upon recovery from the primary infection. The results show that in chickens kept on the lower doses of F-2 and T-2 toxins, the parasite infection ran a similar course to that in the control groups, and the animals became resistant to re-infection. However, when higher doses (2.0–6.0 mg kg⁻¹) of T-2 toxin were used, a depression of weight gain was observed with some other physiological parameters (PCV, weight of bursa, weight of thymus, skin thickness in PHA-P skin test) also indicating toxic effect and, simultaneously, the oocyst output decreased significantly and the patent period was slightly prolonged. Although certain modifications of the immune response could be revealed, the chickens became resistant to re-infection. Only early (1 week of age) parasite infection and 6 mg kg⁻¹ T-2 toxin in the feed significantly depressed body weight gain and immunity.     

The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis

A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12 Δ42–52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917–922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12: (Pro)₇, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:Δ42–52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)₇, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:Δ42–52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bounds in hr Asite with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro₇ sequence retains sufficient flexibility for full activity.     

Gsh-2, a murine homeobox gene expressed in the developing brain

A novel murine dispersed homeobox gene, designated Gsh-2, is described. Analysis of cDNA sequence, including the full open reading frame, reveals an encoded homeodomain that is surprisingly similar to those of the Antennapedia-type clustered Hox genes. In addition, the encoded protein includes polyhistidine and polyalanine tracts, as observed for several other genes of developmental significance. In situ hybridizations showed Gsh-2 expression in the developing central nervous system, including the ganglionic eminences of the forebrain, the diencephalon, which gives rise to the thalamus and hypothalamus, and in the hindbrain. Furthermore, a random oligonucleotide selection and PCR amplification procedure was used to define a target DNA binding sequence, CNAATTAG, as a first step towards the identification of downstream target genes.     

Multiple gene genealogies and species recognition in the ectomycorrhizal fungus Paxillus involutus

Paxillus involutus (basidiomycetes, Boletales) is a common ectomycorrhizal fungus in the Northern Hemisphere. The fungus displays significant variation in phenotypic characters related to morphology, physiology, and ecology. Previous studies have shown that P. involutus contains several intersterility groups and morphological species. In this study, we have used concordance of multiple gene genealogies to identify genetically isolated species of P. involutus. Fragments from five protein coding genes in 50 isolates of P. involutus collected from different hosts and environments in Europe and one location in Canada were analysed using phylogenetic methods. Concordance of the five gene genealogies showed that P. involutus comprises at least four distinct phylogenetic lineages: phylogenetic species I (with nine isolates), II (33 isolates), III (three isolates), and IV (five isolates). The branches separating the four species were long and well supported compared with the species internodes. A low level of shared polymorphisms was observed among the four lineages indicating a long time since the genetic isolation began. Three of the phylospecies corresponded to earlier identified morphological species: I to P. obscurosporus, II to P. involutus s. str., and III to P. validus. The phylogenetic species had an overlapping geographical distribution. Species I and II differed partly in habitat and host preferences.     

Cytopathology and release of an RNA virus from a strain of Trichomonas vaginalis

A strain of Trichomonas vaginalis infected with a double-stranded RNA virus showed pronounced cytopathology in the form of giant syncytia generated by the recruitment of single cells. The giant cells ultimately lysed, releasing virus into the culture medium. In the infected cells, clusters of electron-dense particles resembling viral structures were found in the cytoplasm. In addition, distinctive inclusions composed of similar particles were present in the nuclei of some cells. Double-stranded viral RNA of 5.5 kbp was demonstrated in both cytoplasmic and nuclear fractions from these cells. Viral particles collected from the cell-free culture supernatant were of the same shape and size as the RNA virus isolated from a strain of T. vaginalis described previously (Wang & Wang, Journal of Biological Chemistry, 260: 3697–3702, 1985; Wang & Wang, Proceedings of the National Academy of Sciences of the U.S.A. 83: 7956–7986) which does not show this cytopathology.     

Molecular phylogeny of Pseudokeronopsis (Protozoa, Ciliophora, Urostylida), with reconsideration of three closely related species at inter- and intra-specific levels inferred from the small subunit ribosomal RNA gene and the ITS1-5.8S-ITS2 region sequences

In order to address the confused taxonomy of morphotypes within the genus Pseudokeronopsis , the small subunit ribosomal RNA (SS rRNA) gene and the internal transcribed spacer (ITS1)-5.8S-ITS2 region for 13 populations/clones of morphologically similar Pseudokeronopsis species, Pseudokeronopsis flava , Pseudokeronopsis rubra and Pseudokeronopsis carnea , were sequenced and compared at inter- and intra-specific levels. Different geographical/time interval isolates belonging to the same species were slightly divergent, while clones from the same strain had identical SS rRNA gene sequences. Compared with the SS rRNA gene sequence, the sequence of the ITS1-5.8S-ITS2 region of Pseudokeronopsis revealed higher diversity, with nucleotide dissimilarities of 3.9–4.9% at the intra-specific level and 8.0–10.5% at the inter-specific level. There were several minor differences among ITS2 secondary structures of three Pseudokeronopsis species, while three Pseudok. carnea populations were identical. Phylogenetic analyses using multiple algorithms and different species selections confirm that Pseudok. flava , Pseudok. rubra and Pseudok. carnea form a monophyletic group within the urostylids. The result also supports the closest relationship of Pseudokeronopsis and Pseudourostyla that forms the so-called Acaudalia Berger (2006) .     

Sequence and characterisation of a ribosomal RNA operon fromAgrobacterium vitis

One of the four ribosomal RNA operons (rrnA) from theAgrobacterium vitis vitopine strain S4 was sequenced.rrnA is most closely related to therrn operons ofBradyrhizobium japonicum andRhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case ofR. sphaeroides. The 16S rRNA sequence of S4 differs from theA. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3 ends of the three otherrrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3 ends of the four repeats and defines two groups:rrnA/rrnB andrrnC/rrnD.     

Strongylus asini (Nematoda, Strongyloidea): Genetic relationships with other strongylus species determined by ribosomal DNA

Genomic DNA was isolated from adult Strongylus asini collected from zebra. The second ribosomal transcribed spacer (ITS-2) was amplified and sequenced using polymerase chain reaction (PCR) based techniques. The DNA sequence was compared with previously published data for 3 related Strongylus species. A PCR-linked restriction fragment length polymorphism method allowed the 4 species to be differentiated unequivocally. The ITS-2 sequence of S. asini was found to be more similar to those of S. edentatus (87.1%) and S. equinus (95.3%) than to that of S. vulgaris (73.9%). This result confirms that S. asini and S. vulgaris represent separate species and supports the retention of the 4 species within 1 genus.     

云南真藓和细叶真藓的核糖体DNA ITS序列分析

从形态特征和地理分布特征比较分析云南真藓和细叶真藓的区别,通过对核糖体DNA的内转录间隔区序列分析研究,对二者进行分子水平鉴定.对包括真藓属及相关属的6种植物的ITS序列进行扩增和测序,使用DNAMAN和MEGA3.0软件对获得的ITS序列数据进行分析.结果表明,云南真藓和细叶真藓的ITS-1和ITS-2区在序列长度,...     

Schistosoma mansoni, NIH-Sm-PR-2 strain, in non-susceptible Biomphalaria glabrata: protection by Echinostoma paraensei

Sullivan J. T., Richards C. S., Lie K. J. and Heyneman D. 1981. Schistosoma mansoni, NIH-Sm-PR-2 strain, in non-susceptible Biomphalaria glabrata: Protection by Echinostoma paraensei. International journal for Parasitology11:481–484. Among seven inbred genetic stocks of Biomphalaria glabrata that are non-susceptible for the NIH-Sm-PR-2 strain of Schistosoma mansoni (PR-2), five stocks revert to nearly complete susceptibility when first infected with Echinostoma paraensei. These include both stocks in which PR-2 sporocysts are normally destroyed within 3–7 days, and stocks in which sporocysts often survive undeveloped for at least 3 weeks. Hence, these five stocks are resistant to but physiologically suitable for the development of PR-2. Of the two remaining stocks, one remains partly non-susceptible to PR-2, since less than 50 % of echinostome-infected snails revert to susceptibility, while the other stock remains completely non-susceptible to PR-2 following echinostome infection, due perhaps to a high level of residual resistance and/or unsuitability.     

Growth phase and growth rate dependence of ribosomal peptidyltransferase activity status in Escherichia coli

Ribosomes from a clinical isolate of E coli were purified and characterized. The structural features of these ribosomes were identical to wild-type E coli ribosomes, with the exception that rRNA in general, but especially 23S rRNA, was degraded as a result of the transition from early to late logarithmic growth phase, on different growth media. Analysis of the ribosomal protein by gel electrophoresis indicated that the L12/L7 molar ratio increases during early logarithmic phase, reaching a maximum value of about 1.6 at midlogarithmic phase, and then falling to 0.7 in late logarithmic phase. Concomitantly with L12/L7 alterations, the activity status of ribosomal peptidyltransferase was found to undergo a striking shift. Reconstitution experiments demonstrated that the two effects are closely related. Moreover, L12/L7 molar ratio as well as peptidyltransferase activity increased with increasing growth rate. In the latter case, however, the acetylation level of L12 protein per se seemed to be inadequate to modulate the peptidyltransferase activity.     

A Conserved Motif in the 5.8S Ribosomal RNA (rRNA) Gene is a Useful Diagnostic Marker for Plant Internal Transcribed Spacer (ITS) Sequences

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has become an important nuclear locus for molecular systematic investigations of angiosperms at the intergenic and interspecific levels. Universal PCR primers are positioned on the conserved rRNA genes (18S, 5.8S, 26S) to amplify the entire ITS spacer region. Recent reports of fungal and algal contaminants, first described as plant ITS sequences, stress the need for diagnostic markers specific for the angiosperm ITS region. This report describes a conserved 14 base pair (bp) motif in the 5.8S rRNA gene that can be used to differentiate between flowering plants, bryophytes, and several orders of algae and fungi, including common plant pathogenic and non-pathogenic fungi. A variant of the motif (found in fungi and algae) contains a convenient EcoRI restriction site that has several applications for eliminating problematic contaminants from plant ITS preparations.