<Emphasis Type="Italic">Helicobacter pylori</Emphasis> Virulence Genotypes in Portuguese Children and Adults with Gastroduodenal Pathology

The aim of this study was to evaluate the prevalence of virulence genotypes, namely cagA, vacA and babA2, of Helicobacter pylori strains isolated from Portuguese adults and children presenting gastroduodenal pathology. One hundred thirty-six strains were studied, 82 isolated from adult patients (50 with nonulcerative gastritis and 32 with active peptic ulcer) and 58 isolated from children (54 with nonulcerative gastritis and 4 with duodenal ulcer). Genotyping of cagA, vacA and babA2 was assessed by polymerase chain reaction. Overall, Helicobacter pylori strains carrying more virulent genotypes were much more prevalent in adults than in children, particularly the type I (vacAs1- and cagA-positive) and the triple-positive (vacAs1-, cagA- and babA2-positive) strains (P<0.001). A subpopulation of adults and children with nonulcerative gastritis was also studied, and differences in the prevalence of virulent genotypes were observed, either for individual genotypes (P=0.017 for cagA, P=0.010 for vacAs1) or in combinations, i.e. the type I genotype (P=0.005) and the triple-positive strains (P=0.031). There was no difference between the two populations in the distribution of babA2 and m1/m2 genotypes. Considering the cohort effect in the epidemiology of Helicobacter pylori infection, these results suggest that different strains might circulate during different periods of time, or that, after infection in childhood, individual strains will undergo changes during the course of infection. Electronic Publication     

The importance of <Emphasis Type="Italic">Helicobacter pylori tnpA</Emphasis>, <Emphasis Type="Italic">tnpB</Emphasis>, and <Emphasis Type="Italic">cagA</Emphasis> genes in various gastrointestinal diseases

The cag (cytotoxin-associated gene) pathogenicity island (cagPAI) is one of the major virulence determinants of Helicobacter pylori (H. pylori). The purpose of this study was to investigate the association of the three genes (tnpA, tnpB, and cagA) in H. pylori isolated from Azerbaijani patients with the different gastrointestinal disease. A total of 362 gastric biopsies were collected from hospitals of Tabriz University of Medical Sciences, and were cultured on Brucella agar. The tnpA, tnpB, and cagA genes were detected by PCR. Of the total 264 H. pylori isolates, tnpA, tnpB, and cagA genes were detected in 120 (45.5%), 56 (21.2%) and 172 (65.2%), respectively. A significant association between tnpA and tnpB genes and clinical outcomes were found (P < 0.05). The cagA status was not related to clinical outcomes in our subjects. The predominant genotype among cag-PAI is the cagA. The prevalence of tnpA, tnpB, and cagA genes are high in patients with gastric cancer, and a significant association is revealed between tnpA and tnpB with gastric cancer.     

Influence of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> infection on the expression of <Emphasis Type="Italic">MLH1</Emphasis> and <Emphasis Type="Italic">MGMT</Emphasis> in patients with chronic gastritis and gastric cancer

The aim of the present study was to evaluate the influence of Helicobacter pylori on MLH1 and MGMT mRNA levels in patients with chronic gastritis and gastric cancer. The study included 217 patients, of which 26 were uninfected, 127 had chronic gastritis and were H. pylori-positive, and 64 had gastric cancer. Bacterial genotypes were evaluated by polymerase chain reaction (PCR), and the expression levels of MLH1 and MGMT were determined by quantitative real-time PCR and immunohistochemistry. There was an association between infection with cagA, vacA s1m1 strains and gastric cancer development. When the gastric epithelium and associated inflammation were examined for expression of MLH1 and MGMT, an overall increase in expression was observed. On the other hand, these levels decrease significantly among gastric cancer patients. The loss of MLH1 and MGMT expression in gastric cancer patients suggests that it is not an early event in H. pylori-associated gastric carcinogenesis.     

Malo-ethanolic fermentation in<Emphasis Type="Italic"> Saccharomyces</Emphasis> and<Emphasis Type="Italic"> Schizosaccharomyces</Emphasis>

Yeast species are divided into the K(+) or K(–) groups, based on their ability or inability to metabolise tricarboxylic acid (TCA) cycle intermediates as sole carbon or energy source. The K(–) group of yeasts includes strains of Saccharomyces, Schizosaccharomyces pombe and Zygosaccharomyces bailii, which is capable of utilising TCA cycle intermediates only in the presence of glucose or other assimilable carbon sources. Although grouped together, these yeasts have significant differences in their abilities to degrade malic acid. Typically, strains of Saccharomyces are regarded as inefficient metabolisers of extracellular malic acid, whereas strains of Sch. pombe and Z. bailii can effectively degrade high concentrations of malic acid. The ability of a yeast strain to degrade extracellular malic acid is dependent on both the efficient transport of the dicarboxylic acid and the efficacy of the intracellular malic enzyme. The malic enzyme converts malic acid into pyruvic acid, which is further metabolised to ethanol and carbon dioxide under fermentative conditions via the so-called malo-ethanolic (ME) pathway. This review focuses on the enzymes involved in the ME pathway in Sch. pombe and Saccharomyces species, with specific emphasis on the malate transporter and the intracellular malic enzyme.Communicated by S. Hohmann     

Prevalence of virulence-associated genotypes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and correlation with severity of gastric pathology in patients from western Sicily,Italy

In a bacterium like Helicobacter pylori, which is characterized by a recombinant population structure, the associated presence of genes encoding virulence factors might be considered an expression of a selective advantage conferred to strains with certain genotypes and, therefore, a potentially useful tool for predicting the clinical outcome of infections. However, differences in the geographical and ethnic prevalence of the H. pylori virulence-associated genotypes can affect their clinical predictive value and need to be considered in advance. In this study we carried out such an evaluation in a group of patients living in Sicily, the largest and most populous island in the Mediterranean Sea. cagA, vacA, babA2, hopQ, oipA, sabA, and hopZ were the H. pylori virulence-associated genes assayed; their presence, expression status or allelic homologs were detected in H. pylori DNA samples and/or isolated strains, obtained by gastric biopsy from 90 Sicilian patients with chronic gastritis, inactive (n = 37), active (n = 26), or active with peptic ulcer (n = 27). Genotypes cagA ⁺, vacAs1, vacAm1, babA2 ⁺, and hopQ I, I/II were identified in 51.8, 80.4, 35.2, 47.3, and 67.7% of the different samples respectively. Only these genotypes were associated with each other and with the active form of chronic gastritis, irrespective of the presence of a peptic ulcer. In our isolates their prevalence was more similar to values observed in the north of Italy and France than to those observed in Spain or other Mediterranean countries that are closer and climatically more similar to western Sicily.     

Hypermethylation of<Emphasis Type="Italic"> Chfr</Emphasis> and<Emphasis Type="Italic"> hMLH1</Emphasis> in gastric noninvasive and early invasive neoplasias

Human tumors are genetically unstable, and the instability exists at two distinct levels—the chromosomal level and the nucleotide level. Chfr and hMLH1 hypermethylation, which may lead to chromosomal instability (CIN) and microsatellite instability (MSI), respectively, was analyzed in gastric noninvasive neoplasias (NIN, Padova international classification) and submucosal invasive adenocarcinomas and in their corresponding non-neoplastic gastric epithelia. Results were compared with microsatellite status, p53 immunoreactivity, and cellular phenotype. Hypermethylation of Chfr and hMLH1 was observed in: 10% (1/10) and 0% (0/10) of low-grade NIN (L-NIN); 63% (5/8) and 63% (5/8) of high-grade NIN, including suspicion for carcinoma without invasion (H-NIN); 36% (5/14) and 57% (8/14) of high-grade NIN, including carcinoma without invasion; and 35% (7/20) and 25% (5/20) of submucosal invasive adenocarcinomas, respectively. Hypermethylation was less frequent in L-NIN than H-NIN (P<0.05) for Chfr and was also less frequent in L-NIN than the others (P<0.05) for hMLH1. We failed to find a significant correlation between Chfr hypermethylation and chromosomal loss of heterozygosity, although hypermethylation of hMLH1 was significantly associated with high-frequency MSI (P<0.01). Expression of p53 was not associated with Chfr or hMLH1 methylation. As for cellular phenotype, hypermethylation of Chfr and hMLH1 was frequent in tumors exhibiting the foveolar epithelial phenotype (50%, 2/4 and 75%, 3/4, respectively) and the ordinary phenotype (40%, 16/40 and 38%, 15/40, respectively), but never in those with the complete-type intestinal metaplastic phenotype (0%, 0/8 for both). In addition, hypermethylation of Chfr and hMLH1 occurred concurrently (P<0.01); methylation was more frequent in patients over 70 years of age (P<0.01), and it was also present in some samples of non-neoplastic gastric epithelia from elderly patients. Thus, some gastric tumors with the foveolar or ordinary phenotype may develop as a result of age-related methylation of Chfr and hMLH1, although Chfr methylation was not associated with CIN.     

Detection of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in bioptates of gastric mucosa of patients with gastritis and gastric ulcers using real-time PCR

Helicobacter pylori is currently believed to play an important role in the pathogenesis of gastritis, gastric and duodenal ulcers, and stomach cancer. The ability of H. pylori to colonize gastric mucosa and cause inflammation is determined by genetic factors, three of which are the best studied: the pathogenicity factors CagA and VacA and the adhesion factor BabA. We developed primer systems and probes for real-time polymerase chain reaction (RT PCR), which allowed us to detect H. pylori in specimens and to perform typing of H. pylori virulence factors CagA, VacA, and BabA. Comparison of two H. pylori diagnostic techniques, histological studies of bioptates and RT PCR, demonstrated the superiority of RT PCR in specificity and sensitivity. Typing of H. pylori demonstrated that 70–80% of strains had the cagA⁺/vacA s1 genotype and 10–20% had the cagA/vacA s2 genotype; babA was detected in 75–85% of all strains. H. pylori typing data did not reveal any correlations between the pathogenicity factors CagA and VacA or the adhesion factor BabA with severity of infection.     

Biochemical characterization of new strains of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis> and<Emphasis Type="Italic"> T. rangeli</Emphasis> isolates from Peru and Mexico

Seven trypanosome stocks isolated have been characterized by lectin agglutination, isoenzyme analysis, and the end products excreted. The stocks were isolated from different geographic areas—one from Mexico (TM5), and six from Peru, four of these isolated from different species of triatoma (TP504, TP702, TP704 and TP706), the other two isolated from the salivary glands of Rhodnius ecuadorensis (TRa605 and TRa606). Additionally, one strain of Trypanosoma cruzi isolated from a human case (strain TC-Maracay) and one strain of T. rangeli (TRa, Cajamarca-Peru strain), characterized and maintained in our laboratory, were used as reference strains. According to statistical study, the stocks were grouped into three clusters: (1) cluster I included the reference strain of T. cruzi (TC-Maracay); (2) cluster II was subdivided into two groups—subcluster IIA for the Mexican isolate (TM5) and subcluster IIB for the Peruvian ones, isolated from the salivary glands of Rhodnius ecuadorensis (TRa 605 and TRa 606) and the reference strain T. rangeli (TRa); these two new isolates were classified as T. rangeli; and (3) cluster III for the rest of the Peruvian isolates, which should be considered at least as a different strain from the T. cruzi strain Maracay. We show that the identification of T. cruzi and T. rangeli in mixed infections is readily achieved by biochemical methods. These findings identified three clusters of Mexican and Peruvian stocks that correlate with geographic origin, although assignment to a T. cruzi linage was not possible.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis>-infection-associated CpG island hypermethylation in the stomach and its possible association with Polycomb repressive marks

Helicobacter pylori infection can induce CpG island (CGI) hypermethylation in gastric mucosa. Recently, genes occupied by Polycomb proteins in embryonic stem cells were shown to be vulnerable to aberrant DNA hypermethylation in cancers. To explore the relationship between H. pylori infection and DNA methylation changes in neoplastic and non-neoplastic stomach, we analyzed 25 CGIs and repetitive DNA elements from 82 chronic gastritis and 69 gastric carcinomas. Twenty-three CGIs showed significantly higher methylation levels in H. pylori-negative gastric carcinoma (n = 28) than in H. pylori-negative chronic gastritis (n = 39; P < 0.05), indicating cancer-associated methylation. Eight CGIs exhibited significantly higher methylation levels in H. pylori-positive chronic gastritis (n = 43) than in H. pylori-negative chronic gastritis (n = 39; P < 0.05). Six CGIs showed both cancer-associated and H. pylori-associated hypermethylation. Six (75%) of the eight H. pylori-associated hypermethylated genes contained at least one of three repressive marks (Suz12 occupancy, Eed occupancy, histone H3 K27 trimethylation), whereas 31% of the remaining cancer-associated hypermethylated genes had at least one mark. The findings suggest that H. pylori infection strongly induces CGI hypermethylation in gastric epithelial cells and that susceptibility to H. pylori-induced DNA hypermethylation may be determined by Polycomb repressive marks in stem or progenitor cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Supported by the Korea Research Foundation Grant (MOEHRD; KRF-2005-041-E00081; G.H.K.) and by the second stage Brain Korea 21 Project.     

Molecular profiles of<Emphasis Type="Italic"> Trypanosoma brucei</Emphasis>,<Emphasis Type="Italic"> T. evansi</Emphasis> and<Emphasis Type="Italic"> T. equiperdum</Emphasis> stocks revealed by the random amplified polymorphic DNA method

A total of 20 random primers (10-mers) were used to amplify RAPD markers from the genomic DNA of four Trypanosoma brucei stocks from East and West Africa, four T. evansi stocks from Africa, Asia and South America and one T. equiperdum stock from Asia. Between 65 and 88 reproducible fragments ranging from 0.25 to 2.15 kb were generated from these stocks depending on the stock/primer combination. The similarity coefficient (SC) among the stocks of T. brucei from Kenya, Nigeria, Tanzania and Zambia ranged from 62.9% to 74.0% (average: 67.6%). The SC among the stocks of T. evansi from Kenya, China and Brazil was 76.4%–95.5% (average: 86.4%), while the SC between T. evansi stock from China and Brazil was 95.5%. For T. evansi and T. equiperdum, the SC among the stocks ranged from 81.2% to 94.4% (average: 87.6%). As for the SC among the stocks of T. brucei and T. evansi, it was found to be from 54.7% to 80.3% (average: 68.0%) and the SC among stocks of T. brucei and T. equiperdum was from 59.4% to 76.9% (average: 68.1%). Our results indicate that the stocks of T. evansi from China and from Brazil are more closely related to the stock of T. equiperdum from China than to the stocks of T. evansi isolated from Kenya and to the stocks of T. brucei. In addition, our results further support the hypothesis that T. evansi stocks from China and Brazil could have arisen from a single lineage. The possible evolution of T. evansi and T. equiperdum is also discussed.     

Prevalence of<Emphasis Type="Italic"> Sarcocystis</Emphasis> species sporocysts in Northern Virginia opossums (<Emphasis Type="Italic">Didelphis virginiana</Emphasis>)

A total of 206 Virginia opossums (Didelphis virginiana) collected from the mid-Michigan region, United States, during a period extending from 1996 to 2002 were sampled for the presence of Sarcocystis spp sporocysts. All isolates were phenotypically identified as Sarcocystis spp and genotyped to the species level by PCR-based techniques. The overall prevalence of Sarcocystis spp in opossums was 18% (37/206). The prevalence of Sarcocystis spp differed significantly with age (P<0.001) and adult opossums were more commonly infected (14.6%; 30/206) than juveniles (3.4%; 7/206). No significant difference in the prevalence of Sarcocystis spp infection was observed between male and female (P<0.15). The highest prevalence was recorded during summer (9.2%; 19/206). PCR-RFLP analyses demonstrated the majority of Sarcocystis isolates to be S. neurona, with some animals co-infected with sporocysts of S. falcatula. Out of the 37 Sarcocystis-infected opossums, 23 (62%) had sporocysts of S. neurona only, four (11%) had sporocysts of S. falcatula only, and eight (22%) had a mixture of S. neurona and S. falcatula sporocysts. These findings indicate that mixed Sarcocystis infections in opossums are common. The propensity for Sarcocystis spp to co-exist in the opossum gut enhances dissemination and environmental contamination with these coccidia. Additionally, this increases the chance for sexual recombination between Sarcocystis spp, given the proclivity of these species to reproduce sexually at high numbers in the intestinal cells of their definitive host.     

Identification of<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu lato,<Emphasis Type="Italic"> Anaplasma</Emphasis> and<Emphasis Type="Italic"> Ehrlichia</Emphasis> Species,and Spotted Fever Group Rickettsiae in Ticks from Southeastern Europe

Prevalence data for tick-borne pathogens are used to assess the risk for human health. In this study the presence and identity of Borrelia burgdorferi sensu lato, Ehrlichia, Anaplasma, and Rickettsia species in Bulgarian Ixodes ricinus ticks and in non-Ixodes ticks from Turkey and Albania was determined by polymerase chain reaction (PCR) and reverse line blot hybridization. In the adult Bulgarian ticks, the prevalence of Borrelia burgdorferi sensu lato infection was approximately 40%, while Borrelia afzelii was the predominant species, representing more than half of all Borrelia-positive ticks. Ehrlichia and Anaplasma species were detected in 35% of the adult Ixodes ricinus ticks and in 10% of the nymphs. Sequence analysis of PCR products reacting with the Anaplasma phagocytophila probe revealed a 16S rRNA gene identical to that of the Anaplasma phagocytophila prototype strain. Ehrlichia and Anaplasma species were found in approximately 7% of the non-Ixodes ticks. Sequence analysis of some of these samples revealed the presence of Anaplasma ovis, Ehrlichia canis, and a species closely resembling Ehrlichia chaffeensis. About half of all adult ticks examined and approximately 20% of all nymphs were infected with Rickettsia species. In Ixodes ricinus ticks, Rickettsia helvetica and a Rickettsia species designated as IRS3 were found in high prevalence. Rickettsia conorii was found in virtually all non-Ixodes tick species from Albania and Turkey. The results of this study show that many tick-borne diseases are most probably endemic in the Balkan area. Furthermore, the results suggest that there is a considerable chance for simultaneous transmission of tick-borne pathogens to human beings.     

Interactions related to non-host snails in the host-finding process of<Emphasis Type="Italic"> Euparyphium albuferensis</Emphasis> and<Emphasis Type="Italic"> Echinostoma friedi</Emphasis> (Trematoda: Echinostomatidae) miracidia

In order to determine whether the miracidia of Euparyphium albuferensis and Echinostoma friedi are sensitive to their host snail (HS) and capable of discriminating between HS and non-host snails (NHS), or whether these NHS can interfere and thus reduce the infection rates (decoy effect), a total of three experiments were conducted with HS, NHS and snail-conditioned water (SCW). Gyraulus chinensis is the HS for E. albuferensis miracidia, while Physa acuta, Radix peregra and Lymnaea fuscus are considered NHS. For E. friedi miracidia R. peregra, G. chinensis and L. fuscus are the HS, while P. acuta is the NHS. The NHS R. peregra produces the greatest decoy effect on Euparyphium albuferensis miracidia, while R. peregra, as the HS of Echinostoma friedi miracidia, is always subject to a NHS decoy effect. However, an increase in E. friedi miracidia infectivity is observed in its HS G. chinensis, in the presence of SCW of P. acuta. These experimental results might explain the low prevalence of snails infected with E. albuferensis miracidia in their natural habitat.     

Phylogeny of sheep and goat<Emphasis Type="Italic"> Theileria</Emphasis> and<Emphasis Type="Italic"> Babesia</Emphasis> parasites

The phylogenetic relationship of Theileria and Babesia species infecting sheep and goats on the basis of their 18S RNA gene structure was addressed in the present study. For this purpose, the complete sequences of the small ribosomal RNA genes of a panel of sheep and goat piroplasm isolates, including T. lestoquardi, T. ovis, T. separata, B. ovis, B. motasi, B. crassa and several novel species, were sequenced and compared. The classification based on the established phylogenetic tree corresponded with traditional systematics and revealed that sheep/goat piroplasm species are of polyphyletic origin. The independent evolution of almost all sheep/goat piroplasms suggests that speciation may have occurred after transfer of the piroplasm-transmitting tick from a primal wild ruminant host to domestic sheep and goats. In accordance with recent reports, our study confirms the existence of at least two additional sheep/goat piroplasm species, designated Theileria sp. 1 (China) and Theileria sp. 2 (China). The recently reported pathogenic sheep/goat Theileria sp. 1 (China) seems to be identical with a Theileria sp. isolated from Japanese serow. Furthermore, our results suggest that T. ovis represents a single species.     

Detection of <Emphasis Type="Italic">Helicobacter pylori</Emphasis>, <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>, and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in the subgingival biofilm of HIV-infected subjects undergoing HAART with chronic periodontitis

This study compared the frequency of Helicobacter pylori, Enterococcus faecalis, and Pseudomonas aeruginosa in the subgingival microbiota of HIV-seropositive and HIV-seronegative subjects with periodontitis or clinically healthy periodontal tissues. Fifty-four subjects were distributed into two HIV-seropositive groups (chronic periodontitis [HCP = 13] and periodontal health [HH = 10]) and two HIV-seronegative groups (chronic periodontitis [CP = 17] and periodontal health [H = 14]). The detection of bacterial species was carried out by polymerase chain reaction (PCR). CP patients showed significantly more periodontal destruction, inflammation, and supragingival plaque than HCP patients (P < 0.05). All species were detected at a higher prevalence in CP and HCP than H individuals (P < 0.01). In the HIV groups, H. pylori was significantly more prevalent in periodontitis compared to healthy patients (P < 0.01). A higher frequency of E. faecalis and P. aeruginosa was observed in the subgingival biofilm of HH than H subjects (P < 0.01). Moreover, E. faecalis was detected significantly more often in HIV-seropositive compared to HIV-seronegative patients, regardless of periodontal status (P < 0.01). These data indicate that H. pylori is frequently detected in the subgingival microbiota of periodontitis subjects. In contrast, HIV-seropositive patients with either periodontitis or periodontal health present a high prevalence of E. faecalis.     

The association of <Emphasis Type="Italic">vacA</Emphasis> genotypes and <Emphasis Type="Italic">Helicobacter pylori</Emphasis>-related gastroduodenal diseases in the Middle East

The variations in the three regions of the Helicobacter pylori vacA gene, the signal (s1 and s2), intermediate (i1 and i2) and middle regions (m1 and m2), are known to cause the differences in vacuolating activities. However, it was unclear whether these vacA genotypes are associated with the development of gastric cancer and peptic ulcer in the Middle East. The aim was to identify the prevalence of vacA genotypes in the Middle East and the association with gastroduodenal diseases. We investigated the relationship of vacA genotypes to H. pylori-related disease development by meta-analysis using previous reports of 1,646 patients from the Middle East. The frequency of the vacA s1, m1 and i1 genotypes in the Middle Eastern strains was 71.5% (1,007/1,409), 32.8% (427/1,300) and 40.7% (59/145), respectively. Importantly, the frequency of vacA s- and m-region genotypes significantly differed between the north and south parts of the Middle East countries (P < 0.001). The vacA genotypes significantly increased the risk of gastric cancer (odds ratio [OR]: 4.02, 95% confidence interval [CI]: 1.98–8.14 for the s1 genotype; 2.50, 1.62–3.85 for m1; 5.27, 1.97–14.1 for s1m1; 15.03, 4.69–48.17 for i1) and peptic ulcers (OR: 3.07, 95% CI: 2.08–4.52 for s1; 1.81, 1.36–2.42 for m1). The cagA-positive genotype frequently coincided with the s1, m1 and i1 genotypes. The vacA s- and m-region genotypes may be useful risk factors for gastrointestinal diseases in the Middle East, similar to European and American countries. Further studies will be required to evaluate the effects of the i-region genotype.     

Association of positional and functional candidate genes<Emphasis Type="Italic"> FGF1</Emphasis>,<Emphasis Type="Italic"> FBN2</Emphasis>, and<Emphasis Type="Italic"> LOX</Emphasis> on 5q31 with intracranial aneurysm

We previously performed a genome-wide linkage study of intracranial aneurysm (IA) and found positive evidence of linkage at chromosomes 5q22–31, 7q11, and 14q22. In the present study, we focus on 5q31, where three candidate genes, fibroblast growth factor 1 (FGF1), fibrillin 2 (FBN2), and lysyl oxidase gene (LOX) lie, and evaluate associations with IA. Genomic DNAs were obtained from 172 IA patients and 192 controls. Association analysis was performed with ten, five, and four single-nucleotide polymorphisms (SNPs) identified in FGF1, FBN2, and LOX, respectively. A difference in allelic frequency was observed for only the SNP at intron 4 in FGF1 (χ²=4.44, df=1, P=0.035). Although a haplotype association was observed with the combination of ten SNPs in FGF1 (χ²=16.04, df=1, P=0.00006), significant haplotype associations were not observed when haplotypes were constructed with the three, two, and four SNPs in FGF1 according to the linkage disequilibrium structure. No associations of FBN2 and LOX with IA were detected in the present study.     

Phylogenetic analysis of<Emphasis Type="Italic"> Theileria</Emphasis> species transmitted by<Emphasis Type="Italic"> Haemaphysalis qinghaiensis</Emphasis>

The phylogenetic relationships between six isolates of Theileria spp. infective to small ruminants, and two isolates of Theileria spp. infective to yak, all transmitted by Haemaphysalis qinghaiensis, together with the Theileria orientalis/sergenti/buffeli group and T. sinensis, were analyzed using the 18S ssrRNA gene sequence. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was used either for direct sequencing or was ligated to the PCR II vector for sequencing. The length of the 18S ssrRNA gene of all Theileria spp. involved in this study was around 1,740 bp. Two phylogenetic trees were inferred based on the 18S ssrRNA gene sequence of the Chinese isolates only, and Chinese isolates and other species of Theileria available in GenBank. In the first tree, the Theileria sp. infective to yaks was found to be T. sinensis. The Theileria sp. infective to small ruminants was found to be composed of two separate species of Theileria. Theileria sp. from Qinghai, Madang, Ningxian and Lintan, which was identical to the unidentified Theileria sp. described previously, is designated Theileria sp (China 1). The Theileria sp. from Longde, Zhangjiachuan and Lintan, which has not been described previously, is designated Theileria sp. (China 2) in order to avoid confusion. In the second tree, Theileria sp. (China 1) was closely related to benign Theileria, such as T. buffeli and T. sergenti, while Theileria sp. (China 2) was separated from other Theileria spp. The results indicate that H. qinghaiensis transmit at least three species of Theileria, two which are infective to sheep and goats, but not yak and one which is infective to yaks and cattle, but not to sheep and goats.     

Three genes specifically expressed during phosphate deficiency in<Emphasis Type="Italic"> Pholiota nameko</Emphasis> strain N2 encode hydrophobins

We previously isolated 31 cDNAs corresponding to pdi [inorganic phosphate (Pi) deficiency-inducible] genes through the differential screening of a cDNA library constructed from the mycelium of Pholiota nameko strain N2 cultured in Pi-depleted medium. Among the cDNAs, pdi251, pdi263 and pdi315 were analyzed here. The deduced amino acid sequences of pdi251, pdi263 and pdi315 showed high similarity to fungal hydrophobins and genes corresponding to these cDNAs encoding P. nameko hydrophobins were designated pnh1, pnh2 and pnh3, respectively. PNH1, PNH2 and PNH3 had a conserved spacing of eight cysteine residues in hydrophobins and a hydropathy pattern characteristic of class I hydrophobins. Phylogenetic analysis showed that PNH1, PNH2 and PNH3 were phylogenetically similar and significantly related to the hydrophobin POH1 specifically expressed in fruiting bodies of Pleurotus ostreatus. Northern blot analysis indicated that, under conditions of Pi deficiency, pnh2 and pnh3 were also induced in strains N4 and N301.Communicated by U. Kück