Expression and regulation of ROR-1 during early avian limb development

ROR-1 is a member of the ROR family of tyrosine kinase like orphan receptors and is highly conserved among various species. We have isolated the chick ROR-1 (cROR-1) and show that cROR-1 expression is high and restricted to the proximal limb region until HH-stage 25. At later stages, expression spreads towards the distal limb region. In order to determine the signals that control cROR-1 expression, factors known to be involved in limb patterning (FGFs, BMPs, SHH, retinoic acid) were applied to the developing limb. Whereas neither FGFs, BMPs, nor SHH affected cROR-1 expression, upregulation could be achieved by ectopic application of retinoic acid to the distal limb region. As retinoic acid also upregulated retinoic acid receptor beta (Rar-), we assume that cROR-1 upregulation is mediated by Rar-. We conclude that ROR-1 signaling is an independently regulated pathway, which is involved in late rather than early limb development.     

Expression of<Emphasis Type="Italic"> Tri15</Emphasis> in<Emphasis Type="Italic"> Fusarium sporotrichioides</Emphasis>

In the fungus Fusarium sporotrichioides, biosynthesis of trichothecene mycotoxins requires at least three genetic loci: a core 12-gene cluster, a smaller two-gene cluster, and a single-gene locus. Here, we describe the Tri15 gene, which represents a fourth locus involved in trichothecene biosynthesis. Tri15 is predicted to encode a Cys₂-His₂ zinc finger protein and is expressed in a manner similar to genes in the core trichothecene gene cluster. However, disruption of F. sporotrichioides Tri15 does not affect production of T-2 toxin, the major trichothecene produced by this fungus. This result suggests that Tri15 is not necessary for the production of toxin. Cultures with exogenously added T-2 toxin have high levels of Tri15 expression and no detectable expression of the trichothecene biosynthetic genes Tri5 and Tri6. The expression analysis is consistent with Tri15 being a negative regulator of at least some of the trichothecene biosynthetic genes. In F. graminearum, Tri15 has been mapped to linkage group 2 and is therefore unlinked to the main trichothecene biosynthetic gene cluster.Communicated by U. Kück     

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.     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Spatial and temporal pattern of<Emphasis Type="Italic"> Wnt-6</Emphasis> expression during chick development

The WNT family of proteins is composed of several members. In the present study we isolated the full length chick Wnt-6 cDNA and analyzed its expression pattern by in situ hybridization during chick development. Wnt-6 expression is observed in the ectoderm from HH-stage 4 onwards. At HH-stages, 7–16 expression can be seen in the ectoderm overlying the segmental plate and the epithelial somite, while the ectoderm overlying the compartmentalized somite is Wnt-6 negative. Expression is also observed at the heart outflow tract and in the ectoderm overlying the pharyngeal arches. From HH-stages 17 to 27, expression is also observed at limb level, both in the dorsal and ventral ectoderm and a stronger expression in the dorsoventral boundary. Furthermore, expression in the ectoderm delimiting the somitic boundaries in the anteroposterior and mediolateral axis at limb level was observed, as well as in the ventral body wall. Expression becomes evident in the inner ear. From HH-stage 30 onwards, expression is restricted to the feather buds and to the gastrointestinal tract.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

No evidence of<Emphasis Type="Italic"> Wolbachia</Emphasis> endosymbiosis with<Emphasis Type="Italic"> Loa loa</Emphasis> and<Emphasis Type="Italic"> Mansonella perstans</Emphasis>

Endosymbiotic Wolbachia bacteria from different filarial species, including major pathogens of humans such as Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus, seem to play an important role in the development, viability and fertility of these worms. Wolbachia trigger inflammatory host responses as well as adverse reactions against standard treatment regimens and are therefore under investigation as novel treatment targets. We investigated whether Wolbachia are also endosymbiotic in Loa loa and Mansonella perstans. In both male and female adult L. loa, we found no evidence of bacteria by light or transmission electron microscopy. Furthermore, Wolbachia-specific PCR was negative in both L. loa and M. perstans microfilariae. The absence of Wolbachia in both filarial species therefore discourages the use of antibiotics as an adjunct or alternative approach to current treatment concepts for both loiasis and mansonelliasis perstans.     

Functional and structural characterisation of <Emphasis Type="Italic">Ag</Emphasis>MNPV <Emphasis Type="Italic">ie1</Emphasis>

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the −492 and −357 versions contains sequence motifs important for the level of the lacZ reporter gene expression. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. The GenBank accession number of the sequence reported in this paper is AF368905.     

<Emphasis Type="Italic">Slc11a1</Emphasis> (<Emphasis Type="Italic">Nramp</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) gene modulates immune-inflammation genes in macrophages during pristane-induced arthritis in mice

Objective and design

Pristane-induced arthritis (PIA) in AIRmax mice homozygous for Slc11a1 R and S alleles was used to characterize the influence of Slc11a1 gene polymorphism on immune responses during disease manifestation. Previous reports demonstrated that the presence of the Slc11a1 S allele increased the incidence and severity of PIA in AIRmax ^SS , suggesting that this gene could interact with inflammatory loci to modulate PIA. We investigated the effects of Slc11a1 alleles on the activation of phagocytes during PIA.

Treatment

Mice were injected intraperitoneally with two doses of 0.5 mL of mineral oil pristane at 60-day intervals. Arthritis development was accompanied for 180 days.

Results

AIRmax ^SS mice showed differential peritoneal macrophage gene expression profiles during PIA, with higher expression and production of H₂O₂, NO, IL-1β, IL-6, TNF-α, and several chemokines. The presence of the Slc11a1 R allele, on the other hand, diminished the intensity of macrophage activation, restricting arthritis development.

Conclusion

Our data demonstrated the fine-tuning roles of Slc11a1 alleles modulating macrophage activation, and consequent PIA susceptibility, in those mouse lines.

     

A role for accessory genes <Emphasis Type="Italic">rI.</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">rI.1</Emphasis> in the regulation of lysis inhibition by bacteriophage T4

Lysis inhibition (LIN) is a known feature of the T-even family of bacteriophages. Despite its historical role in the development of modern molecular genetics, many aspects of this phenomenon remain mostly unexplained. The key element of LIN is an interaction between two phage-encoded proteins, the T holin and the RI antiholin. This interaction is stabilized by RIII. In this report, we demonstrate the results of genetic experiments which suggest a synergistic action of two accessory proteins of bacteriophage T4, RI.-1, and RI.1 with RIII in the regulation of LIN.     

Selective activity of extracts of <Emphasis Type="Italic">Margaritaria discoidea</Emphasis> and <Emphasis Type="Italic">Homalium africanum</Emphasis> on <Emphasis Type="Italic">Onchocerca ochengi</Emphasis>

Background  

The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi, a model parasite for O. volvulus. These plants are used to treat the disease in North West Cameroon.     

Rapid diagnosis of<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> bacteremia using <Emphasis Type="Italic">S. aureus</Emphasis> PNA FISH

In the study presented here, the performance of the S. aureus PNA FISH assay was evaluated using 285 blood cultures (from 104 patients) that had gram-positive cocci resembling staphylococci on Gram stain. The new molecular test is based on a fluorescence in situ hybridization assay using peptide nucleic acid probes targeting Staphylococcus aureus 16S rRNA and is designed for the rapid identification of Staphylococcus aureus directly from positive blood cultures. The sensitivity, specificity, and positive and negative predictive values of the S. aureus PNA FISH for the rapid identification of Staphylococcus aureus directly from positive blood culture bottles were 100, 99.4, 99.2 and 100%, respectively.     

Fate of <Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> and <Emphasis Type="Italic">Cryptosporidium hominis</Emphasis> oocysts and <Emphasis Type="Italic">Giardia duodenalis</Emphasis> cysts during secondary wastewater treatments

This study investigates the fate of Cryptosporidium parvum and C. hominis oocysts and Giardia duodenalis cysts at four Irish municipal wastewater treatment plants (i.e., Plant A, B, C, and D) that utilize sludge activation or biofilm-coated percolating filter systems for secondary wastewater treatment. The fate of these pathogens through the sewage treatment processes was determined based on their viable transmissive stages, i.e., oocysts for Cryptosporidium and cysts for Giardia. Analysis of final effluent indicated that over 97% of viable oocysts and cysts were eliminated, except at Plant C, which achieved only 64% of oocyst removal. A significant correlation between the removal of oocysts and cysts was found at Plants A, B, and D (R = 0.98, P < 0.05). All sewage sludge samples were positive for C. parvum and C. hominis, and G. duodenalis, with maximum concentrations of 20 oocysts and eight cysts per gram in primary sludge indicating the need for further sludge sanitization treatments. This study provides evidence that C. parvum and C. hominis oocysts and G. duodenalis cysts are present throughout the wastewater processes and in end-products, and can enter the aquatic environment with consequent negative implications for public health.