Detection of the deletion of interferon-alpha and beta genes in lymphoblastoid cells by PCR]

The HuIFN-alpha and beta genes were examined by the PCR method in the 11 human lymphoblastoid cell lines. The results showed that the homozygous deletion of HuIFN-alpha and beta genes was detected in 5 of 11 cell lines and in 5 of 11 cell lines, respectively. The deletions of both the HuIFN-alpha and beta genes were observed in 4 of 11 cell lines. One T cell leukemia cell line deleted only HuIFN-alpha gene, while the other T cell leukemia line deleted only HuIFN-beta gene. This suggests that the deletions of HuIFN-alpha and beta genes may be related the development of leukemia or lymphoma.     

Targeting CD9 produces stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis: a novel antiangiogenic therapy

The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis, and appears to be an effective and safe antiangiogenic approach. These results shed light on the biological roles of CD9 and may lead to novel antiangiogenic therapies.     

Comparison of two polymorphic sites in the clock gene cryptochrome in the Taiwan strain of the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae): a possible quick method to estimate the mating time of trapped invading flies

For successful sterile insect technique (SIT), synchronized copulation between invaded females and sterilized males is required. Understanding the mating time of the invaded strain is an aid in synchronizing and thus improving the effectiveness of SIT. We previously demonstrated a relationship between variation at two sites of a circadian clock gene cryptochrome (cry) (cry1212 and cry1865) and circadian behavior in the melon fly Bactrocera cucurbitae (Coquillett). Here we investigated the relationship in two other populations, Taiwan1 (T1) and Taiwan2 (T2), which may re-invade Okinawa. The results showed that T1 exhibited a lower frequency of the S-type allele, which was observed in early mating flies in the strains in Okinawa, than T2 at the site of cry1212. In addition, T1 showed a longer circadian period than T2. We also noted that the cry1212 site showed higher amino acid sequence conservation than cry1865 by comparing CRY1 among five insect species. These results suggest that genotyping of only the cry1212 site of trapped flies enables an immediate estimate of the mating time of the B. cucurbitae population from Taiwan and that cry1212 would be more likely to be involved in determining the mating time than cry1865.     

Single restriction enzyme-assisted megaprimer polymerase chain reaction to fuse two DNA sequences on separate cloning vectors

We describe a simple and versatile method to fuse two DNA sequences on separate cloning vectors in a single polymerase chain reaction (PCR). The method, termed restriction enzyme-assisted megaprimer PCR (REM–PCR), requires that the two cloning vectors share a common sequence and that the DNA sequences to be fused are cloned in the same orientation with respect to the common sequence. Fusion of the two sequences is achieved by mutual priming at the common sequence between two DNA fragments that were generated by restriction enzyme and linearly amplified by repetitive priming in the PCR reaction mixture.     

Review of acquired cystic disease-associated renal cell carcinoma with focus on pathobiological aspects

Acquired cystic disease (ACD)-associated renal cell carcinoma (RCC) is a recently established entity. In this article, we introduce the general view of this new entity. Macroscopically, the disease exclusively occurs in ACD and may arise as a dominant mass or non-dominant masses. Histologically, the tumor is characterized by a microcystic pattern, neoplastic cells with an eosinophilic or oncocytic cytoplasm and frequent intratumoral oxalate crystal deposition. Prominent nucleoli of tumor cells are often observed. Immunohistochemically, neoplastic cells are generally positive for AMACR but negative for cytokeratin 7. Ultrastructurally, neoplastic cells contain abundant mitochondria in the cytoplasm. Genetically, the gain of chromosomes 3, 7, 17 and abnormality of the sex chromosome were frequently observed in several studies. In conclusion, ACD-associated RCC may be widely recognized as a distinct entity in the near future because this tumor is morphologically and genetically different from other renal tumor entities that have been previously established.     

Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated α subunit of the heterotrimeric GTPase Gq

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through the activation of heterotrimeric GTPase (G(q), G(12/13) and G(i))-dependent pathways, by deamidating a glutamine residue in the α subunit of these GTPases. However, the enzymatic characteristics of PMT are yet to be analyzed in detail because the deamidation has only been observed in cell-based assays. In the present study, we developed rat monoclonal antibodies, specifically recognizing the deamidated Gα(q), to detect the actions of PMT by immunological techniques such as western blotting. Using the monoclonal antibodies, we found that the toxin deamidated Gα(q) only under reducing conditions. The C-terminal region of PMT, C-PMT, was more active than the full-length PMT. The C3 domain possessing the enzyme core catalyzed the deamidation in vitro without any other domains. These results not only support previous observations on toxicity, but also provide insights into the enzymatic nature of PMT. In addition, we present several lines of evidence that Gα(11), as well as Gα(q), could be a substrate for PMT.     

Malaria prevalence, risk factors and spatial distribution in a hilly forest area of Bangladesh

Background

Malaria is a major public health concern in Bangladesh and it is highly endemic in the Chittagong Hill Tracts where prevalence was 11.7% in 2007. One sub-district, Rajasthali, had a prevalence of 36%. Several interventions were introduced in early 2007 to control malaria. This study was undertaken to evaluate the impacts of these intensive early stage interventions on malaria in Bangladesh. This prevalence study assesses whether or not high malaria prevalence remains, and if so, which areas and individuals remain at high risk of infection.

Methods and Principal Findings

A 2-stage cluster sampling technique was used to sample 1,400 of 5,322 (26.3%) households in Rajasthali, and screened using a rapid diagnostic test (Falci-vax). Overall malaria prevalence was 11.5%. The proportions of Plasmodium falciparum, Plasmodium vivax and infection with both species were 93.2%, 1.9% and 5.0%, respectively. Univariate, multivariate logistic regression, and spatial cluster analyses were performed separately. Sex, age, number of bed nets, forest cover, altitude and household density were potential risk factors. A statistically significant malaria cluster was identified. Significant differences among risk factors were observed between cluster and non-cluster areas.

Conclusion and Significance

Malaria has significantly decreased within 2 years after onset of intervention program. Both aspects of the physical and social environment, as well as demographic characteristics are associated with spatial heterogeneity of risk. The ability to identify and locate these areas provides a strategy for targeting interventions during initial stages of intervention programs. However, in high risk clusters of transmission, even extensive coverage by current programs leaves transmission ongoing at reduced levels. This indicates the need for continued development of new strategies for identification and treatment as well as improved understanding of the patterns and determinants of parasitaemia.