Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S–S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles

Syncope: experience at a tertiary care hospital in Karachi, Pakistan

Introduction:Our aim was to determine the characteristics of Patients presenting with syncope at a tertiary care hospital in Karachi, Pakistan.Methods: A review of medical records was conducted retrospectively at the Department of Medicine, Aga Khan University Hospital, Karachi. Patients aged 16 and above, admitted from January 2000 to December 2005 with the diagnosis of syncope made by the attending physician were included.Results:A total of 269 Patients were included (75% males, mean age: 57.4 years). Neurogenic (vasovagal) syncope was the most common cause (47%), followed by cardiogenic syncope (18%) and orthostatic syncope (9%). A total of 24% were discharged undiagnosed. Twenty Patients (7.4%) did not have any prodrome. Common prodromal symptoms included dizziness (61%), sweating (25%), palpitations (19%), nausea/vomiting (19%) and visual symptoms (17%). The distribution of symptoms according to cause of syncope revealed only breathlessness to be significantly associated with cardiogenic syncope (p = 0.002). Most Patients with cardiogenic syncope were aged above 40 (98%, p \u3c 0.001), had coronary artery disease (72%, p \u3c 0.001) and abnormal electrocardiogram at presentation (92%, p \u3c 0.001).Conclusion:Despite differences in burden of diseases, our findings were similar to those of published syncope literature. Further studies are needed to develop a protocol to expedite the evaluation and limit the work-up and admission in low-risk Patients

Fanconi anemia genes are highly expressed in primitive CD34(+ )hematopoietic cells

BACKGROUND: Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow failure (BM) and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. METHODS: Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. RESULTS: We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34(- )stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. CONCLUSIONS: These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34(+ )cells correlates with a role at the CD34(+ )differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism

Identification of genes differentially expressed in a resistant reaction to Mycosphaerella pinodes in pea using microarray technology

<p>Abstract</p> <p>Background</p> <p>Ascochyta blight, caused by <it>Mycosphaerella pinodes </it>is one of the most important pea pathogens. However, little is known about the genes and mechanisms of resistance acting against <it>M. pinodes </it>in pea. Resistance identified so far to this pathogen is incomplete, polygenic and scarce in pea, being most common in <it>Pisum </it>relatives. The identification of the genes underlying resistance would increase our knowledge about <it>M. pinodes-</it>pea interaction and would facilitate the introgression of resistance into pea varieties. In the present study differentially expressed genes in the resistant <it>P. sativum </it>ssp. <it>syriacum </it>accession P665 comparing to the susceptible pea cv. Messire after inoculation with <it>M. pinodes </it>have been identified using a <it>M. truncatula </it>microarray.</p> <p>Results</p> <p>Of the 16,470 sequences analysed, 346 were differentially regulated. Differentially regulated genes belonged to almost all functional categories and included genes involved in defense such as genes involved in cell wall reinforcement, phenylpropanoid and phytoalexins metabolism, pathogenesis- related (PR) proteins and detoxification processes. Genes associated with jasmonic acid (JA) and ethylene signal transduction pathways were induced suggesting that the response to <it>M. pinodes </it>in pea is regulated via JA and ET pathways. Expression levels of ten differentially regulated genes were validated in inoculated and control plants using qRT-PCR showing that the P665 accession shows constitutively an increased expression of the defense related genes as peroxidases, disease resistance response protein 39 (DRR230-b), glutathione S-transferase (GST) and 6a-hydroxymaackiain methyltransferase.</p> <p>Conclusions</p> <p>Through this study a global view of genes expressed during resistance to <it>M. pinodes </it>has been obtained, giving relevant information about the mechanisms and pathways conferring resistance to this important disease. In addition, the <it>M. truncatula </it>microarray represents an efficient tool to identify candidate genes controlling resistance to <it>M. pinodes </it>in pea.</p

Small Heat Shock Protein αA-Crystallin Prevents Photoreceptor Degeneration in Experimental Autoimmune Uveitis

The small heat shock protein, αA-crystallin null (αA−/−) mice are known to be more prone to retinal degeneration than the wild type mice in Experimental Autoimmune Uveoretinitis (EAU). In this report we demonstrate that intravenous administration of αA preserves retinal architecture and prevents photoreceptor damage in EAU. Interestingly, only αA and not αB-crystallin (αB), a closely related small heat shock protein works, pointing to molecular specificity in the observed retinal protection. The possible involvement of αA in retinal protection through immune modulation is corroborated by adaptive transfer experiments, (employing αA−/− and wild type mice with EAU as donors and Rag2−/− as the recipient mice), which indicate that αA protects against the autoimmune challenge by modulating the systemic B and T cell immunity. We show that αA administration causes marked reduction in Th1 cytokines (TNF-α, IL-12 and IFN-γ), both in the retina and in the spleen; notably, IL-17 was only reduced in the retina suggesting local intervention. Importantly, expression of Toll-like receptors and their associated adaptors is also inhibited suggesting that αA protection, against photoreceptor loss in EAU, is associated with systemic suppression of both the adaptive and innate immune responses

Syndromes with congenital brittle bones

BACKGROUND: There is no clear definition of osteogenesis imperfecta (OI). The most widely used classification of OI divides the disease in four types, although it has been suggested that there may be at least 12 forms of OI. These forms have been named with numbers, eponyms or descriptive names. Some of these syndromes can actually be considered congenital forms of brittle bones resembling OI (SROI). DISCUSSION: A review of different syndromes with congenital brittle bones published in the literature is presented. Syndromes are classified in "OI" (those secondary to mutations in the type I pro-collagen genes), and "syndromes resembling OI" (those secondary to mutations other that the type I pro-collagen genes, identified or not). A definition for OI is proposed as a syndrome of congenital brittle bones secondary to mutations in the genes codifying for pro-collagen genes (COL1A1 and COL1A2). SUMMARY: A debate about the definition of OI and a possible clinical and prognostic classification are warranted

Quantitative trait loci and candidate gene mapping of aluminum tolerance in diploid alfalfa

Aluminum (Al) toxicity in acid soils is a major limitation to the production of alfalfa (Medicago sativa subsp. sativa L.) in the USA. Developing Al-tolerant alfalfa cultivars is one approach to overcome this constraint. Accessions of wild diploid alfalfa (M. sativa subsp. coerulea) have been found to be a source of useful genes for Al tolerance. Previously, two genomic regions associated with Al tolerance were identified in this diploid species using restriction fragment length polymorphism (RFLP) markers and single marker analysis. This study was conducted to identify additional Al-tolerance quantitative trait loci (QTLs); to identify simple sequence repeat (SSR) markers that flank the previously identified QTLs; to map candidate genes associated with Al tolerance from other plant species; and to test for co-localization with mapped QTLs. A genetic linkage map was constructed using EST-SSR markers in a population of 130 BC(1)F(1) plants derived from the cross between Al-sensitive and Al-tolerant genotypes. Three putative QTLs on linkage groups LG I, LG II and LG III, explaining 38, 16 and 27% of the phenotypic variation, respectively, were identified. Six candidate gene markers designed from Medicago truncatula ESTs that showed homology to known Al-tolerance genes identified in other plant species were placed on the QTL map. A marker designed from a candidate gene involved in malic acid release mapped near a marginally significant QTL (LOD 2.83) on LG I. The SSR markers flanking these QTLs will be useful for transferring them to cultivated alfalfa via marker-assisted selection and for pyramiding Al tolerance QTLs