A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 1 (BBU1)

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR(5000). The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH5000) panel. The retention frequency of individual markers across the panel ranged from 17.8 to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs. Copyright (C) 2007 S. Karger AG, Basel.

Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Identification of polycyclic aromatic hydrocarbons and methoxylated phenols in wood smoke emitted during production of charcoal

More than 130 organic substances in dichloromethane-methanol (4: 1) extracts of particulate matter and the gaseous phase from wood burning for the production of charcoal have been identified by capillary gas chromatography coupled with low-resolution mass spectrometry (GC-MS), use of GC retention indices, and comparison with authentic standards. Many of the substances identified are methoxyphenols (derivatives of syringol and guaiacol), polycyclic aromatic hydrocarbons (PAH), oxidized PAH (oxy-PAH), and levoglucosan, the last being a monosoccharide derivative from the thermal breakdown of cellulose. The amount of unsubstituted PAH was greater than that of methyl- and dimethyl-substituted homologs.

Molecular model of shikimate kinase from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based. © 2002 Elsevier Science (USA). All rights reserved.

Karyotypic and morphological divergence between two cryptic species of Eigenmannia in the Amazon basin with a new occurrence of XX/XY sex chromosomes (Gymnotiformes: Sternopygidae)

Espécies de Eigenmannia estão amplamente distribuídas na região Neotropical, com oito espécies válidas atualmente reconhecidas. Populações de Eigenmannia de três localidades do leste da Amazônia foram investigadas usando técnicas citogenéticas e morfológicas, revelando dois táxons designados aqui comoEigenmannia sp. "A" e Eigenmannia sp. "B". As espécies diferem em três caracteres morfométricos, dois merísticos e um osteológico. Eigenmannia sp. "A" apresenta 2n = 34 (22 m/sm+12st/a) e Eigenmannia sp. "B" apresenta 2n = 38 (14 m/sm+24st/a) e cromossomos sexuais de diferenciação simples, do tipo XX/XY. Em ambas espécies a Heterocromatina Constitutiva (HC) rica em bases A-T está distribuída na região centromérica de todos os cromossomos. Eigenmannia sp. "B" também apresenta blocos de HC na região intersticial dos pares cromossômicos 8, 9 e X que coraram positivamente para CMA₃, indicando regiões ricas em G-C. A NOR está localizada no braço curto do par 17 em Eigenmannia sp. "A" e no braço curto do par 14 em Eigenmannia sp. "B". FISH com sondas de rDNA hibridizaram em regiões de tamanhos diferentes entre os homólogos, sugerindo heteromorfismo. A diferenciação do cromossomo X em Eigenmannia sp. "B" pode ser o resultado de amplificação de sequências repetitivas de DNA.

First cytogenetic characterization of the giant Amazonian catfish Brachyplatystoma filamentosum (Siluriformes, Pimelodidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evolution of the Metazoan Mitochondrial Replicase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The homeostasis of iron, copper, and zinc in paracoccidioides brasiliensis, cryptococcus neoformans var. Grubii, and cryptococcus gattii: a comparative analysis

Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensis Pb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways.

A Novel Stress-Induced Sugarcane Gene Confers Tolerance to Drought, Salt and Oxidative Stress in Transgenic Tobacco Plants

Background: Drought is a major abiotic stress that affects crop productivity worldwide. Sugarcane can withstand periods of water scarcity during the final stage of culm maturation, during which sucrose accumulation occurs. Meanwhile, prolonged periods of drought can cause severe plant losses. Methodology/Principal Findings: In a previous study, we evaluated the transcriptome of drought-stressed plants to better understand sugarcane responses to drought. Among the up-regulated genes was Scdr1 (sugarcane drought-responsive 1). The aim of the research reported here was to characterize this gene. Scdr1 encodes a putative protein containing 248 amino acids with a large number of proline (19%) and cysteine (13%) residues. Phylogenetic analysis showed that ScDR1is in a clade with homologs from other monocotyledonous plants, separate from those of dicotyledonous plants. The expression of Scdr1 in different varieties of sugarcane plants has not shown a clear association with drought tolerance. Conclusions/Significance: The overexpression of Scdr1 in transgenic tobacco plants increased their tolerance to drought, salinity and oxidative stress, as demonstrated by increased photosynthesis, water content, biomass, germination rate, chlorophyll content and reduced accumulation of ROS. Physiological parameters, such as transpiration rate (E), net photosynthesis (A), stomatal conductance (gs) and internal leaf CO2 concentration, were less affected by abiotic stresses in transgenic Scdr1 plants compared with wild-type plants. Overall, our results indicated that Scdr1 conferred tolerance to multiple abiotic stresses, highlighting the potential of this gene for biotechnological applications.

Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome

Arthrogryposisrenal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apicalbasolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotypephenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR. Hum Mutat 33:16561664, 2012. (c) 2012 Wiley Periodicals, Inc.

A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis

The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

Analysis of Schistosoma mansoni genes shared with Deuterostomia and with possible roles in host interactions

Abstract Background: Schistosoma mansoni is a blood helminth parasite that causes schistosomiasis, a disease that affects 200 million people in the world. Many orthologs of known mammalian genes have been discovered in this parasite and evidence is accumulating that some of these genes encode proteins linked to signaling pathways in the parasite that appear to be involved with growth or development, suggesting a complex co-evolutionary process. Results: In this work we found 427 genes conserved in the Deuterostomia group that have orthologs in S. mansoni and no members in any nematodes and insects so far sequenced. Among these genes we have identified Insulin Induced Gene (INSIG), Interferon Regulatory Factor (IRF) and vasohibin orthologs, known to be involved in mammals in mevalonate metabolism, immune response and angiogenesis control, respectively. We have chosen these three genes for a more detailed characterization, which included extension of their cloned messages to obtain full-length sequences. Interestingly, SmINSIG showed a 10-fold higher expression in adult females as opposed to males, in accordance with its possible role in regulating egg production. SmIRF has a DNA binding domain, a tryptophan-rich N-terminal region and several predicted phosphorylation sites, usually important for IRF activity. Fourteen different alternatively spliced forms of the S. mansoni vasohibin (SmVASL) gene were detected that encode seven different protein isoforms including one with a complete C-terminal end, and other isoforms with shorter C-terminal portions. Using S. mansoni homologs, we have employed a parsimonious rationale to compute the total gene losses/gains in nematodes, arthropods and deuterostomes under either the Coelomata or the Ecdysozoa evolutionary hypotheses; our results show a lower losses/gains number under the latter hypothesis. Conclusion: The genes discussed which are conserved between S. mansoni and deuterostomes, probably have an ancient origin and were lost in Ecdysozoa, being still present in Lophotrochozoa. Given their known functions in Deuterostomia, it is possible that some of them have been co-opted to perform functions related (directly or indirectly) to host adaptation or interaction with host signaling processes.

Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

Abstract Background MicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (Saccharum spp.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species. Results In this study, we have computationally identified 19 distinct sugarcane miRNA precursors, of which several are highly similar with their sorghum homologs at both nucleotide and secondary structure levels. The accumulation pattern of mature miRNAs varies in organs/tissues from the commercial sugarcane hybrid as well as in its corresponding founder species S. officinarum and S. spontaneum. Using sugarcane MIR827 as a query, we found a novel MIR827 precursor in the sorghum genome. Based on our computational tool, a total of 46 potential targets were identified for the 19 sugarcane miRNAs. Several targets for highly conserved miRNAs are transcription factors that play important roles in plant development. Conversely, target genes of lineage-specific miRNAs seem to play roles in diverse physiological processes, such as SsCBP1. SsCBP1 was experimentally confirmed to be a target for the monocot-specific miR528. Our findings support the notion that the regulation of SsCBP1 by miR528 is shared at least within graminaceous monocots, and this miRNA-based post-transcriptional regulation evolved exclusively within the monocots lineage after the divergence from eudicots. Conclusions Using publicly available nucleotide databases, 19 sugarcane miRNA precursors and one new sorghum miRNA precursor were identified and classified into 14 families. Comparative analyses between sugarcane and sorghum suggest that these two species retain homologous miRNAs and targets in their genomes. Such conservation may help to clarify specific aspects of miRNA regulation and evolution in the polyploid sugarcane. Finally, our dataset provides a framework for future studies on sugarcane RNAi-dependent regulatory mechanisms.

Endo-PDI is required for TNFα-induced angiogenesis

Protein disulfide isomerase (PDI) and its homologs are oxidoreductases facilitating protein folding in the ER. Endo-PDI (also termed ERp46) is highly expressed in endothelial cells. It belongs to the PDI family but its physiological function is largely unknown. We studied the role of Endo-PDI in endothelial angiogenic responses. Stimulation of human umbilical vein endothelial cells (with TNFα (10ng/ml) increased ERK1/2 phosphorylation. This effect was largely attenuated by Endo-PDI siRNA, whereas JNK and p38 MAP kinase phosphorylation was Endo-PDI independent. Similarly, TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo-PDI siRNA. The action of Endo-PDI was not mediated by extracellular thiol exchange or cell surface PDI as demonstrated by nonpermeative inhibitors and PDI-neutralizing antibody. Moreover, exogenously added PDI failed to restore ERK1/2 activation after Endo-PDI knockdown. This suggests that Endo-PDI acts intracellularly potentially by maintaining the Ras/Raf/MEK/ERK pathway. Indeed, knockdown of Endo-PDI attenuated Ras activation measured by G-LISA and Raf phosphorylation. ERK activation influences gene expression by the transcriptional factor AP-1, which controls MMP-9 and cathepsin B, two proteases required for angiogenesis. TNFα-stimulated MMP-9 and cathepsin B induction was reduced by silencing of Endo-PDI. Accordingly, inhibition of cathepsin B or Endo-PDI siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays. Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo-PDI siRNA. In conclusion, our study establishes Endo-PDI as a novel, important mediator of AP-1-driven gene expression and endothelial angiogenic function