Sequence analysis of the cDNA encoding for SpCTx: a lethal factor from scorpionfish venom ( Scorpaena plumieri )

Background: Lethal factors are multifunctional oligomeric proteins found in the venomous apparatus of Scorpaeniformes fish. These toxins elicit not only an array of biological responses in vitro but also cardiovascular disorders and strong hemolytic, nociceptive and edematogenic activities in vivo. This work describes the cloning and molecular identification of two toxin subunits, denominated Sp-CTx- and Sp-CTx-, from scorpionfish venom ( Scorpaena plumieri ). Methods: The primary structures were deduced after cDNA amplification by PCR with primers from conserved sequences described in Scorpaeniformes toxins. Following DNA sequencing and bioinformatic analysis, the tridimensional structures of both subunits were modeled. Results: The translated sequences (702 amino acids, each subunit) show homology with other lethal factors, while alignment between Sp-CTx- and Sp-CTx- shows 54% identity. The subunits lack N-terminal signal sequences and display masses of approximately 80 kDa each. Both Sp-CTx subunits display a B30.2/SPRY domain at the C-terminal region with typically conserved motifs as described in these toxins. Secondary structure prediction identified six -helices 18 residues long in both and subunits, some of them amphiphilic with their N-terminal flanked by many basic residues, creating a cationic site associated with the cytolytic activity of these toxins. Antimicrobial potential sites were identified in Sp-CTx and share some features with other peptides presenting variable and broad-spectrum activity...

Sequence analysis of the cDNA encoding for SpCTx: a lethal factor from scorpionfish venom ( Scorpaena plumieri )

Lethal factors are multifunctional oligomeric proteins found in the venomous apparatus of Scorpaeniformes fish. These toxins elicit not only an array of biological responses in vitro but also cardiovascular disorders and strong hemolytic, nociceptive and edematogenic activities in vivo. This work describes the cloning and molecular identification of two toxin subunits, denominated Sp-CTx-α and Sp-CTx-ß, from scorpionfish venom ( Scorpaena plumieri ). Methods: The primary structures were deduced after cDNA amplification by PCR with primers from conserved sequences described in Scorpaeniformes toxins. Following DNA sequencing and bioinformatic analysis, the tridimensional structures of both subunits were modeled. Results: The translated sequences (702 amino acids, each subunit) show homology with other lethal factors, while alignment between Sp-CTx-α and Sp-CTx-ß shows 54% identity. The subunits lack N-terminal signal sequences and display masses of approximately 80 kDa each. Both Sp-CTx subunits display a B30.2/SPRY domain at the C-terminal region with typically conserved motifs as described in these toxins. Secondary structure prediction identified six α-helices 18 residues long in both α and ß subunits, some of them amphiphilic with their N-terminal flanked by many basic residues, creating a cationic site associated with the cytolytic activity of these toxins. Antimicrobial potential sites were identified in Sp-CTx and share some features with other peptides presenting variable and broad-spectrum activity. A phylogenetic tree built to represent these toxins supports the proximity between scorpionfish, lionfish and stonefish. Conclusion: The study identified a putative toxin protein whose primary structure is similar to other fish toxins and with potential for production of antivenom against scorpionfish envenomation in Brazil. As a prelude to structure-function studies, we propose that the toxin is structurally related to pore-forming marine toxins.(AU)

Infección de Aedes albopictus (Skuse, 1894) con el genotipo asiático-americano del virus del dengue serotipo 2 en Medellín y su posible papel como vector del dengue en Colombia / Aedes albopictus (Skuse, 1894) infected with the American-Asian genotype of dengue type 2 virus in Medellín suggests its possible role as vector of dengue fever in Colombia

Resumen Introducción. Aedes aegypti y Ae. albopictus son reconocidos vectores de arbovirus como los del dengue, la fiebre amarilla, el chikungunya y el Zika, en regiones tropicales y subtropicales del mundo. En Colombia, la distribución geográfica de Ae. albopictus ha sufrido un incremento y hoy incluye ciudades como Cali y Medellín. Hasta ahora, sin embargo, no se ha recabado información concluyente sobre su infección viral y su capacidad de transmisión a los humanos. Objetivo. Determinar la infección natural por dengue en ejemplares de Ae. albopictus recolectados en un área urbana de Medellín. Materiales y métodos. Se recolectaron individuos de Ae. albopictus en el campus de la Universidad Nacional de Colombia, sede Medellín. Se confirmó su clasificación taxonómica mediante el análisis del gen citocromo oxidasa I (COI), y se extrajo el ARN total para la identificación del virus del dengue y de los respectivos serotipos. La presencia del genotipo DENV se infirió mediante el análisis del gen NS3. Resultados. El análisis del COI corroboró el estatus taxonómico de Ae. albopictus. Uno de los mosquitos procesados fue positivo para DENV-2 y el análisis del NS3 mostró una gran similitud con el genotipo asiático-americano. Conclusión. Se reporta la infección con DENV-2 en Ae. albopictus en Medellín, Colombia. La presencia del genotipo asiático-americano en una zona urbana sugiere su posible circulación entre humanos y en Ae. albopictus, lo cual alerta sobre su eventual papel en la transmisión del DENV-2, y sobre la necesidad de incluir esta especie en la vigilancia entomológica en Colombia.

Abstract Introduction: Aedes aegypti and Ae. albopictus are recognized vectors of dengue, yellow fever, chikungunya and Zika arboviruses in several countries worldwide. In Colombia, Ae. albopictus geographical distribution has increased to include highly populated cities such as Cali and Medellín. Although this species has been frequently found in urban and semi-urban zones in the country, its role as vector of the dengue fever is poorly known. Objective: To identify the presence of Ae. albopictus specimens naturally infected with dengue virus collected in Medellín. Materials and methods: Insects were collected in the Universidad Nacional de Colombia campus in Medellín. Individuals were classified as Ae. albopictus and confirmed by DNA barcode region analysis. Mosquitoes were processed for dengue virus identification, and a fragment of the NS3 gen was sequenced and compared with DENV-2 genotypes reported in the literature. Results: Sequence analysis of COI indicated Ae. albopictus individuals were similar to those recently reported in Colombia, and genetically close to those from other regions worldwide. Among the pools tested one was positive for DENV-2, and the NS3 analysis indicated it belonged to the Asian-American clade. Conclusion: We report the presence Ae. albopictus naturally infected with the Asian-American genotype of DENV-2 in Colombia. The presence of Ae. albopictus specimens carrying the most common genotype infecting humans in a highly populated city such as Medellín indicates its potential role as dengue vector in Colombia and highlights the relevance of including it in current vector surveillance strategies.

Expressed sequence tags in venomous tissue of Scorpaena plumieri (Scorpaeniformes: Scorpaenidae)

Species of the family Scorpaenidae are responsible for accidents and sporadic casualties by the shore they inhabit. The species Scorpaena plumieri from this family populate the Northeastern and Eastern coast of Brazil causing human envenomation characterized by local and systemic symptoms. In experimental animals the venom induces cardiotoxic, hypotensive, and airway respiratory effects. As first step to identify the venom components we isolated gland mRNA to produce a cDNA library from the fish gland. This report describes the partial sequencing of 356 gland transcripts from S. plumieri. BLAST analysis of transcripts showed that 30% were unknown sequences, 17% hypothetical proteins, 17% related to metabolic enzymes, 14% belonged to signal transducing functions and the remaining groups (7-8%) composed by gene related with expressing proteins, regulatory proteins and structural proteins. A considerable number of these EST were not found in available databases suggesting the existence of new proteins and/or functions yet to be discovered. By screening the library with antibodies against a lectin fraction from S. plumieri venom we identified several clones whose DNA sequence showed similarities with lectins found in fish. In silico analysis of these clones confirm the identity of these molecules in the venom gland of S. plumieri. .

Espécies da família Scorpaenidae são responsáveis por acidentes e mortes esporádicas ao longo da costa que habitam. A espécie Scorpaena plumieri desta família povoam a costa Leste e Nordeste do Brasil, causando envenenamento humano caracterizado por sintomas locais e sistêmicos. Em modelos experimentais animais a peçonha induz cardiotoxicidade, efeitos hipotensivos e alterações nas vias aéreas respiratórias. Como primeiro passo para identificar os componentes da peçonha foram isolados os mRNA das glândulas do peixe para produzir uma biblioteca de cDNAs. Esse artigo descreve o sequenciamento parcial de 356 transcritos das glândulas de S. plumieri. Análises em bancos de dados (BLAST) dos transcritos demonstraram que 30% eram sequências desconhecidas, 17% proteínas hipotéticas, 17% relacionadas às enzimas do metabolismo, 14% pertenciam a funções de transdução de sinais e os demais grupos (7-8%) formados por genes relacionados com a expressão de proteínas, proteínas regulatórias e estruturais. Um número considerável destes EST não foi encontrado em bases de dados disponíveis, sugerindo a existência de novas proteínas e/ou funções ainda a serem descobertas. Ao fazer um barrido da biblioteca com anticorpos produzidos contra uma fração das lectinas do veneno de S. plumieri, identificamos vários clones, cuja sequência de DNA mostram semelhanças com lectinas encontradas em peixes. A análise in silico destes clones confirmam a identidade destas moléculas na glândula de peçonha de S. plumieri.

Growth differentiation factor 9 and bone morphogenetic protein 15 expression in previtellogenic oocytes and during early embryonic development of Yellow-tail Kingfish Seriola lalandi

BACKGROUND: During fish oocyte maturation, specific molecules are expressed and accumulated within oocyte until fertilization and embryo development. Special attention have been paid in members of the transforming growth factor (TGF-ß) superfamily; growth differentiation factor 9 (GDF9/gdf9) and bone morphogenetic protein 15 (BMP15/bmp15), which exert regulatory functions during oocyte maturation and follicle development. However, little attention has been paid to the involvement of these molecules during embryogenesis considering its importance for the formation of a good quality egg and subsequent embryo survival. The purpose of this study was to analyze the expression of gdf9 andbmp15 in previtellogenic oocytes and during early embryonic development in Seriola lalandi, a pelagic fish with increasing prospect for its aquaculture development, which however, show high mortality at embryo and larval stages. RESULTS: Through RT-qPCR it was found that gdf9 expression was higher in previtellogenic oocytes decreasing after ovulation. This expression profile agrees with its participation in early stages of the follicular development. The transcripts for bmp15 also showed the highest levels in previtellogenic oocytes, however this expression was lower than obtained with gdf9. Conversely, in recently spawned oocytes mRNA bmp15 levels were highest than observed to gdf9. This, is consequent with the main role proposed for this growth factor at the final fish oocyte maturation: avoid the ovulation of an immature oocyte. During embryo development, low levels of mRNA were detected to gdf9, with an increase in 48 H post-fertilization embryos. The bmp15 expression did not change throughout development and was higher than gdf9 at 16 cells, blastula and appearance embryos stages. CONCLUSIONS: Both (gdf9 and bmp15) expression profiles in previtellogenic oocytes and newly spawned eggs are consistent with the described functions for these growth factors in vertebrate ovarian physiology in early and late stages of the follicular development. So, these genes could be considered as quality biomarkers at these stages. However, further studies of these proteins throughout folliculogenesis, are necessaries to fully understand their functions during the oocyte formation. In addition, the persistent expression of these growth factors during development, allows us to speculate possible roles in embryonic processes, which must also be addressed.

Identification of differentially expressed genes in gauze-exposed omentum of dogs using differential display RT-PCR

Molecular mechanisms governing peritonitis caused by the presence of aseptic gauze have remained unclear. To identify the genes involved, sterile gauze-exposed omentum was collected at 0, 6, 12, 24, and 48 h intervals, and analyzed by differential display RT(reverse transcription)-PCR. Among over 1,200 bands, 230 bands were found differentially expressed. These bands represented the fragment sizes of approximately 200 to 1,500 bp. The eight fragments were expressed differentially in the treatment group but not in the control. The sequences of two bands were similar to those of genes associated with the inflammatory process and a band was related to repair and regeneration process. Another one was related with spermatogonia and the rest four were unknown. Additionally, amplicons corresponding to the full-length sequences of two inflammatory gene fragments were synthesized by rapid amplification of cDNA end PCR. One showed 99% similarity to the major histocompatibility complex class II dog leukocyte antigen-DR beta chain and the other was canis familiaris proteasome beta type 3. Results of the present study suggested that sterile gauze induced the differential expression of genes in the omentum involved in inflammation and healing process.

Suppression subtractive hybridization PCR isolation of cDNAs from a Caribbean soft coral

Transcriptomic studies of marine organisms are still in their infancy. A partial, subtracted expressed sequence tag (EST) library of the Caribbean octocoral Erythropodium caribaeorum and the sea fan Gorgonia ventalina has been analyzed in order to find novel genes or differences in gene expression related to potential secondary metabolite production or symbioses. This approach entails enrichment for potential non-“housekeeping” genes using the suppression subtractive hybridization (SSH) polymerase chain reaction (PCR) method. More than 500 expressed sequence tags (ESTs) were generated after cloning SSH products, which yielded at least 53 orthologous groups of proteins (COGs) and Pfam clusters, including transcription factors (Drosophila Big Brother), catalases, reverse transcriptases, ferritins and various “hypothetical” protein sequences. A total of 591 EST sequences were deposited into GenBank [dbEST: FL512138 - FL512331, GH611838, and HO061755-HO062154]. The results represent proof of concept for enrichment of unique transcripts over housekeeping genes, such as actin or ribosomal genes, which comprised approximately 17 percent of the total dataset. Due to the gene and sequence diversity of some ESTs, such sequences can find utility as molecular markers in current and future studies of this species and other soft coral biogeography, chemical ecology, phylogenetics, and evolution.

Preferential transcription of Paracoccidioides brasiliensis genes: host niche and time-dependent expression

Paracoccidioides brasiliensis causes infection through inhalation by the host of airborne propagules from the mycelium phase of the fungus. This fungus reaches the lungs, differentiates into the yeast form and is then disseminated to virtually all parts of the body. Here we review the identification of differentially-expressed genes in host-interaction conditions. These genes were identified by analyzing expressed sequence tags (ESTs) from P. brasiliensis cDNA libraries. The P. brasiliensis was recovered from infected mouse liver as well as from fungal yeast cells incubated in human blood and plasma, mimicking fungal dissemination to organs and tissues and sites of infection with inflammation, respectively. In addition, ESTs from a cDNA library of P. brasiliensis mycelium undergoing the transition to yeast were previously analyzed. Together, these studies reveal significant changes in the expression of a number of genes of potential importance in the host-fungus interaction. In addition, the unique and divergent representation of transcripts when the cDNA libraries are compared suggests differential gene expression in response to specific niches in the host. This analysis of gene expression patterns provides details about host-pathogen interactions and peculiarities of sites within the host.

Hospitalization due to norovirus and genotypes of rotavirus in pediatric patients, state of Espírito Santo

Viruses are the leading cause for hospitalization due to gastroenteritis worldwide. Group A rotaviruses (RV) are the most prevalent and are assorted in glycoproteins (G) and protease sensitive (P) dual genotypes based on polymorphic genes that encode the external VP7 and VP4 capsid proteins, respectively. Noroviruses (NoV) have increasingly answered by sporadic gastroenteritis. This study aimed to determine the prevalence of NoV and RV in 68 hospitalized children, between July 2004 and November 2006, at a pediatric hospital in Vitória city, state of Espírito Santo, Southeastern Brazil. Nucleic acid was extracted from fecal suspension following the guanidine-silica procedure. Reverse transcriptase-polymerase chain reaction (RT-PCR) and polyacrylamide gel electrophoresis were employed for NoV and RV detection, respectively. RV genotyping was accomplished using RT-PCR followed by heminested multiplex PCR with specific primers for the most prevalent types of G and P. Fecal samples were positive for NoV and RV in 39.7 percent (27/68) and 20.5 percent (14/68), respectively and together were responsible for 60 percent (41/68) of the cases. RV genotypes were: 50 percent G9P[8], 28.7 percent G2P[4], 7.1 percent G1P[8], G2P[8] and G?P[8]. Vomit was a prominent manifestation observed in 92 percent and 85 percent of the NoV and RV cases, respectively. The median hospitalization was 5 and 5.5 days for the patients infected with NoV and RV, respectively. The data showed that NoV prevailed over RV and it also corroborated the emergence of RV G9 genotype followed by G2P[4], reinforcing the need for RV genotype surveillance.

Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum.

A RING zinc finger ankyrin protein gene,designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE.Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4- type RING finger domain was found at the C-terminal region of the AdZFP1 protein,and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869.Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root,stem and leaf of the plant.Semi-quantitative reverse- transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity,cold and heat to some extent.Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

Production of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from Schistosoma mansoni

The number of sequences generated by genome projects has increased exponentially, but gene characterization has not followed at the same rate. Sequencing and analysis of full-length cDNAs is an important step in gene characterization that has been used nowadays by several research groups. In this work, we have selected Schistosoma mansoni clones for full-length sequencing, using an algorithm that investigates the presence of the initial methionine in the parasite sequence based on the positions of alignment start between two sequences. BLAST searches to produce such alignments have been performed using parasite expressed sequence tags produced by Minas Gerais Genome Network against sequences from the database Eukaryotic Cluster of Orthologous Groups (KOG). This procedure has allowed the selection of clones representing 398 proteins which have not been deposited as S. mansoni complete CDS in any public database. Dedicated sequencing of 96 of such clones with reads from both 5' and 3' ends has been performed. These reads have been assembled using PHRAP, resulting in the production of 33 full-length sequences that represent novel S. mansoni proteins. These results shall contribute to construct a more complete view of the biology of this important parasite.

Transcriptome overview of Paracoccidioides brasiliensis proteases

Proteases perform a wide variety of functions inside and outside cells, regulating many biological processes. Infectious microorganisms use proteases, either secreted or attached to their cell surface to weaken and invade their hosts. Therefore, proteases are targets for drugs against a diverse set of diseases. Paracoccidioides brasiliensis is the most prevalent fungal pathogen causing systemic mycosis in Latin America. The development of paracoccidioidomycosis depends on interactions between fungal and host components and proteases have been described as important factors implicated in the mechanism of host colonization by fungi. The primary goal for this study is to present an overview of the transcriptome sequences--identified cDNAs that encode proteases. We obtained a total of 53 cDNAs encoding proteases; 15 were classified as ATP-independent, 12 as ATP-dependent, 22 as proteasome subunits, and 4 as deubiquitinating proteases. The mechanisms and biological activity of these proteases differ in substrate specificity and in catalytic mechanisms.

Molecular cloning and characterization of a glutathione S-transferase encoding gene from Opisthorchis viverrini.

An adult stage Opisthorchis viverrini cDNA library was constructed and screened for abundant transcripts. One of the isolated cDNAs was found by sequence comparison to encode a glutathione S-transferase (GST) and was further analyzed for RNA expression, encoded protein function, tissue distribution and cross-reactivity of the encoded protein with other trematode protein counterparts. The cDNA has a size of 893 bp and encodes a GST of 213 amino acids length (OV28GST). The most closely-related GST of OV28GST among those published for trematodes is a 28 kDa GST of Clonorchis sinensis as shown by multiple sequence alignment and phylogenetic analysis. Northern analysis of total RNA with a gene-specific probe revealed a 900 nucleotide OV28GST transcriptional product in the adult parasite. Through RNA in situ hybridization OV28GST RNA was detected in the parenchymal cells of adult parasites. This result was confirmed by immunolocalization of OV28GST with an antiserum generated in a mouse against bacterially-produced recombinant OV28GST. Both, purified recombinant and purified native OV28GST were resolved as 28 kDa proteins by SDS-PAGE. Using the anti-recOV28GST antiserum, no or only weak cross-reactivity was observed in an immunoblot of crude worm extracts against the GSTs of Schistosoma mansoni, S. japonicum, S. mekongi, Eurytrema spp. and Fasciola gigantica. The enzyme activity of the purified recombinant OV28GST was verified by a standard 1-chloro-2, 4-dinitrobenzene (CDNB) based activity assay. The present results of our molecular analysis of OV28GST should be helpful in the ongoing development of diagnostic applications for opisthorchiasis viverrini.

Allergy to house dust mites and asthma

House dust mites have been shown to be important sources of indoor allergens associated with asthma and other allergic conditions. Asthma is a chronic respiratory disease that affects millions of people worldwide, and numerous scientific studies have shown that the prevalence of asthma is increasing. The most common dust mite species around the world include Dermatophagoides pteronyssinus (Dp), Dermatophagoides farinae (Df), Euroglyphus maynei (Em) and Blomia tropicalis (Bt). Over the past three decades, many important allergens from these species have been identified and characterized at the molecular level. The biological function of several house dust mite allergens has been elucidated, with many of them showing enzymatic activity. However, Bt allergens remain the least studied, even though this mite is very common in tropical and subtropical regions of the world, including Puerto Rico. Therefore, it is very important to include Bt in diagnostic and therapeutic strategies for house dust mite induced allergy and asthma, particularly in areas where Bt exposure and sensitization is high. Recombinant DNA technology, as well as other molecular biology and immunological techniques, have played a fundamental role in advances towards a better understanding of the biology of house dust mites and their role in allergic diseases. This kind of study also contributes to the understanding of the complex immunologic mechanisms involved in allergic reactions. The development of effective diagnostic and therapeutic approaches depends on the continuity of research of house dust mite allergens. The objectives of this review are to describe the most important aspects of house dust mite allergy and to acquaint the scientific community with the latest findings pertaining to house dust mite allergens, particularly those derived from Bt.

Functional equivalence of dihydropyridine receptor a1S and b1a subunits in triggering excitation-contraction coupling in skeletal muscle

Molecular understanding of the mechanism of excitation-contraction (EC) coupling in skeletal muscle has been made possible by cultured myotube models lacking specific dihydropyridine receptor (DHPR) subunits and ryanodine receptor type 1 (RyR1) isoforms. Transient expression of missing cDNAs in mutant myotubes leads to a rapid recovery, within days, of various Ca2+ current and EC coupling phenotypes. These myotube models have thus permitted structure-function analysis of EC coupling domains present in the DHPR controlling the opening of RyR1. The purpose of this brief review is to highlight advances made by this laboratory towards understanding the contribution of domains present in a1S and b1a subunits of the skeletal DHPR to EC coupling signaling. Our main contention is that domains of the a1S II-III loop are necessary but not sufficient to recapitulate skeletal-type EC coupling. Rather, the structural unit that controls the EC coupling signal appears to be the a1S/b1a pair.

Putative pyrophosphate phosphofructose 1-kinase genes identified in sugar cane may be getting energy from pyrophosphate

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrus paradisi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the alpha and beta subunits of this enzyme. The deduced amino acid sequences showed 76 and 80 similarity with the corresponding alpha and beta subunits of C. paradisi. A high degree of similarity was also observed among the PFK b subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum. It appears that alpha and beta are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of b PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the b subunit of the pyrophosphate-dependent enzyme

NS2a-NS2b sequence analysis of DEN-4 virus strains.

To study the genetic variability of DEN-4 Chinese isolates, and to trace the origin of DV4 Chinese isolates, we cloned and sequenced the NS2a-NS2b junction of 5 isolates and prototype DV4 (H-241). Our results show that isolates from the 1990 Guangdong epidemic, which were isolated in the early, middle, and late periods of the epidemic, share the same sequence in the NS2a-NS2b junction. The sequence similarity between isolates from the Guangdong epidemic in 1990 and DV4 H-241 is 96%; these isolates can be grouped into genotype I. The sequence similarity between the isolate from the Guangdong epidemic in 1987 and Dominica strain 814669 is 96%; this isolate can be grouped into genotype II. For the first time, our results show that there are also 2 DV4 genotypes in the Guangdong area of southern China, and these isolates perhaps were introduced from other epidemic areas outside of China.

Identification of amyloid beta-peptide responsive genes by cDNA microarray technology: Involvement of RTP801 in amyloid beta-peptide toxicity

Amyloid beta-peptide (Abeta), a causative molecule in the pathogenesis of Alzheimer's disease and the main component of senile plaques, is known to be neurotoxic in vitro and in vivo. The mechanisms involved in this Ab-mediated neurotoxicity are not fully understood, although there is evidence to suggest the involvement of oxidative stress, alterations in calcium homeostasis, and/or of CDK activators. Many studies have suggested that Ab may exert its toxic effect via the activation of transcription factors. Therefore, we investigated Ab- responsive genes in human neuroblastoma CHP134 cells using 3.1K human DNA microarrays. Among the several genes overexpressed or repressed by Ab, RTP801, Hi95/sestrin 2, and stanniocalcin 2 were confirmed to be Ab-mediated overexpression in the cells by semiquantitative RT-PCR. Transient expression of the sense RTP801 gene in CHP134 cells increased sensitivity to Abeta cytotoxicity and the expression of the antisense RTP801 gene protected the cells from the Abeta toxicity. These results suggest that RTP801 might play important roles in Abeta toxicity and the pathogenesis of Alzheimer's disease.

Growth hormone mRNA expression in the pituitary of Bos indicus and Bos taurus x Bos indicus crossbred young bulls treated with recombinant bovine somatotropin

The effects of breed and of recombinant bovine somatotropin (rbST) treatment on growth hormone gene expression were studied in young bulls. The experiment was completely randomized in a [2 x 2]-factorial arrangement, using two levels of rbST (0 or 250 mg/animal/14 days), and two breed groups (Nelore and Simmental x Nelore crossbred). A cDNA encoding Bos indicus growth hormone was cloned and sequenced for use as a probe in Northern and dot blot analyses. Compared to the Bos taurus structural gene, the Bos indicus cDNA was found to begin 21 bases downstream from the transcription initiation site and had only two discrepancies (C to T at position 144-His and T to C at position 354-Phe), without changes in the polypeptide sequence. However, two amino acid substitutions were found for Bubalus spp., which belong to the same tribe. The rbST treatment did not change any of the characteristics evaluated (body and pituitary gland weights, growth hormone mRNA expression level). Crossbred animals had significantly higher body weight and heavier pituitaries than Nelore cattle. Pituitary weight was proportional to body weight in both breed groups. Growth hormone mRNA expression in the pituitary was similar (P>0.075) for both breed and hormonal treatment groups, but was 31.9 higher in the pure Nelore group, suggesting that growth hormone gene transcription regulation differs among these breeds

Effect of local background intensities in the normalization of cDNA microarray data with a skewed expression profiles

Normalization of the data of cDNA microarray is an obligatory step during microarray experiments due to the relatively frequent non-specific errors. Generally, normalization of microarray data is based on the null hypothesis and variance model. In the Yang's model (Yang et al., 2001), at least two types of noises are included. The one is additive noise and the other is multiplicative noise. Usually, background is considered as one of additive noise to the signal and the variation between the signal pixels is the representative multiplicative noise. In this study, the relation between the signal (spot intensity minus background intensity) and background was observed and the influence of background on normalization as a representative additive factor was investigated. Although the relation has not been considered as a factor affecting the normalization, it could improve the accuracy of microarray data when the normalization was carried out considering signal/background ratio. The background dependent normalization decreased the number of genes whose expression levels were changed significantly and it could make their distribution more consistent through the whole range of signal intensities. In this study, printing pin dependent normalization was also carried out regarding the printing pin as a representative multiplicative noise. It improved the distribution of spots in the Cy3-Cy5 scatter plot, but its effect was slight. These studies suggest that there are some influences of the signals on the local backgrounds and they must be considered for the normalization of cDNA microarray data.