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Item Cyclosporin A inhibits the growth of neonatal MHC-expressing myotubes independent of NFATc1 and NFATc3 in the mechanically overloaded soleus muscle of mice(Scientific Research Publishing, 2011-04) Sakuma, Kunihiro; Yamaguchi, AkihikoABSTRACT The molecular signaling pathway linked to hyper-trophy of the anti-gravity/postural soleus muscle af-ter mechanical overloading has not been identified. Using Western blot and immunohistochemical analy-ses, we investigated whether the amounts of NFATc3, GSK-3?, NFATc1, and neonatal MHC change in the mechanically overloaded soleus muscle after cyc-losporine A (CsA) treatment. Adult male ICR mice were subjected to a surgical ablation of the gas-trocnemius muscle and treated with either CsA (25 mg/Kg) or vehicle once daily. They were sacrificed at 2, 4, 7, 10, and 14 days post-injury. Mechanical over-loading resulted in a significant increase in the wet weight and the cross-sectional area of slow and fast fibers of the soleus muscle in placebo-treated mice but not CsA-treated mice. After 4 days of mechanical overloading, we observed a similar co-localization of neonatal MHC and NFATc3 in several myotubes of both mice. The placebo-treated mice possessed larger myotubes with neonatal MHC than CsA-treated mice. At 7 days, mechanical overloading induced marked expression of neonatal MHC in myotubes and/or myofibers. Such neonatal MHC-positive fibers emerged less often in the hypertrophied soleus mus-cle subjected to treatment with CsA. CsA treatment did not significantly change the amount of GSK-3? protein in the soleus muscle. The modulation of growth in neonatal MHC-positive myofibers by CsA treatment may inhibit the hypertrophic process in the soleus muscle after mechanical overloading.Item Chromosomal aberrations and nucleic acids systems affected by some Egyptian medicinal plants used in treating female pregnant diabetic rats(Scientific Research Publishing, 2011-04) Abdou, Halima S .; Salah, Sherifa H.; El Raouf, Amira A.; Abdel-Rahim, E.A.The influences of medicinal plants Juniperus Phoenicea (Araar), Hyphaene thebaica (Doum), An-astatica hierochuntica (Kafta) and Cleome droserifolia (Sammo) as antidiabetic agents were investigated using female pregnant albino rats. Female rats were injected with 60 mg/kg b.w. alloxan to induce diabe-tes. Diabetic rats treated orally with the methanol extracts of tested plants till the 19 day of gestation. The present studies include the frequencies of chro-mosomal aberrations and nucleic acid system of liver in the female pregnant rats and their embryos. The results showed that injection of alloxan caused highly significant increase in chromosomal aberrations as well as in blood glucose levels as a result of diabetes in pregnant females. It also caused a high incidence of chromosomal deviation in embryos and decreased the liver soluble protein contents of female rats and their embryos. These effects in alloxanized animals were treated and improved by ingestion of the methanol extracts of the tested plants (Araar, Doum, Kafta and Somma) in which under their treatments, the inceased level of blood glu-cose of diabetic rats was deceased. Ingestion with the plants methanolic extracts improved and normalized the effects of diabetes in nucleic acids values of liver tissues. These were accompanied with nucleases (RNAase and DNAase) activities. The inhibited ac-tivities of both DNA ase and RNA ase of pregnant rats and their embryos were stimulated and read-justed around the normal values. Also administration of the plants methanol extracts decreased the per-centage of chromosomal aberrations in the female rats and embryos. It is concluded that there are some biochemical dynamics which might occur in the metabo-lism of glucose, nucleic acids and proteins in order to prevent or to reduce the oxidative stress of diabetes by flavonoides treatment.Item Cloning, purification and characterization of the ribosomal protein L11 from E. coli(Scientific Research Publishing, 2011-04) Todorova, RoumianaA high-expression system of L11 was constructed and investigated its interaction with other elements of the ribosome using physicochemical methods. The gene rplK, coding for the protein L11 from the E. coli 50S ribosomal subunit was amplifyied, cloned and over-expressed. The protein L11 was purified under native and denaturing conditions, refolded and the structure of both proteins was compared. The protein L11 properly refolded from 6M urea after dialysis. Experiments on binding of proteins L11, RRF and EF-G from Escherichia coli were performed by ana-lytical centrifugation and Biacore. Specific binding between protein L11 and RRF by analytical cen-trifugation was not detected probably due to struc-tural reasons. These findings may be helpful in the design of new antibiotics that specifically disrupt the interactions in the “GTP-associated site” of the bac-terial ribosome, as many of them are not effective anymore. A common intrinsically disordered region of protein L11 was found to be the amino acid se-quence 86-97, while the residues 67-74, containing the linker region, are predicted to be disordered by DisEMBL.Item Influence OF anticancer drugs on DNA methylation in liver of female mice(Scientific Research Publishing, 2011-06) Hanafy, Shaden M.; Salem, Tarek A.; El-Aziz, Amal A.; EL-Fiky, Bahgat A.; Shokair, Mahmoud A.Epigenetic changes such as DNA methylation regulate gene expression in normal development. Methotrexate and Adriamycine are antineoplastic drugs that target DNA and enzymes acting on DNA. We aimed to evaluate their cytotoxic effect on cell lines and on female mice to investigate the in vivo influence of both drugs on the DNA methylation and subsequently the protein expression. The total level of DNA methylation showed a significant reduction from 62.2% to 36.7%, 36.6% as compared to control group, when using different doses of MTX and ADR. Hepatic protein pattern revealed five bands with low MW (16 - 6.1 KDa) in acute and LD50 doses. In conclusion DNA methylation is influenced by anticancer drugs in a dose - dependent manner. Some specific protein fragments may be considered as a potential markers associated with high dose of anticancer drugs.Item Effects of genetic and environmental factors and gene-environment interaction on expression variations of genes related to stroke in rat brain(Scientific Research Publishing, 2011-07) Tan, Yuan D.; Fornage, MyriamTo determine if genetic and environmental (dietary) factors and gene-environment interaction impact on the expression variation of genes related to stroke, we conducted microarray experiments using two homozygous rat strains SHRSR and SHRSP fed with high and low dietary salt levels. We obtained expression data of 8,779 genes and performed the ranking analysis of microarray data. The results show that the genetic difference for stroke in rat brain has a strong effect on expression variations of genes. At false discovery rate (FDR) ≤ 5%, 534 genes were found to be differentially expressed between the genotypes resistant and prone to stroke, among which 304 genes were up-regulated in the resistant genotype and down-regulated in the prone genotype and 230 were down-regulated in the former and up-regulated in the latter. In addition, 365 were functional genes for transcription and translation, receptors (in particular, neurotransmitter receptor), channels of ions, transportation, metabolism and enzymes, and functional and structural proteins. Some of these genes are pivotal genes that cause stroke. However, dietary salt levels and GE interaction do not strongly impact on the expression variations of these genes detected on arrays.Item A reduced computational load protein coding predictor using equivalent amino acid sequence of DNA string with period-3 based time and frequency domain analysis(Scientific Research Publishing, 2011-07) Meher, Jayakishan K.; Dash, Gananath N.; Meher, Pramod K.; Raval, Mukesh K.Development of efficient gene prediction algorithms is one of the fundamental efforts in gene prediction study in the area of genomics. In genomic signal processing the basic step of the identification of protein coding regions in DNA sequences is based on the period-3 property exhibited by nucleotides in exons. Several approaches based on signal processing tools and numerical representations have been applied to solve this problem, trying to achieve more accurate predictions. This paper presents a new indicator sequence based on amino acid sequence, called as aminoacid indicator sequence, derived from DNA string that uses the existing signal processing based time-domain and frequency domain methods to predict these regions within the billions long DNA sequence of eukaryotic cells which reduces the computational load by one-third. It is known that each triplet of bases, called as codon, instructs the cell machinery to synthesize an amino acid. The codon sequence therefore uniquely identifies an amino acid sequence which defines a protein. Thus the protein coding region is attributed by the codons in amino acid sequence. This property is used for detection of period-3 regions using amino acid sequence. Physico-chemical properties of amino acids are used for numerical representation. Various accuracy measures such as exonic peaks, discriminating factor, sensitivity, specificity, miss rate, wrong rate and approximate correlation are used to demonstrate the efficacy of the proposed predictor. The proposed method is validated on various organisms using the standard data-set HMR195, Burset and Guigo and KEGG. The simulation result shows that the proposed method is an effective approach for protein coding prediction.Item N–nitrosodiethylamine cytochrome P450 induction and cytotoxicity evaluation in primary cultures of rat hepatocytes(Scientific Research Publishing, 2011-07) Aiub, Claudia A.; Gadermaier, Gabriele; Ferreira, Fátima; Felzenszwalb, Israel; Eckl, Peter; Pinto, Luis F.The primary routes of potential human exposure to N-nitrosodiethylamine (NDEA) are ingestion, inhalation, and dermal contact. Air, diet and smoking contribute to potential human exposure at levels of a few µg of NDEA/day. Potential exposure depends on the ability of the nitrosamines to migrate from the product into the body. The first step in the metabolic degradation of NDEA by cytochrome oxidase (CYPs) enzymes is the introduction of a hydroxyl group and in human esophage and liver CYP2A3 and CYP2E1 participate on this metabolism. Measuring cytotoxicity in female rat primary hepatocytes cultures, were used to understand the CYP induction and metaboli-zation correlated with low NDEA concentrations. We observed that NDEA at different concentrations in the absence of CYPs inducers, was able to induce CYP2B1, CYP2B2, CYP2E1, CYP3A1 and CYP4A3. A positive NDEA synergistic effect on the levels of mRNA, was observed in the presence of pyrazole (300 µM) for CYP2B1 and CYP2B2 and for pregnenolone 16- carbonitrile (0.15 µM) for CYP2E1. Negative NDEA synergistic effects were observed for ethanol (0.3%) for CYP3A1, pyrazol (300 µM) for CYP2A1 and CYP2E1, and phenobarbital (1 mM) for CYP2A1. These facts are extremally important once that these metabolites can be directly related to the primary DNA lesions. We consider that studies to elucidate the biological factors that determine the shape of the dose-response curve are crucial for low-dose extrapolations of risk.Item Characterization of asian and north American avian H5N1(Scientific Research Publishing, 2011-07) Hu, WeiSince the emerge of the highly pathogenic avian H5N1 virus in Asia in 1996, the possibility for this virus to cross species barriers to infect humans and its ability to cause large outbreaks in birds have been a public health concern. This virus has been spreading from Asia to Europe and Africa by migratory birds with North America as its next possible stop. In this study, an ensemble of computational techniques including Random Forests, Informational Spectrum Method, Entropy, and Mutual Information were employed to unravel the distinct characteristics of Asian and North American avian H5N1 in comparison with human and swine H5N1. Critical differences were identified in the HA cleavage and binding sites, the HA receptor selection, the interaction patterns of HA and NA, and NP, PA, PB1, and PB2, and the important sites in the influenza proteins including HA, NA, M1, M2, NS1, NS2, NP, PA, PB1, PB1-F2, and PB2.Item Ghrelin-induced cSrc activation through constitutive nitric oxide synthase-dependent S-nitrosylation in modulation of salivary gland acinar cell inflammatory responses to Porphyromonas gingivalis(Scientific Research Publishing, 2011-07) Slomiany, Bronislaw L.; Slomiany, AmaliaA peptide hormone, ghrelin, recognized for its role in the regulation of nitric oxide production has emerged as an important modulator of oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis. As cSrc kinase plays a major role in controlling the activity of nitric oxide synthase (NOS) system, in this study we investigated the influence of P. gingivalis LPS on the processes of Src activation in rat sublingual gland acinar cells. The LPS-induced enhancement in the activity of inducible (i) iNOS and the impairment in constitutive (c) cNOS were reflected in the suppression in cSrc activity and the extent of its phosphorylation at Tyr416. Further, we show that the countering effect of ghrelin on the LPS-induced changes in cSrc activity and the extent of its phosphorylation was accompanied by a marked reduction in iNOS and the increase in cNOS activation through phosphorylation at Ser1179. Moreover, the effect of ghrelin on cSrc activation was associated with the kinase S-nitrosylation that was susceptible to the blockage by cNOS inhibition. Our findings suggest that P. gingivalis-induced up-regulation in iNOS leads to disturbances in cNOS phosphorylation that exerts the detrimental effect on the processes of cSrc activation through cNOS mediated S-nitrosylation. We also show that the effect of ghrelin on P. gingivalis-induced inflammatory changes are manifested in the enhancement in cSrc activation through S-nitrosylation and the increase in its phosphorylation at Tyr416.Item An investigation of 10 Y-STR loci and the detection of specific haplotype frequencies in Turkish population(Scientific Research Publishing, 2011-07) Rüstemoglu, Aydın; Akay, Güvem G.; Karabulut, Halil G.; Kadıkıran, Ahmet; Bökesoy, IşıkThis study is to survey 10 Y-STR loci in 241 males from Turkey. In this study, the 241 healthy and unrelated males living in different parts of Turkey for at least three generations were included. Genomic DNAs were isolated from peripheral blood samples by standard phenol-chloroform extraction method. 10 Y-STR loci including DYS19, DYS385a/b, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393, and YCAIIa/b were analyzed by using PCR and denaturing PAGE. Allele frequencies, gene diversities and haplotype frequencies were analyzed. Gene diversity per locus varied from 0.5788 (DYS388) to 0.8903 (DYS385a/b). The numbers of haplotypes in minHt recommended by YCC and Ht10 have been 208 and 186, respectively. When our minHt haplotypes frequencies compared with the other seven populations, we have found statistically significant differences between our results and other populations (P < 0.01) except that Czech population (P > 0.05). We suggest that an alternative haplotype designated as aHt maybe alternative to minHt in respect of its Y-STR content with the highest gene diversity value. The aHt haplotype has found a higher discriminatory potential than minHt haplotype with a better Pd combined value (0.9999936 vs 0.9999836) and has higher average gene diversity per locus (0.7834 vs 0.7518) in Turkish population. aHt haplotype can be proposed as an alternative to minHt in paternity testing and forensic medicine applications involving Turkish male population. This study has also provided additional information to the framework of variation involving 10 Y-STR loci as well as a further contribution to the Y-STR database for Turkish male population.Item EGR1 is essential for transcriptional regulation of BMPR2(Scientific Research Publishing, 2011-10) Gaddipati, Radhika; West, James D.; Loyd, James E.; Blackwell, Thomas; Lane, Kirsten A.; Lane, Nicole M.; Lane, Kirk B.In this study, RLM-RACE was used to identify the transcriptional start site 387 bp upstream of the translational start. Evolutionarily conserved transcription factor binding sites were identified, and a series of luciferase reporter constructs driven by BMPR2 promoter elements used to determine their functional relevance. We found the promoter area from 983 bp to 90 bp upstream of the transcriptional start gave maximal activity, greater than longer constructs, with an area between 570 bp and 290 bp upstream of the transcriptional start containing an important repressor element. To characterize this repressor, we used a combination of EMSA, mutation of the EGR1 binding site, transfection with EGR1 and NAB1 constructs, and mutation of the NAB1 binding site within the EGR1 protein. From this we conclude that EGR1 is essential to BMPR2 transcription, but that NAB1 binding to EGR1 causes it to act as a repressor.Item HPV positivity varies with technique and primer set, in formalin-fixed paraffin-embedded benign and malignant breast tissue from Malta(Scientific Research Publishing, 2011-10) Mallia, Roderick; Mangion, Jean-Paul; Camenzuli, Christian; Cassar, Annalisse; Cacciottolo, Paul; Cauchi, John; Borg, Joseph; Ali, Sarfraz; Schembri-Wismayer, PierreThe role of human papillomavirus in breast cancer pathogenesis is an area of considerable debate following differing results from all over the world. This study analyzed paraffin embedded tissue from 19 patients with benign or malignant breast disease. Standard PCR with GP5+/6+ and FAP primers, proved negative in all cases. However, qRT-PCR using the SF10 primer set detected HPV DNA in 2 out of these samples. Whilst our data does not support a major role for HPV in the pathogenesis of breast disease in Malta, it definitely does not exclude it totally. This study also clearly shows that the method of analysis is one of the variables resulting in the range of differing results witnessed worldwide in this area of study.Item Codon bias of influenza a viruses and their hosts(Scientific Research Publishing, 2011-10) Fancher, Keli C.; Hu, WeiThe Influenza A virus remains an annual and worldwide health concern due to its fast evolutionary rate. There are two major forms of Influenza evolution: drift, caused by mutations, and shift, which results from the exchange of genetic information between two gene segments. Using six synonymous codon usage bias indexes (GC content, ENC, SCUO, Codon Volatility, RSCU, and Odds Ratio), this study revealed the evolutionary drift patterns in the Influenza A viruses of avian, human, and swine origins as well as those of their hosts. We found that the variation of GC content across the 11 genes in Influenza A uniquely determines the viral origins in avian, human, and swine hosts. As was previously noticed in the Flaviviridae virus, a codon’s RSCU value of the Influenza viruses is positively correlated to the Odds Ratio of the dinucleotides contained within that codon. Additionally, the RSCU values of avian, human, and swine viruses and their corresponding hosts are similar, which is also true of Odds Ratio. Furthermore, the GC content, ENC, SCUO, and Codon Volatility are similar across the avian, human, and swine hosts; however, the RSCU and Odds Ratio of the hosts are distinct. Our findings expanded the knowledge on codon bias of Influenza viruses and their hosts.Item Gene expression profiling in soybean under aluminum stress: genes differentially expressed between Al-tolerant and Al-sensitive genotypes(Scientific Research Publishing, 2011-10) Duressa, Dechassa; Soliman, Khairy M.; Taylor, Robert W.; Chen, DongquanIt is well documented that aluminum (Al) toxicity is the most important constraint to crop production on acid soils and soybean is one of the most Al sensitive plant species. To advance our understanding of the molecular and genetic mechanisms of Al-tolerance in soybean we compared root tip (1 cm long) transcriptome profiles of an Al-tolerant (PI 416937) and Al-sensitive (Young) soybean genotypes using a combination of DNA microarrays and quantitative real-time PCR gene expression profiling technologies, in a time-course experiment (2, 12, 48, 72 h post Al treatment). We observed many genes differentially expressed between the two genotypes in constitutive and non-constitutive manner. The most likely candidate Al-tolerance genes expressed at high level include the previously reported transcription factors, auxin down regulated-like protein (ADR6-like) and, basic leucine zipper (bZIP 94), sulfur transmembrane transport protein and lipid transfer protein; and several novel genes that include rare cold inducible protein (RCI2B ), GPI-transamidase, malonyl-COA: Isoflavone 7-O-glucoside-6"-O-malontransferase, a cell proliferation protein (WPP2), oleosin protein, pectinestrease inhibitor, and impaired sucrose induction1; whereas genes negatively correlated with Al-tolerance, namely cellulose synthase and calcium transporters were down regulated in Al-tolerant PI 416937 compared to the Al-sensitive Young. The possible mechanisms of how these genes contribute to Al-tolerance trait are discussed. In conclusion, transcriptome profile comparisons of Al-tolerant and Al-sensitive soybean genotypes revealed novel putative Al-tolerance genes. These genes deserve further functional characterization for eventual utilization in developing soybean germplasm adapted to high aluminum soils.Item JnCML-like, an EF-hand motif-containing gene seasonally upregulated in the transition zone of black walnut (Juglans nigra L.)(Scientific Research Publishing, 2011-10) Huang, Zhonglian; Surana, Priyanka; Kihara, Daisuke; Meilan, Richard; Woeste, KeithThe economic value of a black walnut (Juglans nigra L.) tree is strongly determined by the quality and quantity of darkly colored heartwood in its stem. To understand the regulation of heartwood formation, we analyzed the region of heartwood formation in walnut stems (i.e., the transition zone, TZ) for the expression of 80 ESTs. Semi-quantitative RT-PCR and real-time PCR was performed to detect expression changes of candidate genes in the TZ and sapwood of trees harvested in summer and fall. Results revealed that the transcript of a clone containing two presumed EF-hand motifs was expressed at higher levels in the TZ than in other xylem tissues. Analysis of the full-length coding sequence revealed that the black walnut gene JnCML-like is similar to grancalcin-like calcium-binding EF hand proteins in Arabidopsis thaliana (At3g10300) and Zea mays (NM 001153810). A model of the predicted structure of JnCML-like showed it is similar to grancalcin and m-calpain, penta-EF-hand family proteins associated with cell proliferation, differentiation and programmed cell death. JnCML-like transcript was detected in tissue from the region of the pith meristem, and in roots, embryogenic callus, vascular cambium, female flowers, male flowers, green leaves, and partially and fully senescent leaves of black walnut, although transcript abundance varied considerably among these tissues.Item Cytosolic chaperonin CCT possesses GTPase activity(Scientific Research Publishing, 2011-10) Noguchi, Susumu; Toyoshima, Kazuyoshi; Yamamoto, Soh; Miyazaki, Toshio; Otaka, Michiro; Watanabe, Sumio; Imai, Katsunori; Senoo, Haruki; Kobayashi, Ryoji; Jikei, Mitsutoshi; Kawata, Yasushi; Kubota, Hiroshi; Itoh, HideakiCytosolic chaperonin CCT (also known as TRiC) is a hetero-oligomeric cage-like molecular chaperone that assists in protein folding by ATPase cycle-dependent conformational changes. However, role of the nucleo-tide binding and hydrolysis in CCT-assisted protein folding is still poorly understood. We purified CCT by using ATP-Sepharose and other columns, and found that CCT possesses ability to hydrolyze GTP, with an activity level very similar to the ATPase activity. CCT was more resistant to proteinase K treatment in the presence of GTP or ATP. These results suggest that the GTPase activity of CCT may play a role in chaperone-assisted protein folding.Item SPT5 affects the rate of mRNA degradation and physically interacts with CCR4 but does not control mRNA deadenylation(Scientific Research Publishing, 2012-01) Cui, Yajun; Chiang, Yueh-Chin; Viswanathan, Palaniswamy; Lee, Darren J.; Denis, Clyde L.The CCR4-NOT complex has been shown to have multiple roles in mRNA metabolism, including that of transcriptional elongation, mRNA transport, and nuclear exosome function, but the primary function of CCR4 and CAF1 is in the deadenylation and degradation of cytoplasmic mRNA. As previous genetic analysis supported an interaction between SPT5, known to be involved in transcriptional elongation, and that of CCR4, the physical association of SPT5 with CCR4 was examined. A two-hybrid screen utilizing the deadenylase domain of CCR4 as a bait identified SPT5 as a potential interacting protein. SPT5 at its physiological concentration was shown to immunoprecipitate CCR4 and CAF1, and in vitro purified SPT5 specifically could bind to CAF1 and the deadenylase domain of CCR4. We additionally demonstrated that mutations in SPT5 or an spt4 deletion slowed the rate of mRNA degradation, a phenotype associated with defects in the CCR4 mRNA deadenylase complex. Yet, unlike ccr4 and caf1 deletions, spt5 and spt4 defects displayed little effect on the rate of deadenylation. They also did not affect decapping or 5' - 3' degradation of mRNA. These results suggest that the interactions between SPT5/SPT4 and the CCR4-NOT complex are probably the consequences of effects involving nuclear events and do not involve the primary role of CCR4 in mRNA deadenylation and turnover.Item Molecular docking investigation for Indonesian H274Y mutant neuraminidase type 1 with neuraminidase inhibitors(Scientific Research Publishing, 2012-01) Herlambang, Sigit J.; Saleh, RosariThe aim of this study is to get insight the interaction between Indonesian H274Y mutant neuraminidase with four inhibitors. Not only to seek preferable inhibitor to be used, but also to investigate the interaction occurred, especially hydrogen bonds formed. Hydrogen bonds analysis and its interaction energies calculation showed that zanamivir is the most preferable inhibitor with 13 hydrogen bonds formed and –439.96 kcal/mol. Laninamivir would be an alternative inhibitor since it has 10 hydrogen bonds and –307.19 kcal/mol. The investigation of ΔSAS showed almost all active site residues buried when interacted with inhibitors. Only a few residues have an increases ΔSAS. Lipinski rule analysis showed that zanamivir and laninamivir would be best taken by injection or inhalation.Item Molecular surveillance of dengue virus in Bahia State, Brazil(Scientific Research Publishing, 2012-01) Melo, Paulo R.; Domingo, Cistina; Santos, Stênio C.; Cerqueira, Bruno A.; Andrade, Luis J.; Nunes, Juliana B.; Ciuffo, Isolina A.; Tenório, Antônio; Blanton, Ronald E.; Reis, Mitermayer G.Dengue is an important emerging viruses, posing a threat to one-third of the global human population. In 2002, the introduction of DENV-3 in the state of Bahia produced massive epidemic (about 35,000 cases detected) and the first cases of dengue hemorrhagic fever. To understand the nature of the virus circulating at Bahia, E/NS1 sequence was determined for 31 DENV viruses isolated in Bahia during the 2006 and 2007 transmission season, from patients presenting with different degrees of severity. The carboxi-terminal region of the E gene (220 nt) of 31 viruses, isolated from dengue patients with clinical diagnosis of dengue infection were used to determine the genetic variability of dengue 2 (DENV-2) and dengue 3 (DENV-3). Sequence data were used in phylogenetic comparisons with global samples of DENV-2 and DENV-3. DENV-2 sample grouped in the South East Indian genotype, while DENV-3 samples were grouped within Indian genotype. This study is the first report on Bahia isolates during two transmission seasons. Our data confirms reports from other parts of Brazil and different countries showing the DENV-3 (geno-type III) strains circulating in the Americas are closely related, and cluster within the genotype that has been associated with DHF epidemics in different conti-nents.Item Interaction of haptoglobin with hemoglobin octamers based on the mutation αAsn78Cys or βGly83Cys(Scientific Research Publishing, 2012-01) Brillet, Thomas; Marden, Michael C.; Yeh, Joanne I.; Shen, Tong-Jian; Ho, Nancy T.; Kettering, Regina; Du, Shoucheng; Vasseur, Corinne; Domingues-Hamdi, Elisa; Ho, Chien; Baudin-Creuza, VéroniqueOctameric hemoglobins have been developed by the introduction of surface cysteines in either the alpha or beta chain. Originally designed as a blood substitute, we report here the structure and ligand binding function; in addition the interaction with haptoglobin was studied. The recombinant Hbs (rHbs) with mutations alpha Asn78Cys or beta Gly83Cys spontaneously form octamers under conditions where the cysteines are oxidized. Oxygen binding curves and CO kinetic studies indicate a correct allosteric transition of the tetramers within the octamer. Crystallographic studies of the two rHbs show two disulfide bonds per octamer. Reducing agents may provoke dissociation to tetramers, but the octamers are stable when mixed with fresh human plasma, indicating that the reduction by plasma is slower than the oxidation by the dissolved oxygen, consistent with an enhanced stability. The octameric rHbs were also mixed with a solution of haptoglobin (Hp), which binds the dimers of Hb: there was little interaction for incubation times of 15 min; however, on longer timescales a complex was formed. Dynamic light scattering was used to follow the interaction of Hp with the alpha Asn78Cys octamer during 24 hours; a transition from a simple complex of 15 nm to a final size of 60 nm was observed. The results indicate a specific orientation of the αβ dimers may be of importance for the binding to haptoglobin.