Sting_RDB: a relational database of structural parameters for protein analysis with support for data warehousing and data mining

An effective strategy for managing protein databases is to provide mechanisms to transform raw data into consistent, accurate and reliable information. Such mechanisms will greatly reduce operational inefficiencies and improve one's ability to better handle scientific objectives and interpret the research results. To achieve this challenging goal for the STING project, we introduce Sting_RDB, a relational database of structural parameters for protein analysis with support for data warehousing and data mining. In this article, we highlight the main features of Sting_RDB and show how a user can explore it for efficient and biologically relevant queries. Considering its importance for molecular biologists, effort has been made to advance Sting_RDB toward data quality assessment. To the best of our knowledge, Sting_RDB is one of the most comprehensive data repositories for protein analysis, now also capable of providing its users with a data quality indicator. This paper differs from our previous study in many aspects. First, we introduce Sting_RDB, a relational database with mechanisms for efficient and relevant queries using SQL. Sting_rdb evolved from the earlier, text (flat file)-based database, in which data consistency and integrity was not guaranteed. Second, we provide support for data warehousing and mining. Third, the data quality indicator was introduced. Finally and probably most importantly, complex queries that could not be posed on a text-based database, are now easily implemented. Further details are accessible at the Sting_RDB demo web page: http://www.cbi.cnptia.embrapa.br/StingRDB.     

Homology of polytene elements between Drosophila and Zaprionus determined by in situ hybridization in Zaprionus indianus

The drosophilid Zaprionus indianus due to its economical importance as an insect pest in Brazil deserves more investigation into its genetics. Its mitotic karyotype and a line-drawing map of its polytene chromosomes are already available. This paper presents a photomap of Z. indianus polytene chromosomes, which was used as the reference map for identification of sections marked by in situ hybridization with gene probes. Hybridization signals for Hsp70 and Hsr-omega were detected, respectively, in sections 34B and 32C of chromosome V of Z. indianus, which indicates its homology to the chromosomal arm 3R of Drosophila melanogaster and, therefore, to Muller's element E. The main signal for Hsp83 gene probe hybridization was in section 17C of Z. indianus chromosome III, suggesting its homology to arm 3L of D. melanogaster and to element D of Muller. The Ubi probe hybridized in sections 10C of chromosome II and 17A of chromosome III. Probably the 17A is the polyubiquitin locus, with homology to arm 3L of D. melanogaster and to the mullerian D element, as suggested also by Hsp83 gene location. The Br-C gene was mapped in section 1D, near the tip of the X chromosome, indicating its homology to the X chromosome of D. melanogaster and to mullerian element A. The Dpp gene probe hybridized mainly in the section 32A of chromosome V and, at lower frequencies to other sections, although no signal was observed as expected in the correspondent mullerian B element. This result led to the suggestion of a rearrangement including the Dpp locus in Z. indianus, the secondary signals possibly pointing to related genes of the TGF-beta family. In conclusion, the results indicate that chromosomes X, III, V of Z. indianus are respectively correspondents to elements A, D, and E of Muller. At least chromosome V of Z. indianus seems to share synteny with the 3R arm of D. melanogaster, as indicated by the relative positions of Hsp70 and Hsr-omega, although the Dpp gene indicates a disruption of synteny in its distal region.     

The lactate dehydrogenases encoded by the ldh and ldhB genes in Lactococcus lactis exhibit distinct regulation and catalytic properties - comparative modeling to probe the molecular basis

Lactococcus lactis FI9078, a construct carrying a disruption of the ldh gene, converted approximately 90% of glucose into lactic acid, like the parental strain MG1363. This unexpected lactate dehydrogenase activity was purified, and ldhB was identified as the gene encoding this protein. The activation of ldhB was explained by the insertion of an IS905-like element that created a hybrid promoter in the intergenic region upstream of ldhB. The biochemical and kinetic properties of this alternative lactate dehydrogenase (LDHB) were compared to those of the ldh-encoded enzyme (LDH), purified from the parental strain. In contrast to LDH, the affinity of LDHB for NADH and the activation constant for fructose 1,6-bisphosphate were strongly dependent on pH. The activation constant increased 700-fold, whereas the K(m) for NADH increased more than 10-fold, in the pH range 5.5-7.2. The two enzymes also exhibited different pH profiles for maximal activity. Moreover, inorganic phosphate acted as a strong activator of LDHB. The impact of replacing LDH by LDHB on the physiology of L. lactis was assessed by monitoring the evolution of the pools of glycolytic intermediates and cofactors during the metabolism of glucose by in vivo NMR. Structural analysis by comparative modeling of the two proteins showed that LDH has a slightly larger negative charge than LDHB and a greater concentration of positive charges at the interface between monomers. The calculated pH titration curves of the catalytic histidine residues explain why LDH maintains its activity at low pH as compared to LDHB, the histidines in LDH showing larger pH titration ranges.     

The state of the art of adeno-associated virus-based vectors in gene therapy

Background

Genotyping of HBV is generally used for determining the epidemiological relationship between various virus strains and origin of infection mostly in research studies. The utility of genotyping for clinical applications is only beginning to gain importance. Whether HBV genotyping will constitute part of the clinical evaluation of Hepatitis B patients depends largely on the availability of the relevance of the evidence based information. Since Pakistan has a HBV genotype distribution which has been considered less virulent as investigated by earlier studies from south East Asian countries, a study on correlation between HBV genotypes and risk of progression to further complex hepatic infection was much needed

Methods

A total of 295 patients with HBsAg positive were selected from the Pakistan Medical Research Council's (PMRC) out patient clinics. Two hundred and twenty six (77%) were males, sixty nine (23%) were females (M to F ratio 3.3:1).

Results

Out of 295 patients, 156 (53.2%) had Acute(CAH), 71 (24.2%) were HBV Carriers, 54 (18.4%) had Chronic liver disease (CLD) Hepatitis. 14 (4.7%) were Cirrhosis and HCC patients. Genotype D was the most prevalent genotype in all categories of HBV patients, Acute (108), Chronic (39), and Carrier (53). Cirrhosis/HCC (7) were HBV/D positive. Genotype A was the second most prevalent with 28 (13%) in acute cases, 12 (22.2%) in chronics, 14 (19.7%) in carriers and 5 (41.7) in Cirrhosis/HCC patients. Mixed genotype (A/D) was found in 20 (12.8%) of Acute patients, 3 (5.6%) of Chronic and 4 (5.6%) of carriers, none in case of severe liver conditions.

Conclusion

Mixed HBV genotypes A, D and A/D combination were present in all categories of patients except that no A/D combination was detected in severe conditions. Genotype D was the dominant genotype. However, genotype A was found to be more strongly associated with severe liver disease. Mixed genotype (A/D) did not significantly appear to influence the clinical outcome.     

Anastrozole quantification in human plasma by high-performance liquid chromatography coupled to photospray tandem mass spectrometry applied to pharmacokinetic studies

A rapid, sensitive and specific method for quantifying the aromatase inhibitor (anastrozole) in human plasma using dexchlorpheniramine as the internal standard (I.S.) is described herein. The analyte and the I.S. were extracted from 200 microl of human plasma by liquid-liquid extraction using a mixture of diethyl ether:dichloromethane (70:30, v/v) solution. Extracts were removed and dried in the organic phase then reconstituted with 200 microl of acetonitrile:water (50:50; v/v). The extracts were analyzed by high performance liquid chromatography coupled with photospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed isocratically on a Genesis, C18 4 microm analytical column (100 mm x 2.1mm i.d.). The method had a chromatographic run time of 2.5 min and a linear calibration curve ranging from 0.05-10 ng ml(-1). The limit of quantification (LOQ) was 0.05 ng ml(-1). This HPLC-MS-MS procedure was used to assess pharmacokinetic studies.     

Structural and Biological Characterization of Two Crotamine Isoforms IV-2 and IV-3 Isolated from the <Emphasis Type="Italic">Crotalus durissus cumanensis</Emphasis> Venom

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its “in vitro” neurotoxic, myotoxic and lethality (DL₅₀) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (μ-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD₅₀ of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.     

Dopamine levels modulate the updating of tastant values

To survive, animals must constantly update the internal value of stimuli they encounter; a process referred to as incentive learning. Although there have been many studies investigating whether dopamine is necessary for reward, or for the association between stimuli and actions with rewards, less is known about the role of dopamine in the updating of the internal value of stimuli per se. We used a single-bottle forced-choice task to investigate the role of dopamine in learning the value of tastants. We show that dopamine transporter knock-out mice (DAT-KO), which have constitutively elevated dopamine levels, develop a more positive bias towards a hedonically positive tastant (sucrose 400 mM) than their wild-type littermates. Furthermore, when compared to wild-type littermates, DAT-KO mice develop a less negative bias towards a hedonically negative tastant (quinine HCl 10 mM). Importantly, these effects develop with training, because at the onset of training DAT-KO and wild-type mice display similar biases towards sucrose and quinine. These data suggest that dopamine levels can modulate the updating of tastant values, a finding with implications for understanding sensory-specific motivation and reward seeking.     

2,2',4-trihydroxybenzophenone: crystal structure, and anti-inflammatory and antioxidant activities

The crystal structure of '2,2',4-trihydroxybenzophenone' (=(2,4-dihydroxyphenyl)(2-hydroxyphenyl)methanone; 1) was determined, and its molecular structure, along with intra- and intermolecular H-bonds, was analyzed. The anti-inflammatory potential of 1, evaluated by means of the rat-paw-edema assay, with carrageenan as inflammation stimulus, was found to be similar high as that of indomethacin. In contrast, benzophenone proper (2) was hardly active in this assay. Our results indicate that these anti-inflammatory effects are related to the action of kinins and prostaglandins. The radical-scavenging properties of 1 towards DPPH were found to be similar as those of typical phenolics, but somewhat lower than that of ascorbic acid. The structure-activity relationship (SAR) of 1 is discussed.