Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.     

Adenovirus serotype determines association and localization of the large E1B tumor antigen with cellular tumor antigen p53 in transformed cells.

The distribution and stability of the cellular tumor antigen p53 were studied in baby rat kidney cells transformed by region E1 sequences of nononcogenic adenovirus (Ad) type 5 (Ad5) or oncogenic type 12 (Ad12). In transformed cells expressing the large E1B T antigen of Ad5, p53 was associated with this T antigen. The complexed proteins were concentrated in a cytoplasmic body, which has been shown to consist of a cluster of 8-nm filaments (A. Zantema et al., Virology 142:44-58, 1985). In transformed cells expressing the E1B region of Ad12, however, no association between the viral large T antigen and p53 was detectable. In the latter case, both proteins were found almost exclusively in the nucleus. The stability of p53 in both Ad5- and Ad12-transformed cells was increased relative to that in primary cells or cells immortalized by the E1A region only. Thus, the increased stability of p53 in Ad-transformed cells is not caused by association with a viral T antigen, but it correlates with expression of E1B and with morphological transformation.     

Effects of N-nitrosopiperidine substitutions on mutagenicity in Drosophila melanogaster.

N-Nitrosopiperidine (NP) and various derivatives were fed to Drosophila melanogaster males over a wide concentration range in order to assess their mutagenic potency in the induction of X-linked recessive lethals and chromosome loss. NP was effective in inducing lethals, as were its halogen and methyl-substituted derivatives, with the exception of 2,6-dimethyl NP. (Methyl substitutions at the alpha carbon atoms reduce or eliminate mutagenic activity.) Substitution of halogen groups on the piperidine ring enhanced the mutagenic activity, with the 3-chloro compound being the most mutagenic. In contrast, substitutions with a hydroxyl, carboxyl, or keto group resulted in a loss of mutagenicity. None of the compounds tested increased the frequency of chromosome loss or breakage in mature sperm.     

Naturally Acquired Picornavirus Infections in Primates at the Dhaka Zoo

The conditions in densely populated Bangladesh favor picornavirus transmission, resulting in a high rate of infection in the human population. Data suggest that nonhuman primates (NHP) may play a role in the maintenance and transmission of diverse picornaviruses in Bangladesh. At the Dhaka Zoo, multiple NHP species are caged in close proximity. Their proximity to other species and to humans, both zoo workers and visitors, provides the potential for cross-species transmission. To investigate possible interspecies and intraspecies transmission of picornaviruses among NHP, we collected fecal specimens from nine NHP taxa at the Dhaka Zoo at three time points, August 2007, January 2008, and June 2008. Specimens were screened using real-time PCR for the genera Enterovirus, Parechovirus, and Sapelovirus, and positive samples were typed by VP1 sequencing. Fifty-two picornaviruses comprising 10 distinct serotypes were detected in 83 fecal samples. Four of these serotypes, simian virus 19 (SV19), baboon enterovirus (BaEV), enterovirus 112 (EV112), and EV115, have been solely associated with infection in NHP. EV112, EV115, and SV19 accounted for 88% of all picornaviruses detected. Over 80% of samples from cages housing rhesus macaques, olive baboons, or hamadryas baboons were positive for a picornavirus, while no picornaviruses were detected in samples from capped langurs or vervet monkeys. In contrast to our findings among synanthropic NHP in Bangladesh where 100% of the picornaviruses detected were of human serotypes, in the zoo population, only 15% of picornaviruses detected in NHP were of human origin. Specific serotypes tended to persist over time, suggesting either persistent infection of individuals or cycles of reinfection.     

Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE). Part I: Structure guided discovery and optimization of dual targeting agents with potent,broad-spectrum enzymatic activity

The bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) are essential enzymes that control the topological state of DNA during replication. The high degree of conservation in the ATP-binding pockets of these enzymes make them appealing targets for broad-spectrum inhibitor development. A pyrrolopyrimidine scaffold was identified from a pharmacophore-based fragment screen with optimization potential. Structural characterization of inhibitor complexes conducted using selected GyrB/ParE orthologs aided in the identification of important steric, dynamic and compositional differences in the ATP-binding pockets of the targets, enabling the design of highly potent pyrrolopyrimidine inhibitors with broad enzymatic spectrum and dual targeting activity.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

The structural basis for molecular recognition by the vitamin B 12 RNA aptamer

Previous solution structures of ligand-binding RNA aptamers have shown that molecular recognition is achieved by the folding of an initially unstructured RNA around its cognate ligand, coupling the processes of RNA folding and binding. The 3 A crystal structure of the cyanocobalamin (vitamin B12) aptamer reported here suggests a different approach to molecular recognition in which elements of RNA secondary structure combine to create a solvent-accessible docking surface for a large, complex ligand. Central to this structure is a locally folding RNA triplex, stabilized by a novel three-stranded zipper. Perpendicular stacking of a duplex on this triplex creates a cleft that functions as the vitamin B12 binding site. Complementary packing of hydrophobic surfaces, direct hydrogen bonding and dipolar interactions between the ligand and the RNA appear to contribute to binding. The nature of the interactions that stabilize complex formation and the possible uncoupling of folding and binding for this RNA suggest a strong mechanistic similarity to typical protein-ligand complexes.     

The 1.3 A crystal structure of a biotin-binding pseudoknot and the basis for RNA molecular recognition

A pseudoknot-containing aptamer isolated from a pool of random sequence molecules has been shown previously to represent an optimal RNA solution to the problem of binding biotin. The affinity of this RNA molecule is nonetheless orders of magnitude weaker than that of its highly evolved protein analogs, avidin and streptavidin. To understand the structural basis for biotin binding and to compare directly strategies for ligand recognition available to proteins and RNA molecules, we have determined the 1.3 A crystal structure of the aptamer complexed with its ligand. Biotin is bound at the interface between the pseudoknot's stacked helices in a pocket defined almost entirely by base-paired nucleotides. In comparison to the protein avidin, the aptamer packs more tightly around the biotin headgroup and makes fewer contacts with its fatty acid tail. Whereas biotin is deeply buried within the hydrophobic core in the avidin complex, the aptamer relies on a combination of hydrated magnesium ions and immobilized water molecules to surround its ligand. In addition to demonstrating fundamentally different approaches to molecular recognition by proteins and RNA, the structure provides general insight into the mechanisms by which RNA function is mediated by divalent metals.     

A retrospective analysis of factors contributing to calf mortality and dystocia in beef cattle

Records of 2191 calvings from the Clemson University Beef Physiology Unit between 1981 and 1993 were analyzed to determine factors affecting malpresentation, mortality and dystocia. Only 20 (0.91%) parturitions involved malpresentation: posterior presentation (n = 14), leg deviations (n = 3), head deviations (n = 2) and breech birth (n = 1). Dystocia affected calf mortality within 24 h of birth (P < 0.05), with mortality increasing as the severity of dystocia increased. There was an overall 4.5% death loss within 24 h of birth, with 4 and 7% mortality rates for calves from multiparous and primiparous dams, respectively (P < 0.05). Mortality was higher for bull vs heifer calves (P < 0.05). Ninety-four percent of calvings were unassisted, while 6% were assisted births. Dystocia was greater (P < 0.01) in primiparous (17%) than in multiparous dams (4%). In births involving dystocia, 28.1% required mild traction, 69.3% required heavy traction and 2.6% required Cesarean section. Birth weights associated with normal births and mild traction (36 and 36 kg) were lighter than those associated with heavy traction and Cesarean section (40 and 42 kg, respectively; P < 0.05). In conclusion, malpresentations were too few to be of significance, and dystocia influenced mortality within 24 h of birth. Calf birth weight and parity of dam explained most of the observed variations in dystocia.     

DETECTION AND ENUMERATION OF H2S+ BACTERIA: APPLICATION TO SHEWANELLA PUTREFACIENS (CIP)

H₂S⁺ bacteria responsible for the degradation of sulfur-containing amino acids of fish muscle are currently little used to evaluate the microbiological pal quality of fish. Shewanella putrefaciens greatly predominates in this flora, and was therefore used to define a suitable culture method and medium. Inoculations by the Spiral surface method at 25C, with an incubation of 72h, gave the best counts on a medium containing two sources of sulfur (organic and inorganic) for H₂S⁺ bacteria. The culture medium and the NaCl concentration were determinant in the evaluation of this flora. At present there is no standard medium which meets these requirements.