BRCA2 deficiency in mice leads to meiotic impairment and infertility

The role of Brca2 in gametogenesis has been obscure because of embryonic lethality of the knockout mice. We generated Brca2-null mice carrying a human BAC with the BRCA2 gene. This construct rescues embryonic lethality and the mice develop normally. However, there is poor expression of the transgene in the gonads and the mice are infertile, allowing examination of the function of BRCA2 in gametogenesis. BRCA2-deficient spermatocytes fail to progress beyond the early prophase I stage of meiosis. Observations on localization of recombination-related and spermatogenic-related proteins suggest that the spermatocytes undergo early steps of recombination (DNA double strand break formation), but fail to complete recombination or initiate spermiogenic development. In contrast to the early meiotic prophase arrest of spermatocytes, some mutant oocytes can progress through meiotic prophase I, albeit with a high frequency of nuclear abnormalities, and can be fertilized and produce embryos. Nonetheless, there is marked depletion of germ cells in adult females. These studies provide evidence for key roles of the BRCA2 protein in mammalian gametogenesis and meiotic success.     

Computational aspects of host-parasite phylogenies

Computational aspects of host-parasite phylogenies form part of a set of general associations between areas and organisms, hosts and parasites, and species and genes. The problem is not new and the commonalities of exploring vicariance biogeography (organisms tracking areas) and host-parasite co-speciation (parasites tracking hosts) have been recognised for some time. Methods for comparing host-parasite phylogenies are now well established and fall within two basic categories defined in terms of the way the data are interpreted in relation to the comparison of host-parasite phylogenies, so-called a posteriori, eg Brooks' Parsimony Analysis (BPA), or a priori, eg reconciled trees and other model-based methods, as implemented in the program TreeMap; the relative merits of the two philosophies inherent in these two approaches remain hotly debated. This paper reviews the computational methods currently available to analyse host-parasite relationships.     

Evaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction

Background

A detailed understanding of an RNA's correct secondary and tertiary structure is crucial to understanding its function and mechanism in the cell. Free energy minimization with energy parameters based on the nearest-neighbor model and comparative analysis are the primary methods for predicting an RNA's secondary structure from its sequence. Version 3.1 of Mfold has been available since 1999. This version contains an expanded sequence dependence of energy parameters and the ability to incorporate coaxial stacking into free energy calculations. We test Mfold 3.1 by performing the largest and most phylogenetically diverse comparison of rRNA and tRNA structures predicted by comparative analysis and Mfold, and we use the results of our tests on 16S and 23S rRNA sequences to assess the improvement between Mfold 2.3 and Mfold 3.1.

Results

The average prediction accuracy for a 16S or 23S rRNA sequence with Mfold 3.1 is 41%, while the prediction accuracies for the majority of 16S and 23S rRNA structures tested are between 20% and 60%, with some having less than 20% prediction accuracy. The average prediction accuracy was 71% for 5S rRNA and 69% for tRNA. The majority of the 5S rRNA and tRNA sequences have prediction accuracies greater than 60%. The prediction accuracy of 16S rRNA base-pairs decreases exponentially as the number of nucleotides intervening between the 5' and 3' halves of the base-pair increases.

Conclusion

Our analysis indicates that the current set of nearest-neighbor energy parameters in conjunction with the Mfold folding algorithm are unable to consistently and reliably predict an RNA's correct secondary structure. For 16S or 23S rRNA structure prediction, Mfold 3.1 offers little improvement over Mfold 2.3. However, the nearest-neighbor energy parameters do work well for shorter RNA sequences such as tRNA or 5S rRNA, or for larger rRNAs when the contact distance between the base-pairs is less than 100 nucleotides.     

Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency virus type 1 gp120

IgG1 b12 is a broadly neutralizing antibody against human immunodeficiency virus type 1 (HIV-1). The epitope recognized by b12 overlaps the CD4 receptor-binding site (CD4bs) on gp120 and has been a target for vaccine design. Determination of the three-dimensional structure of immunoglobulin G1 (IgG1) b12 allowed modeling of the b12-gp120 interaction in which the protruding third complementarity-determining region (CDR) of the heavy chain (H3) was crucial for antibody binding. In the present study, extensive mutational analysis of the antigen-binding site of Fab b12 was carried out to investigate the validity of the model and to identify residues important for gp120 recognition and, by inference, key to the anti-HIV-1 activity of IgG1 b12. In all, 50 mutations were tested: 40 in H3, 4 each in H2 and L1, and 2 in L3. The results suggest that the interaction of gp120 with H3 of b12 is crucially dependent not only on a Trp residue at the apex of the H3 loop but also on a number of residues at the base of the loop. The arrangement of these residues, including aromatic side chains and side chains that hydrogen bond across the base of the loop, may rigidify H3 for penetration of the recessed CD4-binding cavity. The results further emphasize the importance to gp120 binding of a Tyr residue at the apex of the H2 loop that forms a second finger-like structure and a number of Arg residues in L1 that form a positively charged, shelf-like structure. In general, the data are consistent with the b12-gp120 interaction model previously proposed. At the gene level, somatic mutation is seen to be crucial for the generation of many of the structural features described. The Fab b12 mutants were also tested against the b12 epitope-mimic peptide B2.1, and the reactivity profile had many similarities but also significant differences from that observed for gp120. The paratope map of b12 may facilitate the design of molecules that are able to elicit b12-like activities.     

Human coronavirus 229E: receptor binding domain and neutralization by soluble receptor at 37 degrees C

Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37 degrees C, but not 4 degrees C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37 degrees C that facilitate virus entry.     

Structure of the HERG K+ channel S5P extracellular linker: role of an amphipathic alpha-helix in C-type inactivation

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585-Ile593 and Gly604-Tyr611 of the channel. The Trp585-Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604-Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.     

Acute and chronic leptin treatment mediate contrasting effects on signaling,glucose uptake,and GLUT4 translocation in L6-GLUT4myc myotubes

We have previously shown that in L6-GLUT4myc rat skeletal muscle cells, acute treatment with leptin reduced insulin-stimulated glucose uptake without altering insulin-stimulated GLUT4 translocation. In contrast, we show here that the ability of leptin to increase phosphorylation of its receptor and to reduce insulin-stimulated glucose uptake was lost in cells that were continuously exposed to leptin for 24 h. This desensitization correlated with an increase in expression of suppressor of cytokine signaling-3 (SOCS-3). Time course analysis demonstrated that the transition from acute to chronic effects of leptin occurs after 2 h. The desensitization of leptin action at 24 h was not reversed by 30 min washout period prior to re-exposing cells to leptin. However, despite insulin-stimulated glucose uptake being unaffected upon 24 h preincubation with leptin, a small but significant decrease (37%) in insulin-stimulated GLUT4 translocation and phosphorylation of Akt on T308 was detected. Insulin-stimulated phosphorylation of Akt on S473 or of p38 MAPK were unaffected. These results suggest that the chronic leptin treatment leads to desensitization of leptin signaling yet can simultaneously decrease the ability of insulin to phosphorylate Akt on T308 and translocate GLUT4. However, this does not manifest as a reduction in total glucose uptake into L6 myotubes.     

Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease

Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms of DNA rearrangement in other disorders.     

Characterization of chicken TNFR superfamily decoy receptors,DcR3 and osteoprotegerin

Tumor necrosis factor (TNF) family ligands bind to death domain-containing TNF receptors (death receptors), which can subsequently activate intracellular signaling pathways to initiate caspase activity and apoptotic cell death. Decoy receptors, without intracellular death domains, have been reported to prevent cytotoxic effects by binding to and sequestering such ligands, or by interfering with death receptor trimerization. The chicken death receptors, Fas, TNFR1, DR6, and TVB, are constitutively expressed in a relatively wide variety of hen tissues. In this study, two chicken receptors with sequence homology to the mammalian decoys, DcR3 and osteoprotegerin, were identified and their pattern of expression was characterized. Unlike the previously identified chicken death receptors, the newly characterized decoy receptors show comparatively limited expression among tissues, suggesting a tissue-specific function. Finally, characterization of these chicken receptors further contributes to understanding the evolutionary divergence of TNFR superfamily members among vertebrate species.     

The metabolism of formyl-substituted benzanthracenes to hydroxymethyl metabolites in rat liver in vitro and in vivo

Hydroxylation of benzylic methyl carbon atoms on drugs and carcinogenic polycyclic aromatic hydrocarbons (PAHs) forms benzylic alcohols. Many carcinogenic and mutagenic PAHs bear a primary or secondary benzylic hydroxyl group attached to the meso-region of the molecule. According to the unified theory, PAHs bearing a benzylic hydroxyl group are proximate carcinogenic metabolites. This paper demonstrates that carcinogenic benz[a]anthracenes bearing a formyl group at the meso-region undergo enzymatic reductive metabolism to the corresponding carcinogenic benzylic alcohol in vitro and in vivo. The unified theory would then predict sulfuric acid esterification of such benzylic alcohols as the final common step in their metabolic activation to generate ultimate electrophilic benzylic carbocations. Finally, oxidative metabolism of 7-formylbenz[a]anthracenes gives rise to corresponding carboxylic acids and other oxygenated metabolites that are carcinogenically inert. Thus, oxidative metabolism of meso-region formyl compounds represents an avenue for the elimination of the carcinogen in a detoxified form.