Analysis on sliding helices and strands in protein structural comparisons: a case study with protein kinases

Protein structural alignments are generally considered as 'golden standard' for the alignment at the level of amino acid residues. In this study we have compared the quality of pairwise and multiple structural alignments of about 5900 homologous proteins from 718 families of known 3-D structures. We observe shifts in the alignment of regular secondary structural elements (helices and strands) between pairwise and multiple structural alignments. The differences between pairwise and multiple structural alignments within helical and beta-strand regions often correspond to 4 and 2 residue positions respectively. Such shifts correspond approximately to "one turn" of these regular secondary structures. We have performed manual analysis explicitly on the family of protein kinases. We note shifts of one or two turns in helix-helix alignments obtained using pairwise and multiple structural alignments. Investigations on the quality of the equivalent helix-helix, strand-strand pairs in terms of their residue side-chain accessibilities have been made. Our results indicate that the quality of the pairwise alignments is comparable to that of the multiple structural alignments and, in fact, is often better. We propose that pairwise alignment of protein structures should also be used in formulation of methods for structure prediction and evolutionary analysis.     

The effects of prenatal PCBs on adult female paced mating reproductive behaviors in rats

Polychlorinated biphenyls (PCBs) are a family of toxicants that persist in measurable quantities in human and wildlife tissues, despite their ban in production in 1977. Some PCB mixtures can act as endocrine disrupting chemicals (EDCs) by mimicking or antagonizing the actions of hormones in the brain and periphery. When exposure to hormonally active substances such as PCBs occurs during vulnerable developmental periods, particularly prenatally or in early postnatal life, they can disrupt sex-specific patterning of the brain, inducing permanent changes that can later be manifested as improper sexual behaviors. Here, we investigated the effects of prenatal exposure to the PCB mixture Aroclor (A) 1221 on adult female reproductive behaviors in a dose-response model in the Sprague-Dawley rat. Using a paced mating paradigm that permits the female to set the timing of mating and control contact with the male during copulation, we were able to uncover significant differences in female-typical sexual activities in A1221-exposed females. Specifically, A1221 causes significant effects on mating trial pacing, vocalizations, ambulation and the female's likelihood to mate. The results further demonstrate that the intermediate treatment group has the greatest number of disrupted endpoints, suggestive of non-linear dose responses to A1221. These data demonstrate that the behavioral phenotype in adulthood is disrupted by low, ecologically relevant exposures to PCBs, and the results have implications for reproductive success and health in wildlife and women.     

Structure-activity relationship (SAR) investigations of substituted imidazole analogs as TRPV1 antagonists

A novel series of 4,5-biarylimidazoles as TRPV1 antagonists were designed based on the previously reported 4,6-disubstituted benzimidazole series. The analogs were evaluated for their ability to block capsaicin- or acid-induced calcium influx in TRPV1-expressing CHO cells. These studies led to the identification of a highly potent and orally bioavailable TRPV1 antagonist, imidazole 33.     

Recombinant production of a VL single domain antibody in Escherichia coli and analysis of its interaction with peptostreptococcal protein L

A kappa-light chain from a Fab expression system was truncated by the insertion of a stop codon in the gene sequence to produce a variable light (VL) single domain antibody (dAb). Here, we describe the expression of dAb in the periplasm of Escherichia coli through fermentation in a defined media. Immunoglobulin binding domains from peptostreptococcal protein L (PpL) have been shown to bind specifically to kappa-light chains. We have produced recombinant PpL, at high yield, and this was used to custom-produce PpL-Sepharose affinity columns. Here, we show that the affinity purification of VL dAb by this method is simple and efficient with no apparent loss in protein at any stage. The truncated dAb protein product was confirmed by electrospray mass spectrometry and N-terminal sequencing. When analyzed by SDS-PAGE it was shown to be over 95% pure and produced at yields of 35-65 mg/L of culture medium. The dAb protein produced was shown by NMR and CD to be a folded beta-sheet domain. This domain is bound by PpL with a Kd of approximately 50 nM as determined by stopped-flow fluorimetry.     

Chimeric T helper-B cell peptides induce protective response against Japanese encephalitis virus in mice

Virus neutralizing MAb binding and T helper cell stimulating peptide epitopes from structural and non-structural proteins of Japanese encephalitis virus were delineated. It was observed that priming by T helper peptides potentiated neutralizing antibody response against JE virus. Immunization with chimeric T helper - B cell peptides could thus protect mice from lethal challenge with JE virus.     

Development of a bladder instillation of the indoloquinone anticancer agent EO-9 using tert-butyl alcohol as lyophilization vehicle

The purpose of this research was to develop a stable bladder instillation of EO-9 for the treatment of superficial bladder cancer. First, stability and dissolution studies were performed. Subsequently, the freeze-drying process was optimized by determination of the freeze-drying characteristics of the selected cosolvent/water system and differential scanning calorimetry analysis of the formulation solution. Furthermore, the influence of the freeze-drying process on crystallinity and morphology of the freeze-dried product was determined with x-ray diffraction analysis and scanning electron microscopy, respectively. Subsequently, a reconstitution solution was developed. This study revealed that tert-butyl alcohol (TBA) can be used to both dramatically improve the solubility and stability of EO-9 and to shorten the freeze-drying cycle by increasing the sublimation rate. During freeze drying, 3 TBA crystals were found: TBA hydrate-ice crystals, crystals of TBA hydrate, and a third crystal, probably composed of TBA hydrate crystals containing ≈90% to 95% TBA. Furthermore, it was shown that crystallization of TBA hydrate was inhibited in the presence of both sodium bicarbonate (NaHCO₃) and mannitol. Addition of an annealing step resulted in a minor increase in the crystallinity of the freeze-dried product and formation of the δ-polymorph of mannitol. A stable bladder instillation was obtained after reconstitution of the freeze-dried product (containing 8 mg of EO-9, 20 mg of NaHCO₃, and 50 mg of mannitol per vial) to 20 mL with a reconstitution solution composed of propylene glycol/water for injection (WfI)/NaHCO₃/sodium edetate 60%/40%/2%/0.02% vol/vol/wt/wt, followed by dilution with Wfl to a final volume of 40 mL. Published: August 3, 2007     

Neonatal mortality levels for 193 countries in 2009 with trends since 1990: a systematic analysis of progress, projections, and priorities

Background

Historically, the main focus of studies of childhood mortality has been the infant and under-five mortality rates. Neonatal mortality (deaths <28 days of age) has received limited attention, although such deaths account for about 41% of all child deaths. To better assess progress, we developed annual estimates for neonatal mortality rates (NMRs) and neonatal deaths for 193 countries for the period 1990–2009 with forecasts into the future.

Methods and Findings

We compiled a database of mortality in neonates and children (<5 years) comprising 3,551 country-years of information. Reliable civil registration data from 1990 to 2009 were available for 38 countries. A statistical model was developed to estimate NMRs for the remaining 155 countries, 17 of which had no national data. Country consultation was undertaken to identify data inputs and review estimates. In 2009, an estimated 3.3 million babies died in the first month of life—compared with 4.6 million neonatal deaths in 1990—and more than half of all neonatal deaths occurred in five countries of the world (44% of global livebirths): India 27.8% (19.6% of global livebirths), Nigeria 7.2% (4.5%), Pakistan 6.9% (4.0%), China 6.4% (13.4%), and Democratic Republic of the Congo 4.6% (2.1%). Between 1990 and 2009, the global NMR declined by 28% from 33.2 deaths per 1,000 livebirths to 23.9. The proportion of child deaths that are in the neonatal period increased in all regions of the world, and globally is now 41%. While NMRs were halved in some regions of the world, Africa''s NMR only dropped 17.6% (43.6 to 35.9).

Conclusions

Neonatal mortality has declined in all world regions. Progress has been slowest in the regions with high NMRs. Global health programs need to address neonatal deaths more effectively if Millennium Development Goal 4 (two-thirds reduction in child mortality) is to be achieved. Please see later in the article for the Editors'' Summary     

Expression of estrogen receptor {alpha} in the anteroventral periventricular nucleus of hypogonadal mice

Gonadotropin-releasing hormone-1 (GnRH-1) neurons play critical roles in the development and maintenance of reproductive function in all vertebrates. Due to a truncation in the GnRH-1 gene, hypogonadal (hpg) mice are unable to synthesize GnRH-1 and are infertile. These animals develop in the complete absence of exposure to gonadal steroid hormones, making them an interesting model for understanding brain sexual differentiation and dimorphism. We studied expression of the estrogen receptors (ERs) alpha and beta in the medial anteroventral periventricular nucleus (mAVPV), an important reproductive neuroendocrine brain region, in wild-type and hpg mice of both sexes. Adult wild-type and hpg mice of the same genetic background were used to quantify numbers of ERalpha and ERbeta immunoreactive cells in the mAVPV using a stereologic approach. Quantitative analyses showed that ERalpha cell numbers were significantly higher in hpg than wild-type mice, irrespective of sex. Qualitatively, ERalpha-immunoreactive cells were concentrated more densely along the ventricular zone of the AVPV of wild-type female mice compared with wild-type male mice or hpg male and female mice. No ERbeta-immunoreactive cells were detected in the mAVPV of any mice, a result that was surprising because ERbeta expression is abundant in the mAVPV of rats. These results on ERalpha provide additional evidence that the female brain is not the "default" organizational pattern, because neither ERalpha cell number nor its distribution in hpg mice, which develops with a deficiency of reproductive hormones, resembles that of the wild-type female mouse. Differences in ERalpha expression may be due in part to the absence of gonadal steroid hormones, although they more likely to also involve other factors, potentially GnRH itself.     

Recombination within a nucleotide-binding-site/leucine-rich-repeat gene cluster produces new variants conditioning resistance to soybean mosaic virus in soybeans

The soybean Rsv1 gene for resistance to soybean mosaic virus (SMV; Potyvirus) has previously been described as a single-locus multi-allelic gene mapping to molecular linkage group (MLG) F. Various Rsv1 alleles condition different responses to the seven (G1-G7) described strains of SMV, including extreme resistance, localized and systemic necrosis, and mosaic symptoms. We describe the cloning of a cluster of NBS-LRR resistance gene candidates from MLG F of the virus-resistant soybean line PI96983 and demonstrate that multiple genes within this cluster interact to condition unique responses to SMV strains. In addition to cloning 3gG2, a strong candidate for the major Rsv1 resistance gene from PI96983, we describe various unique resistant and necrotic reactions coincident with the presence or absence of other members of this gene cluster. Responses of recombinant lines from a high-resolution mapping population of PI96983 (resistant) x Lee 68 (susceptible) demonstrate that more than one gene in this region of the PI96983 chromosome conditions resistance and/or necrosis to SMV. In addition, the soybean cultivars Marshall and Ogden, which carry other previously described Rsv1 alleles, are shown to possess the 3gG2 gene in a NBS-LRR gene cluster background distinct from PI96983. These observations suggest that two or more related non-TIR-NBS-LRR gene products are likely involved in the allelic response of several Rsv1-containing lines to SMV.