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We show that receptor induced G protein [beta][gamma] subunit translocation from the plasma membrane to the Golgi allows a receptor to initiate fragmentation and regulate secretion. A lung epithelial cell line, A549, was shown to contain an endogenous translocating G protein y subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating [gamma] subunit. A kinase defective protein kinase D and a phospholipase C [beta] inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with [beta][gamma] translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating [gamma]3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic [beta] cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative [gamma]3 subunit consistent with the Golgi fragmentation induced by [beta][gamma] complex translocation playing a role in secretion. G protein-coupled receptors | live cell imaging | signaling | insulin doi/ 10.1073/pnas. 1003042107

The lipid peroxidation product 4-hydroxynonenal (4-HNE) forms as a consequence of oxidative stress. By electrophilic attack on biological macromolecules, 4-HNE mediates signaling or may cause toxicity. A major route of 4-HNE disposal is via glutathione conjugation, in the mouse catalyzed primarily by glutathione transferase mGSTA4-4. Unexpectedly, mGsta4-null mice, in which 4-HNE detoxification is impaired, have an extended life span. This finding could be explained by the observed activation of the transcription factor Nrf2 in the knockout mice, which in turn leads to an induction of antioxidant and antielectrophilic defenses. Especially, the latter could provide a detoxification mechanism that contributes to enhanced longevity. We propose that disruption of 4-HNE conjugation elicits a hormetic response in which an initially increased supply of 4-HNE is translated into activation of Nrf2, leading to a new steady state in which the rise of 4-HNE concentrations is dampened, but life-extending detoxification mechanisms are concomitantly induced. Key Words: Aging--Life span--Mouse--hydroxynonenal--Glutathione transferase. doi: 10.1093/gerona/glp165

Abstract Background The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP) is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP). The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. Results Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP) and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP. Conclusion The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations.

Studies about the age-dependent development of obesity which precedes the onset of insulin resistance due to disruption of mGsta4, a gene encoding the 4-HNE-conjugating enzyme mGSTA4-4 are reported. The results provide insight into two reciprocal processes, loss of mGSTA4-4 and an increase in the level of 4-HNE which promote and perpetuate the obese state.

Abstract: Apis mellifera intermissa is the native honey bee subspecies of Algeria, and approximates a position among bee races between tropical African and European breeds. This bee is very aggressive, nervous, and produces many broods with many queen cells. It is prone to swarming and exhibits defensive behavior and an abundant use of propolis. In the present study, pure line samples of A. m. intermissa collected from Blida (Algeria; 36°31′N, 2°58′E) with preferable Varroa resistance confirmed their through hygienic cleaning behavior and high temperature adaptation, were selected as a reference sample for full‐genome sequencing. Array comparative genomic hybridization (aCGH) was also performed on the same samples to validate genomic variations. These analyses will be an important source of information for the honey bee research community worldwide. The 240‐Mb genome was annotated with 26 355 transcripts and analyzed. Analysis of 133 pathways indicated an abundance of pathways related to metabolic processes, the biosynthesis of secondary metabolites, and biodegradation of xenobiotics . In addition to simple sequence repeat markers, variant analysis for beneficial trait genes, transposons, and phylogeny was performed. The mitochondrial genome and genes involved in immunity and chemoreception were identified and compared with those from other sequenced insect models. The indels obtained by sequencing were validated by aCGH. The genome sequence, annotation, and analysis of A. m. intermissa provides new understanding of the function of bee genes, and comparison with the genome of other A. mellifera subspecies promises to yield insights into the evolution of the adaptations to high temperature and resistance to Varroa parasite infestation. [ABSTRACT FROM AUTHOR]

Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next-generation sequencing (NGS) technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses characteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in-depth functional annotation methods to identify ∼600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK-STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function-specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development (Cactus, Relish, dorsal, Ank2, baz), Varroa hygiene (NorpA2, Zasp, LanA, gasp, impl3) and Varroa resistance (Pug, pcmt, elk, elf3-s10, Dscam2, Dhc64C, gro, futsch) functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry. [ABSTRACT FROM AUTHOR]

Sufficient knowledge on the root and root canal anatomy is essential for practicing root canal treatment. The mesiobuccal roots of maxillary molar teeth present an endodontic challenge due to their wide variability and complexity of their internal morphological landmarks. A review on the literature indicates that the prevalence of a third mesiobuccal root canal in the mesiobuccal root of maxillary molar teeth may reach 9%, and the root canal configuration usually is type XV (3-2). These reported data reveal the importance of absolute awareness for this anatomical aberration that requires special attention from dental practitioners while commencing root canal treatment in maxillary molar teeth. Hence, this article aims to report and describe the management of a maxillary first molar tooth with three mesiobuccal root canals, but with an unusual configuration. [ABSTRACT FROM AUTHOR]

Human antibodies against specific targets of tumor cells are the most desirable molecules for possible immunotherapy. They could be developed by using the combinatorial antibody library displayed on a phage. We selected four human antibody fragments (scFv) binding to the oncoplacental antigen Heat Stable Alkaline Phosphatase (HSAP, the placental isozyme of alkaline phosphatase) from a synthetic human antibody library. Characterization of these scFvs showed they bound HSAP with moderate affinity but did not have isozyme specificity, as determined by binding to cell lines exhibiting differential expression of isozymes of alkaline phosphatase. The VH sequences of two of these scFvs were similar and although both bound to HSAP only one was cross-reactive with albumin. The sequences revealed a difference in the framework region (FR1) of these antibodies, indicating a role for this region in the determination of specificity. This is also significant considering that the heavy chains generated the diversity of the synthetic library used in this study, and only a single light chain showing binding to BSA was used for the entire library. [ABSTRACT FROM AUTHOR]

Crown fractures are common detrimental consequences of dental traumatic injuries. Early management of such cases is mandatory in order to prevent subsequent pathological changes that could further complicate the treatment. Pulp necrosis, chronic and cystic apical periodontitis can be the fate if these teeth are left untreated. Despite these serious complications, root canal treatment followed by apical surgery is considered a valid treatment option when such cases become complicated with large periapical lesions. However, whether a retrograde filling is essential to be placed or not is still a matter of debate. This case report discusses the orthograde endodontic management, the surgical approach and the clinical outcomes of longstanding crown fractured teeth with large cyst-like periapical lesions with and without retrograde filling. [ABSTRACT FROM AUTHOR]

Aim: This study aims to evaluate the cytotoxicity of a new fast set highly viscous conventional glass ionomer cement (GIC) with L929 fibroblasts. Materials and Methods: The cement capsule was mixed and introduced into a paraffin wax mould. After setting, the cement was incubated in Dulbecco's Modified Eagle's Medium. Six replicates of the material extract were added to the culture medium in 96-well plates. L929 mouse fibroblast cells were added into the wells and then incubated for 48 h. Dimethylthiazol diphenyltetrazolium bromide test was performed for cytotoxicity evaluation. Results: The results showed that this GIC brand did not yield a half-maximal inhibitory concentration value, IC50, as the cell viability was above 50% at all concentrations. Cell viability over 90% was observed at the concentrations of 3.125 and 1.5625 mg/ml. Maximum concentration of the material showed cell viability of 59.4%. Conclusions: This new fast set highly viscous conventional GIC showed low cytotoxicity to mouse fibroblast cells, and it can be suggested as a substitute for dental cements exhibiting a long setting time. [ABSTRACT FROM AUTHOR]