Surface phosphomonoesterase activity of a natural immobilized system: Chroococcidiopsis in an Antarctic desert rock

The phosphomonoesterase activity of a microbialcommunity dominated by Chroococcidiopsis in arock sample from the Antarctic dry desert was comparedwith that of a non-axenic culture of thecyanobacterium isolated from the same rock in order toestablish whether the organism differed in itsproperties when immobilized in the rock. The pHoptimum (9.5) was the same, but the response totemperature and light both differed. In the case oftemperature, the optimum was much higher in culture. Light flux at the low value of 8 mol photonm^-2 s^-1 was slightly inhibitory for acrushed rock sample, but enhanced activity of thecultured material. Although materials both showeda typical (apparent) Michaelis-Menten relationship,the values for Km and V_max differed. Itis suggested that study of natural immobilized systemsis important for the development of procedures to useimmobilized phototrophs for practical purposes.     

Downregulation of calcineurin activity in cervical carcinoma

Background  

Calcineurin (CaN) is an important serine-threonine phosphatase (PP2B), which plays a crucial role in calcium-calmodulin mediated signal transduction events. Calcineurin has been implicated in pathogenesis of various diseases cardiac hypertrophy, diabetic neuropathy and Alzheimer's, however its role in neoplasia remains unclear.     

Analysis of alternative splicing with microarrays: successes and challenges

Recently, DNA microarrays have emerged as potentially powerful tools for analyzing alternative splicing. We briefly review the latest results in this field and highlight the current challenges that they have revealed.     

Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System

Francisella tularensis is a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensis SchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD₁₀₀) doses of F. tularensis LVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensis antigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensis proteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens.     

Morphological,cellular and molecular changes during postovulatory egg aging in mammals

Postovulatory aging is associated with several morphological, cellular and molecular changes that deteriorate egg quality either by inducing abortive spontaneous egg activation (SEA) or by egg apoptosis. The reduced egg quality results in poor fertilization rate, embryo quality and reproductive outcome. Although postovulatory aging-induced abortive SEA has been reported in several mammalian species, the molecular mechanism(s) underlying this process remains to be elucidated. The postovulatory aging-induced morphological and cellular changes are characterized by partial cortical granules exocytosis, zona pellucida hardening, exit from metaphase-II (M-II)arrest and initiation of extrusion of second polar body in aged eggs. The molecular changes include reduction of adenosine 3'',5''- cyclic monophosphate (cAMP) level, increase of reactive oxygen species (ROS) and thereby cytosolic free calcium (Ca²⁺) level. Increased levels of cAMP and/or ROS trigger accumulation of Thr-14/Tyr-15 phosphorylated cyclin-dependent kinase 1 (Cdk1) on one hand and degradation of cyclin B1 through ubiquitin-mediated proteolysis on the other hand to destabilize maturation promoting factor (MPF). The destabilized MPF triggers postovulatory aging-induced abortive SEA and limits various assisted reproductive technologies (ARTs) outcome in several mammalian species. Use of certain drugs that can either increase cAMP or reduce ROS level would prevent postovulatory aging-induced deterioration in egg quality so that more number of good quality eggs can be made available to improve ART outcome in mammals including human.     

A nonapoptotic role for CASP2/caspase 2: Modulation of autophagy

CASP2/caspase 2 plays a role in aging, neurodegeneration, and cancer. The contributions of CASP2 have been attributed to its regulatory role in apoptotic and nonapoptotic processes including the cell cycle, DNA repair, lipid biosynthesis, and regulation of oxidant levels in the cells. Previously, our lab demonstrated CASP2-mediated modulation of autophagy during oxidative stress. Here we report the novel finding that CASP2 is an endogenous repressor of autophagy. Knockout or knockdown of CASP2 resulted in upregulation of autophagy in a variety of cell types and tissues. Reinsertion of Caspase-2 gene (Casp2) in mouse embryonic fibroblast (MEFs) lacking Casp2 (casp2^−/−) suppresses autophagy, suggesting its role as a negative regulator of autophagy. Loss of CASP2-mediated autophagy involved AMP-activated protein kinase, mechanistic target of rapamycin, mitogen-activated protein kinase, and autophagy-related proteins, indicating the involvement of the canonical pathway of autophagy. The present study also demonstrates an important role for loss of CASP2-induced enhanced reactive oxygen species production as an upstream event in autophagy induction. Additionally, in response to a variety of stressors that induce CASP2-mediated apoptosis, casp2^−/− cells demonstrate a further upregulation of autophagy compared with wild-type MEFs, and upregulated autophagy provides a survival advantage. In conclusion, we document a novel role for CASP2 as a negative regulator of autophagy, which may provide important insight into the role of CASP2 in various processes including aging, neurodegeneration, and cancer.     

Purification and Characterization of Antioxidant Peptides from Oyster (Saccostrea cucullata) Hydrolysate and the Anticancer Activity of Hydrolysate on Human Colon Cancer Cell Lines

The focus of the study was to investigate antioxidant activity and characterize antioxidant peptides from oyster (Saccostrea cucullata) protein hydrolysate. The protease hydrolysate of oyster exhibited strong potential to donate hydrogen and was able to scavenge Hydrogen peroxide, Hydroxyl and DPPH radicals. Due to the high antioxidant potential, hydrolysate was purified in Sephadex G-25 gel filtration chromatography. The active peptide fraction was further purified by UPLC-MS. Totally seven antioxidant peptides were collected. Among seven peptides (SCAP 1–7), three peptides (SCAP 1, 3 and 7) had highest scavenging ability on DPPH radicals. The amino acid sequence and molecular mass of purified antioxidant peptides (SCAP1, SCAP3 and SCAP7) were determined by Q-TOF ESI mass spectroscopy and structures of the peptides were Leu-Ala-Asn-Ala-Lys (MW = 515.29 Da), Pro-Ser-Leu-Val-Gly-Arg-Pro–Pro-Val-Gly-Lys-Leu-Thr-Leu (MW = 1,432.89 Da) and Val-Lys-Val-Leu-Leu-Glu-His-Pro-Val-Leu (MW = 1,145.75 Da), respectively. The oyster hydrolysate was tested for cell cytotoxicity on Vero (kidney epithelial cells of the African Green Monkey) and HT-29 (human colon carcinoma) cell lines. It was found that the hydrolysate did not show any cytotoxic effect for Vero cell lines and exerted a significant cytotoxic effect on HT-29 cell lines. We thus conclude that the anticancer and antioxidative hydrolysate from oyster (S. cucullata) may be useful ingredients in food and nutraceutical applications.     

G Protein Beta 5 Is Targeted to D2-Dopamine Receptor-Containing Biochemical Compartments and Blocks Dopamine-Dependent Receptor Internalization

G beta 5 (Gbeta5, Gβ5) is a unique G protein β subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gβ5, but not that of the G beta 1 (Gβ1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gβ5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gβ5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gβ5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gβ5, but not Gβ1. However, the same Gβ5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of β-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gβ5 are not mediated by endogenously expressed R7 RGS proteins.