The Calsequestrin Mutation CASQ2D307H Does Not Affect Protein Stability and Targeting to the Junctional Sarcoplasmic Reticulum but Compromises Its Dynamic Regulation of Calcium Buffering

Mutations in cardiac ryanodine receptor (RYR2) and cardiac calsequestrin (CASQ2) genes are linked to catecholaminergic polymorphic ventricular tachycardia, a life-threatening genetic disease. They predispose young individuals to cardiac arrhythmia in the absence of structural abnormalities. One such mutation that changes an aspartic residue to histidine at position 307 in CASQ2 has been linked to catecholaminergic polymorphic ventricular tachycardia. In this study we made a transgenic mouse model expressing the mutant CASQ2^D307H protein in a CASQ2 null background and investigated if the disease is caused by accelerated degradation of the mutant protein. Our data suggest that the mutant protein can be expressed, is relatively stable, and targets appropriately to the junctional sarcoplasmic reticulum. Moreover, it partially normalizes the ultrastructure of the sarcoplasmic reticulum, which was altered in the CASQ2 null background. In addition, overexpression of the mutant protein does not cause any pathology and/or structural changes in the myocardium. We further demonstrate, using purified protein, that the mutant protein is very stable under chemical and thermal denaturation but shows abnormal Ca²⁺ buffering characteristics at high calcium concentrations. In addition, trypsin digestion studies reveal that the mutant protein is more susceptible to protease activity only in the presence of high Ca²⁺. These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca²⁺ activation.     

Electronic Characterization of Au/DNA/ITO Metal-Semiconductor-Metal Diode and Its Application as a Radiation Sensor

Deoxyribonucleic acid or DNA molecules expressed as double-stranded (DSS) negatively charged polymer plays a significant role in electronic states of metal/silicon semiconductor structures. Electrical parameters of an Au/DNA/ITO device prepared using self-assembly method was studied by using current–voltage (I-V) characteristic measurements under alpha bombardment at room temperature. The results were analyzed using conventional thermionic emission model, Cheung and Cheung’s method and Norde’s technique to estimate the barrier height, ideality factor, series resistance and Richardson constant of the Au/DNA/ITO structure. Besides demonstrating a strongly rectifying (diode) characteristic, it was also observed that orderly fluctuations occur in various electrical parameters of the Schottky structure. Increasing alpha radiation effectively influences the series resistance, while the barrier height, ideality factor and interface state density parameters respond linearly. Barrier height determined from I–V measurements were calculated at 0.7284 eV for non-radiated, increasing to about 0.7883 eV in 0.036 Gy showing an increase for all doses. We also demonstrate the hypersensitivity phenomena effect by studying the relationship between the series resistance for the three methods, the ideality factor and low-dose radiation. Based on the results, sensitive alpha particle detectors can be realized using Au/DNA/ITO Schottky junction sensor.     

Identification of a novel antigen cross‐presenting cell type in spleen

Antigen-presenting cells (APC), like dendritic cells (DC), are essential for T-cell activation, leading to immunity or tolerance. Multiple DC subsets each play a unique role in the immune response. Here, a novel splenic dendritic-like APC has been characterized in mice that has immune function and cell surface phenotype distinct from other, described DC subsets. These were identified as a cell type continuously produced in spleen long-term cultures (LTC) and have an in vivo equivalent cell type in mice, namely ‘L-DC’. This study characterizes LTC-DC in terms of marker phenotype and function, and compares them with L-DC and other known splenic DC and myeloid subsets. L-DC display a myeloid dendritic-like phenotype equivalent to LTC-DC as CD11c^loCD11b^hiMHC-II⁻CD8α⁻ cells, distinct by high accessibility and endocytic capacity for blood-borne antigen. Both LTC-DC and L-DC have strong antigen cross-presentation ability leading to strong activation of CD8⁺ T cells, particularly after exposure to lipopolysaccharide. However, they have weak ability to stimulate CD4⁺ T cells in antigen-specific responses. Evidence is presented here for a novel DC type produced by in vitro haematopoiesis which has distinct antigen-presenting potential and reflects a DC subset present also in vivo in spleen.     

Effect of loading and vitrification solutions on survival of cryopreserved oil palm polyembryoids

The present study evaluates the effect of six loading solutions and five vitrification solutions (VS) and their time of exposure on the survival of oil palm (Elaeis guineensis) polyembryoids in liquid nitrogen (LN). In vitro grown polyembryoids of oil palm were successfully cryopreserved by vitrification with 45% survival. Individual polyembryoids, isolated from 2-month old culture, were precultured in liquid Murashige and Skoog medium supplemented with 0.5 M sucrose for 12 h and treated with a mixture of 10% (w/v) dimethyl sulphoxide (DMSO) plus 0.7 M sucrose for 30 min. Polyembryoids were then subjected to plant vitrification solution-2 (PVS2) (30% (w/v) glycerol plus 15% (w/v) EG plus 15% (w/v) DMSO plus 0.4 M sucrose) exposure for 5 min at 26 ± 2°C and subsequently plunged into LN. Thawed polyembryoids resumed growth within 8 days of culture and shoot development was recorded at 25 days of growth. Scanning electron micrograph revealed that successful regeneration of cryopreserved polyembryoids was due to stabilization of cellular integrity through optimum VS exposure.     

NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis

We previously found that human NK cells lyse Mycobacterium tuberculosis-infected monocytes and alveolar macrophages and upregulate CD8(+) T cell responses. We also found that human NK cells produce IL-22, which inhibits intracellular growth of M. tuberculosis, and that NK cells lyse M. tuberculosis-expanded CD4(+)CD25(+)FOXP3(+) T regulatory cells (Tregs). To determine the role of NK cells during the protective immune response to vaccination in vivo, we studied the NK cell and T cell responses in a mouse model of vaccination with bacillus Calmette-Guérin (BCG), followed by challenge with virulent M. tuberculosis H37Rv. BCG vaccination enhanced the number of IFN-γ-producing and IL-22-producing NK cells. Depletion of NK1.1(+) cells at the time of BCG vaccination increased the number of immunosuppressive Tregs (CD4(+)CD25(hi), 95% Foxp3(+)) after challenge with M. tuberculosis H37Rv, and NK1.1(+) cells lysed expanded but not natural Tregs in BCG-vaccinated mice. Depletion of NK1.1(+) cells at the time of BCG vaccination also increased the bacillary burden and reduced T cell responses after challenge with M. tuberculosis H37Rv. IL-22 at the time of vaccination reversed these effects and enhanced Ag-specific CD4(+) cell responses in BCG-vaccinated mice after challenge with M. tuberculosis H37Rv. Our study provides evidence that NK1.1(+) cells and IL-22 contribute to the efficacy of vaccination against microbial challenge.     

Effect of T. chebula on mitochondrial alterations in experimental myocardial injury

Mitochondria play a central role in molecular events leading to tissue damage in ischemia. The present study examines the role of the alcoholic extract of T. chebula (TCE) pretreatment (50 mg/100 g body weight) to attenuate the isoproterenol (ISO) (20mg/100g body wt, sc) induced alterations on heart mitochondrial ultrastucture and function in experimental rats. ISO induced cardiotoxicity was evidenced by a significant rise in the level of lactate, decrease in enzyme activities of tricarboxylic acid cycle (TCA), mitochondrial respiration, levels of adenosine triphosphate (ATP) and oxidative phosphorylation. TCE intervention significantly attenuated the above alterations by ISO and retained near normal function of the mitochondria. Electron microscopic studies of the mitochondria further support the isoproterenol induced deleterious changes and accredit the protective effect of TCE on mitochondrial structure and energy metabolism.     

Fusobacterium nucleatum ATCC 10953 Requires Actinomyces naeslundii ATCC 43146 for Growth on Saliva in a Three-Species Community That Includes Streptococcus oralis 34

Formation of dental plaque is a developmental process involving initial and late colonizing species that form polymicrobial communities. Fusobacteria are the most numerous gram-negative bacteria in dental plaque, but they become prevalent after the initial commensal colonizers, such as streptococci and actinomyces, have established communities. The unusual ability of these bacteria to coaggregate with commensals, as well as pathogenic late colonizers, has been proposed to facilitate colonization by the latter organisms. We investigated the integration of Fusobacterium nucleatum into multispecies communities by employing two in vitro models with saliva as the sole nutritional source. In flow cell biofilms, numbers of cells were quantified using fluorescently conjugated antibodies against each species, and static biofilms were analyzed by quantitative real-time PCR (q-PCR) using species-specific primers. Unable to grow as single-species biofilms, F. nucleatum grew in two-species biofilms with Actinomyces naeslundii but not with Streptococcus oralis. However, enhanced growth of fusobacteria was observed in three-species biofilms, indicating that there was multispecies cooperation. Importantly, these community dynamics yielded an 18-fold increase in the F. nucleatum biomass between 4 h and 18 h in the flow cell inoculated with three species. q-PCR analysis of static biofilms revealed that maximum growth of the three species occurred at 24 h to 36 h. Lower numbers of cells were observed at 48 h, suggesting that saliva could not support higher cell densities as the sole nutrient. Integration of F. nucleatum into multispecies commensal communities was evident from the interdigitation of fusobacteria in coaggregates with A. naeslundii and S. oralis and from the improved growth of fusobacteria, which was dependent on the presence of A. naeslundii.The human mouth contains microbiologically diverse communities. While collectively humans harbor more than 700 bacterial phylotypes, each individual is estimated to have fewer than 100 such phylotypes (1), and approximately 50% of human oral bacteria have yet to be cultivated. Although biofilm communities on tooth enamel are polymicrobial (3, 20), more than 60 to 90% of the bacteria found in initial plaque on saliva-coated tooth enamel are streptococci (6, 19). Other bacterial genera that are among the initial commensal colonizers include Actinomyces, Veillonella, and Neisseria (6, 16, 19), and these organisms contribute to the polymicrobial nature of initial plaque.The structure of a community is dependent upon the nature of the foundation. An integral feature of an oral bacterial biofilm foundation is the ability to coaggregate, which is defined as cell-cell recognition and binding between genetically distinct bacteria. After routine oral hygiene treatment, freshly cleaned tooth enamel is quickly coated with a salivary pellicle, which provides a set of receptor molecules recognized by primary colonizing bacteria, such as streptococci and actinomyces. Besides recognizing salivary receptors, these bacteria coaggregate and provide a foundation for the subsequent attachment and growth of other bacteria, such as veillonellae, that form close metabolic relationships with streptococci (12, 15). As initial colonizers develop into biofilm communities with anaerobic microenvironments, incorporation of the obligate anaerobic fusobacteria into these communities becomes possible. Fusobacteria as a group coaggregate with all other oral bacteria and have been suggested, therefore, to be a crucial link between primary colonizing species and later colonizing pathogens (13, 14). Thus, a foundation consisting of coaggregating streptococci, actinomyces, and veillonellae populates the tooth surface, and these organisms are recognized by fusobacteria, which colonize and become the dominant gram-negative bacterial species. The new foundation is a substratum containing fusobacterial surface receptors available for recognition by late colonizing pathogens. Supporting the crucial link is clinical evidence that fusobacteria appear in dental plaque after commensal species and before the pathogenic “red” complex consisting of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia (22, 23).Coaggregation partnerships are highly specific. A significant role for coaggregation in the formation of dental plaque biofilms and particularly in accretion of secondary colonizers to the pioneer species in plaque has been proposed (14) and has been demonstrated for the development of a spatially organized community (20). However, coaggregation may also provide some metabolic advantages (e.g., cross feeding and enzyme complementation) to neighboring cells by facilitating physical juxtaposition of partner cells, as has been shown for glucose metabolism of coaggregates of actinomyces and streptococci (7, 8). One aim of the present study was to examine the structures of two- and three-species communities composed of Actinomyces naeslundii, Streptococcus oralis, and Fusobacterium nucleatum in model biofilm systems. The first two species are initial colonizers and are considered commensals, whereas fusobacteria are secondary colonizers and are postulated to be a coaggregation bridge between initial and late colonizers (14). Our second aim was to investigate the integration and growth of fusobacteria in polymicrobial communities.A variety of experimental methods have been developed to study the formation of biofilms. Model systems often rely on the flow of nutrients over a surface on which bacteria are able to attach and grow. In the present study we used two distinct in vitro models, a saliva-fed flow cell and a polystyrene peg immersed in static saliva. Biofilm communities form naturally and are undisturbed (3, 20, 21). The spatial organization of a multispecies community resulting from colonization and growth is preserved and can be examined noninvasively by confocal laser scanning microscopy (CLSM). In the static system, the amount of each species in multispecies biofilms formed on polystyrene pegs can be measured by real-time quantitative PCR (q-PCR). We show here with both models that fusobacteria are unable to grow as single species, but they integrate into commensal streptococcus-actinomyces communities and grow. Integration and growth are required for fusobacteria to become crucial links between commensal communities and later colonizing pathogenic communities. In the three-species community studied here, A. naeslundii is required for F. nucleatum to integrate and grow.     

Identification and characterization of a novel enzyme related to the synthesis of PUFAs derived from <Emphasis Type="Italic">Thraustochytrium aureum</Emphasis> ATCC 34304

In Thraustochytrids, Thraustochytrium aureum ATCC 34304 was able to produce high levels of several polyunsaturated fatty acids. In the present study, a novel gene encoding protein was cloned from the DHA rich microbe, T. aureum ATCC 34304. The functional analysis of a novel gene was demonstrated by its heterologous expression in Pichia pastoris. The gene was able to synthesize C20 and C22 PUFAs, as well as, to mediate different elongations (Δ9, Δ6, and Δ5) and one Δ5 desaturation activities. The conversion rates of the Δ9 elongation (n-3) and Δ5 desaturation products were found to be higher in response to the novel enzyme than the controls (TaElo and Tad5, respectively). The other Δ9 elongation (n-6) and Δ5 elongation products were slightly lower than those of the control (TaElo). The full length of the 1,374 bp gene contained 458 amino acids that showed very limited homology with desaturases and elongases from various organisms. In addition, the rate of synthesis of PUFAs was evaluated at temperatures ranging from 10 to 30°C. The elongation products were found to decrease dramatically and the desaturation products were found to increase dramatically at 10°C. TaNE was confirmed to be a multifunctional enzyme with higher activity towards Δ6 elongations than Δ9, Δ5 elongations, and Δ5 desaturation.