Carbonic Anhydrase in Calcified Endoskeleton: Novel Activity in Biocalcification in Alcyonarian

Carbonic anhydrase (CA) is a key enzyme in the chemical reaction of living organisms and has been found to be associated with calcification in a number of invertebrates including calcareous sponges, but until now no direct evidence has been advanced to show CA activity in alcyonarian corals. However, it is essential to understand the role of CA in the process of biocalcification in alcyonarian. Here we describe the novel activity of CA and its relationship to the formation of calcified hard tissues in alcyonarian coral, Lobophytum crassum. We find that two CA proteins, which were partially purified by electro-elution treatment, can control the morphology of CaCO₃ crystals and one of them is potentially involved in the process of biocalcification. Previously, we isolated CA from the total extract of alcyonarian, and further, we report here a single protein, which has both calcium-binding and CA activities and is responsible for CaCO₃ nucleation and crystal growth. This matrix protein inhibited the precipitation of CaCO₃ from a saturated solution containing CaCl₂ and NaHCO₃, indicating that it can act as a negative regulator for calcification in the sclerites of alcyonarians. The effect of an inhibitor on the enzyme activity was also examined. These findings strongly support the idea that carbonic anhydrase domain in alcyonarian is involved in the calcification process. Our observations strongly suggest that the matrix protein in alcyonarian coral is not only a structural protein but also a catalyst.     

Timekeeping through social contacts: social synchronization of circadian locomotor activity rhythm in the carpenter ant Camponotus paria

In ant colonies a large proportion of individuals remain inside nests for most of their lives and come out only when necessary. It is not clear how, in a nest of several thousand individuals, information about local time is communicated among members of the colony. Central to this seem to be circadian clocks, which have an intrinsic ability to keep track of local time by entraining to environmental light-dark, temperature, and social cycles. Here, the authors report the results of their study aimed at understanding the role of cyclic social interactions in circadian timekeeping of a day-active species of carpenter ant Camponotus paria. The authors found that daily social interactions with visitors (worker ants) was able to synchronize the circadian locomotor activity rhythm of host worker ants and queens, in one-on-one (pair-wise) and multi-individual (group-wise) interactions. Interestingly, the outcome of cyclic social interactions was context specific; when visitor workers socially interacted with host workers one-on-one, host workers considered the time of interaction as subjective day, but when visitor workers interacted with a group of workers and queens, the hosts considered the time of interaction as subjective night. These results can be taken to suggest that members of the ant species C. paria keep track of local time by socially interacting with workers (foragers) who shuttle in and out of the colony in search of food. (Author correspondence: vsharma@jncasr.ac.in ).     

Steroid receptor coactivator-3 is required for progesterone receptor trans-activation of target genes in response to gonadotropin-releasing hormone treatment of pituitary cells

Regulation of gonadotropin production involves interplay between steroids and neuropeptides, and we have examined the effects of gonadotropin-releasing hormones (GnRH I and GnRH II) on progesterone receptor (PR) activation in alphaT3-1 pituitary cells. Treatment with GnRHs activated a progester-one response element (PRE)-luciferase reporter gene, and this was blocked by protein kinase C and protein kinase A inhibitors but not by RU486. Treatment with GnRHs phosphorylated the PR at Ser(294) and increased PR translocation to the nucleus within 1 h. Interactions between the PR and several coactivators were examined, and treatment with GnRHs specifically induced PR-steroid receptor coactivator-3 (SRC-3) interactions within 8 h. In chromatin immunoprecipitation assays, recruitment of PR and SRC-3 by the PREs of the luciferase reporter gene or the gonadotopin alpha-subunit gene promoter was also increased by GnRHs within 8 h, while progesterone-induced recruitment of PR to the PREs occurred in association with much less SRC-3. A small interfering RNA knockdown of type I GnRH receptor levels reduced PR activation by GnRHs, while progesterone-dependent PR activation was unaffected. Moreover, small interfering RNA knockdown of SRC-3 abolished PRE-luciferase trans-activation by the PR in response to GnRHs. Collectively, these data indicate that PR activation by GnRHs in alphaT3-1 cells is type I GnRH receptor-mediated and that trans-activation of PR-responsive genes requires SRC-3 in this context.     

Evolutionary conservation of a 2-kb intronic sequence flanking a tissue-specific alternative exon in the PTBP2 gene

nPTB is a member of the polypyrimidine tract-binding (PTB) protein family, which participates in alternative pre-mRNA processing. Tissue-specific splicing of exon 10 in nPTB (HGMW-approved symbol PTBP2) may play an important role in regulating the functional activity of nPTB in neuronal versus nonneuronal cells. In this study, we found that 297 consecutive intronic nucleotides flanking this alternatively spliced exon 10 were identical between human, green monkey, mouse, rat, and pig, while 207 consecutive intronic nucleotides were identical between human and bird DNA. In addition, a 2-kb sequence spanning this intron region showed 85 and 70% conservation in mammal and bird DNA, respectively. Unexpected intergenic sequence conservation between human and mouse genomes has recently been identified. We have now identified intragenic (intronic) sequence conservation from mammals to birds. The striking conservation of this large segment of flanking intronic sequence suggests an important role in tissue-specific splice site selection and may function in regulating the production of functional nPTB.     

On-farm monitoring of mouse-invasive Salmonella enterica serovar enteritidis and a model for its association with the production of contaminated eggs.

Mice (Mus musculus) captured in henhouses were assessed for the presence of salmonellae in spleens. Of 621 and 526 spleens cultured during the first and second years of collection, 25.0 and 17.9%, respectively, were positive for Salmonella enterica serovar Enteritidis. Contaminated eggs were cultured from nine houses during the first year of sampling, and for eight of these houses, serovar Enteritidis was recovered from the spleens of mice. Rank sum statistical analysis of positive mouse spleens indicated that three overlapping bacterial populations were present. This pattern of infection was repeated when lipopolysaccharide (LPS) variants were used to infect chicks, and the worst infections were associated with isolates producing high-molecular-weight (HMW) LPS. Mouse isolates were capable of producing unprecedented amounts of HMW LPS as indicated by compositional analysis of six isolates that swarmed across 2% agar, which is a type of bacterial migration dependent upon production of HMW LPS. It is suggested that serovar Enteritidis cultured from the spleens of mice caught on farms will detect strains that are enhanced in their ability to contaminate eggs, in part because they are able to produce HMW LPS.     

Binding interactions of the peripheral stalk subunit isoforms from human V-ATPase

The mammalian peripheral stalk subunits of the vacuolar-type H⁺-ATPases (V-ATPases) possess several isoforms (C1, C2, E1, E2, G1, G2, G3, a1, a2, a3, and a4), which may play significant role in regulating ATPase assembly and disassembly in different tissues. To better understand the structure and function of V-ATPase, we expressed and purified several isoforms of the human V-ATPase peripheral stalk: E1G1, E1G2, E1G3, E2G1, E2G2, E2G3, C1, C2, H, a1_NT, and a2_NT. Here, we investigated and characterized the isoforms of the peripheral stalk region of human V-ATPase with respect to their affinity and kinetics in different combination. We found that different isoforms interacted in a similar manner with the isoforms of other subunits. The differences in binding affinities among isoforms were minor from our in vitro studies. However, such minor differences from the binding interaction among isoforms might provide valuable information for the future structural-functional studies of this holoenzyme.     

Regulation of iNOS expression and glutathione levels in rat liver by oxygen tension

SNI-2011 induces the long-lasting increase in the amount of aquaporin-5 (AQP5) in apical plasma membranes (APMs) of rat parotid acini in a concentration-dependent manner. This induction was inhibited by p-F-HHSiD, U73122, TMB-8, or dantrolene but not by bisindolmaleimide or H-7, indicating that SNI-2011 acting at M(3) muscarinic receptors induced translocation of AQP5 via [Ca(2+)](i) elevation but not via the activation of protein kinase C. In contrast, acetylcholine induced a transient translocation of AQP5 to APMs. SNI-2011 induces long-lasting oscillations of [Ca(2+)](i) in the presence of extracellular Ca(2+). Thus, SNI-2011 induces a long-lasting translocation of AQP5 to APMs coupled with persistent [Ca(2+)](i) oscillations.     

Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.     

HCV Specific IL-21 Producing T Cells but Not IL-17A Producing T Cells Are Associated with HCV Viral Control in HIV/HCV Coinfection

BackgroundDecreased hepatitis C virus (HCV) clearance, faster cirrhosis progression and higher HCV RNA levels are associated with Human Immunodeficiency virus (HIV) coinfection. The CD4⁺ T helper cytokines interleukin (IL)-21 and IL-17A are associated with virus control and inflammation, respectively, both important in HCV and HIV disease progression. Here, we examined how antigen-specific production of these cytokines during HCV mono and HIV/HCV coinfection was associated with HCV virus control.MethodsWe measured HCV-specific IL-21 and IL-17A production by transwell cytokine secretion assay in PBMCs from monoinfected and coinfected individuals. Viral control was determined by plasma HCV RNA levels.ResultsIn acutely infected individuals, those able to establish transient/complete HCV viral control tended to have stronger HCV-specific IL-21-production than non-controllers. HCV-specific IL-21 production also correlated with HCV viral decline in acute infection. Significantly stronger HCV-specific IL-21 production was detected in HAART-treated coinfected individuals. HCV-specific IL-17A production was not associated with lower plasma HCV RNA levels in acute or chronic HCV infection and responses were stronger in HIV coinfection. HCV-specific IL-21/ IL-17A responses did not correlate with microbial translocation or fibrosis. Exogenous IL-21 treatment of HCV-specific CD8⁺ T cells from monoinfected individuals enhanced their function although CD8⁺ T cells from coinfected individuals were somewhat refractory to the effects of IL-21.ConclusionsThese data show that HCV-specific IL-21 and IL-17A-producing T cells are induced in HIV/HCV coinfection. In early HIV/HCV coinfection, IL-21 may contribute to viral control, and may represent a novel tool to enhance acute HCV clearance in HIV/HCV coinfected individuals.     

Crystal structure of the streptococcal superantigen SpeI and functional role of a novel loop domain in T cell activation by group V superantigens

Superantigens (SAgs) are potent microbial toxins that bind simultaneously to T cell receptors (TCRs) and class II major histocompatibility complex molecules, resulting in the activation and expansion of large T cell subsets and the onset of numerous human diseases. Within the bacterial SAg family, streptococcal pyrogenic exotoxin I (SpeI) has been classified as belonging to the group V SAg subclass, which are characterized by a unique, relatively conserved ∼15 amino acid extension (amino acid residues 154 to 170 in SpeI; herein referred to as the α3-β8 loop), absent in SAg groups I through IV. Here, we report the crystal structure of SpeI at 1.56 Å resolution. Although the α3-β8 loop in SpeI is several residues shorter than that of another group V SAg, staphylococcal enterotoxin serotype I, the C-terminal portions of these loops, which are located adjacent to the putative TCR binding site, are structurally similar. Mutagenesis and subsequent functional analysis of SpeI indicates that TCR β-chains are likely engaged in a similar general orientation as other characterized SAgs. We show, however, that the α3-β8 loop length, and the presence of key glycine residues, are necessary for optimal activation of T cells. Based on Vβ-skewing analysis of human T cells activated with SpeI and structural models, we propose that the α3-β8 loop is positioned to form productive intermolecular contacts with the TCR β-chain, likely in framework region 3, and that these contacts are required for optimal TCR recognition by SpeI, and likely all other group V SAgs.