Surface-active phospholipase A2 in mouse spermatozoa

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.     

Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

Fish Gut Microbiome: Current Approaches and Future Perspectives

In recent years, investigations of microbial flora associated with fish gut have deepened our knowledge of the complex interactions occurring between microbes and host fish. The gut microbiome not only reinforces the digestive and immune systems in fish but is itself shaped by several host-associated factors. Unfortunately, in the past, majority of studies have focused upon the structure of fish gut microbiome providing little knowledge of effects of these factors distinctively and the immense functional potential of the gut microbiome. In this review, we have highlighted the recently gained insights into the diversity and functions of the fish gut microbiome. We have also delved on the current approaches that are being employed to study the fish gut microbiome with an aim to collate all the knowledge gained and make accurate conclusions for their application based perspectives. The literature reviewed indicated that the future research should shift towards functional microbiomics to improve the maximum sustainable yield in aquaculture.     

Two new fossil woods from the early Miocene of Kutch,Gujarat, India and their significance

The Kutch region of western India (Gujarat State) is today arid to semiarid and characterised by mostly ephemeral streams which carry water during the monsoon. The uneven distribution of rainfall and disturbed topography are the result of climate change during the Cenozoic period. Two fossil woods, namely Bauhinium palaeomalabaricum Prakash and Prasad (Fabaceae) and Ebenoxylon indicum Ghosh and Kazmi (Ebenaceae), are described from Kutch in order to provide insights into the palaeovegetation and palaeoclimate. Because the modern representatives of the present and previously described taxa from the same horizon are thermophilic in nature and grow in evergreen to deciduous forests, a warm and humid climate is interpreted. Furthermore, the finding of some mangrove taxa in the assemblage indicates the lagoonal to intertidal environment at the time of deposition.     

Susceptibility of Murine Norovirus and Hepatitis A Virus to Electron Beam Irradiation in Oysters and Quantifying the Reduction in Potential Infection Risks

Consumption of raw oysters is an exposure route for human norovirus (NoV) and hepatitis A virus (HAV). Therefore, efficient postharvest oyster treatment technology is needed to reduce public health risks. This study evaluated the inactivation of HAV and the NoV research surrogate, murine norovirus-1 (MNV-1), in oysters (Crassostrea virginica) by electron beam (E-beam) irradiation. The reduction of potential infection risks was quantified for E-beam irradiation technology employed on raw oysters at various virus contamination levels. The E-beam dose required to reduce the MNV and HAV titer by 90% (D₁₀ value) in whole oysters was 4.05 (standard deviations [SD], ±0.63) and 4.83 (SD, ±0.08) kGy, respectively. Microbial risk assessment suggests that if a typical serving of 12 raw oysters was contaminated with 10⁵ PFU, a 5-kGy treatment would achieve a 12% reduction (from 4.49 out of 10 persons to 3.95 out of 10 persons) in NoV infection and a 16% reduction (from 9.21 out of 10 persons to 7.76 out of 10 persons) in HAV infections. If the serving size contained only 10² PFU of viruses, a 5-kGy treatment would achieve a 26% reduction (2.74 out of 10 persons to 2.03 out of 10 persons) of NoV and 91% reduction (2.1 out of 10 persons to 1.93 out of 100 persons) of HAV infection risks. This study shows that although E-beam processing cannot completely eliminate the risk of viral illness, infection risks can be reduced.     

Calcitonin Receptor-Zonula Occludens-1 Interaction Is Critical for Calcitonin-Stimulated Prostate Cancer Metastasis

The role of neuroendocrine peptide calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT–CTR axis in non-invasive PC cells induces an invasive phenotype. Here we show by yeast-two hybrid screens that CTR associates with the tight junction protein Zonula Occludens-1 (ZO-1) via the interaction between the type 1 PDZ motif at the carboxy-terminus of CTR and the PDZ3 domain of ZO-1. Mutation of either the CTR C-PDZ-binding motif or the ZO-1-PDZ3 domain did not affect binding of CTR with its ligand or G-protein-mediated signaling but abrogated destabilizing actions of CT on tight junctions and formation of distant metastases by orthotopically implanted PC cells in nude mice, indicating that these PDZ domain interactions were pathologically relevant. Further, we observed CTR-ZO-1 interactions in PC specimens by proximity ligation immunohistochemistry, and identified that the number of interactions in metastatic PC specimens was several-fold larger than in non-metastatic PC. Our results for the first time demonstrate a mechanism by which PDZ-mediated interaction between CTR and ZO1 is required for CT-stimulated metastasis of prostate cancer. Since many receptors contain PDZ-binding motifs, this would suggest that PDZ-binding motif-adaptor protein interactions constitute a common mechanism for cancer metastasis.     

Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.