Evidence for <Emphasis Type="Italic">Wolbachia</Emphasis> symbiosis in microfilariae of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis> from West Bengal,India

Wolbachia are symbiotic endobacteria that infect the majority of filarial nematodes, including Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus. Recent studies have suggested that Wolbachia are necessary for the reproduction and survival of filarial nematodes and have highlighted the use of antibiotic therapy such as tetracycline/doxycycline as a novel method of treatment for infections caused by these organisms. Before such therapy is conceived and implemented on a large scale, it is necessary to assess the prevalence of the endosymbiont in W. bancrofti from different geographical locations. We present data from molecular and electron microscopic studies to provide evidence for Wolbachia symbiosis in W. bancrofti microfilariae collected from two districts (Bankura and Birbhum) of West Bengal, India.     

Synthesis, aggregation behavior and cholesterol solubilization studies of 16-epi-pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid)

Synthesis, aggregation behavior and in vitro cholesterol solubilization studies of 16-epi-pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid, EPCA) are reported. The synthesis of this unnatural epimer of pythocholic acid (3α,12α,16α-trihydroxy-5β-cholan-24-oic acid, PCA) involves a series of simple and selective chemical transformations with an overall yield of 21% starting from readily available cholic acid (CA). The critical micellar concentration (CMC) of 16-epi-pythocholate in aqueous media was determined using pyrene as a fluorescent probe. In vitro cholesterol solubilization ability was evaluated using anhydrous cholesterol and results were compared with those of other natural di- and trihydroxy bile acids. These studies showed that 16-epi-pythocholic acid (16β-hydroxy-deoxycholic acid) behaves similar to cholic acid (CA) and avicholic acid (3α,7α,16α-trihydroxy-5β-cholan-24-oic acid, ACA) in its aggregation behavior and cholesterol dissolution properties.     

Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

Tumor necrosis factor receptor-1-induced neuronal death by TRADD contributes to the pathogenesis of Japanese encephalitis

While a number of studies have documented the neurotropism of Japanese encephalitis virus (JEV), little is known regarding the molecular mechanism of neuronal death following viral infection. The tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) has been suggested to be the crucial signal adaptor that mediates all intracellular responses from TNFR-1. Using mouse (Neuro2a) and human (SK-N-SH) neuroblastoma cell lines, we have shown that the altered expression of TNFR-1 and TRADD following JEV infection regulates the downstream apoptotic cascades. Activation of TRADD led to mitochondria-mediated neuronal apoptosis. As TRADD-knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD in diseases pathology following JEV infection. We circumvented this problem by silencing TRADD expression with small-interfering RNA (siRNA) and have found that TRADD is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with JEV. Interestingly, siRNA against TRADD also decreased the viral load in Neuro2a cells. Furthermore, siRNA against TRADD increased the survival of JEV-infected mice by altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that the engagement of TNFR-1 and TRADD following JEV infection plays a crucial role in neuronal apoptosis.     

A role for clathrin in reassembly of the Golgi apparatus

The Golgi apparatus is a highly dynamic organelle whose organization is maintained by a proteinaceous matrix, cytoskeletal components, and inositol phospholipids. In mammalian cells, disassembly of the organelle occurs reversibly at the onset of mitosis and irreversibly during apoptosis. Several pharmacological agents including nocodazole, brefeldin A (BFA), and primary alcohols (1-butanol) induce reversible fragmentation of the Golgi apparatus. To dissect the mechanism of Golgi reassembly, rat NRK and GH3 cells were treated with 1-butanol, BFA, or nocodazole. During washout of 1-butanol, clathrin, a ubiquitous coat protein implicated in vesicle traffic at the trans-Golgi network and plasma membrane, and abundant clathrin coated vesicles were recruited to the region of nascent Golgi cisternae. Knockdown of endogenous clathrin heavy chain showed that the Golgi apparatus failed to reform efficiently after BFA or 1-butanol removal. Instead, upon 1-butanol washout, it maintained a compact, tight morphology. Our results suggest that clathrin is required to reassemble fragmented Golgi elements. In addition, we show that after butanol treatment the Golgi apparatus reforms via an initial compact intermediate structure that is subsequently remodeled into the characteristic interphase lace-like morphology and that reassembly requires clathrin.     

Melatonin in the regulation of annual testicular events in carp Catla catla: evidence from the studies on the effects of exogenous melatonin, continuous light, and continuous darkness

The physiological significance of melatonin in the regulation of annual testicular events in a major carp Catla catla was evaluated through studies on the effects of graded dose (25, 50, or 100 µg/100 g body wt.) of melatonin exogenously administered for different durations (1, 15, or 30 days) and manipulation of the endogenous melatonin system by exposing the fish to constant darkness (DD) or constant light (LL) for 30 days. An identical experimental schedule was followed during the preparatory (February-March), pre-spawning (April-May), spawning (July-August), and post-spawning (September-October) phases of the annual cycle. Irrespective of the reproductive status of the carp, LL suppressed while DD increased the mid-day and mid-night values of melatonin compared to respective controls. Influences of exogenous melatonin varied in relation to the dose and duration of treatment and the reproductive status of the carp. However, testicular response to exogenous melatonin (at 100 µg, for 30 days) and DD in each reproductive phase was almost identical. Notably, precocious testicular maturation occurred in both DD and melatonin-injected fish during the preparatory phase and in LL carps during the pre-spawning phase. In contrast, testicular functions in both the melatonin-treated and DD fish were inhibited during the pre-spawning and spawning phases, while the testes did not respond to any treatment during the post-spawning phase. In conclusion, this study provided the first experimental evidence that melatonin plays a significant role in the regulation of annual testicular events in a sub-tropical surface-dwelling carp Catla catla, but the influence of this pineal hormone on the seasonal activity of testis varies in relation to the reproductive status of the concerned fish.     

Radiolabeled anti-claudin 4 and anti-prostate stem cell antigen: initial imaging in experimental models of pancreatic cancer

Global expression profiling of pancreatic cancers has identified two cell surface molecules, claudin 4 and prostate stem cell antigen (PSCA), as being overexpressed in the vast majority of cases. Two antibodies, anti-claudin 4 and anti-PSCA, were radiolabeled with iodine 125 ((125)I) for imaging pancreatic cancer xenografts in mice using gamma scintigraphy and single-photon emission computed tomography-computed tomography (SPECT-CT). Immunofluorescence staining of intact and permeabilized Colo357 human pancreatic cancer cells showed strong extracellular staining by both anti-PSCA and anti-claudin 4. Biodistribution studies in claudin 4 and PSCA-expressing Colo357 and PANC-1 subcutaneous xenograft models in mice showed that [(125)I]anti-claudin 4 tumor to muscle ratio uptake was 4.3 in Colo357 at 6 days postinjection and 6.3 in PANC-1 xenografts at 4 days postinjection. Biodistribution of [(125)I]anti-PSCA showed tumor to muscle ratio uptake of 4.9 in Colo357 at 6 days postinjection. Planar gamma scintigraphic imaging in Colo357 xenograft-bearing mice showed clear tumor uptake of [(125)I]anti-claudin 4 by 24 hours postinjection and by 48 hours postinjection for [(125)I]anti-PSCA. SPECT-CT imaging with [(125)I]anti-claudin 4 and [(125)I]anti-PSCA in an L3.6PL orthotopic xenograft model showed strong tumor and spleen uptake at 5 days postinjection. Both anti-claudin 4 and anti-PSCA demonstrate promise as radiodiagnostic and possibly radiotherapeutic agents for human pancreatic cancers.     

Dichotomy in Growth and Invasion from Low- to High-Grade Glioma Cellular Variants

Glial dysfunction outraging CNS plasticity and integrity results in one of the most dangerous cancers, namely glioma, featuring little median survival period and high recurrence. The hallmark properties of proliferation, invasion and angiogenesis with the infiltrated macrophages in glioma are expected to be tightly coupled or cross-linked, but not properly related so far. The present study is aimed to find a relationship between this featured quadrangle from lower to higher grades (HG) of post-operative glioma tissues and their invading subsets. Elevated Ki67-associated proliferation in lower grades (LG) was supported with VEGF dependent angiogenic maintenance which found a decrease unlikely in HG. In contrast, MMP 2 and 9-associated invasions augmented high in HG with the dominant presence of CD204⁺ M2 polarized macrophages and a general increase in global DNMT1-associated methylation. Marked differences found in ECM invading cellular subsets of HG showing high proliferative capacity indicating rationally for recurrence, contrasting the nature of gross tumor tissue of the same grade. Thus in LG, the neoplastic lesion is more inclined to its growth while in higher grade more disposed towards tissue wreckage in support with cellular environmental milieu whereas the cellular variants and subsets of invaded cells showed different trends. Therefore, some operational dichotomy or coupling among cellular variants in glioma is active in determining its low- to high-grade transition and aggressive progression.