Implication of the VirD4 Coupling Protein of the Lvh Type 4 Secretion System in Virulence Phenotypes of Legionella pneumophila

The genome of the Philadelphia-1 strain of Legionella pneumophila, the causative organism of Legionnaires'' disease, encodes two virulence-associated type 4 secretion systems (T4SSs), the Dot/Icm type 4B (T4BSS) and the Lvh type 4A (T4ASS). Broth stationary-phase cultures of most dot/icm mutants are defective in entry and evasion of phagosome acidification. However, those virulence defects can be reversed by incubating broth cultures of dot/icm mutants in water, termed water stress (WS). WS reversal requires the lvh T4ASS locus, suggesting an interaction between the two T4SSs in producing Legionella virulence phenotypes. In the current work, the loss of WS reversal in a dotA Δlvh mutant of strain JR32 was shown to be attributable to loss of the lvh virD4 gene, encoding the putative coupling protein of the T4ASS. Transformation of a dotA Δlvh mutant with virD4 also reversed entry and phagosome acidification defects in broth cultures. In addition, broth cultures of Δlvh and ΔvirD4 mutants, which were dot/icm⁺, showed 5-fold and >6-fold increases in translocation of the Dot/Icm translocation substrates, proteins RalF and SidD, respectively. These data demonstrate that the Lvh T4ASS functions in both broth stationary-phase cultures conventionally used for infection and cultures exposed to WS treatment. Our studies in a dotA Δlvh mutant and in a dot/icm⁺ background establish that VirD4 and the Lvh T4ASS contribute to virulence phenotypes and are consistent with independent functioning of Dot/Icm and Lvh T4SSs or functional substitution of the Lvh VirD4 protein for a component(s) of the Dot/Icm T4BSS.     

Complement component 3 inhibition by an antioxidant is neuroprotective after cerebral ischemia and reperfusion in mice

Oxidative stress after stroke is associated with the inflammatory system activation in the brain. The complement cascade, especially the degradation products of complement component 3, is a key inflammatory mediator of cerebral ischemia. We have shown that pro‐inflammatory complement component 3 is increased by oxidative stress after ischemic stroke in mice using DNA array. In this study, we investigated whether up‐regulation of complement component 3 is directly related to oxidative stress after transient focal cerebral ischemia in mice and oxygen‐glucose deprivation in brain cells. Persistent up‐regulation of complement component 3 expression was reduced in copper/zinc‐superoxide dismutase transgenic mice, and manganese‐superoxide dismutase knock‐out mice showed highly increased complement component 3 levels after transient focal cerebral ischemia. Antioxidant N‐tert‐butyl‐α‐phenylnitrone treatment suppressed complement component 3 expression after transient focal cerebral ischemia. Accumulation of complement component 3 in neurons and microglia was decreased by N‐tert‐butyl‐α‐phenylnitrone, which reduced infarct volume and impaired neurological deficiency after cerebral ischemia and reperfusion in mice. Small interfering RNA specific for complement component 3 transfection showed a significant increase in brain cells viability after oxygen‐glucose deprivation. Our study suggests that the neuroprotective effect of antioxidants through complement component 3 suppression is a new strategy for potential therapeutic approaches in stroke.     

Plasmodium berghei Calcium Dependent Protein Kinase 1 Is Not Required for Host Cell Invasion

Plasmodium Calcium Dependent Protein Kinase (CDPK1) is required for the development of sexual stages in the mosquito. In addition, it is proposed to play an essential role in the parasite’s invasive stages possibly through the regulation of the actinomyosin motor and micronemal secretion. We demonstrate that Plasmodium berghei CDPK1 is dispensable in the parasite’s erythrocytic and pre-erythrocytic stages. We successfully disrupted P. berghei CDPK1 (PbCDPK1) by homologous recombination. The recovery of erythrocytic stage parasites lacking PbCDPK1 (PbCDPK1-) demonstrated that PbCDPK1 is not essential for erythrocytic invasion or intra-erythrocytic development. To study PbCDPK1’s role in sporozoites and liver stage parasites, we generated a conditional mutant (CDPK1 cKO). Phenotypic characterization of CDPK1 cKO sporozoites demonstrated that CDPK1 is redundant or dispensable for the invasion of mammalian hepatocytes, the egress of parasites from infected hepatocytes and through the subsequent erythrocytic cycle. We conclude that P. berghei CDPK1 plays an essential role only in the mosquito sexual stages.     

The Modulatory Effect of Cholesterol Synthesis by Oryza sativa Derived Bioactive Peptide Fractions: An In Vitro Investigation

International Journal of Peptide Research and Therapeutics - A primary risk factor of cardiovascular disease is referred to the high concentration of plasma cholesterol. The alleviation of plasma...     

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit,Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

Background/Aims

The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.

Methodology and Principal Findings

Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2^nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization.

Conclusions

This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.     

Role of co-stimulation in Leishmaniasis

Leishmania are obligate intracellular parasites that cause a wide spectrum of diseases ranging from cutaneous, mucocutaneous and the visceral kind. Persistence or resolution of leishmaniasis is governed by host immune response. Co-stimulation is an important secondary signal that governs the extent, strength and direction of the immune response that follows. Co-stimulation by CD40, B7 and OX40 family has been shown to influence the outcome following Leishmania infection and manipulation of these pathways has shown promise for use in immune therapy of leishmaniasis. In this review, we discuss the roles of CD40, B7 and OX40 co-stimulatory pathways in regulating immunity to Leishmania and their implications in the treatment of this disease.     

Second harmonic imaging of plants tissues and cell implosion using two‐photon process in ZnO nanoparticles

The optical properties of colloidal ZnO nanoparticle (NP) solutions, with size ranging from several nm to around 200 nm, have been tailored to have high optical nonlinearity for bioimaging with no auto‐fluorescence above 750 nm and minimal auto‐fluorescence below 750 nm. The high second harmonic conversion efficiency enables selective tissue imaging and cell tracking using tunable near‐infrared femtosecond laser source ranging from 750‐980 nm. For laser energies exceeding the two‐photon energy of the bandgap of ZnO (half of 3.34 eV), the SHG signal greatly decreases and the two‐photon emission becomes the dominant signal. The heat generated due to two‐photon absorption within the ZnO NPs enable selective cell or localized tissue destruction using excitation wavelength ranging from 710–750 nm. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Spatial mapping of lipids at cellular resolution in embryos of cotton

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry.     

Recipient nonhematopoietic antigen-presenting cells are sufficient to induce lethal acute graft-versus-host disease

The presentation pathways by which allogeneic peptides induce graft-versus-host disease (GVHD) are unclear. We developed a bone marrow transplant (BMT) system in mice whereby presentation of a processed recipient peptide within major histocompatibility complex (MHC) class II molecules could be spatially and temporally quantified. Whereas donor antigen presenting cells (APCs) could induce lethal acute GVHD via MHC class II, recipient APCs were 100-1,000 times more potent in this regard. After myeloablative irradiation, T cell activation and memory differentiation occurred in lymphoid organs independently of alloantigen. Unexpectedly, professional hematopoietic-derived recipient APCs within lymphoid organs had only a limited capacity to induce GVHD, and dendritic cells were not required. In contrast, nonhematopoietic recipient APCs within target organs induced universal GVHD mortality and promoted marked alloreactive donor T cell expansion within the gastrointestinal tract and inflammatory cytokine generation. These data challenge current paradigms, suggesting that experimental lethal acute GVHD can be induced by nonhematopoietic recipient APCs.     

Melt-in-Mouth Pellets of Fexofenadine Hydrochloride Using Crospovidone as an Extrusion–Spheronisation Aid

Microcrystalline cellulose (MCC) is well established as an extrusion spheronisation aid for the preparation of pellets. Crospovidone (Polyplasdone® XL-10) is compared with microcrystalline cellulose for the preparation of melt-in-mouth pellets. Taste-masked fexofenadine hydrochloride was incorporated in the melt-in-mouth formulation. Crospovidone was found to be well suited as extrusion–spheronisation aid for the preparation of melt-in-mouth pellets. The great advantage of crospovidone is, however, the disintegrating properties of the pellets after only a short time of exposure to liquid. Crospovidone was successfully employed as an extrusion–spheronisation aid to produce melt-in-mouth pellets obviating the need of a traditional extrusion–spheronisation aid, MCC. Dual properties of Crospovidone were explored viz. as an extrusion–spheronisation aid and a disintegrant.