Peptidyl-prolyl cis-trans isomerases (immunophilins) and their roles in parasite biochemistry, host-parasite interaction and antiparasitic drug action

Immunophilin is the collective name given to the cyclophilin and FK506-binding protein families. As the name suggests, these include the major binding proteins of certain immunosuppressive drugs: cyclophilins for the cyclic peptide cyclosporin A and FK506-binding proteins for the macrolactones FK506 and rapamycin. Both families, although dissimilar in sequence, possess peptidyl-prolyl cis-trans isomerase activity in vitro and can play roles in protein folding and transport, RNA splicing and the regulation of multi-protein complexes in cells. In addition to enzymic activity, many immunophilins act as molecular chaperones. This property may be conferred by the isomerase domain and/or by additional domains. Recent years have seen a great increase in the number of known immunophilin genes in parasitic protozoa and helminths and in many cases their products have been characterised biochemically and their temporal and spatial expression patterns have been examined. Some of these genes represent novel types: one example is a Toxoplasma gondii gene encoding a protein with both cyclophilin and FK506-binding protein domains. Likely roles in protein folding and oligomerisation, RNA splicing and sexual differentiation have been suggested for parasite immunophilins. In addition, unexpected roles in parasite virulence (Mip FK506-binding protein of Trypanosoma cruzi) and host immuno-modulation (e.g. 18-kDa cyclophilin of T. gondii) have been established. Furthermore, in view of the potent antiparasitic activities of cyclosporins, macrolactones and non-immunosuppressive derivatives of these compounds, immunophilins may mediate drug action and/or may themselves represent potential drug targets. Investigation of the mechanisms of action of these agents may lead to the design of potent and selective antimalarial and other antiparasitic drugs. This review discusses the properties of immunophilins in parasites and the 'animal model'Caenorhabditis elegans and relates these to our understanding of the roles of these proteins in cellular biochemistry, host-parasite interaction and the antiparasitic mechanisms of the drugs that bind to them.     

Persistent high frequencies of varicella-zoster virus ORF4 protein-specific CD4+ T cells after primary infection

Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4+ T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4+ T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-gamma) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4+ T cells. Indeed, CD4+ T cells were found to dominate the overall virus-specific IFN-gamma cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4+ T cells, with implications for disease pathogenesis and future vaccine development.     

Development of transgenic finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.) resistant to leaf blast disease

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R₀ and R₁ progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R₁ lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.     

A pipeline for the identification and characterization of chromatin modifications derived from ChIP-Seq datasets

The advent of massive parallel sequencing of immunopurified chromatin and its determinants has provided new avenues for researchers to map epigenome-wide changes and there is tremendous interest to uncover regulatory signatures to understand fundamental questions associated with chromatin structure and function. Indeed, the rapid development of large genome annotation projects has seen a resurgence in chromatin immunoprecipitation (ChIP) based protocols which are used to distinguish protein interactions coupled with large scale sequencing (Seq) to precisely map epigenome-wide interactions. Despite some of the great advances in our understanding of chromatin modifying complexes and their determinants, the development of ChIP-Seq technologies also pose specific demands on the integration of data for visualization, manipulation and analysis. In this article we discuss some of the considerations for experimental design planning, quality control, and bioinformatic analysis. The key aspects of post sequencing analysis are the identification of regions of interest, differentiation between biological conditions and the characterization of sequence differences for chromatin modifications. We provide an overview of best-practise approaches with background information and considerations of integrative analysis from ChIP-Seq experiments.     

Genetic engineering of crop plants for fungal resistance: role of antifungal genes

Fungal diseases damage crop plants and affect agricultural production. Transgenic plants have been produced by inserting antifungal genes to confer resistance against fungal pathogens. Genes of fungal cell wall-degrading enzymes, such as chitinase and glucanase, are frequently used to produce fungal-resistant transgenic crop plants. In this review, we summarize the details of various transformation studies to develop fungal resistance in crop plants.     

Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology

Until recently, microbial identification in clinical diagnostic laboratories has mainly relied on conventional phenotypic and gene sequencing identification techniques. The development of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) devices has revolutionized the routine identification of microorganisms in clinical microbiology laboratories by introducing an easy, rapid, high throughput, low-cost, and efficient identification technique. This technology has been adapted to the constraint of clinical diagnostic laboratories and has the potential to replace and/or complement conventional identification techniques for both bacterial and fungal strains. Using standardized procedures, the resolution of MALDI-TOF MS allows accurate identification at the species level of most Gram-positive and Gram-negative bacterial strains with the exception of a few difficult strains that require more attention and further development of the method. Similarly, the routine identification by MALDI-TOF MS of yeast isolates is reliable and much quicker than conventional techniques. Recent studies have shown that MALDI-TOF MS has also the potential to accurately identify filamentous fungi and dermatophytes, providing that specific standardized procedures are established for these microorganisms. Moreover, MALDI-TOF MS has been used successfully for microbial typing and identification at the subspecies level, demonstrating that this technology is a potential efficient tool for epidemiological studies and for taxonomical classification.     

4-Thiothymidine sensitization of DNA to UVA offers potential for a novel photochemotherapy

Photochemotherapy, in which ultraviolet radiation (UVR: 280-400 nm) or visible light is combined with a photosensitizing drug to produce a therapeutic effect that neither drug or radiation can achieve alone, is a proven therapeutic strategy for a number of non-malignant hyperproliferative skin conditions and various cancers. Examples are psoralen plus UVA (320-400 nm) radiation (PUVA) and photodynamic therapy (PDT). All existing photochemotherapies have drawbacks - for example the association of PUVA with the development of skin cancer, and pain that is often associated with PDT treatment of skin lesions. There is a clear need to develop alternative approaches that involve lower radiation doses and/or improved selectivity for target cells. In this review, we explore the possibility to address this need by exploiting thionucleoside-mediated DNA photosensitisation to low, non toxic doses of UVA radiation.     

Mon2 is a negative regulator of the monomeric G protein, Arl1

Using site-directed mutants of ARL1 predicted to alter nucleotide binding, we examined phenotypes associated with the loss of ARL1 , including effects on membrane traffic and K (+) homeostasis. The GTP-restricted allele, ARL[Q72L] , complemented the membrane traffic phenotype (CPY secretion), but not the K (+) homeostasis phenotypes (sensitivity to hygromycin B, steady-state levels of K (+) , and accumulation of (86) Rb (+) ), while the XTP-restricted mutant, ARL1[D130N] , complemented the ion phenotypes, but not the membrane traffic phenotype. A GDP-restricted allele, ARL1[T32N] , did not effectively complement either phenotype. These results are consistent with a model in which Arl1 has three different conformations in vivo. We also explored the relationship between ARL1 and MON2 using the synthetic lethal phenotype exhibited by these two genes and demonstrated that MON2 is a negative regulator of the GTP-restricted allele of ARL1 , ARL1[Q72L] . Finally, we constructed several new alleles predicted to alter binding of Arl1 to the sole GRIP domain containing protein in yeast, Imh1, and found that ARL1[F52G] and ARL1[Y82G] were unable to complement the loss of ARL1 with respect to either the membrane traffic or K (+) homeostasis phenotypes. Our study expands understanding of the roles of Arl1 in vivo.     

Cytotoxic effect of Green synthesized silver nanoparticles using Melia azedarach against in vitro HeLa cell lines and lymphoma mice model

This communication explains the biosynthesis of stable silver nanoparticles (AgNPs) from Melia azedarach and its cytotoxicity against in vitro HeLa cells and in vivo Dalton's ascites lymphoma (DAL) mice model. The AgNPs synthesis was determined by UV–visible spectrum and it was further characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and X-ray diffraction (XRD) analysis. Zeta potential analysis revealed stable AgNPs at ?24.9 mV. UV visible spectrum indicated an absorption peak at 436 nm which reflects its specific Surface Plasmon Resonance (SPR). Biosynthesized AgNPs were predominantly cubical and spherical with an average particle size of 78 nm approximately as observed through SEM and DLS analysis, respectively. Cytotoxicity of biosynthesized AgNPs against in vitro Human epithelial carcinoma cell line (HeLa) showed a dose–response activity. Lethal dose (LD₅₀) value was found to be 300 μg/mL of AgNPs against HeLa cell line. Cytotoxicity against normal continuous cell line human breast lactating, donor 100 (HBL 100) was found only in increased concentration of both AgNPs and 5-FU. In addition, in vivo DAL mice model showed significant increase in life span, induction of apoptosis was evidenced by acridine orange and ethidium bromide (AO and EB) staining.