Profiling of 35 Cases of Hb S/Hb E (HBB: c.20A>T/HBB: c.79G>a), Disease and Association with α-Thalassemia and ß-Globin Gene Cluster Haplotypes from Odisha, India.

Hb S/Hb E (HBB: c.20A>T/HBB: c.79G>A) is an uncommon variant of sickle cell disease resulting from coinheritance of Hb S and Hb E. Clinico-hematological and biochemical parameters of 35 cases of Hb S/Hb E disease were studied and compared with 70 matched cases of homozygous sickle cell disease (Hb SS) and Hb S/ß-thalassemia (ß-thal) with IVS-I-5 (G>C) (HBB: c.92+5G>C). The influence of α-thal and that of of ß-globin gene cluster haplotypes among Hb S/Hb E disease was also studied. Statistical analysis was done using GraphPad InStat version 3.06. Of the 35 cases, 20 (57.14%) had a moderate clinical presentation. Mean lactate dehydrogenase (LDH) level, vaso-occlusive crises (VOCs) per year, and annual blood transfusion requirements were significantly lower in Hb S/Hb E cases than in the other two groups. The hemoglobin (Hb) and packed cell volume (PCV) levels were significantly high in Hb S/Hb E cases with α-thal and these cases were associated with microcytic-hypochromic anemia. α-Thalassemia did not influence clinical presentation in Hb S/Hb E cases. The ß-globin gene cluster haplotypes of 70 alleles of Hb S/Hb E revealed an association of five typical haplotypes [Arab-Indian (A-I), Benin, Bantu, Cameroon and Senegal] in 95.71% cases. Hb S/Hb E disease exhibit asymptomatic to moderate phenotypic expression. However, further in-depth studies on Hb S/Hb E will help in reducing the disease burden especially in high-risk countries like India.

In Silico Phylogenetic Analysis and Molecular Modelling Study of 2-Haloalkanoic Acid Dehalogenase Enzymes from Bacterial and Fungal Origin.

2-Haloalkanoic acid dehalogenase enzymes have broad range of applications, starting from bioremediation to chemical synthesis of useful compounds that are widely distributed in fungi and bacteria. In the present study, a total of 81 full-length protein sequences of 2-haloalkanoic acid dehalogenase from bacteria and fungi were retrieved from NCBI database. Sequence analysis such as multiple sequence alignment (MSA), conserved motif identification, computation of amino acid composition, and phylogenetic tree construction were performed on these primary sequences. From MSA analysis, it was observed that the sequences share conserved lysine (K) and aspartate (D) residues in them. Also, phylogenetic tree indicated a subcluster comprised of both fungal and bacterial species. Due to nonavailability of experimental 3D structure for fungal 2-haloalkanoic acid dehalogenase in the PDB, molecular modelling study was performed for both fungal and bacterial sources of enzymes present in the subcluster. Further structural analysis revealed a common evolutionary topology shared between both fungal and bacterial enzymes. Studies on the buried amino acids showed highly conserved Leu and Ser in the core, despite variation in their amino acid percentage. Additionally, a surface exposed tryptophan was conserved in all of these selected models.

In-silico gene co-expression network analysis in Paracoccidioides brasiliensis with reference to haloacid dehalogenase superfamily hydrolase gene.

CONTEXT: Paracoccidioides brasiliensis, a dimorphic fungus is the causative agent of paracoccidioidomycosis, a disease globally affecting millions of people. The haloacid dehalogenase (HAD) superfamily hydrolases enzyme in the fungi, in particular, is known to be responsible in the pathogenesis by adhering to the tissue. Hence, identification of novel drug targets is essential. AIMS: In-silico based identification of co-expressed genes along with HAD superfamily hydrolase in P. brasiliensis during the morphogenesis from mycelium to yeast to identify possible genes as drug targets. MATERIALS AND METHODS: In total, four datasets were retrieved from the NCBI-gene expression omnibus (GEO) database, each containing 4340 genes, followed by gene filtration expression of the data set. Further co-expression (CE) study was performed individually and then a combination these genes were visualized in the Cytoscape 2. 8.3. STATISTICAL ANALYSIS USED: Mean and standard deviation value of the HAD superfamily hydrolase gene was obtained from the expression data and this value was subsequently used for the CE calculation purpose by selecting specific correlation power and filtering threshold. RESULTS: The 23 genes that were thus obtained are common with respect to the HAD superfamily hydrolase gene. A significant network was selected from the Cytoscape network visualization that contains total 7 genes out of which 5 genes, which do not have significant protein hits, obtained from gene annotation of the expressed sequence tags by BLAST X. For all the protein PSI-BLAST was performed against human genome to find the homology. CONCLUSIONS: The gene co-expression network was obtained with respect to HAD superfamily dehalogenase gene in P. Brasiliensis.

A clinical and molecular study of artesunate + sulphadoxine-pyrimethamine in three districts of central and eastern India.

BACKGROUND: Artesunate + sulphadoxine-pyrimethamine (AS + SP) is recommended throughout India as the first-line treatment for uncomplicated falciparum malaria. Due to the presence of several eco-epidemiological zones of malaria and variable drug pressure, it is necessary to evaluate the efficacy of this combination in different regions of India. The objective of this study was to use clinical and molecular methods to monitor the efficacy of AS + SP in three diverse sites. METHODS: The study was undertaken in three high endemic sites of central and eastern India. Patients with uncomplicated falciparum malaria were enrolled and followed for 28 days. Molecular genotyping was conducted for merozoite surface protein (msp1 and msp2) to differentiate between re-infection and recrudescence and for the dhfr and dhps genes to monitor antifolate drug resistance. RESULTS: In all, 149 patients were enrolled at the three sites. The crude cure rates were 95.9%, 100%, and 100% in Ranchi, Keonjhar, and West Garo Hills respectively. PCR-corrected cure rates were 100% at all sites. In dhfr, 27% of isolates had triple mutations, while 46% isolates were double-mutants. The most prevalent mutation was S108N followed by C59R. 164 L mutation was observed in 43/126 (34%) isolates. In dhps, most (76%) of the isolates were wild-type. Only 2.5% (2/80) isolates showed double mutation. dhfr-dhps two locus mutation were observed in 16% (13/80) isolates. Parasite clearance time was not related with antifolate mutations. CONCLUSIONS: AS + SP combination therapy remained effective against falciparum malaria despite common mutations promoting resistance to antifolate drugs. Although the prevalence of double and triple mutations in dhfr was high, the prevalence of dhfr-dhps two locus mutations were low. Even isolates with dhfr triple and dhfr-dhps two locus mutations achieved adequate clinical and parasitological response.

Leishmanial lipid suppresses tumor necrosis factor alpha, interleukin-1beta, and nitric oxide production by adherent synovial fluid mononuclear cells in rheumatoid arthritis patients and induces apoptosis through the mitochondrial-mediated pathway.

OBJECTIVE: Leishmanial lipid is a strong immunosuppressor of host cells. Inhibition of the inflammatory responses of synovial cells through induction of apoptosis is one of the main targets of therapeutic intervention in rheumatoid arthritis (RA). This study was undertaken to examine the antiinflammatory and apoptosis-inducing effects of leishmanial lipid on adherent synovial fluid mononuclear cells (SFMCs) in patients with RA. METHODS: Lipid was extracted from a Leishmania donovani promastigote (MHO/IN/1978/UR6) by the Bligh and Dyer method. Nitric oxide (NO) was measured using the Griess reaction, and enzyme-linked immunosorbent assays for cytokines, NF-kappaB, and cytochrome c were performed. Levels of cytokines, inducible nitric oxide synthase, caspases, Bcl-2, Bax, t-Bid, and cytochrome c in the cell lysate and of NF-kappaB p65 in the nucleus were determined by Western blotting. Microscopic analysis, nuclear staining, DNA fragmentation assay, fluorescence-activated cell sorting, colorimetric assay for caspases, and fluorescent probe for measurement of mitochondrial membrane potential were used to study the leishmanial lipid-induced apoptotic pathway in SFMCs. RESULTS: Leishmanial lipid inhibited the release of tumor necrosis factor alpha, interleukin-1beta, and NO in the culture, decreased their cytosolic protein levels, and decreased NF-kappaB p65 levels in SFMCs, in a dose-dependent manner. It had the reverse effect on interleukin-10 levels. Leishmanial lipid-induced apoptosis involved the activation of caspase 3, caspase 9, and Bax, the release of cytochrome c, the alteration of mitochondrial membrane potential, and the down-regulation of Bcl-2. CONCLUSION: These results suggest that leishmanial lipid has strong antiinflammatory and apoptosis-inducing effects on SFMCs from patients with RA, and that apoptosis occurs via the mitochondrial pathway.

Selective fluorescence zinc ion sensing and binding behavior of 4-methyl-2,6-bis(((phenylmethyl)imino)methyl)phenol: biological application.

Zinc ion fluorescence sensing and the binding properties of 4-methyl-2,6-bis(((phenylmethyl)imino)methyl)phenol (HL) have been investigated. It displays high selectivity for Zn2+ and can be used as zinc ion-selective luminescent probe for biological application under physiological conditions. The increase in emission in the presence of Zn2+ is accounted for by the formation of hexanuclear complex [Zn6(L)2(OH)2(CH3COO)8] characterized by X-ray crystallography. An approximately 6-fold Zn2+-selective chelation-enhanced fluorescence response in HEPES buffer (pH 7.4) is attributed due to the strong coordination of Zn(II) that would impose rigidity and hence decrease the nonradiative decay of the excited state. By incubation of cultured living cells (B16F10 mouse melanoma and A375 human melanoma) with HL, intracellular Zn2+ concentration could be monitored.

A two-dimensional coordination compound as a zinc ion selective luminescent probe for biological applications.

A 2D coordination compound {[Cu2(HL)(N3)]ClO4}infinity (1; H3L = 2,6-bis(hydroxyethyliminoethyl)-4-methyl phenol) was synthesized and characterized by single-crystal X-ray diffraction to be a polymer in the crystalline state. Each [Cu2(HL)(N3)]+ species is connected to its adjacent unit by a bridging alkoxide oxygen atom of the ligand to form a helical propagation along the crystallographic a axis. The adjacent helical frameworks are connected by a ligand alcoholic oxygen atom along the crystallographic b axis to produce pleated 2D sheets. In solution, 1 dissociates into [Cu2(HL)2(H3L)]2H2O (2); the monomer displays high selectivity for Zn2+ and can be used in HEPES buffer (pH 7.4) as a zinc ion selective luminescent probe for biological application. The system shows a nearly 19-fold Zn2+-selective chelation-enhanced fluorescence response in the working buffer. Application of 2 to cultured living cells (B16F10 mouse melanoma and A375 human melanoma) and rat hippocampal slices was also studied by fluorescence microscopy.

Attenuated Leishmanial sphingolipid induces apoptosis in A375 human melanoma cell via both caspase-dependent and -independent pathways.

A fraction of attenuated Leishmanial lipid (ALL) rich in sphingolipids, previously shown to have apoptosis inducing activity in mouse melanoma (B16F10) and human melanoma (A375) cells, was resolved to isolate the bioactive sphingolipid. The mechanism of apoptosis induction by this bioactive attenuated Leishmanial sphingolipid (ALSL) was studied in A375 cells. Apoptosis induced by ALSL in A375 cells was found to be dose and time-dependent. Exposure of cells to ALSL resulted in a rapid increase in reactive oxygen species generation. Pretreatment of cells with the antioxidant N-acetyl-cystein reduced ROS generation and attenuated apoptosis induced by ALSL. Again, ALSL sensitization resulted in the activation of caspase-3 and -9 but not caspase-8. However, inhibitors of these caspases could not protect the cells completely from ALSL-induced apoptosis. N-acetyl-cystein pretreatment was again found to attenuate the activation of caspase-3 and -9. ALSL treatment also resulted in the alteration of mitochondrial membrane potential, and release of pro-apoptotic factors such as cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Furthermore, c-Jun N-terminal kinase was activated that resulted in apoptosis of A375 cells, whereas p38 MAPK was activated to counteract the stress generated in cells in response to ALSL treatment. Taken together, our results indicate that ALSL-induced apoptosis of A375 cells is mediated by both mitochondrial caspase-dependent and -independent pathways and it involves ROS and JNK activation in the mitogen-activated protein kinase cascade.

Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water-perchlorate helical network.

A new octanuclear copper(II) complex has been synthesized and structurally characterized by X-ray crystallography: [Cu(8)(HL)(4)(OH)(4)(H(2)O)(2)(ClO(4))(2)].(ClO(4))(2).2H(2)O (1) (H(3)L=2,6-bis(hydroxyethyliminoethyl)-4-methyl phenol). The complex is formed by the linkage of two terminal bimetallic cationic units and a tetranuclear mu(3)-hydroxo bridged dicubane core by a very short intramolecular hydrogen bond (O-H...O, 1.48(3)A and the angle 175 degrees). The coordination sphere of the terminal copper atoms is square pyramidal, the apical positions being occupied by water and a perchlorate ion. Complex 1 self-assembles to form a new type of water-perchlorate helical network [(H(2)O)(2)(ClO(4))](infinity) involving oxygen atoms of coordinated perchlorate ion and the two lattice water molecules through hydrogen-bonding interaction. The variable temperature-dependent susceptibility measurement (2-300K) of 1 reveals a strong antiferromagnetic coupling, J(1)=-220cm(-1) and J(2)=-98cm(-1) (J(1) and J(2) representing the exchange constant within [Cu(2+)](4) and [Cu(2+)](2) units, respectively). The complex binds to double-stranded supercoiled plasmid DNA giving a K(app) value of 1.2x10(7)M(-1) and displays efficient oxidative cleavage of supercoiled DNA in the presence of H(2)O(2) following a hydroxyl radical pathway.

Activation of the Mitf promoter by lipid-stimulated activation of p38-stress signalling to CREB.

The microphthalmia-associated transcription factor Mitf plays a critical role in regulating many aspects of melanocyte biology. It is required for melanoblast and postnatal melanocyte survival, regulates proliferation, and activates genes associated with differentiation such as tyrosinase and related genes involved in melanogenesis. Identifying the signals that regulate Mitf expression is crucial if we are to understand how cells of the melanocyte lineage respond to environmental cues. Here we show that the Mitf promoter is induced by lipid signalling via the p38 stress-activated kinase pathway that is also activated by a wide range of receptors as well as UV irradiation. Signalling via p38 leads to increased phosphorylation and activation of cyclic adenosine monophosphate response element-binding (CREB) that binds and activates the Mitf promoter via the cyclic adenosine monophosphate (cAMP) response element. Moreover, we also show that activation of p38 mediated by lipids is potentiated by inhibition of the PI3kinase pathway but not by inhibition of protein kinase A (PKA). The results identify a mechanism in which stress signalling via p38 leads to activation of CREB, enhanced Mitf expression and consequently increased tyrosinase expression. The results are relevant for the regulation of melanocytes by Mitf, but also raise the possibility that lipid mediated activation of p38 signalling may represent a potential therapy for vitiligo.

Immunobiological activities of a new nontoxic lipopolysaccharide from Acidiphilium GS18h/ATCC55963, a soil isolate from an Indian copper mine.

A novel nontoxic lipopolysaccharide (LPS) was purified from Acidiphilium strain GS18h/ATCC55963. The chemical composition of the lipid A part of this LPS is distinctly different from that of known lipid A molecules. The LPS was investigated to determine its capacity to provide protection against toxic LPS or endotoxic shock, as has been reported for other nontoxic LPSs (Rhodobacter sphaeroides and Rhodobacter capsulatus), and also the extent and type of immunomodulatory response in terms of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-beta), and IL-6 release as well as NO secretion by stimulated monocyte-macrophage systems. This study demonstrates clearly that mice immunized or primed with this LPS are fully protected against challenge with toxic Escherichia coli LPS. Unlike most of the extensively studied nontoxic LPSs, this LPS induced reactive nitrogen intermediates and released TNF-alpha, IL-beta and IL-6 in both mouse and human monocyte-macrophage systems. However, the extent of the cytokine and lymphokine releasing response was well below the range of the toxic LPS, for example that of E. coli. Owing to its capacity to provide immunostimulation of the host without causing any lethality to ensure protection against endotoxic shock, this LPS appears to have potential therapeutic value.

A sphingolipid rich lipid fraction isolated from attenuated Leishmania donovani promastigote induces apoptosis in mouse and human melanoma cells in vitro.

Lipids, especially sphingolipids, are emerging as inducer of apoptosis in a wide range of immortal cells, potentiating their therapeutic application in cancer. In the present study, a sphingolipid rich lipid fraction (denoted here as ALL), isolated from an attenuated strain of Leishmania donovani promastigote, was tested for its tumoricidal activity taking melanoma, the dreaded form of skin cancer cells, as model. ALL was found to induce chromatin condensation, internucleosomal DNA fragmentation and phosphatidylserine externalization with enhanced cell population in sub-G1 region in both mouse and human melanoma systems, namely B16F10 and A375 respectively. These are the hallmarks of cells undergoing apoptosis. Further analysis demonstrated that ALL treated melanoma cells showed significant increase in ROS generation, mitochondrial membrane potential depolarization, release of cytochrome c, and caspase-3 activation, which are the events closely involved in apoptosis. These findings indicate that one or more bioactive sphingolipid(s)/ceramide(s) present in ALL could be the causative agent(s) for the induction of apoptosis in melanoma cells. Further studies are thus necessary to identify these specific bioactive sphingolipid(s)/ceramide(s) and to establish their mechanism of action, in order to explore their use as anticancer agents.

Human placental protein/peptides stimulate melanin synthesis by enhancing tyrosinase gene expression.

Placental protein/peptides as biological response modifier are well documented, but not much known about melanogenesis. We possibly for the first time, demonstrated melanogenesis in B16F10 mouse melanoma by a placental protein/peptide fraction (PPPF) prepared from a hydroalcoholic extract of fresh term human placenta. This study described the effect of PPPF on the induction of tyrosinase; the key enzyme of melanogenesis to investigate the basis of PPPF induced pigmentation in primary melanocyte and B16F10 melanoma. Tyrosinase induction by PPPF in B16F10 cells was found dose- and time dependent at the level of activity. Tyrosinase, at the level of transcription and protein expression when assessed by RT-PCR and Western blot analyses found to have considerable induction over untreated control. PPPF led to enhanced activation of tyrosinase promoter resulting higher transcription thus substantiating the role of PPPF as a stimulator of melanogenesis. Actinomycin D, the transcriptional inhibitor of protein synthesis, blocked the stimulatory action of PPPF since the induction of tyrosinase and melanin was markedly reduced in presence of this inhibitor. Thus the results suggested that PPPF mediated increase in tyrosinase expression occurred through transcriptional upregulation to stimulate melanogenesis in B16F10 cells and in primary melanocyte also.