Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood.

High quality and sufficient quantity of genomic DNA (gDNA) are the primary requisites of several molecular biologic applications, including clinical studies related to genetics, genomics, gene polymorphism, and DNA fingerprinting. Whole blood is the primary source of gDNA in most of the clinical investigations. Currently, commercial kits are primarily used to achieve these goals. However, the use of kits is limited by the cost and involvement of several centrifugal steps. Other methods reported are either laborious or do not produce high quality or quantity of gDNA or both. Here, we present the data on the development of a centrifugation-free, cost-effective, and user-friendly method for the isolation of human gDNA from the buffy coat of human blood that involves limited numbers of steps with about 15 min of hands-on time per sample.

Expression of arsenic resistance genes in the obligate anaerobe Bacteroides vulgatus ATCC 8482, a gut microbiome bacterium.

The response of the obligate anaerobe Bacteroides vulgatus ATCC 8482, a common human gut microbiota, to arsenic was determined. B. vulgatus ATCC 8482 is highly resistant to pentavalent As(V) and methylarsenate (MAs(V)). It is somewhat more sensitive to trivalent inorganic As(III) but 100-fold more sensitive to methylarsenite (MAs(III)) than to As(III). B. vulgatus ATCC 8482 has eight continuous genes in its genome that we demonstrate form an arsenical-inducible transcriptional unit. The first gene of this ars operon, arsR, encodes a putative ArsR As(III)-responsive transcriptional repressor. The next three genes encode proteins of unknown function. The remaining genes, arsDABC, have well-characterized roles in detoxification of inorganic arsenic, but there are no known genes for MAs(III) resistance. Expression of each gene after exposure to trivalent and pentavalent inorganic and methylarsenicals was analyzed. MAs(III) was the most effective inducer. The arsD gene was the most highly expressed of the ars operon genes. These results demonstrate that this anaerobic microbiome bacterium has arsenic-responsive genes that confer resistance to inorganic arsenic and may be responsible for the organism's ability to maintain its prevalence in the gut following dietary exposure to inorganic arsenic.

Functional role of lysine 12 in Leishmania major AQP1.

Leishmania major aquaglyceroporin (AQP1) is an adventitious metalloid channel that allows the bidirectional movement of arsenite and antimonite. Here we demonstrate that AQP1 is subjected to proteasome-dependent degradation. Treatment of Leishmania promastigotes with the proteasome inhibitor MG132 resulted in increased AQP1 accumulation. Site-directed mutagenesis in AQP1 revealed that alteration of lysine 12 to either alanine or arginine improves protein stability. AQP1 expression is stabilized by mitogen-activated protein kinase 2 (MPK2). Cells expressing a dominant-negative MPK2 mutant exhibited severely reduced AQP1 expression, which could be reversed upon addition of MG132. Interestingly, the dominant-negative MPK2 mutant could not destabilize either AQP1K12A or AQP1K12R. While stabilization of AQP1 by MPK2 leads to its relocalization from flagellum to the entire surface of the parasite, altered AQP1K12A or AQP1K12R was restricted to flagellum only. Our data demonstrate that lysine 12 is targeted for proteasomal degradation of AQP1 and plays an integral role in subcellular localization of AQP1 as well as its interaction with MPK2. This work also raises the possibility that a strategy combining antimonial with a proteasome inhibitor may be an effective combination regimen against diverse forms of leishmaniasis.

Generation of an aquaglyceroporin AQP1 null mutant in Leishmania major.

The Leishmania aquaglyceroporin AQP1 plays an important physiological role in water and uncharged polar solutes transport, volume regulation, osmotaxis, and is a key determinant of antimony resistance. By targeted gene disruption, we generated a Leishmania major promastigote AQP1 null mutant. This required several attempts but a chromosomal null AQP1 mutant was obtained by loss of heterozygosity in the presence of a rescue plasmid encoding AQP1. Growth in the absence of selection led to the loss of the rescuing plasmid, indicating that AQP1 is not essential for Leishmania viability. The AQP1-null mutant was resistant to antimonyl tartrate (SbIII) and arsenite (AsIII) due to a decrease import of these metalloids. It also exhibited alterations in its osmoregulation abilities compared with wild-type cells. This is the first report of the generation of a genetic AQP1 null mutant in Leishmania parasite, confirming its physiological function and role in resistance to antimonials, the therapeutic mainstay against Leishmania.

Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1.

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3'-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species.

Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase.

Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defence against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites coexpressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr-197 and this phosphorylation requires LmjMPK2 activity. Lys-42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild-type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. Leishmania mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild-type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a mitogen-activated protein kinase.

The role of alanine 163 in solute permeability of Leishmania major aquaglyceroporin LmAQP1.

Leishmania major aquaglyceroporin LmAQP1 allows adventitious passage of antimonite, an activated form of the drug Pentostam, which is used as the first line treatment for leishmaniasis. The extracellular C-loop of an aquaglyceroporin confers substrate specificity. Alteration of Glu125 to serine in the Plasmodium falciparum aquaglyceroporin PfAQP has been shown to selectively affect water but not glycerol permeability. The C-loop of LmAQP1 is twelve residues longer than PfAQP, and Ala163 is at an equivalent position as Glu125 of PfAQP. The role of Ala163 in LmAQP1 solute permeability was investigated. Alteration of Ala163 to serine or threonine did not significantly affect conduction of solutes. However, alteration to aspartate, glutamate, and glutamine blocked passage of water, glycerol, and other organic solutes. While LmAQP1 is a mercurial insensitive water channel, mutation of the adjacent threonine (Thr164) to cysteine led to inhibition of water passage by Hg(2+). This inhibition could be reversed upon addition of ß-mercaptoethanol. These data suggest that, unlike Glu125 (PfAQP), Ala163 is not involved in stabilization of the C-loop and selective solute permeability. Ala163 is located near the pore mouth of the channel, and replacement of Ala163 by bulkier residue sterically hinders the passage of solutes. Alteration of Ala163 to serine or threonine affected metalloid uptake in the order, wild-type>A163S>A163T. Metalloid conduction was near completely blocked when Ala163 was mutagenized to aspartate, glutamate, or glutamine. Mutations such as A163S and A163T that reduced the permeability to antimonite, without a significant loss in water or solute conductivity raises the possibility that, subtle changes in the side chain of the amino acid residue in position 163 of LmAQP1 may play a role in drug resistance.

Monitoring of intracellular nitric oxide in leishmaniasis: its applicability in patients with visceral leishmaniasis.

Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 µM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 µM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds.

Elevated levels of tryparedoxin peroxidase in antimony unresponsive Leishmania donovani field isolates.

Enhancement of the anti-oxidant metabolism of Leishmania parasites, dependent upon the unique dithiol trypanothione, has been implicated in laboratory-generated antimony resistance. Here, the role of the trypanothione-dependent anti-oxidant pathway is studied in antimony-resistant clinical isolates. Elevated levels of tryparedoxin and tryparedoxin peroxidase, key enzymes in hydroperoxide detoxification, were observed in antimonial resistant parasites resulting in an increased metabolism of peroxides. These data suggest that enhanced anti-oxidant defences may play a significant role in clinical resistance to antimonials.

Over expression and analysis of O-glycosylated recombinant human granulocyte colony stimulating factor in Pichia pastoris using Agilent 2100 Bioanalyzer.

Recombinant human granulocyte colony stimulating factor (rhGCSF) was expressed in methylotrophic yeast Pichia pastoris under the control of AOX1 promoter after integration of the GCSF gene into P. pastoris genome. Methanol induction of the Pichia integrants yielded only 2mgl(-1) of rhGCSF whereas inclusion of surfactants during induction enhanced the yields to the level of 200-250mgl(-1) in shake flask studies after 72h of induction. Preliminary studies in a bioreactor showed rhGCSF expression levels of 6mg rhGCSF g(-1) methanol day(-1) which is significantly higher to the reported value of 0.4mg rhGCSF g(-1) methanol day(-1) reported till date for Pichia derived rhGCSF. A single step purification protocol of shake flask derived rhGCSF yielded homogenous rhGCSF protein of >99% purity. Even though, purified rhGCSF showed a single band on reducing SDS-PAGE, examination of the same protein on Agilent 2100 Bioanalyzer, revealed two closely unresolved peaks. Such a pattern was also observed for crude rhGCSF preparations. Mutagenesis of the O-glycosylation site of rhGCSF (Thr(133) to Leu(133)) showed a single peak on bioanalyzer, which overlapped with the peak obtained for a non-glycosylated rhGCSF. Our data discloses for the first time the novel use of Agilent Bioanalyzer to detect glycoforms of proteins in crude and purified preparations and such a tool could be easily applied for glycoprotein profiling of monoclonal antibodies and other fusion proteins expressed in mammalian cells. This is the first report of a simple, rapid, sensitive and a cost-efficient tool for detection of glycoproteins.

Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates.

The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.

Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate.

Leishmania parasites lack catalase and therefore, their anti-oxidant system hinges primarily upon non-protein thiols; accordingly, depletion of thiols could potentially serve as an effective drug target. We have developed a flow cytometry based assay using 5-chloromethyl fluorescein diacetate based upon its selective staining of non-protein thiols. Its specificity was confirmed using buthionine sulphoximine (a gamma-glutamyl cysteine synthetase inhibitor), diamide (an oxidizing agent of intracellular thiols) and N-ethylmaleimide (a covalent modifier of cysteine residues) as evidenced by reduction in fluorescence; furthermore, restoration of fluorescence by N-acetyl cysteine corroborated specificity of 5-chloromethyl fluorescein diacetate to measure non-protein thiols. Differences in basal level of thiols in antimony sensitive and antimony resistant Leishmania field isolates were detected. The depletion of non-protein thiols by conventional anti-leishmanial drugs e.g. antimony and miltefosine was demonstrated. Furthermore, fluorescence was unaffected by depletion of ATP in majority of the strains studied, indicating that 5-chloromethyl fluorescein diacetate is not a substrate for the pump operative in most Leishmania donovani strains. Taken together, measurement of 5-chloromethyl fluorescein diacetate fluorescence is an effective method for monitoring non-protein thiols in Leishmania promastigotes.

Over-expression of proteins using a modified pBAD24 vector in E. coli expression system.

A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine-Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/10(6) c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10(6) c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels.

Cytotoxicity of Senecio in macrophages is mediated via its induction of oxidative stress.

In Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The present investigation was undertaken to demonstrate the pro-oxidant effects of an ethanolic extract of Senecio chrysanthemoides (S-EtOH). S-EtOH impaired viability in macrophages, the IC(50) being 13.8+/-1.11 microg/mL. The effect of S-EtOH (1 microg/mL) on generation of reactive oxygen species (ROS) in macrophages was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate (H(2)DCFDA) where it caused a significant increase in the mean fluorescence channel (MFC) from 8.55+/-0.03 to 47.32+/-2.25 (p<0.001). S-EtOH also effected a 3.8-fold increase in extracellular nitric oxide (NO) generation from 4.90+/-0.72 microM to 18.79+/-0.32 microM (p<0.001), a 2.2-fold increase in intracellular NO production, the MFC increasing from 14.95+/-0.48 to 33.34+/-1.66 (p<0.001), and concomitantly depleted non protein thiols as analyzed by flow cytometry using mercury orange, with a reduction in MFC from 632.5+/-49.44 to 407.4+/-12.61 (p<0.01). Additionally, S-EtOH (14 microg/mL, 24h) caused apoptosis as evident by increased Annexin V binding and terminal deoxynucleotidyl transferase mediated dUTP DNA nick end labeling. Taken together, the cytotoxicity of S-EtOH can be partly attributed to its capacity to inflict oxidative damage via generation of both reactive oxygen and nitrogen species culminating in apoptosis.

High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris.

Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (approximately 1 g/l) of extracellular glycosylated rSAK (approximately 18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (approximately 15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.

High yielding recombinant Staphylokinase in bacterial expression system--cloning, expression, purification and activity studies.

Staphylokinase (SAK) is emerging as an important thrombolytic agent. In this report, we describe the cloning, expression, purification and activity studies of the SAK gene of Staphylococcus aureus from a custom synthesised SAK gene. The SAK gene of 411 bp yielded a protein of approximately 15 kDa when expressed under pET21a vector using IPTG as an inducer in BL21 (DE3) pLysE codon Plus cells. The recombinant SAK (rSAK) was soluble in nature and constituted nearly 35% of the total cellular protein as estimated by densitometry scanning. Fermentation studies were carried out to optimize various parameters for maximizing the yield of rSAK and with the optimized medium, the yield of rSAK was nearly 2.8 g/L of fermentation broth, which is highest yield of rSAK expressed in any bacterial system till date. Two simple purification steps of ion-exchange chromatography yielded homogenous rSAK with almost 36% recovery. The purified SAK protein was characterized by MALDI-TOF and by plasminogen activation studies. The rSAK was found to be active by the chromogenic substrate assay method.

Anti-inflammatory effect of allylpyrocatechol in LPS-induced macrophages is mediated by suppression of iNOS and COX-2 via the NF-kappaB pathway.

The crude ethanol extract of Piper betle leaf is reported to possess anti-inflammatory activity which has been suggested to be mediated by allylpyrocatechol (APC). In the present study, we have demonstrated the anti-inflammatory effects of APC (10 mg/kg, p.o.) in an animal model of inflammation. To investigate the mechanism(s) of this anti-inflammatory activity, we examined its effects on the lipopolysaccaride (LPS)-induced production of NO and PGE(2) in a murine macrophage cell line, RAW 264.7. APC inhibited production of NO and PGE(2) in a dose dependent manner as also decreased mRNA expression of iNOS, COX-2, IL-12p40 and TNF-alpha. Since nuclear factor-kappaB (NF-kappaB) appears to play a central role in transcriptional regulation of these proteins, we investigated the effects of APC on this transcription factor. APC inhibited LPS induced nuclear factor-kappaB (NF-kappaB) activation, by preventing degradation of the inhibitor kappaB (IkappaB). Taken together, our data indicates that APC targets the inflammatory response of macrophages via inhibition of iNOS, COX-2 and IL-12 p40 through down regulation of the NF-kappaB pathway, indicating that APC may have therapeutic potential in inflammation associated disorders.

An ethanolic extract of leaves of Piper betle (Paan) Linn mediates its antileishmanial activity via apoptosis.

An unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for its antimicrobial properties but its antileishmanial potential has not been studied. Accordingly, an ethanolic extract of leaves of Piper betle (PB) was tested for its antileishmanial activity that was evidenced in both promastigotes and amastigotes, with IC50 values of 9.8 and 5.45 microg/ml, respectively; importantly, it was accompanied by a safety index of >12-fold. This leishmanicidal activity of PB was mediated via apoptosis as evidenced by morphological changes, loss of mitochondrial membrane potential, in situ labeling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling, and cell-cycle arrest at the sub-G0/G1 phase. Taken together, the data indicate that PB has promising antileishmanial activity that is mediated via programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes.

A major impediment to effective anti-leishmanial chemotherapy is the emergence of drug resistance, especially to sodium antimony gluconate, the first-line treatment for leishmaniasis. Artemisinin, a sesquiterpene lactone isolated from Artemisia annua, is an established anti-malarial compound that showed anti-leishmanial activity in both promastigotes and amastigotes, with IC(50) values of 160 and 22 microM, respectively, and, importantly, was accompanied by a high safety index (>22-fold). The leishmanicidal activity of artemisinin was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, in situ labelling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G(0)/G(1) phase. Taken together, these data indicate that artemisinin has promising anti-leishmanial activity that is mediated by programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.