Anti-proliferative and metastatic protease inhibitory activities of protoberberines: An in silico and in vitro approaches

In metastasis, tumour cells interact with numerous factors and one of them is extracellular matrix (ECM). Earlier studies have emphasised the on role of matrix metalloproteases (MMPs) and cathepsin B in ECM degradation and cancer metastasis. In silico docking approaches are a boon in exploring the physicochemical characteristics like angiogenesis, growth and repair of cancerous cells. The binding affinities of berberrubine, jatrorrhizine and thalifendine with caspase 3, MMP-9, cathepsin B and telomeric DNA were performed using hex 6v and iGEMDOCK v2.1 software tools. The present investigation on berberrubine, jatrorrhizine and thalifendine revealed the formation of potentially stable complexes with caspase 3, cathepsin B and telomeric DNA similar to doxorubicin, a well-known anticancer drug. Further, in vitro approaches were made to study anti-proliferative activity against colon, lymphoma and ovarian cancer cell lines and enzyme inhibition activity against MMP-9 and cathepsin B. The results obtained reveal that these protoberberines alkaloids have potential inhibitory effect on cancer cell proliferation as well as metastatic proteases.     

Molecular Characterization of Echinococcus granulosus Cysts in North Indian Patients: Identification of G1, G3, G5 and G6 Genotypes

Background

Cystic echinococcosis (CE) caused by the Echinococcus granulosus, is a major public health problem worldwide, including India. The different genotypes of E. granulosus responsible for human hydatidosis have been reported from endemic areas throughout the world. However, the genetic characterization of E. granulosus infecting the human population in India is lacking. The aim of study was to ascertain the genotype(s) of the parasite responsible for human hydatidosis in North India.

Methodology/Principal Findings

To study the transmission patterns of E. granulosus, genotypic analysis was performed on hydatid cysts obtained from 32 cystic echinococcosis (CE) patients residing in 7 different states of North India. Mitochondrial cytochrome c oxidase subunit1 (cox1) sequencing was done for molecular identification of the isolates. Most of the CE patients (30/32) were found to be infected with hydatid cyst of either G3 (53.1%) or G1 (40.62%) genotype and one each of G5 (cattle strain) and G6 (camel strain) genotype.

Conclusions/Significance

These findings demonstrate the zoonotic potential of G1 (sheep strain) and G3 (buffalo strain) genotypes of E. granulosus as these emerged as predominant genotypes infecting the humans in India. In addition to this, the present study reports the first human CE case infected with G5 genotype (cattle strain) in an Asian country and presence of G6 genotype (camel strain) in India. The results may have important implications in the planning of control strategies for human hydatidosis.     

Molecular docking studies and in vitro screening of new dihydropyridine derivatives as human MRP1 inhibitors

The overexpression of multidrug resistance protein 1 (MRP1) by tumor cells results in multidrug resistance (MDR) to structurally unrelated anticancer drugs. Circumvention of MDR by combination of chemosensitizers with antitumor compounds is a new field of investigation in cancer chemotherapy. Much effort has been put-in recently to identify the modulators/inhibitors of MRP1 to overcome the MDR. 1,4-Dihydropyridine (DHP) derivatives are indicated to be a new class of MRP1 inhibitors in cancer treatment. Molecular docking studies were carried out on 48 newly synthesized DHP derivatives with the crystal structure of MRP1 to gain some structural insights on the binding mode and possible interactions with the active site of MRP1 (NBD1). The 10 top-ranked molecules were selectively evaluated, experimentally for their MRP1 inhibitory effect using the insect cell membrane MRP1 ATPase assay. The inhibitory capacity (IC(50) concentrations) of the test compounds was compared with the reported IC(50)- or the K(i)-concentrations for benzbromarone, a standard MRP1 inhibitor. Amongst the compounds tested, compounds IA(1) and IIA(5) were found to exhibit a potent MRP1 inhibitory action with IC(50) values of 20±4 and 14±2 μM (mean±SD), respectively as compared to benzbromarone (IC(50)=4 μM). The compound IIA(5), in particular was found to be more potent than IA(1) in accordance with the docking results. These new DHP derivatives possess promising characteristics for their development as MDR reversal agents.     

Trichomonas vaginalis: random amplified polymorphic DNA analysis of isolates from symptomatic and asymptomatic women in India

Trichomonas vaginalis, the causative agent for human trichomoniasis, is a protozoan parasite. Trichomoniasis is the most common non-viral sexually transmitted disease. The infection in women may be asymptomatic or may lead to severe vaginitis, cervicitis and severe sequelae. Despite its high prevalence, the genetic variability and factors leading to symptomatic infection have been poorly understood. One thousand women in childbearing age group were screened for the presence of T vaginalis. Thirty-eight women were found positive for T vaginalis and out of these 22 (57.9%) were having symptomatic infection and 16 (42%) were asymptomatic. Fresh isolates from 15 symptomatic and 15 asymptomatic women were axenised and subjected to random amplified polymorphic DNA (RAPD) analysis with the use of five different random primers (OPD 1-OPD 5). The isolates with similar banding pattern were assigned as a single type. OPD 3 indicated least (nine types) while OPD 4 indicated highest typing (18 types) ability. Phylogenetic analysis using RAPD distance software indicated two distinct lineages; upper branch consisting of only seven symptomatic isolates while lower branch consisting of all the 15 asymptomatic isolates the other eight symptomatic isolates were recorded in separate cluster. The study indicated that RAPD technique might be helpful to delineate the pathogenic mechanism(s) for its virulence; however, further studies on large number of isolates are desired to elucidate the findings.     

20-hydroxyeicosatetraenoic acid (20-HETE): structural determinants for renal vasoconstriction

The effects of natural and synthetic eicosanoids on the diameter of rat interlobular arteries studied in vitro were compared to that of the potent, endogenous vasoconstrictor 20-HETE. Vasoconstrictor activity was optimum for chain lengths of 20-22 carbons with at least one olefin or epoxide between located between C(13)-C(15) and an oxygen substituent at C(20)-C(22). The presence of delta (Zou et al. Am. J. Physiol. 1996, 270, R228; Gebremedhin, D. et al. Am. J. Physiol. 1998, 507, 771)-, delta (Carroll et al. Am. J. Physiol. 1996, 271, R863; Vazquez et al. Life Sci. 1995, 56, 1455)-, or delta (Imig et al. Hypertension 2000, 35, 307; Lopez et al. Amer. J. Physiol. 2001, 281, F420)-olefins had no influence on the vasoconstrictor response whereas the introduction of a C(7)-thiomethylene enhanced potency. A sulfonamide or alcohol, but not a lactone, could replace the C(1)-carboxylate. These data were used to construct a putative binding domain map of the 20-HETE receptor consisting of: (i) a comparatively open, hydrophilic binding site accommodating the C(1)-functionality; (ii) a hydrophobic trough spanning the olefins; (iii) a shallow pocket containing a critical pi-pi binding site in the vicinity of the pi (Ito et al. Am. J. Physiol. 1998, 274, F395; Quigley, R.; Baum, M.; Reddy, K. M.; Griener, J. C.; Falck, J. R. Am. J. Physiol. 2000, 278, F949)-olefin; and (iv) an oxyphilic binding site proximate to the omega-terminus.     

Phylogenetic identity, growth-response time and rRNA operon copy number of soil bacteria indicate different stages of community succession

Bacterial community succession corresponds to changes in the phylogenetic identity, growth-response time and rRNA operon (rrn) copy number of culturable populations. To test this hypothesis, we compared the bacterial fractions culturable from the oxic zone of flooded, unplanted paddy soil microcosms after 1-day (early succession) and 70-day (late succession) incubation periods. The proportion of bacteria that was cultivable on solid media corresponded for early and late succession to 37-40% and 31-35% of total DAPI cell counts, which were 7.40 (+/-0.36) x 10(8) and 5.54 (+/-0.28) x 10(8) cells per gram of dry soil, respectively. In colony-forming curve analysis, late successional bacteria showed a significant delay in their growth response compared with those from early succession. A total of 59 early successional isolates grouped into 16 species-level clusters (SLC) plus three Bacilli-like SLC, while 66 late successional isolates formed 25 SLC plus five Bacilli-like SLC. Except Bacilli-like spp., isolates from early succession always belonged to different SLC than those from late succession. Betaproteobacteria and Gammaproteobacteria were typical of the early stage, while Alphaproteobacteria and Actinobacteria prevailed in late succession. Considering all SLC except those assigned to Bacilli, growth-response time and rrn copy number were significantly correlated with successional stage. Isolates of most early successional SLC (14 of 16) formed visible colonies within 1 (11 SLC) or 2 days (three SLC) and contained >or= 4 rrn copies. In contrast, isolates of late successional SLC (23 of 25) formed visible colonies within 2 days (four SLC) or, in most cases, only within 3-15 days (19 SLC) and contained 相似文献   

Enhancement of clavulanic acid by replicative and integrative expression of ccaR and cas2 in Streptomyces clavuligerus NRRL3585

Clavulanic acid (CA) is an inhibitor of beta-lactamase that is produced from Streptomyces clavuligerus NRRL3585 and is used in combination with other antibiotics in clinical treatments. In order to increase the production of CA, the replicative and integrative expressions of ccaR (encoding for a specific regulator of the CA biosynthetic operon) and cas2 (encoding for the rate-limiting enzyme in the CA biosynthetic pathway) were applied. Six recombinant plasmids were designed for this study. The pIBRHL1, pIBRHL3, and pIBRHL13 were constructed for overexpression, whereas pNQ3, pNQ2, and pNQ1 were constructed for chromosomal integration with ccaR, cas2, and ccaR-cas2, respectively. All of these plasmids were transformed into S. clavuligerus NRRL3585. CA production in transformants resulted in a significantly enhanced amount greater than that of the wild type, a 2.25-fold increase with pIBRHL1, a 9.28-fold increase with pNQ3, a 5.06-fold increase with pIBRHL3, a 2.93-fold increase with pNQ2 integration, a 5.79-fold increase with pIBRHL13, and a 23.8-fold increase with pNQ1. The integrative pNQ1 strain has been successfully applied to enhance production.     

In vitro effect of larval stages of Ascaris lumbricoides on human blood clotting

The effects of larval stages of Ascaris lumbricoides on human blood clotting was studied in vitro. Extracts and excretory/secretory products of third-stage larvae (L3) and late third-stage larvae (LL3) cultured from ova obtained from infected patients were analysed for anti-coagulant activity. Prothrombin time (PT) was prolonged by the addition of either whole extract of L3/LL3 or ES products of L3/LL3 as compared to controls. Partial thromboplastin time with kaolin (PTTK) was also prolonged on the addition of either extracts of ES products of L3/LL3. The prolongation of PTTK was significantly higher with extracts/ES products of L3 when compared to the extracts/ES products of LL3 (p less than 0.005). Thrombin time (TT) was prolonged by extracts of L3/LL3 and their ES products.     

Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH₄ into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH₄ flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using ¹³C-labelled CH₄ directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to ¹³CH₄ for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.