Effect of MMP-2 gene silencing on radiation-induced DNA damage in human normal dermal fibroblasts and breast cancer cells.

INTRODUCTION: Diagnostic and therapeutic ionizing radiation (IR) is one of the well known long term risk factors of breast cancer. Extremely lethal consequences of IR causes double-strand breaks, which are mainly responsible for genomic instability, altered gene expression, and cell death. FINDINGS: This study evaluated the effect of matrix metalloproteinases-2 (MMP-2) gene silencing using MMP-2 shRNA expression plasmids (pMMP-2) on IR induced cytotoxicity and DNA damage by MTT, dead green, γH2AX and comet assays in human normal dermal fibroblasts (HDFs) and MCF-7 human breast cancer cells. IR has decreased the viability of HDFs and MCF-7 cells with increasing IR (2-10Gy). IR induced DNA damage in both HDFs and MCF-7 cells. However, pMMP-2 transfection has increased the viability of irradiated HDFs (10Gy) and significantly decreased the viability of irradiated MCF-7 cells (10Gy). Further, DNA damage in terms of γH2AX foci decreased with pMMP-2 transfection in irradiated HDFs (10Gy) and increased in irradiated MCF-7 cells (10Gy). In addition, MMP-2 gene silencing using pMMP-2 decreased comet tail length in irradiated HDFs but increased in irradiated MCF-7 cells. CONCLUSIONS: The results conclude that pMMP-2 has protected HDFs and sensitized the MCF-7 cells from IR induced DNA damage. This differential response might be due to IR induced MMP-2 distinctive ROS generation in HDFs and MCF-7 cells.

Zinc Transporter Protein (tzn-1) May Also Play a Role in Conidiation Pathway of Neurospora crassa: An Insight Using Proteogenomic Approach.

BACKGROUND: Zinc transporter (tzn-1) of Neurospora crassa plays a crucial role in conidiation pathway, as its removal results in aconidiation which was reported in our earlier studies. OBJECTIVES: The main objective of this study was to analyze the role of tzn-1 in conidiation process, by comparing knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) with wild oak ridge (OR) 74 'A' strain by 'Proteo-genomic' approach. METHODS: To identify the commonly expressed protein spots in knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) by comparing with wild oak ridge (OR) 74 'A' strain. Two sets (Δtzn-1 to wild and Δacon-3 to wild) were analyzed by combining 2- Dimensional gel electrophoresis (2DE) with Matrix Associated Laser Desortion/Ionization mass spectrometry -Peptide Mass Fingerprint (MALDI-PMF). Then, the peptide sequences which were obtained by MASCOT (database software) were identified by FGSC BLASTp search analysis. Finally, to evaluate the expression of the KO mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) in comparison to wild (OR) 74 'A' type was analyzed by Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) studies. RESULTS: 2DE and MALDI-PMF has shown the nine commonly overexpressed protein spots from the two sets (Δtzn-1 to wild and Δacon-3 to wild). Peptide sequences were obtained by MASCOT (database software) analysis and peptide sequences were identified by FGSC BLASTp search. Eight sequences have shown the similarities with the genes involved during the early stages of conidial and sexual development. Our qRT-PCR analysis has shown that tzn-1 gene was upregulated in contrast to acon-3 gene in absence of iron concentration and down regulated with increase in iron concentrations in wild samples. With increase in zinc supplements, the tzn-1 gene is normally regulated and shown contrasting feature in absence of zinc and acon-3 gene is normally regulated both in presence and absence of zinc. At regular time intervals, declined growth rate was observed after 18hours of induction. CONCLUSION: Thus, we conclude that tzn-1 and acon-3 genes were actively participating in early stages of conidial process and metal ions play some crucial role in the development of the organism.

Screening of Potential Lead Molecule as Novel MurE Inhibitor: Virtual Screening, Molecular Dynamics and In Vitro Studies.

BACKGROUND: The prevalence of multi-drug resistance S. aureus is one of the most challenging tasks for the treatment of nosocomial infections. Proteins and enzymes of peptidoglycan biosynthesis pathway are one among the well-studied targets, but many of the enzymes are unexplored as targets. MurE is one such enzyme featured to be a promising target. As MurE plays an important role in ligating the L-lys to stem peptide at third position that is crucial for peptidoglycan synthesis. OBJECTIVE: To screen the potential MurE inhibitor by in silico approach and evaluate the best potential lead molecule by in vitro methods. METHOD: In the current study, we have employed structure based virtual screening targeting the active site of MurE, followed by Molecular dynamics and in vitro studies. RESULTS: Virtual screening resulted in successful screening of potential lead molecule ((2R)-2-[[1-[(2R)- 2-(benzyloxycarbonylamino) propanoyl] piperidine-4-carbonyl]amino]-5-guanidino-pentan). The molecular dynamics of the MurE and Lead molecule complex emphasizes that lead molecule has shown stable interactions with active site residues Asp 406 and with Glu 460. In vitro studies demonstrate that the lead molecule shows antibacterial activity close to standard antibiotic Vancomycin and higher than that of Ampicillin, Streptomycin and Rifampicin. The MIC of lead molecule at 50µg/mL was observed to be 3.75 µg/mL, MBC being bactericidal with value of 6.25 µg/mL, cytotoxicity showing 34.44% and IC50 of 40.06µg/mL. CONCLUSION: These results suggest ((2R)-2-[[1-[(2R)-2-(benzyloxycarbonylamino) propanoyl] piperidine-4-carbonyl]amino]-5-guanidino-pentan) as a promising lead molecule for developing a MurE inhibitor against treatment of S. aureus infections.

Antineoplastic activity of monocrotaline against hepatocellular carcinoma.

Plants are fantastic sources for present day life saving drugs. Monocrotaline a natural ligand exhibits dose-dependent cytotoxicity with potent antineoplastic activity. This study was intended to disclose the therapeutic potential of monocrotaline against hepatocellular carcinoma. The in silico predictions have highlighted the antineoplastic potential, druglikeness and biodegradability of monocrotaline. The in silico docking study has provided an insight and evidence for the antineoplastic activity of monocrotaline against p53, HGF and TREM1 proteins which play a threatening role in causing hepatocellular carcinoma. The mode of action of monocrotaline was determined experimentally by in vitro techniques such as XTT assay, NRU assay and whole cell brine shrimp assay have further supported our in silico studies. The in vitro cytotoxicity of monocrotaline was proved at IC50 24.966 µg/mL and genotoxicity at 2 X IC50 against HepG2 cells. Further, the credible druglike properties with non-mutagenicity, non-toxic on mammalian fibroblast and the potential antineoplastic activity through in vitro experimental validations established monocrotaline as a novel scaffold for liver cancer with superior efficacy and lesser side effects.

Characterization of Ctr family genes and the elucidation of their role in the life cycle of Neurospora crassa.

Transcriptional analysis using qRT-PCR of 62 metal ion transporters during conidial germination of Neurospora crassa showed a significant up regulation of a hypothetical copper transporter gene, tcu-1, that belongs to the Ctr family. Herein we characterised the Ctr family genes (tcu-1, tcu-2 and tcu-3) and deciphered their role in various developmental phases of the N. crassa life cycle. Cross complementation assays in copper uptake mutant of Saccharomyces cerevisiae revealed that tcu-1, tcu-2 and tcu-3 are functional homologs of S. cerevisiae copper transporters. Expression studies of Ctr family members in various developmental phases of N. crassa showed differential expression pattern for high-affinity copper transporter, TCU1. Functional analysis of their gene knockout mutants showed that tcu-1 is essential for saprophytic conidial germination, vegetative growth and perithecia development under copper limited conditions while conidiation remained unaffected.

MrdH, a novel metal resistance determinant of Pseudomonas putida KT2440, is flanked by metal-inducible mobile genetic elements.

We report here the identification and characterization of mrdH, a novel chromosomal metal resistance determinant, located in the genomic island 55 of Pseudomonas putida KT2440. It encodes for MrdH, a predicted protein of approximately 40 kDa with a chimeric domain organization derived from the RcnA and RND (for resistance-nodulation-cell division) metal efflux proteins. The metal resistance function of mrdH was identified by the ability to confer nickel resistance upon its complementation into rcnA mutant (a nickel- and cobalt-sensitive mutant) of Escherichia coli. However, the disruption of mrdH in P. putida resulted in an increased sensitivity to cadmium and zinc apart from nickel. Expression studies using quantitative reverse transcription-PCR showed the induction of mrdH by cadmium, nickel, zinc, and cobalt. In association with mrdH, we also identified a conserved hypothetical gene mreA whose encoded protein showed significant homology to NreA and NreA-like proteins. Expression of the mreA gene in rcnA mutant of E. coli enhanced its cadmium and nickel resistance. Transcriptional studies showed that both mrdH and mreA underwent parallel changes in gene expression. The mobile genetic elements Tn4652 and IS1246, flanking mrdH and mreA were found to be induced by cadmium, nickel, and zinc, but not by cobalt. This study is the first report of a single-component metal efflux transporter, mrdH, showing chimeric domain organization, a broad substrate spectrum, and a location amid metal-inducible mobile genetic elements.

Functional characterization of tzn1 and tzn2-zinc transporter genes in Neurospora crassa.

Previous work from our laboratory involved the description of the Neurospora metal transportome, which included seven hypothetical zinc transporters belonging to the ZIP family. The aim of the present study was to make a comparative functional evaluation of two hypothetical zinc transporters named tzn1 (NCU07621.3) and tzn2 (NCU11414.3). Phenotypic analysis of tzn1 and tzn2 mutants and a double mutant (tzn1tzn2) revealed that the deletion of tzn1 causes aconidiation and a greater defect in growth than the single deletion of tzn2. Supplementation with zinc restores growth but not conidiation in tzn1 and tzn1tzn2. TZN1 complemented a zinc-uptake-deficient Saccharomyces cerevisiae mutant (zrt1zrt2) in zinc-deficient conditions, while tzn2 restored growth upon supplementation with zinc (0.05 mM). Furthermore, the Deltatzn1 mutant was found to have severely reduced zinc content indicating that tzn1 functions as a key regulator of intracellular zinc levels in Neurospora crassa. Zinc uptake studies indicate tzn1 is a specific transporter of zinc, while tzn2 transports both zinc and cadmium. Quantitative RT-PCR showed up-regulation of tzn1 (128-fold) under zinc-depleted conditions and down-regulation (>1,000-fold) in zinc-replete conditions. The present study indicates that the zinc transport proteins encoded by tzn1 and tzn2 are members of the zinc uptake system regulated by zinc status in N. crassa.

Metal transportome of Neurospora crassa.

Neurospora crassa has been the model filamentous fungus for the study of many fundamental cellular mechanisms of transport and metabolism. The recently completed genome sequence of N. crassa has over 10,000 genes without significant matches for a large number of genes (41%) in the sequence databases, indeed presents many challenges for new discoveries. Using transporter database and BLAST searches a total of 65 open reading frames for putative cation transporter genes have been identified in N. crassa. These were further confirmed by characteristic features of the family like transmembrane domains (TOPPRED 2), conserved motifs (Clustal W) and phylogenetic analysis (TREETOP). In Neurospora cation transporter genes constitute nearly 18.3% of the total membrane transport systems, which is higher than E. coli (8.8%), S. cerevisiae (13.7%), S. pombe (17.2%), A. fumigatus (10.1%), A. thaliana (16.8%) and H. sapiens (15.6%). We refer to the complete complement of metal ion transporter genes as "Metal Transportome". There are a total of 33 putative transporters for alkali and alkaline earth metals constituting 18 for calcium (P-ATPase, VIC, CaCA, Mid1), 7 for sodium (P-ATPase, CPA1, CPA2), 4 for potassium (Trk, VIC, KUP), and 4 for magnesium (MIT). Transition metal ion transporters account for 32 transporters including 7 for zinc (ZIP), 6 for copper (Ctr2, Ctr1), 2 each for manganese (Nramp), iron (OFeT), arsenite (ArsAB, ACR3) and other metal ions (ABC and P-ATPase) and 1 each for nickel (NiCoT) and chromate (CHR). N. crassa has 7 linkage groups of which LGI harbors 21 of metal ion transporters and in contrast LGVII has only 2. Studies on metal transportomes of different organisms will help to unravel the role of metal ion transporters in homeostasis.