Comparative Proteomics of Inner Membrane Fraction from Carbapenem-Resistant Acinetobacter baumannii with a Reference Strain

Acinetobacter baumannii has been identified by the Infectious Diseases Society of America as one of the six pathogens that cause majority of hospital infections. Increased resistance of A. baumannii even to the latest generation of β-lactams like carbapenem is an immediate threat to mankind. As inner-membrane fraction plays a significant role in survival of A. baumannii, we investigated the inner-membrane fraction proteome of carbapenem-resistant strain of A. baumannii using Differential In-Gel Electrophoresis (DIGE) followed by DeCyder, Progenesis and LC-MS/MS analysis. We identified 19 over-expressed and 4 down-regulated proteins (fold change>2, p<0.05) in resistant strain as compared to reference strain. Some of the upregulated proteins in resistant strain and their association with carbapenem resistance in A. baumannii are: i) β-lactamases, AmpC and OXA-51: cleave and inactivate carbapenem ii) metabolic enzymes, ATP synthase, malate dehydrogenase and 2-oxoglutarate dehydrogenase: help in increased energy production for the survival and iii) elongation factor Tu and ribosomal proteins: help in the overall protein production. Further, entry of carbapenem perhaps is limited by controlled production of OmpW and low levels of surface antigen help to evade host defence mechanism in developing resistance in A. baumannii. Present results support a model for the importance of proteins of inner-membrane fraction and their synergistic effect in the mediation of resistance of A. baumannii to carbapenem

Gene regulation by long purine tracks in brain related diseases

Purine repeats are randomly distributed in the human genome, however, they show potential role in the transcriptional deregulation of genes. Presence of long tracks of purine repeats in the genome can disturb its integrity and interfere with the cellular behavior by introducing mutations and/or triple stranded structure formation in DNA. Our data revealed interesting finding that a majority of genes carrying purine repeats, of length n≥200, were down regulated and found to be linked with several brain related diseases [1]. The unique feature of the purine repeats found in the present study clearly manifests their significant application in developing therapeutics for neurological diseases

Anticancer activity of HMGA1 promoter targeting triplex forming oligonucleotide in HeLa cell line

219-229High mobility group protein A1 (HMGA1) acts as an architectural transcription factor and regulates transcription of various genes. Upregulation of HMGA1 has been described in a large number of human malignancies and serves as a ‘tumor marker’. Due to its role in neoplastic transformation and tumor progression, hmga1 is considered as a promising therapeutic target. In the present study, we investigated the interaction of triplex forming oligonucleotide (TFO) of 18 bps targeted to hmga1 promoter (-1917 to -1940) and its influence on the expression of HMGA1 in HeLa cells. Stability of DNA triplex was characterized using various biophysical and thermodynamic methods and was confirmed by gel retardation assay using γ-32P [ATP]. Treatment of HeLa cells with hmga1 specific TFO significantly downregulated HMGA1 expression at mRNA, protein levels (~48%) and inhibited cell proliferation as investigated by RT-PCR, Western blot and Flow cytometry. The findings of the study suggest that TFO-mediated inhibition of hmga1 expression can be a promising strategy for modulation of gene expression and for inhibition of cancer cell proliferation. Moreover, DNA triplex-based therapeutic approaches hold promise in combating cancers associated with HMGA1 overexpression

Identification of genes containing expanded purine repeats in the human genome and their apparent protective role against cancer

<p>Purine repeat sequences present in a gene are unique as they have high propensity to form unusual DNA-triple helix structures. Friedreich’s ataxia is the only human disease that is well known to be associated with DNA-triplexes formed by purine repeats. The purpose of this study was to recognize the expanded purine repeats (EPRs) in human genome and find their correlation with cancer pathogenesis. We developed “<i>PuRepeatFinder.pl</i>” algorithm to identify non-overlapping EPRs without pyrimidine interruptions in the human genome and customized for searching repeat lengths, <i>n</i> ≥ 200. A total of 1158 EPRs were identified in the genome which followed <i>Wakeby</i> distribution. Two hundred and ninety-six EPRs were found in geneic regions of 282 genes (EPR-genes). Gene clustering of EPR-genes was done based on their cellular function and a large number of EPR-genes were found to be enzymes/enzyme modulators. Meta-analysis of 282 EPR-genes identified only 63 EPR-genes in association with cancer, mostly in breast, lung, and blood cancers. Protein–protein interaction network analysis of all 282 EPR-genes identified proteins including those in cadherins and VEGF. The two observations, that EPRs can induce mutations under malignant conditions and that identification of some EPR-gene products in vital cell signaling-mediated pathways, together suggest the crucial role of EPRs in carcinogenesis. The new link between EPR-genes and their functionally interacting proteins throws a new dimension in the present understanding of cancer pathogenesis and can help in planning therapeutic strategies. Validation of present results using techniques like NGS is required to establish the role of the EPR genes in cancer pathology.</p

Differential expression of Outer membrane proteins in early stages of meropenem-resistance in Acinetobacter baumannii

ABSTRACT Acinetobacter baumannii has emerged as one of the six most important drug-resistant microbes in the world. Resistance by A. baumannii to β-lactams and in particular to meropenem is a serious concern. In this connection, it is essential to understand the changes in the outer membrane proteome of A. baumannii in the initial stages of resistance. For this we have chosen one low resistant strain with MIC of 32 μg/ml and one intermediate strain with very low MIC of 0.8 μg/ml of meropenem and compared their outer membrane profiles with that of sensitive strain, ATCC 19606 of A. baumannii. Decreased expression of porins, transporters and increased production of metabolic enzymes like Succinyl-CoA synthetase, enoyl-CoA hydratase is a common feature in both intermediate strain and low resistant strains. Interestingly, the differential protein expression levels showed a direct relationship with increasing meropenem resistance. It is clear that initial exposure to meropenem resistance drives A. baumannii to restrict the production of OmpAb, CarO, transporters, while the upregulation of genes of altered CarO, metabolic enzymes, peroxidines and antioxidant protein assist in the survival of the bacterium. Because of these unique features of adaptation combined with high metabolic changes in response to antibiotic pressure, A. baumannii poses challenges in therapeutic strategies

Differential expression of hyaluronic acid binding protein 1 (HABP1)/P32/C1QBP during progression of epidermal carcinoma

Our laboratory has characterized a novel cell surface glycoprotein, Hyaluronic Acid Binding Protein 1 (HABP1), interacting specifically with hyaluronan (HA) and regulating HA-mediated cellular event. The involvement of HA in different stages of carcinoma is well documented. In the present communication, the expression profile of HABP1 was investigated from initiation to progression of epidermal carcinoma in mice, induced by benzo[a]pyrene (B[a]P) exposure. During tumor initiation, HABP1 accumulated in inflammatory subsquamous tissue and with progression, the protein, was also seen to overexpress in papillomatic and acanthotic tissue. With the onset of metastasis, HABP1 overexpression was confined to metastatic islands, while it disappeared gradually from the surrounding mass. Such expression profiles in metastasized tissue were supported by decreased levels of HABP1, both at protein and transcript levels. These observations taken together suggest that the changes in HABP1 level coincide with specific stages of tumor progression, that lead to disruption of its interaction with HA, implying a role in the regulation of tumor metastasis. (Mol Cell Biochem 267: 133-139, 2004)

Interaction of adriamycin with a regulatory element of hmgb1: spectroscopic and calorimetric approach

HMGB1 is a non-histone nuclear protein which plays important role in transcription, variable, diverse and joining (VDJ) recombination, chromatin remodeling, and DNA repair, etc. and its over expression is directly correlated with various human malignancies and inflammatory diseases. Because of the clear association between HMGB1 and cancer, we studied the binding of adriamycin (ADM), a well-known anticancer drug with the promoter region (-165 to -183) of hmgb1 by using a variety of spectroscopic, calorimetric techniques, and in-silico molecular modeling. Changes in UV and CD spectral characteristics (intensity and wavelength) of ADM and DNA associated with an induced peak (300nm) in CD spectrum of DNA and a high binding constant of 2.0x10(5)M(-1) suggest a strong and stable complex formation between DNA and ADM. Scatchard analysis of spectroscopic data indicate that ADM binds to DNA in a non-cooperative nature. Further the quenching of fluorescence emission of ADM and isothermal titration calorimetry of ADM in presence of DNA points out to the intercalative mode of ADM binding to DNA which is enthalpically driven with additional small entropic contribution. Results from molecular modeling, Isothermal titration calorimetry, and Fourier transform infrared spectroscopy reveal that ADM has no marked preference between AT vs. GC base pair in binding to DNA. Therefore, hmgb1 can be considered as a novel potential chemotherapeutic target in treating cancers associated with HMGB1 upregulation