Structural Characterization of an Exopolysaccharide Isolated from Enterococcus faecalis, and Study on its Antioxidant Activity, and Cytotoxicity Against HeLa Cells.

An exopolysaccharide (EPS-I) having the molecular weight ~ 2.6 × 105 Da, was isolated from a Zinc resistant strain of Enterococcus faecalis from costal area. The exopolysaccharide consists of D-mannose, D-glucose, and L-fucose in molar ratio of 9:4:1. The monosaccharide units in the EPS-1 were determined through chemical (total acid hydrolysis and methylation analysis) and spectroscopic (FTIR and 1H NMR experiment) analysis. The mannose-rich EPS-1 showed total antioxidant activity (1 mg mL-1 of EPS-I as functional as approximately to 500 ± 5.2 µM of ascorbic acid) and Fe2+ metal ion chelation activity (EC50 = 405.6 µg mL-1) and hydroxyl radical scavenging activity (EC50 = 219.5 µg mL-1). The in vitro cytotoxicity experiment of EPS-I against cervical carcinoma cell line, HeLa cells showed strong cytotoxic effect (LC50 = 267.3 µg mL-1) and at that concentration, it found almost nontoxic against normal healthy cells (HEK-293).

A concentration dependent auto-relay-recognition by the same analyte: a dual fluorescence switch-on by hydrogen sulfide via Michael addition followed by reduction and staining for bio-activity.

H2S is shown, for the first time, to play an extraordinary dual role due to its nucleophilicity and reducing property with our single chemosensor, PND [4-(piperidin-1-yl) naphthalene-1,2-dione]. The initial nucleophilic attack via Michael addition (a lower concentration of H2S, blue fluorescence) is followed by the reduction of the 1,2-diketo functionality (a higher concentration of H2S, green fluorescence). This chemosensor, which also shows biological response, is remarkably effective in sensing the same analyte (H2S) at its different concentrations in a relay pathway via a fluorescence "off-on-on" mechanism, and this is also supported by DFT calculation and Cyclic voltammograms.

Rapid detection of hydrazine in a naphthol-fused chromenyl loop and its effectiveness in human lung cancer cells: tuning remarkable selectivity via the reaction altered pathway supported by theoretical studies.

Our designed and synthesized chemosensor naphthalene based chromenyl derivative (NAC) [1-(3-hydroxy-3 methyl-3H-benzo[f]chromen-2-yl) ethanone] has been used for fast (<30 s, DL = 0.22 ppb) and selective detection of N2H4 by a new way via the chromenyl ring opening followed by the pyrazole ring formation giving a strong blue fluorescence. The DFT study and the real application in different water samples along with the dipstick method in low cost devices have also been performed here. Human lung cancer cells (NCI-H460) have been used for hydrazinolysis of the NAC in vivo system for detection by the appearance of blue fluorescence and also for the MTT assay showing its remarkable cancer sensitivity.

Identification of an endoplasmic reticulum membrane protein interacting with DNA polymerase beta by a yeast two-hybrid screen.

Base excision repair (BER) is a key pathway for maintaining genomic stability. A key enzyme in the BER pathway is DNA polymerase beta (polbeta). It has been shown that more than 11% of breast, bladder, esophageal, colon, and gastric cancer samples studied so far exhibit polbeta mutation. A truncated form of polbeta, polbetadelta (exon 11 deletion), identified in a colon tumour sample, exhibited dominant negative activity. Using this polbetadelta as bait, we screened a HeLa cDNA library for any interacting protein(s) in the yeast two-hybrid (Y2H) system. Polbetadelta was cloned into a pGBKT7 vector (pGBKT7-polbetadelta). pGBKT7-polbetadelta was transformed into the yeast strain AH109. Then the cDNA library was co-transformed into AH109/pGBKT7-polbetadelta and screened by the selection procedure. The yeast-purified plasmids were transformed into Escherichia coli. Plasmid DNA was isolated from the colonies, purified, digested with Sma I and Sal I, and the fragments were sequenced. Four positive clones were obtained. Out of these, three proteins were already known to interact with polbeta (XRCC1, MGC5306, and AP endonuclease 1). The only member previously not known to interact with polbeta was phosphatidylinositol glycosylase type S (PIGS). PIGS is a 64-kDa membrane protein, encoded in chromosome 17. The PIGS protein interacts also with wild-type polbeta which was confirmed by co-immunoprecipitation and Western blot analysis. The role of the newly identified protein in the dominant negative function of the variant form of polbeta remains to be seen.

Ratiometric and absolute water-soluble fluorescent tripodal zinc sensor and its application in killing human lung cancer cells.

A new "naked-eye" and ratiometric fluorescent zinc sensor (TAQ) of carboxamidoquinoline with 2-chloro-N-(quinol-8-yl)-acetamide as a receptor was designed and synthesized. The sensor shows good water solubility and high selectivity for sensing; about a 15-fold increase in fluorescence quantum yield and a 100 nm red-shift of fluorescence emission upon binding Zn²âº in aqueous HEPES buffer solution are observed. The human lung cancer cell line (A549) activity is also demonstrated.

Structure and biological properties of exopolysaccharide isolated from Citrobacter freundii.

This study aimed to investigate the molecular characterization, antioxidant activity in vitro, cytotoxicity study of an exopolysaccharide isolated from Citrobacter freundii. Firstly, the culture conditions were standardized by the Design of experiments (DoE) based approach, and the final yield of thecrude exopolysaccharide was optimized at 2568 ± 169 mg L-1. One large fraction of exopolysaccharide was obtained from the culture filtrate by size exclusion chromatography and molecular characteristics were studied. A new mannose rich exopolysaccharide (Fraction-I) with average molecular weight ~ 1.34 × 105 Da was isolated. The sugar analysis showed the presence of mannose and glucose in a molar ratio of nearly 7:2 respectively. The structure of the repeating unit in the exopolysaccharide was determined through chemical and 1D/2D- NMR experiments as: Finally, the antioxidant activity, and the cytotoxicity of the exopolysaccharide were investigated and the relationship with molecular properties was discussed as well.

Detection of Somatic Mutation in Exon 12 of DNA Polymerase ß in Ovarian Cancer Tissue Samples

Background: DNA polymerase ß (pol ß) is a key enzyme of base excision repair pathway. It is a 1-kb gene consisting of 14 exons. Its catalytic part lies between exon 8 and exon 14. Exon 12 has a role in deoxyribonucleotide triphosphate selection for nucleotide transferase activity. Methods: Genomic DNA was isolated from ovarian carcinoma samples. Single strand conformation polymorphism method was used to detect mutation in genomic DNA. Results: Twenty-four patients of the 152 pair of tumor samples (15.8%) exhibited a point mutation (C→G) in position 725 in exon 12, which shifts proline to arginine (P242R). Statistical analysis showed a significant (p < 0.001) relationship between pol ß mutation and the age of detection. Conclusion: This is a newly reported somatic mutation of pol ß in ovarian carcinoma patients from India.

HeLa Cells Containing a Truncated Form of DNA Polymerase Beta are More Sensitized to Alkylating Agents than to Agents Inducing Oxidative Stress.

The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta (polß Δ208-304) specific for ovarian cancer. Pol ß Δ208-304 has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. Polß Δ208-304 cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards H2O2 and UV when compared with HeLa cells alone. It has been shown that cell death in Pol ß Δ208-304 transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

Association of a newly identified variant of DNA polymerase beta (polßΔ63-123, 208-304) with the risk factor of ovarian carcinoma in India.

BACKGROUND: DNA polymerase is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. The encoded enzyme is a key to the base excision repair (BER) pathway. It is evident that pol beta has mutations in various cancer samples, but little is known about ovarian cancer. AIM: Identification of any variant form of polß cDNA in ovarian carcinoma and determination of association between the polymorphism and ovarian cancer risk in Indian patients. We used 152 samples to isolate and perform RT-PCR and sequencing. RESULTS: A variant of polymerase beta (deletion of exon 4-6 and 11-13, comprising of amino acid 63-123, and 208-304) is detected in heterozygous condition. The product size of this variant is 532 bp while wild type pol beta is 1 kb. Our study of association between the variant and the endometrioid type shows that it is a statistically significant factor for ovarian cancer [OR=31.9 (4.12-246.25) with p<0.001]. The association between variant and stage IV patients further indicated risk (χ2 value of 29.7, and OR value 6.77 with 95% CI values 3.3-13.86). The correlation study also confirms the association data (Pearson correlation values for variant/stage IV and variant/endometrioid of 0.44 and 0.39). CONCLUSION: Individuals from this part of India with this type of variant may be at risk of stage IV, endometrioid type ovarian carcinoma.

Exon 8-9 mutations of DNA polymerase ß in ovarian carcinoma patients from Haldia, India.

BACKGROUND: Ovarian cancer is the number one killer among all the gynecological cancers. We undertook association study to identify potential alterations in the genomic DNA of a DNA repair gene, DNA polymerase beta (polß), involved in base excision repair (BER), in ovarian carcinomas of patients from Haldia, India. Mutations, splice variants have been reported earlier in different tumors other than ovarian tumors. AIM: In this study we explored the possibility of association of any mutation of pol beta (Exon 8) with prognosis in 152 ovarian cancer samples. RESULTS: Alteration in the exon 8 region (Exon 8:468, AgC; 15.1%) was noted among fifty seven polymorphism positive samples. Alteration in the intervening sequence 8 (IVS8, -25, AgC; 3.9%) was also noted. All alterations are heterozygous in nature. CONCLUSIONS: We found no significant association among the samples from serous type, stage IV, and the polß mutations (P ≤ 0.01). Only a slight tendency of association was evident between IVS8, -25, A to C; and stage III. Further analysis with a larger number of samples is needed.

Association between newly identified variant form of DNA polymerase beta (Δ 208-304) and ovarian cancer.

BACKGROUND: Base excision repair (BER) is a key pathway for maintaining genomic stability. A key enzyme in the BER pathway is DNA polymerase beta (polß), which removes the deoxyribose phosphate group (dRP) and fills in the gap with a nucleotide after the DNA lesion is excised. It has been shown that more than thirty percent of breast, bladder, esophageal, colon, and gastric cancer samples studied so far have exhibited DNA polymerase beta mutation. AIM: To examine the association between polß polymorphism and ovarian cancer, case control study was performed using one hundred fifty two cancer samples and non-metastatic normal samples from the same patients in Indian population. DESIGN: The polß polymorphism was studied in ovarian carcinoma tissues samples initially by RT-PCR followed by sequencing and then by western blot analysis. RESULT: A new type of variant was detected along with the WT allele (polßΔ _{208-304}). Stage IV samples have shown a significant factor for cancer progression in ovarian cancer patients of India [OR=3.58; 95% CI (1.6-7.9); and p=0.001]. The association study involving serous type and the variant showed a tendency towards ovarian carcinogenesis [OR=1.57; 95% CI (0.8-3.1); p=0.19]. The western blot analysis result indicates that the specific deletion appears to be associated with disease progression. CONCLUSION: The result reveals that this variant form of polß is a predisposing factor for stage IV ovarian cancer samples in Indian population.

Association of two polymorphisms of DNA polymerase beta in exon-9 and exon-11 with ovarian carcinoma in India.

BACKGROUND: DNA polymerase beta (polß ) is a key enzyme in the base excision repair pathway. It is 39 kDa protein, with two subunits, one large subunit of 31 kDa having catalytic activity between exon V to exon XIV, and an 8 kDa smaller subunit having single strand DNA binding activity. Exons V to VII have double strand DNA binding activity, whereas exons VIII to XI account for the nucleotidyl transferase activity and exons XII to XIV the dNTP selection activity. AIM: To examine the association between polß polymorphisms and the risk of ovarian cancer, the present case control study was performed using 152 cancer samples and non-metastatic normal samples from the same patients. In this study, mutational analysis of polß genomic DNA was undertaken using primers from exons IX to XIV - the portion having catalytic activity. RESULTS: We detected alteration in DNA polymerase beta by SSCP. Two specific heterozygous point mutations of polß were identified in Exon 9:486, A->C (polymorphism 1; 11.18%) and in Exon 11:676, A->C (polymorphism 2; 9.86%). The correlation study involving polymorphism 1 and 4 types of tissue showed a significant correlation between mucinous type with a Pearson correlation value of 4.03 (p=0.04). The association among polymorphism 2 with serous type and stage IV together have shown Pearson χ2 value of 3.28 with likelihood ratio of 4.4 (p=0.07) with OR =2.08 (0.3- 14.55). This indicates that there is a tendency of correlation among polymorphism 2, serous type and stage IV, indicating a risk factor for ovarian cancer. CONCLUSION: Hence, the results indicate that there is a tendency for polß polymorphisms being a risk factor for ovarian carcinogenesis in India.