Expulsion of plasmid-mediated antibiotic resistance genes in E. coli by ethidium bromide and acridine orange treatment.

Plasmid borne antibiotics resistance is the global threat to healthcare facilities. Such antibiotics resistance is inherited stably within the same bacterial generations and transmitted horizontally to other species of bacteria. The elimination of such resistance plasmid is of great importance to contain dispersal of antibiotics resistance. E. coli strains were identified, screened for the presence of antibiotics resistance by disc diffusion method, and cured by sub-lethal concentrations of Ethidium bromide and Acridine orange. After curing, again antibiotic resistance was determined. Before and after curing, plasmids were extracted by column spin Kit and subjected to 1% agarose gel electrophoresis and antibiotic resistance genes were identified by PCR. The Ethidium bromide was more effective than Acridine orange in eliminating antibiotics resistance and resistance genes bearing plasmids (4, 5, 6, 8, 9, 10 and <10kb). The most frequently eliminated antibiotic resistance was against Imipenem and Meropenem followed by Cefoperazone-sulbactam, Amikacin and cephalosporins in sequence. The loss of antibiotic resistance was associated with the elimination of plasmid-borne antibiotic resistance genes; bla-TEM, bla-SHV, bla-CTX-M, qnrA, qnrB, qnrC and qnrD. Some E. coli strains did not show the removal of antibiotics resistance and plasmids, suggesting the presence of resistance genes on main chromosome and or non-curable plasmids.

Purification and characterization of ß-galactosidase from Aspergillus fumigatus PCSIR-2013.

Extra cellular ß-galactosidase enzyme was purified and characterized from Aspergillus fumigatus PCSIR- 2013. Estimated molecular mass of the enzyme was approximately 95 kDa. by native polyacrylamide gel electrophoresis. Initially, different fermentation parameters were optimized for maximum production of ß-galactosidase. The kinetic study of the partially purified enzyme exhibited that it remained active in broad range of temperature from 25°C to 70°C with an optimum of 60°C. The Km and Vmax were calculated as 9.95mmol/l and 51.78 U/ml/min, respectively. The optimum pH was 5.0, when reaction mixture was incubated for 30 min. The enzyme was very stable in the presence of different metal ions, although Na+ (16%) stimulates the activity at 10mM concentration. In contrast, Ba+2 and Hg+2 have negative effect on enzyme activity and activity decreased to 54% and 19%, respectively. Thermo stability study was revealed that the enzyme retained 72% of its activity at 50°C. Whereas, when enzyme was incubated at 60°C for 120 min, its residual activity was decreased to 42.0%. However, the enzyme was completely inactivated at 80°C after 120 min of pre-incubation. Among different surfactant which incorporated with enzyme, Tween 20 and Triton X-100 both have stimulatory effect and activity increased to 29% and 17%, respectively.

Native wild plants in Karachi, Pakistan: Rich source of antioxidant raw material.

Extracts of nine plants were studied for DPPH radical scavenging and reducing abilities. Pentatropis spiralis, Calotropis procera, Helitropium curassavicum, Withania somnifera and Chenopodium album showed reducing power ranging from 34% to 146%. Suaeda fruticosa, Trianthema portulacastrum, Pluchea lanceolata and Rumex dentatus has excellent antioxidant potential proved by their DPPH scavenging and reducing power. 1000µg/10µl chloroform extract of S. fruticosa gave 92% scavenging with IC50 value less than 0.7µg/10µl while its hexane extract possessed 80% reducing activity at 100µg/10µl concentration. DPPH free radical scavenging by methanolic extract of Trianthema portulacastrum was 60% and 76% at 1000 and 100µg/10µl respectively with IC50 value of 0.03µg/10µl while the reducing activity of 124% at 100µg/10µl. Methanolic extract of P. lanceolata showed 91% and 70% scavenging activity at 1000 and 100µg/10µl with IC50 value of 0.7µg/ 10µl. Reducing power is comparable with the reference BHA standard that is 98% at 100µg/10µl concentration. Rumex dentatus' extracts are excellent DPPH scavengers and hydrogen donators produced 156% reduction. Chloroform extract was inefficient antioxidant. These results make these plants a candidate for future research for treating ailments due to imbalance in free radicals.

New anthrarobin acyl derivatives as butyrylcholinesterase inhibitors: synthesis, in vitro and in silico studies.

To treat Alzheimer's disease (AD), the available candidates are effective only against mild AD or have side effects. So, a study was planned to synthesis new candidates that may have good potential to treat AD. A series of new anthrarobin acyl derivatives (2-8) were synthesized by the reaction of anthrarobin (1) and acetic anhydride/acyl chlorides. The product were characterized by 1H NMR and EI-MS, and evaluated for butyrylcholinesterase (BuChE) inhibition activity. Compounds 5 and 4 showed notable BuChE inhibitory potential with IC50 5.3 ± 1.23 and 17.2 ± 0.47 µM, respectively when compared with the standard eserine (IC50 7.8 ± 0.27 µM), compound 5 showed potent BuChE inhibition potential than the standard eserine. The active compounds 5 and 4 have acyl groups at 2-OH and 10-OH positions which may be responsible for inhibitory potential as this orientation is absent in other products. In silico studies of 5 and 4 products revealed the high inhibitory potential due to stable binding of ligand with the BuChE active sites with docking energy score -18.8779 kcal/mol and -23.1159 kcal/mol, respectively. Subsequently, compound 5 that have potent BuChE inhibitory activity could be the potential candidate for drug development for Alzheimer's disease.

Termiglaucescin, a new polyhydroxy triterpene glucoside from Terminalia glaucescens with antioxidant and anti-inflammatory potential.

Termiglaucescin (1), a new triterpene glucoside, has been isolated from the ethyl acetate extract of the root bark of Terminalia glaucescens Planch. ex Benth, together with 11 known compounds, ß-D-glucopyranosyl 2α,3ß,6ß-trihydroxy-23-galloylolean-12-en-28-oate (2), arjunglucoside I (3), sericoside (4), arjungenin (5), sericic acid (6), arjunetin (7), chebuloside II (8), 3,3',4-tri-O-methylelagic acid (9), 3,3'-di-O-methylelagic acid (10), ß-sitosterol (11) and stigmasterol (12). Compounds 2, 3, 7, 8 and 9 are reported from the plant for the first time. The structures of the isolated compounds were characterized by spectroscopic data interpretations, especially 1D and 2D NMR. The triterpenic isolates showed potent antioxidant and anti-inflammatory activities.

Evaluation of antioxidant and urease inhibition activities of roots of Glycyrrhiza glabra.

The object of this study is to determine the antioxidant activity of extracts from Glycyrrhiza glabra roots. The parent extract is methanolic extract while its sub fractions were prepared in ethyl acetate, chloroform, and n-butanol. The method based on scavenging activity and reduction capability of 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH). Urease inhibition activities of these extracts were also evaluated. Chloroform fraction was the most effective antioxidant with 87.7% activity but the activity is less than the crude methanolic extract i.e. 90%. Chloroform fraction showed the same trend in reducing power as that in radical scavenging activity. However n- butanol extract was devoid of any activity when compared to standard BHA. Crude methanolic fraction and its sub-fractions were also screened for enzyme inhibition activities using jackbean urease as substrate. Significant anti urease activity i.e. 72 % was observed in the ethyl acetate fraction with respect to standard inhibitor thiourea.

Neovascularity patterns in breast carcinoma: correlation of Doppler ultrasound features with sonographic tumour morphology.

OBJECTIVE: To determine the association and correlation between morphologic features of breast cancer on gray scale ultrasound, and vascularity patterns and indices on color and spectral Doppler ultrasound. STUDY DESIGN: Cross-sectional, analytical study. PLACE AND DURATION OF STUDY: Department of Radiology, Dow University of Health Sciences and Civil Hospital, Karachi, from August 2006 to June 2007. METHODOLOGY: Fifty adult female patients with histopathological proven breast cancer underwent ultrasound for evaluation of morphologic features on gray scale mode; vascularity patterns on color Doppler scan and flow indices measurement on spectral Doppler ultrasound. Regression analysis was conducted to determine correlation between the variables and associations of vascularity patterns. RESULTS: The 50 studied patients had predominantly solid tumour in 46 (92%) with depth to width ratio of > 1 in 37 (76%). The mean tumour size was 3.6 +/- 1.34 cm, with irregular margins in 41 (82%), posterior shadowing in 43 (86%) and calcifications in 27 (54%). Type 3 vascularity (multiple peripheral vessels) was the most frequent pattern (n=21, 42%) that showed a positive correlation with solid tumours (r=0.7, p < 0.001). Low resistance spectral waveform was seen in 44 (88%) cases. The average size was 3.69 cm in vascular and 3.1 cm in avascular tumours. Mean resistive index (RI) was 0.67. Mean pulsatility index (PI) was 1.1. RI was significantly high in cystic tumours (mean=0.8, p < 0.001) and significantly low in tumours measuring less than 2 cm (RI=0.18, p < 0.001). CONCLUSION: In this study, multiple peripheral vessels with low resistance flow was the pattern most significantly associated with all appearance of focal breast cancer. Apart from a positive correlation with solid tumours, markedly high RI in cystic tumours and markedly low RI in tumours less than 2 cm, there was no consistent correlation trend difference between Doppler findings and tumour size. Histopathology, therefore, remains the main modality to evaluate the tumour type and characteristics.

Actinomycin D identified as an inhibitor of discoidin domain receptor 2 interaction with collagen through an insect cell based screening of a drug compound library.

Discoidin domain receptors belong to the cell surface receptor tyrosine kinase family and recognize collagens for their activating ligands. They have been implicated for cell growth and migration and their elevated expressions were observed in various human cancers. When we expressed human Discoidin domain receptor 2 (DDR2) in insect cells, the protein was targeted properly into the cell membrane and this could enforce the cells to adhere on culture plate coated with type I collagen. By taking advantage of this, we established a novel insect cell based screening protocol to identify chemicals which inhibit the interaction between DDR2 and collagen. We screened a drug-compound library to select an anti-cancer drug, actinomycin D, as the inhibitory compound. Actinomycin D prevented the activation of DDR2 by type I collagen in human embryonic kidney 293 cells with an IC(50) value of 9 microM, while it did not interfere with the activation of other receptor tyrosine kinases by their ligands. In conclusion we identified a new biological function of actinomycin D and the insect cell based method provides a useful protocol for screening inhibitors against the association of DDR2 with collagen.