Oxyradical-induced GFP damage and loss of fluorescence

Small amounts of highly reactive oxygen species (oxyradicals) are normal by-products of cellular metabolism. However, under certain conditions large amounts of oxyradicals are generated inside cells which may cause extensive cellular damage. Not surprisingly, a large number of disease states have been linked to oxidative stress, including cancer, diabetes, Parkinson's disease, Alzheimer's disease, and heart disease. Previously, we had shown that fluorescence spectroscopy could be used to study the pH-dependence of GFP denaturation with various agents. In this report, we show that GFP readily loses its auto-fluorescence upon exposure to oxyradicals as measured by fluorescence spectroscopy. We further show that oxyradical scavengers can prevent this loss of GFP fluorescence, thus oxyradical-induced loss of GFP fluorescence could be used to screen for antioxidants. We have evaluated various parameters which could affect the sensitivity of this GFP-based oxyradical scavenging assay, such as concentration H(2)O(2) used to produce oxyradicals, pH of the buffer, as well as UV intensity. Surprisingly we found that pH had a very dramatic effect on oxyradical-induced GFP damage. GFP was found to be most susceptible to oxyradical-induced damage at pH 6.5, and least susceptible at pH 8.5. This is the first demonstration that GFP loses its fluorescence upon exposure to oxyradicals. Furthermore, the data presented here suggest that GFP could be used to develop assays to screen for antioxidants or radical scavengers.     

Nonlinear coupling is absent in acute myocardial patients but not healthy subjects

We investigated whether autonomic nervous system imbalance imposed by pharmacological blockades and associated with acute myocardial infarction (AMI) is manifested as modifications of the nonlinear interactions in heart rate variability signal using a statistically based bispectrum method. The statistically based bispectrum method is an ideal approach for identifying nonlinear couplings in a system and overcomes the previous limitation of determining in an ad hoc way the presence of such interactions. Using the improved bispectrum method, we found significant nonlinear interactions in healthy young subjects, which were abolished by the administration of atropine but were still present after propranolol administration. The complete decoupling of nonlinear interactions was obtained with double pharmacological blockades. Nonlinear couplings were found to be the strongest for healthy young subjects followed by degradation with old age and a complete absence of such couplings for the old age-matched AMI subjects. Our results suggest that the presence of nonlinear couplings is largely driven by the parasympathetic nervous system regulation and that the often-reported autonomic nervous system imbalance seen in AMI subjects is manifested as the absence of nonlinear interactions between the sympathetic and parasympathetic nervous regulations.     

Seed Germination of a Halophytic Grass Aeluropus lagopoides

Aeluropus lagopoides(Linn.) Trin. Ex Thw. (Poaceae) is a perennialgrass distributed from coastal Sindh and Balochistan to salineflats of Punjab, Pakistan. Seeds collected from an inland populationofA. lagopoides located on the University of Karachi campuswere germinated under various levels of salinity (0, 100, 200,300, 400 and 500 m M NaCl) and temperature regimes (10/20, 15/25,20/30 and 25/35 °C) in a 12 h dark/12 h light photoperiod.Highest germination was obtained under non-saline conditions,and an increase in NaCl concentration progressively inhibitedgermination. Inhibition of germination was greater at coolertemperatures (10/20 °C) when no seed germinated above aconcentration of 300 m M NaCl. The germination response at moderatetemperatures (20/30 °C) was optimal, with 30% of seeds germinatingin 500 m M NaCl. The rate of germination decreased as salinityincreased. Germination rate was highest at 20/30 °C andlowest at 10/20 °C. Seeds were transferred from salt solutionsto distilled water after 20 d and those from high salinitiesrecovered quickly at warmer temperatures with an optimal responseat 20/30 °C. Copyright 2001 Annals of Botany Company Aeluropus lagopoides, germination, halophyte, Karachi, salinity, temperature     

Genotoxic studies in hypertensive and normotensive rats treated with amiodarone

Amiodarone, a benzofuran derivative, is a very effective antiarrhythmic medication, but has potential to cause side effects. Although its cytotoxicity potential is very well-known, there are few reports about its genotoxicity effects. Since amiodarone has not been investigated in genotoxicity studies, and the spontaneously hypertensive rat (SHR) is a well-characterized model for hypertension, the aim of the present study was to perform cytogenetic analysis on chromosome aberrations in bone marrow cells of SHRs and normotensive Wistar-Kyoto rats (WKYs) that received oral amiodarone treatment for 4 weeks. Amiodarone activity was also monitored using electrocardiograms. The presence of bradycardia in amiodarone-treated rats confirmed that this drug was really active. Metaphase analysis on bone marrow cells showed that there were significant differences in total chromosomal damage and percentage abnormal metaphase between WKY and SHR negative controls. In the SHR negative control, the frequencies of basal chromosomal aberrations and abnormal metaphases were significantly higher (p < 0.05). There were high numbers of chromosomal aberrations in all amiodarone-treated groups, compared with negative controls. In amiodarone-treated groups, the most frequent chromosomal aberration was chromatid breaks. More chromosomal aberrations were found in WKYs that received amiodarone, with a statistically significant difference in comparison with negative controls (p < 0.05). However, in SHR rats there was no significant difference between the amiodarone and negative groups regarding chromosomal damage induction. These results showed that treatment with amiodarone was genotoxic in WKYs, but not in SHRs. Further studies are needed to confirm whether amiodarone is genotoxic or efficient and harmless, among humans undergoing therapy.     

Biosorption of Cu(II) from aqueous solution by Fucus serratus: surface characterization and sorption mechanisms

In this work, the brown alga Fucus serratus (FS) used as a low cost sorbent has been studied for the biosorption of copper(II) ions in batch reactors. Firstly, the characterization of the surface functional groups was performed with two methods: a qualitatively analysis with the study of FT-IR spectrum and a quantitatively determination with potentiometric titrations. From this latter, a total proton exchange capacity of 3.15 mmolg(-1) was extrapolated from the FS previously protonated. This value was similar to the total acidity of 3.56 mmolg(-1) deduced from the Gran method. Using the single extrapolation method, three kinds of acidic functional groups with three intrinsic pK(a) were determined at 3.5, 8.2 and 9.6. The point of zero net proton charge (PZNPC) was found close to pH 6.3. Secondly, the biosorption of copper ions was studied. The equilibrium time was about 350 min and the adsorption equilibrium data were well described by the Langmuir's equation. The maximum adsorption capacity has been extrapolated to 1.60 mmolg(-1). The release of calcium and magnesium ions was also measured in relation to the copper biosorption. Finally, the efficiency of this biosorbent in natural tap water for the removal of copper was also investigated. All these observations indicate that the copper biosorption on FS is mainly based on ion exchange mechanism and this biomass could be then a suitable sorbent for the removal of heavy metals from wastewaters.     

Synthesis and antituberculosis activity of 5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazone derivatives

New series of 5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones 3a-t, 1-methyl-5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones 4a-y and 5-trifluoromethoxy-1-morpholinomethyl-1H-indole-2,3-dione 3-thiosemicarbazones 5a-m were synthesized. The structures of the synthesized compounds were confirmed by spectral data and elemental analysis. The new 5-methyl/trifluoromethoxy-1H-indole-2,3-dione derivatives, along with previously synthesized 5-methyl-1H-indole-2,3-dione 3-thiosemicarbazones 6a-l, were evaluated for in vitro antituberculosis activity against Mycobacterium tuberculosis H37Rv. 5-Methyl-1H-indole-2,3-dione 3-thiosemicarbazones (3b, 3d, 3f, 6c, 6d, and 6f), 5-trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones (3q-s) and 5-trifluoromethoxy-1-morpholinomethyl-1H-indole-2,3-dione 3-thiosemicarbazones (5e and 5j-l) were found to be the most potent inhibitors of M. tuberculosis growth described in this study.     

Carbonic anhydrase inhibitors: synthesis and inhibition studies against mammalian isoforms I-XV with a series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides

A series of 2-(hydrazinocarbonyl)-3-substitutedphenyl-1H-indole-5-sulfonamides possessing various 2-, 3- or 4- substituted phenyl groups with methyl-, halogeno- and methoxy- functionalities, as well as the perfluorophenyl moiety have been synthesized and evaluated as inhibitors of 13 catalytically active, mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms, that is, CA I-CA XV (of human (h) or murine (m) origin). The new compounds were ineffective inhibitors of isozymes hCA III, hCA IV, hCA VA, hCA VB, hCA VI and mCA XIII, moderate inhibitors of hCA I, hCA VII, hCA IX and mCA XV, and excellent, low-nanomolar inhibitors of hCA II and hCA XIV. The substitution pattern of the aromatic group in the 3-position of the indole ring influenced biological activity and isozyme inhibition profiles in this series of sulfonamides. Some of the best and most selective hCA XIV and mCA XV inhibitors ever reported have been identified in this study.     

Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.