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Trivanga Bhasma, a metallic preparation containing Bhasmas of Naga (lead), Vanga (tin) and Yashada (zinc), was studied for repeated dose toxicity in Swiss albino mice to estimate No Observed Effect Level (NOEL) or No Observed Adverse Effect Level (NOAEL). A total of 80 Swiss albino mice of either sex with an average body weight of 28-30 g were equally divided into four groups (Group I, II, III, and IV). Group I served as control and was given vehicle (honey: water in 2:3 ratio) Group II, III, and IV received Trivanga Bhasma @ 7.8, 39.5,and 78 mg/kg body weight for 90 consecutive days. The effect of drug was assessed on body weight, feed and water consumption changes, hematological, and histopathological parameters. At the end of the study, all animals were sacrificed and examined for gross pathological changes. Histopathological evaluation was performed for control and high dose group. Trivanga Bhasma was found to be safe. No significant clinical signs were noted in all groups studied. No major alterations were observed during histopathological evaluation. Hence, dose rate of 78 mg/kg body weight was established as NOAEL. It is suggested to carry out a toxicity study at possible higher doses and in a different species so as to establish target organ of toxicity.

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BACKGROUND: Advanced Kaposi's sarcoma is frequently associated with chronic lymphedema (cLO). The histopathological features of lymphedematous HIV-associated KS (KS) are poorly documented and the co-existence of fibroma-like nodules in lymphedematous KS is under-recognized. The aims of this study were to assess the clinicopathological spectrum and diagnostic difficulties associated with lymphedematous KS and to highlight the clinicopathological profile of fibroma-like nodules. In addition, the pathogenesis of fibroma-like nodules and cLO is revisited. MATERIALS AND METHODS: Prospective 17-month clinicopathological study of all biopsies from patients with lymphedematous KS. RESULTS: Seventy-four biopsies, the majority from the lower limbs, from 41 patients were evaluated. Nineteen, 14, five and three patients had one, two, three or four biopsies each, respectively. In 14 biopsies, there was poor clinicopathological correlation of KS stage. Exclusive lesional KS (patch, plaque, nodule or lymphangioma-like) was identified in 29 biopsies; 23 and eight biopsies demonstrated KS or fibroma-like morphology and the adjacent dermis demonstrated cLO. There was variable intratumoral and peritumoral venous compression and lymphatic dilatation. Fourteen biopsies demonstrated cLO exclusively. Smaller fibroma-like nodules lacked KS spindle cells, whereas >5 mm nodules demonstrated focal KS spindle cell proliferation and aggregation on extensive sectioning. The subcutis of 42 biopsies demonstrated variable fibrosis, hemosiderin deposits, lymphocytes, plasma cells, KS, interstitial granular material and pools of lymph fluid. Subcutaneous abscesses were identified in six biopsies. All biopsies had variable epidermal features of cLO. CONCLUSIONS: cLO influences clinicopathological correlation of KS stage and may also mask the presence of KS and the co-existence of subcutaneous abscesses. Smaller fibroma-like nodules are hypothesized to be a manifestation of cLO that have the potential to acquire the characteristics of KS. Lymphatic and venous obstruction, protein-rich interstitial fluid, tissue hemosiderin and subcutaneous infection are hypothesized to play a combined role in the evolution and perpetuation of cLO.

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We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 degrees C by observing changes in difference molar absorbance at 400 nm (Deltaepsilon400), mean residue ellipticities at 222 nm ([theta]222) and difference mean residue ellipticity at 409 nm (Delta[theta]409). The denaturation is a three-step process when measured by Deltaepsilon400 and Delta[theta]409, and it is a two-step process when monitored by [theta]222. The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO4-induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl2) suggests that LiClO4 induces a unique MG state, i.e., (i) the core in the LiClO4-induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO4-induced process, native state <--> MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions.

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We have recently concluded from the heat-induced denaturation studies that polyols do not affect deltaG(D) degrees (the Gibbs free energy change (deltaG(D)) at 25 degrees C) of ribonuclease-A and lysozyme at physiological pH and temperature, and their stabilizing effect increases with decrease in pH. Since the estimation of deltaG(D) degrees of proteins from heat-induced denaturation curves requires a large extrapolation, the reliability of this procedure for the estimation of deltaG(D) degrees is always questionable, and so are conclusions drawn from such studies. This led us to measure deltaG(D) degrees of ribonuclease-A and lysozyme using a more accurate method, i.e., from their isothermal (25 degrees C) guanidinium chloride (GdmCl)-induced denaturations. We show that our earlier conclusions drawn from heat-induced denaturation studies are correct. Since the extent of unfolding of heat- and GdmCl-induced denatured states of these proteins is not identical, the extent of stabilization of the proteins by polyols against heat and GdmCl denaturations may also differ. We report that in spite of the differences in the structural nature of the heat- and GdmCl-denatured states of each protein, the extent of stabilization by a polyol is same. We also report that the functional dependence of deltaG(D) of proteins in the presence of polyols on denaturant concentration is linear through the full denaturant concentration range. Furthermore, polyols do not affect the secondary and tertiary structures of the native and GdmCl-denatured states.

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It is generally believed that compatible osmolytes stabilize proteins by shifting the denaturation equilibrium, native state <--> denatured state toward the left. We show here that if osmolytes are compatible with the functional activity of the protein at a given pH and temperature, they should not significantly perturb this denaturation equilibrium under the same experimental conditions. This conclusion was reached from the measurements of the activity parameters (K(m) and k(cat)) and guanidinium chloride-induced denaturations of lysozyme and ribonuclease-A in the presence of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at pH 7.0 and 25 degrees C.

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Thermal denaturation curves of lysozyme and ribonuclease-A were determined by measuring their far-UV circular dichroism (CD) spectra in the presence of different concentrations of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at various pH values in the range 7.0--1.9. The denaturation curve at each polyol concentration and pH was analysed to obtain values of T(m) (midpoint of denaturation) and DeltaH(m) (enthalpy change at T(m)), and these DeltaH(m) and T(m) values obtained at different pH values were used to obtain DeltaC(p) (constant-pressure heat capacity change) at each polyol concentration. Using values of DeltaH(m), T(m) and DeltaC(p) in the Gibbs-Helmholtz equation, DeltaG(D) degrees (Gibbs energy change at 25 degrees C) was determined at a given pH and polyol concentration. Main conclusions of this study are that polyols have no significant effect on DeltaG(D) degrees at pH 7.0, and they stabilise proteins in terms of DeltaG(D) degrees against heat denaturation at lower pH values. Other conclusions of this study are: (i) T(m) at each pH increases with increasing polyol concentration, (ii) DeltaH(m) remains, within experimental error, unperturbed in the presence of polyols, and (iii) DeltaC(p) depends on polyol concentration. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of both proteins in their native and denatured states are not perturbed on the addition of polyols.

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Earlier studies have reported that trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, is a universal stabilizer of proteins because it folds unstructured proteins and counteracts the deleterious effects of urea and salts on the structure and function of proteins. This conclusion has been reached from the studies of the effect of TMAO on proteins in the pH range 6.0-8.0. In this pH range TMAO is almost neutral (zwitterionic form), for it has a pK(a) of 4.66 +/- 0.10. We have asked the question of whether the effect of TMAO on protein stability is pH-dependent. To answer this question we have carried out thermal denaturation studies of lysozyme, ribonuclease-A, and apo-alpha-lactalbumin in the presence of various TMAO concentrations at different pH values above and below the pK(a) of TMAO. The main conclusion of this study is that near room temperature TMAO destabilizes proteins at pH values below its pK(a), whereas it stabilizes proteins at pH values above its pK(a). This conclusion was reached by determining the T(m) (midpoint of denaturation), delta H(m) (denaturational enthalpy change at T(m)), delta C(p) (constant pressure heat capacity change), and delta G(D) degrees (denaturational Gibbs energy change at 25 degrees C) of proteins in the presence of different TMAO concentrations. Other conclusions of this study are that T(m) and delta G(D) degrees depend on TMAO concentration at each pH value and that delta H(m) and the delta C(p) are not significantly changed in presence of TMAO.

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