Glomerular morphometry in biopsy evaluation of minimal change disease, membranous glomerulonephritis, thin basement membrane disease and Alport's syndrome

OBJECTIVE: To assess the role of glomerular morphometry in biopsy evaluation in renal disorders in addition to conventional diagnostic procedures. STUDY DESIGN: The study includes 10 cases each of minimal change disease (MCD), idiopathic membranous glomerulonephritis (idiopathic MGN), thin basement membrane disease (TBMD) and Alport's syndrome. Renal biopsies for normal study were obtained from age- and sex-matched autopsy cases without any renal disorder, confirmed histologically and ultrastructurally. Glomerular morphometry was performed by semiautomatic procedure using Quantimet-600 image analysis system (Leica, Cambridge, United Kingdom). RESULTS: Morphometric findings revealed significant increase in glomerular "diameter and area" and "tuft diameter and area" in patients of idiopathic MGN, but no significant difference was found in patients of MCD, TBMD and Alport's syndrome. Evaluation of glomerular volume fractions revealed a decrease in capillary space volume fraction and an increase in "membranes and mesangial matrix" volume fraction in patients with idiopathic MGN. Significant decrease in capillary space volume fraction was also observed in patients of MCD. Patients with Alport's syndrome showed variable changes. CONCLUSION: Glomerular morphometry could be considered as an adjunct to the diagnostic armamentarium of light microscopy, immunofluorescence and electron microscopy because it provides deep insight into quantitative parameters.     

Adenylylation and catalytic properties of Mycobacterium tuberculosis glutamine synthetase expressed in Escherichia coli versus mycobacteria

Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456-464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially ( approximately 25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated ( approximately 30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.     

4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization

The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM < c < mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9.     

Carbon dioxide and poultry waste utilization for production of polyhydroxyalkanoate biopolymers by <Emphasis Type="Italic">Nostoc muscorum</Emphasis> Agardh: a sustainable approach

The new paradigm is to view wastes as resources for sustainable development. In this regard, the feasibility of poultry waste and CO₂ utilization for cultivation of a filamentous nitrogen-fixing cyanobacterium, Nostoc muscorum Agardh, was investigated for production polyhydroxyalkanoates, the biodegradable polymers. This cyanobacterium showed profound rise in biomass yield with up to 10 % CO₂ supply in airstream with an aeration rate of 0.1 vvm. Maximum biomass yield of 1.12 g L^?1 was recorded for 8 days incubation period, thus demonstrating a CO₂ biofixation rate of 0.263 g L^?1 day^?1 at 10 % (v/v) CO₂-enriched air. Poultry litter (PL) supplementation also had a positive impact on the biomass yield. The nutrient removal efficiency of N. muscorum was reflected in the significant reduction in nutrient load of PL over the experimental period. A maximum poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)] copolymer yield of 774 mg L^?1 (65 % of dry cell wt.), the value almost 11-fold higher than the control, was recorded in 10 g L^?1 PL-supplemented cultures with 10 % CO₂ supply under the optimized condition, thus demonstrating that N. muscorum has good potential for CO₂ biomitigation and poultry waste remediation while simultaneously producing eco-friendly polymers.     

Dietary Supplementation with Organoselenium Accelerates Recovery of Bladder Expression,but Does Not Improve Locomotor Function,following Spinal Cord Injury

Selenium is an essential element required for activity of several antioxidant enzymes, including glutathione peroxidase. Because of the critical role of the antioxidant system in responding to traumatic events, we hypothesized that dietary selenium supplementation would enhance neuroprotection in a rodent model of spinal cord injury. Rats were maintained on either a control or selenium-enriched diet prior to, and following, injury. Dietary selenium supplementation, provided as selenized yeast added to normal rat chow, resulted in a doubling of selenium levels in the spinal cord. Dietary selenium reduced the time required for recovery of bladder function following thoracic spinal cord injury. However, this was not accompanied by improvement in locomotor function or tissue sparing.     

Role of Minor Groove Width and Hydration Pattern on Amsacrine Interaction with DNA

Amsacrine is an anilinoacridine derivative anticancer drug, used to treat a wide variety of malignancies. In cells, amsacrine poisons topoisomerase 2 by stabilizing DNA-drug-enzyme ternary complex. Presence of amsacrine increases the steady-state concentration of these ternary complexes which in turn hampers DNA replication and results in subsequent cell death. Due to reversible binding and rapid slip-out of amsacrine from DNA duplex, structural data is not available on amsacrine-DNA complexes. In the present work, we designed five oligonucleotide duplexes, differing in their minor groove widths and hydration pattern, and examined their binding with amsacrine using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Complexes of amsacrine with calf thymus DNA were also evaluated for a comparison. Our results demonstrate for the first time that amsacrine is not a simple intercalator; rather mixed type of DNA binding (intercalation and minor groove) takes place between amsacrine and DNA. Further, this binding is highly sensitive towards the geometries and hydration patterns of different minor grooves present in the DNA. This study shows that ligand binding to DNA could be very sensitive to DNA base composition and DNA groove structures. Results demonstrated here could have implication for understanding cytotoxic mechanism of aminoacridine based anticancer drugs and provide directions to modify these drugs for better efficacy and few side-effects.     

Exacerbation of Brain Pathology After Partial Restraint in Hypertensive Rats Following SiO2 Nanoparticles Exposure at High Ambient Temperature

This investigation examines the possibility that exposure to silica dust of hypertensive individuals may exacerbate brain pathology and sensory motor dysfunction at high environmental temperature. Hypertension was produced in rats (200–250 g) by two-kidney one clip (2K1C) method, and in these animals, SiO₂ nanoparticles (NPs; 50 to 60 nm) were administered at 50 mg/kg, i.p. daily for 1 week. On the 8th day, these rats were subjected to partial restraint in a Perspex box for 4 h either at room temperature (21 °C) or at 33 °C in a biological oxygen demand incubator (wind velocity, 2.6 cm/s; relative humidity, 65 to 67 %). In these animals, behavioral functions, blood–brain barrier (BBB) permeability to Evans blue albumin (EBA) and radioiodine (^[131]-Iodine), brain water content and neuronal injuries were determined. Hypertensive rats subjected to 4 h restraint at room temperature did not exhibit BBB dysfunction, brain edema, neural injury, or alterations in rotarod or inclined plane angle performances. However, when these hypertensive rats were subjected to restraint at 33 °C, breakdown of the cortical BBB (EBA, +38 %; radioiodine, +56 %), brain water (+0.88 %), neuronal damages (+18 %), and behavioral impairment were exacerbated. Interestingly, SiO₂ exposure to these rats further exacerbated BBB breakdown (EBA, 280 %; radioiodine, 350 %), brain edema (4 %), and neural injury (30 %) after identical restraint depending on the ambient temperature. SiO₂ treatment also induced brain pathology and alteration in behavioral functions in normotensive rats after restraint at high temperature. These observations clearly show that hypertension significantly enhances restraint-induced brain pathology, and behavioral anomalies particularly at high ambient temperature and SiO₂ intoxication further exacerbated these brain pathologies and cognitive dysfunctions.     

A Structural Insight into Major Groove Directed Binding of Nitrosourea Derivative Nimustine with DNA: A Spectroscopic Study

Nitrosourea therapeutics occupies a definite place in cancer therapy but its exact mechanism of action has yet to be established. Nimustine, a chloroethyl nitrosourea derivative, is used to treat various types of malignancy including gliomas. The present work focuses on the understanding of nimustine interaction with DNA to delineate its mechanism at molecular level. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) has been used to determine the binding sites of nimustine on DNA. Circular dichroism (CD) spectroscopy has been used to confirm conformational variations in DNA molecule upon nimustine-DNA interaction. Thermodynamic parameters of nimustine-DNA reaction have been calculated by isothermal titration calorimetry. Results of the present study demonstrate that nimustine is not a simple alkylating agent rather it causes major grove-directed-alkylation. Spectroscopic data suggest binding of nimustine with nitrogenous bases guanine (C6 = O6) and thymine (C4 = O4) in DNA major groove. CD spectra of nimustine-DNA complexes point toward the perturbation of native B-conformation of DNA and its partial transition into C-form. Thermodynamically, nimustine-DNA interaction is an entropy driven endothermic reaction, which suggests hydrophobic interaction of nimustine in DNA-major groove pocket. Spectral results suggest base binding and local conformational changes in DNA upon nimustine interaction. Investigation of drug-DNA interaction is an essential part of rational drug designing that also provides information about the drug’s action at molecular level. Results, demonstrated here, may contribute in the development of new nitrosourea therapeutics with better efficacy and fewer side effects.