Enhancement of ethyl propionate synthesis by poly (AAc-co-HPMA-cl-MBAm)-immobilized Pseudomonas aeruginosa MTCC-4713, exposed to Hg2+ and NH4+ ions

A purified alkaline thermo-tolerant bacterial lipase from Pseudomonas aeruginosa MTCC-4713 was immobilized on a poly (AAc-co-HPMA-cl-MBAm) hydrogel. The hydrogel-bound lipase achieved 93.6% esterification of ethanol and propionic acid (300 mM: 100 mM) into ethyl propionate at temperature 65 degrees C in 3 h in the presence of a molecular sieve (3 angstroms). In contrast, hydrogel-immobilized lipase pre-exposed to 5 mM of HgCl2 orNH4Cl resulted in approximately 97% conversion of reactants in 3 h into ethyl propionate under identical conditions. The salt-exposed hydrogel was relatively more efficient in repetitive esterification than the hydrogel-bound lipase not exposed to any of the cations. Moreover, bound lipase exposed Hg2+ or NH4+ ions showed altered specificity towards p-nitrophenyl esters and was more hydrolytic towards higher C-chain p-nitrophenyl esters (p-nitrophenyl laurate and p-nitrophenyl palmitate with C 12 and C 16 chain) than the immobilized lipase not exposed to any of the salts. The later showed greater specificity towards p-nitrophenyl caprylate (C 8).     

Representations of specific acoustic patterns in the auditory cortex and hippocampus

Previous behavioural studies have shown that repeated presentation of a randomly chosen acoustic pattern leads to the unsupervised learning of some of its specific acoustic features. The objective of our study was to determine the neural substrate for the representation of freshly learnt acoustic patterns. Subjects first performed a behavioural task that resulted in the incidental learning of three different noise-like acoustic patterns. During subsequent high-resolution functional magnetic resonance imaging scanning, subjects were then exposed again to these three learnt patterns and to others that had not been learned. Multi-voxel pattern analysis was used to test if the learnt acoustic patterns could be ‘decoded’ from the patterns of activity in the auditory cortex and medial temporal lobe. We found that activity in planum temporale and the hippocampus reliably distinguished between the learnt acoustic patterns. Our results demonstrate that these structures are involved in the neural representation of specific acoustic patterns after they have been learnt.     

Fine needle aspiration cytologic diagnosis of erythema nodosum leprosum: a case report

BACKGROUND: Erythema nodosum leprosum (ENL), the type 2 lepra reaction occurring in lepromatous or borderline lepromatous leprosy, presents clinically with acute manifestations that compel the patient to seek medical attention. Recognition and timely management of these patients is critical in order to avoid permanent disability. Fine needle aspiration cytology (FNAC) is a simple, effective tool that aids in correct diagnosis and management of ENL. CASE: A 30-year-old woman presented with history of fever, reddening of the face, and multiple raised, reddish, painful swellings of the bilateral forearms and legs for 7 days. One year previously, she was diagnosed and treated for lepromatous leprosy with type 2 reaction. After a thorough clinical examination a diagnosis of ENL was made. FNA smears from the forearm swellings showed pus-like material with intact and degenerated polymorphonuclear leukocytes and many foamy macrophages with strong granular acid-fast bacillus (AFB) positivity. A cytologic diagnosis of ENL was given, which was confirmed on histopathologic examination of skin biopsy. CONCLUSION: Cytologic features such as a large number of intact and degenerated neutrophils with foamy macrophages and strong granular AFB positivity, in an appropriate clinical background, allows a confident diagnosis of ENL.     

Interdomain contacts control folding of transcription factor RfaH

Escherichia coli RfaH activates gene expression by tethering the elongating RNA polymerase to the ribosome. This bridging action requires a complete refolding of the RfaH C-terminal domain (CTD) from an α-helical hairpin, which binds to the N-terminal domain (NTD) in the free protein, to a β-barrel, which interacts with the ribosomal protein S10 following RfaH recruitment to its target operons. The CTD forms a β-barrel when expressed alone or proteolytically separated from the NTD, indicating that the α-helical state is trapped by the NTD, perhaps co-translationally. Alternatively, the interdomain contacts may be sufficient to drive the formation of the α-helical form. Here, we use functional and NMR analyses to show that the denatured RfaH refolds into the native state and that RfaH in which the order of the domains is reversed is fully functional in vitro and in vivo. Our results indicate that all information necessary to determine its fold is encoded within RfaH itself, whereas accessory factors or sequential folding of NTD and CTD during translation are dispensable. These findings suggest that universally conserved RfaH homologs may change folds to accommodate diverse interaction partners and that context-dependent protein refolding may be widespread in nature.     

Oral delivery of curcumin bound to chitosan nanoparticles cured Plasmodium yoelii infected mice

Curcumin has been shown to have anti malarial activity, but poor bioavailability and chemical instability has hindered its development as a drug. We have bound curcumin to chitosan nanoparticles to improve its bioavailability and chemical stability. We found that curcumin bound to chitosan nanoparticles did not degrade that rapidly in comparison to free curcumin when such particles were incubated in mouse plasma in vitro at room temperature. The uptake of bound curcumin from chitosan nanoparticles by mouse RBC was much better than from free curcumin. Oral delivery of curcumin bound chitosan nanoparticles to normal mice showed that they can cross the mucosal barrier intact and confocal microscopy detected the nanoparticles in the blood. Curcumin loaded chitosan nanoparticles when delivered orally improved the bioavailability of curcumin in the plasma and RBC. While mice infected with a lethal strain of Plasmodium yoelii (N-67) died between 8 and 9 days post infection, feeding of chitosan nanoparticles alone made them to survive for five more days. Feeding 1mg of native curcumin to infected mice per day for seven days resulted in survival of one third of mice but under the same condition when 1mg of curcumin bound to chitosan nanoparticles was fed all the mice survived. Like chloroquine, curcumin inhibited parasite lysate induced heme polymerization in vitro in a dose dependent manner and curcumin had a lower IC(50) value than chloroquine. We believe that binding of curcumin to chitosan nanoparticles increases its chemical stability and enhances its bioavailability when fed to mice. In vitro data suggest that it can inhibit hemozoin synthesis which is lethal for the parasite.     

Anti-hemolytic and Peroxyl Radical Scavenging Activity of Organoselenium Compounds: An In Vitro Study

Selenium-containing amino acids, selenocystine (CysSeSeCys), methylselenocysteine (MeSeCys), and selenomethionine (SeMet) have been examined for anti-hemolytic and peroxyl radical scavenging ability. Effect of these compounds on membrane lipid peroxidation, release of hemoglobin, and loss of intracellular K⁺ ion as a consequence of peroxyl radicals-induced oxidation of human red blood cells were used to evaluate their anti-hemolytic ability. The peroxyl radicals were generated from thermal degradation of 2,2′-azobis(2-methylpropionamidine) dihydrochloride. Significant delay (t _eff) was observed in oxidative damage in the presence of the selenium compounds. From the IC₅₀ values for the inhibition of hemolysis, lipid peroxidation, and K⁺ ion leakage, the relative anti-hemolytic ability of the compounds were found to be in the order of CysSeSeCys > MeSeCys > SeMet. The anti-hemolytic abilities of the compounds, when compared with sodium selenite (Na₂SeO₃) under identical experimental conditions, were found to be better than Na₂SeO₃. Relative rate constants estimated for the reaction of MeSeCys and SeMet with peroxyl radicals by competition kinetics using ABTS²⁻ as a reference confirmed that all the compounds are efficient peroxyl radical scavengers. Comparison of the GPx-like activity of these compounds, by NADPH–GSH reductase coupled assay, indicated that CysSeSeCys exhibits the highest activity. Based on these results, it is concluded that among the compounds examined, CysSeSeCys, possessing the ability to reduce peroxyl radicals and hydroperoxides showed efficient anti-hemolytic activity.     

Purification and properties of a novel broad substrate specific alcohol oxidase from Aspergillus terreus MTCC 6324

An alcohol oxidase was isolated from the microsome of n-hexadecane grown Aspergillus terreus and purified by ion exchange chromatography. The oxidase was found to act on short chain-, long chain-, secondary-, and aromatic-alcohol substrates with highest affinity for n-heptanol (K(M)=0.498 mM, K(cat)=2.7x10(2) s(-1)). The native protein molecular mass was 269+/-5 kDa and the subunit molecular masses were 85-, 63-, 43-, 27-, and 13-kDa. The isoelectric point of the proteins was within 8.3-8.5. High aggregating property of the protein was demonstrated by AFM, DLS and TEM analyses. Chemical analysis showed the presence of oleic acid and palmitic acid at a ratio of 2:1 in the purified protein. This lipoidic nature of the protein particles was correlated to the high aggregating property. In this flavoenzyme, flavin was non-covalently but avidly associated. Peptide mass fingerprinting studies showed the presence of two FAD binding domains in 63 kDa protein. Among these two FAD binding domain sequences only the YPVIDHEYDAVVVGAGGAGLR peptide shows 45-50% sequence homology with the reported N-terminal sequences of other known alcohol oxidases. Non-redundant database search of 63- and 43-kDa subunits peptide sequences showed no sequence similarity with the other alcohol oxidase protein reported till now.     

Matrix-Assisted Refolding,Purification and Activity Assessment Using a ‘Form Invariant’ Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed ‘form invariant’ assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.