Structure is lost incrementally during the unfolding of barstar

Coincidental equilibrium unfolding transitions observed by multiple structural probes are taken to justify the modeling of protein unfolding as a two-state, N <==> U, cooperative process. However, for many of the large number of proteins that undergo apparently two-state equilibrium unfolding reactions, folding intermediates are detected in kinetic experiments. The small protein barstar is one such protein. Here the two-state model for equilibrium unfolding has been critically evaluated in barstar by estimating the intramolecular distance distribution by time-resolved fluorescence resonance energy transfer (TR-FRET) methods, in which fluorescence decay kinetics are analyzed by the maximum entropy method (MEM). Using a mutant form of barstar containing only Trp 53 as the fluorescence donor and a thionitrobenzoic acid moiety attached to Cys 82 as the fluorescence acceptor, the distance between the donor and acceptor has been shown to increase incrementally with increasing denaturant concentration. Although other probes, such as circular dichroism and fluorescence intensity, suggest that the labeled protein undergoes two-state equilibrium unfolding, the TR-FRET probe clearly indicates multistate equilibrium unfolding. Native protein expands progressively through a continuum of native-like forms that achieve the dimensions of a molten globule, whose heterogeneity increases with increasing denaturant concentration and which appears to be separated from the unfolded ensemble by a free energy barrier.     

Antifungal activity of nanoemulsion from Cleome viscosa essential oil against food-borne pathogenic Candida albicans

Pathogenic and spoilage fungi cause enormous challenges to food related fatal infections. Plant essential oil based classical emulsions can functions as antifungal agents. To investigate the antifungal spectrum, that is the scope of the nanoemulsion composed of Cleome viscosa essential oil and Triton-x-100 fabricated by ultrasonication method. Minimum inhibitory and fungicidal concentration of essential oil nanoemulsion (EONE) was tested against food borne pathogenic C. albicans. The MIC and MFC values ranged from 16.5 to 33 µl/ml with significant reduction on biofilm of C. albicans isolates. The alteration of molecular fingerprints was confirmed by Fourier transformed infrared spectroscopy and subsequent reduction of chitin levels in cell walls was noted by spectroscopic analysis. The EONE and their bioactive compounds cause collateral damage on C. albicans cells.     

Perilipin Controls Lipolysis by Regulating the Interactions of AB-hydrolase Containing 5 (Abhd5) and Adipose Triglyceride Lipase (Atgl)

The mobilization of stored lipid by hormones is a fundamental function of fat cells, and there is strong evidence that perilipin (Plin), a lipid droplet scaffold, and adipose tissue triglyceride lipase (Atgl), a triglyceride-specific lipase, play critical roles. Previous work suggested that Abhd5, a protein activator of Atgl, coordinates with Plin in controlling basal and stimulated lipolysis; however, the underlying mechanism is controversial. The present experiments investigated protein trafficking and interactions among Plin, Atgl, and Abhd5 in live cells. The results demonstrate that Plin binds Abhd5 with high affinity and thereby suppresses the interaction of Abhd5 with Atgl. Sequestration of Abhd5 appears to a major mechanism by which Plin reduces basal lipolysis. Phosphorylation of Plin on serine 492 or serine 517 rapidly releases Abhd5 from Plin, allowing Abhd5 to directly interact with Atgl. Imaging experiments demonstrated that the Plin-dependent interaction of Abhd5 and Atgl occurs mainly, but not exclusively, on lipid droplets that contain Plin.     

Acyclovir-Polyethylene Glycol 6000 Binary Dispersions: Mechanistic Insights

The dissolution and subsequent oral bioavailability of acyclovir (ACY) is limited by its poor aqueous solubility. An attempt has been made in this work to provide mechanistic insights into the solubility enhancement and dissolution of ACY by using the water-soluble carrier polyethylene glycol 6000 (PEG6000). Solid dispersions with varying ratios of the drug (ACY) and carrier (PEG6000) were prepared and evaluated by phase solubility, in vitro release studies, kinetic analysis, in situ perfusion, and in vitro permeation studies. Solid state characterization was done by powder X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) analysis, and surface morphology was assessed by polarizing microscopic image analysis, scanning electron microscopy, atomic force microscopy, and nuclear magnetic resonance analysis. Thermodynamic parameters indicated the solubilization effect of the carrier. The aqueous solubility and dissolution of ACY was found to be higher in all samples. The findings of XRD, DSC, FTIR and NMR analysis confirmed the formation of solid solution, crystallinity reduction, and the absence of interaction between the drug and carrier. SEM and AFM analysis reports ratified the particle size reduction and change in the surface morphology in samples. The permeation coefficient and amount of ACY diffused were higher in samples in comparison to pure ACY. Stability was found to be higher in dispersions. The results suggest that the study findings provided clear mechanical insights into the solubility and dissolution enhancement of ACY in PEG6000, and such findings could lay the platform for resolving the poor aqueous solubility issues in formulation development.     

Molecular Diversity and Evolution of bla TEM Genes Encoding β-Lactamases Resistant to Clavulanic Acid in Clinical E. coli

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and β-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as ``TEM-1B like' and ``TEM-2 like' restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the β-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of β-lactam–β-lactamase inhibitor combinations. Received: 18 March 1996 / Accepted: 15 July 1996     

Membrane-Active Macromolecules Resensitize NDM-1 Gram-Negative Clinical Isolates to Tetracycline Antibiotics

Gram-negative ‘superbugs’ such as New Delhi metallo-beta-lactamase-1 (bla _NDM-1) producing pathogens have become world’s major public health threats. Development of molecular strategies that can rehabilitate the ‘old antibiotics’ and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards bla _NDM-1 Klebsiella pneumonia and bla _NDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards bla _NDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.