Stabilization of an intermediate activation state for transducin by a fluorescent GTP analogue

The GTP-binding protein (G protein), transducin, serves as a key molecular switch in vertebrate vision through the tight regulation of its GTP-binding (activation)/GTP hydrolytic (deactivation) cycle by the photoreceptor rhodopsin. To better understand the structure-function characteristics of transducin activation, we have set out to identify spectroscopic probes that bind to the guanine nucleotide-binding site of this G protein and maintain its ability to interact with its specific cellular target/effector, the cyclic GMP phosphodiesterase (PDE). In this study, we describe the characterization of a fluorescently labeled GTP analogue, BODIPY-FL GTPgammaS (BOD-GTPgammaS), that binds to the alpha subunit of transducin (alpha(T)) in a rhodopsin- and Gbetagamma-dependent manner, similar to the binding of GTP or GTPgammaS, with an apparent dissociation constant of 100 nM. The rhodopsin-dependent binding of BOD-GTPgammaS to alpha(T) is slow, relative to the rate of binding of GTPgammaS, particularly under conditions where rhodopsin must act catalytically to stimulate the exchange of BOD-GTPgammaS for GDP on multiple alpha(T) subunits. This reflects a slower rate of dissociation of rhodopsin and Gbetagamma from alpha(T)-BOD-GTPgammaS complexes, relative to their rates of dissociation from alpha(T)-GTPgammaS. The binding of BOD-GTPgammaS occurs without a change in the intrinsic tryptophan fluorescence of alpha(T), indicating that only a subtle movement of the Switch 2 domain on alpha(T) accompanies the binding of this GTPgammaS analogue. Nevertheless, the BOD-GTPgammaS-bound alpha(T) subunit is able to bind with high affinity to the recombinant, purified gamma subunit of PDE (gamma(PDE)) labeled with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS (K(d) approximately 13 nM)), as well as bind to and stimulate the activity of PDE, albeit less efficiently compared to alpha(T)-GTPgammaS. Taken together, these findings suggest that the binding of BOD-GTPgammaS to transducin causes it to adopt a distinct conformation that appears to be intermediate between the inactive and fully active states of alpha(T), and this fluorescent nucleotide analogue can be used as a reporter group to characterize the interactions of alpha(T) in this conformational state with its biological target/effector.     

SSEP: Secondary structural elements of proteins

SSEP is a comprehensive resource for accessing information related to the secondary structural elements present in the 25 and 90% non-redundant protein chains. The database contains 1771 protein chains from 1670 protein structures and 6182 protein chains from 5425 protein structures in 25 and 90% non-redundant protein chains, respectively. The current version provides information about the alpha-helical segments and beta-strand fragments of varying lengths. In addition, it also contains the information about 3(10)-helix, beta- and nu-turns and hairpin loops. The free graphics program RASMOL has been interfaced with the search engine to visualize the three-dimensional structures of the user queried secondary structural fragment. The database is updated regularly and is available through Bioinformatics web server at http://cluster.physics.iisc.ernet.in/ssep/ or http://144.16.71.148/ssep/.     

An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml(-1)) followed by achromopeptidase (60 U ml(-1)) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.     

Ramachandran plot on the web

A graphics package has been developed to display the main chain torsion angles phi, psi (phi, Psi); (Ramachandran angles) in a protein of known structure. In addition, the package calculates the Ramachandran angles at the central residue in the stretch of three amino acids having specified the flanking residue types. The package displays the Ramachandran angles along with a detailed analysis output. This software is incorporated with all the protein structures available in the Protein Databank.     

Survival of a strain of Bacillus megaterium carrying a lepidopteran-specific gene of Bacillus thuringiensis in the phyllospheres of various economically important plants

The colonizing ability of a transcipient strain of Bacillus megaterium carrying a lepidopteran-specific cryIA (a) gene of Bacillus thuringiensis in the phyllospheres of various economically important plants was studied. Similar experiments were also carried out using the parental B. thuringiensis var. kurstaki strain HD1 for a comparison. While the transcipient remained on the leaves of cotton and okra for more than 28 days, its survival in phyllospheres of mulberry, peanut, chickpea, tomato and rice was rather limited to about 3 – 5 days. The persistence of B. thuringiensis, on the other hand, was extremely short (i.e. less than 4 days) on all the crop plants tested.     

Disseminated rhinosporidiosis masquerading as soft tissue round cell tumour diagnosed by fine needle aspiration cytology

Rhinosporidium seeberi belongs to the eukaryotic class Mesomycetozoea and causes chronic granulomatous lesions known as rhinosporidiosis. Rhinosporidiosis frequently involves the nasal cavity and nasopharynx through transepithelial invasion. Atypical presentations of this disease at other body sites have been reported, including the subcutis, visceral organs, bones, and genitals. Only a few cases of cutaneous and subcutaneous involvement have been reported to date. This chronic granulomatous condition is known for its recurrence following autoinoculation unless the correct diagnosis and appropriate treatment are given. We describe a case of an immunocompetent adult who had undergone fine needle aspiration cytology (FNAC) of mass-like swellings in the right thigh and right calf at another healthcare centre and had been diagnosed with a small round blue cell tumour. FNAC at our centre confirmed a rare case of rhinosporidiosis that was clinically mimicking a soft tissue neoplasm of the lower extremity, and the erroneous interpretation of the prior cytology studies had resulted in misinterpretation of the individually dispersed pathogenic organisms as individual malignant cells. FNAC of rhinosporidiosis can lead to early diagnosis and prompt treatment of this pathogen when it presents at unanticipated body sites.     

Microfluidic models of the human circulatory system: versatile platforms for exploring mechanobiology and disease modeling

The human circulatory system is a marvelous fluidic system, which is very sensitive to biophysical and biochemical cues. The current animal and cell culture models do not recapitulate the functional properties of the human circulatory system, limiting our ability to fully understand the complex biological processes underlying the dysfunction of this multifaceted system. In this review, we discuss the unique ability of microfluidic systems to recapitulate the biophysical, biochemical, and functional properties of the human circulatory system. We also describe the remarkable capacity of microfluidic technologies for exploring the complex mechanobiology of the cardiovascular system, mechanistic studying of cardiovascular diseases, and screening cardiovascular drugs with the additional benefit of reducing the need for animal models. We also discuss opportunities for further advancement in this exciting field.