Localization of melatonin receptor 1 in mouse retina and its role in the circadian regulation of the electroretinogram and dopamine levels

Melatonin modulates many important functions within the eye by interacting with a family of G-protein-coupled receptors that are negatively coupled with adenylate cyclase. In the mouse, Melatonin Receptors type 1 (MT(1)) mRNAs have been localized to photoreceptors, inner retinal neurons, and ganglion cells, thus suggesting that MT(1) receptors may play an important role in retinal physiology. Indeed, we have recently reported that absence of the MT(1) receptors has a dramatic effect on the regulation of the daily rhythm in visual processing, and on retinal cell viability during aging. We have also shown that removal of MT(1) receptors leads to a small (3-4 mmHg) increase in the level of the intraocular pressure during the night and to a significant loss (25-30%) in the number of cells within the retinal ganglion cell layer during aging. In the present study we investigated the cellular distribution in the C3H/f(+/+) mouse retina of MT(1) receptors using a newly developed MT(1) receptor antibody, and then we determined the role that MT(1) signaling plays in the circadian regulation of the mouse electroretinogram, and in the retinal dopaminergic system. Our data indicate that MT(1) receptor immunoreactivity is present in many retinal cell types, and in particular, on rod and cone photoreceptors and on intrinsically photosensitive ganglion cells (ipRGCs). MT(1) signaling is necessary for the circadian rhythm in the photopic ERG, but not for the circadian rhythm in the retinal dopaminergic system. Finally our data suggest that the circadian regulation of dopamine turnover does not drive the photopic ERG rhythm.     

Schizophyllum commune: A New Organism in Eye Infection

Purpose

We report a case of mycotic keratitis caused by a rare fungus Schizophyllum commune.

Methods

Clinical examination, slit-lamp examination, and microbiological evaluation of the corneal ulcer were done, and its treatment outcome was studied. The fungal etiology was established by conventional microbiological techniques, polymerase chain reaction and speciation by DNA sequencing.

Results

Corneal scraping showed the presence of fungal filaments. The fungus was identified as S. commune based on DNA sequence analysis of the internal transcribed spacer region. The organism was susceptible to amphotericin B and voriconazole and demonstrated resistance to anidulafungin, itraconazole, and fluconazole. Therapeutic keratoplasty was performed but there was recurrence of the infection in the graft, which was controlled with topical voriconazole and intracameral amphotericin B. At the end of 3 months, the affected eye had developed phthisis bulbi.

Conclusion

The best of our knowledge, this is the first reported case of keratitis caused by the rare fungus S. commune. Management of these cases is difficult, and surgical procedures may be needed.     

On some biochemical physiognomies of two common Darjeeling tea cultivars in relation to blister blight disease

Abstract

Tea, Camellia sinensis (L.) O. Kuntze, an agro-based industry, is considered as one of the prime sector of exporting resources and thus considered as “cash-crop”. Earlier report shows that tea is native to eastern and northern India, which was cultivated and consumed there since long back. Presently, more scientific reports confirmed the health-benefit traits of tea and awareness increased to a greater extent and in this regard, tea has gained its best worldwide popularity. Darjeeling Tea attained its highest acceptance globally for its pre-eminence in flavour, colour and taste and thus crop improvement is the prime interest to the Indian Scientific Community. Blister blight disease, a common disorder of tea bushes (Exobasidium vexans, a Basidiomycetes fungus) causes drastic damage of tea plantation. Depending on quality production, two common cultivars were released by TRA, Jorhat, Assam viz. Bannockburn – 157 (B-157) and Ambei Valai - 2 (AV-2), of which B-157 is susceptible to the Blister Blight and AV-2 is fairly resistant cultivar. Some biochemical considerations between the two cultivars have been made here for understanding the biochemical reasons behind the resistant characteristics. Plant secondary metabolites, like total phenol, proanthocynadin, total soluble protein provide defending feature against disease onset. AV-2 cultivar shows advantage over B-157 in these regard. Depending on band intensity analysis of native gels, acid phosphatase, catechol oxidase, peroxidase and superoxide dismutase occur in superior amount in AV-2 cultivar than that of B-157. The specific role of these enzymes in blister blight disease compatibility of two cultivars studied has been discussed.     

A naturally occurring amino acid substitution in the voltage-dependent sodium channel selectivity filter affects channel gating

The pore of sodium channels contains a selectivity filter made of 4 amino acids, D/E/K/A. In voltage sensitive sodium channel (Nav) channels from jellyfish to human the fourth amino acid is Ala. This Ala, when mutated to Asp, promotes slow inactivation. In some Nav channels of pufferfishes, the Ala is replaced with Gly. We studied the biophysical properties of an Ala-to-Gly substitution (A1529G) in rat Nav1.4 channel expressed in Xenopus oocytes alone or with a β1 subunit. The Ala-to-Gly substitution does not affect monovalent cation selectivity and positively shifts the voltage-dependent inactivation curve, although co-expression with a β1 subunit eliminates the difference between A1529G and WT. There is almost no difference in channel fast inactivation, but the β1 subunit accelerates WT current inactivation significantly more than it does the A1529G channels. The Ala-to-Gly substitution mainly influences the rate of recovery from slow inactivation. Again, the β1 subunit is less effective on speeding recovery of A1529G than the WT. We searched Nav channels in numerous databases and noted at least four other independent Ala-to-Gly substitutions in Nav channels in teleost fishes. Thus, the Ala-to-Gly substitution occurs more frequently than previously realized, possibly under selection for alterations of channel gating.     

Elucidating the chemical and biochemical applications of Citrus sinensis-mediated silver nanocrystal

Abstract

Synthesis of nanoparticles using biodegradable source is safer and echo-friendly. Here, we describe the synthesis of polycrystalline silver nanocrystals using Citrus sinensis acting as both reducing and capping agents. After exposing the silver ions to orange extract, rapid reduction of silver ions led to the formation of stable silver nanocrystals due to the reducing and stabilizing properties of orange fruit juice. The synthesized silver nanocrystals were characterized using various analytical techniques like UV–vis spectroscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscopy (TEM). The biochemical activity of the synthesized nanocrystals was studied in the light of affinity to bovine serum albumin using several biophysical methods like absorbance, fluorescence and circular dichroism spectroscopy. Cytotoxic activity of these nanocrystals was also studied against Hep-2 cell line using fluorescence microscopy. It was also found that the synthesized nanocrystals can sense mercuric ion down to 50?µM in the presence of a number of cations. Furthermore, we established that the silver nanoparticles can effectively catalyse the reduction of methylene blue by ascorbic acid. The present study will enrich our knowledge on the chemical and biochemical activities of green-synthesized silver nanocrystals.     

Structural Features of a Cold-adapted Alaskan Bacterial Lipase

Abstract

The three dimensional model of cold-adapted Alaskan psychrotroph Pseudomonas species (Strain B11-1) lipase has been constructed by homology modeling based on the crystal structure of acetyl esterase from Rhodococcus species and refined by molecular dynamics methods. Our model locates the substrate-binding cavity and further suggests that Ser-155, Asp-250, and His-280 are the members of the catalytic triad. Substrate specificity of the modeled lipase has been examined by docking experiments, which indicates that the ester of C₆ fatty acid has the highest affinity for the enzyme. Our model also identifies the oxyanion hole that plays an important role in the stabilization of the tetrahedral intermediate during catalysis. Comparison of this cold-adapted lipase with the crystal structure of a thermophilic Bacillus stearothermophilus P1 lipase supported the assumption that cold-adapted enzymes have a more flexible three-dimensional structure than their thermophilic counterparts. The conformational flexibility of this modeled cold-adapted lipase at low temperature probably originates from a combination of factors compared to its thermophilic counterpart, i.e., lower number of salt bridges and cation-π interactions, increase in the non-polar surface area exposed to solvent. Our study may help in understanding the structural features of a cold- adapted lipase and can further be used in engineering lipase that can function at or near extreme temperatures with considerable biotechnological potential.     

A single nucleotide polymorphism in transcobalamin II (I5V) induces structural changes in the protein as revealed by molecular modeling studies

Cobalamin is an essential micronutrient in mammals. Deficiencies of this micronutrient have been implicated as risk factors for various complex diseases. Cobalamin is transported to the cells by the transport protein transcobalamin II (TCII), and hence genetic variations (like single nucleotide polymorphisms) in TCII could be perceived to affect the binding of cobalamin to TCII, thereby modulating the intracellular concentrations of cobalamin. To understand whether three nonsynonymous mutations in TCII (I5V, P241R, and R381Q) alter the structure of the protein which could potentially affect cobalamin binding, we performed molecular dynamics simulation in silico. Superimposition of active sites of the four simulated models (wild type and three variants) with the human TCII crystal structure revealed that the distance between the Nε nitrogen atom of His-173 and the cobalt ion of cobalamin deviated considerably in the I5V model as compared to wild type and other variants. His-173 directly coordinates with the cobalt ion of cobalamin. Further, from our dynamic cross-correlation and principal component analysis it appears that in the I5V model the β-domain moves apart from the α-domain creating a wide gap between the two domains. This might facilitate the initial binding of cobalamin in the I5V model as cobalamin enters the binding site through the gap between the two domains. These observations were not found in the other variants. We thus speculate that binding of cobalamin will be more facile in the I5V variant.     

Metabolic flux analysis of CHO cell metabolism in the late non‐growth phase

Chinese hamster ovary (CHO) cell cultures are commonly used for production of recombinant human therapeutic proteins. Often the goal of such a process is to separate the growth phase of the cells, from the non‐growth phase where ideally the cells are diverting resources to produce the protein of interest. Characterizing the way that the cells use nutrients in terms of metabolic fluxes as a function of culture conditions can provide a deeper understanding of the cell biology offering guidance for process improvements. To evaluate the fluxes, metabolic flux analysis of the CHO cell culture in the non‐growth phase was performed by a combination of steady‐state isotopomer balancing and stoichiometric modeling. Analysis of the glycolytic pathway and pentose phosphate pathway (PPP) indicated that almost all of the consumed glucose is diverted towards PPP with a high NADPH production; with even recycle from PPP to G6P in some cases. Almost all of the pyruvate produced from glycolysis entered the TCA cycle with little or no lactate production. Comparison of the non‐growth phase against previously reported fluxes from growth phase cultures indicated marked differences in the fluxes, in terms of the split between glycolysis and PPP, and also around the pyruvate node. Possible reasons for the high NADPH production are also discussed. Evaluation of the fluxes indicated that the medium strength, carbon dioxide level, and temperature with dissolved oxygen have statistically significant impacts on different nodes of the flux network. Biotechnol. Bioeng. 2011; 108:82–92. © 2010 Wiley Periodicals, Inc.