Nature of the chromophore binding site of bacteriorhodopsin: the potential role of Arg82 as a principal counterion

The nature of the chromophore binding site of light-adapted bacteriorhodopsin is analyzed by using modified neglect of differential overlap with partial single and double configuration interaction (MNDO-PSDCI) molecular orbital theory to interpret previously reported linear and nonlinear optical spectroscopic measurements. We conclude that in the absence of divalent metal cations in close interaction with Asp85 and Asp212, a positively charged amino acid must be present in the same vicinity. We find that models in which Arg82 is pointed upward into the chromophore binding site and directly stabilizes Asp85 and Asp212 are successful in rationalizing the observed one-photon and two-photon properties. We conclude further that a water molecule is strongly hydrogen bonded to the chromophore imine proton. The chromophore "1Bu*+" and "1Ag*-" states, despite extensive mixing, exhibit significantly different configurational character. The lowest-lying "1Bu*+" state is dominated by single excitations, whereas the second-excited "1Ag*-" state is dominated by double excitations. We can rule out the possibility of a negatively charged binding site, because such a site would produce a lowest-lying "1Ag*-" state, which is contrary to experimental observation. The possibility that Arg82 migrates toward the extracellular surface during the photocycle is examined.     

Circadian Variation in Circulating Levels of Melatonin,Testosterone and Estradiol-17β in a Tropical Freshwater Catfish,Clarias batrachus,during Different Phases of the Annual Reproductive Cycle

In the present investigation, daily variations in plasma levels of melatonin, testosterone and estradiol-17β were analysed in the tropical freshwater catfish, Clarias batrachus during early and late-preparatory, pre-spawning, late-spawning and post-spawning phases of its annual reproductive cycle. Plasma melatonin levels exhibited a significant circadian rhythm during all the reproductive phases but peaks were invariably at night. The amplitude of melatonin was high during the early-preparatory phase and low in the pre-spawning phase. Testosterone also displayed significant rhythm during all the studied phases except post spawning phase. Estradiol-17β revealed diurnal rhythm only during preparatory and pre-spawning phases. The peak in the levels of plasma testosterone and estradiol-17β were recorded during the photophase. During the late-preparatory phase, the rhythm of testosterone coincided with the rhythm of estradiol-17β.     

Aseptic rearing of Epiphyas postvittana (Lepidoptera: Tortricidae) on a meridic diet

The light brown apple moth, Epiphyas postvittana (Walker), was reared aseptically from newly hatched larvae to adults for three generations on a meridic diet. Growth and development on this diet were comparable with that on oligidic diets and on host‐plants. At 25 ± 1°C, 50–60% R.H., and 12‐h photophase, the average larval period was approx. 28 days, pre‐pupal and pupal 11–12 days, and pre‐oviposition approx. 2 days; the incuva‐tion period of eggs was approx. 7 days. The average developmental period from egg to adult was thus about 49 days; 89 % of larvae reached the adult stage. The average fecundity was 598 eggs per female, and the sex ratio was nearly 1:1. There were no differences in the rate of development or in fecundity of insects reared on media with and without a mould inhibitor.     

Processing of Apple Pomace for Bioactive Molecules

The growth of horticulture industries worldwide has generated huge quantities of fruit wastes (25%–40% of the total fruits processed). These residues are generally a good source of carbohydrates, especially cell wall polysaccharides and other functionally important bioactive molecules such as proteins, vitamins, minerals and natural antioxidants. “Apple pomace” is a left-over solid biomass with a high moisture content, obtained as a by-product during the processing of apple fruits for juice, cider or wine preparation. Owing to the high carbohydrate content, apple pomace is used as a substrate in a number of microbial processes for the production of organic acids, enzymes, single cell protein, ethanol, low alcoholic drinks and pigments. Recent research trends reveal that there is an increase in the utilization of apple pomace as a food processing residue for the extraction of value added products such as dietary fibre, protein, natural antioxidants, biopolymers, pigments and compounds with unique properties. However, the central dogma is still the stability, safety and economic feasibility of the process(s)/product(s) developed. This review is mainly focused on assessing recent research developments in extraction, isolation and characterization of bioactive molecules from apple pomace, along with their commercial utilization, in food fortification.     

Differentiation dynamics of mammary epithelial stem cells from Korean holstein dairy cattle under ECM-free conditions

The “stem cells” are commonly defined as “cells capable of self-renewal through replication and differentiating into specific lineages”. The mammary gland contains functional stem/progenitor cells. The current study was planned with the objectives to study the differentiation dynamics of Korean Holstein mammary epithelial stem cells (KHMESCs) under the optimum culture conditions. Lineage negative KHMESCs isolated from mammary tissue of lactating cows have shown the typical differentiation dynamics with formation of lobulo–alveolar structures in in vitro culture. This suggests the existence of bipotential mammary epithelial stem cells in the mammary gland. The strong mRNA expression of pluripotency factors indicates stemness, whereas expression of milk protein genes and epithelial cell-specific gene indicate their differentiation capabilities. Further, immunostaining results have shown the differentiation capabilities of KHMESCs into both luminal and basal lineages under the extracellular matrix (ECM, matrigel) free environment. However, under matrigel, the differentiation process was comparatively higher than without matrigel. Immunostaining results also suggested that differentiated cells could secrete milk proteins such as β-casein. To our knowledge, these data represent the first report on the differentiation dynamics and establishment of mammary epithelial stem cells from Korean Holstein with typical stemness properties. It was observed that isolated KHMESCs had normal morphology, growth pattern, differentiation ability, cytogenetic and secretory activity even without ECM. Therefore, it is concluded that established KHMESCs could be used for further studies on Korean Holstein dairy cows related to lactation studies, as non-GMO animal bioreactors and stem cell-based management of bovine mastitis including post-mastitis damage.     

Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: An uptake and processing study in the midgut of Penaeus monodon

Several oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral delivery was not elucidated. In this study the fate of these components and of live WSSV, orally intubated to black tiger shrimp (Penaeus monodon) was investigated by immunohistochemistry, employing antibodies specific for VP28 and haemocytes. The midgut has been identified as the most prominent site of WSSV uptake and processing. The truncated recombinant VP28 (rec-VP28), formalin-inactivated virus (IVP) and live WSSV follow an identical uptake route suggested as receptor-mediated endocytosis that starts with adherence of luminal antigens at the apical layers of gut epithelium. Processing of internalized antigens is performed in endo-lysosomal compartments leading to formation of supra-nuclear vacuoles. However, the majority of WSSV-antigens escape these compartments and are transported to the inter-cellular space via transcytosis. Accumulation of the transcytosed antigens in the connective tissue initiates aggregation and degranulation of haemocytes. Finally the antigens exiting the midgut seem to reach the haemolymph. The nearly identical uptake pattern of the different WSSV-antigens suggests that receptors on the apical membrane of shrimp enterocytes recognize rec-VP28 efficiently. Hence the truncated VP28 can be considered suitable for oral vaccination, when the digestion in the foregut can be bypassed.     

The Light Skin Allele of SLC24A5 in South Asians and Europeans Shares Identity by Descent

Skin pigmentation is one of the most variable phenotypic traits in humans. A non-synonymous substitution (rs1426654) in the third exon of SLC24A5 accounts for lighter skin in Europeans but not in East Asians. A previous genome-wide association study carried out in a heterogeneous sample of UK immigrants of South Asian descent suggested that this gene also contributes significantly to skin pigmentation variation among South Asians. In the present study, we have quantitatively assessed skin pigmentation for a largely homogeneous cohort of 1228 individuals from the Southern region of the Indian subcontinent. Our data confirm significant association of rs1426654 SNP with skin pigmentation, explaining about 27% of total phenotypic variation in the cohort studied. Our extensive survey of the polymorphism in 1573 individuals from 54 ethnic populations across the Indian subcontinent reveals wide presence of the derived-A allele, although the frequencies vary substantially among populations. We also show that the geospatial pattern of this allele is complex, but most importantly, reflects strong influence of language, geography and demographic history of the populations. Sequencing 11.74 kb of SLC24A5 in 95 individuals worldwide reveals that the rs1426654-A alleles in South Asian and West Eurasian populations are monophyletic and occur on the background of a common haplotype that is characterized by low genetic diversity. We date the coalescence of the light skin associated allele at 22–28 KYA. Both our sequence and genome-wide genotype data confirm that this gene has been a target for positive selection among Europeans. However, the latter also shows additional evidence of selection in populations of the Middle East, Central Asia, Pakistan and North India but not in South India.     

Cardiovascular effects of transdermally delivered bupranolol in rabbits: effect of chemical penetration enhancers

Bupranolol is a promising candidate for transdermal drug delivery system (TDDS) development. The effect of permeation enhancers on the in vivo delivery and beta-blocking effect of reservoir type TDDS was studied in comparison with intravenous BPL in rabbits. The beta-blocking effect was quantified by measuring the inhibition of isoprenaline induced tachycardia in rabbits after BPL administration via transdermal and intravenous routes. The reservoir type TDDS containing a hydroxypropyl cellulose gel and polyethylene membrane was used as a control device. In comparison, the TDDS containing skin penetration enhancers, either 2-pyrrolidone or partially methylated beta cyclodextrin (PMbetaCD) were evaluated. The control device (no enhancer) produced about 52% inhibition of isoprenaline induced tachycardia at 2 h and the effect continued over 24 h application period, however, the devices with 2-pyrolidone or PMbetaCD produced about 85% inhibition of isoprenaline induced tachycardia at 3 h and the same effect continued over 24 h application period. Likewise, the AUC of these devices were significantly higher than that of control device. The intravenous bupranolol showed rapid decline in the pharmacodynamic effect with time indicating its rapid elimination. The in vivo delivery of bupranolol (as estimated by a mass balance study) from the devices made with pyrolidone or PMbetaCD was 3-fold higher than that of control. The results of this study strongly suggest that the penetration enhancers in the TDDS increased the in vivo delivery of BPL, thereby increased the beta-blocking activity of BPL by 50-60% higher than control, enabling the reduction of the TDDS patch size, accordingly.     

Effect of nutrient depletion on beta-carotene and glycerol accumulation in two strains of Dunaliella sp

The response of two strains of Dunaliella, a beta-carotene accumulating halotolerant alga, was evaluated under sulphate, nitrate and phosphate limitation. All these factors decreased the growth rate and chlorophyll content but, increased the beta-carotene content of the two isolates of Dunaliella, D1, obtained from GTCC and D2 an indigenous strain isolated from Sambhar salt lake, India. Both the strains exhibited accumulation of beta-carotene and glycerol under the different nutrient limiting conditions. A maximum accumulation of 3.99 pg/cell of beta-carotene was observed under phosphate depletion. However, nutrient depletion did not significantly affect the glycerol accumulation in these cells. D2, the indigenous isolate, was found to be a better accumulator of beta-carotene than D1.