Biological rhythmicity of the plasma antioxidant defence in lambs following supplementation of micronutrients or providing shelter in temperature-controlled microenvironment in summer

Antioxidant defence is one of the important biological adaptations to thermal stress in animals. Micronutrients play crucial roles in the bioactivity of several antioxidant systems. Shelter management by providing a thermo-comfort micro-environment to animals may also combat the heat stress during summer. Hence, the aim of the present study was to assess the biological ability of Malpura lambs to counter thermal stress following supplementation of micronutrients and providing shelter in a temperature-controlled climatic chamber during the hot day-hours in summer. Twenty-one lambs of 3 - 5 months age were equally divided in to three group viz. Group1: control, Group2: micronutrient- supplemented and Group3: housing in thermo-neutral climatic chamber. Lambs in group1 and 2 were maintained in a shed having free access to hot wind while the lambs in group 3 were kept inside a climatic chamber (temperature and relative humidity about 30 °C and 40%, respectively) between 10 am to 4:30 pm. All the lambs were provided identical diet consisting of 2.5 kg concentrate and ad libitum quantities of Cenchrus ciliaris hay and clean water. Group3 lambs were additionally supplemented with 0.150 g micronutrient pellet per animal per day for a period of 30 days. Blood samples were collected at 0, 15, 30 days and plasma antioxidants activity were estimated. The catalase activity in the micronutrient-pellet supplemented group was significantly (p < 0.05) higher than the control both at day15 and day30. The superoxide dismutase activity was increased significantly (p < 0.05) at day30 than the day0 in both the micronutrient-supplemented and climatic chamber groups. In conclusion, both the micronutrient supplementation and housing in a thermo-comfort environment enhance the antioxidant defence and biological adaptation in lambs during thermal stress in summer.     

An efficient protocol for genetic transformation and shoot regeneration of turmeric (Curcuma longa L.) via particle bombardment

Turmeric (Curcuma longa L.) is an important spice crop plant that is sterile and cannot be improved by conventional breeding. An efficient method for stable transformation for turmeric, C. longa L., was developed using particle bombardment. Callus cultures initiated from shoots were bombarded with gold particles coated with plasmid pAHC25 containing the bar and gusA genes each driven by the maize ubiquitin promoter. Transformants were selected on medium containing glufosinate. Transgenic lines were established on selection medium from 50% of the bombarded calluses. Transgenic shoots regenerated from these were multiplied and stably transformed plantlets were produced. Polymerase chain reaction (PCR) and histochemical GUS assay confirmed the stable transformation. Transformed plantlets were resistant to glufosinate.     

A comparison of in vitro with in vivo flowering in bamboo: Bambusa arundinacea

Seedling explants of Bambusa arundinacea were cultured in a Murashige & Skoog (MS) based liquid medium, supplemented with sucrose (2), coconut water (5) and 6-benzylaminopurine (2.2 μM). In 3–6 months about 70 of the cultures flowered. A comparison was made between in vitro and in vivo flowering. Though smaller in size, in vitro florets were morphologically comparable to the in vivo florets. Anthesis in in vivo flowering took place in the morning hours. It was more or less synchronized and was dependent on the atmospheric temperature and humidity. The lemma and palea opened to expose both androecium and gynoecium to the pollinating agent (wind). In in vitro flowering, some florets opened as in their in vivo counterparts, some did not open but the anthers protruded from the tip of the partially opened lemma and palea. Anthesis was not synchronized under in vitro conditions. Pollen fertility in in vivo and in vitro flowerings were approximately 93 and 31 respectively. Studies by scanning electron microscopy showed some discrepancies in the pollen wall development in vitro. The trifid stigmas of in vivo florets were highly feathery with many papillae and withered soon after pollination or within few hours. The stigmas of in vitro developed florets were smaller with fewer and stouter papillae. They remained turgid for relatively longer periods. Seed production in in vivo flowering was profuse whereas in in vitro flowering seeds were produced only when many florets opened at the same time, in the same culture vessel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pyruvate Induces Transient Tumor Hypoxia by Enhancing Mitochondrial Oxygen Consumption and Potentiates the Anti-Tumor Effect of a Hypoxia-Activated Prodrug TH-302

Background

TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.

Methodology/Results

The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O₂), with minimal effect under aerobic conditions (21% O₂). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm³. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm³), significantly delayed tumor growth.

Conclusions/Significance

Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.     

Malcofaunal Diversity of Chilika Lake,Odisha, India

Investigation during the period of 3 years from 2007 to 2010 on the malacofauna of Chilika lake revealed the occurrence of 126 molluscan taxa belonging to 56 families, 18 orders of three classes in the bottom sediment. Of these 61 species belonged to Bivalvia, 64 species belonged to Gastropoda and one species belonged to Polyplacophora. Maximum Bivalvia and Gastropoda taxa were found in the outer channel region of the lake. The dominating species were Crassostrea cuttackensis, Saccostrea cucullata, Brachidontes undulatus, Meretrix meretrix among bivalves and Cerethideopsilla cingulata, Bullia vittata, Nassarious stolatus, Indothias lacera, Natica tigrina, Turritella attenuata were from the gastropods. Occurrence of a large number of marine taxa is most probably associated with the opening of new lagoon during 1st August 2008.     

New Insights into Polychaete Traces and Fecal Pellets: Another Complex Ichnotaxon?

Neoichnological observations help refine paleoichnological records. The present study reports extensive observations on the distribution, morphology, occurrence and association of burrows and fecal pellets of the polychaete Nereis diversicolor in the Kundalika Estuary on the west coast of India. Our holistic study of these modern-day traces suggests it to be a complex trace arising from domichnial, fodinichnial and possibly pascichnial behavior of polychaetes. The study for the first time reports extensive fecal pellet production, distribution and their preservation as thick stacks in modern estuarine environment. These observations testify the fossilization potential of pellets and provide an explanation to their origin in the geological record. Their occurrence as strings associated with mounds not only suggests pascichnial behaviour of polychaetes but also allows the assignment of post-Paleozoic Tomaculum to the activity of polychaete worms. The production of fecal pellets in such large quantities plays a major role in increasing the average grain size of the substrate of these estuarine tidal flats, thereby improving aeration within the substrate.     

Ethanol-sensitive sites on the human dopamine transporter

Previous studies have shown that ethanol enhanced [(3)H]dopamine uptake in Xenopus oocytes expressing the dopamine transporter (DAT). This increase in DAT activity was mirrored by an increase in the number of transporters expressed at the cell surface. In the present study, ethanol potentiated the function of DAT expressed in HeLa cells but inhibited the function of the related norepinephrine transporter (NET). Chimeras generated between DAT and NET were examined for ethanol sensitivity and demonstrated that a 76-amino acid region spanning transmembrane domains (TMD) 2 and 3 was essential for ethanol potentiation of DAT function. The second intracellular loop between TMD 2 and 3 of DAT, which differs from that of NET by four amino acids, was explored for possible sites of ethanol action. Site-directed mutagenesis was used to replace each of these residues in DAT with the corresponding residue in NET, and the resulting cRNA were expressed in Xenopus oocytes. We found that mutations G130T or I137F abolished ethanol potentiation of DAT function, whereas the mutations F123Y and L138F had no significant effect. These results identify novel sites in the second intracellular loop that are important for ethanol modulation of DAT activity.     

Comparative Analysis of Predicted Plastid-Targeted Proteomes of Sequenced Higher Plant Genomes

Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding plastid biology, preferentially based on model systems is deficient. New plant genomes are expected to enable the identification of potentially new plastid-targeted proteins that will aid in studying novel roles of plastids.     

Potential of Piperazinylalkylester Prodrugs of 6-Methoxy-2-Naphthylacetic Acid (6-MNA) for Percutaneous Drug Delivery

Piperazinylalkyl ester prodrugs (4a–5d) of 6-methoxy-2-naphthylacetic acid (6-MNA) (1) were synthesized and evaluated in vitro for the purpose of percutaneous drug delivery. These ionizable prodrugs exhibited varying aqueous solubilities and lipophilicities depending on the pH of the medium. The prodrugs (4a–5c) showed higher aqueous solubility and similar lipophilicity at pH 5.0 and lower aqueous solubility and higher lipophilicity at pH 7.4 in comparison to 6-MNA. The chemical and enzymatic hydrolyses of the prodrugs was investigated in aqueous buffer solutions (pH 5.0 and 7.4) and in 80% human serum (pH 7.4) at 37°C. The prodrugs showed moderate chemical stability (t_1/2 = 6–60 h) but got readily hydrolyzed enzymatically to 6-MNA with half-life ranging from 10–60 min. In the in vitro permeation study using rat skin, the flux of 6-MNA and the prodrugs was determined in aqueous buffers of pH 5.0 and 7.4. The prodrug (5b) showed 7.9- and 11.2-fold enhancement in skin permeation compared to 6-MNA (1) at pH 5.0 and 7.4, respectively. It was concluded that the parent NSAIDs having favorable pharmacokinetic and pharmacodynamic properties coupled with increased skin permeability of their prodrugs could give better options for the treatment of rheumatic diseases.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0240-6) contains supplementary material, which is available to authorized users.KEY WORDS: 6-MNA, NSAID, piperazinylalkylester, prodrug, skin permeation