A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.     

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2⁺ neural progenitor, A2B5⁺ astrocyte/ glial progenitor, NG2⁺ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.     

Deoxyribonucleases (DNases) in the cortex and endosome from the marine sponge <Emphasis Type="Italic">Tethya aurantium</Emphasis>

The presence and activity of deoxyribonucleases in the cortex and endosome sections from a sponge, the sea orange Tethya aurantium, were investigated. The maximal enzyme activity in sponge homogenate was detected at pH 4.27, pH 7.0 and pH 8.5–8.75. Among different specimens, several distinct patterns of neutral DNase isozymes were observed in the cortex section. In each investigated specimen the highest neutral DNase activity belonged to high molecular weight proteins (up to75 kDa). The acid DNases showed a low level of enzyme activity. In the endosome section the acid DNase activity was up to ten times higher than in the cortex and the presence of DNase II-like protein was detected. Neutral DNase, which expressed the highest enzyme activity in all the investigated specimens, has a molecular weight of 20 kDa and belongs to the DNase I-like family. The results indicate that the activity of neutral and acid DNases is related to sponge sections and their biological functions. The cortex, as the sponge section that communicates with the environment, expresses high interindividual variability and heterogeneity of neutral DNases, while the endosome section, where the intracellular digestion is localized, is a site of high acid DNase activity.     

Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia

Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.     

G protein betagamma subunits interact with alphabeta- and gamma-tubulin and play a role in microtubule assembly in PC12 cells

The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.     

Manganese biomining: A review

Biomining comprises of processing and extraction of metal from their ores and concentrates using microbial techniques. Currently this is used by the mining industry to extract copper, uranium and gold from low grade ores but not for low grade manganese ore in industrial scale. The study of microbial genomes, metabolites and regulatory pathways provide novel insights to the metabolism of bioleaching microorganisms and their synergistic action during bioleaching operations. This will promote understanding of the universal regulatory responses that the biomining microbial community uses to adapt to their changing environment leading to high metal recovery. Possibility exists of findings ways to imitate the entire process during industrial manganese biomining endeavor. This paper reviews the current status of manganese biomining research operations around the world, identifies factors that drive the selection of biomining as a processing technology, describes challenges in exploiting these innovations, and concludes with a discussion of Mn biomining’s future.     

Modulation of PPAR subtype selectivity. Part 2: Transforming PPARα/γ dual agonist into α selective PPAR agonist through bioisosteric modification

A novel series of oxime containing benzyl-1,3-dioxane-r-2-carboxylic acid derivatives (6a-k) were designed as selective PPARα agonists, through bioisosteric modification in the lipophilic tail region of PPARα/γ dual agonist. Some of the test compounds (6a, 6b, 6c and 6f) showed high selectivity towards PPARα over PPARγ in vitro. Further, highly potent and selective PPARα agonist 6c exhibited significant antihyperglycemic and antihyperlipidemic activity in vivo, along with its improved pharmacokinetic profile. Favorable in-silico interaction of 6c with PPARα binding pocket correlate its in vitro selectivity profile toward PPARα over PPARγ. Together, these results confirm discovery of novel series of oxime based selective PPARα agonists for the safe and effective treatment of various metabolic disorders.     

Restoration of ultrastructural and biochemical changes in alloxan-induced diabetic rat sciatic nerve on treatment with Na3VO4 and Trigonella—a promising antidiabetic agent

Vanadium has been reported to have broad pharmacological activity both in vitro and in vivo. Vanadium compound, sodium orthovanadate, Na₃VO₄, is well known for its hypoglycaemic effects. However, Na₃VO₄ exerts these effects at relatively high doses (0.6 mg/ml) and exhibit several toxic effects. In the present study lower doses of Na₃VO₄ (0.2 mg/ml) are combined with Trigonella foenum graecum seed powder (TSP), another hypoglycaemic agent, to reduce its toxicity without compromising its antidiabetic potential. The efficacy of the lower doses of Na₃VO₄ has been investigated in restoring the altered glucose metabolism and histological structure in the sciatic nerves in 21 and 60 days alloxan diabetic rats. A portion of the glucose was found to be channelled from the normal glycolytic route to polyol pathway, evident by the reduced hexokinase activity and increased polyol pathway enzymes aldose reductase and sorbitol dehydrogenase activity causing accumulation of sorbitol and fructose in diabetic conditions. Ultrastructural observation of the sciatic nerve showed extensive demylination and axonal loss after eight weeks of diabetes induction. Blood glucose levels increased in diabetic rats were normalized with the lower dose of vanadium and Trigonella treatment. The treatment of the diabetic rats with vanadium and Trigonella prevented the activation of the polyol pathway and sugar accumulations. The sciatic nerves were also protected against the structural abnormalities found in diabetes with Trigonella foenum graecum as well as Na₃VO₄. Results suggest that lower doses of Na₃VO₄ may be used in combination with TSP as an efficient antidiabetic agent to effectively control the long-term complications of diabetes in tissues like peripheral nerve.     

Nitrate-dependent salicylate degradation by Pseudomonas butanovora under anaerobic conditions

Nitrate-dependent salicylate degradation by the denitrifying Pseudomonas butanovora was investigated and the molar ratio of the cometabolism under anaerobic circumstances was determined. The bacterium was able to utilize salicylate as an electron donor for the reduction of nitrate. Salicylate was eliminated via catechol, which is degraded by means of catechol 2,3-oxygenases (meta-cleavage), forming 2-hydroxymuconic semialdehyde. The molar ratios of NO(3)(-)-N:salicylate existing during the experiment accorded well with the assumed 1:1 molar ratio. The tolerances of the growth, the salicylate degradation and the denitrification of P. butanovora to various heavy metal ions were also studied. Although the strain was tolerant to Pb(2+) and Cu(2+) up to 1 mM in complete medium, salicylate utilization took place only up to a concentration of 0.1 mM for both heavy metal ions. Of the heavy metal ions investigated, Cd(2+) (at a concentration of 0.05 mM) displayed the highest inhibitory effect on salicylate degradation by P. butanovora.