Gold-coated capillary based 2,4-dichlorophenoxyacetic acid chemi-luminescent assays: possibilities towards multianalysis

The application of gold-coated glass capillaries for the design of a sensitive chemiluminescent immunoassay for 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The gold coating on the glass capillaries has been partially characterized and its effect on enhancing the signal intensity has been measured. A simple photo-multiplier tube-based photon detector is used for this purpose. At least three times improvement in the signal intensity is observed compared to uncoated glass capillaries, with a consequent improvement in the sensitivity of detection. Using such gold-coated glass capillaries, 2,4-D in the range 10(-9) to 10(-13) mol/l is detectable at a precision of +/-15% (CV%) and a limit of detection of 10(-15) mol/l is achievable. The possibility of using such gold-coated capillaries with a portable multianalytical set-up for field studies is also demonstrated.     

Fish in Reservoirs

In all reservoirs the fish fauna is recruited from their riverine antecedents. The nature of the riverine environment, created by its unstable hydrological regime, favours the evolution of generalist species. Of these species, only a few are preadapted to lacustrine conditions. When riverine fish communities are trapped in a reservoir, most of the fish stay close to the shore, the mouth of tributaries and in shallows. The pelagic and deep waters are poorly utilized and the yield of fish is below the natural productivity of these water bodies, especially in the case of deep and large reservoirs. This occurs unless the dammed river contains fish species from families which are preadapted for lacustrine conditions. A positive correlation between the percentage of pelagic fish and the total yield of fish in reservoirs indicates that the pelagial represents a vacant habitat, only seldom invaded by riverine species. This situation probably applies to the deep water of reservoirs too, although sufficient data to demonstrate this are not available. The littoral region of reservoirs is utilized by some generalists of euryoecious species derived from the indigenous riverine fauna which may be preadapted for lacustrine conditions. If such preadapted lacustrine species are not present, the introduction of lacustrine species or those preadapted for the lacustrine environment is desirable under certain circumstances. However, caution must be exercised in any introduction.     

Immunosuppressant FK506: Focusing on neuroprotective effects following brain and spinal cord injury

The secondary damage that follows central nervous system (CNS) injury is a target for neuroprotective agents aimed at tissue and function sparing. FK506, a clinically used immunosuppressant, acts neuroprotectively in rat models of brain and spinal cord injury and ischemia. Evidence of in vivo experimental studies highlights the neuroprotective role of FK506 by its direct impact on various cell populations within the CNS. The participation of FK506 in modulation of post-traumatic inflammatory processes is a further potential aspect involved in CNS neuroprotection. In this review we provide an overview of the current laboratory research focusing on the multiple effects of FK506 on neuroprotection following CNS injury.     

High-throughput knockout screen in fission yeast

We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/. We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.     

Proteome alterations in gamma-irradiated human T-lymphocyte leukemia cells

Analyses of the protein expression profiles of irradiated cells may be beneficial for identification of new biomolecules of radiation-induced cell damage. Therefore, in this study we exploited the proteomic approach to identify proteins whose expression is significantly altered in gamma-irradiated human T-lymphocyte leukemia cells. MOLT-4 cells were irradiated with 7.5 Gy and the cell lysates were collected at different times after irradiation (2, 5 and 12 h). The proteins were separated by two-dimensional electrophoresis and quantified using an image evaluation system. Proteins exhibiting significant radiation-induced alterations in abundance were identified by peptide mass fingerprinting. We identified 14 proteins that were either up- or down-regulated. Cellular levels of four of the proteins (Rho GDP dissociation inhibitor 1 and 2, Ran binding protein 1, serine/threonine protein kinase PAK2) were further analyzed by two-dimensional immunoblotting to confirm the data obtained from proteome analysis. All identified proteins were classified according to their cellular function, including their participation in biochemical and signaling pathways. Taken together, our results suggest the feasibility of the proteome method for monitoring of cellular radiation responses.     

Control of Shugoshin function during fission-yeast meiosis

Meiosis consists of a single round of DNA replication followed by two consecutive nuclear divisions. During the first division (MI), sister kinetochores must orient toward the same pole to favor reductional segregation. Correct chromosome segregation during the second division (MII) requires the retention of centromeric cohesion until anaphase II. The spindle checkpoint protein Bub1 is essential for both processes in fission yeast . When bub1 is deleted, the Shugoshin protein Sgo1 is not recruited to centromeres, cohesin Rec8 does not persist at centromeres, and sister-chromatid cohesion is lost by the end of MI. Deletion of bub1 also affects kinetochore orientation because sister centromeres can move to opposite spindle poles in approximately 30% of MI divisions. We show here that these two functions are separable within the Bub1 protein. The N terminus of Bub1 is necessary and sufficient for Sgo1 targeting to centromeres and the protection of cohesion, whereas the C-terminal kinase domain acts together with Sgo2, the second fission-yeast Shugoshin protein, to promote sister-kinetochore co-orientation during MI. Additional analyses suggest that the protection of centromeric cohesion does not operate when sister kinetochores attach to opposite spindle poles during MI. Sgo1-mediated protection of centromere cohesion might therefore be regulated by the mode of kinetochore attachment.     

The role of iron in type 2 diabetes in humans

The role of micronutrients in the etiology of type 2 diabetes is not well established. Several lines of evidence suggest that iron play may a role in the pathogenesis of type 2 diabetes. Iron is a strong pro-oxidant and high body iron levels are associated with increased level of oxidative stress that may elevate the risk of type 2 diabetes. Several epidemiological studies have reported a positive association between high body iron stores, as measured by circulating ferritin level, and the risk of type 2 diabetes and of other insulin resistant states such as the metabolic syndrome, gestational diabetes and polycystic ovarian syndrome. In addition, increased dietary intake of iron, especially that of heme iron, is associated with risk of type 2 diabetes in apparently healthy populations. Results from studies that have evaluated the association between genetic mutations related to iron metabolism have been inconsistent. Further, several clinical trials have suggested that phlebotomy induced reduction in body iron levels may improve insulin sensitivity in humans. However, no interventional studies have yet directly evaluated the effect of reducing iron intake or body iron levels on the risk of developing type 2 diabetes. Such studies are required to prove the causal relationship between moderate iron overload and diabetes risk.     

C-Nucleoside Analogues of Nicotinamide Mononucleotide (NMN)

Abstract

5-(β-D-Ribofuranosyl)nicotinamide (lie) and 6-(β-D-ribo-furanosyDpicolinamide (IId) and their corresponding o-isomers (III) were synthesized from ribonolactone. The C-nucleoside lie was further converted to its 5′-monophosphate Up which is isosteric to NMN Ip).