Pre symptomatic detection of wheat leaf rust in the susceptible cv Skalmeje and the resistant cv Esket by means of UV laser-induced fluorescence

In modern agriculture there is a great demand for a rapid and objective screening method for stress resistance, because so far, the resistance of new cultivars is tested in time- and money consuming field experiments. Based on fluorescence ratios, and lifetime of fluorophores measured by fluorescence spectroscopy, we have postulated that an early discrimination of susceptible and resistant wheat cultivars to the leaf rust pathogen Puccinia triticina can be accomplished. As representative for leaf rust resistant and leaf rust susceptible wheat genotypes the cultivars Esket and Skalmeje, respectively, were chosen. Plants were grown under controlled environment conditions and inoculated with the leaf rust pathogen at the second-leaf-stage by single-droplet application. Fluorescence measurements were carried out from two to four days after inoculation (dai) by using a compact fibre-optic fluorescence spectrometer with nanosecond time-resolution. Experimental results indicated that UV laser-induced spectral characteristics as well as determination of fluorescence lifetime are suited to detect leaf rust two dai. For this purpose several ratios and wavelength can be considered. In general, the tested cultivars showed distinct responses to the pathogen development. In this context the ratio F451/F687 measured three dai and mean lifetimes at 500 nm and 530 nm are suited to differentiate the resistant Esket from the susceptible Skalmeje genotypes.     

SIRT6 protects against pathological damage caused by diet‐induced obesity

The NAD+‐dependent SIRT6 deacetylase is a therapeutic candidate against the emerging metabolic syndrome epidemic. SIRT6, whose deficiency in mice results in premature aging phenotypes and metabolic defects, was implicated in a calorie restriction response that showed an opposite set of phenotypes from the metabolic syndrome. To explore the role of SIRT6 in metabolic stress, wild type and transgenic (TG) mice overexpressing SIRT6 were fed a high fat diet. In comparison to their wild‐type littermates, SIRT6 TG mice accumulated significantly less visceral fat, LDL‐cholesterol, and triglycerides. TG mice displayed enhanced glucose tolerance along with increased glucose‐stimulated insulin secretion. Gene expression analysis of adipose tissue revealed that the positive effect of SIRT6 overexpression is associated with down regulation of a selective set of peroxisome proliferator‐activated receptor‐responsive genes, and genes associated with lipid storage, such as angiopoietin‐like protein 4, adipocyte fatty acid‐binding protein, and diacylglycerol acyltransferase 1, which were suggested as potential targets for drugs to control metabolic syndrome. These results demonstrate a protective role for SIRT6 against the metabolic consequences of diet‐induced obesity and suggest a potentially beneficial effect of SIRT6 activation on age‐related metabolic diseases.     

Plasma homocysteine is regulated by phospholipid methylation

Mild hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine, a non-protein amino acid, is formed from S-adenosylhomocysteine and partially secreted into plasma. A potential source for homocysteine is methylation of the lipid phosphatidylethanolamine to phosphatidylcholine by phosphatidylethanolamine N-methyltransferase in the liver. We show that mice that lack phosphatidylethanolamine N-methyltransferase have plasma levels of homocysteine that are approximately 50% of those in wild-type mice. Hepatocytes isolated from methyltransferase-deficient mice secrete approximately 50% less homocysteine. Rat hepatoma cells transfected with phosphatidylethanolamine N-methyltransferase secrete more homocysteine than wild-type cells. Thus, phosphatidylethanolamine N-methyltransferase is an important source of plasma homocysteine and a potential therapeutic target for hyperhomocysteinemia.     

Heterogeneity effects in the binding of all-trans retinal to bacterio-opsin

The special trimeric structure of bacteriorhodopsin (bR) in the purple membrane of Halobacterium salinarum, and especially, the still controversial question as to whether the three protein components are structurally and functionally identical, have been subject to considerable work. In the present work, the problem is approached by studying the reconstitution reaction of the bR apo-protein with all-trans retinal, paying special attention to the effects of the apo-protein/retinal (P:R) ratio. The basic observation is that at high P:R values, the reconstitution reaction proceeds via two distinct, fast and slow, pathways associated with two different pre-pigment precursors absorbing at 430 nm (P(430)) and 400 nm (P(400)), respectively. These two reactions, exhibiting 2:1 (P(430)/P(400)) amplitude ratios, are markedly affected by the P:R value. The principal feature is the acceleration of the P(400) --> bR transition at low P:R ratios. The data are interpreted in terms of a scheme in which the added retinal first occupies two protein retinal traps, R(1) and R(2), from which it is transferred to two spectroscopically distinct binding sites corresponding to the two pre-pigments, P(430) and P(400), respectively. Two noncovalently bound retinal molecules occupy two P(430) sites of the bR trimer, while one (P(400)) occupies the third. Binding is completed by generating the retinal-protein covalent bond. Analogous experiments were also carried out with an aromatic bR chromophore and with the D85N bR mutant. The accumulated data clearly point out the heterogeneity of the binding reaction intermediates, in which two are clearly distinct from the third. However, CD spectroscopy strongly suggests that even the two P(430) sites are not structurally identical. The heterogeneity of the P intermediates in the binding reaction can be accounted for, either by being induced by cooperativity or by an intrinsic heterogeneity that is already present in the apoprotein. The question as to whether the final reconstituted pigment, as well as native bR, are nonhomogeneous should be the subject of future studies.     

THE PHYLOGENETIC RELATIONSHIP OF PFIESTERIA PISCICIDA, CRYPTOPERIDINIOPSOID SP. AMYLOODINOUM OCELLATUM AND A PFIESTERIA-LIKE DINOFLAGELLATE TO OTHER DINOFLAGELLATES AND APICOMPLEXANS

The taxonomic relationship between heterotrophic and parasitic dinoflagellates has not been studied extensively at the molecular level. In order to investigate these taxonomic relationships, we sequenced the small subunit (SSU) ribosomal RNA gene of Pfiesteria piscicida (Steidinger et Burkholder), a Pfiesteria -like dinoflagellate, Cryptoperidiniopsoid sp., and Amyloodinium ocellatum (Brown) and submitted those sequences to GenBank. Pfiesteria piscicida and Cryptoperidiniopsoid sp. are heterotrophic dinoflagellates, purportedly pathogenic to fish, and A. ocellatum, a major fish pathogen, has caused extensive economic losses in both the aquarium and aquaculture industries. The pathogenicity of the Pfiesteria -like dinoflagellate is unknown at this time, but its growth characteristics and in vitro food preferences are similar to those of P. piscicda. The SSU sequences of these species were aligned with the other full-length dinoflagellate sequences, as well as those of representative apicomplexans and Perkinsus species, the groups most closely related to dinoflagellates. Phylogenetic analyses indicate that Cryptoperidiniopsoid sp., P. piscicida, and the Pfiesteria -like dinoflagellate are closely related and group into the class Blastodiniphyceae, as does A. ocellatum. None of the species examined were closely related to the apicomplexans or to Perkinsus marinus, the parasite that causes "Dermo disease" in oysters. The overall phylogenetic analyses largely supported the current class and subclass groupings within the dinoflagellates.     

A general platform for antibody purification utilizing engineered-micelles

We introduce a new concept and potentially general platform for antibody (Ab) purification that does not rely on chromatography or specific ligands (e.g., Protein A); rather, it makes use of detergent aggregates capable of efficiently capturing Ab while rejecting hydrophilic impurities. Captured Ab are then extracted from the aggregates in pure form without co-extraction of hydrophobic impurities or aggregate dissolution. The aggregates studied consist of conjugated “Engineered-micelles” built from the nonionic detergent, Tween-20; bathophenanthroline, a hydrophobic metal chelator, and Fe²⁺ions. When tested in serum-free media with or without bovine serum albumin as additive, human or mouse IgGs were recovered with good overall yields (70–80%, by densitometry). Extraction of IgGs with 7 different buffers at pH 3.8 sheds light on possible interactions between captured Ab and their surrounding detergent matrix that lead to purity very similar to that obtained via Protein A or Protein G resins. Extracted Ab preserve their secondary structure, specificity and monomeric character as determined by circular dichroism, enzyme-linked immunosorbent assay and dynamic light scattering, respectively.     

Activity patterns of rodents: the physiological ecology of biological rhythms

To date, most research in the field of biological rhythms has been performed on nocturnal rodents under laboratory conditions. This research has made much progress in recent years. It is now time to investigate the adaptive value of the studied molecular mechanisms under natural conditions. Here we review relevant studies of rodent activity patterns. We also review a case study of temporal partitioning between spiny mice. We conclude that the response to environmental stimuli, using a system composed of a rigid master circadian oscillator and more flexible mechanisms such as peripheral oscillators with weak coupling, masking responses, and downstream switching mechanisms, is adaptive since it enables an animal to reset its activity phase without the cost of shifting the phase of the entire circadian system. We suggest that these mechanisms play a significant role in determining activity patterns under natural conditions, and are important for understanding the ecology and evolution of activity rhythms.