The homologous recombination system of Ustilago maydis

Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of U. maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins.     

Decision tree-driven tandem mass spectrometry for shotgun proteomics

Mass spectrometry has become a key technology for modern large-scale protein sequencing. Tandem mass spectrometry, the process of peptide ion dissociation followed by mass-to-charge ratio (m/z) analysis, is the critical component for peptide identification. Recent advances in mass spectrometry now permit two discrete, and complementary, types of peptide ion fragmentation: collision-activated dissociation (CAD) and electron transfer dissociation (ETD) on a single instrument. To exploit this complementarity and increase sequencing success rates, we designed and embedded a data-dependent decision tree algorithm (DT) to make unsupervised, real-time decisions of which fragmentation method to use based on precursor charge and m/z. Applying the DT to large-scale proteome analyses of Saccharomyces cerevisiae and human embryonic stem cells, we identified 53,055 peptides in total, which was greater than by using CAD (38,293) or ETD (39,507) alone. In addition, the DT method also identified 7,422 phosphopeptides, compared to either 2,801 (CAD) or 5,874 (ETD) phosphopeptides.     

Rice grain zinc concentrations as affected by genotype, native soil-zinc availability, and zinc fertilization

The development of rice (Oryza sativa L.) cultivars with a higher Zn content in their grains has been suggested as a way to alleviate Zn malnutrition in human populations subsisting on rice in their daily diets. This study was conducted to evaluate the effects of native soil Zn status and fertilizer application on Zn concentrations in grains of five rice genotypes that had previously been identified as either high or low in grain Zn. Genotypes were grown in field trials at four sites ranging in native soil-Zn status from severely deficient to high in plant available Zn. At each site a −Zn plot was compared to a +Zn plot fertilized with 15 kg Zn ha⁻¹. Results showed that native soil Zn status was the dominant factor to determine grain Zn concentrations followed by genotype and fertilizer. Depending on soil-Zn status, grain Zn concentrations could range from 8 mg kg⁻¹ to 47 mg kg⁻¹ in a single genotype. This strong location effect will need to be considered in estimating potential benefits of Zn biofortification. Our data furthermore showed that it was not possible to simply compensate for low soil Zn availability by fertilizer applications. In all soils fertilizer Zn was taken up as seen by a 50–200% increase in total plant Zn content. However, in more Zn deficient soils this additional Zn supply improved straw and grain yield and increased straw Zn concentrations by 43–95% but grain Zn concentrations remained largely unchanged with a maximum increase of 6%. Even in soils with high Zn status fertilizer Zn was predominantly stored in vegetative tissue. Genotypic differences in grain Zn concentrations were significant in all but the severely Zn deficient soil, with genotypic means ranging from 11 to 24 mg kg⁻¹ in a Zn deficient soil and from 34 to 46 mg kg⁻¹ in a high Zn upland soil. Rankings of genotypes remained largely unchanged from Zn deficient to high Zn soils, which suggests that developing high Zn cultivars through conventional breeding is feasible for a range of environments. However, it may be a challenge to develop cultivars that respond to Zn fertilizer with higher grain yield and higher grain Zn concentrations when grown in soils with low native Zn status.     

Ameliorated hepatic insulin resistance is associated with normalization of microsomal triglyceride transfer protein expression and reduction in very low density lipoprotein assembly and secretion in the fructose-fed hamster

To determine whether reduction of insulin resistance could ameliorate fructose-induced very low density lipoprotein (VLDL) oversecretion and to explore the mechanism of this effect, fructose-fed hamsters received placebo or rosiglitazone for 3 weeks. Rosiglitazone treatment led to normalization of the blunted insulin-mediated suppression of the glucose production rate and to a approximately 2-fold increase in whole body insulin-mediated glucose disappearance rate (p < 0.001). Rosiglitazone ameliorated the defect in hepatocyte insulin-stimulated tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2 and the reduced protein mass of IRS-1 and IRS-2 induced by fructose feeding. Protein-tyrosine phosphatase 1B levels were increased with fructose feeding and were markedly reduced by rosiglitazone. Rosiglitazone treatment led to a approximately 50% reduction of VLDL secretion rates (p < 0.05) in vivo and ex vivo. VLDL clearance assessed directly in vivo was not significantly different in the FR (fructose-fed + rosiglitazone-treated) versus F (fructose-fed + placebo-treated) hamsters, although there was a trend toward a lower clearance with rosiglitazone. Enhanced stability of nascent apolipoprotein B (apoB) in fructose-fed hepatocytes was evident, and rosiglitazone treatment resulted in a significant reduction in apoB stability. The increase in intracellular mass of microsomal triglyceride transfer protein seen with fructose feeding was reduced by treatment with rosiglitazone. In conclusion, improvement of hepatic insulin signaling with rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist, is associated with reduced hepatic VLDL assembly and secretion due to reduced intracellular apoB stability.     

Characteristics of protectant synthesis of infective juveniles of Steinernema carpocapsae and importance of glycerol as a protectant for survival of the nematodes during osmotic dehydration

Two hypotheses on the synthesis of the protectants glycerol and trehalose of the infective juveniles (IJs) of Steinernema carpocapsae during osmotic dehydration were tested and utilised to evaluate the function and importance of glycerol on survival of the nematodes during osmotic dehydration. This was achieved by comparing the changes in survival, morphology, behaviour and levels of glycerol, trehalose and permeated compounds of the IJs dehydrated in seven hypertonic solutions at two temperature regimes: (1) 5 °C for 15 days; and (2) 23 °C for 1 day followed by 5 °C for another 14 days. The results substantiate both hypotheses tested: (1) the permeability of the IJs to various compounds, such as sucrose or ethylene glycol, when they are dehydrated in hypertonic solutions of these compounds; and (2) suppression of the synthesis of protectant glycerol but not trehalose when IJs are dehydrated at low temperature. The results also showed that: (1) although trehalose was the preferred dehydration protectant, glycerol played an important role in rapidly balancing the osmotic pressure when IJs were exposed in hypertonic solutions; (2) the presence of glycerol was essential for the IJs to survive and function properly even under moderate osmotic dehydration, especially when IJs were dehydrated in salt solutions; and (3) some exogenous compounds permeated into IJs during osmotic dehydration such as ethylene glycol, may function in the same way as glycerol and significantly improve the survival and function of the IJs. The results indicate that each of the protectants glycerol and trehalose has a specific function and neither is replaceable by the other.     

Comparison of three fecal steroid metabolites for pregnancy detection used with single sampling in bighorn sheep (Ovis canadensis)

We conpared three fecal steroid metabolite assays for their usefulness in detecting pregnalcy among free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from Bighorn Canyon National Recreation Area, Wyoming and Montana (USA) and captive bighorn ewes at ZooMontana in Billings, Montana. Fecal samples were collected from 11 free-ranging, radio-collared bighorn ewes in late January-May 2001 and from 20 free-ranging, radio-collared ewes in late March to mid-May 2002. Free-ranging ewes were monitored the following spring to determine whether or not they lambed. In addition, two captive ewes were studied at ZooMontana. With three exceptions, free-ranging bighorn ewes that produced lambs had nonspecific progesterone metabolite (iPdG) levels of >1800 ng/g feces and iPdG levels >7000 ng/gm feces when samples were collected between early March and mid-May. Samples collected earlier in the year were inconclusive. One false negative was suspected to be the result of sample collection error. Of the captive ewes, nonspecific pregnanediol-3alpha-glucuronide (PdG) and iPdG followed a predictable curve over the course of the 180-day pregnancies. We conclude that estrone conjugates are not useful in diagnosing pregnancy; however, fecal steroid analysis of PdG and iPdG can be used to accurately determine pregnancy and reproductive function in bighorn sheep. This holds great potential as a noninvasive technique for understanding the role of reproductive disease in wild bighom sheep.     

The required role of endogenously produced lipoxin A4 and annexin-1 for the production of IL-10 and inflammatory hyporesponsiveness in mice

The appropriate development of an inflammatory response is central for the ability of a host to deal with any infectious insult. However, excessive, misplaced, or uncontrolled inflammation may lead to acute or chronic diseases. The microbiota plays an important role in the control of inflammatory responsiveness. In this study, we investigated the role of lipoxin A4 and annexin-1 for the IL-10-dependent inflammatory hyporesponsiveness observed in germfree mice. Administration of a 15-epi-lipoxin A4 analog or an annexin-1-derived peptide to conventional mice prevented tissue injury, TNF-alpha production, and lethality after intestinal ischemia/reperfusion. This was associated with enhanced IL-10 production. Lipoxin A4 and annexin-1 failed to prevent reperfusion injury in IL-10-deficient mice. In germfree mice, there was enhanced expression of both lipoxin A4 and annexin-1. Blockade of lipoxin A4 synthesis with a 5-lipoxygenase inhibitor or Abs against annexin-1 partially prevented IL-10 production and this was accompanied by partial reversion of inflammatory hyporesponsiveness in germfree mice. Administration of BOC-1, an antagonist of ALX receptors (at which both lipoxin A4 and annexin-1 act), or simultaneous administration of 5-lipoxygenase inhibitor and anti-annexin-1 Abs, was associated with tissue injury, TNF-alpha production, and lethality similar to that found in conventional mice. Thus, our data demonstrate that inflammatory responsiveness is tightly controlled by the presence of the microbiota and that the innate capacity of germfree mice to produce IL-10 is secondary to their endogenous greater ability to produce lipoxin A4 and annexin-1.     

Biclustering as a method for RNA local multiple sequence alignment

Motivations: Biclustering is a clustering method that simultaneously clusters both the domain and range of a relation. A challenge in multiple sequence alignment (MSA) is that the alignment of sequences is often intended to reveal groups of conserved functional subsequences. Simultaneously, the grouping of the sequences can impact the alignment; precisely the kind of dual situation biclustering is intended to address. RESULTS: We define a representation of the MSA problem enabling the application of biclustering algorithms. We develop a computer program for local MSA, BlockMSA, that combines biclustering with divide-and-conquer. BlockMSA simultaneously finds groups of similar sequences and locally aligns subsequences within them. Further alignment is accomplished by dividing both the set of sequences and their contents. The net result is both a multiple sequence alignment and a hierarchical clustering of the sequences. BlockMSA was tested on the subsets of the BRAliBase 2.1 benchmark suite that display high variability and on an extension to that suite to larger problem sizes. Also, alignments were evaluated of two large datasets of current biological interest, T box sequences and Group IC1 Introns. The results were compared with alignments computed by ClustalW, MAFFT, MUCLE and PROBCONS alignment programs using Sum of Pairs (SPS) and Consensus Count. Results for the benchmark suite are sensitive to problem size. On problems of 15 or greater sequences, BlockMSA is consistently the best. On none of the problems in the test suite are there appreciable differences in scores among BlockMSA, MAFFT and PROBCONS. On the T box sequences, BlockMSA does the most faithful job of reproducing known annotations. MAFFT and PROBCONS do not. On the Intron sequences, BlockMSA, MAFFT and MUSCLE are comparable at identifying conserved regions. AVAILABILITY: BlockMSA is implemented in Java. Source code and supplementary datasets are available at http://aug.csres.utexas.edu/msa/     

Taking organelles apart, putting them back together and creating new ones: lessons from the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions.