Analysis of the sucrose synthase gene family in Arabidopsis

The properties and expression patterns of the six isoforms of sucrose synthase in Arabidopsis are described, and their functions are explored through analysis of T-DNA insertion mutants. The isoforms have generally similar kinetic properties. Although there is variation in sensitivity to substrate inhibition by fructose this is unlikely to be of major physiological significance. No two isoforms have the same spatial and temporal expression patterns. Some are highly expressed in specific locations, whereas others are more generally expressed. More than one isoform is expressed in all organs examined. Mutant plants lacking individual isoforms have no obvious growth phenotypes, and are not significantly different from wild-type plants in starch, sugar and cellulose content, seed weight or seed composition under the growth conditions employed. Double mutants lacking the pairs of similar isoforms sus2 and sus3, and sus5 and sus6, are also not significantly different in these respects from wild-type plants. These results are surprising in the light of the marked phenotypes observed when individual isoforms are eliminated in crop plants including pea, maize, potato and cotton. A sus1/sus4 double mutant grows normally in well-aerated conditions, but shows marked growth retardation and accumulation of sugars when roots are subjected to hypoxia. The sucrose synthase activity in roots of this mutant is 3% or less of wild-type activity. Thus under well-aerated conditions sucrose mobilization in the root can proceed almost entirely via invertases without obvious detriment to the plant, but under hypoxia there is a specific requirement for sucrose synthase activity.     

Long-Term Patterns in the Population Dynamics of Daphnia longispina,Leptodora kindtii and Cyanobacteria in a Shallow Reservoir: A Self-Organising Map (SOM) Approach

The recognition of long-term patterns in the seasonal dynamics of Daphnia longispina, Leptodora kindtii and cyanobacteria is dependent upon their interactions, the water temperature and the hydrological conditions, which were all investigated between 1999 and 2008 in the lowland Sulejow Reservoir. The biomass of cyanobacteria, densities of D. longispina and L. kindtii, concentration of chlorophyll a and water temperature were assessed weekly from April to October at three sampling stations along the longitudinal reservoir axis. The retention time was calculated using data on the actual water inflow and reservoir volume. A self-organising map (SOM) was used due to high interannual variability in the studied parameters and their often non-linear relationships. Classification of the SOM output neurons into three clusters that grouped the sampling terms with similar biotic states allowed identification of the crucial abiotic factors responsible for the seasonal sequence of events: cluster CL-ExSp (extreme/spring) corresponded to hydrologically unstable cold periods (mostly spring) with extreme values and highly variable abiotic factors, which made abiotic control of the biota dominant; cluster CL-StSm (stable/summer) was associated with ordinary late spring and summer and was characterised by stable non-extreme abiotic conditions, which made biotic interactions more important; and the cluster CL-ExSm (extreme/summer), was associated with late spring/summer and characterised by thermal or hydrological extremes, which weakened the role of biotic factors. The significance of the differences between the SOM sub-clusters was verified by Kruskal-Wallis and post-hoc Dunn tests. The importance of the temperature and hydrological regimes as the key plankton-regulating factors in the dam reservoir, as shown by the SOM, was confirmed by the results of canonical correlation analyses (CCA) of each cluster. The demonstrated significance of hydrology in seasonal plankton dynamics complements the widely accepted pattern proposed by the plankton succession model for lakes, the PEG (Plankton Ecology Group), and may be useful for the formulation of management decisions in dam reservoirs.     

Mortality,Rehospitalisation and Violent Crime in Forensic Psychiatric Patients Discharged from Hospital: Rates and Risk Factors

ObjectivesTo determine rates and risk factors for adverse outcomes in patients discharged from forensic psychiatric services.MethodWe conducted a historical cohort study of all 6,520 psychiatric patients discharged from forensic psychiatric hospitals between 1973 and 2009 in Sweden. We calculated hazard ratios for mortality, rehospitalisation, and violent crime using Cox regression to investigate the effect of different psychiatric diagnoses and two comorbidities (personality or substance use disorder) on outcomes.ResultsOver mean follow-up of 15.6 years, 30% of patients died (n = 1,949) after discharge with an average age at death of 52 years. Over two-thirds were rehospitalised (n = 4,472, 69%), and 40% violently offended after discharge (n = 2,613) with a mean time to violent crime of 4.2 years. The association between psychiatric diagnosis and outcome varied—substance use disorder as a primary diagnosis was associated with highest risk of mortality and rehospitalisation, and personality disorder was linked with the highest risk of violent offending. Furthermore comorbid substance use disorder typically increased risk of adverse outcomes.ConclusionViolent offending, premature mortality and rehospitalisation are prevalent in patients discharged from forensic psychiatric hospitals. Individualised treatment plans for such patients should take into account primary and comorbid psychiatric diagnoses.     

Glutathione S-transferase fusion proteins as an affinity reagent for rapid isolation of specific sequence directly from genomic DNA.

We describe a DNA binding assay for isolation of specific sequence(s) recognized by protein of interest directly from genomic or cosmid DNA. In our assay, the protein is fused to the glutathione-S-transferase and bound to glutathione-Sepharose beads. Then the immobilized fusion protein can be used to search for DNA fragment(s) that interact specifically with the protein of interest. As an example of such an approach, we identified and cloned a few prokaryotic oriC regions using the initiator DnaA protein fused to the glutathione-S-transferase.     

Identification of Amino Acid Residues of ERH Required for Its Recruitment to Nuclear Speckles and Replication Foci in HeLa Cells

ERH is a small, highly evolutionarily conserved nuclear protein of unknown function. Its three-dimensional structure is absolutely unique and it can form a homodimer through a β sheet surface. ERH has been shown to interact, among others, with PDIP46/SKAR and Ciz1. When coexpressed with the latter protein, ERH accumulates in replication foci in the nucleus of HeLa cells. Here, we report that when ERH is coexpressed with PDIP46/SKAR in HeLa cells, it is recruited to nuclear speckles, and identify amino acid residues critical for targeting ERH to both these subnuclear structures. ERH H3A Q9A shows a diminished recruitment to nuclear speckles but it is recruited to replication foci. ERH E37A T51A is very poorly recruited to replication foci while still accumulating in nuclear speckles. Consequently, ERH H3A Q9A E37A T51A is recruited neither to nuclear speckles nor to replication foci. The lack of interactions of these three ERH forms with PDIP46/SKAR and/or Ciz1 was further confirmed in vitro by GST pull-down assay. The residues whose substitutions interfere with the accumulation in nuclear speckles are situated on the β sheet surface of ERH, indicating that only the monomer of ERH can interact with PDIP46/SKAR. Substitutions affecting the recruitment to replication foci map to the other side of ERH, near a long loop between the α1 and α2 helices, thus both the monomer and the dimer of ERH could interact with Ciz1. The construction of the ERH mutants not recruited to nuclear speckles or replication foci will facilitate further studies on ERH actions in these subnuclear structures.     

Mutations in radial spoke head genes and ultrastructural cilia defects in East-European cohort of primary ciliary dyskinesia patients

Primary ciliary dyskinesia (PCD) is a rare (1/20,000), multisystem disease with a complex phenotype caused by the impaired motility of cilia/flagella, usually related to ultrastructural defects of these organelles. Mutations in genes encoding radial spoke head (RSPH) proteins, elements of the ciliary ultrastructure, have been recently described. However, the relative involvement of RSPH genes in PCD pathogenesis remained unknown, due to a small number of PCD families examined for mutations in these genes. The purpose of this study was to estimate the involvement of RSPH4A and RSPH9 in PCD pathogenesis among East Europeans (West Slavs), and to shed more light on ultrastructural ciliary defects caused by mutations in these genes. The coding sequences of RSPH4A and RSPH9 were screened in PCD patients from 184 families, using single strand conformational polymorphism analysis and sequencing. Two previously described (Q109X; R490X) and two new RSPH4A mutations (W356X; IVS3_2-5del), in/around exons 1 and 3, were identified; no mutations were found in RSPH9. We estimate that mutations in RSPH4A, but not in RSPH9, are responsible for 2-3% of cases in the East European PCD population (4% in PCD families without situs inversus; 11% in families preselected for microtubular defects). Analysis of the SNP-haplotype background provided insight into the ancestry of repetitively found mutations (Q109X; R490X; IVS3_2-5del), but further studies involving other PCD cohorts are required to elucidate whether these mutations are specific for Slavic people or spread among other European populations. Ultrastructural defects associated with the mutations were analyzed in the transmission electron microscope images; almost half of the ciliary cross-sections examined in patients with RSPH4A mutations had the microtubule transposition phenotype (9+0 and 8+1 pattern). While microtubule transposition was a prevalent ultrastructural defect in cilia from patients with RSPH4A mutations, similar defects were also observed in PCD patients with mutations in other genes.     

Free hemoglobin induction of pulmonary vascular disease: evidence for an inflammatory mechanism

Cell-free hemoglobin (Hb) exposure may be a pathogenic mediator in the development of pulmonary arterial hypertension (PAH), and when combined with chronic hypoxia the potential for exacerbation of PAH and vascular remodeling is likely more pronounced. We hypothesized that Hb may contribute to hypoxia-driven PAH collectively as a prooxidant, inflammatory, and nitric oxide (NO) scavenger. Using programmable micropump technology, we exposed male Sprague-Dawley rats housed under room air or hypoxia to 12 or 30 mg per day Hb for 3, 5, and 7 wk. Blood pressure, cardiac output, right ventricular hypertrophy, and indexes of pulmonary vascular remodeling were evaluated. Additionally, markers of oxidative stress, NO bioavailability and inflammation were determined. Hb increased pulmonary arterial (PA) pressure, pulmonary vessel wall stiffening, and right heart hypertrophy with temporal and dose dependence in both room air and hypoxic cohorts. Hb induced a modest increase in plasma oxidative stress markers (malondialdehyde and 4-hydroxynonenal), no change in NO bioavailability, and increased lung ICAM protein expression. Treatment with the antioxidant Tempol attenuated Hb-induced pulmonary arterial wall thickening, but not PA pressures or ICAM expression. Chronic exposure to low plasma Hb concentrations (range = 3-10 μM) lasting up to 7 wk in rodents induces pulmonary vascular disease via inflammation and to a lesser extent by Hb-mediated oxidation. Tempol demonstrated a modest effect on the attenuation of Hb-induced pulmonary vascular disease. NO bioavailability was found to be of minimal importance in this model.     

Interferon signaling in the liver during hepatitis C virus infection

Hepatitis C virus is a global health concern, estimated to infect 2-3% of the world's population. Inter-individual differences in the course of infection and response to therapy, highlighted by recent genomewide association studies, point to the crucial role of the host immune system in the efficient control of infection. Ongoing progress in the studies of the role of innate immunity during hepatitis C virus infection has improved our understanding of the intricacies of the host-virus interactions. In this review, we summarize and discuss the current knowledge concerning interferon signaling in the liver during acute and chronic hepatitis C virus infection and its implications for the outcome of interferon-α-based antiviral therapies.