Pichia pastoris protease-deficient and auxotrophic strains generated by a novel, user-friendly vector toolbox for gene deletion.

Targeted gene knockouts play an important role in the study of gene function. For the generation of knockouts in the industrially important yeast Pichia pastoris, several protocols have been published to date. Nevertheless, creating a targeted knockout in P. pastoris still is a time-consuming process, as the existing protocols are labour intensive and/or prone to accumulate nucleotide mutations. In this study, we introduce a novel, user-friendly vector-based system for the generation of targeted knockouts in P. pastoris. Upon confirming the successful knockout, respective selection markers can easily be recycled. Excision of the marker is mediated by Flippase (Flp) recombinase and occurs at high frequency (≥95%). We validated our knockout system by deleting 20 (confirmed and putative) protease genes and five genes involved in biosynthetic pathways. For the first time, we describe gene deletions of PRO3 and PHA2 in P. pastoris, genes involved in proline, and phenylalanine biosynthesis, respectively. Unexpectedly, knockout strains of PHA2 did not display the anticipated auxotrophy for phenylalanine but rather showed a bradytroph phenotype on minimal medium hinting at an alternative but less efficient pathway for production of phenylalanine exists in P. pastoris. Overall, all knockout vectors can easily be adapted to the gene of interest and strain background by efficient exchange of target homology regions and selection markers in single cloning steps. Average knockout efficiencies for all 25 genes were shown to be 40%, which is comparably high.

Determinants of the hyperdynamic circulation and central hypovolaemia in cirrhosis.

BACKGROUND: Patients with advanced cirrhosis often develop a hyperdynamic circulation with central hypovolaemia. The events that initiate the systemic haemodynamic abnormalities and the coupling of these factors to splanchnic haemodynamics are still unclear. Objective On the basis of a large population of patients with cirrhosis to identify splanchnic and clinical characteristics associated with the development of the hyperdynamic circulation and survival. METHODS: We included 410 patients with cirrhosis. In all patients, a full haemodynamic investigation was performed. The data were analysed using regression analyses, principal components analyses, and Cox proportional hazards analyses. RESULTS: Multivariate regression analyses showed that higher cardiac output was independently associated with higher hepatic venous pressure gradient (HVPG) and higher hepatic blood flow (HBF) (p<0.00001). Higher heart rate was independently associated with presence of ascites and higher HVPG (p<0.0001). Central blood volume and circulation time were independently associated with higher HBF and lower postsinusoidal resistance, respectively (p<0.0001). Systemic vascular resistance was independently associated with lower HVPG (p<0.0001). The final Cox proportional hazards model showed that decreased survival was independently associated with higher age (p=0.003), lower blood haemoglobin concentration (p=0.0006), higher plasma creatinine (p=0.01), higher plasma alkaline phosphatase (p=0.007), lower right atrial pressure (p=0.004), and higher heart rate (p=0.002). CONCLUSION: The development of the hyperdynamic circulation and central hypovolaemia are mainly explained by changes in portal pressure and HBF. Together with indicators of liver dysfunction, central hypovolaemia is associated with poorer prognosis.

High-constitutive HuR phosphorylation at Ser 318 by PKC{delta} propagates tumor relevant functions in colon carcinoma cells.

Overexpression of the messenger RNA (mRNA)-binding protein HuR is an important feature of many tumors and in most cases correlates with a high-grade malignancy. Since phosphorylation of HuR by protein kinase C δ (PKCδ) at serine (Ser) 318 implies an important mode in HuR regulation, we studied its functional role in dysregulated HuR and related functions in colon carcinoma cells. Coimmunoprecipitation experiments revealed a high-constitutive association of nuclear PKCδ with HuR. Using a phospho-Ser 318-specific HuR antibody, we found a strong increase in nuclear HuR phosphorylation in DLD-1 cells when compared with nontransformed CCD 841 colon epithelial cells. Importantly, a strong increase in HuR phosphorylation at Ser 318 was also found in tissue specimen from human colon carcinomas. Employing ribonucleoprotein-immunoprecipitation, we show that DLD-1 cells displayed a strong and constitutive RNA binding of HuR to cyclooxygenase-2 (COX-2) and cyclin A encoding mRNAs that was strongly impaired by rottlerin, an inhibitor of novel PKCs. Accordingly, rottlerin accelerated the decay of COX-2 and cyclin A encoding mRNAs concomitant with a reduced expression of both genes. Functionally, migration and invasion is similarly impaired in PKCδ- or HuR-small interfering RNA-depleted cells and in tumor cells transfected with a nonphosphorylatable serine-to-alanine 318 HuR construct. Conversely, expression of a phosphomimetic Ser 318 aspartic acid (D) HuR caused a significant increase in migration and proliferation of CCD 841 cells. Our data suggest that the increased HuR phosphorylation at Ser 318 by PKCδ reflects an important regulatory paradigm for aberrant HuR functions and emphasize the antitumorigenic potential of PKCδ inhibitory strategies.

Respiratory nitrogen metabolism and nitrosative stress defence in ϵ-proteobacteria: the role of NssR-type transcription regulators.

ϵ-Proteobacteria form a globally ubiquitous group of ecologically significant organisms and comprise a diverse range of host-associated and free-living species. To grow by anaerobic respiration, many ϵ-proteobacteria reduce nitrate to nitrite followed by either nitrite ammonification or denitrification. Using the ammonifying model organisms Wolinella succinogenes and Campylobacter jejuni, the electron transport chains of nitrate respiration, respiratory nitrite ammonification and even N2O (nitrous oxide) respiration have been characterized in recent years, but knowledge on nitrosative stress defence, nitrogen compound-sensing and corresponding signal transduction pathways is limited. The potentially dominant role of NssR (nitrosative stress-sensing regulator)-type transcription regulators in ϵ-proteobacterial nitrogen metabolism is discussed.

Plant species effects on soil macrofauna density in grassy arable fallows of different age.

The density of soil macrofauna groups in nine grassy arable fallows of different age were investigated in a factorial design with the factors 'plant species' (legume: Medicago sativa, herb: Taraxacum officinale, grass: Bromus sterilis) and 'age class' (A1: 2-3/3-4, A2: 6-8/7-9, A3: 12-15/13-16 years in 2008/2009). Four plots were selected randomly at each fallow. In May 2008 and May 2009, within each plot five M. sativa, T. officinale and B. sterilis plants were extracted with their associated soil using steel cylinders. The material from each plant species was used for extraction of soil macrofauna and for determination of environmental parameters. The main results were (i) the density of the saprophagous macrofauna was significantly higher in B. sterilis than in M. sativa and T. officinale samples indicating that this group possibly benefited from the particularly high amount of fine roots in the B. sterilis samples; (ii) densities of Gastropoda and predatory beetles were highest in the 7-9 yr old fallows indicating that predators may have benefited from the increased availability of their prey in the medium stage of grassland succession; (iii) focusing on the results of the CCAs (2008, 2009), the water content had the strongest influence of the measured soil parameters on the structure of the soil macrofauna assemblages.

Effects of treatment with ß-blocker and aldosterone antagonist on central and peripheral haemodynamics and oxygenation in cirrhosis.

OBJECTIVES: Patients with cirrhosis often exhibit abnormalities in cardiovascular regulation and oxygenation. Many of these patients are treated with ß-blockers and aldosterone antagonists that may influence the regulation of systemic haemodynamics, but the specific effects on systemic haemodynamics and oxygenation are less studied. We therefore investigated systemic haemodynamics and oxygenation in patients with cirrhosis before and after 3 weeks of treatment with propranolol or spironolactone. MATERIALS AND METHODS: Twenty-two patients with cirrhosis were allocated into three groups as follows: a ß-blocker group (n=7), a spironolactone group (n=8) and a control group (n=7). At baseline, and after 3 weeks, we measured the arterial blood pressure (BP), heart rate, Q-T frequency-corrected interval, baroreflex sensitivity, cardiac output, peripheral arterial inflow (ABFin), transcutaneous oxygenation and hormonal status. We also assessed immediate effects of propranolol in the ß-blocker group. RESULTS: Short-term and long-term ß-blockade significantly reduced BP (-7%) and heart rate (-15%) (P<0.01). Short-term ß-blocker treatment improved the Q-T frequency-corrected interval but reduced the transcutaneous oxygenation (P<0.05). Baroreflex sensitivity and brain natriuretic peptide increased after short-term and after 3 weeks of ß-blocker treatments (P<0.01). After 3 weeks, ß-blockers reduced cardiac output and ABFin by 13 and 26%, respectively (P<0.05-0.01). In the spironolactone group, BP decreased by 8% (P<0.05) and renin increased by 370% (P<0.01). No changes were seen in the control group. CONCLUSION: Short-term and long-term treatments with ß-blockers and aldosterone antagonist modestly affect haemodynamics and oxygenation. Careful monitoring, especially in patients with low arterial blood pressure, if cardiac dysfunction is suspected, should follow the administration of ß-blockers.

Parameters of soluble immune activation in vivo correlate negatively with the proliferative capacity of peripheral blood mononuclear cells in vitro in HIV-infected patients.

OBJECTIVE: In vitro peripheral blood mononuclear cells (PBMCs) of HIV-infected subjects show defective immune responses including impaired proliferative responses after stimulation with antigens and mitogens. The cytokine interferon-gamma, which seems to play an important role in HIV infection, induces neopterin formation and in parallel also tryptophan degradation by the enzyme indoleamine 2,3-dioxygenase. DESIGN: : In this study, interferon-gamma-mediated pathways were examined in the plasma of patients and evaluated for associations with the proliferative responses of PBMC to mitogens in vitro. METHODS: To assess the actual status of immune system activation in patients, plasma concentrations of interferon-gamma, neopterin, tryptophan, and kynurenine were measured by enzyme-linked immunosorbent assay or by high-performance liquid chromatography; the ratio of kynurenine to tryptophan was calculated. Viral load, CD4 counts, and the in vivo immune activation status were compared with the in vitro responses of PBMC isolated from the same patients. PBMCs were stimulated with the mitogens concanavalin A, phytohemagglutinin, and pokeweed mitogen, and their proliferation was assessed by H-thymidine incorporation. RESULTS: The in vitro proliferative capacity of PBMC was associated with viral load, CD4 counts, and also tryptophan degradation. Particularly, high neopterin concentrations were observed to be the best predictor of impaired proliferative response in logistic regression analysis. CONCLUSIONS: The higher the degree of immune activation in vivo, the lower is the proliferative capacity of PBMC in vitro.