Factors underpinning the responsiveness and higher levels of virus resistance realised in potato genotypes carrying virus-specific R genes

Responses to Potato virus A (PVA, genus Potyvirus) segregate to three phenotypic groups in a diploid cross between Solanum tuberosum subsp. andigena and a highly interspecific potato hybrid. The aim of this study was to compare gene expression between the progeny genotypes which react with hypersensitive response (HR) to PVA, allow PVA accumulation in inoculated leaves but restrict PVA infection to the inoculated leaf by blocking systemic movement [non-necrotic resistance (nnr)], or are susceptible (S) and systemically infected with PVA. Expression levels of ca 10 000 genes were compared using probes arranged in a microarray format, and real-time RT-PCR was applied for quantitative comparison of the expression of selected defense-related genes (DRGs). Results showed that a few DRGs were autoactivated in HR genotypes at an early stage of plant growth in the absence of PVA infection, which was not observed in the two other phenotypic groups (nnr and S). More detailed studies on the DRGs encoding a beta-1,3-glucanase, a chitinase and a basic PR-1b protein showed that autoactivation of the genes was not evident in vitro and up to 2 weeks of growth in soil in a controlled growth cabinet but was apparent 2 weeks later. Hence, autoinduction of these DRGs in the HR genotypes could be associated with growth stage, environmental factors or both. Furthermore, a number of other DRGs were induced in the inoculated leaves of HR genotypes as a response to infection with PVA, which was not observed in nnr and S genotypes. These results provide some novel information about factors underpinning the higher levels of virus resistance realised in potato genotypes carrying virus-specific R genes and suggest that part of the resistance is attributable to additional ‘minor’ genes functioning simultaneously, hence adding to the overall responsiveness and level of resistance against infection. These results also imply that some genotypes might be more responsive to chemical induction of pathogen and pest resistance, which could be considered in screening of progenies in plant-breeding programs.     

Quantitative real-time PCR detection of toxic Nodularia cyanobacteria in the Baltic Sea

A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml(-1) of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.     

A validated method for the detection and quantitation of 50 drugs of abuse and medicinal drugs in oral fluid by gas chromatography-mass spectrometry

Oral fluid is of increasing interest as an alternative sample matrix in drugs of abuse analysis. Compared to the conventional sample matrix blood, sample volumes of oral fluid are smaller and the concentrations of some drugs can be much lower. This imposes some restrictions on the analysis method, which has to be able to detect and quantify multiple analytes from a small sample volume at low concentrations. A sensitive multi-component method for quantitative determination of 50 drug compounds from oral fluid samples collected with the StatSure SalivaSampler? device was developed. The compounds analysed include cannabis, cocaine, amphetamines, opioids, benzodiazepines and other psychoactive medicines. Both liquid–liquid-extraction (LLE) and solid-phase-extraction (SPE) are employed in the sample pre-treatment and the samples are analysed using gas chromatography–mass spectrometry (GC–MS) with the mass selective detector (MSD) operating in either electron ionization (EI) or negative-ion chemical ionization (NICI) mode. The method was fully validated, and the validated parameters included linearity, selectivity, accuracy, precision, and extraction efficiency. Stability of the collected samples during storage at ?18 °C was also studied, and even after over a year's storage all analyte concentrations were more than 60% of the original concentrations. The described method is suitable for routine analysis of oral fluid samples and it has been applied to analysis of more than 4000 oral fluid samples collected anonymously from volunteer road users in Finland during 2007–2009 as a part of the EU project DRUID (Driving under the Influence of Drugs, Alcohol and Medicines). At the moment the developed method is the most comprehensive validated analysis method for oral fluid samples.     

Different metabolic responses of human brown adipose tissue to activation by cold and insulin

We investigated the metabolism of human brown adipose tissue (BAT) in healthy subjects by determining its cold-induced and insulin-stimulated glucose uptake and blood flow (perfusion) using positron emission tomography (PET) combined with computed tomography (CT). Second, we assessed gene expression in human BAT and white adipose tissue (WAT). Glucose uptake was induced 12-fold in BAT by cold, accompanied by doubling of perfusion. We found a positive association between whole-body energy expenditure and BAT perfusion. Insulin enhanced glucose uptake 5-fold in BAT independently of its perfusion, while the effect on WAT was weaker. The gene expression level of insulin-sensitive glucose transporter GLUT4 was also higher in BAT as compared to WAT. In conclusion, BAT appears to be differently activated by insulin and cold; in response to insulin, BAT displays high glucose uptake without increased perfusion, but when activated by cold, it dissipates energy in a perfusion-dependent manner.     

Riedel's thyroiditis in a patient with multiple sclerosis

Our patient developed Riedel's thyroiditis soon after having an exacerbation of multiple sclerosis (MS). MS has been associated with other autoimmune diseases, including thyroiditis, and both Hashimoto's thyroiditis and subacute thyroiditis have been described in connection with MS. Yet, we are not aware of any other patient reported to have concomitant MS and Riedel's thyroiditis. The association between MS and Riedel's thyroiditis remains obscure but may reflect an autoimmune disorder common to both diseases.     

Cu(II) complex formation with xylitol in alkaline solutions

The formation of four Cu(II)-xylitol complexes was observed in aqueous alkaline solutions (11.0< or =pH< or =14.0, I=1.0, 20 degrees C) by means of direct current polarography and VIS spectrophotometry. Mononuclear hydroxy complexes, CuXyl(OH)- (log beta=17.7 +/- 0.5), CuXyl(OH)2(2-) (log beta=20.2 +/- 0.3) and CuXyl2(OH)2(4-) (log beta=22.4 +/- 0.3), are formed at high ligand-to-metal ratios (L:M> or =10), whereas dinuclear complex Cu2Xyl (log beta=29.2 +/- 0.3) is the predominant species at low ligand-to-metal ratio (L:M=0.5). Diffusion coefficients and molar absorptivities of the complex species were determined. pH variable 13C NMR suggested that pKa values of xylitol are rather similar and equal to 13.8 +/- 0.2, 13.9 +/- 0.1 and 13.9 +/- 0.2 for OH-groups adjacent to (C-1,C-5), (C-3) and (C-2,C-4) carbon atoms, respectively.     

Mechanisms of Inhibitory Amino Acid Release in the Brain Stem Under Normal and Ischemic Conditions

This article deals with the release of GABA, glycine and taurine from the brain stem under normal conditions and in ischemia. The release mechanisms, the effects of glutamate and adenosine receptors, and the roles of nitric oxide and second messengers are reviewed.     

Quality control for unfolded proteins at the plasma membrane

Cellular protein homeostasis profoundly depends on the disposal of terminally damaged polypeptides. To demonstrate the operation and elucidate the molecular basis of quality control of conformationally impaired plasma membrane (PM) proteins, we constructed CD4 chimeras containing the wild type or a temperature-sensitive bacteriophage λ domain in their cytoplasmic region. Using proteomic, biochemical, and genetic approaches, we showed that thermal unfolding of the λ domain at the PM provoked the recruitment of Hsp40/Hsc70/Hsp90 chaperones and the E2-E3 complex. Mixed-chain polyubiquitination, monitored by bioluminescence resonance energy transfer and immunoblotting, is responsible for the nonnative chimera-accelerated internalization, impaired recycling, and endosomal sorting complex required for transport-dependent lysosomal degradation. A similar paradigm prevails for mutant dopamine D4.4 and vasopressin V2 receptor removal from the PM. These results outline a peripheral proteostatic mechanism in higher eukaryotes and its potential contribution to the pathogenesis of a subset of conformational diseases.     

Coronary flow reserve and heart failure in experimental coxsackievirus myocarditis. A transthoracic Doppler echocardiography study

The objective of this study was to apply transthoracic Doppler echocardiography (TTDE) in mice to study coronary flow reserve (CFR), an index of coronary microvascular function, in mild and severe forms of experimental viral myocarditis. Regarding methodology, BALB/c mice were infected with cardiotropic coxsackieviruses causing either a mild (Nancy strain) or a severe (Woodruff strain) myocarditis. Left ventricular dimensions, fractional shortening, and CFR (ratio of left coronary artery flow velocity during maximal adenosine-induced vasodilatation to rest) were measured by TTDE before infection and again 1 or 2 wk after infection. As a result, the resting flow velocity did not change after infection. In contrast, CFR reduced significantly 1 wk after infection with either virus variant [from 2.5 (SD 0.3) to 1.4 (SD 0.1) in severe and from 2.4 (SD 0.4) to 2.1 (SD 0.3) in mild myocarditis], being significantly lower in the severe than mild myocarditis. CFR remained low in severe myocarditis 2 wk after infection. Fractional shortening decreased to the same levels 1 wk after infection with either virus variant [from 0.54 (SD 0.02) to 0.43 (SD 0.03) in severe and from 0.51 (SD 0.03) to 0.44 (SD 0.02) in mild myocarditis, P < 0.05]. However, 2 wk after infection, mice with severe myocarditis had enlarged left ventricles and lower fractional shortening [0.31 (SD 0.03)] than mice with mild myocarditis [0.47 (SD 0.02), P < 0.01]. In conclusion, CFR measured with TTDE is reduced in coxsackievirus myocarditis in mice. Low CFR is associated with progressive heart failure, indicating that dysfunction of coronary microcirculation is a determinant of poor outcome in viral myocarditis.