Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth

Hydroxyl radicals (*OH), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for *OH generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize ( Zea mays L.) coleoptiles and sunflower ( Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause *OH generation by POD in vitro. The effects of NADH on wall loosening, growth, and *OH production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of *OH in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the *OH-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the *OH-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O2*-) (and H2O2 utilized by POD for) *OH production in the cell wall.     

Zero-Heat-Flux Thermometry for Non-Invasive Measurement of Core Body Temperature in Pigs

Hypothermia is a severe, unpleasant side effect during general anesthesia. Thus, temperature surveillance is a prerequisite in general anesthesia settings during experimental surgeries. The gold standard to measure the core body temperature (T_core) is placement of a Swan-Ganz catheter in the pulmonary artery, which is a highly invasive procedure. Therefore, T_core is commonly examined in the urine bladder and rectum. However, these procedures are known for their inaccuracy and delayed record of temperatures. Zero-heat-flux (ZHF) thermometry is an alternative, non-invasive method quantifying T_core in human patients by applying a thermosensoric patch to the lateral forehead. Since the porcine cranial anatomy is different to the human’s, the optimal location of the patch remains unclear to date. The aim was to compare three different patch locations of ZHF thermometry in a porcine hypothermia model. Hypothermia (33.0°C T_core) was conducted in 11 anesthetized female pigs (26-30kg). T_core was measured continuously by an invasive Swan-Ganz catheter in the pulmonary artery (T_pulm). A ZHF thermometry device was mounted on three different defined locations. The smallest average difference between T_pulm and T_ZHF during stable temperatures was 0.21 ± 0.16°C at location A, where the patch was placed directly behind the eye. Also during rapidly changing temperatures location A showed the smallest bias with 0.48 ± 0.29°C. Location A provided the most reliable data for T_core. Therefore, the ZHF thermometry patch should be placed directly behind the left temporal corner of the eye to provide a non-invasive method for accurate measurement of T_core in pigs.     

Insulin-Like Growth Factor 1 (IGF-1) in Parkinson's Disease: Potential as Trait-, Progression- and Prediction Marker and Confounding Factors

Introduction

Biomarkers indicating trait, progression and prediction of pathology and symptoms in Parkinson''s disease (PD) often lack specificity or reliability. Investigating biomarker variance between individuals and over time and the effect of confounding factors is essential for the evaluation of biomarkers in PD, such as insulin-like growth factor 1 (IGF-1).

Materials and Methods

IGF-1 serum levels were investigated in up to 8 biannual visits in 37 PD patients and 22 healthy controls (HC) in the longitudinal MODEP study. IGF-1 baseline levels and annual changes in IGF-1 were compared between PD patients and HC while accounting for baseline disease duration (19 early stage: ≤3.5 years; 18 moderate stage: >4 years), age, sex, body mass index (BMI) and common medical factors putatively modulating IGF-1. In addition, associations of baseline IGF-1 with annual changes of motor, cognitive and depressive symptoms and medication dose were investigated.

Results

PD patients in moderate (130±26 ng/mL; p = .004), but not early stages (115±19, p>.1), showed significantly increased baseline IGF-1 levels compared with HC (106±24 ng/mL; p = .017). Age had a significant negative correlation with IGF-1 levels in HC (r = -.47, p = .028) and no correlation in PD patients (r = -.06, p>.1). BMI was negatively correlated in the overall group (r = -.28, p = .034). The annual changes in IGF-1 did not differ significantly between groups and were not correlated with disease duration. Baseline IGF-1 levels were not associated with annual changes of clinical parameters.

Discussion

Elevated IGF-1 in serum might differentiate between patients in moderate PD stages and HC. However, the value of serum IGF-1 as a trait-, progression- and prediction marker in PD is limited as IGF-1 showed large inter- and intraindividual variability and may be modulated by several confounders.     

An approach to quality management in structural biology: biophysical selection of proteins for successful crystallization

Aggregation, incorrect folding and low stability are common obstacles for protein structure determination, and are often discovered at a very late state of protein production. In many cases, however, the reasons for failure to obtain diffracting crystals remain entirely unknown. We report on the contribution of systematic biophysical characterization to the success in structural determination of human proteins of unknown fold. Routine analysis using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) was employed to evaluate fold and stability of 263 purified protein samples (98 different human proteins). We found that FTIR-monitored temperature scanning may be used to detect incorrect folding and discovered a positive correlation between unfolding enthalpy measured with DSC and the size of small, globular proteins that may be used to estimate the quality of protein preparations. Furthermore, our work establishes that the risk of aggregation during concentration of proteins may be reduced through DLS monitoring. In summary, our study demonstrates that biophysical characterization provides an ideal tool to facilitate quality management for structural biology and many other areas of biological research.     

Function of the 23 kDa extrinsic protein of Photosystem II as a manganese binding protein and its role in photoactivation

The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage-repair cycle and delivers Mn to repaired PSII.     

Recruitment of human cytomegalovirus immediate-early 2 protein onto parental viral genomes in association with ND10 in live-infected cells

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise co-localizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.     

Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Nasal Carriage in a Random Sample of Non-Hospitalized Adult Population in Northern Germany

Objective

The findings from truly randomized community-based studies on Staphylococcus aureus nasal colonization are scarce. Therefore we have examined point prevalence and risk factors of S. aureus nasal carriage in a non-hospitalized population of Braunschweig, northern Germany.

Methods

A total of 2026 potential participants were randomly selected through the resident''s registration office and invited by mail. They were requested to collect a nasal swab at home and return it by mail. S. aureus was identified by culture and PCR. Logistic regression was used to determine risk factors of S. aureus carriage.

Results

Among the invitees, 405 individuals agreed to participate and 389 provided complete data which was included in the analysis. The median age of the participants was 49 years (IQR: 39–61) and 61% were females. S. aureus was isolated in 85 (21.9%; 95% CI: 18.0–26.2%) of the samples, five of which were MRSA (1.29%; 95% CI: 0.55–2.98%). In multiple logistic regression, male sex (OR = 3.50; 95% CI: 2.01–6.11) and presence of allergies (OR = 2.43; 95% CI: 1.39–4.24) were found to be associated with S. aureus nasal carriage. Fifty five different spa types were found, that clustered into nine distinct groups. MRSA belonged to the hospital-associated spa types t032 and t025 (corresponds to MLST CC 22), whereas MSSA spa types varied and mostly belonged to spa-CC 012 (corresponds to MLST CC 30), and spa-CC 084 (corresponds to MLST CC 15).

Conclusion

This first point prevalence study of S. aureus in a non-hospitalized population of Germany revealed prevalence, consistent with other European countries and supports previous findings on male sex and allergies as risk factors of S. aureus carriage. The detection of hospital-associated MRSA spa types in the community indicates possible spread of these strains from hospitals into the community.     

Chances and Limitations of Wild Bird Monitoring for the Avian Influenza Virus H5N1 — Detection of Pathogens Highly Mobile in Time and Space

Highly pathogenic influenza virus (HPAIV) H5N1 proved to be remarkably mobile in migratory bird populations where it has led to extensive outbreaks for which the true number of affected birds usually cannot be determined. For the evaluation of avian influenza monitoring and HPAIV early warning systems, we propose a time-series analysis that includes the estimation of confidence intervals for (i) the prevalence in outbreak situations or (ii) in the apparent absence of disease in time intervals for specified regional units. For the German outbreak regions in 2006 and 2007, the upper 95% confidence limit allowed the detection of prevalences below 1% only for certain time intervals. Although more than 25,000 birds were sampled in Germany per year, the upper 95% confidence limit did not fall below 5% in the outbreak regions for most of the time. The proposed analysis can be used to monitor water bodies and high risk areas, also as part of an early-warning system. Chances for an improved targeting of the monitoring system as part of a risk-based approach are discussed with the perspective of reducing sample sizes.     

H‐Cadherin expression reduces invasion of malignant melanoma

Melanocytic behavior, survival, and proliferation are regulated through a complex system of cell–cell adhesion molecules. Pathologic changes leading to development of malignant melanoma, upset the delicate homeostatic balance between melanocytes and keratinocytes and can lead to altered expression of cell–cell adhesion and cell–cell communication molecules. Malignant transformation of melanocytes frequently coincides with loss of E‐cadherin expression. We now show loss of another member of the superfamily of classical cadherins, H‐cadherin (CDH13), which may be involved in the development of malignant melanoma. The provided data show that H‐cadherin expression is lost in nearly 80% of the analyzed melanoma cell lines. Knockdown of H‐cadherin using siRNA increases invasive capacity in melanocytes. Functional assays show that the re‐expression of H‐cadherin decreases migration and invasion capacity, as well as anchorage‐independent growth in comparison to control melanoma cells. Furthermore, melanoma cells, which re‐express H‐cadherin via stable transfection show a reduction in rate of tumor growth in a nu/nu mouse tumor model in comparison to the parental control transfected cell lines. Our study presents for the first time the down‐regulation of H‐cadherin in malignant melanomas and its possible functional relevance in maintenance healthy skin architecture.