Functional Somatic Syndromes: Skin Temperatures and Activity Measurements Under Ambulatory Conditions

Functional somatic syndromes are mostly associated with pain and emotional distress. As one marker for the autonomic stress response, the distal skin temperature decreases during psychological stress. In patients with functional somatic syndromes, the distal skin temperature under baseline conditions (without stress induction) is usually lower than in healthy subjects, which could be due to the sustained presence of pain-related stress in such patients. The aim of our study was to investigate whether patients with functional somatic syndromes show altered skin temperatures also under everyday life conditions. 14 patients with functional somatic syndromes and 14 matched healthy control subjects were investigated under ambulatory conditions over six consecutive days. During this time, distal and proximal skin temperatures were continuously recorded and sleep-wake cycles were monitored by actimetry and sleep-wake diaries. Unexpectedly, the patients showed higher distal skin temperatures than control subjects in the afternoon. The objective temperature data did not match the patients’ subjective experience: ratings of thermal comfort did not vary between the two groups. Moreover, similar levels of daytime activity were recorded in the two samples, even though patients reported more tiredness and more body tension than controls. We interpret the observed dissociation between objective skin temperature measurements and subjective ratings of the bodily thermal comfort as support for the notion of an alexisomia account (reduced bodily awareness) for functional somatic syndromes. Moreover, findings indicate that subjective complaints of tiredness and tension do not necessarily result in physical avoidance behaviour.     

Development of Adenoviral Delivery Systems to Target Hepatic Stellate Cells In Vivo

Hepatic stellate cells (HSCs) are known as initiator cells that induce liver fibrosis upon intoxication or other noxes. Deactivation of this ongoing remodeling process of liver parenchyma into fibrotic tissue induced by HSCs is an interesting goal to be achieved by targeted genetic modification of HSCs. The most widely applied approach in gene therapy is the utilization of specifically targeted vectors based on Adenovirus (Ad) serotype 5. To narrow down the otherwise ubiquitous tropism of parental Ad, two modifications are required: a) ablating the native tropism and b) redirecting the vector particles towards a specific entity solely present on the cells of interest. Therefore, we designed a peptide of the nerve growth factor (NGF_p) with specific affinity for the p75 neurotrophin receptor (p75NTR) present on HSCs. Coupling of this NGF_p to vector particles was done either via chemical conjugation using bifunctional polyethylene glycol (PEG) or, alternatively, by molecular bridging with a fusion protein specific for viral fiber knob and p75NTR. Both Ad vectors transmit the gene for the green fluorescent protein (GFP). GFP expression was monitored in vitro on primary murine HSCs as well as after systemic administration in mice with healthy and fibrotic livers using intravital fluorescence microscopy. Coupling of NGF_p to Ad via S11 and/or PEGylation resulted in markedly reduced liver tropism and an enhanced adenoviral-mediated gene transfer to HSCs. Transduction efficiency of both specific Ads was uniformly higher in fibrotic livers, whereas Ad.GFP-S11-NGF_p transduce activated HSCs better than Ad.GFP-PEG-NGF_p. These experiments contribute to the development of a targeted gene transfer system to specifically deliver antifibrotic compounds into activated HSCs by systemically applied adenoviral vector modified with NGF_p.     

The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies

Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots?, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.     

Glycogen synthase kinase (GSK) 3beta directly phosphorylates Serine 212 in the regulatory loop and inhibits microtubule affinity-regulating kinase (MARK) 2

MARK/Par-1, a kinase family with diverse functions particularly in inducing cell polarity, can phosphorylate microtubule-associated proteins in their repeat domain and cause their detachment from microtubules, and thereby microtubule destabilization. Because of its role in abnormal phosphorylation of the Tau protein in Alzheimer disease, we searched for regulatory kinases. MARK family kinases can be activated by phosphorylation of a conserved threonine (Thr-208 in MARK2), and inactivated by phosphorylation of a serine (Ser-212), both in the activation loop of the catalytic domain. Activation is achieved by the kinases MARKK/TAO1 or LKB1, although the inactivating kinase was unknown. We show here that GSK3beta serves the role of the inhibitory kinase. Because GSK3beta can also phosphorylate Tau at sites outside the repeat domain, the activation of GSK3beta, and concomitant inactivation of MARK can shift the pattern of pathological phosphorylation of Tau protein in Alzheimer disease.     

Environmental History and the Desertification of the Karoo,South Africa

Abstract

This paper reviews the desertification debate in South Africa and emphasizes the methods that have been used to understand the environmental history of the semi–arid rangelands in the eastern Karoo during the last 500 years. These mixed grass/dwarf shrub rangelands, with mean annual rainfall totals typically between 300–400 mm, are described and the main driving variables discussed. A brief historical account of the European settlement of the region is also presented. The desertification debate has focussed on three main issues. Firstly, because arid and semi–arid lands appear to be heavily influenced by climate, the changing climatic regime during the Holocene and particularly during the last two hundred years has been the subject of intensive investigation. While all studies conclude that there is no evidence in the historical record to support the popular perception that rainfall totals have decreased this century, the question of changing rainfall seasonality has not been adequately explored. Although mean annual temperatures over the last 50 years have not changed there have been significant increases in mean monthly maximum temperatures and significant decreases in mean monthly minimum temperatures for some of the stations investigated. The second issue of great interest in the South African desertification literature is that of the nature of pre–colonial environments. A wide variety of archaeological, historical and ecological techniques have been used, including analyses of fossil mammal bones in owl pellets, fossil pollen in hyrax middens, notes from traveller's accounts of the region and stable carbon isotopes in soil as well as fossil mammal bones. While all authors agree that the eastern Karoo was more grassy at some stage in the past there is disagreement as to both the timing and cause of the changes to a more shrubby vegetation. The final issue of great concern to the desertification debate in South Africa concerns the rate of change during the last 100 years. Satellite imagery, matched ground and aerial photography, survey data and an analysis of historical stock records cannot agree as to whether the Karoo is degrading or not. Certainly, the classic view of an annually expanding desert margin has been replaced in recent years by a more realistic understanding of the seasonal dynamics of the vegetation. The recent trend to detailed modelling of the demographic process in key species holds much promise for our understanding of the degradation process. The vibrant community of researchers, employing a range of archaeological, historical and ecological techniques, will make important contributions to South Africa's National Action Plan to Combat Desertification.     

Mechanistic inferences from the crystal structure of fumarylacetoacetate hydrolase with a bound phosphorus-based inhibitor

Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetate as the final step of Phe and Tyr degradation. This unusual reaction is an essential human metabolic function, with loss of FAH activity causing the fatal metabolic disease hereditary tyrosinemia type I (HT1). An enzymatic mechanism involving a catalytic metal ion, a Glu/His catalytic dyad, and a charged oxyanion hole was previously proposed based on recently determined FAH crystal structures. Here we report the development and characterization of an FAH inhibitor, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid (HMPOBA), that competes with the physiological substrate with a K(i) of 85 microM. The crystal structure of FAH complexed with HMPOBA refined at 1.3-A resolution reveals the molecular basis for the competitive inhibition, supports the proposed formation of a tetrahedral alkoxy transition state intermediate during the FAH catalyzed reaction, and reveals a Mg(2+) bound in the enzyme's active site. The analysis of FAH structures corresponding to different catalytic states reveals significant active site side-chain motions that may also be related to catalytic function. Thus, these results advance the understanding of an essential catabolic reaction associated with a fatal metabolic disease and provide insight into the structure-based development of FAH inhibitors.     

Assessment of the Total Inflammatory Potential of Bioaerosols by Using a Granulocyte Assay

Occupational health symptoms related to bioaerosol exposure have been observed in a variety of working environments. Bioaerosols contain microorganisms and microbial components. The aim of this study was to estimate the total inflammatory potential (TIP) of bioaerosols using an in vitro assay based on granulocyte-like cells. A total of 129 bioaerosol samples were collected in the breathing zone of workers during their daily working routine at 22 biofuel plants. The samples were analyzed by traditional assays for dust, endotoxin, fungal spores, (1→3)-β-d-glucan, total number of bacteria, the enzyme N-acetyl-β-d-glucosaminidase (NAGase; primarily originating from fungi), Aspergillus fumigatus, and mesophilic and thermophilic actinomycetes; the samples were also assayed for TIP. In a multilinear regression four factors were significant for the TIP values obtained: endotoxin (P < 0.0001), fungal spores (P < 0.0001), (1→3)-β-d-glucan (P = 0.0005), and mesophilic actinomycetes (P = 0.0063). Using this model to estimate TIP values on the basis of microbial composition, the correlation to the measured values was r = 0.91. When TIP values obtained in the granulocyte assay were related to the primary working area, we found that bioaerosol samples from personnel working in straw storage facilities showed high TIP values (≈50 times the TIP of unstimulated controls). In contrast, bioaerosol samples from personnel with work functions in offices or laboratories showed low TIP values (≈5 times the TIP of the unstimulated control). This indicates, as expected, that these areas were less contaminated. In conclusion, the granulocyte assay reacts to multiple contaminants in the environmental samples and can be used to obtain a measurement of TIP. Therefore, potential occupational health effects related to inflammation of the airways in a working environment can be estimated using this assay.For several years reports have related increased prevalence of respiratory symptoms to the exposure to bioaerosols containing, e.g., organic dust particles, actinomycetes, endotoxin, and fungal spores. Occupational health effects of bioaerosols have been reported in swine confinement houses and poultry farms as well as during hay handling. Airway diseases are frequent occupational disorders among farmers in many countries around the world (19, 23). During mechanical handling, biofuels such as straw and wood chips release high amounts of various microbial components such as, e.g., actinomycetes, fungal spores, and endotoxin (11, 25). Studies of personnel exposure to bioaerosols at biofuel plants reveal high concentrations of different microbial components and exposure levels higher than the suggested occupational exposure limits (10).Exposure limits or suggested exposure limits are usually based on studies where traditional microbial quantification methods have been applied and are normally related to the concentration of a single contaminant (e.g., endotoxin) (17) or to gravimetric measures of dust. Furthermore, it is recognized that exposure to various microbial components may cause inflammation in the airways (4, 5, 15, 16, 20, 34, 35). Consequently, both the quantitative and the qualitative composition of bioaerosols may be of importance. In optimal settings, risk assessment of bioaerosols is based on the presence and concentration of a variety of microbial components. However, such analyses are tedious and costly. Therefore, an assay taking multifactorial contaminations into account may be highly relevant, and ideally this assessment should also correlate to airway inflammation. The objective of this work is to find a rapid and cost-efficient alternative to microbial analyses in characterizing bioaerosols from occupational settings.Normally, airways and alveoli contain a relatively small number of granulocytes, but the pulmonary vasculature represents the largest reservoir of granulocytes in the human body. From this pool, granulocytes can be rapidly recruited upon microbial challenge (26). This recruitment is crucial for the immune response of the host against pathogens. Consequently, we speculate that the production of reactive oxygen species (ROS) of granulocytes can be used as readout for bioaerosol exposure. However, isolated granulocytes are short-lived, with a half-life of only a few hours (33). For this reason a granulocyte-like cell line can represent a practical alternative to freshly isolated cells.In 2006 a granulocyte assay based on a cell line was developed mainly to assess microbial contamination in medicines (32). The assay was shown to be very sensitive to a wide range of microorganisms (32) and severalfold more sensitive to fungi than monocyte assays (e.g., the Mono Mac 6 assay) (18, 32). Furthermore, the assay showed sensitivity very similar to that of freshly isolated granulocytes toward a variety of microorganisms and microbial components (our unpublished observations). This indicates that the granulocyte assay is suitable to assess the inflammatory potential of bioaerosols or similar environmental samples.The granulocyte assay is based on differentiated HL-60 cells. To assess the immunomodulatory effect of a given environmental sample, the production of ROS from the granulocyte-like cells is measured in a luminol-dependent chemiluminometric assay. The induced ROS production is used as a measure of the total inflammatory potential (TIP) of the sample. The term TIP as described herein refers to the inflammatory effects induced in the granulocyte assay by the bioaerosols tested. These effects can be induced by both viable and dead microorganisms as well as related cell wall debris. Furthermore, the term covers inflammatory effects induced by other organic and inorganic components of the bioaerosols, such as pollen, particles, viruses, soot, etc., that are known to have or speculated to have inflammatory effects in the granulocyte assay.To examine the potential of the granulocyte assay to measure the TIP of an environmental sample, we examined 129 bioaerosol samples collected at 22 biofuel plants. In occupational settings the presence of fungi, Aspergillus fumigatus, β-glucan, endotoxin, actinomycetes, and dust has been related to airway symptoms (4, 5, 15, 16, 20, 34, 35). Therefore, we have quantified these components in the sampled bioaerosols. Furthermore, we have quantified N-acetyl-β-d-glucosaminidase (NAGase) activity because this enzyme is associated with fungi and has been shown to correlate significantly with the total number of fungal spores counted by microscopy (9, 14). Our hypothesis is that the TIP values obtained in the granulocyte assay can be used as a rapid way to evaluate the bioaerosol exposure and thus could indicate where a more extensive characterization of microbial parameters would be necessary.     

Potential age of aquatic Oligochaeta

The maximum lifetime of species with exclusively sexual reproduction covers 5–15 years or even more in aquaria, although most specimens die consecutively earlier. The zooids of paratomic species live some weeks or months, their clones usually less than a year, rarely for some years. In the species with facultative architomy (fragmentation) the clonal age also exceeds that of the individual, both lasting for several years. The actual lifetime of worms in nature may be shorter than in cultures.