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Hygro-mechanical phenomena induced by moisture loading of a historical wooden panel painting from the 16th century are investigated in this study. Due to swelling and shrinking strains resulting from an inappropriate and restraining support frame, damage in terms of ruptures on the painting itself and large curvature have developed. A brief history of the painting and its conservation status are summarised. A two-phase multi-Fickian diffusion model for moisture transport in wood and a consistently coupled hygro-mechanical simulation are applied to explore the influence of different realistic scenarios of climatic change on the panel painting itself and its supporting frame. The hereby gained results can be used to predict the general mechanical behaviour, and help to identify critical parts of the structure and risky climatic situations, which could increase damage. New methods of conservation in terms of innovative stabilising wood frames and adjusted climatic conditions can efficiently be evaluated with the help of the presented approach.

Perineuronal nets of extracellular matrix have been shown to characterize the microenvironment of individual neurons and the chemoarchitecture of brain regions such as basal forebrain nuclei. Previous work has also demonstrated that neurons in the human cerebral cortex ensheathed by perineuronal nets rarely undergo cytoskeletal changes in Alzheimer’s disease, suggesting a neuroprotective effect of extracellular matrix components. It is not known, however, whether or not perineuronal nets are absent in the microenvironment of the cholinergic basal forebrain neurons that are involved early in the cascade of neurodegeneration in humans. Therefore, the present study was undertaken to examine the distribution patterns of perineuronal nets in the basal forebrain of the higher primates, rhesus monkey and human. Cytochemical staining was performed with the lectin Wisteria floribunda agglutinin and a polyclonal antibody to core proteins of chondroitin sulfate proteoglycans in the perfusion-fixed tissue of rhesus monkeys. In human brains, perineuronal nets were only stained with the immunoreaction for chondroitin sulfate proteoglycans. The results showed similar characteristics in distribution patterns of perineuronal nets in the medial septum, the diagonal band of Broca, the basal nucleus of Meynert (Ch1–Ch4), the lateral septum, the caudate–putamen, and the globus pallidus in both species. Double-labelling revealed that the vast majority of cholinergic neurons, labelled either with antibodies to choline acetyltransferase or the low-affinity neurotrophin receptor p75 NTR , were not ensheathed by perineuronal nets. A small subpopulation of net-associated neurons in close proximity to or intermingled with cholinergic neurons of the Ch1–Ch4 cell groups was found to be immunoreactive for parvalbumin. In the caudate–putamen, a large number of the parvalbumin-positive neurons were surrounded by perineuronal nets, whereas in the external and internal segments of the globus pallidus the coincidence of both markers was nearly complete. The study demonstrates that perineuronal nets of extracellular matrix are associated with different types of non-cholinergic neurons in the primate basal forebrain. The absence of nets around cholinergic basal forebrain neurons may be related to their slow modulatory activity but may also contribute to their susceptibility to degeneration in Alzheimer’s disease.

Determination of matrix metalloproteinase-9 (MMP-9) in human cerebrospinal fluid (CSF) to study blood–brain barrier impairment and immune cell migration in inflammatory neurological diseases recently became a matter of major interest. Regularly, MMP-9 was determined qualitatively or semi-quantitatively by zymography (gelatin gel electrophoresis) or quantitatively by enzyme immunoassay (EIA). As yet, it was not possible by either method to detect MMP-9 in CSF of controls (patients without pathologically increased CSF parameters). We developed an ultrasensitive two-side enzyme-linked immunosorbent assay (ELISA) which allows for the first time to measure reliably MMP-9 concentrations in CSF of controls. This ELISA uses a monoclonal as capture and a polyclonal as detector antibody. The detection limit of the assay is below 10 pg/ml and the assay range is 15–2000 pg/ml. Intra-assay precision is 2.5% for low and 3.7% for high, inter-assay precision is 11% for low and 10.7% for high values, respectively. The determination of the MMP-9 concentration in 50 control CSF gave the following results: range, 22–146 pg/ml; median, 76 pg/ml. The measurement of native and recombinant MMP-9 was carried out with three commercially available ELISAs, most widely employed in MMP-9 research, and compared to the newly developed one. All ELISAs recognize recombinant MMP-9 by factors of 5–20 less sensitively than native MMP-9.

Although multiple sclerosis (MS) is considered primarily as a demyelinating disease, neuronal damage is abundant and correlates with the neurological deficit. Therefore, we investigated the frequency and characteristics of human T cells specific for synapsin-a neuronal protein highly conserved among species. Synapsin specific T cell responses were detected at a frequency similar to that of MBP specific T cells in MS patients, one patient with acute demyelinating encephalomyelitis (ADEM) and controls. Long-term T cell lines specific for synapsin exhibited a CD3(+), CD4(+), CD8(-) phenotype and produced high amounts of tumor-necrosis-factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) after antigen specific stimulation, whereas lymphotoxin (LT), interleukin-4 (IL-4) and interleukin-10 (IL-10) were detectable in smaller quantities.

Matrix metalloproteinase-9 (MMP-9) was investigated by enzyme-linked immunosorbent assay (ELISA) and zymography in 111 paired CSF and serum samples from patients with various neurological disorders. In 20 patients with blood-brain barrier (BBB) impairment but normal CSF cell count, elevated levels of MMP-9 were not observed by ELISA measurement. Another 11 patients characterized in the same way, exhibited only slightly increased MMP-9 levels. In contrast, in 12 patients with intact BBB but elevated CSF cell count, MMP-9 was increased too. It was shown by the more sensitive zymography that MMP-9 increased if CSF cell count exceeded five cells per microl. Spearman rank statistics revealed that MMP-9 concentration in CSF correlated with CSF cell count (r=0.755; P

Glatiramer acetate (GA), represents an established treatment of relapsing/remitting multiple sclerosis (MS). The mechanisms responsible for the effect of GA are not fully understood. We generated GA-, myelin basic protein (MBP)- and purified protein derivative (PPD)-specific T cell lines from three MS patients and one healthy donor. The GA-specific lines were CD3+, CD4+, CD8- and produced tumor-necrosis-factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), interleukin-6 (IL-6) and interleukin-10 (IL-10) after stimulation with GA in the presence of irradiated peripheral blood mononuclear cells. MBP-specific T cell lines showed an identical phenotype and secreted TNF-alpha, IFN-gamma, IL-4, IL-10, but not IL-6. Co-culture experiments demonstrated, that GA-specific T cell lines have the capability to suppress the proliferation of MBP-specific T cell lines.

Perineuronal nets (PNs) are known as chondroitin sulphate-rich, lattice-like coatings of the extracellular matrix. In the cortex of mammalian species investigated so far, they were mainly found around GABAergic neurons, but to a lesser degree also around pyramidal cells. Previous investigations in the rat revealed similar distribution patterns of fast-firing neurons expressing both the Kv3.1b subunit of voltage-gated potassium channels and the calcium-binding protein parvalbumin. In the present study, triple fluorescence labelling was applied for the simultaneous demonstration of PNs with the N-acetylgalactosamine-specific Wisteria floribunda agglutinin (WFA), parvalbumin-immunoreactivity (ir) with a monoclonal antibody and of Kv3.1b-ir with several rabbit antibodies. Subsets of non-pyramidal neurons - enwrapped by PNs and expressing parvalbumin and Kv3.1b - were detected in the rat and monkey neocortex and hippocampus. In the rat, faintly stained PNs were additionally found around several layer II/III and V pyramidal cells immunonegative for Kv3.1b, but contacted by Kv3.1b-containing boutons. In the monkey, more intensely labelled PNs frequently occurred around pyramidal cells which themselves appeared to be Kv3. 1b-immunopositive. We also observed minor Kv3.1b-ir and parvalbumin-ir cortical cell populations which were devoid of PNs; occasionally, nets were detected around neurons lacking both immunoreactivities. By confocal laser scanning microscopy, Kv3.1b-ir and WFA-binding sites were found adjoining at the soma and proximal dendritic surface, while lectin-binding sites usually extended on more distal dendritic segments and the axon initial segments which failed to express detectable Kv3.1b-ir. This spatial relationship of both markers was also confirmed by combined WFA-gold labelling and Kv3.1b-immunoperoxidase staining at the electron microscopic level. The data are used for a critical examination of current hypotheses concerning the functional role of PNs. We conclude that PNs may serve as rapid local buffers of excess cation changes in the extracellular space. Somatic membranes of fast-spiking neurons seem to be a main, but not the only source of such changes.

Two-site enzyme-linked immunosorbent assays (ELISA) have been established for the specific and sensitive determination of two membrane proteins of the small synaptic vesicles (SSV), namely: peripheral synapsin I and integral synaptophysin. The ELISA used highly specific capture monoclonal antibodies (mAB) and polyclonal antibodies (pAB) as detectors. For synapsin I, the mAB were newly generated, whereas for synaptophysin, the commercially available mAB SY38 was applied. In order to calibrate the ELISA and to raise pAB, both proteins were purified in the mg-range. Synapsin I was purified by conventional means from human and porcine brain and synaptophysin was purified by immunoaffinity chromatography from porcine brain. Using the ELISA, neither synapsin I nor synaptophysin could be determined in serum or cerebrospinal fluid (CSF) from healthy donors or patients suffering various neurological disorders or pheochromocytomas. For this reason, the degradation of both proteins in serum and CSF was investigated. With the exception of synaptophysin measured in serum, both proteins exhibited fast rates of degradation. Despite the negative results in human body fluids, the two ELISA are appropriate for the quantification of these membrane proteins in neuronal or neuroendocrine cell extracts or preparations of SSV.

Although meninges represent a major site of biosynthesis, beta-trace protein (beta-trace) has not been studied in the cerebrospinal fluid (CSF) of meningitis patients. We measured beta-trace in lumbar CSF of normal controls (n = 27) and in patients with various neurological diseases (n = 92) by an immunonephelometric assay. The mean concentration of beta-trace in CSF of control patients was 16.6+/-3.6 mg/l. In bacterial meningitis (n = 41), CSF beta-trace was significantly decreased (8.7+/-3.9 mg/l; P< 0.001), whereas in spinal canal stenosis it was elevated (29.2+/-10.3 mg/l; P= 0.002). In viral meningoencephalitis (n = 12), beta-trace CSF concentrations were normal. Beta-trace concentrations remained below the normal range even after curing of bacterial meningitis, and normalisation of CSF leucocytes and blood-CSF barrier function. Beta-trace may be a useful tool for studying the pathophysiology of bacterial meningitis.

Thin sputter deposited layers with a nominal composition of Nd2Fe14B (thickness 120 to 400 nm) on SiSiO2 were analysed by the combined application of RBS and heavy ion ERDA. These methods, based on elastic Coulomb scattering, are advantageous for composition analysis of thin compound layers due to their known cross sections. For high accuracy it is necessary that the spectral contributions of each element can be separated from each other and from the substrate background. This is difficult for light elements with RBS. The light element concentration however can be measured by ERDA with heavy incident ions. With 200 MeV 127I ions from the Munich MP tandem the light elements as well as the Fe recoils were detected using a gas telescope. The Fe peak was used as cross reference to the RBS measurements. With 36 MeV 35Cl ions at the 5 MV Rossendorf tandem too little energy is transferred to the Fe recoils to be identified with the Bragg ionisation chamber (BIC) detector. Therefore a thin aluminium layer was evaporated onto the Nd2Fe14B layer. The aluminium peak is well separated both in the RBS and in the ERDA spectra and is used as cross reference here.

Various human secretions (intestinal secretion, saliva, nasal mucus, lacrimal fluid) have been found to inhibit the binding of antibodies to their antigens. Various characteristics (e.g. time, pH, temperature dependence, affinity and size exclusion chromatography) suggested that the inhibitory activity was attributable to an enzyme. Further investigations revealed that this enzyme reacted with the Fab portion of immunoglobulin G, specifically with the heavy chain. It is assumed that it represents a novel immunoglobulin-specific protease since similar results were not obtained with proteolytic enzymes from human digestive organs e.g. pepsin, trypsin and chymotrypsin. Finally, investigating saliva it was demonstrated that the putative protease was not identical to enzymes from periodontal bacteria which are proteolytic for the Fc portion of immunoglobulins. The findings could be of general importance in the design of immunoassays which are to be applied to human (and possibly animal) secretions.

The chemical codings of neurons that project from the small intestine, caecum, proximal colon, distal colon and rectum to the coeliac ganglion of the guinea pig were investigated. The coeliac ganglion was injected with the retrogradely transported dye Fast Blue, and each of the regions was examined 6 days later in wholemounts that had been prepared for immunohistochemical localisation of pairs of antigens. In both the small and large intestines, all intestinofugal neurons were immunoreactive (IR) for choline acetyltransferase (ChAT). In each region of the large intestine, the largest population, representing 50-60% of retrogradely labelled neurons in each region, was immunoreactive for ChAT, bombesin (BN), calbindin (Calb) and nitric oxide synthase (NOS). Most intestinofugal neurons in the small intestine contain bombesin and VIP-IR along with ChAT-IR but none contain either Calb or NOS. Thus, nerve endings of enteric origin in the coeliac ganglion that contain NOS-IR or Calb-IR come from the large intestine and those with bombesin-IR but not NOS-IR are from the small intestine. The gastric wall was injected with Fast Blue in order to label noradrenergic (NA) neurons in the coeliac ganglion and to determine, by localisation of NOS and bombesin-IR, whether they receive inputs from the small and large intestine. Some NA neurons received inputs from the large intestine (and perhaps also from the small intestine) and some received inputs exclusively from the small intestine. Most NA neurons that received intestinofugal inputs had the chemical code NA/-; some were immunoreactive for somatostatin (NA/SOM neurons), but those with IR for neuropeptide Y (NA/NPY) rarely received intestinofugal inputs.

In the rat choline acetyltransferase (ChAT)-like immunoreactivity (ChAT-LI) was demonstrated in the dorsal root ganglion (DRG), in the superficial spinal cord and in the subepithelial layer of the ureter using immunohistochemical techniques. In the L1 DRG, 66% of the neurones were ChAT-LI. They did not express neurofilament immunoreactivity (RT97 negative) but could also contain calcitonin gene-related peptide-like immunoreactivity (CGRP-LI). In the superficial spinal cord and in the subepithelial plexus of the ureter - both areas where high numbers of fine afferent fibres have been demonstrated - CGRP-LI and ChAT-LI were co-distributed, indicating that ChAT can be found in the peripheral and central endings of small afferents. The data provide morphological evidence that a substantial proportion of afferent fibres are cholinergic.

A specific, sensitive, and reliable sandwich-ELISA (enzyme-linked immunosorbent assay) has been established for the determination of choline acetyltransferase (CHAT) from porcine brain. The detection limit of the assay was 30 micrograms/l and the assay was linear up to 300 micrograms/l. The within-day and day-to-day coefficients of variation were found to be 3.3% and 4.7% respectively for low CHAT concentrations (30 micrograms/l) and 3.1% and 3.4% respectively for high levels of CHAT (300 micrograms/l). The immunoassay was more sensitive than the radiometric assay of Fonnum which is widely used for the measurement of enzyme activity. In the assay monoclonal antibody was adsorbed to the polystyrene surface of the immunoplate as the capture reagent. Using a standard peroxidase protocol the immobilized antigen was detected with a highly specific anti-CHAT antiserum raised in rabbits. Two monoclonal antibodies were available for antigen binding. One of the two--A10.29B4--reacted preferentially with the active enzyme the other one--B3.9B3--reacted only with a degraded form. The polyclonal antiserum recognized both native and denatured enzyme. The effectiveness of employing the two monoclonal antibodies separately or in combination was demonstrated by measurement of porcine CHAT diluted in human cerebrospinal fluid and serum. After some minor modifications the sandwich-ELISA could be used for the determination of CHAT from the central nervous system of the rat.

Summary The peroxidase isoenzyme-groups of Nicotiana tabacum L. (A1 acidic, A2 slightly acidic, C1 basic) were purified from leaves and cell suspension cultures to apparent homogeneity as revealed by SDS-PAGE. The relative molecular masses were 38,000 (A1), 45,000 (A2) and 40,000 (C1). The final and most important purification step was a HPLC with hydrophobic interaction. The RZ-values (A 403nm /A 275nm ) achieved were 2.3 (A1), 2.9 (A2) and 3.4 (C1). Antisera were raised in rabbits against these highly purified peroxidases. By Western blotting and by the Ouchterlony double diffusion method the reactivity of the antisera and the immunological inhibition of the enzymes were studied. By the development of an ELISA the cross reactions of the antisera with the various peroxidase isoenzymes were quantitatively evaluated. The most interesting finding is that A2 reacts hardly with the antisera against the other two isoenzyme-groups. A1 and C1 show higher degrees of cross reaction with the other antisera. The results are discussed in relation to earlier findings on immunology of plant peroxidases.

β-Trace, a protein that represents a major constituent of human cerebrospinal fluid with unknown function has been purified to apparent homogeneity by gel chromatography and electrophoresis. After sodium dodecylsulfate electrophoresis on polyacrylamide gels (SDS-PAGE) and Western blotting, the N-terminal sequence (28 amino acids) has been determined. The high degree of idendity with the corresponding sequences of prostaglandin D synthetase from rat (68%) and human (93%) suggests a strong relationship if not identity with this enzyme. Thus, 30 years after its discovery β-trace might unravel as a well-known protein of the prostaglandin metabolism.

Summary Activity of ethylene formation from 1-aminocyclopropane-1-carboxylic acid (ACC) was compared to peroxidase (POD) activity in crude extracts of etiolated pea shoots ( Pisum sativum L.) as well as in Sephadex and Concanavalin A-Sepharose (ConA) fractions of these extracts. In crude extracts the stability of POD activity was higher than that of ethylene formation. Mercaptoethanol and sodiumazide inhibited both activities, but other inhibitors of ethylene formation (hydroxylammoniumchloride, catalase, EDTA, KCN, CuSO 4 , CoCl 2 ) had a weak or no effect at all on POD activity. High-molecular weight fractions (FI) from Sephadex of pea crude extracts formed ethylene from ACC and exhibited POD activity. By mixing high molecular weight fractions with low molecular weight fractions from the Sephadex column ethylene formation was stimulated. A similar effect could be reached by substituting FI fractions with horse radish peroxidase (HRP), but ethylene production was very low. After ConA fractionation the highest ethylene formation was found to be in the POD containing eluate, but in addition we found ethylene formation in effluent fractions which showed no POD activity. Mn 2+ otherwise has a stimulating effect on ethylene production from Sephadex fractions as well as from HRP but not on the crude extract. It appears that peroxidase itself does not play a major role in decarboxylation or ring cleavage of ACC, but that it might be involved in a more complex way by providing radicals, H 2 O 2 or otherwise unknown factors.

Summary The present communication describes the determination of apparent Michaelis-constants of three peroxidase-isoenzyme-groups for several artificial (chemicals which are active in the peroxidase catalysed reaction) and natural substrates (compounds which are found in plants and which possibly act as electrondonors for H 2 O 2 -reduction by peroxidases in vivo ). G I (cell wall located group) has lowest Michaelis-constants for artificial, G III (protoplast located group) for natural substrates. Scopoletin is no substrate for G III , but for G I particularly and to some extent for G II . Maximal oxidation rates of different flavan-derivatives are highest for G I or G II and lowest for G III . The in vitro polymerisation of cumaryland coniferylalcohol to lignin-like-substances is catalysed by G I and G II to a substantial rate but hardly by G III . All the isoenzyme groups exhibit indole-3-acetic oxidase but no phenol-oxidase-activity. The results are discussed in connection with several hypotheses of peroxidase-function and with the localisation of peroxidase-isoenzyme-groups in tissues of tobacco.

Isolation of cell wall-bound malate dyhydrogenase (MDH, EC 1.1.1.37) from tobacco was obtained by treating purified cell walls with pectinase and hemicellulase. By electrophoretic separation it was demonstrated that the wall-bound isoenzymes of MDH are different from the cellular ones.

Summary Separation and purification of three peroxidase-isoenzyme groups (GI, fast migrating anodic; GII, slow migrating anodic; GIII, slow migrating cathodic) was accomplished by ion-exchange chromatography. Chromatographic separation of the groups was controlled by disk-electrophoresis and by isoelectric focusing. Molecular weights were estimated by SDS-gelelectrophoresis and by thin layer chromatography. The results of both methods are similar. GI has a molecular weight of about 27,000, GII of 43,000 and GIII of 47,000. By affinity chromatography with Concanavalin A-sepharose it was shown that the enzymes of the three peroxidase groups are all glycoproteins. While heat inactivation of GI was measured at 50°C, inactivation of GII and GIII did not take place before 70°C. Absorption spectra of the three groups were typical for ferriperoxidases with the maximum of the Soret band at 401 nm.

H2O2 formed in the process of NADH-oxidation mediated by peroxidase (POD) and cofactors is measured by a newly developed enzymatic method. This method allows detection of very small quantities of H2O2 (10−5 M). Although this method was developed predominantly to study H2O2-formation in vitro it might also be useful for determining H2O2 content in plant saps and extracts. The amount of H2O2-formed in the course of NADH-oxidation is within certain limits proportional to the amount of available NADH and not dependent on the amount of enzyme and other cofactors. The stoichiometric relation of NADH/H2O2 was shown to be 0.5 which coincides with the expected theoretical values.