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Zusammenfassung Gelenkkapselproben von 15 an sekundarem Kreuzbandris erkrankten Hunden wurden mittels eines in vitro Tests (Gross-Lapiere) auf Kollagenaseaktivitat untersucht. In 13 Fallen wurde eine erhohte Enzymaktivitat festgestellt. EDTA und 5-Fluorouracil hemmten die Kollagenaseaktivitat teilweise, wahrend O-Phenantrolin und das Antibiotikum Erythromycin® die Enzymaktivitat in vitro vollstandig blockierten. Auf Grund dieser Befunde wird derzeit die enzymhemmende Wirkung in vivo bei der chronischen Gonarthritis und dem postoperativen Verlauf von Kreuzbandrissen untersucht. Da auch das klinische und histologische Bild von Kniegelenken mit sekundarem Kreuzbandris in vielen Fallen grose Ahnlichkeit mit einer rheumatoiden Arthritis aufweist, wird auf Grund der erhohten Kollagenaseaktivitat ein immunologischer Prozes vermutet. Summary Collagenase activity in the ruptured cruciate ligament of the dog and its inhibition in vitro Joint capsule samples from 15 dogs with secondary cruciate ligament tears were examined by in vitro tests (Gross-Lapiere) for collagenase activity. In 13 cases an increased enzyme activity was found. EDTA and 5-fluorouracil partially inhibited the collagenase activity, whereas O-phenantrolin and erythromycin completely blocked the in vitro enzyme activity. On the basis of these findings the authors are examining the enzyme-inhibiting effect in vivo in chronic gonarthritis and the post-operative course in cruciate ligament rupture. Since the clinical and histological picture of the knee joints with secondary cruciate ligament rupture are in many cases very similar to those in rheumatoid arthritis, the raised collagenase activity may well represent an immunological process. Resume Activite de la collagenase lors d'une dechirure du ligment croise chez le chien et son inhibition in vitro On a examine l'activite de la collagenase sur des echantillons de capsule articulaire de 15 chiens atteints d'une dechirure secondaire du ligament croise au moyen d'un test in vitro (Gross-Lapiere). Une augmentation de l'activite enzymatique a ete constatee dans 13 cas. EDTA et 5-fluorouracil ont inhibe en partie l'activite de la collagenase alors que O-phenantroline et l'antibiotique Erythromycine® ont completement bloque in vitro l'activite de l'enzyme. On a examine sur la base de ces resultats l'action inhibitrice de l'enzyme in vivo lors de gonarthrite chronique et dans l'evolution post-operatoire d'une dechirure du ligament croise. Etant donne que l'image clinique et histologique de l'articulation du genou avec une dechirure secondaire du ligament croise ressemblait dans beaucoup de cas a une arthrite rheumatismale, on a suspecte un processus immunologique sur la base de l'activite augmentee de la collagenase. Resumen La actividad de la colagenasa en la rotura del ligamento cruzado del perro y su inhibicion in vitro Muestras de capsulas articulares de 15 perros enfermos de rotura secundaria del ligamento cruzado se examinaron en pruebas in vitro (Gross-Lapiere) en cuanto a la actividad de la colagenasa. En 13 casos se comprobo que la actividad enzimatica se hallaba aumentada. EDTA y 5-fluorouracilo inhibian en parte la actividad de la colagenasa, mientras que O-fenantrolina y el antibiotico eritromicina® bloqueaban por completo la actividad enzimatica in vitro. A la vista de estos hallazgos, se esta estudiando la accion enzimoinhibidora in vivo en la gonartritis cronica y en el curso postoperatorio de las desgarraduras del ligamento cruzado. Puesto que tambien la imagen clinica e histologica de las articulaciones femorotibiorrotulianas con rotura secundaria del ligamento cruzado tienen una semejanza grande con la artritis reumatoide, se sospecha que se trata de un proceso inmunologico en cuenta a la actividad aumentada de la colagenasa.

Down syndrome (DS) is the most common genetic disorder with mental retardation and a host of deranged proteins has already been described. Protein hunting leads to rapid accumulation of aberrant proteins and proteomics methods not only allow unambiguous identification of proteins, they are also a powerful tools to identify new or predicted proteins. We applied two-dimensional gel electrophoresis with in-gel digestion of proteins and subsequent MALDI-TOF mass-spectrometrical identification and quantification of spots using specific software on cortical brain samples from 7 controls and 7 samples from fetal DS at the early second trimester. Nine hypothetical proteins were identified: three of them (4833418L03Rik protein Q9D614, mitochondrial inner membrane protein Q16891 and Nit protein 2 Q8WUF0) were significantly and about doublefold reduced in fetal DS brain. Hypothetical proteins CGI 99, FLJ10463, 70 kDa WD-repeat tumor rejection antigen homolog, KSRP, Hypothetical protein 49.6 kDa and Elongin A were comparable between groups. Domain analysis of deranged structures revealed a t_SNARE domain for the Rik protein, indicating involvement of this protein in the exocytotic-synaptic machinery impaired in DS, a CN hydrolase domain for Nit protein 2, possibly reflecting aberrant nitrilase-related metabolism and handling and an inner mitochondrial protein, extending knowledge on the mitochondrial deficit in in fetal DS early in life.

Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the molecular mechanisms of the various phenotypes of DS could be due to overexpression of gene(s) on chromosome 21, several groups have challenged this gene dosage effect hypothesis. The near completion of the sequencing of human chromosome 21 provides unprecedented opportunities to understand the molecular pathology of DS, however, functional information on gene products is limited so far. We therefore evaluated the levels of six proteins whose genes are encoded on chromosome 21 (trefoil factor 1, trefoil factor 2, trefoil factor 3, coxsackie virus and adenovirus receptor, carbonyl reductase 1 and interferon- alpha receptor) in fetal cerebral cortex from DS and controls at the early second trimester using Western blot analysis. None of the investigated proteins showed overexpression in DS compared to controls suggesting that these proteins are not involved in abnormal development of fetal DS brain and that DS phenotype can not be simply explained by the gene dosage effect hypothesis. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 and these studies may provide a better understanding of genotype-phenotype correlation in DS.

Previous experiments have shown that a 10-min forced swimming session triggers the release of vasopressin from somata and dendrites, but not axon terminals, of neurons of the hypothalamic-neurohypophysial system. To further investigate regulatory mechanisms underlying this dissociated release, we forced male Wistar rats to swim in warm (20 degrees C) water and monitored release of the potentially inhibitory amino acids gamma amino butyric acid (GABA) and taurine into the hypothalamic supraoptic nucleus using microdialysis. Forced swimming caused a significant increase in the release of taurine (up to 350%; P0.05 vs. prestress release), but not GABA. To reveal the physiological significance of centrally released taurine, the specific taurine antagonist 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide was administered into the supraoptic nucleus via retrodialysis. Administration of this antagonist caused a significant increase in the release of vasopressin within the supraoptic nucleus and into the blood both under basal conditions and during stress (up to 800%; P0.05 vs. basal values), without affecting hypothalamic or plasma oxytocin. Local administration of the GABA(A) receptor antagonist bicuculline, in contrast, failed to influence vasopressin secretion at either time point. In a separate series of in vivo electrophysiological experiments, administration of the same dosage of the taurine antagonist into the supraoptic nucleus via microdialysis resulted in an increased electrical activity of identified vasopressinergic, but not oxytocinergic, neurons. Taken together our data demonstrate that taurine is released within the supraoptic nucleus during physical/emotional stress. Furthermore, at the level of the supraoptic nucleus, taurine inhibits not only the electrical activity of vasopressin neurons but also acts as an inhibitor of both central and peripheral vasopressin secretion during different physiological states.

Kainic acid (KA), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. No global brain protein expression pattern induced by KA-treatment has been reported yet. We therefore studied the effect of systemic KA administration on the levels of brain proteins. Rats were injected placebo or KA intraperitoneally and brain was taken after one week. The mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies and the levels of selected proteins were quantified using specific software. Heat shock protein HSP 27 was exclusively detected in brains of animals treated with KA, whereas the glucose regulated protein GRP 78 was downregulated. The levels of neurofilaments and alpha-internexin were significantly decreased and a fragment of tubulin alpha-1 chain was manifold increased in KA-brains. The mitochondrial enzymes dihydrolipoamide dehydrogenase, ATP synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase M1 was increased following KA treatment. We conclude that the concomitant determination of the brain proteins indicates altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption, and mitochondrial derangement by systemic KA administration. This report confirms and extends previous studies on the effect of KA on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological, and neuropathological proteome studies.

Low-abundance gene products are of interest in proteomic studies, because they are probably involved in disease-related changes and their altered levels or modifications may carry significant biological information. Detection of low-abundance proteins of a proteome is one of the major limitations of proteomics and a scientific challenge. We investigated the changes in the levels of low-abundance rat brain cytosolic proteins after administration of kainic acid, a potent neurotoxin and excitatory amino acid. The cytosolic proteins from controls and animals treated with kainic acid were fractionated on an ion-exchange column. The fractions collected were analyzed by 2D electrophoresis, and the proteins with altered levels were identified by matrix-assisted laser desorption ionization or ion-spray MS. We found a manifold decrease in annexin VII, heat-shock cofactor HOP/p60 and SP-22 and a manifold increase in heparin-binding protein p30. The results suggest, respectively, the involvement of an apoptotic pathway, recruitment of the heat-shock protein machinery, generation of an antioxidant response, and, probably, induction of repair mechanisms. Three of the four proteins with altered levels had not been previously detected in the cytosolic fraction, and detection of the altered levels was possible only after the protein-enriching step.

NADH: ubiquinone oxidoreductase (complex I), one of the most complicated multi-protein enzyme complexes, is important for energy metabolism because it is the initial enzyme of the mitochondrial respiratory chain. Deficiency of complex I is frequently found in various tissues of patients with neurodegenerative disease. Here we studied the protein levels of complex I 24- and 75-kDa subunits in several brain regions from patients with Down syndrome (DS) and Alzheimer's disease (AD). We determined protein levels of complex I 24-, 75-kDa subunits and mitochondrial marker proteins mitochondrial matrix protein P1 (hsp60) and aconitate hydratase from seven brain regions of patients with DS, AD and controls. Proteins were separated by two-dimensional (2-D) gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Complex I 24-kDa subunit was significantly reduced in occipital cortex and thalamus in patients with DS and temporal and occipital cortices in patients with AD. Complex I 75-kDa subunit was significantly reduced in brain regions from patients with DS (temporal, occipital and caudate nucleus) and AD (parietal cortex). Reductions of two subunits of complex I may lead to the impairment of energy metabolism and result in neuronal cell death (apoptosis), a hallmark of both neurodegenerative disorders.

Voltage-dependent anion-selective channel proteins (VDACs) are pore-forming proteins found in the other mitochondrial membrane of all eukaryotes and in brain postsynaptic membranes. VDACs regulate anion fluxes of a series of metabolites including ATP, thus regulating mitochondrial metabolic functions. We determined protein levels of VDACs in individual post-mortem brain regions of patients with Down Syndrome (DS) and Alzheimer's disease (AD) using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). VDAC1 (SWISS-PROT accession number P21796) and VDAC2 (P45880) were unambiguously identified and quantified, but VDAC3 was not found. The spots representing VDAC1 were separated with different p/s (p/7.5, 8.5, and 10.0) probably caused by post-translational modifications as, e.g., phosphorylation. In DS cerebellum, total VDAC1 protein was elevated significantly whereas VDAC2 did not show any significant alterations. In AD brains, VDAC1 p/10.0 was significantly reduced in temporal, frontal, and occipital cortex with the p/7.5 form elevated in occipital cortex. Total VDAC1 was significantly decreased in frontal cortex and thalamus. VDAC2 was significantly elevated in temporal cortex only. The biological meaning of our results may be derangement of voltage-dependent anion-selective channel function and reflecting impaired glucose, energy, and intermediary metabolism as well as apoptotic mechanisms.

1. The role of oxidative stress in the pathogenesis of the diabetic state is being investigated extensively. Although oxidative stress has been reported in terms of glycoxidation, protein oxidation and DNA oxidation in diabetes mellitus, oxidation parameters have not been determined in parallel on the same study population.2. We studied 24 patients with diabetes mellitus (14 patients with Type I diabetes with a mean age of 62.3+/-6.3 years and 10 patients with Type II diabetes aged 67.3+/-5.9 years) and compared them with age-matched non-diabetic controls. Urinary o-tyrosine, 8-hydroxy-2'-deoxyguanosine and pentosidine measurements by HPLC were made on two occasions (t1 and t2).3.A clear statistical difference was found between diabetic patients and controls at t1 or t2 for 8-hydroxy-2'-deoxyguanosine and pentosidine, but not for o-tyrosine. No significant correlations were found between clinical and other laboratory parameters except high-density lipoprotein and uric acid. We revealed significantly increased glycoxidation and DNA oxidation in patients with Type I and Type II diabetes, but protein oxidation was not different from controls.4. The finding of increased glycoxidation reflects increased oxidation of the carbohydrate moiety, whereas the increased levels of oxidized DNA may also be interpreted as due to increased DNA repair. The increased 8-hydroxy-2'-deoxyguanosine does not indicate the generation of an individual active oxygen species, but DNA could have been oxidized simply by alkenals from lipid peroxidation, as e.g. malondialdehyde. As no difference in protein oxidation (i.e. o-tyrosine) between diabetics and controls could be revealed, the oxidation of DNA by hydroxyl radical attack is unlikely, as o-tyrosine was proposed as a marker for hydroxyl radical attack. Therefore, the message is that increased glycoxidation can be confirmed, protein oxidation does not appear to take place and increased DNA oxidation is still not proven, as increased 8-hydroxy-2'-deoxyguanosine may simply reflect repair.

Non-enzymatic glycosylation is a post-translational process that is increased in the diabetic state. In order to determine whether the non-enzymatic reaction can be followed by multiple reflection infrared spectroscopy, we performed studies on the non-enzymatic glycosylation of arginine and collagen. Spectra clearly showed that a reaction between the amino acid and glucose and the collagen and glucose occurred. The major finding was the disappearance of the 1722 cm-1 peak derived from the carbonyl function of glucose after reaction with arginine, indicating that the carbonyl grouping had been blocked by the reaction, a finding in the reduction of carbonyl functions agreement with Schiff base formation. This interpretation is confirmed by changes of the 1219 cm-1 band. When glucose binds to collagen, the presence of the glucose derived 1026 cm-1 band is evident in the spectrum of the glycosylated molecule. This simple non-destructive method provides a useful tool with which to follow non-enzymatic glycosylation.

Sprague-Dawley rats, white, female, 150–180g were fed for a period of one month with D-proline (n = 7), L-proline (n = 7), L-lysine (n = 6) in a mean dosage of 50 mg/kg body weight/ day orally in tap water; 10 healthy rats served as controls. The animals were sacrificed after one month by guillotinization.

12519 Background: Medulloblastoma (MB) is the most common malignant childhood brain tumor. Because of its high risk of leptomeningeal dissemination, standard postoperative treatment includes craniospinal irradiation and chemotherapy. Such treatment produces significant sequelae including neurocognitive dysfunction, endocrine and growth disturbances. These therapy-induced morbidities warrant a novel less toxic approach possibly targeting key molecules within specific pathways of medulloblastoma biology. Recent reports identified high neurotrophin receptor TrkC mRNA expression as a powerful independent predictor of a favorable survival outcome in MB patients. However, the role of activated TrkC receptors in the development and biology of MB remains unclear. Methods: To determine downstream effector proteins of TrkC signaling, the medulloblastoma cell line DAOY was stably transfected with a vector containing the full-length TrkC cDNA sequence or an empty vector control. Accounting for the complexity of ligand-induced changes in cellular pathways and effector proteins, we investigated proteomic changes at multiple time points for up to 48 hrs following neurotrophin-3 induced TrkC receptor activation. Results: We identified 18 proteins differentially expressed: Caldesmon; Cathepsin D; Metastasis inhibition factor nm23; Multidrug resistance-associated protein Mgr1-Ag; Vimentin; Vinculin; Superoxide dismutase (Mn); Glutathione S-transferase P; Stathmin, Lamin A/C, Valosin-containing protein, Annexin A1; ULIP protein; DJ-1 protein; Fascin; Mitofilin; Heterogeneous nuclear ribonucleoprotein H and K. Conclusion: The proteins affected play substantial roles in differentiation, migration, invasion, proliferation, apoptosis and drug resistance. Almost all of the proteins have been described as being essential in the pathogenesis of different solid tumors, but have not been related to MB pathogenesis so far. No significant financial relationships to disclose.

Glomerular basement membranes (GBM) were isolated from the kidneys of adult and newborn Sprague-Dawley rats. The isolated GBM were degraded by trypsin and the degradation products were characterized by immunoelectrophoresis applying an anti-rat GBM serum from the rabbit. GBM of the adult rat showed two precipitation lines, GBM from the newborn rat presented a single line in immunoelectrophoretical characterization. GBM of 6 adult and 6 newborn rats were measured on electron microscopy revealing significant differences of thickness. The mean thickness of the adult rat GBM was 2,060 Å (2.06 × 10-7 m), the SD was ± 83 Å; of the newborn rat mean GBM was 1,500 Å (1.5 × 10-7 m), the SD was ± 71 Å. Our results indicate that the immature GBM differs from the mature structure qualitatively and quantitatively.

Mit Hilfe eines biologischen Assays wurde die koUagenolytische Aktivitat von 10 invasiven Gebarmutterhalskrebsen untersucht. Bei 7 Kar-zinomen konnte eine Kollagenolyse festgestellt werden. Um den Ur-sprung dieser kollagenolytischen Aktivitat zu eruieren, wurden zusatz-lich Hemmstudien mit Athylendiamintetraacetat und normalem menschlichem Serum durchgefuhrt. Normales menschliches Serum hemmte die Kollagenolyse um durchschnittlich 14,9%, EDTA um 9,1%. D-Penicillamin und 1.10-o-Phenanthrolin als spezifische Kollagenasehemmstoffe fuhrten zu einem volligen Stop der Reaktion. Auf-grund unserer Ergebnisse kann angenommen werden, das die koUagenolytische Aktivitat des Zervixkarzinoms nicht vom Serum oder von polymorphkernigen Leukozyten abstammt, sondern von den Tumor -zellen selbst gebildet wird.

A method to eluat donororganspecific antibodies enabling direct and specific access to the pattern of humoral rejection after lung allotransplantation is demonstrated. The antibodies were evaluated quantitatively and qualitatively by elution of lung-grafts immunoglobulins and by a consequent investigation of the eluates using the passive haemagglutination- and indirect immunflourescence- test against donors' lung-antigen. Humoral antidonorlung-antibodies could be proved in all rejected grafts belonging to sensitized as well as to unsensitized recipients. There can be seen a highly significant correlation when comparing the results of the passive haemagglutination and indirect immunfluorescence test (r = 0,93, p less than or equal 0,01). On the other hand negative results of the investigations of eluates by lungantigens of other dogs show the specifity of humoral graft rejection. The suprising fact that there does not exist an essential difference between the results of sensitized and nonsensitized animals reveals a humoral immunresponse in hyperacute as well as in acute rejection.

Mit Hilfe eines biologischen Kollagenasetests wurde die kollagenolytische Aktivitat normaler peripherer menschlicher Lymphozyten und von mit Epstein-Barr-Virus infizierten lymphoblastoiden Zellinien b

The beneficial effect of methionine supplementation to low sulphur amino acid diets has been demonstrated in the human newborn suffering from protein malnutrition.