Combined superoxide dismutase mimetic and peroxynitrite scavenger protects against neointima formation after endarterectomy in association with decreased proliferation and nitro-oxidative stress.

OBJECTIVE: Reactive oxygen and nitrogen species (e.g., peroxynitrite) may trigger neointima formation leading to restenosis. In a rat carotid endarterectomy (CEA) model, we investigated the effects of the manganese(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP), a superoxide dismutase (SOD) mimetic and peroxynitrite scavenger on neointima formation. METHODS: CEA was performed in male Sprague-Dawley rats. Animals received either vehicle (control group; n=15) or 15 mg kg(-1) day(-1) MnTBAP intraperitoneally for 3 weeks (treatment group; n=13). Four groups of carotids were analysed: the left, uninjured carotids (sham) and the right, injured carotids (control CEA) from the control group, the right, injured carotids from the treatment group (CEA+MnTBAP) and an additional group of carotids that were harvested 1h following endarterectomy. The analysis of carotid arteries was performed by histology, immunohistochemistry and real-time polymerase chain reaction (PCR). Plasma malondialdehyde (MDA) levels were measured by lipid hydroperoxidase assay. RESULTS: Stenosis rate (10.5+/-8.1% vs. 45.4+/-28.3%), the percentage of proliferating cell nuclear antigen-positive cells (13.4+/-7.1% vs. 23.3+/-11.0%) and nitrotyrosine immunoreactivity (5.8+/-1.9 vs. 8.0+/-2.0) were significantly reduced in the vascular wall of the CEA+MnTBAP group compared with control CEA group. Ratio of Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL)-positive nuclei was significantly lower after antioxidant therapy (41.7+/-26.7% vs. 64.9+/-18.5%). Plasma MDA levels increased after endarterectomy (11.7+/-4.8 vs. 4.1+/-2.0 micromol l(-1)) and reduced in the treatment group (3.2+/-2.1 micromol l(-1)). No significant gene regulation after MnTBAP treatment could be noted. CONCLUSIONS: MnTBAP decreased neointima formation, which was associated with reduced vascular smooth muscle cell proliferation and attenuated local and systemic nitro-oxidative stress.

Serum resistin levels of obese and lean children and adolescents: biochemical analysis and clinical relevance.

OBJECTIVE: It was hypothesized that resistin links obesity with diabetes, but this has not been studied in children and adolescents to date. PATIENTS: We determined serum resistin levels of 135 obese (body mass index, 32.0 +/- 6.2 kg/m2; age, 12.6 +/- 3.4 yr) and 201 lean children (body mass index, 18.7 +/- 2.4 kg/m2; age, 12.5 +/- 2.5 yr) by a newly developed and extensively evaluated in-house immunoassay. These results were controlled for their association with markers of puberty, obesity, and insulin sensitivity. RESULTS: The analytical evaluation of our assay revealed different resistin isoforms with major peaks of higher than 660 and 55 kDa in the size exclusion chromatography. Using this assay system we found no difference in the resistin levels of obese compared with lean subjects (P = 0.48). However, resistin was significantly higher in girls than in boys (6.74 +/- 2.42 vs. 5.79 +/- 2.45; P < 0.001). Interestingly, in both obese and lean children, resistin correlated with age (P < 0.01), Tanner stage, and testosterone and estradiol levels (P < 0.05). In contrast, no significant correlation was found with parameters of insulin resistance such as homeostasis model assessment, insulin sensitivity index, or insulin, proinsulin, and glucose concentrations in obese subjects. CONCLUSIONS: Resistin appears to be not the main link between obesity and insulin resistance in children and adolescents but because of its association with Tanner stage, it may be related to the maturation of children during pubertal development. Additionally, we have demonstrated the presence of different molecular isoforms of resistin in human blood, and this may raise problems in comparing data from diverse assay systems.

Lack of neuronal NOS has consequences for the expression of POMC and POMC-derived peptides in the mouse pituitary.

The relevance of NO in neuroendocrine signalling has been investigated by analysis of cellular expression of pro-opiomelanocortin (POMC) and the POMC-derived peptides beta-endorphin, alpha-melanocyte stimulating hormone and adrenocorticotropin. Expression patterns were studied in the pituitary gland of 150-day old wild-type and neuronal-NOS (nNOS) knock-out mice by using immunohistochemistry, in situ hybridization and Northern blot analysis. Remaining NO-generating capacities in the knock-out mice were demonstrated by immunohistochemical localization of inducible, endothelial and neuronal NOS isoforms. Quantitative analysis revealed that cellular expression of POMC mRNA was drastically reduced in the pituitary of knock-out mice in comparison to controls. In situ hybridization studies demonstrated that this reduction was most pronounced in the intermediate lobe, while the anterior lobe was much less affected. Immunostaining for the proteolytic fragments of POMC was significantly reduced in the intermediate lobe cells of knock-out mice. A moderate reduction of immunostaining for these peptides was also observed in adenopituitary cells of nNOS knock-out mice. Our data demonstrate that the lack of nNOS substantially affects cellular levels of pituitary opioid peptides, which may have consequences for the response of these animals to stress and pain.

Down-regulation of E-cadherin gene expression by collagen type I and type III in pancreatic cancer cell lines.

E-cadherin-mediated cell-cell adhesion is reduced in epithelial tumors, which is thought to be a prerequisite to acquire invasive properties. We observed that several pancreatic carcinoma cell lines with high metastatic potential expressed normal levels of E-cadherin and possessed functional E-cadherin/catenin adhesion complexes. When the cell lines PANC-1, BxPC-3, and PaTu8988s were cultured either on type I or type III collagen, E-cadherin gene expression was repressed, and E-cadherin and catenin protein concentrations were reduced. In contrast, growth on fibronectin and collagen type IV had no influence. Collagen type I- or type III-dependent reduction of E-cadherin expression led to decreased cell-cell adhesion, increased proliferation, and migratory activity as well as morphological transformation. Overexpression of activated c-Src in PANC-1 cells mimicked collagen-induced E-cadherin down-regulation and changed the elevated cell proliferation and migration. Conversely, treatment of cells with the Src-inhibitors PP1 or herbimycin A resulted in complete suppression of collagen type I-induced E-cadherin decrease. Our data demonstrate that specific collagens are able to promote metastatic behavior by down-regulation of E-cadherin gene expression in a Src-kinase-dependent manner. This points toward a novel mechanism for substrate-dependent signaling and underlines the significance of extracellular matrix environment for tumor growth and invasiveness.

Expression pattern and further characterization of human MAGED2 and identification of rodent orthologues.

In a search for genes involved in X-linked mental retardation we have analyzed the expression pattern and genomic structure of human MAGED2. This gene is a member of a new defined MAGE-D cluster in Xp11.2, a hot spot for X-linked mental retardation. Rat and mouse orthologues have been isolated. In contrast to the genes of the MAGE-A, MAGE- B and MAGE-C clusters, MAGED2 is expressed ubiquitously. High expression was detected in specific brain regions and in the interstitium of testes. Five SNPs in the coding region of human MAGED2 were characterized and their allele frequencies determined in a German and Turkish population.

TGFbeta1 represses proliferation of pancreatic carcinoma cells which correlates with Smad4-independent inhibition of ERK activation.

Transforming growth factor beta (TGFbeta) is a tumor suppressor acting as inhibitor of cell cycle progression of epithelial cells. We show that treatment of the pancreatic carcinoma cell lines PANC-1 and BxPC-3 with TGFbeta1 inhibits both growth factor-induced activation of the extracellular signal-regulated kinase 2 (ERK2) and translocation of the kinase to the nucleus. TGFbeta1 causes a concentration-dependent reduction of cell proliferation in both cell lines. By measuring ERK activation, we can show that TGFbeta1 is able to repress ERK activation induced by mitogenic stimuli such as EGF. This inhibitory effect of TGFbeta1 is not mediated by suppression of Ras or c-Raf-1 activation, but mediated by TGFbeta1-induced activation of a serine-threonine phosphatase, as demonstrated by inhibition of phosphatases by treatment with okadaic acid. Results obtained in the Smad4-deficient pancreatic carcinoma cell line BxPC-3, demonstrate that TGFbeta1-induced growth inhibition is mediated by a Smad4-independent prevention of ERK2 activation. In contrast to the effects of TGFbeta1 on epithelial cells, mesenchymal NIH3T3 fibroblasts exhibit elevated ERK2 activation and increased cell proliferation in response to TGFbeta1 treatment. Smad4-independent phosphatase-mediated inhibition of mitogen-activated ERK2 represents a novel effector pathway contributing to suppression of epithelial pancreatic carcinoma cell proliferation by TGFbeta1, in addition to the well-known Smad-induced tumor suppressor activity of TGFbeta. Oncogene (2000) 19, 4531 - 4541.

Differentially displayed genes in neuroblastoma cells treated with a mitochondrial toxin: evidence for possible involvement of ICAM-1 in 3-nitropropionic acid-mediated neurodegeneration.

The mitochondrial toxin 3-nitropropionic acid (3-NPA) causes neurodegeneration in the basal ganglia and neurological symptoms resembling Huntington's disease (HD) when applied to primates or rodents, and therefore might be used as an animal model for this disorder. For that reason, the molecular mechanisms involved in 3-NPA-induced neurodegeneration are of considerable interest. In our model, murine neuroblastoma cells (Neuro-2a) were treated with different doses of 3-NPA, and changes in gene expression were analyzed by means of mRNA differential display (DDRT-PCR). Using 18 primer combinations, we have identified a set of 33 candidate cDNAs deriving from 29 excised DDRT bands whose expression appeared to be changed in response to the 3-NPA insult (mostly elevated). DNA sequencing revealed that novel, as well as previously described genes, are included in this panel. Amongst the known cDNAs, the differential mRNA expression of the ribosomal proteins S6 and L40, of the protein kinase A (PKA) catalytic beta subunit and of the intercellular adhesion molecule ICAM-1 could be verified using Northern hybridization and RT-PCR, respectively. Furthermore, ICAM-1 expression could also be shown to increase at the protein level, which points to a possible function for this molecule in neuronal cells in the course of neurodegeneration. The results may prove useful in elucidating the multiple processes causing neurodegeneration subsequent to lesions by mitochondrial toxins and excitotoxins as well.

Capillary electrophoretic determination of ferric dimethyldithiocarbamate as iron(III) chelate of EDTA.

A simple and sensitive capillary electrophoretic method with UV detection has been developed for the determination of Ferbam (ferric dimethyldithiocarbamate) in boric acid buffer after its acidic decomposition and complexation with EDTA as Fe-EDTA- complex. The determination is dependent on the pH and the nature of the buffer solutions. In this method the detection limit (S/N = 3) is 1.8 x 10(-6) mol/L (0.7 mg/kg) of Ferbam. The relative standard deviation for the analysis of 50 microg/ml was found to be 2.9%. The method was successfully applied for the analysis of wheat grain samples spiked with Ferbam. The applicability of capillary electrophoresis as a useful tool for the analysis of Ferbam is demonstrated.

Neuroendocrine protein 7B2 is essential for proteolytic conversion and activation of proprotein convertase 2 in vivo.

The 7B2 protein is widely distributed in neural and endocrine tissues. Its biological function was found to be related to the processing enzyme proprotein convertase 2 (PC2), a mammalian subtilisin/kexin-like endoproteinase that cleaves at specific single or multiple basic amino-acid residues. In order to examine the proposed function of 7B2 on PC2 in in vivo models, we first compared the distribution of 7B2 and PC2 mRNAs in the rat brain. Expression of 7B2 mRNA was found to be pan-neuronal, but additionally, we observed 7B2 mRNA in ependymal cells and in the subcommissural organ. Although the expression of PC2 mRNA was exclusively neuronal, it was more restricted, sparing some regions expressing high levels of 7B2. This finding suggests that 7B2 has an additional function in non-PC2-expressing cells. No evidence of PC2-positive/7B2-negative cells could be obtained in the adult rat brain. However, in the developing rat brain (E17), such regions were easily observed, showing higher levels of pro-PC2 (75 kD). Similarly, in the animal model of insulin-induced hypoglycemic shock, where adrenomedullary 7B2 expression is decreased, the ratio of pro-PC2 to mature PC2 (75 kD:68 kD) was observed to be increased. Finally, the human neuroepithelioma SK-N-MCIXC expresses PC2 but not 7B2. Accordingly, only inactive pro-PC2 forms were observed: 75-kD intracellular and 71-kD extracellular. After stable transfection of SK-N-MCIXC cells with 27-kD pro-7B2, mature and active (68-kD) PC2 was secreted into the medium. Our data demonstrate a critical role of 7B2 in the proteolytic conversion and activation of PC2 in vivo.

Antioxidant enzymes in malignant prostate cell lines and in primary cultured prostatic cells.

The antioxidant enzymes catalase, glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were determined in the androgen-response LNCaP and androgen-nonresponsive PC-3 and DU 145 cells as well as in prostatic epithelial cell cultures of benign and malignant human prostatic tissue. There were no differences between the enzyme activities of the human primary cell cultures from cancerous tissue and their normal counterparts. The enzyme activities of the three permanent cell lines were either higher (SOD, catalase, GR) or lower (GST, GPx) than in the primary cell cultures. In LNCaP cells catalase and GR were significantly higher, GST, in contrast, was significantly lower than in PC-3 and DU 145 cells. GST in PC-3 and DU 145 cells, and SOD in all the three cell lines showed no significant differences. Catalase, GPx and GR values were significantly different in the three permanent cell lines. The different enzymatic equipment of the prostate cancer cell lines provides the basis for experimental testing of new concepts of cancer treatment with the help of systematic modulations of the antioxidant defence systems in prostate cancer.

[Spinal seeding metastasis of a WHO grade III oligo-astrocytoma]. / Spinale Abtropfmetastase eines Oligoastrozytoms Grad III WHO.

3 1/2 years after two operations and radiation therapy of a biparietally, parasagittaly localised grade III oligoastrocytoma, a 34-year-old patient developed symptoms of the spinal cord. By performing magnetic resonance tomography and laminectomy, multiple metastases of the anaplastic part of the primary tumour could be identified. Spinal seedings of a tumour of this grading are even rarer than those sporadically reported on corresponding complications of a multiform glioblastoma. Risk factors for the development of such a complication are youth of the patient, primary site of the tumour near the midline and anaplastic parts of the tumour in adults. If such a constellation exists, one should definitely consider the possibility of a spinal seeding in a grade III glioma, especially because in these younger patients thus would be of greater relevance for therapy than in patients with multiform glioblastoma.

Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions.

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.

[Production and characterization of monoclonal antibodies to the secretory components of human IgA]. / Herstellung und Charakterisierung von monoklonalen Antikörpern gegen die sekretorische Komponente des humanen IgA.

The monoclonal antibodies BL-HSC/1-3 are characterized by means of indirect radioimmunotechnique with regard to their binding pattern to a set of human proteins. The results suggest a binding specificity of the three monoclonals for A-determinants of the human secretory component. However, we could not point out a different binding pattern to the immunoglobulin-bound and the free molecule, respectively. By use of appropriate immunoassays, the monoclonal antibody BL-HSC/3 is the most suitable one for the detection of the secretory component and secretory IgA in body fluids. The protein band labeled by BL-HSC/3 in the immunoblotting analysis corresponds to a molar mass of 79,000 D according to the data known for the human secretory component.

S A G--sucrose medium for red blood cell preservation.

A preservation solution for buffy coat-free red cell concentrates (RCC) was tested. It contained sodium chloride (150), glucose (50), adenine (1.25 mmol/l) and additionally sucrose (30 mmol/l). After 35 days of storage about 50% of the initial ATP were found. The 24-hours post-transfusion survival rate amounted to 76%. The hemolysis rate was 0.25% and only 2% of the red cell membrane were released as microvesicles when plastic bags were used for storage. Sucrose can be replaced by mannitol or sorbitol at the same concentration. Guanosine (0.4 mmol per 1 RCC) slightly but not significantly improved the maintenance of ATP and the morphology as well as the membrane stability.

Preservation of resuspended red cell concentrate. Metabolism and survival rate of stored red cells.

The sucrose-poor, electrolyte-rich SAG-sucrose preservation solution for red cell concentrates (RCC) proves to be superior to the sucrose-rich electrolyte-poor CDS-AG solution. After 35 days about 50% of the initial ATP were found and the 24 hours posttransfusion survival rate amounted to 76%. The replacement of sucrose by mannitol or sorbitol did not influence the metabolism of red cells. Guanosine-final concentration 0.4 mmol/l RCC-slightly improved the maintenance of ATP and morphology.

[Effects of different saccharose and electrolyte concentrations on preserved erythrocytes]. / Zum Einfluss unterschiedlicher Saccharose- und Elektrolytkonzentrationen auf konservierte Erythrozyten.

Preservation solutions for buffy coat-free red cell concentrates with sucrose concentrations from 234 decreasing up to 15 mmol per 1 solution were tested. The hemolysis rate increased from 0.5 up to 1.9% by decreasing the sucrose concentration. The red cell volume was unchanged at low sucrose concentrations. No differences were noticed in ATP content and morphological changes. A considerable extracellular pH shift at high sucrose concentration exists only at the beginning of storage. A sucrose concentration of 30-50 mmol/l solution (3-5 mmol per unit red cell concentrate) at an ionic strength of 0.16 proves to be most suitable.

[Importance of secretory IgA in human body fluids. I. Development of an enzyme immunoassay for secretory IgA]. / Zur Bedeutung von sekretorischem IgA in menschlichen Körperflüssigkeiten. I. Aufbau eines Enzymimmunassays für sekretorisches IgA.

This paper describes an enzyme-immunoassay for the quantitative estimation of secretory IgA(S-IgA). As solid phase a monoclonal antibody against human secretory component was used, a polyclonal peroxidase labeled anti-IgA serum as conjugate. Thus, the method was specific for S-IgA. The test was done in microtiter plates, the upper limit was 0.5 ng S-IgA/ml. The standards of our test system ran in normal ranges, the coefficients of variation were 6.9% in the within-day-test and 12.2% in the day-to-day-test. The described test system is effective and sensitive. It is suitable for clinical use.

Purification and characterization of a nuclear protein kinase from rat liver and a hepatoma that is capable of activating poly(A) polymerase.

The cyclic nucleotide-independent protein kinase which is separated from poly(A) polymerase during its purification from nuclei of rat liver and Morris hepatoma 3924A was purified essentially to homogeneity. Liver nuclear poly(A) polymerase was dissociated from protein kinase by phosphocellulose column chromatography. In contrast, protein kinase copurified with the hepatoma poly(A) polymerase on the phosphocellulose column. Neither liver nor hepatoma kinase was stimulated by spermine or inhibited by heparin. These enzymes did not utilize GTP as phosphoryl donor, or histones or tyrosine-containing [Val5]-angiotensin II as phosphoryl acceptors. The apparent Km with respect to ATP was similar for the liver (4.7 microM) and hepatoma (11 microM) kinases, and the apparent Km with respect to casein was identical (0.6 microgram/microliter) for these enzymes. Both enzymes were capable of phosphorylating poly(A) polymerase and stimulating both tumor and liver poly(A) polymerase activity. However, in addition to their different chromatographic properties, the two kinases differed in molecular weight (liver, 37,000; hepatoma, 56,000), in their response to various divalent metal ions, and in their ability to phosphorylate hepatoma poly(A) polymerase (Km 7.9 and 30 microgram/microliter for liver and hepatoma enzymes, respectively). These latter characteristics distinguished the liver and hepatoma protein kinases from each other as well as from the previously described NI protein kinase.

[Erythrocyte concentrates, low in buffy coat: production, preservation and evaluation of its quality]. / Ubersicht Das buffy-coat-arme Erythrozytenkonzentrat Herstellung, Konservierung und Qualitätsbewertung.

Buffy coat-poor packed red cells were prepared from fresh ACD-, ACD-AG- and EDTA-blood, than resuspended with a preservation solution, containing glucose, adenine, guanosine, sucrose, citric acid and sodium citrate and stored at 4 degrees C for 6 weeks. The survival rate of resuspended red cells from ACD-AG-blood amounted to 77% after 6 weeks of storage. The ATP content of resuspended red cells was approximately 25% lower than in ACD-AG whole blood during storage caused probably by increased ATP consuming reactions at the red cell membrane. The P2G-content of resuspended red cells from ACD- and ACD-AG-blood decreased above 50% of the normal level during the first week, as fast as in ACD- and ACD-AG whole blood. The P2G-breakdown in red cells from EDTA-blood was delayed for a week due to the higher pH as in CPD blood. Additions of xylitol, inorganic phosphate, and bicarbonate in 6, 5 and 20 mM final concentrations in the red cell suspensions and an increased pH at the same time delayed the breakdown of ATP and P2G. Packed red cells can be administered fast enough at hematocrits to 0.60 that will be achieved by adding 50 to 100 ml preservation solution. Leukocytes and thrombocytes were reduceds to 70 to 80%. With increasing rate of reduction a higher loss of red cells occured. Buffy coat-poor red cell concentrate contains only few microaggregates. It diminishes the risc of febrile transfusion reactions and delays the appearance of alloimmunisation. The circulatory overload of patients is less frequent than after transfusions of red cell resuspensions containing a large resuspension volume.