Roles for NF-kappaB in nerve cell survival, plasticity, and disease.

Here we review evidence of roles for NF-kappaB in the regulation of developmental and synaptic plasticity, and cell survival in physiological and pathological settings. Signaling pathways modulating NF-kappaB activity include those engaged by neurotrophic factors, neurotransmitters, electrical activity, cytokines, and oxidative stress. Emerging findings support a pivotal role for NF-kappaB as a mediator of transcription-dependent enduring changes in the structure and function of neuronal circuits. Distinct subunits of NF-kappaB may uniquely affect cognition and behavior by regulating specific target genes. NF-kappaB activation can prevent the death of neurons by inducing the production of antiapoptotic proteins such as Bcl-2, IAPs and manganese superoxide dismutase (Mn-SOD). Recent findings indicate that NF-kappaB plays important roles in disorders such as epilepsy, stroke, Alzheimer's and Parkinson's diseases, as well as oncogenesis. Molecular pathways upstream and downstream of NF-kappaB in neurons are being elucidated and may provide novel targets for therapeutic intervention in various neurological disorders.

[Glutamic acid decarboxylase autoantibody concentration and glucose tolerance in late pregnancy]. / Glutamat-Azetat-Dekarboxylase-Autoantikörper-Konzentration und Glukosetoleranz in der Spätschwangerschaft.

Genetic and immunological factors may play a role as possible causes for gestational diabetes. Autoantibodies to glutamic acid decarboxylase (GADA) are frequently found in patients with insulin dependent diabetes, but have only rarely been analyzed with regard to the carbohydrate tolerance in pregnancy. An oral glucose tolerance test (oGTT) with 75 g glucose was performed in 110 pregnant patients during the third trimenon. Glucose (glucose dehydrogenase method) and insulin (RIA) concentrations were measured after 0, 30, 60, 120, and 180 minutes. Patients were divided into five groups of increasing glucose intolerance based on the highest glucose concentration reached during the oGTT. GADA were measured using a quantitative enzyme-immunoassay. Only a single patient showed pathologically elevated GADA, and her oGTT results were within the normal range. GADA in subjects with normal pathological glucose tolerance showed no significant difference (276.6 +/- 151.6 and 263.0 +/- 107.1 mU/ml respectively). There was a tendency of positive correlations between high GADA-levels and higher concentrations of insulin as well as an increased insulin-glucose-index. These findings suggest that pregnant patients with higher GADA-levels may have an increased insulin resistance. In conclusion, the concentration of GADA was not found to be helpful in evaluating the current metabolic situation in gestational diabetes. It remains unclear whether elevated GADA during pregnancy have a prognostic value regarding the manifestation of overt diabetes mellitus later in life.

[Changes in insulin-glucose quotients in a glucose tolerance test in the third trimester]. / Veränderungen der Insulin-Glukose-Quotienten während eines Glukosetoleranztests im III. Trimenon.

OBJECTIVE: In comparison a higher insulin-glucose-index is usually associated with an increased insulin resistance. In the present study changes in insulin-glucose-indices were examined in relation to a defined glucose tolerance in the last trimester of pregnancy. MATERIAL AND METHODS: 249 pregnant women were challenged with a 75 gm oral glucose tolerance test (oGTT). Serum samples for glucose (glucose-dehydrogenase-method) and insulin measurements (RIA) were drawn before and at 30, 60, 120, and 180 minutes after glucose load during oGTT. Patients were assigned to five groups with increasing glucose intolerance according to maximal glucose levels during the test. RESULTS: There were no significant differences in insulin-glucose-indices prior to glucose load. Pregnant women with gestational diabetes were shown to have significantly lower insulin-glucose-indices in the early and intermediate phase of the challenge test while the indices were higher in the final phase of the test. CONCLUSIONS: Women with gestational diabetes demonstrated an initial delay in insulin secretion in combination with a higher insulin-glucose-index, corresponding to an increased insulin resistance, only in the end of the test. These characteristics may possibly be a cause of the observed disorder in glucose metabolism in these patients.

Dose distribution of low-dose subcutaneous recombinant interleukin-2 affects the secondary release of interferon-gamma in vivo.

Human recombinant interleukin-2 (rIL-2) induced interferon-gamma (IFN-gamma) release in vivo was studied in 16 renal cell carcinoma patients treated with low-dose s.c. rIL-2. The s.c. administration of rIL-2 resulted in a significant increase in circulating IFN-gamma in all patients within 6 to 8 hours as measured by enzyme-linked immunosorbent assay (ELISA). Total IFN-gamma release, as expressed by the area under the concentration curve (AUC), and IFN-gamma serum peaks following repetitive s.c. rIL-2 injection showed a direct dose distribution dependancy, whereby significantly higher levels of secondary IFN-gamma were achieved in patients treated with 10 million IU rIL-2/m2 q 12 hours when compared with patients treated with 20 million IU rIL-2/m2 q 24 hours. IFN-gamma release was suppressed significantly in one patient who had been pretreated with corticosteroids, while prior immunotherapy with rIL-2 had no measurable effect on secondary IFN-gamma release in this study. Cumulative secondary IFN-gamma secretion, as expressed by the AUC, and IFN-gamma serum peak concentrations in response to s.c. rIL-2 did not correlate with response to therapy or survival of rIL-2 treated renal cell carcinoma patients.

Nitric oxide modulates synaptic vesicle docking fusion reactions.

Nitric oxide (NO) stimulates calcium-independent neurotransmitter release from synaptosomes. NO-stimulated release was found to be inhibited by Botulinum neurotoxins that inactivate the core complex of synaptic proteins involved in the docking and fusion of synaptic vesicles. In experiments using recombinant proteins, NO donors increased formation of the VAMP/SNAP-25/syntaxin 1a core complex and inhibited the binding of n-sec1 to syntaxin 1a. The combined effects of these activities is predicted to promote vesicle docking/fusion. The sulfhydryl reagent NEM inhibited the binding of n-sec1 to syntaxin 1a, while beta-ME could reverse the NO-enhanced association of VAMP/SNAP-25/syntaxin 1a. These data suggest that post-translational modification of sulfhydryl groups by a nitrogen monoxide (likely to be NO+) alters the synaptic protein interactions that regulate neurotransmitter release and synaptic plasticity.

Recurrent gain of chromosome arm 7q in low-grade astrocytic tumors studied by comparative genomic hybridization.

Consistent tumor-specific chromosomal aberrations have not been described in low-grade astrocytic tumors. The most frequent genetic alterations are mutations of the TP53 tumor suppressor gene and/or loss of heterozygosity (LOH) on 17p that occur in about 30% of the cases in adult patients but that are uncommon in childhood tumors. We used comparative genomic hybridization (CGH) to map DNA copy number alterations in 18 primary low-grade astrocytic tumors (ten adult patients and eight children). A gain of chromosome arm 7q was the most frequent event detected in five of ten astrocytomas (50%) from adult patients, followed by DNA amplification on chromosome arm 8q and gain on 12p (two cases). Loss of chromosomal regions on 1p, 4q, and the X chromosome was observed in two of ten cases [including one patient afflicted with Turner syndrome (45,X)]. In contrast, no consistent changes were observed in low-grade astrocytomas in children. A loss of the X chromosome was the sole aberration detected in two of eight cases using DNA extracted from the normal brain tissue. The findings suggest that a gain of 7q is an early event in the initiation of astrocytomas in adult patients. The discrepant findings in low-grade astrocytic tumors in adults compared to tumors in children support the the hypothesis that there might be different mechanisms responsible for tumor development.

In vivo tumor necrosis factor-alpha as indicator of biologic and clinical response to low-dose SC recombinant interleukin 2.

The effect of low-dose human recombinant interleukin-2 (rIL-2) on the induction of secondary tumor necrosis factor-alpha (TNF-alpha) in vivo was studied in 16 patients with metastatic renal cell carcinoma. In all patients s.c. rIL-2 resulted in a significant increase in TNF-alpha serum levels within 4 to 8 hours, as determined by enzyme-linked immunosorbent assay (ELISA). TNF-alpha serum concentrations remained elevated up to 24 hours following single s.c. administration of rIL-2. Total secondary TNF-alpha release, as assessed by the area under the curve (AUC), appeared to be independent of dose distribution of rIL-2 (10 million IU rIL-2 q12 hours versus 20 million IU rIL-2 q24 hours). rIL-2 induced TNF-alpha release was significantly higher in patients who had received prior rIL-2 immunotherapy, while steroids resulted in a significant suppression of TNF-alpha release. Secondary TNF-alpha release was statistically associated with progression-free survival of renal cell carcinoma patients and may be a prognostic factor in patients receiving rIL-2.

An ADP-ribosyltransferase as a potential target for nitric oxide action in hippocampal long-term potentiation.

Recent studies of long-term potentiation (LTP) in the CA1 region of the hippocampus have demonstrated that nitric oxide (NO) may be involved in some forms of LTP and have suggested that postsynaptically generated NO is a candidate to act as a retrograde messenger. However, the molecular target(s) of NO in LTP remain to be elucidated. The present study examined whether either of two potential NO targets, a soluble guanylyl cyclase or an ADP-ribosyltransferase (ADPRT; EC 2.4.2.31) plays a role in LTP. The application of membrane-permeant analogs of cGMP did not produce any long-lasting alterations in synaptic strength. In addition, application of a cGMP-dependent protein kinase inhibitor did not prevent LTP. We found that the CA1 tissue from hippocampus possesses an ADPRT activity that is dramatically stimulated by NO and attenuated by two different inhibitors of mono-ADPRT activity, phylloquinone and nicotinamide. The extracellular application of these same inhibitors prevented LTP. Postsynaptic injection of nicotinamide failed to attenuate LTP, suggesting that the critical site of ADPRT activity resides at a nonpostsynaptic locus. These results suggest that ADP-ribosylation plays a role in LTP and are consistent with the idea that an ADPRT may be a target of NO action.

Inhibition of hippocampal heme oxygenase, nitric oxide synthase, and long-term potentiation by metalloporphyrins.

Four potent metalloporphyrin inhibitors of heme oxygenase were used to assess whether carbon monoxide production was required for induction of LTP in the CA1 region of the hippocampus. Although the metalloporphyrins produced a similar and substantial inhibition of heme oxygenase activity in hippocampal slices, only two compounds reduced the amount of LTP elicited by tetanic stimulation (chromium mesoporphyrin IX and zinc protoporphyrin IX). Both chromium mesoporphyrin IX and zinc protoporphyrin IX inhibited nitric oxide synthase in the hippocampus; tin mesoporphyrin IX and zinc deuteroporphyrin IX bis glycol neither reduced LTP induction nor inhibited NOS activity, although they did inhibit heme oxygenase. None of these metalloporphyrins reversed established LTP. Thus, together these data do not support carbon monoxide as a mediator in either LTP induction or expression/maintenance and emphasize further the nonselectivity of some metalloporphyrins.

Nitric oxide stimulates Ca(2+)-independent synaptic vesicle release.

A new fluorescence method using the dye FM1-43 was used to examine exocytotic release from hippocampal synaptosomes. Nitric oxide caused a marked transient stimulation of vesicle release. Several structurally unrelated nitric oxide donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, 3-morpholino-sydnonimine, and acidified sodium nitrite, were effective. Release stimulated by nitric oxide and KCl were comparable in time course, using both the fluorescence assay and [3H]L-glutamate to monitor neurotransmitter release. Activation of guanylyl cyclase was not responsible for nitric oxide-stimulated release. Unlike release stimulated by KCl or A23187, nitric oxide-stimulated release was found to be independent of a rise in intrasynaptosomal Ca2+. Indo-1/AM measurements indicated that nitric oxide actually decreased intracellular Ca2+, and the Ca2+ channel blocker Cd2+ did not affect nitric oxide-stimulated vesicle release. Nitric oxide does, however, appear to act on the Ca(2+)-sensitive pool of vesicles. Nitric oxide may be the first physiological mediator that induces vesicle exocytosis without raising Ca2+ and may provide an interesting new tool for the study of molecules involved in vesicle exocytosis.

Comparative genomic hybridization of human malignant gliomas reveals multiple amplification sites and nonrandom chromosomal gains and losses.

Nine human malignant gliomas (2 astrocytomas grade III and 7 glioblastomas) were analyzed using comparative genomic hybridization (CGH). In addition to the amplification of the EGFR gene at 7p12 in 4 of 9 cases, six new amplification sites were mapped to 1q32, 4q12, 7q21.1, 7q21.2-3, 12p, and 22q12. Nonrandom chromosomal gains and losses were identified with overrepresentation of chromosome 7 and underrepresentation of chromosome 10 as the most frequent events (1 of 2 astrocytomas, 7 of 7 glioblastomas). Gain of a part or the whole chromosome 19 and losses of chromosome bands 9pter-23 and 22q13 were detected each in five cases. Loss of chromosome band 17p13 and gain of chromosome 20 were revealed each in three cases. The validity of the CGH data was confirmed using interphase cytogenetics with YAC clones, chromosome painting in tumor metaphase spreads, and DNA fingerprinting. A comparison of CGH data with the results of chromosome banding analyses indicates that metaphase spreads accessible in primary tumor cell cultures may not represent the clones predominant in the tumor tissue.

In vivo time and dose dependency of interleukin-6 secretion in response to low-dose subcutaneous recombinant interleukin-2.

Serum concentrations of Interleukin-6 (IL-6) were determined in renal cell carcinoma patients treated with low-dose subcutaneous human recombinant interleukin-2 (rIL-2). In all patients, administration of rIL-2 resulted in a significant increase in IL-6 serum levels to peak values within 4 to 6 hours as measured by enzyme-linked immunosorbent assays (ELISA). Repetitive administration of rIL-2 induced significantly lower IL-6 serum peaks when compared to the initial administration of rIL-2. Cumulative IL-6 release, as expressed by the area under the concentration curve (AUC), appeared to be independent of rIL-2 dose distribution (10 million IU rIL-2/m2 versus 20 million IU rIL-2/m2), and IL-6 serum peaks showed no direct dose dependency. Prior rIL-2 immunotherapy had no measurable effect on rIL-2 induced IL-6 release, while steroids resulted in a significant suppression of secondary IL-6 did not correlate with response to rIL-2 therapy or survival of rIL-2 treated renal cell carcinoma patients.

Clinical and preclinical evaluation of recombinant PEG-IL-2 in human.

High dose interleukin-2 alone or in combination with lymphokine activated killer (LAK) cells has demonstrated antitumor activity in a variety of malignant diseases. The currently formulated recombinant human interleukin-2 (IL-2) has limited solubility and short circulatory half life resulting in limited bioavailability. To improve the bioavailability of IL-2 the protein was covalently bound to activated Polyethylenglycol (PEG). We designed a phase I/II trial to evaluate the bioactivity of PEG-IL-2 in man, given as intravenous (iv) bolus injection every two weeks, and to determine safety, efficacy, and the maximum tolerated dose (MTD) in patients with advanced malignancies. Assessment of cytokine levels, phenotypic analyses and differential blood counts were performed to investigate the effects of PEG-IL-2 in-vivo. To compare in-vitro PEG-IL-2 activity to activities of IL-2 we evaluated proliferation, cytotolytic activity, morphology, and phenotype of cytokine activated lymphocytes. Among seven patients treated with PEG-IL-2, there was no objective remission, three patients exhibited stabilisation of disease. Four patients presented with further disease progression. Treatment-related toxicity was mild to moderate (mainly WHO grades I and II) in patients receiving dose levels up to 10 x 10(6) IU/m2 (maximum tolerated single dose in the outpatient setting). No toxic deaths occurred. In comparison to IL-2, the pharmacokinetic profile of PEG-IL-2 exhibited increased plasma levels and a decreased clearance (alpha and beta half-life estimates of 4 and 14 hours, respectively). The analysis of a variety of immunologic parameters demonstrated that PEG-IL-2 has significant biologic activity both in vitro, and in man.

Experience of long term treatment and different dosage regimens of isosorbide 5-mononitrate.

The results of 2 clinical studies of controlled-release isosorbide 5-mononitrate (Imdur) in patients with angina pectoris are presented. In an open study in 106 patients the antianginal efficacy of controlled-release isosorbide 5-mononitrate 60 mg once daily was demonstrated by a progressive reduction in the use of short-acting glyceryl trinitrate and in the number of anginal attacks over 6 months. The only significant side effect was headache, which generally disappeared rapidly (after an average of 5 to 6 days). 30 patients were treated in a 1-week single-blind crossover study comparing controlled-release isosorbide 60 mg once daily with a conventional formulation of the drug 20 mg 3 times daily. Both regimens produced similar antianginal and anti-ischaemic effects when the patients were tested by standard ergometry, and the only significant side effect (transient headache) was equally frequent with both regimens.

Growth of Scenedesmus obliquus in relation to the supply of iron.

Equal amounts of iron in the form of iron citrate or Fe-EDTA were added to the mineral media. The effect of the two salts upon cell-, substance-and chlorophyll-production of Scenedesmus obliquus cultivated in light-dark-regimes of 15:9 hours was studied.In bubbling cultures at 75 000 Lux the number of cells and the dry substance increased by a factor of about 16-19 with Fe-EDTA and by a factor of 10-12 with iron citrate. With both salts it was possible to get a production of daughter cells twice within one light-dark-cycle of 24 hours.The chlorophyll content of cells and substance cultivated in Fe-EDTA-medium was smaller than that of those cultivated in iron citrate-medium. The possible relations between the amount of chlorphyll and the production of substance are discussed.