NMDA-induced acetylcholine release in mouse striatum: role of NO synthase isoforms

Striatal cholinergic interneurons are stimulated by glutamatergic inputs from thalamus and cortex via NMDA receptors. The present microdialysis study was designed to characterize the role of nitric oxide (NO) in this process and to identify the NO synthase (NOS) isoform responsible for this effect. For this purpose, we studied the effects of NMDA and 3-morpholino sydnonimine (SIN-1) perfusions on the release of acetylcholine (ACh) in mouse striatum. In wild-type C57/Bl6 mice, perfusion of NMDA (100 micro m) induced a two-fold stimulation of ACh release. This effect was attenuated in mice lacking endothelial NOS but was completely absent in mice lacking neuronal NOS. Local perfusion of SIN-1 (300 micro m), an NO donor, increased ACh release by more than two-fold in all three mouse lines. We conclude that NO synthesized by neuronal NOS provides a nitrergic link in the glutamatergic stimulation of striatal cholinergic interneurons.     

Ethical and regulatory issues arising from proteomic research and technology

Over the last two decades, medical research has begun to make extensive use of products of human origin in therapeutics, oncology, and most recently, in genetic diseases. This has raised many ethical issues involving patient rights, including issues of consent. Besides informed consent, researchers should address several topics when designing studies using human tissues. Reward for the patient should be kept minimal. Sample transfer should be organized along non-profit lines, at least in Europe. Sampling procedures should be at no risk for human volunteers, and at minimal risk for patients. Biosafety aspects should be addressed, in particular when international collaborations are intended or when collaboration is existing between academia and industry. Regulations on importation and exportation of human tissues should be observed. Data acquisition and storage should be addressed in accordance with national data protection regulations, in particular when using computerized databases. If follow-up information is to be taken, the authorization for such information should be requested. The right for patient's information (or for no information) on the research results should also be addressed. The issues of validation and patenting should be also solved, usually by informing the patient that he/she will have no commercial rights on potential research results. The patient should be told if the samples are transferred to another research laboratory or private company. Samples and related data should be destroyed on request at any time point during the course of the study. If possible, traceability of the donor should be ensured.     

Changes of the hepatic proteome in murine models for toxically induced fibrogenesis and sclerosing cholangitis

We investigated the changes in the hepatic proteome in murine models for toxic-induced fibrogenesis and sclerosing cholangitis. A comprehensive comparison of protein changes observed is made and the mechanistical basis of the expression changes is discussed. Hepatic fibrosis was induced by repetitive intraperitoneal CCl4 treatment of BALB/c mice or developed spontaneously in BALB/c-ATP-binding cassette, subfamily B, member 4 (Abcb4) knock out mice. Fibrosis was verified by a morphometric score and assessment of hydroxyproline content of liver tissue, respectively. The innovative difference in-gel electrophoresis (DIGE) technique was used to analyse protein expression levels of the mouse proteome. Results were confirmed by Western blotting and real-time RT-PCR. In CCl4-induced fibrosis 20 out of 40 and in BALB/c-Abcb4(-/-) mice 8 out of 28 differentially expressed proteins were identified utilizing DIGE. Only two proteins, selenium-binding protein (Sbp2) and carbonic anhydrase 3, have been unidirectionally expressed (i.e. down-regulated) in both models. Relevant differences in the pathogenesis of toxically induced liver fibrosis and sclerosing cholangitis exist. The only novel protein with regard to liver fibrosis depicting a unidirectional expression pattern in both animal models was Sbp2. An explicit protein function could not be clarified yet.     

Copepods act as a switch between alternative trophic cascades in marine pelagic food webs

A recent meta‐analysis indicates that trophic cascades (indirect effects of predators on plants via herbivores) are weak in marine plankton in striking contrast to freshwater plankton ( Shurin et al. 2002 , Ecol. Lett., 5, 785–791). Here we show that in a marine plankton community consisting of jellyfish, calanoid copepods and algae, jellyfish predation consistently reduced copepods but produced two distinct, opposite responses of algal biomass. Calanoid copepods act as a switch between alternative trophic cascades along food chains of different length and with counteracting effects on algal biomass. Copepods reduced large algae but simultaneously promoted small algae by feeding on ciliates. The net effect of jellyfish on total algal biomass was positive when large algae were initially abundant in the phytoplankton, negative when small algae were dominant, but zero when experiments were analysed in combination. In contrast to marine systems, major pathways of energy flow in Daphnia‐dominated freshwater systems are of similar chain length. Thus, differences in the length of alternative, parallel food chains may explain the apparent discrepancy in trophic cascade strength between freshwater and marine planktonic systems.     

gamma-Vinyl GABA (4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: effects on brain GABA metabolism in mice

Abstract— γ-Vinyl GABA (4-amino-hex-5-enoic acid, RMI 71754) is a catalytic inhibitor of GABA-T in vitro. When given by a peripheral route to mice, it crosses the blood-brain barrier and induces a long-lasting, dose-dependent, irreversible inhibition of brain GABA transaminase (GABA-T). Glutamate decarboxylase (GAD) is only slightly affected even at the highest doses used. γ -Vinyl GABA has little or no effect on brain succinate semialdehyde dehydrogenase, aspartate transaminase and alanine transaminase activities. GABA-T inhibition is accompanied by a sustained dose-dependent increase of brain GABA concentration. From the rate of accumulation of GABA it was estimated that GABA turnover in brain was at least 6.5 μmol/g/h. Based on recovery of enzyme activity the half-life of GABA-T was found to be 3.4 days, that of GAD was estimated to be about 2.4 days. γ -Vinyl GABA should be valuable for manipulations of brain GABA metabolism.     

Induction of labour with prostaglandin E2 gel in cases of intrauterine fetal death.

In established intrauterine fetal death, 20 patients were treated with prostaglandin E2 gel administered extraamniotically. The results were compared with those of another group of 20 patients who had received combined treatment. In this group, one or more of the following agents had been administered :- i.v. oxytocin, 20% NaCl solution or Premarin instilled intraamniotically, introduction of a balloon catheter or Rivanol administered extraamniotically. Average induction-abortion interval for the PG group was about 12 hours while for the second group it was about 30 hours. The side effects observed were slight in both groups. The results show that administration of PG-gel can be used with advantage in fetal demise because of the relatively short induction-abortion intervals obtained, the insignificant side effects and the low dose of PG required.     

Endobrevin/VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells

Mast cells are important players in innate immunity and mediate allergic responses. Upon stimulation, they release biologically active mediators including histamine, cytokines and lysosomal hydrolases. We used permeabilized rat basophilic leukaemia cells as model to identify R-SNAREs (soluble NSF (N-ethylmaleimide-sensitive fusion protein)) mediating exocytosis of hexosaminidase from mast cells. Of a complete set of recombinant mammalian R-SNAREs, only vesicle associated membrane protein (VAMP8)/endobrevin consistently blocked hexosaminidase release, which was also insensitive to treatment with clostridial neurotoxins. Thus, VAMP8, which also mediates fusion of late endosomes and lysosomes, plays a major role in hexosaminidase release, strengthening the view that mast cell granules share properties of both secretory granules and lysosomes.     

Effect of norethisterone acetate on estrogen metabolism in postmenopausal women.

Dominance of estradiol metabolism at the D-ring over the A-ring metabolism may play a role in the pathophysiology of human breast carcinogenesis. Currently, the influence of progestins on breast cancer risk is debated when added to postmenopausal estradiol replacement therapy. However, nothing is known about the action of progestins on estradiol metabolism. Therefore, the effect of oral and transdermal estradiol/norethisterone acetate (NETA) was investigated on the ratio of the main D-ring metabolite 16alpha-hydroxyestrone (16-OHE1) to the main Aring metabolite 2-hydroxyestrone (2-OHE1). The ratio of 16-OHE1 to 2-OHE1 after transdermal hormone replacement therapy (HRT) was 0.43 before treatment, 0.35 after estradiol and 0.52 after estradiol + NETA. The ratio after oral HRTwas 0.94 before treatment, 0.86 after estradiol and 2.30 after estradiol + NETA. Because of the high variations, no statistical significance could be calculated. Since there was a tendency to an increase after oral estradiol + NETA treatment, the individual patient profiles were examined. Here, three patients in the oral treatment group showed a significant increase of the ratio after the estradiol/NETA phase. In conclusion, transdermal NETA in HRT did not elicit any change in estrogen metabolism after 2 weeks' treatment. However, oral NETA may in some cases have an impact on estradiol metabolism which should be further evaluated.     

The effect of endogenous estradiol metabolites on the proliferation of human breast cancer cells

Evidence is accumulating that estradiol metabolites may be involved in carcinogenesis as some metabolites exert proliferative and others anti-proliferative properties on human cancer cells. The present study is the first to investigate the effect of 14 endogenous estradiol metabolites on the proliferation of the human breast cancer cell line, MCF-7, in comparison with the effect of the parent substance 17beta-estradiol with special concern on high pharmacological concentrations. The steroids were tested in the range from 10(-8) to 10(-5) M on MCF-7 cells which were incubated for nine days. Estradiol and almost all A-ring metabolites displayed biphasic reactions on cell proliferation, i.e. stimulatory at low concentrations and inhibitory at the highest concentration, 10(-5) M. The D-ring metabolites did not show such clear biphasic patterns, in most of them the stimulatory effect prevailed at the highest dosage used. The strongest inhibitory effect was seen for the A-ring metabolite 2-methoxyestradiol at the concentrations of 10(-6) and 10(-5) M and the strongest stimulatory effect was noted for the D-ring metabolite estriol at the same concentrations.The results indicate that some A-ring metabolites might be suitable for breast cancer treatment when used in high dosages. This is of special interest, since many of these metabolites have very weak estrogenic activity.