Involvement of voltage-dependent calcium channels (VDCC) in the action of GnRH on GtH release in common carp (Cyprinus carpio L): comparison with K+ action.

The involvement of different types of voltage-dependent calcium channels (VDCC) in the stimulatory action of GnRH (in comparison with K+) on maturational gonadotropin (GtH) release was investigated using superfused carp pituitary cells. The action of these 2 stimulants was not modified either by D600 or nifedipine (drugs blocking L-type of VDCC). Cadmium (Cd2+), which blocks all types of VDCC indifferently, provoked a dose-dependent stimulation of GtH secretion. Cd2+ action was not altered by addition of sGnRH in any of the doses. Similar results were obtained using K+ as a secretagogue, but only the highest dose of Cd2+ (200 mumol/l) was able to completely block K+ action. Low doses (0.1 and 1 mumol/l) of the L-type VDCC activator BAY-K8644 did not change basal GtH secretion and had no effect on sGnRH-stimulated GtH secretion. Surprisingly, doses (10 mumol/l and higher) of BAY-K8644 evoked dose-dependent inhibition of GtH secretion. On the other hand, a higher concentration (20 mumol/l) of nifedipine provoked a stimulation of GtH release. Our results indicate that the stimulatory action of GnRH and K+ involves activation of a certain type of cadmium-sensitive VDCC (probably T- or N-type VDCC) whereas dihydropyridine and diphenylalkylamine sensitive VDCC (L-type VDCC) does not participate in this phenomenon. The inhibitory action of BAY-K8644 and, on the other hand, the stimulatory action of nifedipine indicate that L-type VDCC probably play a role in other physiological pathways regulating GtH release in carp.     

Effects of N-nitrosopiperidine substitutions on mutagenicity in Drosophila melanogaster.

N-Nitrosopiperidine (NP) and various derivatives were fed to Drosophila melanogaster males over a wide concentration range in order to assess their mutagenic potency in the induction of X-linked recessive lethals and chromosome loss. NP was effective in inducing lethals, as were its halogen and methyl-substituted derivatives, with the exception of 2,6-dimethyl NP. (Methyl substitutions at the alpha carbon atoms reduce or eliminate mutagenic activity.) Substitution of halogen groups on the piperidine ring enhanced the mutagenic activity, with the 3-chloro compound being the most mutagenic. In contrast, substitutions with a hydroxyl, carboxyl, or keto group resulted in a loss of mutagenicity. None of the compounds tested increased the frequency of chromosome loss or breakage in mature sperm.     

Mutagenicity of N-nitrosopyrrolidine derivatives in Salmonella (Ames) and Escherichia coli K-12 (343/113) assays

The mutagenicity of nitrosopyrrolidine (NPYR) and its derivatives was determined by use of the Ames Salmonella assay. A clear specificity to revert the missense stain of TA1535 and a requirement for the phenobarbital-induced rat-liver activation system (S9 mix) were noted. 3,4-Dichloronitrosopyrrolidine was more mutagenic than NPYR, whereas 3-hydroxynitrosopyrrolidine was weakly mutagenic. The carcinogenic nitroso-3-pyrrolidine was not mutagenic under the test conditions. The noncarcinogenic derivatives (2,5-dimethylnitrosopyrrolidine, nitrosoproline and 4-hydroxynitrosoproline) were not mutagenic. Liquid preincubation assays were not any more effective than the pour-plate assays. Selected derivatives of NPYR were tested in the Escherichia coli K-12 (343/113) assay A specificity to revert the missense mutation at the arg locus and a dependence on phenobarbital-induced rat-liver S9 mix were noted with NPYR and its derivatives. 3,4-Dibromonitrosopyrrolidine, which was not mutagenic in Salmonella, was effective in E. coli, and the weakly carcinogenic NPRL was a weak mutagen resulting in a 2-fold enhancement in the E. coli arginine reversion assay.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

The influence of gabaergic drugs upon LH secretion from the pituitary of the common carp: an in vitro study

In the present study, the perifusions of whole pituitary glands of spermiating male common carp were performed in the presence of several GABAergic drugs. Muscimol (agonist of GABA(A) receptors) and bicuculline (the antagonist of the same type of GABA receptors) did not modify basal LH release. LH basal secretion was not modified when pituitaries were perifused with baclofen--an agonist of GABAB receptors. On the other hand, baclofen at doses of 10(-8) and 10(-4) M significantly decreased GnRH-A-induced LH release to about 86% and 88% of LH levels in control group, respectively. In our previous study we have shown that GABA decreased basal and GnRH-A-stimulated in vivo and in vitro LH release. In conclusion, it can be suggested that in the mature male carp GABA exerts an inhibitory influence on GnRH-stimulated LH release, probably through the inhibition of the GnRH action on gonadotropes. This inhibition seems to be mediated by the B type of GABA receptors.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Assessment of tissue damaging effects of mixed micellar absorption enhancers on the intestinal mucosa of common carp (Cyprinus carpio), African catfish (Clarias gariepinus) and rainbow trout (Oncorhynchus mykiss) as a consequence of enhanced intestinal absorption of sGnRH-a

Non-ionic surfactant–polyoxyethylene sorbitan monooleate (Tween-80) and oleic acid are food and pharmaceutical ingredients for oral and parenteral delivery and generally regarded as safe (GRAS). They have the potential as an intestinal absorption enhancer for the development of oral drug delivery systems. However, their safety in terms of mucosal integrity has yet to be evaluated. Therefore, the purpose of the present work was to study the tissue damaging effects of Tween-80, oleic acid and of their mixed micellar formulation (oleic acid + Tween-80). This was investigated at 2 h and 24 h after rectal delivery and compared with the topical effect of polyoxyethylene 9 lauryl ether (Polydocanol). The same experiment was carried out on three species with distinct feeding habits: the common carp, Cyprinus carpio (L.); the African catfish, Clarias gariepinus (Burchell); and the rainbow trout, Oncorhynchus mykiss (Walb.). Based on the findings of this study it was concluded that Tween-80 (4%), or its mixed micelle with oleic acid (0.6%) can be considered as a safe formulation, inducing only a moderate alteration of the intestinal mucosa, comparable to the effect of an isotonic saline. By contrast, in the three species, the same dose of Polydocanol, or of its mixed micelle with oleic acid, induced severe tissue damage of the intestinal mucosa, still present after 24 h. Mixed micelles of oleic acid with Tween-80 were also demonstrated as increasing the intestinal absorption of the salmon gonadotropin releasing hormone analogue (sGnRH-a) in catfish, C. gariepinus , and consequently stimulating GtH II secretion. This effect was compared with the action of other drugs considered as intestinal absorption enhancers.