ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.     

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M₃ subtype. Acute exposure to phorbol esters (1 μM phorbol 12,13-dibutyrate, PDB, or 0.1 μM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 μM) and H₇ [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 μM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [³H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Migraine and depression

Neurological Sciences - Investigations of migraine comorbidity have confirmed its association with diverse psychiatric conditions. This association appears to be stronger for major depression and...     

Loss of heterozygosity at 18q21 region in gastric cancer involves a number of cancer-related genes and correlates with stage and histology,but lacks independent prognostic value

Identification of neuroendocrine differentiation in tumours has important implications for prognosis and therapy. The aim of the present study was to evaluate monoclonal antibodies against synaptic vesicle protein 2 (SV2) as histopathological markers for neuroendocrine differentiation in tumours of the gastrointestinal tract and pancreas. Paraffin blocks from 211 gastrointestinal tumours were examined by immunocytochemistry, using a monoclonal antibody against SV2. Virtually all endocrine tumours of the gastrointestinal tract (11/11 gastric, 53/53 ileal, 16/21 appendiceal, and 22/22 rectal) and pancreas (24/24) were positively labelled. SV2 labelling was also demonstrated in gastrointestinal pacemaker cell tumours (8/8), while adenocarcinomas of the gastrointestinal tract and pancreas were negative, with the exception of occasional adenocarcinomas demonstrating weak SV2 labelling (stomach 1/22, rectum 1/29, and pancreas 0/21). Western blotting of tumour biopsies confirmed expression of SV2 in endocrine tumours of the gastrointestinal tract and pancreas. No relationship was observed between SV2 expression in tumours and hormone production or malignant potential. In conclusion, SV2 is expressed in neuroendocrine tumours of the gastrointestinal tract and pancreas, but not in non-endocrine tumours. The SV2 monoclonal antibody can therefore be used as a general marker for neuroendocrine differentiation in gastrointestinal and pancreatic tumours.     

Cluster headache: remission following ascending aorta/innominate artery bypass surgery. Case report

A 51-year-old man with cluster headache has been free from cluster attacks for over two years following ascending aorta/innominate artery bypass surgery for correction of an atherosclerotic narrowing at the origin of the innominate artery. Surgery may have been a causal factor in the remission of these attacks.

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Sommario A distanza di oltre due anni da un intervento di bypass tra aorta ascendente ed arteria anonima un uomo di 51 anni con cefalea a grappolo non ha più riferito ulteriori episodi algici. Viene avanzata l'ipotesi che l'intervento abbia svolto un ruolo causale nella remissione di tali episodi.

     

Development of NADPH-diaphorase activity in the central nervous system of the cichlid fish, Tilapia mariae

The distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was studied in the cichlid fish Tilapia mariae during ontogenesis by the histochemical reaction of NADPH-diaphorase that indicates, in aldehyde-fixed tissue, the presence of nitric oxide synthase, which is the enzyme responsible for nitric oxide production. The first appearance of NADPH-diaphorase-positive neurons has a striking bilateral symmetry and occurs 20 h after fertilization (stage 8) in the olfactory placodes and in the neural tube where two clusters of positive neurons were seen in the diencephalon and in the rhombomere r4 of the hindbrain. Two days after fertilization (stage 10), the clusters of positive neurons showed labeled axons. The two longitudinal fiber bundles that arose from the diencephalic positive neurons ran caudally in the tract of the postoptic commissure. At stage 12 (3.5 days after fertilization), new populations of NADPH-diaphorase-positive neurons appeared in the telencephalon, in some diencephalic nuclei, and in the hypothalamus. Several trigeminal motor neurons showed strong NADPH-diaphorase activity, whereas the optic tectum and cerebellum were completely free of enzymatic activity. In the hindbrain, clusters of positive neurons were seen in the octavolateral region and in the region defined by the exit of the vagus nerve. In the cervical spinal cord, some ventral putative motor neurons were labeled. At stage 14 (5.5 days after fertilization), several periventricular neurons of the optic tectum and some neurons of the cerebellar lamina were labeled. Dorsal neurons, including a few large superficial neurons were also labeled in the cervical spinal cord. NADPH-diaphorase activity was seen in the neuropil area of the telencephalon, the target of olfactory inputs, and in the sensory dorso-lateral area of the spinal cord.