The protein phosphatase inhibitor okadaic acid (OA) dosedependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 ± 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 mM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 ± 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 ± 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 mM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Shortchain ceramide (30 mM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 ± 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.

Nitric oxide is a biologically active molecule involved in the regulation of main functions of the body and is produced in different cellular types by the action of molecular isoforms of the enzyme nitric oxide synthase (NOS). The presence of the isoforms defined as neuronal, testicular, endothelial and inducible has been studied in the testis of adult pigs by means of immunohistochemical techniques. Western blot analysis and a biochemical quantitative method were also performed to confirm the morphological data and to assess nitric oxide tissue activity.The NOS isoforms were contained in interstitial (Leydig), sustentacular (Sertoli), peritubular, endothelial and macrophage cells, as well as in spermatogonia and spermatids. Positive Leydig cells were numerous, isolated or grouped in clusters and differed in staining intensity. Testicular extracts were found to contain four types of proteins whose molecular weight was very similar to those of the NOS isoforms. The biochemical measurement of the NO 2 tissue content gave the result of 155.25 ± 21.9 nmol/mg.On the basis of a detailed bibliographic review, the findings here obtained led us to hypothesize that a) the sources of nitric oxide in the pig testis are numerous, and b) the oxide could be involved in the regulation of the following testicular functions: steroidogenesis, vasodilatation, peristalsis/permeability of seminiferous tubules and development/apoptosis of germ cells.

The immunohistochemical techniques known as EnVision™+ System (EVS) and Mirror Image Complementary Antibody (MICA) were recently introduced into laboratory practice because of their high sensitivity. In this paper these techniques were compared and their sequences combined to obtain a new method possibly more sensitive than the original ones. The immunohistochemical staining employing the avidin-biotin complex (ABC), largely used as routine, was adopted as a term of comparison. Samples from the small and large intestine of pigs and sheep were fixed in Bouin and embedded in Paraplast. The primary antibodies utilized were directed against the neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) and chromogranin A (Cr A). Targets of these antibodies were nerve structures of the intestinal wall, as well as endocrine cells scattered in the mucosa of the bowel, defined neuroendocrine cells or paraneurons. The EVS method appeared as slightly superior to the MICA method regarding sensitivity of detection. The EVS/MICA (combined) method resulted four/eight times more effective than the original techniques regarding sensitivity of detection and staining intensity, both at low and high dilutions of the primary antibodies. Of these latter, immunopositive structures were still clearly identifiable, at a dilution of 1:256,000. Such efficiency could be explained by the high number of revealing molecules of peroxidase contained in the new sequence. The application of the combined method is recommended when a small quantity of tissue antigens needs to be detected immunohistochemically