Efficiency of two different transfection reagents for use with human NTERA2 cells.

The teratocarcinoma cell line NTERA2 is recently used in a wide range of researches (from developmental biology to toxicology, for their ability to be induced to neural differentiation. In order to study the genetic potential of these cells, it is needed to use methods for gene silencing and/or mRNA interference, allowing cell viability and further differentiation. To check these features, we simultaneously tested the transfection efficiency of NTERA2, A549 and HeLa cells with Metafectene PRO (Biontex, Germany) and another optimal transfection reagent currently used in our Laboratory, using as a reporter gene the DsRed2 vector (Clontech, Mountain View, CA). Under our culture conditions for NTERA2 and HeLa cells, Metafectene PRO transfection method was found to possess high throughput performance, that allows low concentration rate and low exposure time to excitation light source, thus reducing both toxicity and phototoxicity.

Expression of the tissue-specific transcription factor Pit-1 in the lancelet, Branchiostoma lanceolatum.

Lancelets, known also as amphioxus, are protochordates that share common archetypal features with vertebrates. Recently, several developmental and molecular biology studies have pointed out homologies between anatomical structures of lancelets and vertebrates. We have studied the head region of the lancelet, Branchiostoma lanceolatum, by means of scanning electron microscopy, immunocytochemistry, and Western blotting techniques, to localize the pituitary-specific transcription factor, Pit-1. Immunoreactive Pit-1 protein has been found in cells of two typical structures of the lancelets, the Kölliker's and Hatschek's pits. Moreover, the frontal eye complex, neurons, and the rostral nerves show Pit-1 immunoreactivity. A band of 33 kilodaltons has been resolved in lancelet extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and after Western blotting, the bands have been probed by a monoclonal antibody to rat Pit-1. Our results demonstrate that Pit-1 is expressed in both neurones and receptosecretory epithelial cells of adult lancelets, and that the cells lining the two pits display ultrastructural and immunocytochemical features typical of chemoreceptosecretory/olfactory- and adenohypophyseal-like structures.

Evidence for the presence of the tissue-specific transcription factor Pit-1 in lancelet larvae.

Recent molecular studies have noted the affinity among cephalochordates and vertebrates. In particular, a cluster of vertebrate-like homeobox genes regulates the development of the lancelet Branchiostoma lanceolatum. A previous study has outlined the expression pattern of the pituitary-specific transcription factor Pit-1 in adult lancelets. Pit-1 belongs to the POU family of transcription factors, which, like homeotic proteins, are members of the helix-turn-helix superfamily of proteins. POU is an acronym for Pit-1, Oct-1 and Oct-2, and Unc-86. In the present work, we investigated the head region of premetamorphic larvae of B. lanceolatum, by means of scanning electron microscopy, wholemount and tissue sections immunocytochemistry, and Western blotting assay, to verify the presence and distribution of Pit-1. Immunoreactive Pit-1 protein was detected in the rostral nerves and in a cluster of photoreceptor cells of the frontal eye. At the same time, an electrophoretic band of 33 kDa was shown from extracts of premetamorphic larvae and recognized by a monoclonal antibody to rat Pit-1. On the basis of the immunocytochemical and electrophoretic results, we can assume that Pit-1 may play a neuromodulatory role in the larval central nervous system. Moreover, the spatial and temporal distribution of Pit-1 protein in larva and adult lancelets agrees only in part with that described in embryonic and adult mice, suggesting different molecular controls of regional identity in the nervous system of cephalochordates and vertebrates.

Opioids and testicular activity in the frog, Rana esculenta.

The presence and activity of brain, pituitary and testicular beta-endorphin (beta-EP)-like material have been studied in the frog, Rana esculenta, using reverse-phase high-pressure liquid chromatography, coupled with radioimmunoassay and immunocytochemistry. In-vivo and in-vitro treatments with naltrexone were carried out to assess the putative physiological activity of opioid peptides. beta-EP(1-31) and (1-27), together with their acetylated forms, have been identified in brain, pituitary and testis. In particular, beta-EP(1-31) concentrations peaked during July in the brain and pituitary, whilst in testes maximum concentrations were found in April and November. beta-EP immunoreactivity was present in the brain within the nucleus preopticus and nucleus infundibularis ventralis while positive fibres in the retrochiasmatic regions projected to the median eminence. In the testis, interstitial cells, canaliculi of the efferent system, spermatogonia and spermatocytes showed positive immunostaining for beta-EP. In intact animals, naltrexone treatment increased plasma and testicular androgen levels and this effect was confirmed in in-vitro incubations of minced testes. Naltrexone also induced a significant increase in germ cell degeneration. Our results indicated that an opioid system modulates the hypothalamus-pituitary-gonadal axis in the frog, Rana esculenta and, for the first time, we have shown that the testicular activity of a non-mammalian species may be regulated by opiates locally.

Opioid peptides and testicular activity in the lizard Podarcis s. sicula Raf.

In mammals endorphinergic systems have been shown to modulate reproductive processes and beta-endorphin (beta-EP) has been found to influence sexual functions, acting at the hypothalamus-pituitary-gonadal axis level. Using immunocytochemical and in vitro studies, evidence for a diffuse pro-opiomelanocortin-related opioid system in the lizard Podarcis s. sicula was produced. In the testis, beta-EP immunoreactivity showed seasonal variation, being most pronounced in the interstitial cells of sexually quiescent lizards (December). Reverse-phase high-performance liquid chromatography, coupled with radioimmunoassay and immunocytochemistry, showed that beta-EP and acetyl beta-EP increased during December, while their concentrations were low during April, when the highest testicular activity occurred. Using in vivo studies, it was found that naltrexone treatment, blocking pituitary opioid receptor, increased androgen levels in the plasma and in the testis. It was also found with in vitro studies that the endogenous opioid system inhibits gonadotrophin release and therefore androgen production by the testis. The data reported here provide evidence for the physiological role played by opioid peptides at the pituitary level to regulate the seasonal reproductive activity of the lizard Podarcis s. sicula.

Immunocytochemical Localization of Glucagon-Related Peptides in the Cerebral Ganglion of the Protochordate Ascidian, Styela plicata.

Abstract The central nervous system of the protochordate ascidian Styela plicata has been investigated by means of immunocytochemical techniques in order to localize peptides of the glucagon/secretin family. In particular, glucagon- and peptide histidine isoleucine-containing neurons were localized in the cortex of the cerebral ganglion, and numerous nerve cell bodies containing vasoactive intestinal polypeptide occurred mainly in the medullary zone. Moreover, coexistence of glucagon/peptide histidine isoleucine and glucagon/vasoactive intestinal polypeptide was detected in a few cortical neurons. The localization of peptide histidine isoleucine-like peptides in the central nervous system and alimentary tract supports the occurrence of a 'brain-gut axis' in protochordates as well as in vertebrates and some invertebrates, while glucagon- and vasoactive intestinal polypeptide-like substances can be considered exclusively as neuropeptides.

CCK-like peptides in the neural complex of a protochordate.

The neural complex of the ascidian Styela plicata has been investigated by means of cytochemical and immunocytochemical methods. In the cerebral ganglion, using a mammalian antibody to synthetic CCK-8, immunoreactive neurons and nerve fibers have been localized; at the same time immunofluorescent cells are scattered in some glandular lobules of the neural gland. The possible functions of a CCK-8-like peptide in ascidians is suggested and discussed.

Immunocytochemical localization and biochemical characterization of melanotropin-like peptides in the gonads of a protochordate.

The compound gonads of the protochordate ascidian Styela plicata were investigated by immunocytochemistry, HPLC, and radioimmunoassay to verify the presence of melanotropin-like peptides, alpha-MSH-like immunoreactivity is localized in the follicular cells and in the perinuclear cytoplasm of different types of ovaric follicles, as well as in the spermatogonia and spermatocytes of testicular lobules. The ascidian immunoreactive peptides occurring in the gonads consist of alpha-MSH and ACTH(1-13)-NH2 and their amounts are higher in summer than in winter.

Ovarian melanotropic peptides and adaptation in two teleostean species: Sparus aurata L. and Dicentrarchus labrax L.

The ovarian tissue of Dicentrarchus labrax and Sparus aurata displays two immunoreactive peaks that correspond to the elution time of human des-acetyl alpha-MSH [ACTH(1-13)-amide] and human alpha-MSH. In view of the close identity between the primary structure of fish and human alpha-MSH, these data demonstrate that two MSH-related peptides are present both in sea bream and sea bass ovary. alpha-MSH-like immunoreactivity was found within both granulosa and thecal layers of mature follicles, as well as in the cytoplasm of oogonia of sea bream and sea bass ovary. Gonadal content of ACTH(1-13)-amide and alpha-MSH display differences with regard to season, showing the highest peptide levels in reproductive animals. Moreover, the alpha-MSH content is significantly higher in the ovary of fish farm animals, whereas that of ACTH(1-13)-amide prevails in wild fish ovary.

Pro-opiomelanocortin (POMC) expression and immunolocalization of POMC-related peptides in the ovary of Protopterus annectens, an African lungfish.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha MSH) and beta-endorphin were used to localize pro-opiomelanocortin (POMC)-derived peptides in the ovary of the African lungfish Protopterus annectens by immunohistochemistry. Immunoreactivity was observed in the granulosa and the internal theca of the vitellogenic follicles. No immunoreactivity was observed in immature follicles. Using human POMC cDNA as the hybridization probe POMC-like mRNA was identified in situ in cells of the granulosa and internal theca of the vitellogenic follicles. No labeling was observed in primordial follicles. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of the opiate hormones.

Acetyl salmon endorphin-like immunoreactivity in the ovary of two teleostean species: changes with environmental conditions.

The presence of salmon acetylated endorphin (acetyl sEP) in the ovary of seabream and sea bass was investigated through immunocytochemical and biochemical techniques in order to compare aquatic species with terrestrial ones. Endorphin-like immunoreactivity was found in the cytoplasm of oogonia and similar immunostaining was present in the granulosa layer of mature follicles. In both pituitary and ovarian extracts of the two teleostean species, acetyl sEP-like immunoreactivity was distributed over three main peaks, the second one corresponding to the elution time of the reference synthetic peptide. Serial dilutions of HPLC fraction II of the ovaries of both fishes ran parallel with the standard curve obtained with reference peptide. The ovarian content of acetyl sEP, obtained by calculating the integrated area of the fraction II peak, indicates large and highly significant (p < 0.01) differences in the amount of peptide found in ovarian tissues of wild seabream in comparison with that of farmed fish. Increased peptide values in wild animals with respect to farmed fish were also found in the sea bass. These data indicate that not only the pituitary, but also the ovary is sensitive to environmental cues, and strongly suggest the role of opioid peptides in adaptation.

Proopiomelanocortin (POMC) mRNA and POMC-derived peptides immunolocalization in the skin of Protopterus annectens, an African lungfish.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin were used to localize, by immunohistochemistry, proopiomelanocortin (POMC)-derived peptides in the skin excised from different regions of the African lungfish Protopterus annectens. Immunoreactivity was observed in the epidermis mainly in the germinal layer. Using human POMC cDNA as hybridization probe, POMC-like mRNA was identified in situ in epidermal cells. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of opiate hormones.

Expression of pro-opiomelanocortin (POMC) in the cerebral ganglion and ovary of a protochordate.

The distribution of neurones expressing POMC mRNA in the cerebral ganglion of the protochordate ascidian, Styela plicata, was investigated using a non-radioactive in situ hybridization technique. Nerve cell bodies of mono and bipolar types expressing POMC mRNA, were observed mainly in the outer layer of the ganglion. Discrete groups of neurones containing POMC mRNA were also localized in the inner portion of the ganglion, and few small monopolar perykaria expressing POMC mRNA were visible at the emergence of the main nerve trunks. POMC mRNA labeling was also found at level of the cytoplasm of previtellogenic and vitellogenic oocytes, and of follicular cells. Our results demonstrate the expression of one or more genes in the cerebral ganglion and ovary, that may be similar to one or more regions of the mammalian POMC gene. Therefore POMC-related molecules seem to be involved in neuromodulatory pathways and regulatory mechanisms of the oogenesis of ascidians.

5-HT2 presynaptic receptors mediate inhibition of glutamate release from cerebellar mossy fibre terminals.

'Giant' synaptosomes originating from mossy fibre terminals and having sedimentation properties different from those of standard synaptosomes were obtained from rat cerebellum. Exposure of superfused giant synaptosomes to 15 mM KCl caused the release of endogenous glutamate in a largely (about 80%) calcium-dependent manner. The K(+)-evoked overflow of glutamate was inhibited in a concentration-dependent manner by 5-hydroxytryptamine (5-HT) and by the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), but not by the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The effects of 5-HT and DOI were quite potent, already reaching significant inhibition (about 25%) at 10 nM. The 5-HT2 receptor antagonist ketanserin counteracted the inhibitory effect of 5-HT. In cerebellar slices, ketanserin increased on its own the calcium-dependent K(+)-evoked release of glutamate and this effect was not prevented by tetrodotoxin (TTX). The results support the idea that cerebellar mossy fibres use glutamate as a transmitter and show that the release of glutamate can be inhibited via presynaptic heteroreceptors of the 5-HT2 type probably localized on the mossy fibre terminals.

Occurrence of melanotropin- and corticotropin-like material in the cerebral ganglion of a protochordate.

The cerebral ganglion of the ascidian Styela plicata has been investigated using cytochemical and immunocytochemical methods. Peptidergic neurons are present mainly in the cortical zone and they contain alpha-MSH or ACTH-like immunoreactive substances. Coexistence of the two pituitary hormones is also demonstrated in some neurons by means of the double immunofluorescence method. The possible physiological role of these peptides in the ascidian central nervous system is discussed as well as the conservative characteristics of alpha-MSH- and ACTH-like molecules.

Immunoreactive Phe-Met-Arg-Phe-NH2-like peptides in the brain of the antarctic icefish, Chionodraco hamatus.

The distribution of Phe-Met-Arg-Phe-NH2 (FMRFamide)-like immunoreactive peptides was investigated in the brain of the antarctic icefish, Chionodraco hamatus, using the indirect immunofluorescence technique. Three main groups of immunoreactive perikarya were respectively localized in the nucleus entopeduncularis of the telencephalon, the nucleus preopticus periventricularis of the hypothalamus, and within the nucleus oculomotorius of the mesencephalon. Delicate FMRFamide positive nerve fibers were distributed in several brain regions of the forebrain and brainstem. In particular, these fibers densely innervated the caudal part of the dorsomedial pallium, the hypothalamus, the thalamus, the mesencephalic tegmentum and the optic tectum. The distribution pattern of the FMRFamide-like immunoreactivity was compared with that reported in previous studies in other teleost species. The anatomical organization of the FMRFamide-like immunoreactive peptidergic system in the brain of Chionodraco hamatus suggests that a FMRFamide-like peptide may play a role as a neuromodulator in fish adapted to the extreme Antarctic environment.

The tissue-specific transcription factor Pit-1 is expressed in the spinal cord of the lancelet, Branchiostoma lanceolatum.

The spinal cord of the lancelet Branchiostoma lanceolatum was studied by using a monoclonal antibody to the rat tissue-specific transcription factor, Pit-1. Our previous studies have demonstrated Pit-1 immunoreactivity in different nervous and endocrine structures of the head region of adults and in the rostral central nervous system (CNS) of larval lancelet. Our present results show the presence of Pit-1-like protein in dorso-lateral nerve cells and ependymocytes of the adult spinal cord. Using double immunofluorescence techniques, we have revealed the coexistence of the glial fibrillary acidic protein (GFAP) with Pit-1 in groups of laterally located ependymocytes. The occurrence of GFAP, a specific marker of mammalian astrocytes and radial glia, in some lancelet ependymocytes confirms that glial elements are also present in protochordates. Furthermore, other ependymocytes, located in the roof of the central canal and containing Pit-1-like protein exclusively, could be considered as ependymal tanycytes.

Localization of interleukin-1 beta mRNA in the cerebral ganglion of the protochordate, Styela plicata.

There is evidence that interleukin 1 beta (IL-1 beta) plays an important role in several biological functions in mammals where it is synthesized by cells of haematological, dermal and neural origin. Moreover, production of cytokine-like molecules has been demonstrated in some blood cells of non-mammalian vertebrates and invertebrates in which also nerve cells are demonstrated to be IL-1 beta immunoreactive. The purpose of the present study is to demonstrate the IL-1 beta mRNA expression in nerve cells of the ascidian Styela plicata by use of non-isotopic in situ hybridization technique. The expression of IL-1 beta messenger was demonstrated in monopolar neurons in the cortical layer of the cerebral ganglion. The neuronal expression of cytokine-like molecules in tunicates suggests that IL-1 beta is an ancestral and functionally conserved molecule, and that a neuroimmune axis appeared early during the metazoan phylogeny.

Ovarian opioids and the reproductive cycle of the frog Rana esculenta.

In mammals, proopiomelanocortin-related peptides are involved in reproductive processes both at the hypothalamo-pituitary and ovarian levels. Using immunocytochemical, biochemical and physiological "in vitro" studies, we provide here evidence for a diffuse POMC-related opioid system in the frog Rana esculenta. Ovarian beta-endorphin (beta-EP) is expressed in thecal cells and changes during the reproductive cycle in an inverse relationship with follicular development. Seasonal changes in the ovary are different to those in the brain or in the pituitary. The ratio of acetylated vs native beta-EP in the ovary also changes over the reproductive period, affecting the biological activity of the peptide. During both the reproductive spring period and the summer post-reproductive phase pMol amounts of beta-EP stimulate follicular androgen secretion in vitro, in a naloxone-reversible way. In either period, an inhibition of estradiol, possibly mediated via other factors, is the result of opioid action. In conclusion, these data demonstrate for the first time the widespread presence of beta-EP-related peptides in the frog Rana esculenta. Both immunocytochemical and biochemical evidence, as well as in vitro responses, support a physiological role for beta-EP in ovarian seasonality during the reproductive cycle of this amphibian.

Presence and distribution of immunoreactive FMRFamide- and bovine pancreatic polypeptide-like peptides in a protochordate ascidian.

FMRFamide and bovine pancreatic polypeptide (BPP) immunoreactivities were detected in the cerebral ganglion and in the alimentary tract of the protochordate ascidian Styela plicata. Neurons expressing immunostain to FMRFamide were mainly found in the cortical zone of the ganglion and a network of immunoreactive beaded nerve fibers was present in the neuropil. Co-localization of FMRFamide- and BPP-like materials was demonstrated in few nerve cell bodies and both peptides likely play a neurotransmitter role. Parallel, endocrine-like cells containing FMRFamide-like materials were distributed among the esophageal and gastric epithelia, whereas no PP immunostaining was found. The tetrapeptide could be involved in the regulation of the digestive processes by means of endocrine and/or paracrine mechanisms.