Carlo Di Cristo

Involvement of D-aspartic acid in the synthesis of testosterone in rat testes.

D-Aspartic acid (D-Asp) is an endogenous amino acid which occurs in many marine and terrestrial animals. In fetal and young rats, this amino acid occurs prevalently in nervous tissue, whereas at sexual maturity it occurs in endocrine glands and above all in pituitary and testes. Here, we have studied if a relationship exists between the presence of D-Asp and the hormonal activity. The following results were obtained: 1) Both D-Asp and testosterone are synthesized in rat testes in two periods of the animals life: before birth, about the 17th day after fertilization and, after birth, at sexual maturity. 2) Immunocytochemical studies have demonstrated that this enantiomer is localized in Leydig and Sertoli cells. 3) In vivo experiments, consisting of i.p. injection of D-Asp to adult male rats, demonstrated that this amino acid accumulates in pituitary and testis (after 5 h, the accumulation was of 12 and 4-fold over basal values, respectively); simultaneously, luteinizing hormone, testosterone and progesterone significantly increased in the blood (1.6-fold, p < 0.05; 3.0-fold, p < 0.01 and 2.9-fold, p < 0.01, respectively). 4) Finally, in vitro experiments, consisting of the incubation of D-Asp with isolated testes also demonstrated that this amino acid induces the synthesis of testosterone. These results suggest that free D-Asp is involved in the steroidogenesis.

Cephalopod Genomics: A Plan of Strategies and Organization

The Cephalopod Sequencing Consortium (CephSeq Consortium) was established at a NESCent Catalysis Group Meeting, “Paths to Cephalopod Genomics-Strategies, Choices, Organization,” held in Durham, North Carolina, USA on May 24–27, 2012. Twenty-eight participants representing nine countries (Austria, Australia, China, Denmark, France, Italy, Japan, Spain and the USA) met to address the pressing need for genome sequencing of cephalopod mollusks. This group, drawn from cephalopod biologists, neuroscientists, developmental and evolutionary biologists, materials scientists, bioinformaticians and researchers active in sequencing, assembling and annotating genomes, agreed on a set of cephalopod species of particular importance for initial sequencing and developed strategies and an organization (CephSeq Consortium) to promote this sequencing. The conclusions and recommendations of this meeting are described in this white paper.

NMDA receptor stimulation induces temporary α-tubulin degradation signaled by nitric oxide-mediated tyrosine nitration in the nervous system of Sepia officinalis ☆

Biochemical and immunohistochemical evidence is reported, showing basal protein nitration in specific regions of the optic lobes of Sepia officinalis, mainly in the fiber layers of the plexiform zone. SDS-PAGE analysis of optic lobe extracts revealed an intense 3-nitrotyrosine immunoreactive band identified as alpha-tubulin by immunoprecipitation and partial purification. Stimulation of NMDA receptors resulted in a selective decrease in alpha-tubulin levels within 30 min with partial recovery after 4 h. The effect was suppressed by the NO synthase (NOS) inhibitor L-nitroarginine. Incubation of optic lobes with free 3-nitrotyrosine resulted likewise in a selective loss of alpha-tubulin, due apparently to incorporation of the amino acid into the C-terminus of detyrosinated alpha-tubulin to give the nitrated protein purportedly more susceptible to degradation. Overall, these results point to a novel potential physiologic role of NO and free 3-nitrotyrosine in the control of the alpha-tubulin tyrosination/detyrosination cycle and turnover in Sepia nervous tissue.

N-methyl-D-aspartate receptor-like immunoreactivity in the brain of Sepia and Octopus

Ionotropic glutamate receptors have been subdivided into N‐methyl‐D‐aspartate (NMDA) and AMPA/kainate classes. NMDA receptor subunit 2A and 2B immunoreactivity is shown to be present in specific regions of the central nervous system (CNS) of the cephalopod molluscs Sepia officinalis and Octopus vulgaris. An antibody that recognizes both mammalian NMDAR2A and NMDAR2B subunits equally was used. SDS‐PAGE/Western blot analysis performed on membrane proteins revealed an immunoreactive band at 170 kDa for both species. Immunoreactive bands from both Octopus and Sepia brains disappeared when the antibody was preabsorbed with membrane proteins from rat hippocampus or from their own brains. The same antibody was then used for immunohistochemical staining of serial sections of the CNS to reveal localized specific staining of cell bodies and fibers in several lobes of the brain. Staining was found in lower motor centers, in some higher motor centers, in learning centers, and in the optic lobes. Immunopositivity was also found in the areas of brain that control the activity of the optic gland, a gonadotropic endocrine gland. These findings suggest that glutamate, via NMDA receptors, may be involved as a signaling molecule in motor, learning, visual, and olfactory systems in the cephalopod brain. J. Comp. Neurol. 477:202–219, 2004.

Tubulin nitration in human gliomas

Immunohistochemical and biochemical investigations showed that significant protein nitration occurs in human gliomas, especially in grade IV glioblastomas at the level of astrocytes and oligodendrocytes and neurones. Enhanced alpha-tubulin immunoreactivity was co-present in the same elements in the glioblastomas. Proteomic methodologies were employed to identify a nitrated protein band at 55 kDa as alpha-tubulin. Peptide mass fingerprinting procedures demonstrated that tubulin is nitrated at Tyr224 in grade IV tumour samples but is unmodified in grade I samples and in non-cancerous brain tissue. These results provide the first characterisation of endogenously nitrated tubulin from human tumour samples.

The presence of APGWamide in Octopus vulgaris: a possible role in the reproductive behavior

The concerted action of many neuropeptides has been implicated in the nervous control of specific behaviors in many molluscs. In the present study, the presence of amidated tetrapeptide Ala-Pro-Gly-Trp-NH2 (APGWamide) in those lobes that are involved in the control of reproductive behavior in Octopus vulgaris has been investigated. APGWamide immunoreactivity was mainly confined to the posterior olfactory lobule and in the inferior frontal system. These areas are involved in Octopus in the processing of either chemotactile sense or olfaction. From these lobes, immunoreactive fibers reached other lobes of the central nervous system (CNS) which could be indirectly involved in the reproductive behavior. APGWamide immunoreactivity was also present in the glandular cells of the oviducal gland in the female reproductive system. These results constitute the first detailed immunolocalization of APGWamide in cephalopods and open a new insight into the possible effects that both distant and close chemical stimuli can exert on neuropeptidergic circuitries, which may affect the reproductive behavior of cephalopods.

Role of FMRFamide in the reproduction of Octopus vulgaris: molecular analysis and effect on visual input

As a part of continuous research on the neurobiology of the cephalopods in general, and the neuroendocrine control of reproduction in Octopus vulgaris in particular, the presence, the molecular analysis and the effect of FMRFamide on the screening-pigment migration in the visual system have been analysed. FMRFamide immunoreactive fibres are present in the outer plexiform layer of the retina as well as in the plexiform zone of the deep retina. These fibres presumably come from optic and olfactory lobes. We isolated an incomplete Octopus FMRFamide cDNA which encodes an amino terminal truncated precursor containing several FMRFamide-related peptides (FaRPs) showing a high degree of identity with the FaRPs encoded in the precursor of Sepia officinalis, except for the presence of an Rpamide related peptide, present only in cnidarians. Finally, stimulation of isolated retina demonstrated that the effect of this tetrapeptide, coupled with dopamine, is the induction of an extreme adaptation of the retina to the light condition. This situation de facto inhibits sexual maturation. Our results on the effect of FMRFamide on the retina confirm the suggested hypothesis that this peptide plays an inhibitory role on the activity of optic gland.

Neuropeptidergic control of Octopus oviducal gland.

Abstract The oviducal gland of the female of Octopus vulgaris lies about halfway along the oviduct. Progesterone and 17β-estradiol receptors have been immunolocalized in the nuclei of the cells of the glandular compartment of previtellogenic glands. We also have evidence of FMRFamide-like and cGnRH-I-like immunoreactivity in the nerve endings that reach the oviducal gland. Moreover, we have recently shown APGWamide immunoreactivity in the glandular cells of the inner part of the oviducal gland. Here we report a review on these findings as well as our latest studies on the effect that neuropeptides may exert on the secretory activity of the oviducal gland. cAMP seems to be a possible second messenger involved in such a process. We discuss the findings of a neuropeptidergic action on the glandular cells of oviducal gland in a more complex frame of molecules, such as steroids, biogenic amines and neuromodulators, controlling the activity of the gland.

Control of GnRH expression in the olfactory lobe of Octopus vulgaris

Abstract In the cephalopod mollusk Octopus vulgaris , the gonadotropic hormone released by the optic gland controls sexual maturity. Several lobes of the central nervous system control the activity of this gland. In one of these lobes, the olfactory lobe, a gonadotropin releasing hormone (GnRH) neuronal system has been described. We assume that several inputs converge on the olfactory lobes in order to activate GnRH neurons and that a glutamatergic system mediates the integration of stimuli on these neuropeptidergic neurons. The presence of N -methyl- d -aspartate (NMDA) receptor immunoreactivity in the neuropil of olfactory lobes and in the fibers of the optic gland nerve, along with the GnRH nerve endings strongly supports this hypothesis. A distinctive role in the control of GnRH secretion has also been attributed, in vertebrates, to nitric oxide (NO). The lobes and nerves involved in the nervous control of reproduction in Octopus contain nitric oxide synthase (NOS). Using a set of experiments aimed at manipulate a putative l -glutamate/NMDA/NO signal transduction pathway, we have demonstrated, by quantitative real-time PCR, that NMDA enhances the expression of GnRH mRNA in a dose–response manner. The reverting effect of a selective antagonist of NMDA receptors (NMDARs), 2-amino-5-phosphopentanoic acid ( D -APV), confirms that such an enhancing action is a NMDA receptor-mediated response. Nitric oxide and calcium also play a positive role on GnRH mRNA expression. The results suggest that in Octopus l -glutamate could be a key molecule in the nervous control of sexual maturation.In the cephalopod mollusk Octopus vulgaris, the gonadotropic hormone released by the optic gland controls sexual maturity. Several lobes of the central nervous system control the activity of this gland. In one of these lobes, the olfactory lobe, a gonadotropin releasing hormone (GnRH) neuronal system has been described. We assume that several inputs converge on the olfactory lobes in order to activate GnRH neurons and that a glutamatergic system mediates the integration of stimuli on these neuropeptidergic neurons. The presence of N-methyl-d-aspartate (NMDA) receptor immunoreactivity in the neuropil of olfactory lobes and in the fibers of the optic gland nerve, along with the GnRH nerve endings strongly supports this hypothesis. A distinctive role in the control of GnRH secretion has also been attributed, in vertebrates, to nitric oxide (NO). The lobes and nerves involved in the nervous control of reproduction in Octopus contain nitric oxide synthase (NOS). Using a set of experiments aimed at manipulate a putative l-glutamate/NMDA/NO signal transduction pathway, we have demonstrated, by quantitative real-time PCR, that NMDA enhances the expression of GnRH mRNA in a dose-response manner. The reverting effect of a selective antagonist of NMDA receptors (NMDARs), 2-amino-5-phosphopentanoic acid (D-APV), confirms that such an enhancing action is a NMDA receptor-mediated response. Nitric oxide and calcium also play a positive role on GnRH mRNA expression. The results suggest that in Octopusl-glutamate could be a key molecule in the nervous control of sexual maturation.

Progesterone receptor in the liver and oviduct of the lizard Podarcis sicula

In this paper we report the presence of a (3)H-Progesterone ((3)H-P) binding moiety, which has the characteristics of a true receptor, in the liver of the female of the lizard Podarcis sicula. (3)H-P binding studies show the presence of one type of binding site with an average Kd value of 6.2 +/- 2.0 nM in the cytoplasm and 6.3 +/- 1.1 nM in the nucleus. Competition experiments showed that progesterone (P) was the best competitor, while testosterone, deoxycorticosterone (DOC), corticosterone, 17 alpha-hydroxyprogesterone; R5020; RU486 and RU26988-5 were poor competitors. We have also investigated the immunological characteristics of progesterone receptor (PR) in both the liver and the oviduct of Podarcis sicula, by Western blotting using the monoclonal antibody PR22 raised against the PR isoforms A and B of chicken. One imunoreactive band of about 70 kDa was detected in cytoplasmic and nuclear extracts of both the liver and the oviduct. PR immunoreactivity was present in the liver during the quiescent phase. In the oviduct PR immunoreactivity increased from the recovery to the full grown phase. P treatment of estrogen-primed females did not affect the presence of PR in the liver, while brought about a PR increase in the oviduct. This study suggests that PR is expressed differently in the liver and the oviduct of Podarcis sicula throughout the reproductive cycle. PR might fulfill different requirements in relation to the different physiological functions of the tissue during the reproductive cycle.