NCI-ELMO: A New Method To Quickly and Accurately Detect Noncovalent Interactions in Biosystems.

Accurate determinations of noncovalent interactions in biological systems are fundamental to rationalize the structure and to get insights into the functions and the dynamics of macromolecules. Here we propose a new tool for the efficient identification of noncovalent interactions in proteins. The noncovalent interaction (NCI) method, a well-established strategy to detect noncovalent interactions in chemical systems, is coupled with the libraries of extremely localized molecular orbitals (ELMOs), which allow instantaneous reconstruction of quantum mechanically rigorous electron distributions of polypeptides and proteins. Test calculations performed on different interactions show that the new NCI-ELMO strategy always outperforms the original NCI method based on the promolecular approximation, while it is in close agreement with original NCI analyses based on fully quantum mechanical calculations. The new technique allows for unraveling the network of interactions in biological systems and to rapidly monitor their evolutions with time, with possible consequences on the design of new drugs.

Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary.

In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH.

Neuroendocrine system of the digestive tract in Rhamdia quelen juvenile: an immunohistochemical study.

In this work, an immunohistochemical study was performed to determine the distribution and relative frequencies of some neuromodulators of the digestive tract of silver catfish (Rhamdia quelen). The digestive tract of silver catfish was divided into six portions; the oesophagus, stomach, intestine (ascendant, descendant and convoluted segments), and rectum. Immunohistochemical method using a pool of specific antisera against-gastrin, -cholecystokinin-8, -leu-enkephalin, -neuropeptide Y, -calcitonin gene-related peptide (CGRP), and -vasoactive intestinal peptide (VIP) was employed. Immunoreactivity to all antisera was identified in neuroendocrine cells (NECs) localized in the gut epithelium, although no reaction was observed in the oesophagus or stomach. The morphology of NECs immunopositive to each antibody was similar. They were slender in shape, with basally located nucleus, and their main axis perpendicular to the basement membrane. The number of NECs immunoreactive to all antisera was higher in the ascendant and descendant intestine, exhibiting a decreasing trend toward distal segments of the gut. In addition, immunoreactivity to CGRP and VIP was observed in the myenteric plexus and nerve fibers distributed in the mucosal, submucosal and muscular layers. The higher number of immunopositive NECs in the ascendant and descendant intestine may indicate the primary role of these segments in the control of food intake by means of orexigenic and anorexigenic peripheral signals.

Evidence for changes in brain enkephalin contents associated to male rat sexual activity.

Indirect evidence suggests that ejaculation might activate endogenous opioid systems, which exert an inhibitory influence on male rat sexual behaviour. The objective of the present study was to search for putative long-term changes in the contents of immunoreactive (IR) Met-enkephalin (IR-Met), Leu-enkephalin (IR-Leu) and opioid octapeptide Met--Arg(6)--Gly(7)--Leu(8) (IR-Oct) in specific brain areas, after the execution of different amounts of sexual activity. Additionally, basal contents of these enkephalins were compared between sexually active (SA) and persistent sexually inactive (SI) rats. Immunoreactivity to enkephalins was determined by radioimmunoanalysis, in the frontal cortex, the hypothalamus and midbrain of SA and SI rats, as well as 24 or 48 h after males had one ejaculation or copulated to exhaustion. Twenty-four hours after sexual activity, there was a generalised increase in enkephalin contents that returned to control values at the 48 h measurement in all brain areas, but the hypothalamus, where IR-Met and IR-Oct remained elevated. No differences in the magnitude of the changes were found between rats that ejaculated once and sexually satiated males. IR-Oct concentration in the hypothalamus of SI rats appeared significantly higher than in SA animals, with no differences in IR-Met and IR-Leu. Results give direct evidence of the activation of endogenous opioid systems by male rat sexual activity. The occurrence of long lasting increases in the contents of IR-Met and IR-Oct in the hypothalamus of rats that copulated was detected. Finally, an intrinsically elevated octapeptide concentration in the hypothalamus of SI rats was found.

Secretion of a neuropeptide-metabolizing enzyme similar to endopeptidase 22.19 by glioma C6 cells.

An endopeptidase capable of metabolizing a number of neuropeptides and generating [Met5] and [Leu5] enkephalin from enkephalin-containing peptides is secreted by glioma C6 cells. This neutral endopeptidase that is likely to be a thiol protease, has a Mr of 71KDa and is effective only towards oligopeptides. Its specificity towards neuropeptides is identical to that of soluble endopeptidase 22.19. Moreover, when a partially purified preparation of enkephalin-generating enzyme secreted by glioma C6 cells was submitted to immunoblotting, an antiserum against purified brain endopeptidase 22.19 recognized a single band at Mr of 71 KDa. These data suggest that the soluble endopeptidase 22.19 may be secreted by glioma C6 cells thus allowing its participation in the biotransformation of opioid peptides in the CNS.

Different subsets of displaced ganglion cells in the pigeon retina exhibit cholecystokinin-like and enkephalin-like immunoreactivities.

Immunohistochemical and retrograde tracing techniques were combined in order to identify chemically specific displaced ganglion cells in the pigeon retina. About 15% of the displaced ganglion cells that were retrogradely labeled following injections of different tracers into the accessory optic nucleus were shown to contain cholecystokinin8-like immunoreactivity. These cells were medium to large (15-30 microns) and located mostly in the peripheral retina. Another population of about 9% of the retrogradely labeled displaced ganglion cells was shown to contain leucine-enkephalin-like immunoreactivity. These cells were medium-sized (11-18 microns) and distributed almost evenly throughout the retina. These two types of displaced ganglion cells represent together only about 0.1% of the total number of ganglion cells in the pigeon retina. Taken together with previous results, these data indicate that the displaced ganglion cells of the avian retina may comprise several chemically specific cell types. The present results also contribute information on the chemical heterogeneity of retinal ganglion cells.

Efeitos das encefalinas sobre o aparelho digestivo / Enkephalins effects on digestive system

A identificaçäo, localizaçäo e propriedades digestivas das encefalina-metionina e encefalina-leucina säo discutidas neste artigo. Interaçöes sinérgicas e antagônicas säo também apresentadas.

An enkephalin degrading aminopeptidase of human brain preserved during the vertebrate phylogeny.

1. A soluble human brain aminopeptidase which hydrolyses the Tyr-Gly bond of Met-enkephalin and Leu-enkephalin was identified in the brains of the following vertebrates: mammals (Callithrix jacchus and Rattus norvegicus), bird (Gallus domesticus), reptile (Tupinambis teguixin), amphibia (Bufo paracnemis), fish (Sarotherdon niloticus) and elasmobranchy (Galeocerdo cuvieri). 2. The properties of this enzyme are: molecular weight near 100,000 Da, isoelectric point near 4.9, optimum pH near 7.5, activation by dithiothreitol, strong inhibition by Cu2+, Zn2+, Ni2+, puromycin and bacitracin, hydrolysis of enkephalins and basic and neutral aminoacid-beta-naphythylamide substrates. 3. The results indicate the preservation of this human brain aminopeptidase during the course of vertebrate phylogeny.

Adrenal proenkephalin-derived peptides during postnatal development in spontaneously hypertensive rats.

Adrenal enkephalin and enkephalin-containing peptides were studied during postnatal development in normotensive (WKY) and spontaneously hypertensive rats (SHR). The effect of chronic treatment with the ganglionic blocker chlorisondamine (5 mg/kg) was also assessed. Free enkephalin immunoreactivity and total enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin containing fragments, were quantitated in the adrenal glands at 11 days and 7, 16, and 24 weeks of age. Both total and free metenkephalin were significantly diminished in the adrenal of SHR when compared to WKY at all ages tested. The analysis of the chromatographic profile showed that SHR displayed reduced levels of high and low molecular weight materials at 11 days and 16 weeks of age; however intermediate compounds were high in the glands of these animals. Similar increased values for free met-enkephalin were found in adrenals of WKY and SHR after ganglionic blocker treatment, which means that the relative increase was larger in SHR than WKY; while for total enkephalin the relative increase and the concentration reached in SHR was about half of those presented in WKY. These and other results presented suggest that the basic alteration of the adrenal proenkephalin system of SHR may be due to a genetic reduction of proenkephalin levels. Otherwise, the free enkephalin decrease could be related to changes in nervous input to the adrenal gland.