Nuclear magnetic resonance and circular dichroism study of metastin (Kisspeptin-54) structure in solution.

KISS1 was first discovered as a metastasis suppressor, but also plays crucial roles in the onset of puberty. The KISS1 gene encodes a secreted protein of 145 amino acids that exhibits no sequence similarity with any known proteins. KISS1 protein is proteolytically processed to generate a number of so-called kisspeptins (KP), the most well characterized is known as KP-54 or metastin. KP-54 is carboxy-terminally amidated and binds to and activates the KISS1 receptor (KISS1R). The current studies were undertaken in order to determine structure of KP-54 using nuclear magnetic resonance and circular dichroism. KP-54 is mostly disordered both in water and in trifluoroethanol/water mixed solvent, with no structural motifs. In sodium dodecyl sulfate micelles, KP-54 remains mostly disordered except for a small increase in helical propensity (from 3.7% in water to 9.9% in micelles). Despite this apparent lack of structure, KP-54 is biologically active. The intrinsic disorder of KP-54 may confer advantages in its ability to recognize and bind a wide range of target proteins.

CORCEMA refinement of the bound ligand conformation within the protein binding pocket in reversibly forming weak complexes using STD-NMR intensities.

We describe an intensity-restrained optimization procedure for refining approximate structures of ligands within the protein binding pockets using STD-NMR intensity data on reversibly forming weak complexes. In this approach, the global minimum for the bound-ligand conformation is obtained by a hybrid structure refinement method involving CORCEMA calculation of intensities and simulated annealing optimization of torsion angles of the bound ligand using STD-NMR intensities as experimental constraints and the NOE R-factor as the pseudo-energy function to be minimized. This method is illustrated using simulated STD data sets for typical carbohydrate and peptide ligands. Our procedure also allows for the optimization of side chain torsion angles of protein residues within the binding pocket. This procedure is useful in refining and improving initial models based on crystallography or computer docking or other algorithms to generate models for the bound ligand (e.g., a lead compound) within the protein binding pocket compatible with solution STD-NMR data. This method may facilitate structure-based drug design efforts.

Saturation transfer difference NMR and computational modeling of a sialoadhesin-sialyl lactose complex.

The siglecs are a family of I-type lectins binding to sialic acids on the cell surface. Sialoadhesin (siglec-1) is expressed at much higher levels in inflammatory macrophages and specifically binds to alpha-2,3-sialylated N-acetyl lactosamine residues of glycan chains. The terminal disaccharide alpha-D-Neu5Ac-(2-->3)-beta-D-Gal is thought to be the main epitope recognized by sialoadhesin. To understand the basis of this biological recognition reaction we combined NMR experiments with a molecular modeling study. We employed saturation transfer difference (STD) NMR experiments to characterize the binding epitope of alpha-2,3-sialylated lactose, alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-D-Glc 1 to sialoadhesin at atomic resolution. The experimental results were compared to a computational docking model and to X-ray data of a complex of sialyl lactose and sialoadhesin. The data reveal that sialoadhesin mainly recognizes the N-acetyl neuraminic acid and a small part of the galactose moiety of 1. The crystal structure of a complex of sialoadhesin with sialyl lactose 1 was used as a basis for a modeling study using the FlexiDock algorithm. The model generated was very similar to the original crystal structure. Therefore, the X-ray data were used to predict theoretical STD values utilizing the CORCEMA-STD protocol. The good agreement between experimental and theoretical STD values indicates that a combined modeling/STD NMR approach yields a reliable structural model for the complex of sialoadhesin with alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-D-Glc 1 in aqueous solution.

Cytoarchitectonic pattern of the hypothalamus in the catfish, Clarias batrachus (Linn.).

The hypothalamus of the catfish, Clarias batrachus has been investigated to reveal the organization of various parvocellular (aldehyde fuchsin-negative) nuclear complexes and to suggest homologies. The hypothalamus of C. batrachus can be divided into the preoptic area, postoptic area, tuberal area and the laterally placed inferior lobes. Application of cytoarchitectonic criteria permits the delineation of 9 distinct nuclear complexes in the preoptic area extending along the periventricular margin of the preoptic recess. The postoptic area is represented by only two nuclear groups, the nucleus postopticus lateralis and the nucleus of the horizontal commissure. The tuberal area consists of as many as 14 nuclei, some are divisible into subnuclei, arranged in an elaborate pattern. The nucleus lateralis tuberis is located in the mid-ventral margin of the tuberal area and revealed two subdivisions, the pars anterior and pars posterior. At the mid-tuberal level, the nucleus anterior tuberis is well-developed and revealed dorsal, ventral and lateral subdivisions. The paraventricular organ is encircled by nucleus of the paraventricular organ. The conspicuous lateral recesses are flanked dorsally by nucleus recessus lateralis superior and ventrally by nucleus recessus lateral by nucleus recessus lateralis superior and ventrally by nucleus recessus lateralis inferior. In the caudal tuberal area, in close proximity of the pituitary, the nucleus arcuatus hypothalamicus is described for the first time. The recessus posterioris is encircled by nucleus recessus posterioris. The inferior lobes are massive and each consists of 7 well-defined nuclear complexes. The nucleus lobi inferioris centralis is represented by giant-sized cells clustered in the center of the lobes. The periventricular nucleus of the inferior lobes occupies the area surrounding the recesses of the inferior lobes. The hypothalamus shows 4 circumventricular organs. The organum vasculosum laminae terminalis is situated along the ventral wall of the preoptic recess. While the paraventricular organ is located in the dorsal wall of the third ventricle, the posterior recess organ is located in the postero-ventral wall of the third ventricle. The saccus vasculosus is small, capsule-like and located caudally in between the two mamillary nuclei.

The response of nucleus preopticus neurosecretory cells to ovarian pressure in the frog, Rana tigrina.

Intraovarian pressure (IOP) of 5, 15, and 25 mm Hg was administered in the frog, Rana tigrina, and the response of the nucleus preopticus (NPO) pars magnocellularis was investigated with aldehyde fuchsin (AF) stain and immunocytochemical method using neurophysin (NP) antisera. The 5 mm Hg IOP treatment resulted in cell and cell nuclear hypertrophy (P less than 0.001); discrete signs of de novo synthesis of AF-positive and NP-immunoreactive material in the perikarya and remarkable increases in the number and size of "Herring bodies" in the processes were observed. Stimulatory response after 15 mm Hg IOP treatment was characterized by dramatic augmentation of the AF-positive and NP-immunoreactive material in the processes; the engorged and coalescing Herring bodies totally predominated the lateral preoptic area. IOP of 25 mm Hg resulted in extensive loss of secretory material; the processes revealed the presence of vacuoles indicative of the rapid anterograde transport of the neurosecretory material. Furthermore, the application of IOP seemed to promote the transport of NP-immunoreactive material toward the anterior preoptic area and amygdala pars medialis and the release of secretory material into the cerebrospinal fluid. No changes were observed in the NPO when treatment was preceded by the transection of ipsilateral dorsal spinal nerve roots or the spinal cord. The results suggest the existence of an afferent neural pathway from the ovary capable of conducting the stretch signals to the NPO and triggering the synthesis and release of neurohypophysial hormones.

The intrinsic organization of the nucleus preopticus in the catfish, Clarias batrachus (Linn.).

The organization of magnocellular neurons of the nucleus preopticus (NPO) of the catfisch, Clarias batrachus is described with the help of Nissl and aldehyde fuchsin stains and rapid Golgi technique. The NPO consists of a bilaterally located paraventricular group (PV) on either side of the preoptic recess and a supraoptic group (SO) dorsal to the optic tract. The PV shows a vertical laminar arrangement--smaller cells located adjacent to the ependyma and larger ones farther away. On the basis of the pattern of arrangement, the PV is divisible into 4 subdivisions-1: composed of small, spindle-shaped, bipolar, vertically oriented neurons characterized with short processes; subdivision 2: formed of large multipolar neurons with dendrites extending in different directions and some processes projecting into the cerebrospinal fluid (CSF); subdivision 3: consists of larger perikarya forming the lateral margin of the PV, characteristically, the processes of these neurons extend ventrolaterally and penetrate the optic tract; subdivision 4: situated posterodorsally to the above subdivisions of the PV and shows very large neurons, generally multipolar and exhibiting processes with projections to medial (MFB) and lateral (LFB) forebrain bundles, CSF, telencephalon, and towards the caudal diencephalon and tegmentum. The SO consists of three subdivisions, also arranged progressively lateralwards in order of increasing size. Processes of the lateral-most subdivision communicate with the MFB, LFB and extend into the optic tract. The NPO of C. batrachus seems comparable with the paraventricular and supraoptic nuclei of amniotes. Further the nucleus shows a complex organization and parcellation of SO and PV. By virtue of the occurrence of processes, connectivities and interneurons, the nucleus appears to play a pivotal role in receiving and integrating a wide spectrum of information to eventually influence a variety of neuroendocrine functions.

Pattern of acetylcholinesterase activity in the hypothalamus of the cobra, Naja naja.

Application of acetylcholinesterase (AChE) method to the hypothalamus of the cobra Naja naja revealed a clearly discernible pattern of activity in the nuclear groups. The magnocellular neurons distributed in various subdivisions showed strong reaction. Among the parvocellular groups, intense enzyme activity was observed in the cells of nucleus subfornicalis, nucleus hypothalamicus medialis, dorsal and ventral subdivisions of the nucleus hypothalamicus lateralis and the nucleus mamillaris. The paraventricular organ displayed strong amorphous activity accompanied by the intense reaction in the nucleus of the paraventricular organ. The neurons of the lateral preoptic area, nucleus periventricularis posterior, nucleus hypothalamicus dorsomedialis, nucleus lateralis recessus infundibuli and nucleus medialis recessus infundibuli showed heterogenous population revealing varying degrees of staining intensity. The perikarya of the nucleus suprachiasmaticus, nucleus hypothalamicus ventromedialis and nucleus arcuatus showed light staining. The rest of the nuclear groups were unstained. Whereas, lateral forebrain bundles and supraoptic decussation showed some AChE activity in the fibers, anterior commissure and optic chiasma were distinctly negative. The AChE-positive neuropil was confined to the lateral preoptic, and circumscribed periventricular tuberal areas. In the median eminence, a layer of fine AChE-reactive granules restricted to the subependymal zone was found; the ependymal layer was equipped with large, intensely positive somata.

Hypothalamic innervation of the pituitary in the catfish, Clarias batrachus (L.): a retrograde horseradish peroxidase study.

Intrahypophysial administration of horseradish peroxidase (HRP) resulted in extensive labelling of the cells of nucleus preopticus and nucleus lateralis tuberis. Besides, isolated retrogradely labelled neurons were observed in the nucleus preopticus periventricularis in the preoptic area, nucleus of the horizontal commissure, nucleus hypothalamicus ventralis in the rostral tuberal area, and nucleus arcuatus hypothalamicus in the caudal tuberal area. A few labelled cells were also observed in the mamillary region.

The response of nucleus preopticus neurosecretory cells to ovarian pressure in the teleost, Clarias batrachus (Linn.).

The changes undergone by the neurosecretory cells of the nucleus preopticus (NPO) were investigated histologically and immunocytochemically following pressure stimulation of the ovaries in the teleost, Clarias batrachus. Application of intraovarian pressure of 10 mm Hg revealed marked hypertrophy of the cells, while pressures of 20 and 30 mm Hg resulted in hypertrophy and exhaustion of the cells. No changes were observed when the treatment was preceded by transection of the spinal cord. The results suggest the existence of a pressure-excitable neural pathway from the ovary capable of eliciting profound cytological changes in the NPO.