Anchoring TRP to the INAD macromolecular complex requires the last 14 residues in its carboxyl terminus

Drosophila transient-receptor-potential (TRP) is a Ca²⁺ channel responsible for the light-dependent depolarization of photoreceptors. TRP is anchored to a macromolecular complex by tethering to inactivation-no-afterpotential D (INAD). We previously reported that INAD associated with the carboxyl tail of TRP via its third post-synaptic density protein 95, discs-large, zonular occludens-1 domain. In this paper, we further explored the molecular basis of the INAD interaction and demonstrated the requirement of the last 14 residues of TRP, with the critical contribution of Gly1262, Val1266, Trp1274, and Leu1275. We also revealed by pull-down assays that the last 14 residues of TRP comprised the minimal sequence that competes with the endogenous TRP from fly extracts, leading to the co-purification of a partial INAD complex containing INAD, no-receptor-potential A, and eye-protein kinase C (PKC). Eye-PKC is critical for the negative regulation of the visual signaling and was shown to phosphorylate TRP in vivo . To uncover the substrates of eye-PKC in the INAD complex, we designed a complex-dependent eye-PKC assay, which utilized endogenous INAD complexes isolated from flies. We demonstrate that activated eye-PKC phosphorylates INAD, TRP but not no-receptor-potential A. Moreover, phosphorylation of TRP is dependent on the presence of both eye-PKC and INAD. Together, these findings indicate that stable kinase-containing protein complexes may be isolated by pull-down assays, and used in this modified kinase assay to investigate phosphorylation of the proteins in the complex. We conclude that TRP associates with INAD via its last 14 residues to facilitate its regulation by eye-PKC that fine-tunes the visual signaling.     

Lassoing and Corralling Rooted Phylogenetic Trees

The construction of a dendogram on a set of individuals is a key component of a genomewide association study. However, even with modern sequencing technologies the distances on the individuals required for the construction of such a structure may not always be reliable making it tempting to exclude them from an analysis. This, in turn, results in an input set for dendogram construction that consists of only partial distance information, which raises the following fundamental question. For what (proper) subsets of a dendogram’s leaf set can we uniquely reconstruct the dendogram from the distances that it induces on the elements of such a subset? By formalizing a dendogram in terms of an edge-weighted, rooted, phylogenetic tree on a pre-given finite set X with |X|≥3 whose edge-weighting is equidistant and subsets Y of X for which the distances between every pair of elements in Y is known in terms of sets of 2-subsets of X, we investigate this problem from the perspective of when such a tree is lassoed, that is, uniquely determined by the elements in . For this, we consider four different formalizations of the idea of “uniquely determining” giving rise to four distinct types of lassos. We present characterizations for all of them in terms of the child-edge graphs of the interior vertices of such a tree. Our characterizations imply in particular that in case the tree in question is binary, then all four types of lasso must coincide.     

Interstitial cells of Cajal in pancreas

We show here (presumably for the first time) a special type of cell in the human and rat exocrine pancreas. These cells have phenotypic characteristics of the enteric interstitial cells of Cajal (ICC). To identify pancreatic interstitial cells of Cajal (pICC) we used routine light microscopy, non-conventional light microscopy (less than 1 mum semi-thin sections of Epon-embedded specimens cut by ultramicrotomy and stained with Toluidine blue), transmission electron microscopy (TEM), and immunocytochemistry. The results showed that pICC can be recognized easily by light microscopy, particularly on semi-thin sections, as well as by TEM. Two-dimensional reconstructions from serial photos suggest a network-like spatial distribution of pICC. pICC represent 3.3+/-0.5% of all pancreatic cells, and seem to establish close spatial relationships with: capillaries (43%), acini (40%), stellate cells (14%), nerve fibres (3%). Most of pICC (88%) have 2 or 3 long processes (tens of mum) emerging from the cell body. TEM data show that pICC meet the criteria for positive diagnosis as ICC (e.g. numerous mitochondria, 8.7+/-0.8% of cytoplasm). Immunocytochemistry revealed that pICC are CD117/c-kit and CD34 positive. We found pICC positive (40-50%) for smooth muscle alpha-actin or S-100, and, occasionally, for CD68, NK1 neurokinin receptor and vimentin. The reactions for desmin and chromogranin A were negative in pICC. At present, only hypotheses and speculations can be formulated on the possible role of the pICC (e.g., juxtacrine and/or paracrine roles). In conclusion, the quite-established dogma: "ICC only in cavitary organs" is overpassed.     

Probing the activation sequence of NMDA receptors with lurcher mutations

N-methyl-d-aspartate (NMDA) receptor activation involves a dynamic series of structural rearrangements initiated by glutamate binding to glycine-loaded receptors and culminates with the clearing of the permeation pathway, which allows ionic flux. Along this sequence, three rate-limiting transitions can be quantified with kinetic analyses of single-channel currents, even though the structural determinants of these critical steps are unknown. In inactive receptors, the major permeation barrier resides at the intersection of four M3 transmembrane helices, two from each GluN1 and GluN2 subunits, at the level of the invariant SYTANLAAF sequence, known as the lurcher motif. Because the A7 but not A8 residues in this region display agonist-dependent accessibility to extracellular solutes, they were hypothesized to form the glutamate-sensitive gate. We tested this premise by examining the reaction mechanisms of receptors with substitutions in the lurcher motifs of GluN1 or GluN2A subunits. We found that, consistent with their locations relative to the proposed activation gate, A8Y decreased open-state stability, whereas A7Y dramatically stabilized open states, primarily by preventing gate closure; the equilibrium distribution of A7Y receptors was strongly shifted toward active states and resulted in slower microscopic association and dissociation rate constants for glutamate. In addition, for both A8- and A7-substituted receptors, we noticed patterns of kinetic changes that were specific to GluN1 or GluN2 locations. This may be a first indication that the sequence of discernible kinetic transitions during NMDA receptor activation may reflect subunit-dependent movements of M3 helices. Testing this hypothesis may afford insight into the activation mechanism of NMDA receptors.     

Interstitial Cajal-like cells in rat mesentery: an ultrastructural and immunohistochemical approach

Interstitial Cajal-like Cells (ICLC) were recently recognized in a plethora of non-digestive organs. Here, we describe a cell type of rat mesentery sharing ultrastructural and immunohistochemical features with ICLC. Mesenteric ICLC were demonstrated by transmission electron microscopy (TEM) and further tested by light microscope immunohistochemistry. The cell described here fulfils the TEM diagnostic criteria accepted for ICLC: location in the connective interstitium; close vicinity to nerves, capillaries and other interstitial cells; characteristic long, moniliform cell processes; specialized cell-to-cell junctions; caveolae; mitochondria at 5-10% of cytoplasmic volume; rough endoplasmic reticulum at about 1-2%; intermediate and thin filaments, microtubules; undetectable thick filaments. The processes of this mesenteric ICLC were particularly long, with a mean length of 24.91 microm (10.27-50.83 micorm), and a convolution index of 2.32 (1.37-3.63) was calculated in order to measure their potential length. Mean distances versus main target cells of ICLC-nerve bundles, vessels, adipocytes and macrophages-were 110.69, 115.80, 205.07 and 34.65 nm, respectively. We also tested the expression of CD117/c-kit, CD34, vimentin, alpha-smooth muscle actin, nestin, NK-1, tryptase and chymase and the antigenic profile of the mesenteric ICLC was comparable if not identical with that recently observed in ICLC from other extra-digestive tissues. Due to the peculiar aspect of the mesenteric ICLC processes it can be hypothesized that these cells form a three-dimensional network within the mesentery that is at the same time resistant and deformable following stretches consequent to intestine movements, mainly avoiding blood vessels closure or controlling blood vessels rheology. It remains, however, to be established if and how such cells are connected with the archetypal enteric ICC.     

Physicochemical and Preclinical Evaluation of a Novel Buccal Measles Vaccine

The aim of this study is to develop an orally disintegrating film (ODF) containing a microparticulate measles vaccine formulation for buccal delivery. The measles vaccine microparticles were made with biocompatible and biodegradable bovine serum albumin (BSA) and processed by spray drying. These vaccine microparticles were incorporated in the ODF, consisting of Lycoat RS720®, Neosorb P60W® and Tween 80. The yield of the microparticles was approximately 85–95%, w/w. The mean size of the vaccine microparticles was 3.65?±?1.89 μm and had a slightly negative surface charge of 32.65?±?2.4 mV. The vaccine particles were nontoxic to normal cells at high concentrations (500 μg/2.5?×?10⁵ cells) of vaccine particles. There was a significant induction of innate immune response by vaccine microparticles which was observed in vitro when compared to blank microparticles (P?<?0.05). The vaccine microparticles also significantly increased the antigen presentation and co-stimulatory molecules expression on antigen presenting cells, which is a prerequisite for Th1 and Th2 immune responses. When the ODF vaccine formulation was dosed in juvenile pigs, significantly higher antibody titers were observed after week 2, with a significant increase at week 4 and plateauing through week 6 comparative to naïve predose titers. The results suggest that the ODF measles vaccine formulation is a viable dosage form alternative to noninvasive immunization that may increase patient compliance and commercial distribution.     

Effects of alpha-MSH on kainic acid induced changes in core temperature in rats

The effects of intraperitoneal (i.p.) administration of kainic acid (KA) and alpha-melanocyte-stimulating hormone (alpha-MSH) alone or in combination, on core temperature of freely moving rats were examined. KA or saline was administered once (10 mg/kg) and alpha-MSH or saline was given repeatedly i.e. 10 min before and 10, 30 and 60 min after the administration of saline or KA. Two doses of alpha-MSH were used: 0.5 and 2.5 mg/kg. KA alone produced a biphasic effect on core temperature, i.e. an initial short-lasting hypothermia followed by hyperthermia that lasted about 6 h. The higher dose of alpha-MSH had a potentiating effect on KA-induced hypothermia, while the lower dose of alpha-MSH increased the hyperthermia produced by KA. alpha-MSH administered alone produced a late (3 h), dose-dependent increase in core temperature. It is conceivable that repeated administration of alpha-MSH in the doses used in our study may cause a cumulative effect in raising body temperature for a limited period of time.The previously described interactions between KA and alpha-MSH, respectively, with dopaminergic and serotoninergic systems may account for the effects on core temperature in rats observed in our study.     

A Novel Effect in Phycomyces Phototropism : Positive Bending and Compensation Spectrum in Far UV

A novel effect—positive phototropic bending under far UV irradiation (between 260 and 305 nanometers) at low intensities—is reported. Natural compensation points (intensities which cause no bending under unilateral irradiation) have been determined for different wavelengths. The curve connecting these points, the compensation spectrum, divides the intensity-wavelength plane into areas of negative and positive tropism. It is further shown that a highly asymmetrical pattern of light stimulus within the sporangiophore underlies the symmetrical growth response at each compensation point. This suggests that some unknown additional factor is involved in perceiving a UV stimulus at the level of the photoreceptor. It is also demonstrated here that positive tropism in the UV range is due to a lens effect. We conclude that the hypothesis of optical attenuation of the stimulus (considered until now as the most plausible explanation of negative tropism in the UV spectral range) must be dismissed. The results presented here represent the first application of our quantitative theoretical consideration of spatial factors in phototropism heretofore neglected by others.     

Variations of chromosomes 2 and 3 gene expression profiles among pulmonary telocytes,pneumocytes, airway cells,mesenchymal stem cells and lymphocytes

Telocytes (TCs) were identified as a distinct cellular type of the interstitial tissue and defined as cells with extremely long telopodes (Tps). Our previous data demonstrated patterns of mouse TC‐specific gene profiles on chromosome 1. The present study focuses on the identification of characters and patterns of TC‐specific or TC‐dominated gene expression profiles in chromosome 2 and 3, the network of principle genes and potential functional association. We compared gene expression profiles of pulmonary TCs, mesenchymal stem cells, fibroblasts, alveolar type II cells, airway basal cells, proximal airway cells, CD8⁺T cells from bronchial lymph nodes (T‐BL), and CD8⁺ T cells from lungs (T‐LL). We identified that 26 or 80 genes of TCs in chromosome 2 and 13 or 59 genes of TCs up‐ or down‐regulated in chromosome 3, as compared with other cells respectively. Obvious overexpression of Myl9 in chromosome 2 of TCs different from other cells, indicates that biological functions of TCs are mainly associated with tissue/organ injury and ageing, while down‐expression of Pltp implies that TCs may be associated with inhibition or reduction of inflammation in the lung. Dominant overexpression of Sh3glb1, Tm4sf1 or Csf1 in chromosome 3 of TCs is mainly associated with tumour promotion in lung cancer, while most down‐expression of Pde5 may be involved in the development of pulmonary fibrosis and other acute and chronic interstitial lung disease.