Detection of kappa and delta opioid receptors in skin--outside the nervous system

Opioid receptors (OR) are widely expressed in the central nervous system (CNS). Opioid antinociception might be initiated by activation of OR outside the CNS, indicating targeting of peripheral OR could be useful in the treatment of chronic pain. This study was designed to detect OR in skin tissues of healthy volunteers at both mRNA and protein levels. Skin samples from 10 healthy individuals were investigated. Total isolated RNAs were reverse transcribed, amplified and quantified by real-time PCR. Tissue and skin fibroblast OR protein was detected by immunohistochemistry, Western blot, and immunofluorescence. All skin tissue samples expressed delta- (DOR) and kappa-OR (KOR) mRNAs. Using immunohistochemistry, DOR and KOR were localized in skin fibroblast-like and mononuclear cells. Skin fibroblasts in culture expressed DOR and KOR mRNA. Using immunofluorescence, both DOR and KOR proteins were expressed predominantly on the cell membrane with minor staining in the cytoplasm. We suggest that enhanced expression of DOR and KOR in skin justifies the exploration of selective novel delta and kappa agonists for local pain treatment.     

Alpha-conotoxin residues that interact at close range with gamma-tyrosine-111 and mutant delta-tyrosine-113 on the Torpedo nicotinic acetylcholine receptor

The alpha-conotoxins MI and GI display stronger affinities for the alphagamma agonist site on the Torpedo californica electrocyte nicotinic acetylcholine receptor (ACHR) than for the alphadelta agonist site, while alpha-conotoxin SI binds with the same affinity to both sites. Prior studies reported that the arginine at position 9 on GI and the tyrosine at position 111 on the receptor gamma subunit were responsible for the stronger alphagamma affinities of GI and MI, respectively. This study was undertaken to determine if the alpha-conotoxin midchain cationic residues interact with Torpedo gammaY111. The findings show that lysine 10 on MI is responsible for the alphagamma selectivity of MI and confirm the previously reported importance of R9 on GI and on the SI analogue, SIP9R. The results also show that gammaY111 contributes substantially to the selective alphagamma high affinity of all three peptides. Double-mutant cycle analyses reveal that, in the alphagamma site, K10 on MI and R9 on SIP9R interact with the aromatic ring of gammaY111 to stabilize the high-affinity complex, while in contrast, R9 on GI does not. The substitution of Y for R at position 113 on the delta subunit converts the alphadelta site into a high-affinity site for MI, GI, and SIP9R through the interacting of deltaY113 with K10 on MI and with R9 on both GI and SIP9R. The overall data show that the residues in the two sites with which MI interacts, other than at gamma111/delta113, are either the same or similar enough to exert equivalent effects on MI, indicating that MI binds in the same orientation at the alphagamma and alphadelta sites. Similar findings show that SIP9R probably also binds in the same orientation at the wild-type alphagamma and alphadelta sites. The finding that R9 on GI interacts closely with deltaR113Y but not with gammaY111 means that GI binds in different orientations at the alphagamma and alphadelta sites. This report also discusses the molecular basis of the difference in the MI high-affinity sites on Torpedo and embryonic mouse muscle ACHRs.     

Mechanical regulation of HB-GAM expression in bone cells

Bone adaption upon mechanical stimulation is accompanied by changes in gene expression. In this context we investigated the influence of mechanical loading on heparin binding growth associated molecule (HB-GAM) expression, an extracellular matrix molecule which in cell culture has been shown to stimulate the differentiation of osteoblasts. We obtained information on the participating signal transduction pathways using a mitogenic loading regimen. Specific inhibitors of various signal transduction pathways were added to loaded cells and to unloaded controls. By semi-quantitative PCR studies we demonstrated a rapid decrease of HB-GAM expression in primary osteoblasts and SaOs-2 cells by 20-30% upon mechanical loading within 30min. We showed that the RGD-integrin interaction is involved in the regulation of HB-GAM expression. Furthermore, integrity of the cytoskeleton, stretch-activated, and voltage-sensitive Ca(2+) channels as well as gap junctional communication are necessary for the downregulation of HB-GAM expression by mechanical loading.     

SOCS-1 and SOCS-3 inhibit IFN-alpha-induced expression of the antiviral proteins 2,5-OAS and MxA

Although the use of IFN-alpha in combination with ribavirin has improved the treatment efficacy of chronic hepatitis C virus (HCV) infection, 20-50% of patients still fail to eradicate the virus depending on the HCV genotype. Recently, overexpression of HCV core protein has been shown to inhibit IFN signaling and induce SOCS-3 expression. Aim of this study was to examine the putative role of SOCS proteins in IFN resistance. By Western blot analysis, a 4-fold induction of STAT-1/3 phosphorylation by IFN-alpha was observed in mock-transfected HepG2 clones. In contrast, IFN-induced STAT-1/3 phosphorylation was considerably downregulated by SOCS-1/3 overexpression. In mock-transfected cells, IFN-alpha induced 2',5'-OAS and myxovirus resistance A (MxA) promoter activity 40- to 80-fold and 10- to 35-fold, respectively, and this effect was abrogated in SOCS-1/3 overexpressing cells. As detected by Northern blot technique, IFN-alpha potently induced 2',5'-OAS and MxA mRNA expression in the control clones. Overexpression of SOCS-1 completely abolished both 2',5'-OAS and MxA mRNA expression, whereas SOCS-3 mainly inhibited 2',5'-OAS mRNA expression. Our results demonstrate that SOCS-1 and SOCS-3 proteins inhibit IFN-alpha-induced activation of the Jak-STAT pathway and expression of the antiviral proteins 2',5'-OAS and MxA. These data suggest a potential role of SOCS proteins in IFN resistance during antiviral treatment.     

Shear stress inhibits while disuse promotes osteocyte apoptosis

Cell apoptosis operates as an organizing mechanism in biology in addition to removing effete cells. We have recently proposed that during bone remodeling, osteocyte apoptosis steers osteonal alignment in relation to mechanical loading of the whole bone [J. Biomech. 36 (2003) 1453]. Here we present evidence that osteocyte apoptosis in cell culture is modulated by shear stress. Under static culture conditions, serum starved osteocytes exposed phosphatidylserine (PS) on their cell membrane 6x more often than periosteal fibroblasts and 3x more often than osteoblasts. Treatment with shear stress reduced the number of osteocytes that exposed PS by 90%, but did not affect the other cell types. Fluid shear stress of increasing magnitude, dose-dependently stimulated Bcl-2 mRNA expression in human bone cells, while shear stress did not change Bax expression. These data suggest that disuse promotes osteocyte apoptosis, while mechanical stimulation by fluid shear stress promotes osteocyte survival, by modulating the Bcl-2/Bax expression ratio.     

Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: II. Investigation of the ManNAc kinase functionality

N-Acetylmannosamine (ManNAc) is the physiological precursors to all sialic acids that occur in nature. As variations in the sialic acid decoration of cell surfaces can profoundly affect cell-cell, pathogen-cell, or drug-cell interactions, the enzymes that convert ManNAc into sialic acid are attractive targets for the development of drugs that specifically interrupt sialic acid biosynthesis or lead to modified sialic acids on the surface of cells. The first step in the enzymatic conversion of ManNAc into sialic acid is phosphorylation, yielding N-acetylmannosamine-6-phosphate. The enzyme that catalyzes this conversion is the N-acetylmannosamine kinase (ManNAc kinase) as part of the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Here, we employed saturation transfer difference (STD) NMR experiments to study the binding of ManNAc and related ligands to the ManNAc kinase. It is shown that the configuration of C1 and C4 of ManNAc is crucial for binding to the enzyme, whereas the C2 position not only accepts variations in the attached N-acyl side chain but also tolerates inversion of configuration. Our experiments also show that ManNAc kinase maintains its functionality, even in the absence of Mg(2+). From the analysis of the STD NMR-derived binding epitopes, it is concluded that the binding mode of the N-acylmannosamines critically depends on the N-acyl side chain. In conjunction with the relative binding affinities of the ligands obtained from STD NMR titrations, it is possible to derive a structure-binding affinity relationship. This provides a cornerstone for the rational design of drugs for novel therapeutic applications by altering the sialic acid decorations of cell walls.     

Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: I. Investigation of the UDP-GlcNAc 2-epimerase functionality

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme for the biosynthesis of sialic acids. As terminal components of glycoconjugates, sialic acids are associated with a variety of pathological processes such as inflammation and cancer. For the first time, this study reveals characteristics of the interaction of the epimerase site of the enzyme with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives thereof at atomic resolution. Saturation transfer difference NMR experiments were crucial in obtaining ligand binding epitopes and to rank ligands according to their binding affinities. Employing a fragment based approach, it was possible to assign the major component of substrate recognition to the UDP moiety. In particular, the binding epitopes of the uridine moieties of UMP, UDP, UDP-GalNAc, and UDP-GlcNAc are rather similar, suggesting that the binding mode of the UDP moiety is the same in all cases. In contrast, the hexopyranose units of UDP-GlcNAc and UDP-GalNAc display small differences reflecting the inability of the enzyme to process UDP-GalNAc. Surprisingly, saturation transfer difference NMR titrations show that UDP has the largest binding affinity to the epimerase site and that at least one phosphate group is required for binding. Consequently, this study provides important new data for rational drug design.     

Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects

Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBDelta/Delta mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junBDelta/Delta osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16(INK4a) levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage-osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity.     

A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis

Measures of parasitemia by intraerythrocytic hematozoan parasites are normally expressed as the number of infected erythrocytes per n erythrocytes and are notoriously tedious and time consuming to measure. We describe a protocol for generating rapid counts of nucleated erythrocytes from digital micrographs of thin blood smears that can be used to estimate intensity of hematozoan infections in nonmammalian vertebrate hosts. This method takes advantage of the bold contrast and relatively uniform size and morphology of erythrocyte nuclei on Giemsa-stained blood smears and uses ImageJ, a java-based image analysis program developed at the U.S. National Institutes of Health and available on the internet, to recognize and count these nuclei. This technique makes feasible rapid and accurate counts of total erythrocytes in large numbers of microscope fields, which can be used in the calculation of peripheral parasitemias in low-intensity infections.