Neurokinin A and neurokinin B in the human retina

Very recently, the authors found levels of neurokinin (NK) A-like immunoreactivities in the human retina which were more than five times higher than those of substance P (SP). The present study aimed to find out how many of these immunoreactivities can be attributed to NKA and NKB and then the exact distribution pattern of both NKA and NKB was evaluated in the human retina and compared with that of SP. For this purpose, NKA-like immunoreactivities were characterized in the human retina by reversed phase HPLC followed by radioimmunoassay using the K12 antibody which recognizes both NKA and NKB. Furthermore, the retinae from both a 22- and 70-year-old donor were processed for double-immunofluorescence NKA/SP and NKB/SP. The results showed that NKA contributes to approximately two thirds and NKB to approximately one third of the immunoreactivities measured with the K12 antibody. NKA was found to be localized in sparse amacrine cells in the proximal inner nuclear layer, in displaced amacrine cells in the ganglion cell layer with processes ramifying in stratum 3 of the inner plexiform layer and also in sparse ganglion cells. By contrast, staining for NKB was only observed in ganglion cells and in the nerve fiber layer. Double-immunofluorescence revealed cellular colocalization of NKA with SP and also of NKB with SP. Thus, the levels of NKA and NKB are more than three and two times higher than those of SP, respectively. Whereas the distribution pattern of NKA is typical for neuropeptides, the localization of NKB exclusively in ganglion cells is atypical and unique.     

Cyclic AMP inhibits p38 activation via CREB-induced dynein light chain

The mitogen-activated protein kinase p38 plays a critical role in inflammation, cell cycle progression, differentiation, and apoptosis. The activity of p38 is stimulated by a variety of extracellular stimuli, such as the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), and subjected to regulation by other intracellular signaling pathways, including the cyclic AMP (cAMP) pathway. Yet the underlying mechanism by which cAMP inhibits p38 activation is unknown. Here we show that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of p38 activation. cAMP inhibits p38 activation via the protein kinase A-CREB pathway. The inhibition is mediated by the CREB target gene Dlc, whose protein product, DLC, interferes with the formation of the MKK3/6-p38 complex, thereby suppressing p38 phosphorylation activation by MKK3/6. The inhibition of p38 activation by cAMP leads to suppression of NF-kappaB activity and promotion of apoptosis in response to TNF-alpha. Thus, our results identify DLC as a novel inhibitor of the p38 pathway and provide a molecular mechanism by which cAMP suppresses p38 activation and promotes apoptosis.     

Supported bilayer lipid membranes modified with a phosphate ionophore

This article reports the electrical responses of a phosphate ionophore, the cyclic polyamine 3-decyl-1,5,8-triazacyclodecane-2,4-dione (N₃-cyclic amine) incorporated into metal supported bilayer lipid membranes (s-BLM). Teflon coated silver wire was used as a support. In a potentiometric mode, the ionophore had a response that was linearly related to the logarithm of HPO₄²⁻ concentration and was also dependant on pH. Selectivity coefficients for other anions compared to HPO₄²⁻ ions, determined by the separate solution method, fell within the range 1.73 × 10⁻⁴ to 6.38 × 10⁻².     

Solution Structure of Factor I-like Modules from Complement C7 Reveals a Pair of Follistatin Domains in Compact Pseudosymmetric Arrangement

Factor I-like modules (FIMs) of complement proteins C6, C7, and factor I participate in protein-protein interactions critical to the progress of a complement-mediated immune response to infections and other trauma. For instance, the carboxyl-terminal FIM pair of C7 (C7-FIMs) binds to the C345C domain of C5 and its activated product, C5b, during self-assembly of the cytolytic membrane-attack complex. FIMs share sequence similarity with follistatin domains (FDs) of known three-dimensional structure, suggesting that FIM structures could be reliably modeled. However, conflicting disulfide maps, inconsistent orientations of subdomains within FDs, and the presence of binding partners in all FD structures led us to determine the three-dimensional structure of C7-FIMs by NMR spectroscopy. The solution structure reveals that each FIM within C7 contains a small amino-terminal FOLN subdomain connected to a larger carboxyl-terminal KAZAL domain. The open arrangement of the subdomains within FIMs resembles that of first FDs within structures of tandem FDs but differs from the more compact subdomain arrangement of second or third FDs. Unexpectedly, the two C7-FIMs pack closely together with an approximate 2-fold rotational symmetry that is rarely seen in module pairs and has not been observed in FD-containing proteins. Interfaces between subdomains and between modules include numerous hydrophobic and electrostatic contributions, suggesting that this is a physiologically relevant conformation that persists in the context of the parent protein. Similar interfaces were predicted in a homology-based model of the C6-FIM pair. The C7-FIM structures also facilitated construction of a model of the single FIM of factor I.The membrane attack complex (MAC)² is the terminal product of the complement cascade and is therefore a fundamental component of mammalian innate immunity. The formation of this multi-protein complex is triggered by proteolytic cleavage of complement component C5. This is followed swiftly by a remarkable, although little understood, self-assembly process involving multiple sequential protein-protein recognition events. MAC assembly culminates in the formation of a pore traversing the targeted cell membrane (1). Accumulation of multiple MACs in a membrane triggers cell-dependent responses and may result in cell lysis (2). The key to progress in understanding MAC formation will be three-dimensional structural information for each of its component proteins, namely C5b, C6, C7, C8, and C9.Classical, alternative, and lectin pathways of complement activation converge at a step in which C5 is cleaved to release activated C5b. Immediately following C5b formation, C6 and C7 bind sequentially; the C5b6 complex is soluble and relatively stable (3), but soluble C5b67 has a brief half-life and is proposed to attach rapidly to target membrane surfaces (4, 5). Subsequently, C8 binds to the nascent complex, inserting into the target membrane and causing disruptive rearrangements of the lipid bilayer. Finally the mature MAC, C5b6789_n, forms by recruitment of between 10 and 16 copies of C9 that insert in the membrane to form the pore. Notably, once C5b is generated, MAC assembly requires no additional enzymatic triggers; this implies that individual components encompass highly specific, complementary binding sites that become exposed during MAC formation.Complement proteins C6, C7, C8 (α and β subunits), and C9 comprise the “MAC family” (Fig. 1a) (6). Family members share, in addition to a large central membrane attack complex perforin domain (7–9), several tandemly arranged, cysteine-rich modules of less than 80 amino acid residues each. These smaller modules include thrombospondin type I (10), low density lipoprotein receptor class A (11) and modules similar in sequence to epidermal growth factor (Fig. 1a). C6 and C7 each contain an additional four modules at their carboxyl termini: two ∼60-residue complement control protein modules (12, 13), followed by two cysteine-rich modules composed of ∼75 residues each; these are the factor I-like modules (FIMs) (also known as factor I membrane attack complex domains (14, 15)), so named because of their apparent relatedness to an amino-terminal domain of complement factor I (fI) (Fig. 1b).Open in a separate windowFIGURE 1.Modular composition of the proteins of the membrane attack complex (MAC). a, the MAC family of proteins aligned, domain-wise, with C6. b, the domain structure of fI. The heavy chain contains the amino-terminal domains and the light chain comprises a serine protease domain. An intramolecular disulfide bond between light and heavy chain (Cys³⁰⁹–Cys⁴³⁵) and a proposed interdomain disulfide between the amino-terminal region and first low density lipoprotein domain (Cys¹⁵–Cys²³⁷) are shown as diagonal lines. The domains were defined using the SMART data base (16, 17). TSP, thrombospondin type 1; LDL, low density lipoprotein receptor type A; MACPF, membrane attack complex perforin domain; EGF, epidermal growth factor; CCP, complement control protein; FIM, factor I-like module; CD5, CD5-like; SP, serine protease domain.Latent C5 was shown, in vitro, to bind reversibly to both C6 and C7 prior to activation. These interactions are distinct from and precede irreversible binding of C6 and subsequently C7 to C5b (18). It is hypothesized that the C56 and C57 preactivation complexes ensure that C6 and C7 are maintained proximal to C5 in the plasma. This may be significant because activated C5b is labile (19, 20), hence swift assembly of C5b67 is advantageous. Within this preactivation complex, critical interactions occur between the carboxyl-terminal C345C domain of C5, C5-C345C (21), and the carboxyl-terminal FIM pair of both C6 and C7 (22, 23). The involvement of these domains in MAC formation was demonstrated using recombinant proteins, where either C7-FIMs or C5-C345C inhibited the binding of C7 to C5b6 and inhibited complement-mediated erythrocyte lysis (23). The FIMs of C6, however, although shown to promote MAC assembly, do not appear to be essential for MAC formation (22). C7-FIMs have a stronger affinity than C6-FIMs for C5-C345C, suggesting that C7-FIMs may displace C6-FIMs during MAC assembly (23). Thus, interactions between C5- C345C and FIMs are key to the early assembly of MAC, and their structural basis is an important target of investigations.The structure of the C5-C345C domain is well established (24, 25); however, there has been no three-dimensional structural information available for any of the FIMs or for any other domains within C6 or C7. The closely related FIM within fI has been postulated to resemble a follistatin domain (26). Intriguingly, however, disulfide mapping of human C6 isolated from plasma appeared to exclude that possibility (27). The three-dimensional arrangement of the neighboring FIMs, and the extent of interactions between them, has also been a mystery.We previously described a protein construct comprising the carboxyl-terminal pair of FIMs from human C7 (18), which folds homogeneously and binds to C5 in surface plasmon resonance assays. Here we report the solution structure of this consecutive pair of FIMs. This new structure reveals that, despite previous evidence to the contrary, each FIM adopts a follistatin-like fold, and the two FIMs are intimately associated to form a homodimer-like, pseudosymmetrical carboxyl terminus of C7. This work, therefore, serendipitously provides the first published structure of a follistatin-domain pair in the absence of ligand and suggests that conformational changes within FIM pairs accompany ligand binding. Novel structures of the FIMs from both C6 and fI have been modeled based upon our NMR-derived solution structure of the C7-FIMs.     

Inhibitory activity of prostaglandin E2 production by the synthetic 2′-hydroxychalcone analogues: Synthesis and SAR study

A series of 2′-hydroxychalcones has been synthesized and screened for their in vitro inhibitory activities of cyclooxygenase-2 catalyzed prostaglandin production from lipopolysaccharide-treated RAW 264.7 cells. Structure–activity relationship study suggested that inhibitory activity against prostaglandin E₂ production was governed to a greater extent by the substituent on B ring of the chalcone, and most of the active compounds have at least two methoxy or benzyloxy groups on B ring. The relationship between chalcone structures and their PGE₂ inhibitory activities was also interpreted by docking study on cyclooxygenase-2.     

Proteomic analysis of responses to osmotic stress in laboratory and sake-brewing strains of Saccharomyces cerevisiae

The difference in responses to osmotic stress between the laboratory and sake-brewing strains of Saccharomyces cerevisiae at the translational level was compared by two-dimensional polyacrylamide gel electrophoresis. Proteins, whose production was significantly changed by the osmotic stress, were identified by peptide mass fingerprinting. In the laboratory strain, translation of Hor2p, the protein responsible for glycerol biosynthesis, and Ald6p, related to acetate biosynthesis, was induced under high osmotic pressure conditions. In addition, production of proteins related to translation and stress response was also changed under this condition. On the other hand, in the sake-brewing strain, translation of Hor2p, Hsp26p, and some stress-related proteins was upregulated. The change in the production of enzymes related to glycolysis and ethanol formation was small; however, the production of enzymes related to glycerol formation increased in both strains. These results suggest that enhancement of glycerol formation due to enhancement of the translation of proteins, such as Hor2p, is required for growth of S. cerevisiae under high osmotic pressure condition. This is the first report on the analysis of responses of a sake-brewing strain to high osmotic pressure stress based on proteomics.