Human galanin expresses amphipathic properties that modulate its vasoreactivity in vivo

The purpose of this study was to determine whether human galanin, a pleiotropic 30-amino acid neuropeptide, expresses amphipathic properties in vitro and, if so, whether these properties modulate its vasoactive effects in the intact peripheral microcirculation. We found that human galanin aggregates in an aqueous solution and forms micelles with a critical micellar concentration (CMC) of 0.4 microM. In addition, the peptide interacted with model membrane as indicated by long and significant increase of the surface pressure of the biomimetic monolayer membrane in vitro. Interactions of human galanin with sterically stabilized phospholipid micelles (SMM) were not associated with a significant change in peptide conformation. Using intravital microscopy, we found that suffusion of human galanin alone elicited significant concentration-dependent vasoconstriction in the intact hamster cheek pouch. This response was amplified when human galanin in SSM was suffused onto the cheek pouch. The effects of human galanin alone and in SSM were mediated by galanin receptors because galantide, a galanin receptor antagonist, abrogated galanin-induced vasoconstriction. Collectively, these data show that human galanin expresses amphipathic properties in the presence of phospholipids which in turn amplifies its vasoactive effects in the intact peripheral microcirculation.     

Conformation-dependent effects of VIP on nociception in rats

The purpose of this study was to determine whether intrathecal injection of aqueous (random coil) vasoactive intestinal peptide (VIP) and VIP self-associated with sterically stabilized phospholipid micelles (alpha-helix VIP) at the lower lumbar vertebral level modulates foot withdrawal latency to low and high rate noxious radiant skin heating in anesthetized rats. We found that intrathecal random coil VIP evoked a significant bimodal, concentration-dependent response, early potent antinociception followed by hyperalgesia, during exposure to low and high rates of skin heating (P<0.05). Intrathecal alpha-helix VIP elicited a qualitatively similar response to that of random coil VIP except that the rate of decay of antinociception was faster and slower at low and high rates of skin heating, respectively. In addition, a low concentration of alpha-helix VIP evoked a potent late antinociception not observed with random coil VIP. Taken together, these data indicate that VIP modulates somatosensory processing in the lumbosacral spinal cord of rats in a complex fashion, and that this response is dependent, in part, on the conformation of VIP in the vicinity of target cells in the peripheral nervous system.     

Transmembrane domains in the functions of Fc receptors

In the present study, we use a novel method, PHDhtm, to predict the exact locations and extents of the transmembrane (TM) domains of multisubunit immunoglobulin Fc-receptors. Whereas most previous studies have used single residue hydrophobicity plots for characterizing of these domains, PHDhtm utilizes a system of neural networks and the evolutionary information contained in multiple alignments of related sequences to predict the above. Present PHDhtm application predicts TM domains of immunoglobulin Fc-receptors that in many cases differ significantly from those derived by using earlier methods. Comparisons of helical wheel projections of the presently derived TM domains from PHDhtm with those produced earlier reveal different hydrophobic moments as well as hydrophobic and hydrophilic surfaces. These differences probably alter the character of subunit association within the receptor complexes. This new algorithm can also be used for other membrane protein complexes and may advance both understanding the principles underlying such complexes formation and design of peptides that can interfere with such TM domain association so as to modulate specific cellular responses.     

EspH,a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. During infection, EPEC and EHEC use a type III secretion system (TTSS) to translocate effector proteins into the infected cells and thereby modify specific host functions. These include transient filopodium formation which is Cdc42-dependent. Filopodia formation is followed by assembly of actin pedestals, the process enhanced by inhibition of Cdc42. We discovered that orf 18 of the enterocyte effacement locus encodes a new effector, which we termed EspH. We show that EspH is translocated efficiently into the infected cells by the TTSS and localizes beneath the EPEC microcolonies. Inactivation of espH resulted in enhanced formation of filopodia and attenuated the pedestals formation. Furthermore, overexpression of EspH resulted in strong repression of filopodium formation and heightened pedestal formation. We also demonstrate that overexpression of EspH by EHEC induces marked elongation of the typically flat pedestals. Similar pedestal elongation was seen upon infection of COS cells overexpressing EspH. EspH transiently expressed by the COS cells was localized to the membrane and disrupted the actin cytoskeletal structure. Our findings indicate that EspH is a modulator of the host actin cytoskeleton structure.     

Ovalbumin increases macromolecular efflux from the in situ nasal mucosa of allergic hamsters

Gao, Xiao-Pei, Syed R. Akhter, and Israel Rubinstein.Ovalbumin increases macromolecular efflux from the in situ nasal mucosa of allergic hamsters. J. Appl.Physiol. 84(1): 169-176, 1998.The purpose ofthis study was to determine whether bradykinin mediatesovalbumin-induced increase in macromolecular efflux from the nasalmucosa of ovalbumin-sensitized hamsters in vivo and, if so, whether theL-arginine/nitric oxidebiosynthetic pathway transduces, in part, this response. We found thatsuffusion of ovalbumin onto the in situ nasal mucosa ofovalbumin-sensitized hamsters, but not of controls, elicited asignificant time- and concentration-dependent increase in clearance offluorescein isothiocyanate-labeled dextran (mol mass, 70 kDa;P < 0.05). HOE-140, but notdes-Arg⁹,[Leu⁸]-bradykinin,andN^G-L-argininemethyl ester (L-NAME), but notN^G-D-argininemethyl ester, significantly attenuated ovalbumin-induced responses.L-Arginine, but notD-arginine, abolished the effects ofL-NAME.L-NAME also significantlyattenuated bradykinin-, but not adenosine- induced increase inmacromolecular efflux from the in situ nasal mucosa. Overall, thesedata suggest that ovalbumin increases macromolecular efflux from the insitu nasal mucosa of ovalbumin-sensitized hamsters, in part, byproducing bradykinin with subsequent activation of theL-arginine/nitric oxidebiosynthetic pathway.

     

A DJ-1 Based Peptide Attenuates Dopaminergic Degeneration in Mice Models of Parkinson's Disease via Enhancing Nrf2

Drugs currently used for treating Parkinson''s disease patients provide symptomatic relief without altering the neurodegenerative process. Our aim was to examine the possibility of using DJ-1 (PARK7), as a novel therapeutic target for Parkinson''s disease. We designed a short peptide, named ND-13. This peptide consists of a 13 amino acids segment of the DJ-1-protein attached to 7 amino acids derived from TAT, a cell penetrating protein. We examined the effects of ND-13 using in vitro and in vivo experimental models of Parkinson''s disease. We demonstrated that ND-13 protects cultured cells against oxidative and neurotoxic insults, reduced reactive oxygen species accumulation, activated the protective erythroid-2 related factor 2 system and increased cell survival. ND-13 robustly attenuated dopaminergic system dysfunction and in improved the behavioral outcome in the 6-hydroxydopamine mouse model of Parkinson''s disease, both in wild type and in DJ-1 knockout mice. Moreover, ND-13 restored dopamine content in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model. These findings validate DJ-1 as a promising therapeutic target in Parkinson''s disease and identify a novel peptide with clinical potential, which may be significant for a broader range of neurological diseases, possibly with an important impact for the neurosciences.     

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda,Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes.Extensive hemocyte aggregates (''islets'') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.