Tertiary structural changes associated with iron binding and release in hen serum transferrin: a crystallographic and spectroscopic study

The iron binding and release of serum transferrin are pH-dependent and accompanied by a conformational change between the iron-bound (holo-) and iron-free (apo-) forms. We have determined the crystal structure of apo-hen serum transferrin (hAST) at 3.5A resolution, which is the first reported structure to date of any full molecule of an apo-serum transferrin and studied its pH-dependent iron release by UV-vis absorption and near UV-CD spectroscopy. The crystal structure of hAST shows that both the lobes adopt an open conformation and the relative orientations of the domains are different from those of apo-human serum transferrin and human apolactoferrin but similar to that of hen apo-ovotransferrin. Spectroscopic analysis reveals that in hen serum transferrin, release of the first iron starts at a pH approximately 6.5 and continues over a broad pH range (6.5-5.2). The complete release of the iron, however, occurs at pH approximately 4.0. The near UV-CD spectra show alterations in the microenvironment of the aromatic residues surrounding the iron-binding sites.     

Phylogenetic analysis on genera of Corallobothriinae (Cestoda: Proteocephalidea) from North American ictalurid fishes, using partial sequences of the 28s ribosomal gene

Partial sequences of the 28S rDNA (ribosomal gene) were obtained from a total of 11 populations of 5 species (in 3 genera) of North American corallobothriine proteocephalideans. These included Corallobothrium fimbriatum (3 populations), Corallobothrium parafimbriatum (1 population), Corallotaenia minutia (1 population), Megathylacoides giganteum (2 populations), and Megathylacoides lamothei (4 populations). These sequences were used in a phylogenetic analysis to test the monophyly of Corallobothriinae and to investigate the interrelationships of the North American species. The results indicate that Corallobothriinae, as conventionally understood, is not monophyletic and that only the North American corallobothriines, parasites of ictalurid catfishes, form a monophyletic group. Corallobothrium parafimbriatum is sister taxon to a clade that includes Corallotaenia intermedia and C. minutia and not to its congener C. fimbriatum. Also, M. giganteum from Mexico appears to be more closely related to M. lamothei than to its conspecific in Canada. This and the amount of sequence divergence indicate possible cryptic speciation in its endemic host, the Lerma catfish, Ictalurus dugesi.     

Auriculostoma astyanace n. gen., n. sp. (Digenea: Allocreadiidae), from the banded astyanax, Astyanax fasciatus (Characiformes: Characidae), from Nicaragua, with a reevaluation of neotropical Crepidostomum spp

Auriculostoma n. gen. (Trematoda: Allocreadiidae) is proposed for Auriculostoma astyanace n. sp. from the intestine of the characid fish Astyanax fasciatus in the Atlantic coastal drainages of Nicaragua. The new genus differs from all papillose allocreadiid genera, except Bunoderella, in possessing 2 pairs of muscular oral papillae (instead of 1 or 3), of which the ventrolateral pair is moderately developed and the dorsolateral papillae are long and auricular. Auriculostoma differs from Bunoderella Schell, 1964, in having lateral vitelline follicles, completely separated or confluent only in the posttesticular region, a uterus limited to the pretesticular region or with a few eggs at the level of the testes, and a long cirrus sac that overlaps the acetabulum or usually reaches posteriorly to the ovarian level. Three other allocreadiid species, all from South American freshwater fishes and each of which had previously been placed in Crepidostomum, are transferred to Auriculostoma based on the presence of the diagnostic muscular oral papillae. These include Crepidostomum platense Szidat, 1954, C. macrorchis Szidat, 1954, and C. stenopteri Ma?é-Garzón and Gascón, 1973. Diagnostic features for Auriculostoma also include mainly pretesticular uterus, lateral vitellaria with variation in posttesticular confluence, and tandem testes. The genus appears to be typically associated with neotropical siluriforms (catfishes) and characiforms (tetras).     

Initial cleavage of the human immunodeficiency virus type 1 GagPol precursor by its activated protease occurs by an intramolecular mechanism

Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.     

Genetic control of leaf-blade morphogenesis by the <Emphasis Type="Italic">INSECATUS</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis>

To understand the role of INSECATUS (INS) gene in pea, the leaf blades of wild-type, ins mutant and seven other genotypes, constructed by recombining ins with uni-tac, af, tl and mfp gene mutations, were quantitatively compared. The ins was inherited as a recessive mutant allele and expressed its phenotype in proximal leaflets of full size leaf blades. In ins leaflets, the midvein development was arrested in distal domain and a cleft was formed in lamina above this point. There was change in the identity of ins leaflets such that the intercalary interrupted midvein bore a leaf blade. Such adventitious blades in ins, ins tl and ins tl mfp were like the distal segment of respective main leaf blade. The ins phenotype was not seen in ins af and ins af uni-tac genotypes. There was epistasis of uni-tac over ins. The ins, tl and mfp mutations interacted synergistically to produce highly pronounced ins phenotype in the ins tl mfp triple mutant. The role(s) of INS in leaf-blade organogenesis are: positive regulation of vascular patterning in leaflets, repression of UNI activity in leaflet primordia for ectopic growth and in leaf-blade primordium for indeterminate growth of rachis, delimitation of proximal leaflet domain and together with TL and MFP homeostasis for meristematic activity in leaflet primordia. The variant apically bifid shape of the affected ins leaflets demonstrated that the leaflet shape is dependent on the venation pattern.     

PI 3 kinase-dependent Akt kinase and PKCepsilon independently regulate interferon-gamma-induced STAT1alpha serine phosphorylation to induce monocyte chemotactic protein-1 expression

Monocyte chemotactic protein-1 (MCP-1) recruits activated phagocytes to the site of tissue injury. Interferon-gamma (IFN-gamma) present in the microenvironment of glomerulus acts on mesangial cells to induce local production of MCP-1. The mechanism by which IFN-gamma stimulates expression of MCP-1 is not clear. We therefore examined the role of PI 3 kinase signaling in regulating the IFN-gamma-induced MCP-1 expression in mesangial cells. Blocking PI 3 kinase activity with Ly294002 attenuated IFN-gamma-induced MCP-1 protein and mRNA expression. IFN-gamma increased Akt kinase activity in a PI 3 kinase-dependent manner. Expression of dominant negative Akt kinase inhibited serine phosphorylation of STAT1alpha, without any effect on its tyrosine phosphorylation, and decreased IFN-gamma-induced expression of MCP-1. These data for the first time indicate a role for PI 3 kinase-dependent Akt kinase in MCP-1 expression. We have recently shown that along with Akt, PKCepsilon is a downstream target of PI 3 kinase in IFN-gamma signaling. Similar to dominant negative Akt kinase, dominant negative PKCepsilon also inhibited serine phosphorylation of STAT1alpha without any effect on tyrosine phosphorylation. Dominant negative PKCepsilon also abrogated MAPK activity, resulting in decrease in IFN-gamma-induced MCP-1 expression. Furthermore, Akt and PKCepsilon are present together in a signaling complex. IFN-gamma had no effect on this complex formation, but did increase PKCepsilon-associated Akt kinase activity. PKCepsilon did not regulate IFN-gamma-induced Akt kinase. Finally, expression of dominant negative Akt kinase blocked IFN-gamma-stimulated MAPK activation. These data provide the first evidence that PI 3 kinase-dependent Akt and PKCepsilon activation independently regulate MAPK activity and serine phosphorylation of STAT1alpha to increase expression of MCP-1.     

P311-induced myofibroblasts exhibit ameboid-like migration through RalA activation

We previously showed that P311, an intracellular protein involved in cell migration, is found in human wound myofibroblast precursors (proto-myofibroblasts) and myofibroblasts. Furthermore, by binding to the TGF-beta1 latency associated protein (LAP), P311 induced NIH 3T3 cells to transform into non-fibrogenic myofibroblasts characterized by lack of TGF-beta1 production. Here we demonstrate that P311-induced myofibroblasts migrate in an ameboid rather than a mesenchymal pattern. Ameboid migration is characterized by lack of focal adhesions and stress fibers, absence of integrins and MMPs clustering/activation and changes in small GTPases activity, all leading to increased cell motility. P311-induced ameboid migration depended on activation of the GTPase RalA and was reverted to mesenchymal-type migration by RalA RNA interference. Ameboid migration was conserved in cells plated on fibrin, the initial wound matrix, but was switched back to mesenchymal-type migration by collagen I, the main ECM component in late stages of wound healing. TGF-beta1, the major stimulus of collagen production during wound repair, also reversed the ameboid phenotype to mesenchymal. Our studies therefore suggest that, by inducing RalA activity, P311 promotes a motile proto-myofibroblast and myofibroblast phenotype specifically adapted to rapidly populate the initial wound matrix.     

Two chimeric regulators of G-protein signaling (RGS) proteins differentially modulate soybean heterotrimeric G-protein cycle

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1-4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1-4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks.