The Gly-Gly linker region of the insect cytokine growth-blocking peptide is essential for activity

Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe3) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe3 is a binding determinant of the N-terminal domain.     

Age-related macular degeneration: a high-resolution genome scan for susceptibility loci in a population enriched for late-stage disease

Age-related macular degeneration (AMD) is a complex multifactorial disease that affects the central region of the retina. AMD is clinically heterogeneous, leading to geographic atrophy (GA) and/or choroidal neovascularization (CNV) at advanced stages. Considerable data exists in support of a genetic predisposition for AMD. Recent linkage studies have provided evidence in favor of several AMD susceptibility loci. We have performed a high-resolution (5-cM) genome scan of 412 affected relative pairs that were enriched for late-stage disease (GA and/or CNV). Nonparametric linkage analysis was performed using two different diagnostic criteria and also by dividing the affected individuals according to GA or CNV phenotype. Our results demonstrate evidence of linkage in regions that were suggested in at least one previous study at chromosomes 1q (236-240 cM in the Marshfield genetic map), 5p (40-50 cM), and 9q (111 cM). Multipoint analysis of affected relatives with CNV provided evidence of additional susceptibility loci on chromosomes 2p (10 cM) and 22q (25 cM). A recently identified Gln5345Arg change in HEMICENTIN-1 on chromosome 1q25 was not detected in 274 affected members in the restricted group with AMD, 346 additional patients with AMD, and 237 unaffected controls. Our results consolidate the chromosomal locations of several AMD susceptibility loci and, together with previous reports, should facilitate the search for disease-associated sequence variants.     

Receptor-operated Ca2+ influx and its association with the Src family in secretagogue-stimulated pancreatic acini

We investigated signal transduction between receptor-operated Ca(2+) influx (ROCI) and Src-related nonreceptor protein tyrosine kinase (PTK) in rat pancreatic acini. CCK and the Ca(2+) ionophore enhanced the Src-related PTK activity, whereas the high-affinity CCK-A receptor agonists, fibroblast growth factor (FGF), and the protein kinase C (PKC) activator had no or little effect. This increase was abolished by eliminating [Ca(2+)](o), loading of the intracellular Ca(2+) chelator, and administering the PTK inhibitor genistein. While genistein inhibited extracellular Ca(2+) or Mn(2+) entry induced by CCK and carbachol, it did not affect intracellular Ca(2+) release and oscillations. CCK dose-dependently increased the Src phosphotransferase activity, which was abolished by inhibitors of G(q) protein, phospholipase C (PLC), and Src, but not by the calmodulin kinase (CaMK) inhibitor. Intensities of the Src band and amounts of tyrosine phosphorylated Src were enhanced by CCK stimulation. Thus, Src cascades appear to be coupled to the low-affinity CCK-A receptor and utilize G(q)-PLC pathways for their activation, independent of PKC and CaMK cascades. The low-affinity CCK-A receptor regulates ROCI via mediation of Src-related PTK and activates Src pathways to cause [Ca(2+)](o)-dependent pancreatic exocytosis.     

Lipoxygenase-catalyzed polymerization of phenolic lipids suggests a new mechanism for allergic contact dermatitis induced by urushiol and its analogs

Lipoxygenase was found to catalyze the oxidative polymerization of phenolic lipids containing a (Z,Z)-pentadiene in the side chain, the model compounds of urushiol and its analog, yielding methanol-soluble and insoluble polymers. The structural analysis of the resulted polymers suggested that the polymerization occurred at both the phenol and the unsaturated side chain. The key step of the polymerization was the generation of the hydroperoxide at the unsaturated side chain by lipoxygenase. The decomposition of hydroperoxide and concomitant dehydrogenation of phenol ring catalyzed by lipoxygenase might produce radicals that could be coupled to form cross-linked polymers. This lipoxygenase-mediated reaction implies a new mechanism for contact allergy of urushiol and its analogs.     

beta-Helix is a likely core structure of yeast prion Sup35 amyloid fibers

Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.     

Peptide binding to Geminin and inhibitory for DNA replication

Geminin binds to Cdt1 to ensure that DNA replication occurs only once during the cell cycle. To identify the peptide that binds to Geminin and thereby modifies the latter's ability to alter the DNA replication activity in human cancer cells, we screened a phage display library of random peptides in successive cycles of phage library panning and found one peptide sequence that bound to the 31-111 amino acid residues of Geminin. Delivery of this peptide sequence into the nucleus of HCT116 human colon cancer cells resulted in the suppression of BrdU incorporation. These results provide new insights into the function of Geminin and further validate Geminin as a potential therapeutic target in tumors.     

Identification and characterization of taxilin isoforms

The syntaxin family is implicated in intracellular vesicle traffic. We have recently identified taxilin, a novel syntaxin-binding protein, which has a long coiled-coil region in its C-terminal half. A database search has revealed the presence of two other molecules having a long coiled-coil region homologous to that of taxilin in mammals. Then, we here attempted to isolate and characterize the two molecules. Both the two molecules stoichiometrically interacted with several syntaxin family members. Then, we renamed original taxilin alpha-taxilin and named the two molecules beta- and gamma-taxilins, respectively. Beta-taxilin was a human homologue of chicken MDP77. Gamma-taxilin was an uncharacterized protein and Northern blot analysis revealed that gamma-taxilin was ubiquitously expressed. Beta- and gamma-taxilins preferentially interacted with syntaxin-1a and -4, respectively. The taxilin family members mutually interacted with the syntaxin family members. These results indicate that there is the taxilin family composed of at least three members in mammals.     

Comparative Analysis of the Ribosomal Componentsof the Hydrogenosome-Containing Protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.     

Circulation of type 1 vaccine-derived poliovirus in the Philippines in 2001

In 2001, highly evolved type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from three acute flaccid paralysis patients and one contact from three separate communities in the Philippines. Complete genomic sequencing of these four cVDPV isolates revealed that the capsid region was derived from the Sabin 1 vaccine strain but most of the noncapsid region was derived from an unidentified enterovirus unrelated to the oral poliovirus vaccine (OPV) strains. The sequences of the cVDPV isolates were closely related to each other, and the isolates had a common recombination site. Most of the genetic and biological properties of the cVDPV isolates were indistinguishable from those of wild polioviruses. However, the most recently identified cVDPV isolate from a healthy contact retained the temperature sensitivity and partial attenuation phenotypes. The sequence relationships among the isolates and Sabin 1 suggested that cVDPV originated from an OPV dose given in 1998 to 1999 and that cVDPV circulated along a narrow chain of transmission. Type 1 cVDPV was last detected in the Philippines in September 2001, and population immunity to polio was raised by extensive OPV campaigns in late 2001 and early 2002.     

Glycyrrhizin inhibits neutrophil-associated generation of alternatively activated macrophages

Patients with severe burn injuries are extremely susceptible to infection, and the host's antibacterial responses are frequently suppressed by alternatively activated macrophages (M2Mphi), commonly demonstrated in these patients. An immunosuppressive subset of neutrophils (PMN-II), demonstrated in the peripheral blood of thermally injured patients, has been described as an inducer of M2Mphi. In the present studies, the inhibitory effect of glycyrrhizin (GL) on M2Mphi generation stimulated by PMN-II was examined. M2Mphi were generated from resident Mphi (R-Mphi, lower chamber) after cultivation with PMN-II (upper chamber) in a dual-chamber transwell. However, M2Mphi were not generated from R-Mphi when the same transwell cultures were performed in the presence of GL. M2Mphi were not generated from R-Mphi after cultivation with PMN-II previously treated with GL, while R-Mphi previously treated with GL converted to M2Mphi after they were cultured with PMN-II in transwells. Interleukin-10 and CCL2 released from PMN-II were shown to be effector molecules responsible for the generation of M2Mphi. However, these soluble factors were not produced by PMN-II treated with GL. These results indicate that GL inhibits PMN-II-stimulated M2Mphi generation through the inhibition of CCL2/interleukin-10 production by PMN-II.