Fluorescence correlation spectroscopy analysis of the hydrophobic interactions of protein 4.1 with phosphatidyl serine liposomes

Fluorescence correlation spectroscopy (FCS) was applied to examine the interactions between a protein and a membrane lipid. The protein 4.1-phosphatidyl serine (PS) interactions served as the model system to demonstrate the membrane lipid-protein interactions. This protein was labeled with rhodamine and its interactions with PS-liposomes were measured by FCS. The present results clearly demonstrated that a small protein molecule, protein 4.1, interacts specifically with a large particle, a PS-liposome. This interaction appears to be hydrophobic and not electrostatic, since the bound protein 4.1 did not dissociate in solution and was specifically released from PS-liposomes by treatment with phospholipase A(2) (PLA(2)). In the present study, using FCS we could demonstrate that the serine residue of PS is required for protein 4.1 to bind to PS-liposomes and that the bound protein 4.1 is closely associated with the fatty acid of the PS molecule in the liposomes.     

VHY, a novel myristoylated testis-restricted dual specificity protein phosphatase related to VHX

The human DUSP15 gene encodes an uncharacterized 235-amino acid member of the subfamily of small dual specificity protein phosphatases related to the Vaccinia virus VH1 phosphatase. Similar to VHR-related MKPX (VHX) (DUSP22), the predicted protein has an N-terminal myristoylation recognition sequence, and we show here that both are indeed modified by the attachment of a myristate to Gly-2. In recognition of this relatedness to VHX, we refer to the DUSP15-encoded protein as VH1-related member Y (VHY). We report that VHY is expressed at high levels in the testis and barely detectable levels in the brain, spinal cord, and thyroid. A VHY-specific antiserum detected a protein with an apparent molecular mass of 26 kDa, and histochemical analysis showed that VHY was readily detectable in pachytene spermatocytes (midstage of meiotic division I) and round spermatids and weakly in Leydig cells (somatic cells outside of the seminiferous tubules). When expressed in 293T or NIH-3T3 cells, VHY was concentrated at the plasma membrane with some staining of vesicular structures in the Golgi region. Mutation of the myristoylation site Gly-2 abrogated membrane location. Finally, we demonstrate that VHY is an active phosphatase in vitro. We conclude that VHY is a new member of a subgroup of myristoylated VH1-like small dual specificity phosphatases.     

Scanning mutagenesis of the alpha repeats and of the transmembrane acidic residues of the human retinal cone Na/Ca-K exchanger

The Na/Ca-K exchanger (NCKX) utilizes the inward sodium gradient and outward potassium gradient for Ca(2+) extrusion; two distinct NCKX isoforms are expressed in the outer segments of retinal rod (NCKX1) and cone (NCKX2) photoreceptors, respectively, where NCKX extrudes Ca(2+) that enters photoreceptors via the cGMP-gated channels. We carried out the first systematic NCKX mutagenesis study in which 96 residues were mutated in the human cone NCKX2 cDNA, and functional consequences of these mutations were measured; the residues selected for mutagenesis are conserved between rod and cone NCKX, the large majority are also conserved in NCKX paralogs found in lower organisms, and finally, they include the few residues conserved between members of the NCKX and members of the NCX (potassium-independent Na/Ca exchange) gene families. Twenty-five residues were identified for which mutagenesis reduced NCKX function to <20% of wild-type cone NCKX2 activity, while protein expression and plasma membrane targeting were not affected. Three classes of residues were found to be most sensitive toward mutagenesis: acidic (glutamate/aspartate) residues, polar (serines/threonine) residues, and glycine residues. These results are discussed with respect to residues that may contribute to the NCKX cation binding site(s).     

PIG-W is critical for inositol acylation but not for flipping of glycosylphosphatidylinositol-anchor

Many cell surface proteins are anchored to a membrane via a glycosylphosphatidylinositol (GPI), which is attached to the C termini in the endoplasmic reticulum. The inositol ring of phosphatidylinositol is acylated during biosynthesis of GPI. In mammalian cells, the acyl chain is added to glucosaminyl phosphatidylinositol at the third step in the GPI biosynthetic pathway and then is usually removed soon after the attachment of GPIs to proteins. The mechanisms and roles of the inositol acylation and deacylation have not been well clarified. Herein, we report derivation of human and Chinese hamster mutant cells defective in inositol acylation and the gene responsible, PIG-W. The surface expressions of GPI-anchored proteins on these mutant cells were greatly diminished, indicating the critical role of inositol acylation. PIG-W encodes a 504-amino acid protein expressed in the endoplasmic reticulum. PIG-W is most likely inositol acyltransferase itself because the tagged PIG-W affinity purified from transfected human cells had inositol acyltransferase activity and because both mutant cells were complemented with PIG-W homologs of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The inositol acylation is not essential for the subsequent mannosylation, indicating that glucosaminyl phosphatidylinositol can flip from the cytoplasmic side to the luminal side of the endoplasmic reticulum.     

Distribution of aquaporin 1 in the rat pancreatic duct system examined with light- and electron-microscopic immunohistochemistry

The pancreatic duct is the major site for the secretion of pancreatic fluid, but the pathway of water transport in this system is not known. Recently, intense signal for mRNA of aquaporin 1 (AQP1) water channels was detected in isolated rat interlobular ducts. Therefore, we performed light- and electron-microscopic (EM) immunohistochemistry for AQP1 in the rat pancreatic ducts. AQP1 immunoproducts were not observed in the acinar cells, centroacinar cells or intercalated ducts. In the smaller intralobular ducts less than 10 microm in diameter (the lumen plus duct cells), most cells were immunonegative. AQP1-positive cells appeared in intralobular ducts 10-15 microm in diameter. In small and medium-sized interlobular ducts 15-70 microm in diameter surrounded by periductal connective tissue 2-40 microm thick, most cells were AQP1 positive with various degrees of immunoreactivity. In the larger interlobular ducts, the expression of AQP1 was variable, ranging from immunopositive to negative. In the main pancreatic duct, most cells were negative for AQP1. EM immunohistochemistry of the intralobular and small interlobular ductal epithelial cells showed that the AQP1 immunoproducts were more abundant in the basolateral membrane than in the apical membrane, though they were present in both membranes. In the medium-sized interlobular ducts, AQP1 immunoproducts were distributed densely along the apical, lateral interdigitation and basal membrane of the epithelial cells. In the various sizes of interlobular ducts, immunoproducts were associated not only with the plasma membrane, but also with the caveolae and vesicle-like structures. Secretin did not induce any significant difference in AQP1 expression and cellular and subcellular localization. These results indicate that the expression and subcellular localization of AQP1 vary considerably depending on the duct size, which may reflect water transport characteristics in the different divisions of the pancreatic duct system.     

Contribution of IL-18 to Th1 response and host defense against infection by Mycobacterium tuberculosis: a comparative study with IL-12p40

The present study was conducted to critically determine the protective role of IL-18 in host response to Mycobacterium tuberculosis infection. IL-18-deficient (knockout (KO)) mice were slightly more prone to this infection than wild-type (WT) mice. Sensitivity of IL-12p40KO mice was lower than that of IL-12p40/IL-18 double KO mice. IFN-gamma production caused by the infection was significantly attenuated in IL-18KO mice compared with WT mice, as indicated by reduction in the levels of this cytokine in sera, spleen, lung, and liver, and its synthesis by spleen cells restimulated with purified protein derivatives. Serum IL-12p40 level postinfection and its production by peritoneal exudate cells stimulated with live bacilli were also significantly lower in IL-18KO mice than WT mice, suggesting that attenuated production of IFN-gamma was secondary to reduction of IL-12 synthesis. However, this was not likely the case, because administration of excess IL-12 did not restore the reduced IFN-gamma production in IL-18KO mice. In further studies, IL-18 transgenic mice were more resistant to the infection than control littermate mice, and serum IFN-gamma level and its production by restimulated spleen cells were increased in the former mice. Taken together, our results indicate that IL-18 plays an important role in Th1 response and host defense against M. tuberculosis infection although the contribution was not as profound as that of IL-12p40.     

Relation between Reactivity to the NS-4 Region Peptides of Hepatitis C Virus (HCV) and Clinical Features among Patients Infected with HCV Genotype 1b

Nearly all patients infected with hepatitis C virus (HCV) genotype 1b have reactivity to the core (c22-3) or non-structural (NS)-3 region (c33c) protein in a second-generation recombinant immunoblot assay (RIBA-2). However, reactivities to the NS-4 region antigens (5-1-1, c100-3) vary among patients. To clarify whether differences in serological reactivities to the NS-4 antigens are associated with the clinical features or response to interferon (IFN) therapy of patients infected with hepatitis C virus (HCV) genotype 1b, we clinically investigated 115 such patients. Positive reactions to 5-1-1 and c100-3 were seen in 75.7 and 79.1%, respectively, of the patients. There were no differences between the patients with and those without antibodies to NS-4 region antigens (5-1-1, c100-3) with regard to age, duration of HCV infection, severity of liver disease and virus load. Fifty-one of the patients were treated with recombinant IFN-α, and 17 of the 51 patients showed sustained response to the therapy. The sustained response was more frequently seen in the patients positive for antibodies to both 5-1-1 and c100-3 as compared with those negative for either or both antibodies (41.0% vs. 8.3%, P < 0.05).     

Linear and Conformation-Dependent Antigenic Sites on the Nucleoprotein of Rabies Virus

A set of 29 monoclonal antibodies (MAbs) specific for the rabies virus nucleoprotein (N protein) was prepared and used to analyze the topography of antigenic sites. At least four partially overlapping antigenic sites were delineated on the N protein of rabies virus by competitive binding assays. Indirect immunofluorescent antibody tests using MAbs with a series of rabies and rabies-related viruses showed that epitopes shared by various fixed and street strains of rabies virus were mainly localized at antigenic sites II and III, while epitopes representing the genus-specific antigen of Lyssavirus were widely presented at sites I, III and IV. All but one of seven MAbs specific for antigenic sites I, IV and bridge site (I and II) reacted with the antigen that had been denatured by sodium dodecyl sulfate or 2-mercaptoethanol, as well as with the denatured N protein in Western blotting assays. However, none of the MAbs against antigenic sites II and III reacted with the denatured antigen. These data indicate that antigenic sites I and IV, and sites II and III on the N protein of rabies virus are composed of linear and conformation-dependent epitopes, respectively.     

A Unique Mutation of Glycoprotein Gene of the Attenuated RC-HL Strain of Rabies Virus,a Seed Virus Used for Production of Animal Vaccine in Japan

Although the RC-HL strain of rabies virus is avirulent in adult mice, the amino acid at position 333 of its G protein is arginine, which is thought to be necessary for virulence in adult mice upon intracerebral inoculation of the virus. This result suggests that besides arginine at position 333, some other positions of G protein might also be involved in determining the virulence of rabies virus.     

Demonstration of W chromosome-specific repetitive DNA sequences in the domestic fowl,Gallus g. domesticus

Evidence is presented to demonstrate the presence of W chromosome-specific repetitive DNA sequences in the female White Leghorn chicken, Gallus g. domesticus, based on two different experimental approaches. First, ³H-labelled, female chicken DNA was hybridized with excess, unlabelled, mercurated, male DNA, and unhybridized single-stranded ³H-DNA (³H-SHU-DNA) was recovered by SH-Sepharose and hydroxyapatite column chromatography. Approximately 24% of the hybridizable ³H-SHU-DNA was female-specific and localized on the W chromosome. The second approach was to examine female-specific DNA fragments among the digests of chicken DNA with various restriction endonucleases. Among them, we found that digestion with XhoI produced two prominent female-specific bands of 0.60 kb (= kilobase pairs) and 1.1 kb. The 0.60 kb fragment was isolated and ³H-labelled by nick-translation. Female-specificity of the ³H-XhoI—0.60 kb DNA was judged to be at least 95% under the conditions of hybridization with membrane filter-bound DNA. Presence of amplified XhoI—0.60 kb DNA on the W chromosome seems to be limited to different lines of G. g. domesticus and no such repeat was detected in three species belonging to other genera in the order Galliformes and in three species belonging to other avian orders.