Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains

Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane–bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane–bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase that is essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of the two domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to how the C-terminal lipase domain recognizes autophagic bodies to degrade them.     

Phospholamban of cardiac sarcoplasmic reticulum consists of two functionally distinct proteolipids

Phosphorylation of phospholamban by either a cAMP-dependent or a calmodulin-dependent kinase stimulates the Ca²⁺ transporting activity of cardiac sarcoplasmic reticulum membranes. It has now been found that phospholamban consists of 2 distinct proteins; one is the specific substrate for the cAMP-dependent phosphorylation, and the other for the calmodulin-dependent kinase. In spite of functional diversity, the 2 polypeptides share a number of properties. Among them, the proteolipid character, M_r, resistance to trypsinization, and subunit composition.     

Hydrolysis of galactosylceramide is catalyzed by two genetically distinct acid beta-galactosidases

Two genetically distinct acid beta-galactosidases are apparently involved in the hydrolysis of galactosylceramide in fibroblasts. These beta-galactosidases were activated by different bile salts. The classical galactosylceramidase (galactosylceramidase I, EC 3.2.1.46) was activated by sodium taurocholate, while the other galactosylceramidase (galactosylceramidase II) was activated by sodium cholate. The former was genetically lacking in globoid cell leukodystrophy (GLD) and the latter in GM1 gangliosidosis. Galactosylceramidase II cross-reacted with antibody raised against purified GM1 ganglioside beta-galactosidase (EC 3.2.1.23) from the human placenta. The purified beta-galactosidase had galactosylceramidase II activity, which was competitively inhibited by GM1 ganglioside. Thus, galactosylceramidase II seems to be identical to GM1 ganglioside beta-galactosidase and lactosylceramidase II. Galactosylceramidase II had a very low affinity for galactosylsphingosine. In the galactosylceramide-loading tests using fibroblasts from patients with GLD and GM1 gangliosidosis, both cell lines hydrolyzed the incorporated galactosylceramide, with lower rates than control fibroblasts but higher than the fibroblasts from patients with I-cell disease, in which both galactosylceramidase I and II were deficient. These results indicate that galactosylceramide is hydrolyzed by two genetically distinct beta-galactosidases and explain well that galactosylsphingosine but not galactosylceramide accumulates in the brain of patients with GLD.     

Expression of two distinct cytolytic mechanisms among murine CD4 subsets

A TNF (TNF-alpha and TNF-beta)-sensitive target, L929, and two TNF-resistant targets, P815 and LK were used to compare the cytolytic activity among subsets of CD4+ (Th) clones. Cytolytic activity was induced with either Con A, CD3-mAb, or Ag-pulsed LK cells. Six Th1 clones are strongly cytolytic against all three targets. In contrast, Th2 clones are either noncytolytic or weakly cytolytic. Although there is an apparent correlation between TNF production, killing of L929 cells, and the killing of TNF-resistant targets, an anti-TNF serum (capable of neutralizing both TNF-alpha and TNF-beta) selectively inhibits CD4 clones to lyse L929 cells, whereas the lysis of P815 or LK cells was unaffected. The continuous presence of noncytotoxic levels of Actinomycin D (AcD) and cycloheximide, but not mitomycin C, cyclosporin A (CsA), or cholera toxin (ChT) inhibits the lysis of Ag-pulsed, Ia-bearing LK cells; indicating a requirement for de novo synthesis of RNA and protein for cytolytic activity. Although pretreatment with AcD, CsA, or ChT strongly inhibits production of IL-2, TNF and IFN-gamma, only clones pretreated with AcD lose cytolytic activity against Ag-pulsed, Ia-bearing LK cells. These observations support a model of TNF-independent killing of TNF-resistant targets. The TNF-independent cytolytic activity does not correlate with serine esterase activity released into media upon activation of CD4 clones. Moreover, the effects of metabolic inhibitors on serine esterase release do not correlate with their effects on cytolytic activity. Collectively, the data demonstrate that activated CD4 cells express two distinct cytolytic activities; a TNF (and IFN-gamma)-mediated cytotoxicity and a TNF (and IFN-gamma)-independent cytolytic activity. Both pathways require de novo synthesis of RNA and protein and appear to be independent of granule enzyme release. Only the TNF-independent cytolytic activity is resistant to CsA and ChT inhibition.     

Allele and genotype frequencies of polymorphic FMO3 gene in two genetically distinct populations

The aims of this study were to analyze flavin-containing monooxygenase 3 (FMO3) polymorphisms and allele and genotype frequencies in 256 Han Chinese and 50 African-American individuals, to compare the allele and genotype frequencies of these populations with those of other world populations. For Han Chinese, genotyping of three common single nucleotide polymorphisms, E158K, V257M and E308G was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). For African-Americans, genotyping of all coding exons was performed by modified PCR-single strand conformational polymorphism (SSCP). Evolutionary rates of FMO3 were estimated computationally. We found that there were significant differences in allele and genotype frequencies among Han Chinese, African-Americans and other world populations. In Han Chinese, the minor allele frequencies (MAFs) were 0.229 (E158K), 0.203 (V257M) and 0.148 (E308G), respectively. In African-Americans, MAFs were 0.48 (E158K), 0.05 (V257M) and 0 (E308G), respectively. There was rapid evolution during the divergence of primate FMO3. This is the first report comparing FMO alleles and genotypes between Han Chinese and African-Americans. A Han Chinese population database has been established for three gene polymorphisms. The data presented here justify further pharmacogenetic studies for potentially optimizing recommended drug dosages and evaluating relationships with disease processes.     

Comparative studies on the growth dynamics of two genetically distinct isolates of Giardia duodenalis in vitro.

The in vitro growth behaviour of the intestinal protozoan Giardia duodenalis was studied in detail and comparisons were made between two genetically and biologically distinct cloned isolates. Replicates of each clone were grown at six different initial cell concentrations and in culture media at four different pH values. Significant differences in in vitro growth were found between the two isolates, BAH12 and P1. BAH12 had a specific narrow pH requirement, with satisfactory growth only obtained at pH 6. The mean generation time of BAH12 at pH 6 between days 1 and 3 was 10.8 h, compared to an average of 6 h for the same period for P1, both at pH 6 and pH 7. Comparative health of cultures was assessed during both the pH and growth experiments using a suite of six variables. Consistent changes in the health of cultures over time were found to reflect growth behaviour over time. These results provide the first detailed evidence that genetically different isolates of Giardia may differ in such fundamental biological parameters as growth rate and pH requirements. These differences may have important epidemiological and taxonomic implications.     

The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.     

Characterization of two genetically distinct type I-like collagens from lamprey (Entosphenus japonicus)

1. Type I-like collagens were isolated by limited pepsin digestion from various tissues of lamprey, a member of the cyclostomes. 2. Characterization of these collagens revealed the tissue-specific existence of two genetically distinct molecular species, each possessing the typical heterotrimeric nature of (alpha 1)2 alpha 2; one was designated skin collagen which existed in dermis and the other was designated body collagen which was distributed in muscle, intestine and cartilage. 3. The body collagen resembled invertebrate Type I-like collagens in many respects, whereas the skin collagen had a primordial form of vertebrate Type I collagen.     

Role of two distinct gammadelta T cell subsets during West Nile virus infection.

gammadelta T cells respond rapidly following West Nile virus (WNV) infection, limiting viremia and invasion of the central nervous system and thereby protecting the host from lethal encephalitis. Here, we investigated the role of two major subpopulations of peripheral gammadelta T cells, Vgamma1(+) and Vgamma4(+) cells, in host immunity against WNV infection. We found initially that aged mice were more susceptible to WNV infection than young mice. Following WNV challenge, Vgamma1(+) cells in young mice expanded significantly whereas Vgamma4(+) cells expanded modestly. In contrast, aged mice exhibited a slower and reduced response of Vgamma1(+) cells but maintained a higher content of Vgamma4(+) cells. Vgamma1(+) cells were the major gammadelta subset producing IFN-gamma during WNV infection. Mice depleted of Vgamma1(+) cells had an enhanced viremia and higher mortality to WNV encephalitis. Vgamma4(+) cells had a higher potential for producing tumor necrosis factor-alpha (TNF-alpha), a cytokine known to be involved in blood-brain barrier compromise and WNV entry into the brain. Depletion of Vgamma4(+) cells reduced TNF-alpha level in the periphery, accompanied by a decreased viral load in the brain and a lower mortality to WN encephalitis. These results suggest that Vgamma1(+) and Vgamma4(+) cells play distinct roles in protection and pathogenesis during WNV infection.     

A dimer satellite sequence in bonnet monkey DNA consists of distinct monomer subunits

A family of 342 nucleotide fragments was isolated from total bonnet monkey DNA by the restriction endonuclease HaeIII and its base sequence was determined. This family was found to consist of a dimer of two related but distinct nucleotide sequences. Both sequences are closely related to previously reported sequences from African green monkey and human DNA. The two bonnet monkey sequences are unequally divergent from the African green monkey sequence, and have fewer bases in common with each other than they do with African green monkey. Restriction of the dimer with other endonucleases confirms the inequality of the two monomers.     

The receptor for interferon-gamma on human peripheral blood monocytes consists of multiple distinct subunits.

The interaction of interferon-gamma (IFN gamma) (a product of activated T lymphocytes) and monocytes is essential for immune responsiveness, host defense, and chronic inflammation. In this report we define the IFN gamma receptor (IFN gamma R) on human monocytes as a receptor complex consisting of at least three subunits. Solubilization and immunoprecipitation of [35S]methionine- and [35S]cysteine-labeled monocytes were optimized by controlling the detergent concentration during solubilization and washing of the immunoprecipitates. This enabled subunits to be coimmunoprecipitated by several different anti-IFN gamma R antibodies raised against the 90-kDa cloned binding protein. Immunoprecipitation under stringent (1% sodium dodecyl sulfate) conditions resulted in the visualization of only the 80-90-kDa binding protein. Under less stringent conditions at least two coimmunoprecipitated subunits (molecular mass of 200 and 38 kDa) were consistently associated with the 80-kDa (90-92 kDa reduced) binding protein. The 38-kDa subunit was shown to be distinct from the 80-kDa subunit by proteolytic fragment analysis. Cross-linking of 125I-rIFN gamma to monocytes yielded receptor-IFN gamma complexes consistent with the existence of multiple subunits.     

The ebg operon consists of at least two genes

The ebg operon of Escherichia coli includes a second gene designated ebgB. The ebgB gene product is a 79,000-molecular-weight protein and is expressed coordinately with the ebgA gene product, ebg beta-galactosidase. Insertion of the transposable elements Tn5 and Tn9 into ebgA eliminates the expression of ebgB, suggesting that ebgB is distal to ebgA. Ultraviolet light mapping confirms that gene order. The function of the ebgB gene product is unknown.     

The T8 antigen is a multimeric complex of two distinct subunits on human thymocytes but consists of homomultimeric forms on peripheral blood T lymphocytes

Monoclonal antibodies allow for the detection of structures on the cell surface of human cytotoxic thymus-derived lymphocytes, which are involved in their function. Among these cell surface components, T8 is of particular interest, since it is required during the recognition of target cells by a subset of human cytotoxic T lymphocytes. The only other cell types on which T8 has been detected are functionally inert thymocytes. Here we demonstrate that T8 isolated from peripheral blood T lymphocytes is different from thymocyte T8. On peripheral blood T lymphocytes T8 was found in multimeric forms of a 34-kDa glycoprotein. These forms were also found on thymocytes, but in addition multimeric complexes of the 34-kDa T8 with a 46-kDa protein were detected. Preliminary chemical analyses showed that the 34-kDa and the 46-kDa forms differ in both their protein and carbohydrate structures. The 46-kDa structure was found to be distinct from the major thymocyte antigen T10. The interchain disulfide bridges between the 34-kDa T8 polypeptide are located in the 24-kDa CNBr fragment, which contains a hydrophobic region. Two (14 kDa and 20 kDa) of the three CNBr fragments of the 46-kDa T8 subunit were found to be involved in interchain disulfide bridging with the 34-kDa T8 species. We suggest that the switch from heteromultimers to homomultimers may occur concomitantly with the last step in T lymphocyte maturation.     

CD4+CD45RA+ and CD4+CD45RA- T cell subsets in man maintain distinct function and CD45RA expression persists on a subpopulation of CD45RA+ cells after activation with Con A.

We have previously shown that Con A-induced suppressor T cells belong to the CD45RA+ subset. After unseparated T cells are activated with Con A, CD45RA expression increases to a maximum (Day 2), and then decreases significantly, but does not disappear entirely (Day 9), while CD29 expression increases steadily. In the present study, we examined the fate of these cell surface molecules on isolated CD4+CD45RA+ and CD4+CD45RA- cells following activation with Con A, and their relationship to the regulatory functions of these subsets. After activation of CD4+CD45RA+ cells with Con A, CD45RO and CD29 antigen expression rapidly increases (greater than 90%). While CD45RA expression is downregulated, approximately 40% of the cells continue to express low-density CD45RA in a stable fashion through Day 21. Despite these phenotypic changes, cells originally CD45RA+ continue to suppress IgG synthesis and provide only minimal B cell help. Furthermore, when cells originally CD45RA+ were sorted on the basis of continued presence, or loss of CD45RA antigen 14 days after activation, both populations demonstrated potent suppression and minimal help. In contrast, after activation with Con A, CD4+CD45A- cells maintain stable phenotype and provide significant help and minimal suppression. Immunoprecipitation of the CD45RA antigen from Day 14 activated CD4+CD45RA+ cells confirms the continued presence of the 205-kDa isoform, but reveals a significant decrease in the 220-kDa isoform. These results suggest that after activation with Con A, cells originally CD45RA+ remain functionally distinct from cells originally CD45RA-, and that CD45RA antigen persists on a subpopulation of CD45RA+ cells after activation with Con A.     

Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit

The key step of N-glycosylation of proteins in the endoplasmic reticulum is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). It transfers the lipid-linked core-oligosaccharide to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains. Biochemical and genetic approaches have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1p, Swp1p, Stt3p, Ost1p, Ost2p, Ost4p, Ost5p, Ostp3 and Ost6p. By blue native polyacrylamide electrophoresis we show that yeast OST consists of two isoforms with distinct functions differing only in the presence of the two related Ost3 and Ost6p proteins. The OST6-complex was found to be important for cell wall integrity and temperature stress. Ost3p and Ost6p are not essential for OST activity, and can in part displace each other in the complex when overexpressed, suggesting a dynamic regulation of the complex formation.     

Host resistance to cyclosporine induced syngeneic graft-versus-host disease. Requirement for two distinct lymphocyte subsets

Cyclosporine is crucial for the prevention of organ allograft rejection and allogeneic graft-vs-host disease (GVHD). Despite its potent immunosuppressive activity, cyclosporine elicits a T cell-mediated autoimmune syndrome after autologous or syngeneic bone marrow transplantation, which has been termed syngeneic GVHD (SGVHD). Recent studies have shown that for disease manifestation, a cytoxan and radiation-sensitive T cell dependent host resistance mechanism must be eliminated, allowing the clonal expansion of autoreactive cells. This report characterizes the autoregulatory lymphocyte population, present in normal animals, capable of inhibiting the adoptive transfer of SGVHD. First, twice the number of unfractionated splenocytes from normal animals to those from autoimmune donors ensured complete inhibition of the adoptive transfer of immune reactivity. Second, the phenotype of this host resistance mechanism in normal splenocytes involves dual regulatory T cell subsets. A helper/inducer subset (W3/25+) must be cotransferred with a cytotoxic/suppressor subset (OX8+) in a ratio that approximates the normal ratio in normal unfractionated splenocytes in order to affect inhibition of the transfer of SGVHD. Moreover the specific inducer regulatory activity resides in the OX22-, W3/25+ subset of Th cells.