Protein kinase C phosphorylation of desmin at four serine residues within the non-alpha-helical head domain

We reported that phosphorylation by either cAMP-dependent protein kinase or protein kinase C (Ca2+/phospholipid-dependent enzyme) in vitro induces disassembly of the desmin filaments (Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., and Sato, C. (1988) J. Biol. Chem. 263, 5970-5978). For this subunit protein, Ser-29, Ser-35, and Ser-50 within the non-alpha-helical head domain were shown to be the sites of phosphorylation for cAMP-dependent protein kinase (Geisler, N., and Weber, K. (1988) EMBO J. 7, 15-20). In the present work, we identified the sites of desmin phosphorylated in vitro by other protein kinase which affects the filament structure. The protein kinase C-phosphorylated desmin was hydrolyzed with trypsin, and the phosphorylated peptides were isolated by reverse-phase chromatography. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-12, Ser-29, Ser-38, and Ser-56 were phosphorylated by protein kinase C. All four sites are located within the non-alpha-helical head domain of desmin. Ser-12, Ser-38, and Ser-56, specifically phosphorylated by protein kinase C, have arginine residues at the carboxyl-terminal side (Arg-14, Arg-42, and Arg-59, respectively). Ser-29 phosphorylated by both protein kinase C and cAMP-dependent protein kinase has arginine residues at the amino and carboxyl termini (Arg-27 and Arg-33). These findings support the view that the head domain-specific phosphorylation strongly influences desmin filament structure; however, each protein kinase differed with regard to site recognition on this domain.     

IgG3 is the major source of cryoglobulins in mice

A total of 20 of 23 IgG3 mAb derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice was shown to generate cryoglobulins which were composed exclusively of IgG3. Although three IgG3 mAb failed to develop cryoglobulins, they were able to bind nonspecifically to any IgG3 molecules as efficiently as cryoprecipitable IgG did. The direct role of the gamma 3 constant region for the generation of cryoglobulins was demonstrated by the following findings: 1) the cryoglobulin activity was independent of the specificity of the IgG3 mAb, 2) no mAb other than those of the IgG3 subclass, including IgM rheumatoid factors (RF), generated cryoglobulins, and 3) the cryoglobulin activity was gained after the Ig class switch of mAb from IgM to IgG3. Analysis of Ig components in three different sources of cryoglobulins, either induced by the injection of bacterial LPS or by the infection with Plasmodium yoelii in BALB/c mice or developed spontaneously in MRL/MpJ-lpr/lpr mice, revealed the selective concentration of IgG3 in these cryoglobulins; greater than 99%, 73% and 58% of IgG recoverable from these three cryoglobulins, respectively, were IgG3. This further attests to the major role of IgG3 in the generation of cryoglobulins in mice. In addition, the enhanced formation and even induction of IgG3 cryoglobulins in the presence of IgM anti-IgG3 RF mAb, and the enrichment of IgM RF in LPS- or malaria-induced cryoglobulins indicated that IgM RF can be involved in the generation of cryoglobulins by interacting with noncryoprecipitable IgG3 as well as cryoprecipitable IgG3.     

Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2

We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.     

Biphasic effects of alcohols on the phase transition of poly(L-lysine) between alpha-helix and beta-sheet conformations.

Poly(L-lysine) exists as a random-coil at neutral pH, an alpha-helix at alkaline pH, and a beta-sheet when the alpha-helix poly(L-lysine) is heated. The present Fourier-transform infrared (FTIR) study showed that short-chain alcohols (methanol, ethanol, and 2-propanol) partially transformed alpha-helix poly(L-lysine) to beta-sheet when their concentrations were low. At higher concentrations, however, these alcohols reversed the reaction, and the alcohol-induced beta-sheet was transformed back to alpha-helix structure. The reversal occurred at 1.40 M methanol, 0.96 M ethanol, and 0.55 M 2-propanol. The alcohol effects on the secondary structure were further investigated by circular dichroism (CD) on the thermally induced beta-sheet poly(L-lysine). Methanol, ethanol, and 1-propanol, but not 1-butanol, shifted the negative mean-residue ellipticity at 217 nm of the beta-sheet poly(L-lysine) to the positive side at low concentrations of the alcohols and to the negative side at high concentrations. With 1-butanol, only the positive-side shift was observed. The positive-side shift at low concentrations of alcohols indicates enhancement of the hydrophobic interactions among the side chains of the polypeptide in the beta-sheet conformation. The negative-side shift indicates a partial transformation to alpha-helix. The shift from the positive to negative side occurred at 7.1 M methanol, 4.6 M ethanol, and 3.1 M 1-propanol. The alcohol concentrations for the beta-to-alpha transition were higher in the CD study than in the IR study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Mutations in the Pigment Cell-Specific Gene Located at the Brown Locus in Mouse

The pigment cell-specific gene, located at the brown (b)-locus in mouse, encodes the protein that determines the type of melanin synthesized. This protein is known as tyrosinase-related protein, but here we tentatively term it b-locus protein to avoid confusions with the related sequence cross-hybridizing to the tyrosinase gene. In order to identify the mutation at the b-locus, we have cloned and characterized the b-locus protein gene of BALB/c mouse (b/b, c/c). The gene is about 18 kb long and organized into 8 exons and 7 introns. Sequence analysis of the b-locus protein gene reveals four base changes within the protein-coding regions: two missense mutations and two silent mutations. Two missense mutations result in the Cys to Tyr substitution at position 86 (codon 110) and the Arg to His substitution at position 302 (codon 326) of a b-locus protein molecule. Using allele-specific amplification, we confirmed that these missense mutations are actually present in the genomic DNA of two b-mutant strains examined, BALB/c and DBA/2 (b/b, C/C) mice, suggesting that these mutations are specific for the mutant mice at the b-locus. Moreover, we are able to show that the b-locus protein containing Tyr 86 is not reactive with the anti-b-locus protein monoclonal antibody, TMH-1, in transient expression assays.     

Differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary T-lymphocyte and macrophage cultures.

Two primary cell targets for human immunodeficiency virus type 1 (HIV-1) infection in vivo are CD4+ T lymphocytes and monocyte-derived macrophages (MDM). HIV-1 encodes envelope glycoproteins which mediate virus entry into these cells. We have utilized infected and radiolabelled primary peripheral blood mononuclear cell (PBMC) and MDM cultures to examine the biochemical and antigenic properties of the HIV-1 envelope produced in these two cell types. The gp120 produced in MDM migrates as a broad, diffuse band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels compared with that of the more homogeneous gp120 released from PBMCs. Glycosidase analyses indicated that the diffuse appearance of the MDM gp120 is due to the presence of asparagine-linked carbohydrates containing lactosaminoglycans, a modification not observed with the gp120 produced in PBMCs. Neutralization experiments, using isogeneic PBMC and MDM-derived macrophage-tropic HIV-1 isolates, indicate that 8- to 10-fold more neutralizing antibody, directed against the viral envelope, is required to block virus produced from MDM. These results demonstrate that HIV-1 released from infected PBMC and MDM cultures differs in its biochemical and antigenic properties.     

Endo.SK1: an inducible site-specific endonuclease from yeast mitochondria

Site-specific endonucleases have been found in various eukaryotic organelles such as mitochondria, chloroplasts and nuclei. These endonucleases initiate site-specific or homologous gene conversion in mitochondrial and nuclear DNA. Here, we report a new site-specific endonuclease activity, Endo.SK1, identified in mitochondria of strain SK1, a homothallic diploid strain ofSaccharomyces cerevisiae. Nucleotide sequences around the Endo.SK1-cleavage sites are different from those of known yeast site-specific endonucleases. The Endo.SK1 activity is, at least partly, specified by a gene in the SK1-derived mitochondria. A novel feature of the Endo.SK1 activity is its inducibility: the endonuclease activity was induced by ca. 40-fold by transfer of cells from a glucose medium into an acetate medium, and was then repressed. This transient induction was independent of the ploidy level of the cells, and coincided with induction of fumarase, a mitochondrial enzyme involved in the TCA cycle. Co-induction and co-repression of the mitochondrial site-specific endonuclease activity and a respiration-related enzyme indicate that the endonuclease activity is regulated in response to physiological conditions, and suggest a possible role for the endonuclease in mitochondrial DNA metabolism.     

Failure to Reject an Allografted Tumor after Elimination of Macrophages in Mice

After an i.p. transplantation of an allogeneic tumor (Meth A) to C57BL/6 mice, a macrophage (MΦ)-rich, non-T, non-NK cell population is induced as the major infiltrate and cytotoxic cells. We here evaluated the role of the MΦs in the rejection of allografted Meth A cells and characterized the MΦs in comparison with other well-known MΦs. At all time intervals after transplantation, the highest cytotoxic activities against Meth A tumor were obtained with the MΦ-rich population. In addition, the lymphocyte-rich population had a significant but low cytotoxic activity, whereas two other population types, granulocytes and large granular cells, were inactive. When the MΦ-rich or the T cell-depleted MΦ-rich population was i.p. transplanted simultaneously with Meth A cells into untreated C57BL/6 mice, the tumor cells were rejected without growth. After specific elimination of MΦs by in vivo application of dichloromethylene diphosphonate-containing liposomes, the cytotoxic activity against Meth A cells was hardly induced at the transplantation site of Meth A cells and the allografted Meth A tumor continued to grow, indicating that a type of MΦ is the effector cell essential for the rejection. In contrast to other well-known MΦs, the cytotoxic activity against Meth A cells was cell-to-cell contact dependent and soluble factor (e.g., NO and TNF-α) independent. Moreover, the cytotoxic activity of the MΦs (H-2^b) against ⁵¹Cr-labeled Meth A (H-2^d) cells was inhibited by the addition of unlabeled H-2^d, but not H-2^a, H-2^k or H-2^b, lymphoblasts as well as Meth A cells, implying the specific interaction of the MΦs with H-2^d cells.     

A novel gene family defined by human dihydropyrimidinase and three related proteins with differential tissue distribution

We have isolated cDNA clones encoding dihydropyrimidinase (DHPase) from human liver and its three homologues from human fetal brain. The deduced amino acid (aa) sequence of human DHPase showed 90% identity with that of rat DHPase, and the three homologues showed 57–59% aa identity with human DHPase, and 74–77% aa identity with each other. We tentatively termed these homologues human DHPase related protein (DRP)-1, DRP-2 and DRP-3. Human DRP-2 showed 98% aa identity with chicken CRMP-62 (collapsin response mediator protein of relative molecular mass of 62 kDa) which is involved in neuronal growth cone collapse. Human DRP-3 showed 94–100% aa identity with two partial peptide sequences of rat TOAD-64 (turned on after division, 64 kDa) which is specifically expressed in postmitotic neurons. Human DHPase and DRPs showed a lower degree of aa sequence identity with Bacillus stearothermophilus hydantoinase (39–42%) and Caenorhabditis elegans unc-33 (32–34%). Thus we describe a novel gene family which displays differential tissue distribution: i.e., human DHPase, in liver and kidney; human DRP-1, in brain; human DRP-2, ubiquitously expressed except for liver; human DRP-3, mainly in heart and skeletal muscle.