Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution.

Although the evolutionary success of polyploidy in higher plants has been widely recognized, there is virtually no information on how polyploid genomes have evolved after their formation. In this report, we used synthetic polyploids of Brassica as a model system to study genome evolution in the early generations after polyploidization. The initial polyploids we developed were completely homozygous, and thus, no nuclear genome changes were expected in self-fertilized progenies. However, extensive genome change was detected by 89 nuclear DNA clones used as probes. Most genome changes involved loss and/or gain of parental restriction fragments and appearance of novel fragments. Genome changes occurred in each generation from F2 to F5, and the frequency of change was associated with divergence of the diploid parental genomes. Genetic divergence among the derivatives of synthetic polyploids was evident from variation in genome composition and phenotypes. Directional genome changes, possibly influenced by cytoplasmic-nuclear interactions, were observed in one pair of reciprocal synthetics. Our results demonstrate that polyploid species can generate extensive genetic diversity in a short period of time. The occurrence and impact of this process in the evolution of natural polyploids is unknown, but it may have contributed to the success and diversification of many polyploid lineages in both plants and animals.

An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.

We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed. Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles.

Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells.

Human melanoma cells can process the MAGE-1 gene product and present the processed nonapeptide EADPTGHSY on their major histocompatibility complex class I molecules, HLA-A1, as a determinant for cytolytic T lymphocytes (CTLs). Considering that autologous antigen presenting cells (APCs) pulsed with the synthetic nonapeptide might, therefore, be immunogenic, melanoma patients whose tumor cells express the MAGE-1 gene and who are HLA-A1+ were immunized with a vaccine made of cultured autologous APCs pulsed with the synthetic nonapeptide. Analyses of the nature of the in vivo host immune response to the vaccine revealed that the peptide-pulsed APCs are capable of inducing autologous melanoma-reactive and the nonapeptide-specific CTLs in situ at the immunization site and at distant metastatic disease sites.

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules.

We have investigated the relationship of the so-called small dense core vesicle (SDCV), the major catecholamine-containing neurosecretory vesicle of sympathetic neurons, to synaptic vesicles containing classic neurotransmitters and secretory granules containing neuropeptides. SDCVs contain membrane proteins characteristic of synaptic vesicles such as synaptophysin and synaptoporin. However, SDCVs also contain membrane proteins characteristic of certain secretory granules like the vesicular monoamine transporter and the membrane-bound form of dopamine beta-hydroxylase. In neurites of sympathetic neurons, synaptophysin and dopamine beta-hydroxylase are found in distinct vesicles, consistent with their transport from the trans-Golgi network to the site of SDCV formation in constitutive secretory vesicles and secretory granules, respectively. Hence, SDCVs constitute a distinct type of neurosecretory vesicle that is a hybrid of the synaptic vesicle and the secretory granule membranes and that originates from the contribution of both the constitutive and the regulated pathway of protein secretion.

Identification of endothelin 1 and big endothelin 1 in secretory vesicles isolated from bovine aortic endothelial cells.

Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA. Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA. Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximately 100 nm in diameter. Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles. Immunofluorescence staining of whole BAECs using a specific ET-1-(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy. This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme.

Library of libraries: approach to synthetic combinatorial library design and screening of "pharmacophore" motifs.

Construction of synthetic combinatorial libraries is described that allows for the generation of a library of motifs rather than a library of compounds. Peptide libraries based on this strategy were synthesized and screened with model targets streptavidin and anti-beta-endorphin antibody. The screens resulted in observation of expected motifs providing evidence of the effectiveness of the suggested approach.

Induction of intracellular cAMP by a synthetic retroviral envelope peptide: a possible mechanism of immunopathogenesis in retroviral infections.

A synthetic heptadecapeptide, CKS-17, represents the highly conserved amino acid sequences occurring within the transmembrane envelope protein of many animal and human retroviruses. CKS-17 has been demonstrated to exhibit suppressive properties for numerous immune functions. We have recently shown that CKS-17 acts as an immunomodulatory epitope causing an imbalance of human type 1 and type 2 cytokine production and suppression of cell-mediated immunities. cAMP, an intracellular second messenger, plays an important role in regulation of cytokine biosynthesis--i.e., elevation of intracellular cAMP levels selectively inhibits type 1 cytokine production but has no effect or enhances type 2 cytokine production. Here, we demonstrate that CKS-17 induces dramatic rises in the intracellular cAMP levels of a human monocyte cell line and of human peripheral blood mononuclear cells in a time- and dose-dependent manner. A peptide corresponding to the reverse sequence of CKS-17, used as control, has no effect on intracellular cAMP levels. The cAMP-inducing ability of CKS-17 is significantly blocked by SQ-22536, an inhibitor of adenylate cyclase. These results indicate that CKS-17, a highly conserved component of the transmembrane proteins of immunosuppressive retroviruses, induces increased intracellular levels of cAMP via activation of adenylate cyclase and suggest that this retroviral envelope peptide may differentially modulate type 1 and type 2 cytokine production through elevation of intracellular cAMP levels.

Redistribution of synaptic vesicles and their proteins in temperature-sensitive shibire(ts1) mutant Drosophila.

From an extract of Drosophila melanogaster head homogenates, a membrane fraction can be isolated that has the same sedimentation properties as vertebrate synaptic vesicles and contains Drosophila synaptotagmin. The fraction disappears from homogenates of temperature-sensitive (ts) mutant shibire(ts1) (shi(ts1)) flies paralyzed by exposure to non-permissive temperatures, and reappears on return to permissive temperatures. Since reversible, temperature-dependent depletion of synaptic vesicles is known to occur in shibire(ts1) flies, we conclude that the fraction we have identified contains synaptic vesicles. We have examined the fate of synaptic vesicle membrane proteins in shibire flies at nonpermissive temperatures and found that all of these vesicle antigens are transferred to rapidly sedimenting membranes and codistribute with a plasma membrane marker by both glycerol velocity and metrizamide density sedimentation and by confocal microscopy. Three criteria were used to establish that other neuron-specific antigens--neuronal synaptobrevin and cysteine-string proteins--are legitimate components of synaptic vesicles: cosedimentation with Drosophila synaptotagmin, immunoadsorption, and disappearance of these antigens from the vesicle fractions in paralyzed shibire flies.

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

Rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library.

Peripheral blood leukocytes incubated with a semisynthetic phage antibody library and fluorochrome-labeled CD3 and CD20 antibodies were used to isolate human single-chain Fv antibodies specific for subsets of blood leukocytes by flow cytometry. Isolated phage antibodies showed exclusive binding to the subpopulation used for selection or displayed additional binding to a restricted population of other cells in the mixture. At least two phage antibodies appeared to display hitherto-unknown staining patterns of B-lineage cells. This approach provides a subtractive procedure to rapidly obtain human antibodies against known and novel surface antigens in their native configuration, expressed on phenotypically defined subpopulations of cells. This approach does not depend on immunization procedures or the necessity to repeatedly construct phage antibody libraries.

Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein.

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.