Antibodies to pollen exines

A polyclonal antiserum and monoclonal antibodies have been prepared to purified pollen exines of Calocedrus decurrens Florin. The location of the antigen is in the exine, as shown by light-and electron-microscopic immunocytochemistry. The greatest reduction in antibody binding follows treatment of the exine with chemicals known to alter sporopollenin. These results provide evidence that sporopollenin is antigenic. Exines of ten species of gymnosperms and angiosperms also bound the polyclonal antiserum, indicating similarity of sporopollenin structure.     

Phosphatidylinositol linkage of a truncated form of the platelet-derived growth factor receptor

The platelet-derived growth factor (PDGF) receptor is usually anchored to the plasma membrane through a membrane-spanning hydrophobic amino acid sequence that splits the molecule into two approximately equal pieces, an amino-terminal external domain that contains the binding site for PDGF and a carboxyl-terminal cytoplasmic domain that includes the tyrosine kinase coding sequences. Here we report the expression of a truncated PDGF receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. Unexpectedly, this form of the receptor that lacks a hydrophobic membrane-anchoring sequence was bound to the membrane and was not secreted into the culture media. Conventional methods to dissociate noncovalent protein-protein interactions failed to release the protein from the membrane. When the transmembrane and cytoplasmic sequences were artificially deleted from the PDGF receptor, the truncated extracellular domain was anchored to the membrane through phospholipids and could be released by phospholipase C treatment. This truncated form of the receptor bound PDGF with an affinity 5-20-fold lower than the full-length receptor.     

Crystallization and preliminary diffraction data of Escherichia coli ADP glucose pyrophosphorylase

ADP glucose pyrophosphorylase from Escherichia coli has been crystallized from polyethylene glycol 8000 solutions. The crystals are: orthorhombic, a = 155(2), b = 153(2), c = 174(2) A, space group P2(1)2(1)2(1), four tetrameric molecules/unit cell. This gives a solvent fraction of about 75% consistent with the relatively poor diffraction quality of crystals (5.0-A resolution) and their sensitivity to x-ray exposure damage. Ways of circumventing the former and improving the latter are proposed.     

A novel function of glutamine in cell culture: utilization of glutamine for the uptake of cystine in human fibroblasts

Transport and metabolism of glutamine has been investigated in human diploid fibroblasts, IMR-90. Glutamine was taken up via System ASC (Na+-dependent amino acid transport system especially reactive with short or polar side chain amino acids). In the routine culture medium the cells contained a large quantity of glutamate; its major source was shown to be glutamine in the medium. Previously we described a transport system that mediates the entrance of cystine in exchange for the exit of glutamate (Bannai, 1986). Since the cystine taken up is reduced to cysteine and the cysteine readily exits to the medium where it is oxidized to cystine, a cystine-cysteine cycle across the plasma membrane has been postulated. When the cells were cultured in glutamate/glutamine-free medium, intracellular glutamate decreased, depending on the amount of cystine in the medium; in the absence of cystine, glutamate decreased very slowly. When the cells were cultured in ordinary medium, glutamine in the medium decreased, and glutamate in the medium increased. Both changes were well correlated with cystine concentration in the medium. These results are consistent with the view that the intracellular glutamate, of which the source is glutamine in the medium, is released from the cells into the medium in order to take up cystine and thereby to rotate the cystine-cysteine cycle. In the routine culture one-third to one-half of the total consumption of glutamine seems to be used for the uptake of cystine.     

Analysis of human C8 with monoclonal antibodies. Characterization of a monoclonal antibody that recognizes free C8 alpha-gamma subunit

The eighth component of human C is essential for the formation of the membranolytic C attack complex. C8 has a unique structure in that two covalently linked chains, C8 alpha and C8 gamma, are associated non-covalently with the third chain, C8 beta. In order to study the structure and assembly of the C8 molecule, a panel of mAb has been produced against the C component C8. Eight of these mAb had reactivity to the C8 alpha-gamma subunit, whereas four reacted with C8 beta. One of the C8 alpha-gamma mAb, C8A2, had specificity for an epitope on the C8 alpha-chain and exhibited no cross-reactivity to any of the other terminal C components, including C8 beta. C8A2 inhibited the hemolytic activity of the C8 alpha-gamma subunit but had no effect on the activity of fluid phase whole C8 or C8 within membrane-bound C5b-8. Functional experiments suggest that C8A2 inhibits C8 alpha-gamma activity by interfering with its interaction with the C8 beta-chain. In an enzyme immunoassay using the C8A2 mAb, free C8 alpha-gamma subunit could be detected in both homozygous and heterozygous C8 beta-deficient serum. However, only low level binding was observed when homozygous C5- and C7-deficient sera were tested. Thus the mAb, C8A2, recognizes an epitope expressed on the C8 alpha-gamma subunit but not on intact C8 and can detect free C8 alpha-gamma in the presence of native C8.     

Expression of C gamma 4 T cell receptors and lack of isotype exclusion by dendritic epidermal T cell lines

Although four murine C gamma gene segments (C gamma 1, 2, 3, and 4) are known to exist, the large majority of expressed gamma-chains have been shown to be of the C gamma 1 isotype and no evidence exists for the expression of more than one receptor by gamma delta TCR-bearing cells. We investigated the nature of the TCR expressed on a number of murine dendritic epidermal T cell-derived cell lines by using both Northern blot and immunoprecipitation analyses. One of these CD3+ cell lines (T195) expresses C gamma 4, V gamma 1, and delta mRNA, and its CD3-associated TCR complex can be precipitated by both anti-C gamma 4 and anti-delta sera, indicating that this receptor is a C gamma 4/delta heterodimer. Furthermore, we show that two cell lines (Y245, Y93) express two distinct TCR gamma-chains, one derived from the C gamma 4 locus, whereas the second gamma-chain is probably derived from the C gamma 2 locus. Together with the previous demonstration of C gamma 1/delta TCR on a number of dendritic epidermal T cell lines (DETC), these results indicate that such DETC are capable of expressing a variety of gamma delta TCR and that, in some DETC, isotype exclusion of gamma-chain expression does not occur.     

Characterization of envelope antigens of influenza A (H3N2) virus isolated during 1983-1985 epidemics and from sporadic cases of infection

Ten strains of influenza A (H3N2) virus isolated from an outbreak in 1983, and ten strains isolated in 1985 from sporadic cases of infection were included in the study. For characterization of envelope antigens were used the polyclonal and monoclonal antibodies tested in the reaction of haemagglutinin inhibition, neuraminidase inhibition, and by lectin test. The strains but slightly different in the tests with polyclonal antibodies could clearly be classified to 3-4 groups using 5 monoclonal antibodies to H antigen of A/Bangkok 1/79 and A/Philippines 2/82 strains. Strains from the 1983 epidemics represent a more homogeneous group of which only one of ten strains failed to react with monoclonals of the strains A/Bangkok and A/Philippines. Strains from sporadic cases of infection in 1985, except for two strains, did not react at all with the monoclonal discriminating A/Bangkok and A/Philippines. The other strains could be classified to three groups, i.e. whether they agreed with 4, 2 or none of the A/Philippines H antigen epitopes. Alterations of neuraminidase are less apparent, and cannot be defined by means of normal immune sera. With the use of monoclonal antibodies the strains under study do not react any more with the strains of 1968-1973 influenza virus; yet the monoclonals to A/Texas/77 strain still do recognize one or two epitopes of the 1983-1985 strains.     

Comparison of 16S rRNA sequences from the family Pasteurellaceae: phylogenetic relatedness by cluster analysis

The taxonomy of the family Pasteurellaceae has remained controversial despite investigations of biochemistry, serology, and nucleic acid relatedness. In an attempt to resolve some of this confusion, we have partially sequenced the 16S rRNAs of seven members of the family, representing all three genera. The sequences were aligned, similarity scores calculated, and single, average and complete linkage cluster analysis of the resulting distance matrix performed. In this way, an evolutionary branching pattern of these closely related species was reconstructed, and the approximate phylogenetic position of the family determined. Actinobacillus (Haemophilus) actinomycetemcomitans clustered with Haemophilus instead of Actinobacillus, supporting transfer of this species to the genus Haemophilus. Thus cluster analysis of phylogenetic relatedness was found to be particularly useful for studying closely related organisms, and could be performed using a microcomputer.     

Detection of private and recurrent idiotopes on natural anti-tubulin antibodies by monoclonal anti-idiotopic antibodies

Two monoclonal anti-idiotopic antibodies (anti-Id) were raised in mice against a human monoclonal IgA,K displaying a monospecific anti-tubulin (anti-alpha- and anti-beta-tubulin) activity. One anti-Id (IgG,K) recognized a private idiotope, TID 3.2, present only on the IgA,K immunogen, close to or within the antigen-combining site. The other anti-Id (IgM,K) recognized a recurrent idiotope, TID 7.1, outside the paratope and present in normal human and BALB/c mouse serum, on 2 of 11 polyspecific human monoclonal immunoglobulins and on 6 of 11 murine natural monoclonal auto-antibodies exhibiting a widespread anticytoskeletal protein-binding activity. Both the idiotopes were absent on two induced anti-tubulin antibodies exhibiting a monospecific anti-alpha- and anti-beta-tubulin specificity. Utilizing competitive and additivity immunoassays, we could show that the polyspecific human and mouse anticytoskeletal antibodies tested, whether bearing the TID 7.1 Id or not, appeared to compete in variable degrees for epitopes on the tubulin molecule recognized by the monoclonal IgA,K but distinct from the epitopes recognized by the induced monospecific anti-tubulin antibodies. The high incidence of the recurrent TID 7.1 idiotope in man and mouse suggests an important physiologic and perhaps regulatory function of this idiotope. Furthermore our data suggest that a restricted family of germ-line genes, highly conserved during phylogeny, may encode for these idiotope-bearing Ig molecules.     

Pre-lysosomal divergence of alpha 2-macroglobulin and transferrin: a kinetic study using a monoclonal antibody against a lysosomal membrane glycoprotein (LAMP-1)

We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes.