Analysis of the membrane-interacting domain of the sea urchin sperm adhesive protein bindin

We have investigated the domain of the bindin polypeptide that selectively associates with gel-phase phospholipid vesicles. We found that small trypsin fragments of bindin retain the ability to selectively associate with gel-phase vesicles. The primary amino acid sequence of bindin suggests that these peptides are derived from the central portion of the polypeptide between residues 77 and 126, which is the most hydrophobic region of bindin. We have also employed 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and novel, radioiodinated, photoactivatable derivatives of the polar head group of phosphatidylethanolamine (ASD-PE and ASA-PE) to identify membrane-associated polypeptide segments after the transfer of radiolabel from the probe to the bindin polypeptide. After photolysis, bindin was selectively labeled only from probes incorporated in gel-phase vesicles. The labeling of bindin was much more efficient from the head group probes ASA-PE and ASD-PE (8 and 2% of the total label, respectively) in comparison to the hydrophobic probe TID (less than 0.02% of the total label), suggesting that bindin is localized within the polar part of the bilayer. Protease mapping experiments with V8 protease, trypsin, and endoprotease Lys-C suggest that some of the probe label is distributed along the amino-terminal portion of bindin between residues 1 and 76 and the rest of the label is restricted to the segments between residues 77 and 126 which also selectively bind to gel-phase vesicles. The carboxyl-terminal portion of bindin between residues 127 and 236 is not labeled.     

The time-dependent distribution of 125I-asialo-orosomucoid-horseradish peroxidase and 131I-immunoglobulin A among three endosomal subfractions isolated from rat liver.

Three discrete endosomal fractions showing a time-dependent uptake of radioactive ligand were partially purified from rat liver. The 3,3'-diaminobenzidine (DAB)-induced density-shift protocol of Courtoy, Quintart & Baudhuin [(1984) J. Cell Biol. 98, 870-876] was used to study the distribution among these three endosomal fractions of two ligands with different intracellular destinations. Rats received both 125I-asialo-orosomucoid-horseradish peroxidase (125I-ASOR-HRP) and 131I-dIgA simultaneously by intraportal injection. The liver was fractionated at various times after injection, the three ligand-containing endosomal fractions (A, B and C) were separated and each was subjected separately to the DAB-induced density-shift procedure in which only vesicles containing 125I-ASOR-HRP are increased in density. Information on whether 131I-dIgA was co-localized or segregated from 125I-ASOR-HRP was obtained. The two ligands in the A fraction were partly segregated and partly co-localized, and this distribution appeared to be relatively unchanged with time. The two ligands in the B fraction were co-localized at all times studied. We have tentatively identified the B fraction as a compartment in which vesicle fusion has occurred. The two ligands in the C fraction were also partly co-localized and partly segregated, but the 131I-dIgA became increasingly segregated with time. This represents the first report of the purification of an endosomal subfraction specifically involved in the accumulation of multiple ligands.     

Variation in adhesion and cell surface hydrophobicity in Candida albicans white and opaque phenotypes

A previous study had established that a select group of pathogenic isolates of Candida albicans was capable of switching heritably, reversibly and at a high frequency (10^–2 to 10^–3) between two phenotypes (white or opaque) readily distinguishable by the size, shape, and color of colonies formed on agar at 25°C. This paper describes experiments designed to determine the ability of these two phenotypes to attach to buccal epithelial cells (BECs) and plastic, and to compare the cell surface hydrophobicities of white and opaque phenotypes from three clinical isolates. White cells were found to be significantly more adhesive to BECs, and a strong correlation was also found between phenotype adhesiveness and the percentage of BECs to which C. albicans had attached. The percentage of BECs with one or more attached C. albicans was approximately 90% for the white phenotype and approximately 50% for the opaque phenotype. Opaque cells, in contrast, were twice as hydrophobic as white cells, and the percentage of opaque cells bound to BECs by coadhesion was also double that of white cells. The differences in adhesion to plastic between the two phenotypes were not statistically significant and there was no distinct trend to suggest which phenotype might be more adhesive to plastic. These results indicate that several factors are involved in the adhesion of C. albicans to plastic, and confirm the hypothesis that cell surface hydrophobicity is of minor importance in direct adhesion to epithelial cells but that it may contribute to indirect attachment to epithelial cells by promoting yeast coadhesion. Moreover, the data presented in this paper also revealed that under identical growth conditions, adhesion of C. albicans was significantly altered depending on the phenotypic state of the organism tested. Therefore, because C. albicans can switch at a high frequency to various phenotypes in vitro, it may be that in future adhesion studies involving Candida the phenotypic state of the organism at the time of testing will have to be determined. Otherwise, the results, even within the same laboratory, may be difficult to interpret.     

Analysis of the oligosaccharides from the roots of Arnica montana L., Artemisia absinthium L., and Artemisia dracuncula L.

The oligosaccharides extracted from the roots of Arnica montana L., Artemisia absinthium L. and Artemisia dracunculus L. have been analysed by thin-layer and gel-permeation chromatography to assess their applicability as ‘guide’ substances for pharmacological activity. Differences observed in the oligosaccharide component composition of such extracts might be more related to the vegetative stage of the plants at time of harvest than to the species themselves. In addition to a series of non-reducing oligofructosides, a series of reducing inulin-type oligosaccharides was found at the initial stages of growth, whereas in later stages of growth only non-reducing oligofructosides were present. These differences have been related to different stages of biosynthetic activity within the plants.     

Aerial dispersal of the two-spotted spider mite (Tetranychus urticae) from field corn

Field data from 48 plots monitored during diverse weather conditions in two separate years were subjected to multiple regression analysis to determine which factors were related to spider-mite aerial dispersal. With the number of aerially dispersing mites as the dependent variable, partial regression coefficients (b) for mite population density and percent corn-leaf area infested with mites were positive, while those for percentNeozygites-infected mites and hours per week 90% r.h. were negative. When an aerial dispersal index (number of aerially dispersing mites/mites per plant) was used as the dependent variable, the partial regression coefficient for percent leaf area infested was positive, while coefficients for hours per week 90% r.h. and percentNeozygites-infected mites were negative. Mite aerial dispersal was greatest in predator-suppressed field plots under dry weather conditions. Mite aerial dispersal was substantially reduced in plots where moist weather conditions induced epizootics ofNeozygites floridana before corn plants became entirely infested with mites.     

Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University     

Yolk proteins from nematodes, chickens, and frogs bind strongly and preferentially to left-handed Z-DNA

Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left-handed Z-DNA probe, brominated poly(dG-dC). We estimate that the nematode proteins bind to Z-DNA with an association constant of at least 10(4) (M-1) and that this association constant is at least 40-50-fold higher than the association constant to B-DNA. Thus, yolk proteins have a higher Z-DNA specificity than most of the Z-DNA binding proteins previously isolated from other sources. Although yolk protein binding to Z-DNA is poorly competed by a wide variety of nucleic acids, the interaction is strongly competed by the phospholipids cardiolipin and phosphatidic acid (500-1000-fold better than by the same mass of B-DNA). We suggest that Z-DNA interacts with the yolk protein phospholipid binding site. In general, our results emphasize the danger of using physical properties to infer biological function. In particular, our results should raise serious questions about the biological relevance of previously isolated Z-DNA binding proteins.     

Specific immune regulation of chronic-relapsing experimental allergic encephalomyelitis in mice

These studies were designed to examine immunologic means of regulating the clinical course of murine chronic-relapsing experimental allergic encephalomyelitis (R-EAE). We asked whether induction of specific immune tolerance to the major CNS myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), could inhibit the development of R-EAE. Neuroantigen-specific tolerance was induced in SJL/J mice in a dose-dependent manner by the i.v. injection of mouse spinal cord homogenate-coupled syngeneic splenocytes (MSCH-SP) on day -7 relative to immunization on days 0 and +7. Sham-tolerized controls developed significant MBP- and PLP-specific DTH responses before the onset of clinical R-EAE. In contrast, MSCH-SP tolerized mice exhibited a dramatically reduced incidence of clinical and histologic signs of disease which correlated with the failure to develop MBP- and PLP-specific DTH responses. In 10 separate experiments, 118/149 (79%) of control mice, but only 22/137 (16%) of tolerized mice developed clinical R-EAE. Tolerance took time to develop and lasted at least 4 wk as mice injected with Ag-coupled splenocytes on day -1 relative to immunization remained susceptible to R-EAE, whereas mice injected on days -7, -14, or -28 were resistant. Tolerance induction required neuroantigens as injection of splenocytes coupled with a syngeneic mouse kidney homogenate failed to significantly alter the incidence of R-EAE or the development of neuroantigen-specific DTH responses. Thus, induction of R-EAE can be specifically and significantly regulated after the i.v. injection of splenocytes coupled with a crude, heterogeneous mixture of neuroantigens (i.e. MSCH).     

Structural polymorphism of the human platelet Fc gamma receptor

A variable T lymphocyte proliferative response to murine IgG1 anti-T3 monoclonal antibodies, in which most North American Caucasians respond whereas a minority do not, is well established. This is most likely the result of a genetic polymorphism manifested by 1) the inability of the monocyte 40-kDa IgG FcR of some individuals to bind murine IgG1, and 2) a distinctive trimorphic pattern on IEF of the monocyte 40-kDa FcR, one form being seen in all individuals who do not respond and another form (or a combination of both forms) being seen in those who do respond. We have evaluated the IEF patterns of the platelet 40-kDa FcR and find that in every individual tested the pattern for platelet FcR correlates with that seen for the monocyte 40-kDa FcR pattern. Furthermore, the platelets of those individuals whose "nonresponder" monocyte 40-kDa FcR did not mediate a murine IgG1 anti-T3 response did not respond with an aggregation reaction to murine IgG1 immune complexes (opsonized E). In contrast, platelets from donors possessing "responder" monocytes displayed positive "aggregation" responses to E coated with murine IgG1 antibody. However, the platelet FcR structural polymorphism described earlier did not correlate with the donor-specific variability in capacity of platelets to respond functionally to aggregated human IgG described in an earlier paper. Rather, the variation in capacity of platelets from individual donors to respond functionally to aggregated human IgG was related to the quantitative expression of platelet FcR. These data indicate that the molecular mechanisms responsible for the platelet 40-kDa FcR structural polymorphism are quite different from the mechanisms governing the variation in quantitative expression of the receptor.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.