Identification of two regions in apolipoprotein B100 that are exposed on the cytosolic side of the endoplasmic reticulum membrane.

Protease protection assays of apolipoprotein B100 (apoB) in digitonin-permeabilized HepG2 cells indicated that multiple domains of apoB are exposed to the cytosol through an extensive portion of the secretory pathway. The intracellular orientation of apoB in the secretory pathway was confirmed by immunocytochemistry using antibodies recognizing specific domains of apoB in streptolysin-O (STP-O)- and saponin-permeabilized HepG2 cells. Lumenal epitopes on marker proteins in secretory pathway compartments (p63, p53, and galactosyltransferase) were not stained by antibodies in STP-O-treated cells, but were brightly stained in saponin-treated cells, confirming that internal membranes were not perforated in STP-O-treated cells. An anti-apoB peptide antibody (B4) recognizing amino acids 3221-3240 caused intense staining in close proximity to the nuclear membrane, and less intensely throughout the secretory pathway in STP-O-permeabilized cells. Staining with this antibody was similar in STP-O- and saponin-treated cells, indicating that this epitope in apoB is exposed to the cytosol at the site of apoB synthesis and throughout most of the remaining secretory pathway. Similar results indicating a cytosolic orientation were obtained with monoclonal antibody CC3.4, which recognizes amino acids 690-797 (79-91 kD) in apoB. Two polyclonal antibodies made to human LDL and two monoclonal antibodies recognizing amino acids 1878-2148 (D7.2) and 3214-3506 (B1B6) in apoB did not produce a strong reticular signal for apoB in STP-O-treated cells. The anti-LDL and B1B6 antibodies produced almost identical punctate patterns in STP-O-treated cells that overlapped with LAMP-1, a membrane marker for lysosomes. These observations suggest that the B1B6 epitope of apoB is exposed on the surface of the lysosome. The results identify two specific regions in apoB that are exposed to the cytosol in the secretory pathway.

Temperature dependence of phonon-defect interactions: phonon scattering vs. phonon trapping.

The interactions between thermal phonons and defects are conventionally described as scattering processes, an idea proposed almost a century ago. In this contribution, ab-initio molecular-dynamics simulations provide atomic-level insight into the nature of these interactions. The defect is the Si|X interface in a nanowire containing a δ-layer (X is C or Ge). The phonon-defect interactions are temperature dependent and involve the trapping of phonons for meaningful lengths of time in defect-related, localized, vibrational modes. No phonon scattering occurs and the momentum of the phonons released by the defect is unrelated to the momentum of the phonons that generated the excitation. The results are extended to the interactions involving only bulk phonons and to phonon-defect interactions at high temperatures. These do resemble scattering since phonon trapping occurs for a length of time short enough for the momentum of the incoming phonon to be conserved.

Influence of the T-helper epitope on the titre and affinity of antibodies to B-cell epitopes after co-immunization.

We have assessed the influence of different T-helper cell epitopes on the level and affinity of antibody to B-cell epitopes induced following co-immunization with free peptides mimicking epitopes from measles and respiratory syncytial virus envelope proteins. The responses obtained following co-immunization have been compared to those obtained following immunization with chimeric synthetic peptide immunogens in which the epitopes were covalently coupled. The results show that covalent linkage of the B- and T-cell epitopes is not necessary for the generation of T-cell dependent antibody responses to non-immunogenic B-cell epitopes. In addition the induction of memory B-cells required adjuvant but subsequent stimulation of these memory cells did not. The responses obtained were non-MHC restricted since co-immunization resulted in the production of antibody responses to B-cell epitopes in a panel of five inbred mouse strains but there were differences in the ability of different T-cell epitopes to provide help for antibody production to the same B-cell epitope. The affinity of antibodies to the B-cell epitopes induced following immunization with chimeric T:B peptides was higher than that obtained following co-immunization. These results indicate the value of co-immunization for the induction of antibody responses to B-cell epitopes across MHC differences and suggest that this strategy may be of value in the development of synthetic peptide vaccines. However, modifications of the approach need to be developed to ensure the production of antibody of the highest possible affinity.

Mutations in BTD causing biotinidase deficiency.

Biotinidase (BTD) is the only enzyme that can cleave biocytin, a product of the proteolytic digestion of holocarboxylases. Profound BTD deficiency (less than 10% mean normal activity in serum) is an autosomal recessive disorder that can result in neurological and cutaneous abnormalities. Both the cDNA and the genomic DNA of normal BTD gene have been isolated and characterized. The BTD gene is localized to chromosome 3p25. Thus far 61 mutations in three of the four exons of the BTD and one mutation in an intron gene that cause profound BTD deficiency have been reported. Mutations occur at different frequencies in symptomatic children than they do in children ascertained by newborn screening. Two mutations, 98-104del7ins3 and R538C, were present in 52% or 31 of 60 alleles found in symptomatic patients. Three other mutations, A755G, Q456H, and 511 G>A; 1330G>C (double mutation), accounted for 52% of the alleles detected by newborn screening in the United States. Two asymptomatic adults, parents of children with profound BTD deficiency detected by newborn screening, have been described. Additional different mutations have been found in Turkish, Saudi Arabian, and Japanese children with profound BTD deficiency. Partial BTD deficiency (10-30% of mean normal serum activity) is predominantly caused by the single 1330G>C mutation that results in D444H on one allele in combination with one of the mutations causing profound deficiency on the other allele. Four intragenic polymorphisms, three neutral and one amino acid change, have also been found. Although a preponderance of mutations causing the production of truncated BTD protein occurs in symptomatic children with profound deficiency, preliminary studies fail to demonstrate clear genotype-phenotype correlations.

An inhibition enzyme immunoassay for estimating relative antibody affinity and affinity heterogeneity.

A method to measure the relative affinity of antibodies using an inhibition enzyme immunoassay is described. It is validated using monoclonal antibodies of defined affinity characteristics and by comparison with conventional methods of affinity measurement. The method allows measurement of the relative affinity of low levels of antibody, and the calculation of an empirical estimate of the heterogeneity of affinity in antibody populations.

Influence of antibody affinity on the performance of different antibody assays.

The effect of antibody affinity on the performance of 5 commonly used assays was studied. The assays used were measurement of antigen binding capacity in a Farr type assay, haemagglutination, solid-phase radioimmunoassay (SP-RIA), solid-phase ELISA and precipitation. The first 4 assays were all more sensitive for high affinity antibodies. Precipitation was not related to affinity, suggesting that factors secondary to antigen-antibody binding may be more important in determining the level of precipitate formation. The effect of epitope density of the antigen was also investigated in the SP-RIA and ELISA. Affinity dependence was more marked when antigen of low epitope density was used and this dependence could be reduced in the ELISA by choosing a low OD endpoint. Thus, the most reliable way to estimate antibody content by the ELISA may be to determine a low OD endpoint titre against antigen of high epitope density. When epitope density per molecule cannot be increased, an alternative approach to the problem is to increase epitope density by covalent coupling of antigen to the solid-phase rather than by adsorption.

Immunological analysis of the protective responses to the chimeric synthetic peptide representing T- and B-cell epitopes from the fusion protein of measles virus.

The role of different adjuvant formulations and routes of immunization on the antibody responses and protection induced in mice was determined by a synthetic peptide representing T- and B- cell epitopes from the measles virus (MV) fusion (F) protein (TTB peptide) which has previously been shown to induce protective responses against MV encephalitis in mice. When the peptide TTB was administered in Freud's complete adjuvant (FCA), Freud's incomplete adjuvant (FIA), alum or with IL-2, anti-peptide antibody responses and anti-MV antibody responses were detected. Interestingly, immunization with TTB without adjuvant resulted in the induction of anti-peptide antibody responses which did not cross react with the MV. The use of FIA as an adjuvant led to a significantly higher IgG2a antibody response compared with FCA and alum, whereas alum led to a significantly lower IgG3 response. Immunization with TTB in FCA, FIA and alum led to the generation of high affinity antibodies (with alum generating the highest affinity), whereas immunization of peptide with IL-2 or in PBS resulted in the induction of antibodies of lower affinity. Only the FCA, FIA and alum formulations led to the induction of protective responses in mice against MV-induced encephalitis. When the subcutaneous route (s.c.) of immunization was compared with the intraperitoneal route (i.p.), s.c. immunization with the TTB peptide led to higher anti-peptide and anti-MV antibody responses and higher affinity antibodies compared to those induced following i.p. immunization. Mice receiving the TTB peptide via the s.c. route had a higher percentage survival after MV challenge than those immunized via the i.p. route. These results show that the nature of the adjuvant used as well as the route of immunization play an important role in the induction of protective anti-TTB peptide antibody responses against MV-induced encephalitis in mice.

The effect of 16,16-dimethyl prostaglandin E2 on proliferation of an intestinal goblet cell line and its synthesis and secretion of mucin glycoproteins.

The effect of 16,16'-dimethyl prostaglandin E2 (dmPGE2) on the human colonic adenocarcinoma derived mucus-secreting goblet cell line HT29-18N2 was investigated. The proliferation rate of HT29-18N2 was increased by exposure to 10 or 100 microM dmPGE2. Exposure to 10 or 100 microM dmPGE2 caused a significant decrease in the rate of radiolabeled glucosamine incorporation into newly synthesized glycoproteins during an 8 or 24 h exposure. At concentrations as low as 1 microM, dmPGE2 accelerated the secretion of mucin glycoproteins as assessed by the release of newly synthesized radiolabeled glycoproteins, a mucin-specific enzyme-linked immunoassay and a whole-mount immunofluorescence assay. A 1 h exposure to dmPGE2 did not, however, result in a morphometrically detectable decrease in intracellular mucous granule stores or elicit any other readily detectable morphological change. The experimental results suggest elevated levels of PGs may contribute to the previously recognized decreases in intracellular mucin stores and shifts in the types of mucins species present at sites of mucosal inflammation in ulcerative colitis patients.

Bradykinin modulates mucin secretion but not synthesis from an intestinal goblet cell line.

The effect of the inflammatory mediator bradykinin on glycoprotein synthesis and mucin secretion in the human colonic adenocarcinoma cell line HT29-18N2 was examined. Bradykinin, at a threshold of 0.01 microM, accelerated the rate of mucin discharge as assessed by a mucin-specific ELISA. Using immunofluorescence microscopy, a thick meshwork of extracellular mucus was observed over bradykinin-treated monolayers but not mock-treated controls. Morphometric analysis of bradykinin-treated monolayers revealed no decreases in intracellular mucin stores or any other easily discernable morphological alteration. The ability of the cyclooxygenase inhibitors indomethacin and naproxen to decrease the response to bradykinin by approximately 68% indicates the effect is mediated, at least partially, through the generation of prostaglandins. Bradykinin did not alter the rate of incorporation of 3H-glucosamine into newly synthesized glycoproteins. Bradykinin-accelerated mucin secretion may be linked to the depletion of intracellular mucin stores in the inflammatory bowel disease ulcerative colitis.

Selective secretion and replenishment of discrete mucin glycoforms from intestinal goblet cells.

Antibodies against MUC2, MUC3, and MUC5AC peptide epitopes stained the secretory contents of all goblet cells in the human colon-derived HT29-18N2 cell line. In contrast, four carbohydrate-specific monoclonal antibodies stained mucin glycoforms in consistent subsets of goblet cells. Cholinergic agonist-evoked decreases in total mucin stores were not always mirrored by proportional changes in mucin glycoforms in the same monolayers. Selective secretion of mucin glycoforms did not result from differences in receptor distribution, since cholinergic stimulation was found to increase intracellular free calcium in all cells and selective secretion was also observed when the cells were directly stimulated with the protein kinase C activator phorbol myristate acetate. The results demonstrate that goblet cells cycle through transient periods in which their exocytotic response is unresponsive to cholinergic or protein kinase C-mediated stimuli. Goblet cells replenished intracellular mucin stores to control levels within 1 h, but the relative proportion of mucin glycoforms was not always restored until 24 h after stimulation.

Intrathymic distribution of the two avian thymic parvalbumins.

Although parvalbumins are generally viewed as intracellular Ca2- buffers/transporters, avian species express two thymus-specific isoforms that may play alternative biological roles. These proteins, known as avian thymic hormone (ATH) and chicken parvalbumin 3 (CPV3), are conjectured to influence thymopoiesis. In this paper, we compare their intrathymic distributions. Isoform-specific monoclonal antibodies against ATH and CPV3 were labeled with fluorescein and Cy5, respectively, and then used to probe paraffin sections of chicken thymus tissue. Confocal microscopy of the stained sections reveals that ATH and CPV3 are both confined to the thymic cortex and that they are frequently coexpressed by a subset of epithelial cells. However, their expression patterns are not completely superimposible. Cortical epithelial cells are also observed that stain for just one of the two avian parvalbumin isoforms, albeit at lower frequency. Significantly, a subset of cortical thymocytes exhibits peripheral staining for one or both of the proteins. This result may imply the existence of plasma membrane receptors for the two proteins on select T-cell precursors. Alternatively, it may signal low level expression of the thymic parvalbumins by the thymocytes population.

Immune responses in mice following immunization with chimeric synthetic peptides representing B and T cell epitopes of measles virus proteins.

The immunogenicity of chimeric peptides produced by collinear synthesis to contain both T and B cell epitopes from the fusion protein and the haemagglutinin of measles virus was studied in mice. The T cell epitope used was from the fusion protein (residues 288 to 302), which has been shown to be promiscuous in its binding to mouse major histocompatibility complex molecules. This epitope was coupled by (i) a glycine-glycine spacer to a B cell epitope from the fusion protein (residues 404 to 414) and (ii) either its amino or carboxy terminus to a neutralizing antibody epitope from the haemagglutinin (residues 188 to 199). The results obtained show that such chimeric peptides can indeed function as complete immunogens in a range of mouse strains of different H-2 haplotype, and can induce the production of antibodies which bind to the fusion protein and to measles virus. Furthermore, it was shown that the orientation of the T cell epitope with respect to the B cell epitope had a significant effect upon the immunogenicity and antigenic specificity of the chimera. This work gives further support to the concept of rationally designed synthetic peptide vaccines.

A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response.

A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses.

Plant cells contain two functionally distinct vacuolar compartments.

The plant cell vacuole has multiple functions, including storage of proteins and maintenance of an acidic pH where proteases will have maximal activity. It has been assumed that these diverse functions occur in the same compartment. Here, we demonstrate that antibodies to two different tonoplast intrinsic proteins, alpha-TIP and TIP-Ma27, label vacuole membranes of two different compartments within the same cell. These compartments are functionally distinct, because barley lectin, a protein stored in root tips, is exclusively contained within the alpha-TIP compartment, while aleurain, a protease that serves as a marker for an acidified vacuolar environment, is exclusively contained within the TIP-Ma27 compartment. As cells develop large vacuoles, the two compartments merge; this may represent a process by which storage products in the alpha-TIP compartment are exposed to the acidic lytic TIP-Ma27 compartment for degradation.

High-affinity antibody induced by immunization with a synthetic peptide is associated with protection of cattle against foot-and-mouth disease.

Previous work has shown that the synthetic peptide C-C-(200-213)-P-P-S-(141-158)-P-C-G, in which residues 200-213 and 141-158 correspond to immunogenic regions of the VP1 protein of foot-and-mouth disease virus (FMDV), is capable of inducing high levels of neutralizing antibody but only inconsistent protection of cattle against virulent FMDV challenge. The possibility exists that differences in affinity may well underlie the observed variations in biological effectiveness of the anti-peptide antibodies in immunized animals. This has been investigated by assessing the affinity for peptide and whole virus of the anti-peptide antibodies in sera from protected and non-protected cattle using both fluid-phase and solid-phase assays. The results obtained show that the affinities of serum antibodies for peptide and virus in protected cattle were significantly higher than those in non-protected animals. Thus in order to assess vaccine efficacy, particularly in the case where synthetic immunogens are employed, consideration should be given to the determination of antibody affinity in addition to other parameters of the antibody response.

The affinity of IgG antibodies to gag p24 and p17 in HIV-1-infected patients correlates with disease progression.

The affinity of anti-gag antibody was studied for up to 9 years (1984-1993) in sera from 15 HIV-1+ patients with haemophilia. On the basis of their 1993 clinical status patients were divided into two groups: (i) patients who remained asymptomatic (n = 9); and (ii) those who progressed to AIDS between late 1987 and 1993. The affinity constants of antibody for p24 and p17 were determined by a double isotope fluid-phase radioimmunoassay; and the relationships between antibody affinity and titre, patient clinical course, CD4 cell counts and p24 antigenaemia were analysed. The affinity of p24- and p17-specific antibody was up to 100 times greater in asymptomatic patients than in patients who progressed to AIDS. Patients who developed AIDS either lost or failed to develop high-affinity antibodies early in the infection. Asymptomatic patients maintained high-affinity antibodies for several years; however, in some of these patients the affinity of anti-p24 and p17 antibodies subsequently fell later in the study period. The presence of low-affinity antibody and progressive reduction in the titre of specific antibody were earlier predictors of disease onset than CD4 cell counts. The failure to either develop or maintain high affinity gag-specific antibody suggests an early impairment of T helper function in individuals who progressed to AIDS. The presence of antibody of high affinity could be essential in controlling virus replication and the onset of AIDS.

Modulation of bovine papillomavirus DNA replication by phosphorylation of the viral E1 protein.

E1 is the DNA replication origin recognition protein for bovine papillomavirus (BPV), and it carries out enzymatic functions required for initiation of viral DNA replication. Cellular mechanisms likely play a role in regulating BPV DNA replication. We are investigating the role of phosphorylation of E1 on viral replication in vivo and on E1 activity in vitro. Serine 109 is a phosphoacceptor in vivo and is targeted by protein kinase A and protein kinase C in vitro. A viral genome carrying a serine 109 to alanine mutation replicates more efficiently than wild-type in vivo in a transient replication assay. Furthermore, purified mutant protein, while having wild-type levels of ATPase activity, is able to bind more origin-containing DNA than wild-type E1. Phosphorylation therefore appears to play a selective role in modulating a specific E1 function during viral DNA replication.

Adherence of Vibrio cholerae to cultured differentiated human intestinal cells: an in vitro colonization model.

Choleragenic vibrios adhered to and multiplied on monolayers of the highly differentiated mucin-secreting cell line HT29-18N2. Their adherence followed first-order kinetics, was dependent on the concentration of vibrios, and was partially inhibited by lipopolysaccharide. Comparison of genetically modified vibrios showed that flagella, an active toxR gene, and the virulence cassette were not essential for initial binding. Inactivation of the hemagglutinin/protease increased binding. This highly differentiated human intestinal cell line provides a versatile new approach for studying major events occurring during intestinal colonization: adherence, multiplication, and detachment.