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Biomedical subjects

D G Klapper

Publications and source records attributed to D G Klapper.

At least 55 records · Page 3Linked to original sources
PubMed1986

Arthropathic properties of cell wall polymers from normal flora bacteria.

Peptidoglycan-polysaccharide (PG-PS) fragments were purified from cell walls of group D streptococci (Streptococcus faecium, strains ATCC 9790 and F-24) with a protocol which minimizes autolytic activity and tested for ability to induce arthritis in rats. PG-PS fragments from cell walls of other normal flora bacteria (Peptostreptococcus productus, and Propionibacterium acnes), group A streptococci, and pseudomurein-PS fragments from cell walls of Methanobacterium formicicum, were similarly purified and tested. Upon intraarticular injection into rat ankles, all PG-PS polymers induced acute inflammation; pseudomurein-PS fragments were approximately five times less active than the PG-PS preparations. After intraperitoneal injection, P. acnes PG-PS induced a minimal acute arthritis, Peptostreptococcus productus PG-PS induced a moderately severe acute joint inflammation followed by a mild chronic arthritis, and both group A and group D streptococcal PG-PS induced severe acute arthritis which evolved into chronic, erosive joint disease; pseudomurein-PS fragments were without effect, consistent with a crucial role for the PG moiety of PG-PS. Chronic arthritis induced by group D streptococcal PG-PS subsided after 60 days, whereas that induced by group A streptococcal PG-PS was still active after 128 days. The arthropathic properties of this modest number of common normal flora bacteria suggest that different PG-PS structures derived from the normal flora have the potential to induce a wide range of responses, from transient acute to chronic erosive joint disease.

Amino Acids↗
PubMed1985

Beta-epidermal growth factor is the des-asparaginyl form of the polypeptide.

Reversed-phase high performance liquid chromatography of mouse epidermal growth factor (EGF) yielded two major forms, alpha- and beta-EGF, and a minor component, gamma-EGF. All three forms exhibited receptor-binding activity. Analysis of native alpha- and beta-EGF by mass spectrometry and partial Edman degradation led us to propose that alpha-EGF has a primary structure equivalent to that originally reported for EGF and that beta-EGF is the des-asparaginyl form of the polypeptide. When the purified alpha- and beta-polypeptides were cultured with human embryonic palatal mesenchymal cells stimulation of cell proliferation was observed at concentrations as low as 0.01 ng/ml with maximal stimulation occurring at about 1 ng/ml. Essentially no difference was noted in the mitogenic potency of the two forms. This suggests that the NH2-terminal region of EGF is not critical for mitogenic activity.

Amino Acid Sequence↗
PubMed1985

Ra5G, a homologue of Ra5 in giant ragweed pollen: isolation, HLA-DR-associated activity and amino acid sequence.

Recent studies [Marsh et al. (1982) J. exp. Med. 155, 1439-1451; Coulter (1983) M.Sc. thesis, McGill University, Montreal, Canada; Coulter et al. (1983) in Genetic and Environmental Factors in Clinical Allergy (Edited by Marsh D.G., Blumenthal M.N. and Santilli J., Jr), University of Minnesota Press, Minneapolis, MN] have shown a highly significant association between HLA-Dw2/DR2 and host sensitivity to the 5000-D, 4-disulfide bonded protein Ra5S of short ragweed pollen. To extend these findings, we isolated Ra5G, an Ra5S-like protein, from giant ragweed pollen by gel and ion-exchange chromatography. The protein was homogeneous by polyacrylamide gel electrophoresis (pH 4.3), reverse-phase high-performance liquid chromatography, and antigenic assays. Its mol. wt and amino acid composition (including 8 half-cystine residues) were closely similar to Ra5S, but the two proteins had little or no antigenic or allergenic cross-reactivity. In a study of 200 ragweed-sensitive individuals, host sensitivity simultaneously to Ra5G and Ra5S was significantly associated with the DR2 allele. The amino acid sequence of Ra5G was determined and showed close homology with Ra5S. The potential function of a highly homologous decapeptidyl sequence stretch is discussed in relation to Ir gene control of immune response to the 2 proteins.

Allergens↗
PubMed1985

Immunochemical and genetic studies of Amb.t. V (Ra5G), an Ra5 homologue from giant ragweed pollen.

Giant ragweed pollen allergen Amb.t. V (Ra5G), a homologue of short ragweed pollen Amb.a. V (Ra5S), was isolated in ultrapure form from a 16-min extract of ragweed pollen by a combination of molecular sieving through an Amicon hollow fiber cartridge (H1P5), cation-exchange chromatography, and gel filtration. The size was found to be 4400 daltons (D) by amino acid analysis and 6000 D by SDS-PAGE, and the pI was 8.3 as determined by isoelectric focusing. There was no cross-reactivity detected between the two Amb. V antigens by immunodiffusion and IEP with the use of hyperimmune antisera raised against crude or highly purified antigens. Cross-reactivity between the two Amb. V antigens was further investigated by inhibition double antibody radioimmunoassay by using the sera of nine selected ragweed-allergic patients who had recently been immunized with either mixed short-giant ragweed pollen extract or with short ragweed extract alone and who had IgG antibodies (Ab) to Amb.t. V and generally to Amb.a. V. Unlabeled Amb.t. V did not inhibit the binding of 125I-Amb.a. V to the IgG Ab in any of the sera tested. Conversely, unlabeled Amb.a. V produced some inhibition of the binding of 125I-Amb.t. V to the patients' IgG Ab, primarily in those patients who had received immunotherapy with short ragweed alone. This weak cross-reactivity was probably a result of the primary structural homology between the two protein allergens. The sera from two groups of ragweed-allergic individuals were investigated for the presence of IgG and IgE Ab to Amb.t. V. The presence of IgG Ab was found to be associated both with previous (or current) immunotherapy with giant ragweed extract and with HLA-Dw2. The HLA association is of interest in view of the previously established association between Dw2 and response toward the homologue Amb.a. V. The result suggests the existence of a similar genetic control at the primary level of antigenic recognition of the two Amb. V antigens.

Allergens↗
PubMed1983

Intramolecular heterogeneity of ligand binding by two IgM antibodies derived from murine hybridomas.

The ligand binding properties of two murine anti-DNP IgM hybridoma antibodies were analyzed. These IgM antibodies, designated NP3-17 C1-20 and SP2/0 I-64 C1-12, each displayed an average of only five high affinity binding sites for the DNP moiety. Reductive 7S subunits of both of these proteins each adsorbed to and were hapten eluted from DNP affinity columns. These subunits, when examined by equilibrium dialysis, each contained an average of one high affinity binding site. Structural analysis of these molecules indicated each to be homogeneous. Results obtained from trypsin hydrolysis of each molecule indicated that differences in conformation of the binding sites may account for the observed binding heterogeneity.

Aminocaproates↗
PubMed1983

The polypeptide structure of the Sm and RNP nuclear antigens.

Sm and nuclear ribonucleoprotein are ubiquitous nuclear antigens towards which important autoantibodies are directed in systemic lupus erythematosus and other human autoimmune syndromes. Using physicochemical techniques and affinity adsorptions, we have purified the polypeptide components of these antigens. The Sm antigen contained polypeptide chains of 15,000 and 17,000 mol. wt. The RNP antigen, which is known by immunochemical techniques to contain the Sm antigen, had the same two polypeptides as well as a larger one of 85,000 mol. wt. This larger peptide was quite labile and apparently broke down into smaller components with manipulation. In addition, the process of affinity purification of the Sm polypeptides gave a product which had increased positive charge. Amino acid analysis of the Sm polypeptides confirmed the presence of relatively large numbers of basic residues. The purified Sm antigen provided an effective reagent for the investigation of autoreactivity to Sm. The differences in structure from our results and those published by others are probably accounted for by the lability of the constituent polypeptides.

Amino Acids↗
PubMed1983

Sequence analysis of somatomedin-C: confirmation of identity with insulin-like growth factor I.

Somatomedin-C (Sm-C) was purified from Cohn fraction IV of human plasma by previously published methods. Purity was established by SDS polyacrylamide electrophoresis followed by silver staining of the gel. Amino acid analysis of an acid hydrolysate revealed no significant discrepancies from the amino acid composition of insulin-like growth factor I (IGF-I). The first 24 residues beginning at the amino terminal glycine were identical to the corresponding residues in IGF-I. Tryptic and chymotryptic degradation followed by determination of the amino acid composition and sequence of the resultant peptides was used to complete the primary structure of Sm-C. The results of these studies document that Sm-C and IGF-I are identical peptides, thus supporting previous observations that Sm-C and IGF-I are qualitatively and quantitatively indistinguishable in radioligand and biological assay systems.

Amino Acid Sequence↗
PubMed1983

Isolation and properties of a new short ragweed pollen allergen, Ra6.

Two chromatographic forms (A and B) of the rapidly released short ragweed (Ambrosia elatior) pollen allergen Ra6 were isolated to antigenic ultrapurity by a combination of membrane filtration and ion exchange and gel filtration chromatography. Both Ra6 forms had a m.w. of 11,500 by gel filtration chromatography and 8000 by SDS-PAGE, and each consisted of two cathodically migrating bands on agarose electrophoresis at pH 8.5. Amino-terminal amino acid sequencing analysis detected two similar sequences (sequence 1 and sequence 2), which were present in different proportions in each of the chromatographic forms of Ra6. Among a ragweed-positive population sample (n = 101), 21% had specific IgE antibody (ab) and 42% specific IgG ab toward Ra6A; among ragweed-negative subjects (n = 260), 10.8% had IgG ab. We were unable to discriminate between Ra6A and Ra6B immunologically with the use of hyperimmune animal antisera or IgE and IgG ab from five Ra6-allergic humans. It appears that Ra6 is composed of at least four closely related isoallergens of similar size but slightly different charges and amino acid sequences, which appeared to be antigenically and allergenically indistinguishable according to present evidence.

Allergens↗
PubMed1982

Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells.

Cultured hamster trachea epithelial cells were selected as an in vitro model system to study Bordetella pertussis in the respiratory tract. DNA synthesis by serum-stimulated tracheal cells, in contrast to other cell types tested, was inhibited by the supernatant from log-phase B. pertussis broth cultures. A sensitive microassay with these tracheal cells permitted the development of a chromatographic purification scheme based on aggregation of the biological activity under salt-free conditions. The active fraction from this first stage of purification caused a dose-dependent inhibition of DNA synthesis without a similar effect on RNA or protein synthesis. Organ cultures of hamster tracheal rings, when exposed to this partially purified fraction, developed epithelial cytopathology comparable to that seen during B. pertussis infection. Ciliary activity showed and eventually ceased as ciliated cells were extruded from the ring, leaving an intact but mostly nonciliated epithelium. Further purification of this biological activity was achieved with preparative-scale high-voltage paper electrophoresis. Based on ninhydrin staining and the radioactive profile of material purified from radiolabeled B. pertussis cultures, four fractions were eluted from the paper by descending chromatography. Only component B caused a dose-dependent inhibition of cultured tracheal cell DNA synthesis and epithelial cytopathology in tracheal rings. Combination experiments also demonstrated enhanced inhibition by component B in the presence of component G (oxidized glutathione), a copurifying molecule from the growth medium. Amino acid analysis (five residues), glycine (two residues), cysteine (two residues), and diaminopimelic acid (one residue), as well as muramic acid and glucosamine.

Amino Acids↗
PubMed1982

Soluble peptidoglycan-polysaccharide fragments of the bacterial cell wall induce acute inflammation.

Peptidoglycan (PG)-polysaccharide (PS) polymers derived from group A streptococcal cell walls were solubilized by M-1 mutanolysin (endo-N-acetylmuramidase) and phage-associated lysin (N-acetylmuramyl-l-alanine amidase). Fragments were isolated by ultrafiltration and a series of gel filtrations and were injected intravenously into Sprague-Dawley rats. No fragments with a molecular weight of less than 5 x 10(6) were able to induce arthritis by systemic injection. However, the enzyme-derived fragments displayed a new biological activity. High-molecular-weight PG-PS fragments ( congruent with500,000) derived from mutanolysin digests induced a severe edematous reaction in the front and hind limbs. The response started 5 to 10 min postinjection, reached maximum intensity in approximately 30 min, and disappeared by 10 h. The smallest dose capable of eliciting the response was 0.31 mug/g of body weight. Low-molecular-weight PG-PS ( congruent with30,000) derived from the mutanolysin digests and the PG-PS fragments isolated from phage-associated lysin digests also induced edema; however, a higher dose was required to elicit the same response as that produced by high-molecular-weight PG-PS fragments. The active fragments contained rhamnose, glucosamine, muramic acid, alanine, glutamic acid, and lysine in various molar ratios. PG-PS fragments obtained by sonic degradation of cell walls (molecular weight >/=5.3 x 10(6)), as well as enzyme-treated PG preparations and muramyl dipeptide, failed to elicit the response. These findings indicate that PG-PS fragments of sizes too small to be arthritogenic can affect the vascular endothelium to induce a rapidly developing edema. Fragments with this biological property could have a key role in the pathogenesis of experimental arthritis by influencing the tissue distribution of arthritogenic PG-PS.

Animals↗
PubMed1982

Two isoallergens of short ragweed component Ra5.

Two isoallergenic forms of short ragweed (Ambrosia elatior) allergen Ra5 were isolated in ultrapure form by a combination of molecular sieving (through Amicon hollow-fiber cartridges), cation-exchange chromatography, and gel filtration. The two forms, Ra5A and Ra5B, exhibited charge differences by CM-Sephadex chromatography and agarose electrophoresis at pH 8.5. The A form as identical to conventional Ra5 by its physical and chemical properties and was more cationic than the previously undescribed B form. Ra5A and Ra5B had identical m.w. of 5000 by SDS-PAGE. Their amino-terminal sequences of 30 residues showed no differences from each other or from the previously published sequence of the major form of Ra5. The two forms were antigenically indistinguishable when using hyperimmunized animal antisera to Ra5 and crude ragweed extract. Although the IgG and IgE ab of most Ra5-responder patients could not discriminate between the two Ra5 forms, significant small differences in binding to Ra5A and Ra5B were consistently observed in 10 to 15% of the patients studied. Further analysis of the Ra5A preparation suggested a purity of at least 99.5% and probably greater than or equal to 99.9%.

Allergens↗
PubMed1980

Amino acid sequence of ragweed allergen Ra3.

The complete amino acid sequence of ragweed pollen allergen Ra3 has been determined. The molecule consists of 101 amino acid residues and to date is the only allergen isolated from Ambrosia elatior (short ragweed) which contains carbohydrate. This particular preparation of allergen has a single, unique amino acid sequence, but there is evidence suggesting that, like Ra5, Ra3 isolated from pollen collected in diverse geographical areas shows amino acid sequence variation. The complete amino acid sequence was derived by utilizing only 10--12 mg of material (approximately 1 microM) as a result of recent technical innovations such as DEAE--glass ion exchangers and Polybrene as a useful sequencing aid. This is the second pollen allergen active in man which has been sequenced, and information resulting from these data should be useful in dissecting the molecular mechanisms involved in atopic allergy.

Allergens↗
PubMed1980

Ala-Gly- and Val-Asp-[Arg8]-vasopressin: bovine storage forms of arginine vasopressin with natriuretic activity.

Extracts of fresh-frozen bovine neurohypophysis were purified by chromatographic techniques to isolate and characterize the components that produce natriuresis in nondiuretic dogs. Two compounds with natiuretic properties similar to those of synthetic arginine vasopressin accounted for most of the natriuretic activity and appeared to be the prevalent vasopressin-like molecules in the extract. These peptides were Ala-Gly-[Arg8]-vasopressin and Val-Asp-[Arg8]-vasopressin; the natriuretic potency of each appeared to be similar to synthetic arginine vasopressin and could be observed with doses in the range of 50 picomoles. In the dog the most conspicuous difference between synthetic arginine vasopressin and the new vasopressin peptides was the smaller pressor responses to natriuretic doses of the new compounds.

Amino Acid Sequence↗
PubMed1980

Purification of somatomedin-C from human plasma: chemical and biological properties, partial sequence analysis, and relationship to other somatomedins.

Somatomedin-C (SM-C) was purified from Cohn fraction IV of human plasma by a series of steps which included cation-exchange chromatography, gel filtration, isoelectric focusing, and reverse-phase high-pressure liquid chromatography. The peptide isolated contained 10,142 units/mg, as judged by a radioimmunoassay, representing a 780,000-fold purification from native plasma. The isolated peptide was basic (pI 8.1-8.5) and was judged to be no less than 90% pure. The best fit for integral amino acids was obtained with 78 residues. Limited sequence data were obtained on the N terminus and on five fragments obtained by tryptic digestion after blocking the lysine residues. Three of these fragments and the N terminus could be aligned with portions of insulin-like growth factor I (IGF-I). Of the 25 residues so aligned, 22 were identical with IGF-I. In addition, two tryptic fragments were obtained which are not present in the sequence of IGF-I. SM-C and IGF-I produced identical curves of displacement in radioimmunoassay and radioreceptor assays for SM-C and in an insulin radioreceptor assay. The potencies of SM-C and IGF-I in these assays differed significantly from IGF-II, somatomedin-A, and several preparations of multiplication stimulating activity. At a concentration of 1 ng/mL, SM-C stimulated the progression of Balb/c 3T3 cells into DNA synthesis and when injected in vivo restored mitosis in lens epithelium of hypophysectomized frogs.

Amino Acid Sequence↗
PubMed1980

Genetic locus (nmp-1) affecting the principal outer membrane protein of Neisseria gonorrhoeae.

An increase in the apparent molecular weight of the principal outer membrane protein (POMP) of Neisseria gonorrhoeae is associated with introduction of the penB2 genetic marker, which results in low-level, relatively nonspecific antibiotic resistance. Limited proteolysis of the two forms of POMP showed that they had few if any peptides in common. The nonspecific antibiotic resistance of penB2 was separated from the change in POMP by genetic transformation and by isolation of spontaneous penB mutants that showed no change in POMP. The genetic locus involved in the change from one POMP to another, which we have designated nmp-1, is closely linked to, but not identical with, penB2.

Bacterial Proteins↗
PubMed1979

Detection of antibodies in human sera to streptococcal groups A and C carbohydrates by a radioimmunoassay.

Human antibodies to streptococcal groups A and C carbohydrates were measured quantitatively with a radioimmunoassay using tyrosylated 125I carbohydrate. Analysis of acute and convalescent sera from people with groups A or C streptococcal pharyngeal infection or persistent carriage revealed a significant rise in antibodies. Inhibition reactions with the cold carbohydrate indicated the specificity of the elicited antibodies. In some instances, group C as well as group A antibodies occurred after group A pharyngeal infections. Further clinical and epidemiological studies are required to determine the significance of group-specific antibodies. Antistreptolysin-O (ASO) rises were observed in individuals with group C antibody responses after persistent group C pharyngeal carriage. For this reason, epidemiological surveys that rely primarily on ASO surveys to determine the incidence of group A streptococcal infections must be interpreted with caution, at least in the developing countries, where group C pharyngeal carriage is common.

Antibodies, Bacterial↗
PubMed1977

Phylogeny of immunoglobulin structure and function; characterization of the cysteine-containing peptide involved in the pentamerization of shark IgM.

Nurse shark (Ginglymostoma cirratum) immunoglobulins were studied in an attempt to further define the relationship between the naturally occurring monomeric 7S and the pentameric 19S forms of extracellular IgM. A peptide containing the cysteine involved in the formation of intersubunit disulfide bonds linking 7S monomers into pentamers was isolated from the H chain of the 19S molecule and characterized. A similar peptide was also isolated from the H chain of the naturally occurring 7S molecule. These observations serve to substantiate previous claims that the two shark proteins belong to the same immunoglobulin class.

Amino Acid Sequence↗