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W J Knowles

Publications and source records attributed to W J Knowles.

14 recordsLinked to original sources
PubMed1994

Autoantigenic properties of native and denatured glutamic acid decarboxylase: evidence for a conformational epitope.

The frequency of antibodies to GAD (anti-GAD) in insulin-dependent diabetes mellitus (IDDM) varies greatly according to the type of assay employed. We therefore examined the immunoassay characteristics of diabetic sera using GAD purified from porcine brain and shown to contain both isoforms. Sera from 38 patients with IDDM, including 1 patient with both stiff-man syndrome (SMS) and IDDM, were studied for anti-GAD by radioimmunoprecipitation (RIP), Western blotting, and dot-blotting. The sera were selected according to reactivity in the RIP assay. There was a good correlation between potency of the RIP reaction at the screening dilution of 1:2 and the endpoint dilution in the assay which ranged from 1:2 to 1:30,000 for IDDM sera, and 1:300,000 for the SMS serum. Of the 38 sera positive for anti-GAD by RIP, only 6 had antibodies detectable by Western blotting, and all gave an RIP titer of at least 1:250. The low frequency of antibodies by Western blotting was explicable by denaturation of the antigen. Thus, using a dot-blotting assay in which reactivity to untreated "native" GAD was compared with reactivity to GAD after denaturation by reduction with 2-mercaptoethanol and boiling, 20 of the 38 IDDM sera reacted unequivocally with the native GAD compared with only 2 that reacted with denatured GAD after reduction and boiling. The sera were tested for their capacity to inhibit the catalytic activity of GAD, but only the high-titer serum from the patient with SMS did so. Our study further validates the RIP assay for anti-GAD and establishes that anti-GAD exists in IDDM over a wide range of titers that correlate with other assay characteristics, and also indicates that the conformational autoantibody epitope on GAD is susceptible to alteration under denaturing conditions.

Adolescent↗
PubMed1992

Antibodies to glutamic acid decarboxylase discriminate major types of diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is marked by circulating antibodies to a 64,000-M(r) islet cell antigen identified as glutamic acid decarboxylase (GAD). We describe a radioimmunoprecipitation assay with GAD isolated from pig brain. The sera tested were from 80 patients with IDDM including 26 with disease of recent onset and 54 with disease of longer duration (3-42 yr), 20 with non-insulin-dependent diabetes mellitus (NIDDM), and 55 nondiabetic subjects. Conventional assays for islet cell cytoplasmic antibodies were performed concurrently. The level of antibody in serum was expressed in units based on percentage reactivity of a standard reference serum. The frequency of antibody to GAD in IDDM was 69% in short-duration cases and 59% in long-duration cases. The latter was substantially higher than the frequency of islet cell cytoplasmic antibody. Antibodies to GAD were elevated (means +/- 3 SD) in 5% NIDDM cases and in none of the nondiabetic subjects. A simple laboratory test with a defined autoantigen has substantial implications for population screening and early diagnosis of IDDM and for better understanding of its pathogenesis.

Adolescent↗
PubMed1990

Spatiotemporal segregation of endothelial cell integrin and nonintegrin extracellular matrix-binding proteins during adhesion events.

Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs.

Acid Phosphatase↗
PubMed1989

Synthetic peptides mimic the assembly of transmembrane glycoproteins.

The composition of the intramembranous domains of many receptors are remarkably uniform, yet there is evidence that many transmembrane proteins associate together to form specific noncovalent homo- or heterocomplexes within the membrane. We have synthesized peptides corresponding to transmembrane domains of glycophorin A, glycophorin C, and the interleukin 2-receptor Tac antigen to study the interactions between transmembrane domains in vitro. Synthetic transmembrane glycophorin A peptide formed a complex with native glycophorin and glycoproteins of erythrocyte and K562 cell membranes that was reversible, specific, and could be demonstrated in a natural bilayer system in the absence of detergents. Synthetic glycophorin C and interleukin 2-receptor Tac antigen transmembrane peptides, although similar in amino acid composition, did not interact with glycophorin and did not inhibit the binding of the synthetic glycophorin A transmembrane peptide to native glycophorin. It is proposed that the transmembrane segments of receptor proteins contain not only the structural information necessary for insertion and anchoring but specific binding sites that mediate interactions between transmembrane glycoproteins.

Amino Acid Sequence↗
PubMed1986

Abnormal spectrin in hereditary elliptocytosis.

An abnormal alpha subunit of erythrocyte spectrin has been described in hereditary pyropoikilocytosis (HPP), a rare hemolytic anemia characterized by erythrocyte budding and fragmentation. In HPP spectrin, the N terminal domain of the alpha subunit (alpha I T80) shows increased susceptibility to tryptic digestion, resulting in cleavage to a 50,000-d peptide, presumably due to a change in primary structure of the alpha I domain which alters conformation and generates the new cleavage site. The functional result of this conformational alteration is marked impairment of spectrin oligomer formation in vitro, consistent with the established role of alpha I T80 in spectrin self-association. In the present study, we demonstrate an abnormal spectrin alpha subunit in two kindreds with hereditary elliptocytosis (HE) that is qualitatively identical to HPP spectrin. Clinical expression of HE in these families ranges from mild elliptocytosis without hemolysis to severe poikilocytic hemolytic anemia clinically resembling HPP. In all affected individuals, a fraction of alpha I T80 is abnormal, as shown by its cleavage during mild tryptic digestion to the 50 kd peptide described in HPP; the fraction of alpha I T80 affected is directly proportional to the severity of clinical expression of HE. Spectrin oligomer formation is likewise impaired to a degree which correlates with hematologic disease. One of the HE kindreds studied demonstrated polymorphism in the spectrin alpha II domain, previously described as a frequent occurrence in blacks. This family also demonstrates a variant alpha III domain in spectrin that has not previously been described. We conclude that the abnormality in the alpha I domain originally described in HPP spectrin is shared by a subset of patients with HE; the severity of clinical expression, ranging from mild nonhemolytic HE to poikilocytic hemolytic anemia, is related to the fractional quantity of the alpha subunit that is affected.

Adolescent↗
PubMed1984

Common structural polymorphisms in human erythrocyte spectrin.

Restricted tryptic digestion of erythrocyte spectrin at 4 degrees C followed by two-dimensional (isoelectric-focusing/sodium dodecyl sulfate) polyacrylamide electrophoresis yields highly reproducible maps of approximately 50 peptides with molecular weights between 80,000 and 12,000. Based on molecular weight and isoelectric point (pI), each unique alpha- and beta-subunit domain can be identified and compared with spectrin peptides from other individuals. The alpha-subunit of spectrin from 60 Caucasian donors contains a 46,000-mol-wt tryptic domain, called alpha II-T46, Type 1; more extensive tryptic digestion of this domain generates peptides with molecular weights of 35,000, 30,000, 25,000, and 16,000. Spectrin from 29 of 37 black donors representing 14 kindreds shows variation in the molecular weight and/or pI of peptides from the alpha II domain. In the most common form, Type 2, alpha II tryptic peptides are increased in molecular weight by 4,000, and the pI becomes more basic. Other alpha II variants are characterized by either the 4,000 increase in molecular weight (Type 3) or by the basic shift in pI (Type 4). When limit peptide maps of intermediate-sized tryptic and CNBr peptides from the alpha II-domain Types 1 and 2 are compared, a consistent alteration in the chromatographic mobility of one limit peptide is observed. Polymorphism in the alpha II subunit of spectrin did not itself produce anemia, nor did it appear to alter the expression of an underlying hereditary spherocytosis or elliptocytosis. In six family studies, the alpha II 46,000-mol-wt variations observed were consistent with Mendelian inheritance.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis.

The structural and functional properties of spectrin from normal and hereditary pyropoikilocytosis (HPP) donors from the two unrelated families were studied. The structural domains of the spectrin molecule were generated by mild tryptic digestion and analyzed by two-dimensional electrophoresis (isoelectric focusing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The alpha I-T80 peptide (Mr 80,000) is not detectable in two related HPP donors; instead, two new peptides (Mr 50,000 and 21,000) are generated and have been identified as fragments of the normal alpha I-T80. A third sibling has reduced levels of both the normal alpha I-T80 and the two new peptides. A similar analysis of spectrin from another HPP family indicates that their spectrins contain reduced amounts of the alpha I-T80 and the 50,000 and 21,000 fragments of the alpha I domain. The HPP donor also has other structural variations in the alpha I, alpha II, and alpha III domains. The alpha I-T80 domain of normal spectrin has been shown to be an important site for spectrin oligomerization (J. Morrow and V.T. Marchesi. 1981. J. Cell Biol. 88: 463-468), and in vitro assays indicate that HPP spectrin has an impaired ability to oligomerize. Ghost membranes from HPP donors are also more fragile than membranes from normal erythrocytes when measured by ektacytometry. In both the oligomerization and fragility assays, the degree of impairment is correlated with the amount of normal alpha I-T80 present in the spectrin molecule. We believe that a structural alteration in the alpha I-T80 domain perturbs normal in vivo oligomerization of spectrin, producing a marked decrease in erythrocyte stability.

Adult↗
PubMed1982

A structural model of human erythrocyte spectrin. Alignment of chemical and functional domains.

Proteolytic susceptibility has been used to probe the structure of human erythrocyte spectrin. Nine unique polypeptide segments have been defined by mild trypsin digestion (0 degrees C) and analyzed by two-dimensional peptide mapping techniques. These peptide segments, referred to operationally as chemical domains, exhibited varying degrees of sensitivity to further proteolytic cleavage. One region (beta I) which contained the phosphorylated amino acids of the beta subunit was quite sensitive to proteolysis and was rapidly degraded to numerous small peptides. Overlap peptides produced by enzymatic and chemical cleavages were used to align each domain in the appropriate spectrin subunit. The molecular weights of the largest unique peptides from both subunits sum to the approximate weight of the intact molecule. Similarly, summation of the two-dimensional peptide maps of the intermediate sized peptides approximates the two-dimensional maps of the intact spectrin subunits, indicating that most or all of the molecule is represented. These results suggest that spectrin is composed of multiple, ordered, largely alpha-helical domains that are connected by small protease-sensitive segments. A comprehensive structural model is presented.

Electrophoresis, Cellulose Acetate↗
PubMed1982

Monoclonal antibodies as probes of domain structure of the spectrin alpha subunit.

A library of nine monoclonal antibodies which bind to the alpha subunit of human erythrocyte spectrin has been established. The specificity of these antibodies confirms the alignment and uniqueness of each of the five previously identified peptide domains in this subunit and establishes the identity of additional smaller proteolytic peptide fragments. This immunochemical approach is complementary to the identification of peptide relationships by two-dimensional chymotryptic peptide mapping, and the results of both methods are in complete agreement.

Antibodies, Monoclonal↗
PubMed1980

Identification of proteolytically resistant domains of human erythrocyte spectrin.

Digestion of purified human erthrocyte spectrin with proteolytic enzymes at 0 degrees C results in the production of intermediate-size peptides that resist further cleavage at 0 degrees C. By two-dimensional peptide analysis of these intermediate peptides it has been determined that five unique peptides are produced by tryptic cleavage of the alpha subunit of spectrin (band 1); these have apparent molecular weights of 80,000, 46,000, 46,000, 41,000, and 30,000 and account for 97% of the alpha subunit. Similarly, four unique peptides having apparent molecular weights of 74,000, 65,000, 33,000, and 38,000 account for 90% of the beta subunit (band 2). By examining larger peptide fragments, the linear alignment of the unique peptides along each of the spectrin subunits has been established. These results indicate that spectrin is composed of two nonidentical subunits, each containing multiple proteolytically resistant domains. These domains, which may be largely alpha-helical, seem to be connected by small protease-sensitive segments. The proteolytic resistance of these domains is not influenced by the multimeric state of the spectrin molecule.

Chymotrypsin↗
PubMed1980

Identification of functional domains of human erythrocyte spectrin.

Isolated human erythrocyte spectrin is a dimer of two unique polypeptide chains. The dimer (alpha beta) undergoes reversible salt- and temperature-dependent association to form (alpha beta)2 tetramers. Spectrin also binds with high affinity to a protein receptor on the cytoplasmic surface of erythrocyte membrane vesicles. By cleavage of spectrin at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid, a 50,000-dalton peptide fragment has been isolated which inhibits the binding of spectrin to erythrocyte membrane vesicles. This peptide arises from a terminal region of the beta chain. An 80,000-dalton peptide generated by restricted trypsin digestion binds preferentially to dimeric spectrin. This peptide arises from a terminal portion of the alpha chain. Multiple peptides involved in noncovalent associations between the chains have also been identified. These associations indicate that the two subunits of spectrin are aligned parallel to one another and that the tetramer formation site and the high-affinity membrane binding site are in close proximity to one another.

Binding Sites↗
PubMed1977

A simple method of obtaining an enriched fraction of tegumental brush border from Hymenolepis diminuta.

A method for isolating an enriched preparation of tegumental brush border from the tapeworm, Hymenolepis diminuta, is described. Combining incubation of whole tapeworms in Krebs-Ringer/tris-maleate solution containing a hemolytic saponin, low shear-force agitation, and differential centrifugation, a pellet is obtained at 2,500 g which contains a significant concentration of surface brush border. The content of brush border in this fraction is identified by the presence of numerous microvilli, increased specific radioactivity after surface tagging with 3H-Concanavalin A, and relatively little mitochondrial contamination (succinic dehydrogenase). Based on morphological criteria, fractions sedimenting with greater force contain dense vesicles and mitochondria from the outer portion of the tegument.

Cestoda↗