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R C Williams

Publications and source records attributed to R C Williams.

At least 127 records · Page 7Linked to original sources
PubMed1993

Unambiguous classification of microtubule-ends in vitro: dynamic properties of the plus- and minus-ends.

To understand the mechanism of dynamic instability of microtubule growth and shortening, one needs a means of reliably determining the polarity of the microtubules under investigation. Sea urchin sperm-tail axonemal fragments nucleate the growth of both plus-ended and minus-ended microtubules, but their polarity is not apparent by video-enhanced DIC microscopy. The polarity of a microtubule is usually assessed by observing differences between the rates and lengths of growth and shortening excursions of the two ends. In practice, though, a significant fraction of the population of microtubules displays characteristics intermediate between the average characteristics of either end, thereby escaping classification. Excluding these "intermediate" microtubules from the measured populations introduces bias into the understanding of microtubule dynamic instability. We circumvent this problem by making use of the plus-end directed movement of the microtubule-dependent molecular motor kinesin to determine the polarity of any given microtubule unambiguously. Carboxylated-microspheres coated with kinesin, which are clearly visible by DIC microscopy, were used to determine the polarity of a microtubule. The dynamics were then observed. Kinesin was found to have no marked effect on dynamic instability. By this technique, we show that the distributions of properties that describe microtubule dynamic instability (rates and lengths of growth and shortening as well as frequencies of interconversion between these phases) of plus-ends overlap to a significant extent with those of minus-ends. It is this overlap that obscures the usual classification of the ends. Therefore, models describing microtubule dynamic instability need to incorporate the broad and overlapping range of properties of the two ends.

Animals↗
PubMed1993

New technologies for the diagnosis of periodontal disease.

Historically the initial onset of periodontal disease in an individual was considered slow but continuously progressive over time. It was inferred that an individual would experience progressive destruction of the attachment structures until edentulous. However, several studies in the early 1970s contested the traditional concept of continuous disease progression. These studies suggested that periodontal disease progressed by recurrent acute episodes. In specific individuals, the loss of attachment was faster than the concept of slowly progressing disease. There were also sites in patients that progressed slower (or not at all) than anticipated on the basis of radiographic and clinical history of previous disease progression. These findings clearly indicated the need to explore the nature of periodontal disease progression and to institute a major effort into innovative methods to diagnose periodontal diseases. Dentists and researchers could more effectively prevent and treat periodontal disease and relate pertinent research findings to specific disease initiation and progression.

Alveolar Bone Loss↗
PubMed1993

Conformational dependency of human IgG heavy chain-associated Gm allotypes.

Human IgG allotypic markers Gm(a)[Glm(1)], Gm(x)[Glm(2)]; Gm(f)[Glm(4)], Gm(b)[G3m(5) and (11)] and Gm(g)[G3m(21)] were studied after chemical modification of IgG histidines by diethylpyrocarbonate, tyrosines by N-acetylimidazole and lysines by formaldehyde and sodium borohydride. Degrees of substitution were estimated by trinitrobenzenesulfonic acid assay. IgG of known Gm phenotype isolated from serum of hyperimmune anti-tetanus toxoid donors was studied. Histidyl modification resulted in virtually complete loss of Gm(a) and Gm(g) antigenicity but preservation of Gm(x), Gm(b) and Gm(f). Reconstitution of the histidyl residues using hydroxylamine resulted in virtually complete restoration of Gm(a) and Gm(g) antigenicity. Histidine modification resulted in no significant decrease in ELISA anti-tetanus antibody activity. Alteration of tyrosyl residues using N-acetylimidazole considerably diminished Gm(a) and Gm(f) expression. This effect was reversed by hydroxylamine treatment. Moreover, chemical alteration of tyrosyl residues produced a complete loss of Gm(g) antigenicity which was only partially restored after deacylation. A urinary H chain fragment containing the VH region directly linked to C gamma 3 which contained the Gm(a) specific and Gm(x) specific amino acid residues was positive for Gm(a) but negative for Gm(x). Another urinary H chain fragment containing only the C gamma 3 domain was negative for both Gm(a) and (x). These findings indicate that Gm allotypic markers may depend on conformational determinants in which strongest expression for Gm(a) and (x) depends on structures expressed by C gamma 3 linked to C gamma 2 domains. Although RFs react with the region encompassing the C gamma 2-C gamma 3 interface, Gm-specificities of such reactions are affected allosterically through single or double amino acid substitutions at a relatively distant site.

Amino Acid Sequence↗
PubMed1993

Rheumatoid factors from patients with rheumatoid arthritis react with Des-Lys58-beta 2m, modified beta 2-microglobulin.

Ten polyclonal IgM rheumatoid factor (RF) preparations, affinity-purified from IgG columns, from patients with rheumatoid arthritis were studied for their ELISA reactivity with native beta 2m in parallel with Lys58-cleaved beta 2m and Des-Lys58-beta 2m, the latter representing cleavage products of the native molecule present in some pathologic human sera. Most RF showed positive reactions with the native form of beta 2m but reduced reactivity for the cleaved forms of beta 2m. Reactions between cleaved beta 2m and RF, in solution, were demonstrated by inhibition of RF binding to native beta 2m by preincubation with a range of concentrations of Des-Lys58-beta 2m. By contrast, eight of nine murine MoAbs to human beta 2m showed approximately equivalent binding to native beta 2m, Lys58-cleaved beta 2m, and Des-Lys58-beta 2m. Reactions between individual human RF and the altered forms of beta 2m (Lys58-cleaved beta 2m and Des-Lys58-beta 2m) appeared to parallel the previously determined beta 2m single amino acid specificities, in that RF showing strong reactivity with Lysine 58 also showed a significant diminished reactivity with the Des-Lys58-beta 2m lacking the critical lysine residue. The present studies demonstrate that while human RF react with Lys58-cleaved beta 2m or Des-Lys58-beta 2m, preferential reactivity is observed for native unaltered beta 2m.

Amino Acid Sequence↗
PubMed1993

The effect of a mouthrinse based on nisin, a bacteriocin, on developing plaque and gingivitis in beagle dogs.

The aim of this study was to evaluate the effect of nisin, an antimicrobial peptide, on the development of plaque and gingivitis in beagle dogs when compared with 0.12% chlorhexidine and a placebo. 16 female beagle dogs 1 year of age were brought to optimum gingival health by scaling, root planing and polishing. At the conclusion of the pretreatment phase, the dogs were divided into 4 groups for the application of the test agents and were placed on a plaque promoting diet of Purina Dog Chow softened with water. Test agents included 100 micrograms/ml nisin and 300 micrograms/ml nisin formulated in a vehicle containing 1 mM NaEDTA; negative control comprised of exactly the same formulation but omitting nisin; and 0.12% chlorhexidine as Peridex. Throughout the treatment period, formulations were applied 2x daily to premolar teeth in each quadrant for 1 min using a Monojet syringe. The development of plaque and gingivitis was monitored at 15, 27, 39, 53, 74 and 88 days during the treatment phase using standard measurements of gingival index, plaque index, stain index and bleeding to probing. Throughout the treatment phase, plaque accumulation increased in all groups, but the rate of plaque build-up was less in groups treated with either nisin or chlorhexidine formulations compared with the placebo treated group. The gingival index of dogs in all groups increased throughout the study period. However, from day 27 onward, the groups receiving nisin had lower gingival index scores than did the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1993

Nonsteroidal antiinflammatory drugs as inhibitors of periodontal disease progression.

Recent interest in the control and modulation of periodontal disease has focused on the potential benefits of blocking the host response mechanisms involved in the progression of the disease. In addition to recent advances in the identification and control of etiologic bacteria, investigators have indicated promising results using nonsteroidal antiinflammatory drugs (NSAIDs) as inhibitors of the inflammatory destruction in periodontal disease. This article examines research efforts over the last 20 years describing the role of prostaglandins in periodontal disease and the effect of NSAIDs on the progression of gingival inflammation and alveolar bone loss.

Alveolar Bone Loss↗
PubMed1993

Periodontal regeneration of human infrabony defects. III. Diagnostic strategies to detect bone gain.

Assessment of actual treatment outcome by simple and available diagnostic tests represents an important aspect in the evaluation of periodontal regenerative procedures. This report focuses on the diagnostic accuracy of different strategies to detect the original extent of bone loss and bone gain in deep vertical defects following guided tissue regeneration. Characteristics of probing attachment level and radiographic bone linear measurements were compared to a gold standard obtained as intrasurgical clinical measurements at baseline and at the 1 year re-entry operation. Radiographic linear measurements underestimated bone loss (CEJ-BD) in 55% of cases while the estimate was within 1 mm in 25% of cases. The goodness of agreement could be significantly described by a general linear model incorporating measures of defect morphology. The original level of bone loss could be best detected by an approach which consisted of adding 1.5 mm to the measured attachment level loss. Such an approach correctly estimated bone loss within 1 mm in 85% of sites. In terms of ability to detect bone gain, radiographic measurements underestimated bone gain in 55% and overestimated it in 15% of sites. Diagnostic accuracy was assessed at different threshold values of true gain. The sensitivity and specificity plots intersected at a threshold value of 2.7 mm bone gain. Conversely, measures of probing attachment level gain agreed with the gold standard within 1 mm 45% of times. Intersection of the specificity and sensitivity plots occurred at 3 mm. Classification tables are presented for both diagnostic tests at different threshold values.2+é

Adolescent↗
PubMed1993

Monoclonal IgM rheumatoid factors generated from synovial B cells of rheumatoid arthritis patients react with beta 2-microglobulin. Monoclonal RF react with beta 2m.

Four of 15 monoclonal human IgM rheumatoid factors (RF) derived from synovial B cells of patients with rheumatoid arthritis showed positive ELISA reactions with human beta 2-microglobulin. These findings were different from those previously noted using IgM RF derived from monoclonal Waldenstrom's paraproteins or the IgM components of mixed cryoglobulins, and resembled the anti-beta 2 microglobulin specificity of polyclonal IgM RF from patients with rheumatoid arthritis. Reactions of monoclonal IgM synovial RF with overlapping 7-mers of beta 2m sequence indicated major regions of positive reactivity at positions 57-64 and 89-95 which were maintained in the presence of high salt (300 mM NaCl) conditions. Glycine substitution of each residue within RF-reactive beta 2m regions indicated that tryptophanes at position 60 and 95, lysine at 58, phenylalanine at 62, valine at 93 and arginine at 97 constituted important single amino acids for the reactive epitopes. These findings indicate that clonally restricted human IgM RF derived from diseased tissues of patients with RA show anti-beta 2m reactivity similar to polyclonal RF from the same patients. This particular fine specificity is not present in monoclonal RF derived from patients with Waldenstrom's or mixed cryoglobulins showing anti-gamma-globulin activity.

Amino Acid Sequence↗
PubMed1993

Antigenic epitopes on beta 2-microglobulin reacting with monoclonal antibodies.

Monoclonal antibody-reactive antigenic regions on the 99 amino acid sequence of human beta 2-microglobulin were examined by using nine anti-beta 2 m monoclonal antibodies (mAbs) and Geysen pins coated with overlapping heptamers derived from the primary sequence. Reactive linear sequences were then restudied with glycine substitution for each successive residue. All nine mAbs showed a similar profile, with peak reactivity at residues 57-63 SKDWSFY and 87-97 LSQPKIVKWDR. Glycine substitution established the tryptophans at positions 60 and 95 as well as the lysines at 58, 91, and 94 as immunodominant residues important for mAb reactivity. Other adjacent residues (serine 57, phenylalanine 62, tyrosine 63, isoleucine 92, valine 93, and proline 90) also make important contributions to beta 2 m antigenic epitopes. Rabbit antipeptide antibodies against the beta 2 m peptides SKDWSFY and LSQPKIVKWDR produced quantitative precipitin curves with beta 2 m in solution. Addition of iodine 125-labeled beta 2 m to antipeptide antibody followed by protein A precipitation and sodium dodecyl sulfate gel electrophoresis with autoradiography showed that labeled beta 2 m reacted in solution phase with anti-SKDWSFY or anti-LSQPKIVKWDR antibody. These findings establish residues 57-63 and 87-97 in human beta 2 m as major reactive antigenic sites for monoclonal as well as polyclonal anti-beta 2 m antibodies.

Amino Acid Sequence↗
PubMed1992

Serologic cross-reactivities poorly reflect allelic relationships in the HLA-B12 and HLA-B21 groups. Dominant epitopes of the alpha 2 helix.

Previous analysis has emphasized the correlation between primary structures of class I HLA molecules and their patterns of serologic cross-reactivity. Here we describe the structures of two serologic groups of HLA-B alleles for which this is not the case. HLA-B45, an allele associated with black populations, is serologically paired with B44 in the B12 group; its structure, however, is divergent from that of B44 but closely related to B50. The BN21 (B*4005) allele is associated with native Americans and is serologically grouped with B50 in the B21 group; its structure, however, is more closely related to alleles of the B40 group. The B44 and B45 serologically cross-reactive molecules differ at seven functional positions of the Ag recognition site; the B50 and BN21 molecules differ at four such residues. These differences are predicted to alter peptide presentation and be capable of eliciting strong alloreactive T cell responses. For these pairs of B12 and B21 Ag, serology appears dominated by epitopes formed by short sequences of the alpha 2 helix which have been shuffled by recombination between alleles. The implications of these results for HLA matching in transplantation are discussed.

Alleles↗
PubMed1992

Differential mapping of Fc gamma-binding and monoclonal antibody-reactive epitopes on gE, the Fc gamma-binding glycoprotein of herpes simplex virus type 1.

The entire 396 residue extracellular sequence of gE the HSV-1 Fc gamma-binding glycoprotein has been studied to determine epitopes binding to two mAb II-481 and 88S previously demonstrated to react with gE at or near the Fc gamma-binding regions. Overlapping 7-mers constructed from the established sequence were tested with mAb II-481 and 88S along with their Fab fragments. Control mAb of the same IgG 2b subclass as well as whole rabbit and human IgG and Fc were also tested for binding to overlapping linear sequences using the ELISA pin assay to map Fc gamma-binding regions. Six sequences PKTSWRRVS, GLYTLSV, QVASVVLVVQP, PAPPRSWP, CLYHPQLP, and ASTWTSRL were found that constituted major regions binding to the two different mAb of the same specificity. Glycine substitution for each residue within these sequences indicated that arginine 29, tryptophane 70, valine 144, valine 157, arginine 208, histidine 283, and arginine 305 constituted important portions of the II 481 mAb-reactive epitope. Many of the same regions along with one other, GPLHPSW, appeared to be involved in Fc gamma binding. Substitution of glycine for each residue indicated that histidine 67, tryptophane 70, valine 71, valine 157, valine 158, valine 160, valine 161, tryptophane 210, serine 279, cysteine 280, leucine 281, tyrosine 282, histidine 283, proline 284, glutamine 285, proline 287, tryptophane 302, and arginine 305 were important for Fc gamma-binding. Inhibition by gE peptides of rosetting of E sensitized with rabbit IgG antibody around HSV-1-infected cells, as well as inhibition of rosetting using F(ab)2 fragments of rabbit antibodies to these peptides was used to assay relative contributions of all seven regions to Fc gamma-binding activity. Our results provide a tentative map of mAb binding and Fc gamma-reactive sites on gE. mAb and Fc gamma binding of a limited number of individual antigenic amino acids widely distributed among the separate reactive regions suggest that many of the same separate residues contribute both to antigenicity as well as to Fc gamma-binding activity.

Amino Acid Sequence↗
PubMed1992

Heteroclitic polyclonal and monoclonal anti-Gm(a) and anti-Gm(g) human rheumatoid factors react with epitopes induced in Gm(a-), Gm(g-) IgG by interaction with antigen or by nonspecific aggregation. A possible mechanism for the in vivo generation of rheumatoid factors.

Heteroclitic rheumatoid factors (RF) are specific for allotypic determinants, e.g., Gm(a) or Gm(g) on allogeneic, but not autologous IgG. All polyclonal RF we isolated from nine rheumatoid arthritis patients with circulating Gm(a-), (b+), (g-), (f+) IgG displayed dual heteroclitic activity for the Gm(a) and Gm(g) allotypes, as shown by using appropriate RBC agglutination assays and affinity columns bearing Gm(a+) or Gm(g+) IgG. To investigate possible mechanisms underlying the in vivo generation of heteroclitic RF, we tested the ability of nonspecifically and immune-specifically aggregated Gm(a-), (g-) IgG to function as targets for RF from Gm(a-), (g-) patients with rheumatoid arthritis. Heat aggregation (63 degrees C for 20 min) or binding to Ag (as in tetanus toxoid-antitetanus toxoid complexes) induced a "functional" Gm(a+) and/or (g+) phenotype in Gm(a-), (g-) IgG from five healthy subjects and five rheumatoid patients, as suggested by the ability of these altered IgG to function as efficient targets for six heteroclitic RF in direct binding and competitive inhibition experiments. That heterocliticity and dual Gm(a), Gm(g) specificity can be features of a single antibody molecule was formally demonstrated by analysis of a monoclonal RF (IgM mAb 61) generated from a Gm(a-), (g-) rheumatoid patient. RF mAb 61 displayed a high affinity (Kd, 10(-7) M) for IgG Fc fragment of Gm(a+) and (g+) IgG or aggregated autologous Gm(a-), (g-) IgG but did not bind to native autologous IgG. To investigate the molecular basis of the acquired Gm(a) phenotype, PBMC from five Gm(a-) patients with rheumatoid arthritis and two Gm(a-) normal subjects arthritis and two Gm(a-) normal subjects were cultured in vitro after activation with PWM. In most instances, these PBMC produced IgG that behaved as Gm(a+) in sensitive ELISA. Application of the polymerase chain reaction (PCR), using probes specific for the nucleotide sequence coding for the Gm(a) tetrapeptide, to the amplification of DNA from the in vitro-stimulated Gm(a-) normals or rheumatoid patients' PBMC provided no evidence for Gm(a) nucleotide sequences. The present data suggest that acquisition of the Gm(a) determinant by Gm(a-) IgG may result from subtle changes in the CH2-CH3 RF-binding region. Such changes would occur when Gm(a) IgG are complexed with Ag or nonspecifically altered, thereby providing a possible explanation for the induction of heteroclitic RF in Gm(a-) rheumatoid arthritis patients.

Antibodies, Monoclonal↗
PubMed1992

Rheumatoid factors from patients with rheumatoid arthritis react with beta 2-microglobulin.

IgM rheumatoid factors (RF) from 18 sera of rheumatoid arthritis (RA) patients isolated from monomeric IgG affinity columns showed strongly positive ELISA reactions with human beta 2-microglobulin (beta 2m), as well as with recombinant beta 2m. When the same RA sera were adsorbed to beta 2m-Sepharose affinity columns, eluted material showed predominant IgM anti-Fc of IgG and anti-beta 2m reactivity. Inhibition reactions with "RF" obtained from IgG affinity columns showed slightly higher reactivity of RF for Fc over beta 2m; however, when RF from the same RA serum had been adsorbed to and eluted from beta 2m affinity columns, beta 2m showed greater inhibition than Fc for RF reacting with either beta 2m or Fc on ELISA plates. Thus two overlapping populations of RF were identified in RA sera showing reactivity with both beta 2m and Fc of IgG. When RF were isolated from IgG columns, affinity was slightly higher for Fc than beta 2m. Conversely, RF eluted from beta 2m Sepharose reacted slightly more with beta 2 m than Fc. Trypsin digests of a polyclonal RA IgM RF showed no beta 2m reactivity in Fc mu 5 fragments. Fab mu RF retained slight anti-Fc IgG but no residual anti-beta 2m activity. Monoclonal human IgM, IgG, or IgA RF either from mixed cryoglobulins or EBV-stimulated RA lymphoid cell lines showed negative or occasional weakly positive anti-beta 2m activity. Overlapping 7-mer peptide ELISA analysis of the entire 99-amino acid sequence of beta 2m showed a major RF-reactive linear hydrophilic sequence at positions 56-60 which included a 3-amino acid exact homology to positions 401, 403, and 404 of the C gamma 3 domain. A peptide encompassing this sequence produced 90% inhibition of RF binding to whole beta 2m. Substitution of neutral glycines for each amino acid throughout the reactive epitope at positions 56-66 indicated that lysine at position 58 aspartic acid at 59, and tryptophane at 60 represented major portions of the RF-reactive epitope. These findings indicate that human RF derived from patients with RA react with other epitopes besides those present on IgG Fc, including epitopes on human beta 2m. For many years serum RF3 found in patients with RA have been regarded as premier examples of autoantibodies to autologous IgG.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Unusual HLA-B alleles in two tribes of Brazilian Indians.

The Kaingang and Guarani are culturally and linguistically distinct tribes of southern Brazil. Like all Amerindian groups they show limited HLA polymorphism, which probably reflects the small founder populations that colonized America by overland migration from Asia 11,000-40,000 years ago. We find the nucleotide sequences of HLA-B alleles from the Kaingang and Guarani to be distinct from those characterized in caucasian, oriental and other populations. By comparison, the HLA-A and C alleles are familiar. These results and those reported in the accompanying paper on the Waorani of Ecuador reveal that a marked evolution of HLA-B has occurred since humans first entered South America. New alleles have been formed through recombination between pre-existing alleles, not by point mutation, giving rise to distinctive diversification of HLA-B in different South American Indian tribes.

Alleles↗
PubMed1992

Microtubules grow and shorten at intrinsically variable rates.

Microtubules were assembled from pure tubulin with axonemal pieces serving as nuclei. They were observed by video-enhanced differential-interference-contrast light microscopy. Their lengths were measured from videotaped images at frequent intervals (0.13-5 s). Error analysis indicated that the uncertainty in measuring a single length was quite small; the 95% confidence limit approximated the microscope's limit of resolution. Rates of growth and shortening of the dynamically unstable microtubules, obtained from the length-versus-time data, were found to be highly variable. The variability was far too large to be attributed to known random error of measurement and must be a property of the microtubules. Further experiments were aimed at finding its structural cause. The variability of rates exhibited by a single microtubule was as great as that of the whole population. The locations at which a growing microtubule changed its rate of growth were not related to the locations at which rates changed during its subsequent shortening. The cause of the variability must therefore be both small relative to the size of a microtubule and transient relative to its lifetime. Fluctuations in configuration of the microtubule's cap appear to be the likeliest source.

Animals↗
PubMed1992

The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules.

In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.

Amides↗