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Biomedical subjects

J A Dent

Publications and source records attributed to J A Dent.

At least 37 records · Page 2Linked to original sources
PubMed1993

Optimization of a recombinant von Willebrand factor fragment as an antagonist of the platelet glycoprotein Ib receptor.

The binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) Ib receptor is one of the initial events in thrombus formation. Previous studies have shown that RG12986, a reduced and alkylated recombinant fragment of vWF (Ser445-Val733), can inhibit binding of native vWF to GP Ib and offers potential as an anti-thrombotic agent. We have now evaluated a series of deletion mutants of RG12986 and found that reduced and alkylated rvWF508-704 is close to the minimal sequence with optimal RG12986-like activity (IC50 for inhibition of GP Ib-dependent platelet aggregation in the absence of modulators: 0.022 microM +/- 0.01, n = 3) and that it too binds directly to GP Ib. Under in vitro conditions, with no exogenous modulators present and in the absence of shear stress, oxidized rvWF508-704 (containing a disulfide bond between Cys508 and Cys659) is approximately 5-fold less active than reduced and alkylated rvWF508-704; the two fragments, however, display comparable activity in the presence of the modulator botrocetin. The smaller rvWF508-704 fragment offers distinct advantages over RG 12986. In particular, removal of non-active NH2 and COOH terminal sequences may reduce the risk of antigenicity and may contribute to rendering the molecule mostly monomeric in solution, as opposed to the monomer-dimer equilibrium previously described for RG12986.

Chromatography, High Pressure Liquid↗
PubMed1993

von Willebrand factor mutation enhancing interaction with platelets in patients with normal multimeric structure.

Variant von Willebrand disease designated as type I New York or type Malmö is characterized by enhanced ristocetin-induced platelet agglutination with normal von Willebrand factor multimeric distribution in plasma. We have studied four such patients belonging to three unrelated families and found in all of them a unique cytosine-to-thymine transition changing the codon for Pro503 (CCG) to Leu (CTG). In three patients the mutant allele also had a silent mutation in the codon for Ser500 (TCG-->TCA). Both nucleotide changes are present in the von Willebrand factor pseudogene; however, the characterization of distinctive markers where the gene and pseudogene differ, as well as the examination of amplified cDNA derived from platelet mRNA, confirmed that the abnormality occurs in the von Willebrand factor gene of the patients. Moreover, recombinant expression of the isolated glycoprotein Ib-binding domain of von Willebrand factor provided direct evidence that the Pro503-->Leu mutation is responsible for enhanced platelet reactivity to lower ristocetin concentrations. These results define a new structural element affecting the affinity of von Willebrand factor for glycoprotein Ib and establish the molecular basis of a variant form of von Willebrand disease.

Adult↗
PubMed1993

Continuous passive motion in hand rehabilitation.

This paper reviews the literature comparing the results obtained in applying regimes involving motion with those involving rest following injury or surgery. The deleterious effects of immobilisation are compared to those obtained under conditions of passive motion and intermittent passive motion. It is concluded that continuous passive motion (CPM) represents an improvement on intermittent motion. Models of CPM machines are described and some results presented. It is proposed that "intelligent" CPM would represent a further improvement in technique and a prototype machine for this purpose and some preliminary results are described.

Animals↗
PubMed1993

Which tests to choose when assessing hand function.

In an attempt to simplify the choice of the many tests available for the assessment of hand function nine commonly used tests assessing different modalities of hand function have been evaluated on a small group of patients. The tests are judged by how well they fulfil the criteria of being simple and quick to use, easy to replicate, relevant to the needs of the patient, objective and inexpensive to acquire. The five tests which most fulfilled these criteria have been used to form a concise and practical hand assessment pro forma.

Activities of Daily Living↗
PubMed1992

Herbert screw: results of a single-centre trial.

We report the results of a single-centre trial of the Herbert screw in the treatment of acute fractures, fracture-dislocations and non-unions of the carpal scaphoid. In the trial, 22 patients were studied with a maximum follow-up of 2.5 years. Of the patients, 84 per cent had minimal or no pain, and 94 per cent were happy with their result or significantly improved. There was an average loss of 34 per cent of radial deviation of the wrist and of 10 per cent in cylinder grip strength. On radiographic examination, 86 per cent achieved satisfactory union. However, only 73 per cent of type D2 fractures united. Overall, the subjective and objective clinical results achieved were more satisfactory than the radiographic results would suggest.

Adult↗
PubMed1992

Host cell factors controlling vimentin organization in the Xenopus oocyte.

To study vimentin filament organization in vivo we injected Xenopus oocytes, which have no significant vimentin system of their own, with in vitro-synthesized RNAs encoding Xenopus vimentins. Exogenous vimentins were localized primarily to the cytoplasmic surface of the nucleus and to the subplasma membrane "cortex." In the cortex of the animal hemisphere, wild-type vimentin forms punctate structures and short filaments. In contrast, long anastomosing vimentin filaments are formed in the vegetal hemisphere cortex. This asymmetry in the organization of exogenous vimentin is similar to that of the endogenous keratin system (Klymkowsky, M. W., L. A. Maynell, and A. G. Polson. 1987. Development (Camb.). 100:543-557), which suggests that the same cellular factors are responsible for both. Before germinal vesicle breakdown, in the initial stage of oocyte maturation, large vimentin and keratin filament bundles appear in the animal hemisphere. As maturation proceeds, keratin filaments fragment into soluble oligomers (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787-797), while vimentin filaments remain intact and vimentin is hyperphosphorylated. To examine the role of MPF kinase in the M-phase reorganization of vimentin we deleted the conserved proline of vimentin's single MPF-kinase site; this mutation had no apparent effect on the prophase or M-phase behavior of vimentin. In contrast, deletion of amino acids 19-68 or 18-61 of the NH2-terminal "head" domain produced proteins that formed extended filaments in the animal hemisphere of the prophase oocyte. We suggest that the animal hemisphere cortex of the prophase oocyte contains a factor that actively suppresses the formation of extended vimentin filaments through a direct interaction with vimentin's head domain. During maturation this "suppressor of extended filaments" appears to be inactivated, leading to the formation of an extended vimentin filament system.

Amino Acid Sequence↗
PubMed1991

Independent assembly and secretion of a dimeric adhesive domain of von Willebrand factor containing the glycoprotein Ib-binding site.

von Willebrand factor (vWF) is a multimeric glycoprotein that supports platelet adhesion on thrombogenic surfaces as part of the normal hemostatic response to vascular injury. We have employed a domain-specific expression strategy to analyze the biosynthetic processing steps and minimum structural requirements for assembly of the platelet receptor glycoprotein Ib-binding domain of vWF. A chimeric cDNA that codes for the vWF signal peptide and a segment of vWF internal primary sequence, residues 441-730, directs the secretion of a functional vWF fragment from mammalian cells. The recombinant molecule intrinsically assembles through intermolecular disulfide bond formation into a dimeric adhesive domain without contributions from other regions of vWF, including propeptide, previously indicated as essential for vWF multimer assembly. Prevention of N-linked glycosylation on the recombinant domain does not impair dimer formation or the ability to support platelet aggregation. These results identify a minimum structural element for vWF subunit assembly and provide new insights into the processing steps to produce vWF multimers and adhesive domains.

Animals↗
PubMed1991

Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor.

von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.

Cloning, Molecular↗
PubMed1991

Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit.

In this report we demonstrate that proteolytic cleavage of the constituent subunit is one of the causes determining the heterogeneous size distribution of plasma von Willebrand factor (vWf) multimers. As shown by two-dimensional nonreduced/reduced agarose/polyacrylamide gel electrophoresis, the structure of circulating vWf molecules may deviate from that represented by assemblage of a variable number of identical subunits. Indeed, even though the largest multimers in normal plasma appear to be composed predominantly of intact 225-kD subunits, those of intermediate and smaller size contain also 189-, 176-, and 140-kD proteolytic fragments. Different subunit composition patterns are repeated regularly in multimers of increasing molecular mass, yielding series of bands with similar structure. One of these series consists of molecules without evidence of proteolytic fragmentation, and its smallest member appears to be a dimer of 225-kD subunits. Type IIA von Willebrand disease, characterized by absence of the largest multimers, displays a pattern wherein the fragments of 176 and 140 kD are relatively increased, that of 189 kD is markedly decreased or absent, but the composition of individual multimers is otherwise similar to that of species seen also in normal plasma. In contrast to those in the circulation, all normal platelet vWf multimers contain only intact subunit. These results suggest that proteolytic cleavage of plasma vWf subunits occurs after release from cellular sites, whereas platelet vWf stored in alpha-granules is protected from proteolysis. These findings provide information that may be relevant for understanding the normal processing of vWf multimers and for elucidating the pathogenesis of some of the congenital and acquired structural abnormalities of this molecule.

Edetic Acid↗
PubMed1990

Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor.

Proteolytic cleavage of the von Willebrand factor subunit may be important for processing and/or function of the molecule and is altered in certain subtypes of von Willebrand disease. It results in the generation of two main fragments with apparent molecular masses of 140 kDa and 176 kDa from the 225-kDa subunit. We have now obtained chemical evidence to locate the protease-sensitive bond between residues Tyr-842 and Met-843, a site that appears to reflect the specificity of calcium-dependent neutral proteases (calpains). Antibodies were raised against four synthetic peptides that represented sequences immediately preceding or following or including the cleavage site. One antibody (against the fragment from Ala-837 through Asp-851) reacted only with the intact subunit, and its epitope included the cleavage site. All others reacted specifically with either the 140-kDa or the 176-kDa fragment, demonstrating their origin from a single cleavage. In samples of purified von Willebrand factor from four of five patients with type IIA von Willebrand disease, the anti-peptide antibodies showed markedly decreased reactivity with either the 140-kDa or the 176-kDa fragment, suggesting the existence of distinct molecular abnormalities clustered around the cleavage site. Thus, in the majority of type IIA patients, a common pathogenetic mechanism may lead to the disappearance of the larger multimers as a consequence of structural changes that may expose a sensitive bond to the action of specific proteases. These studies demonstrate the use of anti-peptide antibodies directed at a relevant structural domain for the immunochemical differentiation of normal and mutant molecules.

Amino Acid Sequence↗
PubMed1989

Type II H von Willebrand disease: new structural abnormality of plasma and platelet von Willebrand factor in a patient with prolonged bleeding time and borderline levels of ristocetin cofactor activity.

In this study a new variant of type II von Willebrand disease is identified by multimeric analyses of increasing resolving power. Prior to multimeric analysis, the patient was misdiagnosed as carrying an undefined abnormality in platelet function because of his normal von Willebrand factor antigen (vWF:Ag) and low borderline ristocetin cofactor (Ricof) levels. Absence of the largest multimers from the patient's plasma and platelets was shown in a low-resolution system, but all the multimers were present in his relatives. An abnormality in the complex multimeric structure was demonstrated in both plasma and platelets with high-resolution agarose gels. The plasma of the proband and of several family members shows a broader central band with a minor, faster moving satellite band differing from the typical "triplet pattern" observed with normal plasma. Platelets show a "doublet" that runs with a mobility different from the "doublet" in normals. Therefore the proband may be either a homozygote or double heterozygote for this new abnormality. Treatment with desmopressin (DDAVP) on several occasions corrected the prolonged bleeding time of the patient only transiently. Factor VIII increased significantly, but vWF:Ag and Ricof responded poorly. We conclude that this vWF abnormality is different from those observed in the other variants (II A-G) previously described. Therefore the proposed designation for this new variant is type II H.

Adult↗
PubMed1989

Invasive pituitary adenoma presenting with nasal obstruction.

We report two cases of massive invasive pituitary adenoma with the unusual presentation of nasal obstruction. In both cases the diagnosis was only made when the obstructing tissue was removed. Cases reported previously have usually presented with severe focal neurological signs or a long history of neurological symptoms. These two cases are unusual in having minimal neurological deficits despite the extension of tumour into the nasopharynx and the middle and posterior cranial fossae. These extensions were well illustrated by computerised tomography. This unusual presentation is discussed with reference to the literature.

Adenoma, Chromophobe↗
PubMed1989

A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus.

We have developed a whole-mount immunocytochemical method for Xenopus and used it to map the expression of the intermediate filament protein vimentin during early embryogenesis. We used two monoclonal antibodies, 14h7 and RV202. Both label vimentin filaments in Xenopus A6 cells, RV202 reacts specifically with vimentin (Mr, 55 x 10(3] on Western blots of A6 cells and embryos. 14h7 reacts with vimentin and a second, insoluble polypeptide of 57 x 10(3) Mr found in A6 cells. The 57 x 10(3) Mr polypeptide appears to be an intermediate filament protein immunochemically related to vimentin. In the whole-mount embryo, we first found vimentin at the time of neural tube closure (stage 19) in cells located at the lateral margins of the neural tube. By stage 26, these cells, which are presumably radial glia, are present along the entire length of the neural tube and in the tail bud. Cells in the optic vesicles express vimentin by stage 24. Vimentin-expressing mesenchymal cells appear on the surface of the somites at stage 22/23; these cells appear first on anterior somites and on progressively more posterior somites as development continues. Beginning at stage 24, vimentin appears in mesenchymal cells located ventral to the somites and associated with the pronephric ducts; these ventral cells first appear below the anterior somites and later appear below more posterior somites. The dorsal fin mesenchyme expresses vimentin at stage 26. In the head, both mesodermally-derived and neural-crest-derived mesenchymal tissues express vimentin by stage 26. These include the mesenchyme of the branchial arches, the mandibular arch, the corneal epithelium, the eye, the meninges and mesenchyme surrounding the otic vesicle. By stage 33, vimentin-expressing mesenchymal cells are present in the pericardial cavity and line the vitelline veins. Vimentin expression appears to be a marker for the differentiation of a subset of central nervous system cells and of head and body mesenchyme in the early Xenopus embryo.

Animals↗
PubMed1988

von Willebrand disease "Vicenza" with larger-than-normal (supranormal) von Willebrand factor multimers.

When normal volunteers or patients with type I von Willebrand disease (VWD) are given desmopressin (DDAVP), a set of larger-than-normal (supranormal) von Willebrand factor (VWF) multimers, similar to those present in VWF-containing cells such as platelets megakaryocytes and endothelial cells, appear transiently in postinfusion plasma. In two kindreds with mild lifelong bleeding symptoms transmitted as an autosomal dominant trait, all ten symptomatic members (but none of the five asymptomatic members) had a supranormal multimeric structure for plasma VWF, apparently identical to that seen for postdesmopressin normal plasma. Plasma factor VIII coagulant activity (VIII:C), VWF antigen (VWF:Ag), ristocetin-induced platelet agglutination, and ristocetin cofactor (RiCof) activity were low. Platelet VWF:Ag and RiCof levels (tested for three patients only) were normal. Bleeding times were normal or slightly prolonged. The patients' platelet multimeric structure was the same as that for normal platelets. After desmopressin infusion the plasma VWF multimeric structure remained supranormal as for preinfusion plasma, with VIII:C VWF:Ag and RiCof increasing markedly over baseline values and disappearing at a normal rate. Examination of the VWF subunit composition from three of these patients indicated that proteolytic processing of their VWF did not differ from normal. This study describes the first variant of VWD with a supranormal multimeric structure.

Blood Platelets↗