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K J Smith

Publications and source records attributed to K J Smith.

At least 163 records · Page 9Linked to original sources
PubMed1996

Factor VII central. A novel mutation in the catalytic domain that reduces tissue factor binding, impairs activation by factor Xa, and abolishes amidolytic and coagulant activity.

Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patient's factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of Phe328 to Ser. The patient was homozygous for this mutation, whereas each parent of the patient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S factor VII exhibited <1% factor VII procoagulant activity and a 2-fold decreased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg315-Lys316 peptide bond in intact F328S factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F328S factor VIIa to cleave substrates may result from the apparent formation of a hydrogen bond between Tyr377 and Asp338, a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings suggest that Phe328, which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis.

Adult↗
PubMed1996

Determination of the structure of the N-terminal splice region of the cyclic AMP-specific phosphodiesterase RD1 (RNPDE4A1) by 1H NMR and identification of the membrane association domain using chimeric constructs.

A 25-residue peptide representing the membrane targeting N-terminal splice region of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1) was synthesized, and its structure was determined by 1H NMR. Two independently folding helical regions were identified, separated by a highly mobile "hinge" region. The first helical region was formed by an N-terminal amphipathic alpha-helix, and the second consisted of multiple overlapping turns and contained a distinct compact, hydrophobic, tryptophan-rich domain (residues 14-20). Chimeric molecules, formed between the N-terminal region of RD1 and the soluble bacterial protein chloramphenicol acetyltransferase, were used in an in vitro system to determine the features within the splice region that were required for membrane association. The ability of RD1-chloramphenicol acetyltransferase chimera to become membrane-associated was not affected by deletion of any of the following regions: the apolar section (residues 2-7) of the first helical region, the polar part of this region together with the hinge region (residues 8-13), or the polar end of the C-terminal helical region (residues 21-25). In marked contrast, deletion of the compact, hydrophobic tryptophan-rich domain (residues 14-20) found in the second helical region obliterated membrane association. Replacement of this domain with a hydrophobic cassette of seven alanine residues also abolished membrane association, indicating that membrane-association occurred by virtue of specific hydrophobic interactions with residues within the compact, tryptophan-rich domain. The structure of this domain is well defined in the peptide, and although the region is helical, both the backbone and the distribution of side chains are somewhat distorted as compared with an ideal alpha-helix. Hydrophobic interactions, such as the "stacked" rings of residues Pro14 and Trp15, stabilize this domain with the side chain of residue Leu16 adopting a central position, interacting with the side chains of all three tryptophan residues 15, 19, and 20. These bulky side chains thus form a hydrophobic cluster. In contrast, the side chain of residue Val17 is relatively exposed, pointing out from the opposite "face" of the peptide. Although it appears that this compact, tryptophan-rich domain is responsible for membrane association, at present the target site and hence the specific interactions involved in membrane targeting by the RD1 splice region remain unidentified.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Production and crystallization of MHC class I B allele single peptide complexes.

Major histocompatibility complex class I B alleles, HLA B8, B53 and B3501 have been cloned, expressed, refolded and crystallized in specific complexes with a number of different 8-mer and 9-mer peptides. For some of these crystallization was initiated by cross-seeding between different B allele complexes. All crystallize in the space group P212121, with similar unit cell dimensions of approximately 52 A X 81 A X 112 A, contain one complex per asymmetric unit and diffract to approximately 2.0 A resolution.

Alleles↗
PubMed1996

Structural integrity of the gamma-carboxyglutamic acid domain of human blood coagulation factor IXa Is required for its binding to cofactor VIIIa.

This report describes the analysis of a novel mutant human factor IX protein from a patient with hemophilia B (factor IX activity <1%; factor IX antigen 45%). Enzymatic amplification of all eight exons of the factor IX gene followed by direct sequence analysis reveals a single nucleotide change (a guanine --> adenine transition) in exon 2 at nucleotide 6409 which results in a glycine --> arginine substitution at amino acid 12 in the gamma-carboxyglutamic acid rich (Gla) domain of the mature protein. Factor IX was isolated by immunoaffinity chromatography from plasma obtained from the proband. The purified protein is indistinguishable from normal factor IX by polyacrylamide gel electrophoresis. Characterization of the variant in purified component assays reveals that it is activated normally by its physiologic activator factor XIa, but its phospholipid-dependent activation by the factor VIIa-tissue factor complex is diminished. In the presence of phospholipid and 5 mM Ca2+, the activities of variant and normal plasma-derived factor IX are similar; however, in the presence of activated factor VIIIa (intrinsic tenase complex), the normal augmentation of the cleavage of the specific substrate of factor IX, factor X, is not observed. The determination of the association constants for normal and variant factor IXa with factor VIIIa shows that the affinity of the activated variant factor IX for the cofactor factor VIIIa is 172-fold lower than normal. Competition studies using active site-inactivated factor IXas in the intrinsic tenase complex confirm that the defect in the variant protein is in its binding to factor VIIIa. We conclude that the structural integrity of the Gla domain of human factor IX is critical for the normal binding of factor IXa to factor VIIIa in the intrinsic tenase complex. In addition, a glycine at amino acid 12 is necessary for normal activation of factor IX by the factor VIIa-tissue factor complex.

Adenine↗
PubMed1996

Expression of the APC tumor suppressor protein in oligodendroglia.

Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene have been linked to familial polyposis, an inherited predisposition to colon cancer, and a high percentage of sporadic colon adenomas. Although this gene is best known for its role in development of bowel neoplasms, in recent studies we have found that APC mRNA levels are greatly enriched in brain compared with peripheral tissues. To help define its role in the nervous system, in this study we have determined its cellular localization immunohistochemically in adult rat brain sections and have detected intense APC immunoreactivity in oligodendrocytes. Since prominent APC immunostaining is detected in cell bodies of mature oligodendrocytes, these antibodies may provide a useful addition to available oligodendrocyte markers. Although the cellular function of APC remains undefined, previous biochemical studies have demonstrated that APC is associated with catenins, cytoplasmic proteins involved in regulating cell-cell adhesion. We propose that, in addition to its critical role in ensuring normal maturation of colonic epithelial cells, the APC tumor suppressor protein also regulates the adhesive properties of oligodendrocytes.

Adenomatous Polyposis Coli↗
PubMed1996

Changes in the distribution of a calcium-dependent ATPase during demyelination and remyelination in the central nervous system.

A calcium-adenosine triphosphatase (Ca(2+)-ATPase) activity expressed by CNS nerve fibres has been examined during demyelination and remyelination in rats, 21-26 days after an intraspinal injection of ethidium bromide. The Ca(2+)-ATPase distribution was determined cytochemically, using a technique believed primarily to reflect the presence of ecto-ATPases. We confirm that in normal nerve fibres Ca(2+)-ATPase activity was present on the external surface of the myelin sheath, and on the axolemma at the nodes of Ranvier. Labelling of the internodal axolemma was restricted to small, scattered, punctate regions. However, following demyelination the Ca(2+)-ATPase activity was expressed continuously along both the exposed, previously internodal axolemma of entirely naked axons, and it was particularly prominent at sites of contact between axons and glial-cell processes. During remyelination (which in this lesion is accomplished predominantly by Schwann cells) the proportion of the axonal surface exhibiting Ca(2+)-ATPase activity decreased in concert with the progressive thickening of the new myelin sheath. The non-myelin forming plasmalemma of Schwann cells was positive for the Ca(2+)-ATPase activity, but activity was abruptly lost at the site of compaction between the inner and outer leaflets of the forming myelin sheath. Ecto-ATPase activity is a property of some cell adhesion molecules, and it follows that the changes observed in the distribution of ATPase activity in this study may reflect changes in the axolemma which are important for the successful repair of the lesion by remyelination. The ATPase activity may, for example, reflect the changing distribution of molecules important in aiding axo-glial recognition and the establishment of axo-glial contacts.

Animals↗
PubMed1996

Blood-brain barrier permeability in astrocyte-free regions of the central nervous system remyelinated by Schwann cells.

The patency of the blood-brain barrier was examined during the development and repair of focal demyelinating lesions induced in the dorsal columns of rats by the intraspinal injection of ethidium bromide, with or without concomitant irradiation. Blood-brain barrier integrity was determined by the intravenous injection of horseradish peroxidase or by the immunofluorescent localization of endogenous albumin. Following repair, the central area of the lesions was remyelinated by Schwann cells and lacked astrocytes. In unirradiated lesions, demyelination was established at one week and the lesion was largely repaired by remyelination by 12 weeks. Horseradish peroxidase extravasation was absent at one day after injection, but was present at three days and throughout the period of repair. With one exception, all animals which exhibited regions of demyelination also exhibited horseradish peroxidase extravasation. No horseradish peroxidase was seen in lesions where all the demyelinated axons had been repaired by remyelination, and strong albumin immunofluorescence was also absent from such lesions. Albumin immunoreactivity was also absent from normal spinal cords, although it was prominent in normal sciatic nerves and dorsal roots. Irradiation of lesions resulted in a delay in the repair by remyelination, and repair of the blood-brain barrier was similarly delayed. Promotion of Schwann cell remyelination has been suggested as a potential therapy for central demyelinating disorders such as multiple sclerosis; however, central regions remyelinated by Schwann cells lack astrocytes, cells which have been implicated in the induction and maintenance of the blood-brain barrier. Since blood-brain barrier opening may be an early step in the production of new lesions, a defective barrier could allow such remyelinated regions to act as foci for further lesion development. We conclude, however, that the remyelination of central demyelinating lesions by Schwann cells is accompanied by recovery of properties of an intact blood-brain barrier, despite the lack of astrocytes. The present findings support the idea that promotion of remyelination by Schwann cells may form an effective therapy for central demyelinating diseases.

Animals↗
PubMed1996

Granulocyte colony-stimulating factor as a marker for bacterial infection in neonates.

OBJECTIVE: To evaluate granulocyte colony-stimulating factor (G-CSF) as an early marker of bacterial or fungal infection in neonates. STUDY DESIGN: We measured G-CSF levels in infants of varying gestational and postnatal ages. We separated the infants into three groups: group 1, positive bacterial or fungal blood culture result; group 2, negative blood culture result but evidence of clinical sepsis; and group 3, negative blood culture result and no or weak evidence of sepsis. Comparison of mean G-CSF levels by group was accomplished by an analysis of variance. RESULTS: One hundred seventy-six evaluations for sepsis were done for 156 infants with gestational ages ranging from 24 to 43 weeks; 50% of these infants were less than 35 weeks of gestational age. The mean G-CSF levels of groups 1 and 2 were significantly higher than those of group 3. The mean G-CSF level of each group was 2278 pg/ml (group 1), 1873 pg/ml (group 2), and 280 pg/ml (group 3) (p < 0.001). On the basis of a cutoff level of 200 pg/ml, the sensitivity of the test was 95%, specificity 73%, positive predictive value 40%, and negative predictive value 99%. CONCLUSION: G-CSF levels represent a sensitive marker of infection in neonates of all gestational ages.

Bacteremia↗
PubMed1996

Bacillus piliformis infection (Tyzzer's disease) in a patient infected with HIV-1: confirmation with 16S ribosomal RNA sequence analysis.

Bacillus piliformis is a long, rod-shaped bacterium that has never been grown in cell-free medium and whose taxonomic classification is uncertain. B. piliformis is the causative agent of Tyzzer's disease, which is frequently reported in laboratory, wild, and domesticated animals. The spectrum and severity of this disease is wide in animals. Although many infections are rapidly fatal, subclinical infections are also common. To date, there have been no reports of B. piliformis infection in human beings, although elevated antibody levels have been reported in pregnant women. We describe the first case of human B. piliformis infection, in a man with HIV-1 infection and chronic, localized, crusted verrucous lesions. The diagnosis was confirmed by ribosomal RNA sequencing. The spectrum of organisms leading to infection and the spectrum of diseases caused by these organisms continue to expand, as new infections are identified and as patients with HIV-1 live longer with more severe immune suppression. The extreme difficulty in culturing B. piliformis and the lack of clinical and histopathologic experience with this organism in human beings mean that B. piliformis is potentially another infectious agent to be considered in human beings. Also, when an infectious organism is a strong clinical consideration, silver stains may be of use when results of routine bacterial staining are negative.

AIDS-Related Opportunistic Infections↗
PubMed1996

Histologic and immunohistochemical features in biopsy sites in which bovine collagen matrix was used for hemostasis.

BACKGROUND: Bovine collagen matrix (Helistat and Helatene), which is used primarily for hemostasis, decreases wound contracture in skin biopsy sites and promotes wound granulation. OBJECTIVE: Our purpose was to evaluate excision specimens of previous biopsy sites to determine whether there are histologic differences between cases in which bovine collagen matrix was used for hemostasis and those in which it was not used. METHODS: Thirty-two diagnostic punch biopsies were done with hemostasis obtained with bovine collagen matrix in 24 biopsies and by pressure alone in eight. The biopsy sites were reexcised at various time intervals, and the histologic and immunohistochemical features were evaluated and compared. RESULTS: In specimens in which collagen matrix was used, migration of stromal and epithelial cells along the bovine collagen matrix was evident by 2 days and had progressed rapidly by 4 days. Around and within the matrix hyaluronic acid increased during the first 8 to 10 days. Initially stromal cells stained with CD34 and reticulum fibers were present at 8 to 10 days. AT 12 to 15 days, factor XIIIa+ stromal cells were present within the matrix, and host collagen fibers were present and well-oriented within the degenerating matrix. Biopsy specimens allowed to heal without the matrix showed a delayed and an uneven increase in hyaluronic acid. Furthermore, the granulation tissue within the dermis showed no organization with a persistent acute inflammatory infiltrate and increased muscle actin-positive stromal cells as scar formation proceeded. CONCLUSION: Bovine collagen products appear to provide a matrix that promotes thrombosis. The collagen matrix also promotes migration and attachment of stomal and epithelial cells, thereby organizing and accelerating wound healing.

Actins↗
PubMed1996

Clinical and histopathologic features of hair loss in patients with HIV-1 infection.

BACKGROUND: Hair loss is common in patients with HIV-1 infection, and in black patients this loss may be associated with straightening. Possible causes are frequently present in patients with HIV-1. These causes include chronic HIV-1 infection itself and recurrent secondary infections, nutritional deficiencies, immunologic and endocrine dysregulation, and exposure to multiple drugs. However, histopathologic features have rarely been reported in these patients. OBJECTIVE: The objective was to evaluate the changes in the hairs of a group of these patients and to identify the light microscopic and ultrastructural changes in the hairs and the histologic changes in the scalp. METHODS: Hair plucks and pulls with scanning electron microscopy of the hairs were done on 10 patients with late-stage HIV-1 infection. In addition, scalp biopsy specimens were examined in both vertical and transverse sections. RESULTS: All patients had telogen effluvium. Numerous apoptotic or necrotic keratinocytes were seen in the upper external root sheath follicular epithelium in addition to a mild to moderate perifollicular mononuclear cell infiltrate often containing eosinophils. Variable dystrophy of the hair shafts was also a consistent feature. CONCLUSION: Although telogen effluvium is a common response to a wide spectrum of biologic stresses, the presence of apoptotic or necrotic keratinocytes within the upper end of the external root sheath epithelium and dystrophy of hairs may be markers of hair loss in patients with HIV-1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed1996

An altered position of the alpha 2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501.

The crystal structure of the human major histocompatibility complex class I B allele HLA B*3501 complexed with the 8-mer peptide epitope HIV1 Nef 75-82 (VPLRPMTY) has been determined at 2.0 angstrom resolution. Comparison with the crystal structure of the closely related allele HLA B*5301 reveals the structural basis for the tyrosine specificity of the B*3501 F pocket. The structure also reveals a novel conformation of the 8-mer peptide within the binding groove. The positions of the peptide N and C termini are nonstandard, but the classic pattern of hydrogen bonding to nonpolymorphic MHC class I residues is maintained, at the N terminus by addition of a water molecule, and at the C terminus by a substantial shift in the alpha 2 helix.

Alleles↗
PubMed1996

Bound water structure and polymorphic amino acids act together to allow the binding of different peptides to MHC class I HLA-B53.

The structure of the human MHC class I molecule HLA-B53 complexed to two nonameric peptide epitopes (from the malaria parasite P. falciparum and the HIV2 gag protein) has been determined by X-ray crystallography at 2.3 angstrom resolution. The structures reveal the architecture of a Pro-specific B pocket common to many HLA-B alleles. Relative to other alleles, the B53 peptide-binding groove is widened by a significant (up to 1.25 angstrom) shift in the position of the alpha 1 helix. Within this groove, bound water molecules, acting in concert with the side chains of polymorphic residues, provide the functional malleability of the MHC, which enables the high affinity/low specificity binding of multiple peptide epitopes.

Amino Acid Sequence↗
PubMed1996

A randomized, double-blind comparison of donor tolerance of 400 mL, 200 mL, and sham red cell donation.

BACKGROUND: Volume replacement could allow the safe collection of twice the normal amount of red cells in a standard donation. Studies in small numbers of donors have shown that a temporary decrease in red cell mass is well tolerated when donors give twice the usual amount (170-225 mL) of red cells in a standard 405- to 495-mL donation. Sham-donation control groups have not been included in previous studies of increased red cell donation, and perceptions of donation effects could have been biased. STUDY DESIGN AND METHODS: In the study reported here, 17 male and 13 female volunteers were randomly assigned to make a sham donation, 1-unit donation, or 2-unit donation on an automated blood cell separator. Donor tolerance was assessed by ambulatory heart rate monitoring and by a poststudy interview. Hemoglobin, hematocrit, ferritin, serum iron, total iron-binding capacity, red cell 2,3 DPG, and serum erythropoietin were measured before and after donation for comparison of the erythropoietic responses in the three study groups. RESULTS: Red cells collected totaled 206 +/- 10 mL in the 1-unit group and 414 +/- 21 mL in the 2-unit group. Changes in heart rate, systolic blood pressure, and diastolic blood pressure with donation and changes in heart rate recorded by ambulatory monitoring did not differ for the experimental groups. Postdonation changes from baseline values were evaluated on Days 2, 7, and 14. Changes in hemoglobin were significantly different between groups (p < 0.017) in all postdonation tests. There were differences between groups in erythropoietin response, red cell 2,3 DPG, ferritin levels, and hemoglobin synthesis. Hemoglobin synthesis and mean changes in 2,3 DPG, erythropoietin, ferritin, and postdonation hemoglobin were greater in the 2-unit group than in the 1-unit group. CONCLUSION: Donor tolerance of red cell donations of 414 +/- 21 mL, a volume of red cells twice that in a standard 450-mL blood donation, does not differ from donor tolerance of standard or sham donations. Physiologic adjustments and the hematopoietic response to reduced red cell mass were greater in the 2-unit group, but the donation of 1 unit or 2 units did not cause detectable symptoms of reduced oxygen-carrying capacity.

Blood Donors↗
PubMed1996

Cutaneous histopathologic features in weanling pigs after exposure to three different doses of liquid sulfur mustard.

Sulfur mustard (2,2' dichlorodiethyl sulfide, HD) is a chemical warfare agent that is easily produced, and may be used against civilian populations as well as against military troops. However, good therapeutic and prophylactic measures await a better understanding of the pathophysiology of lesions produced by this agent. Because the skin remains is one of the principal routes for HD exposure and damage, the study of HD-induced skin lesions is of major interest. Blister formation is a characteristic of HD-induced skin lesions in humans. Attempts have been made to find an animal model that produces cutaneous microblisters after exposure to the naturally occurring liquid as well as vaporized HD. Weanling pigs were exposed to three different doses of liquid HD. Histopathologic findings showed microblister formation as well as variable apoptosis and/or necrosis of epidermal keratinocytes and vascular endothelium. Pig skin is morphologically similar to human skin. In the pig, the epidermal lipids, the density of hair follicles, the presence of sweat glands, the proliferation kinetics, and the antigenicity are all closer to human skin than are rodent models. All these features may be important in lesions induced by HD, and may mean that the pig is a superior model for studying the pathophysiology of HD-induced cutaneous lesions.

Animals↗
PubMed1996

Bacillary angiomatosis in an immunocompetent child.

Bacillary angiomatosis (BA) is a pathological process characterized by prominent vascular proliferation secondary to organisms of the genus Rochalimaea. BA has been most commonly associated with HIV-1+ patients, but has also been reported rarely in other immune-suppressed patients and in a small group of patients with no demonstrated immune suppression. Even in immune-suppressed children, BA is extremely rare. We report a 5-year-old girl with no apparent immune suppression and no risk factors for HIV-1+ disease, who presented with a skin lesion that histopathologically was diagnostic of BA.

Abscess↗