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K J Smith

Publications and source records attributed to K J Smith.

At least 19 recordsLinked to original sources
PubMed1992

The binding of human factor IX to endothelial cells is mediated by residues 3-11.

We have used chimeras and point mutations of recombinant coagulation factor IX to examine factor IX's specific interaction with bovine endothelial cells. Previously (Toomey, J. R., Smith, K. J., Roberts, H. R., and Stafford, D. W. (1992) Biochemistry 31, 1806-1808), we restricted the region of factor IX responsible for binding to endothelial cells to its Gla domain. Molecular modeling of the Gla domain of factor IX using the coordinates of the Gla domain of bovine prothrombin-(1-145) (Soriano-Garcia, M., Padmanabhan, K., deVos, A. M., and Tulinsky, A. (1992) Biochemistry 31, 2554-2566) reveals two major surface determinants whose sequences differ among factors IX, X, and VII. A chimeric protein comprised of the Gla domain of factor VII with the remainder of the molecule of factor IX did not bind to the endothelial cell binding site. We changed residues 33, 34, 35, 39, and 40 to those of factor IX without restoring endothelial cell binding. Replacement of amino acid residues 3-10 with those of factor IX restored normal binding. With the knowledge that specific binding was localized to the first 11 amino acids, point mutations were made at residues predicted to be on the surface in this region of the factor IX molecule. Changing lysine 5 to alanine (K5A) or valine 10 to lysine (V10K) resulted in loss of binding with total retention of in vitro clotting activity. The lysine 5 to arginine (K5R) mutation also was fully active in vitro but displayed 3-fold tighter binding. In addition to defining the sequence of factor IX necessary for binding to endothelial cells, these results suggest that the binding site is not phospholipid but instead is specific, and in all likelihood, protein.

Amino Acid Sequence↗
PubMed1992

Is CNS trauma a prerequisite for the elongation of CNS axons into denervated peripheral nerve?

The demonstration that some central nervous system (CNS) axons can regenerate when provided with a suitable environment raises the possibility of new treatments for CNS injury. However, at present the conditions for optimal regeneration are not well understood. For example, the methods used in previous studies have entailed CNS trauma as part of the research protocol (e.g. that resulting from the implantation of peripheral nerve grafts), and so the role of neuronal or axonal injury in the regrowth observed has been difficult to establish. To determine whether such injury is necessary for the central reinnervation of denervated peripheral nerve, the L5 dorsal root has been chronically denervated in rats by freeze-thawing its dorsal root ganglion (DRG), and the root has been left attached to either traumatized or non-traumatized spinal cord. The trauma induced was quite mild, and resulted from several vertical insertions of a fine needle. Two to 4 months later, retrogradely transported horseradish peroxidase (HRP) was used to label spinal neurons which sent axons into the denervated roots. HRP-labelled neurons were found in each of the spinal cords subjected to trauma, but no labelled neurons were observed in any of the non-traumatized cords. The number of HRP-labelled neurons in individual spinal cords was positively correlated with the degree of spinal cord trauma. We conclude first that the chronic and intimate presence of a denervated PNS tissue in continuity with the spinal cord is not, in itself, a sufficient stimulus to induce its reinnervation by CNS axons. Second, we conclude that under the conditions of this experiment CNS trauma is a prerequisite for the reinnervation of denervated peripheral nervous tissue by CNS axons.

Animals↗
PubMed1992

Conduction properties of central nerve fibers remyelinated by Schwann cells.

Demyelination of central axons arises from a number of conditions, including multiple sclerosis and spinal cord compression. The demyelination disrupts conduction and leads directly to the production of symptoms. Repair of the demyelination by peripheral myelinating cells could potentially relieve the symptoms, but the conduction properties of central axons remyelinated by Schwann cells have yet to be studied in detail. This paper examined the conduction properties of such axons. Large focal demyelinating and remyelinating lesions were induced in the dorsal columns of rats by the intraspinal injection of ethidium bromide. Recordings of compound action potentials conducted through these lesions were then made at various recovery times. Thus the changing conduction properties of the affected fibers could be correlated with the different stages of lesion development. During the early stages of demyelination there was widespread conduction block, with no evidence of appreciable conduction occurring with prolonged latency or refractory period of transmission (RPT). However, with the onset of remyelination by Schwann cells, conduction was restored in many axons, and most, if not all, of the affected axons eventually showed successful conduction through the lesion. Initially the conduction was characterized by very prolonged latency, long RPT, and an inability to conduct fast trains of impulses. These deficits became less prominent as remyelination progressed. In chronically remyelinated axons the RPT was restored to within normal limits, although some deficit in both conduction velocity and the ability to conduct trains of impulses persisted. Since these deficits were not severe we conclude that remyelination of central demyelinated axons by Schwann cells should be effective in promoting the restoration of normal function.

Action Potentials↗
PubMed1992

Hemophilia B caused by five different nondeletion mutations in the protease domain of factor IX.

Factor IX is a multidomain protein and is the proenzyme of a serine protease, factor IXa, essential for hemostasis. In this report, we describe the molecular basis of hemophilia B (deficiency of factor IX activity) in five patients who have neither deletions nor rearrangements of the factor IX gene. By enzymatic amplification and sequencing of all exons and promoter regions, the following causative mutation in the protease domain of factor IX was identified in each patient: IXSchmallenberg: nucleotide 31,215G----T, Ser365Ile; IXVarel: nucleotide 31,214A----G, Ser365Gly; IXMechtal: nucleotide 31,211G----C, Asp364His; IXDreihacken: nucleotide 30,864G----A, Arg248Gln; and IXMonschau: nucleotide 30,855A----T, Glu245Val. In IXVarel, nucleotide 31,213T was also replaced by C, which results in a silent mutation (GAT----GAC) at Asp-364. Thus, this patient has a double base-pair substitution of TA to CG at nucleotides 31,213 and 31,214 but only a single amino acid change of Ser-365 to Gly. This patient also developed an antibody to factor IX during replacement therapy, which suggests that deletion of the factor IX gene is not necessary for development of the antibody in hemophilia B patients. The levels of plasma factor IX antigen in the patients ranged from 40% to 100% except for IXDreihacken (Arg248Gln), in which case it was approximately 4% of normal. The Ser365Gly and Ser365Ile mutants are nonfunctional because of lack of the active site serine residue. Mutant Asp364His is inactive because it cannot form the hydrogen bond between the carboxylate group of Asp-364 and the alpha-amino group of Val-181 generated after activation. As observed in other homologous serine proteases, this hydrogen bond is essential for maintaining the correct active site conformation in normal factor IXa (IXaN). Purified Arg248Gln had approximately 41% and Glu245Val had approximately 17% of the activity of normal factor IX (IXN) in a partial thromboplastin time (aPTT) assay. In immunodot blot experiments, the isolated Glu245Val mutant did and the Arg248Gln mutant did not bind to an anti-IXN monoclonal antibody that has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. We have recently shown that a high-affinity calcium binding site exists in the protease domain of IXN; among the proposed Ca(2+)-binding ligands is the carboxyl group of Glu-245. Further, a part of the epitope for the above antibody was shown to be contained in the 231 to 265 residue segment of factor IX.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1992

The endothelial cell binding determinant of human factor IX resides in the gamma-carboxyglutamic acid domain.

The blood coagulation factor IX(a) binds specifically to a site on endothelial cells with a Kd of 2.0-3.0 nM. A number of previous studies have attempted to define the region(s) of factor IX(a) that mediate this interaction. These studies suggested that there are two regions of factor IX(a), the gamma-carboxyglutamic acid (Gla) domain and the epidermal growth factor like (EGF-like) domains, that mediate high-affinity binding to endothelial cells. Recently, however, the participation of the EGF1 domain has been excluded from the interaction. This indicated that if there was an EGF component of factor IX contributing to the binding affinity, then it must be in the second EGF-like domain. In order to further evaluate this relationship, we performed competitive binding experiments between 125I plasma factor IX and a set of six chimeric proteins composed of portions of factor VII and factor IX. Our data suggest that the high-affinity interaction between factor IX and the endothelial cell binding site is mediated by the factor IX Gla domain and that the factor IX EGF domains are not involved in binding specificity.

1-Carboxyglutamic Acid↗
PubMed1992

Molluscum contagiosum. Ultrastructural evidence for its presence in skin adjacent to clinical lesions in patients infected with human immunodeficiency virus type 1. Military Medical Consortium for Applied Retroviral Research.

BACKGROUND AND DESIGN: Molluscum contagiosum in acquired immunodeficiency disease, although not life threatening, is often a marker of late-stage disease and may lead to disfiguring cutaneous lesions. Although most current therapy results in at least temporary clearing of individual lesions, lesions frequently recur and new lesions arise. Examination of hematoxylin-eosin-stained histologic sections in two patients showed changes suggestive of viral infection in the epidermis 0.5 cm and 1 cm lateral to obvious clinical lesions. These areas were clinically free of any lesions. Both routine histopathologic examination and ultrastructural examination were performed in two patients infected with human immunodeficiency virus type 1 (HIV-1) and three non-HIV-1-infected patients. RESULTS: All patients showed histologic changes diagnostic of molluscum contagiosum. In addition, the sections from HIV-1-infected patients showed areas of acanthosis, hyperkeratosis, and nuclear atypia. Electron microscopy of these areas revealed rare viral organisms in these areas. Similar acanthotic, hyperkeratotic areas were not seen in the biopsy specimens from the non-HIV-1-infected patients and no viral particles were found in the epidermis around the lesions. CONCLUSION: Viral structures consistent with molluscum contagiosum are present within the clinically normal epidermis around lesions of molluscum contagiosum in some HIV-1-infected patients. This may explain the large number of lesions seen in these patients and the difficulty in controlling the spread and recurrence of molluscum contagiosum in HIV-1-infected patients.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Verrucous lesions secondary to DNA viruses in patients infected with the human immunodeficiency virus in association with increased factor XIIIa-positive dermal dendritic cells. The Military Medical Consortium of Applied Retroviral Research Washington, D.C.

BACKGROUND: Hyperkeratotic lesions caused by varicella-zoster, herpes simplex, or cytomegalovirus occur in patients infected with human immunodeficiency virus type 1 (HIV-1). We have also observed this type of lesion with molluscum contagiosum. OBJECTIVES: These cases were studied to determine whether there are any pathologic changes unique to these lesions. METHODS: The cases were studied by routine microscopic examination and immunohistochemistry. RESULTS: Each case showed changes diagnostic of the viral infection, which was confirmed by immunohistochemical stains for herpes simplex and cytomegalovirus. In the dermis there were fewer inflammatory cells than expected, but there was an increase in factor XIIIa-positive dendritic cells. CONCLUSION: Varicella-zoster, herpes simplex virus, cytomegalovirus, and molluscum contagiosum can cause verrucous lesions in HIV-1-infected patients. These lesions may be related to an increase in factor XIIIa-positive dendritic cells.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Spindle cell neoplasms coexpressing cytokeratin and vimentin (metaplastic squamous cell carcinoma).

Spindle cell squamous carcinoma (SCSC) and atypical fibroxanthoma (AFX) are both spindle cell neoplasms (SCN) that usually arise in areas of solar or ionizing radiation of elderly patients. Both lesions have a similar biologic behavior. In addition, the morphologic and ultrastructural similarities found in AFX and the spindle cell component of SCSC, have led some investigators to conclude that these tumors have a similar cell of origin. We studied 15 SCNs with no evidence of epithelial origin and no morphologic epithelial component, that showed immunohistochemical and ultrastructural evidence that would support metaplastic changes of a squamous cell carcinoma to a neoplasm with mesenchymal characteristics.

Aged↗
PubMed1992

Recent advances and controversies concerning adnexal neoplasms.

This article reviews the diagnostic clinical and histologic features of a group of adnexal tumors, including papillary eccrine adenoma, adenoid cystic carcinoma, Merkel's cell carcinoma, aggressive digital papillary adenoma (and adenocarcinoma), Bowen's disease, intraepidermal epithelioma, microcystic adnexal carcinoma and related tumors, and subcutaneous trichoepithelioma. These are adnexal tumors either often not recognized, because of their rarity, or the origin, biologic potential, and classification of which have been controversial.

Adenocarcinoma, Papillary↗
PubMed1992

Iontophoresis of vinblastine into normal skin and for treatment of Kaposi's sarcoma in human immunodeficiency virus-positive patients. The Military Medical Consortium for Applied Retroviral Research.

BACKGROUND: Patients who test positive for human immunodeficiency virus type 1 (HIV-1) and who have disfiguring and/or painful cutaneous lesions of Kaposi's sarcoma (KS) may not be candidates for systemic chemotherapy and/or immunotherapy. Intralesional vinblastine sulfate, as a single-agent chemotherapeutic drug, has been used with some success to treat KS in patients who are HIV-1 positive. However, some patients may not tolerate the pain associated with injection of vinblastine. Transcutaneous iontophoresis of vinblastine was evaluated for therapy of KS in HIV-1-infected patients. Prior to therapy of patients, we iontophoresed vinblastine into the normal skin of volunteers who were not infected with HIV-1 to document the clinical and histologic features that occurred. OBSERVATIONS: Iontophoresis produced a localized erythematous papular eruption in non-HIV-infected volunteers but not in HIV-1-infected patients. Histologic changes in the biopsy specimens taken from non-HIV-infected volunteers consisted primarily of scattered necrotic keratinocytes and a mild to moderate superficial lymphohistiocytic infiltrate. Thirty-one lesions of KS were treated with partial to complete clearing and symptomatic improvement. CONCLUSION: Clinical and histologic features of iontophoresed normal skin suggest an immunologic mechanism of action. Iontophoresis of vinblastine for KS is well tolerated and results in symptomatic improvement as well as varying degrees of clearing of the lesions.

Adult↗
PubMed1992

Cutaneous histopathologic, immunohistochemical, and clinical manifestations in patients with hemophagocytic syndrome. Military Medical Consortium for Applied Retroviral Research (MMCARR).

BACKGROUND AND DESIGN: The hemophagocytic syndrome (HPS) is characterized by fever, wasting, generalized lymphadenopathy, hepatosplenomegaly, and pancytopenia, often with associated coagulopathy. The most common cutaneous manifestations are panniculitis and purpura. Cytophagic histiocytic panniculitis fits within the spectrum of HPS, and the most consistent histopathologic feature in HPS is a proliferation of mature histiocytes that exhibit prominent erythrophagocytosis and cytophagocytosis. The clinical spectrum, the underlying causes, and the histopathologic features found in HPS are broad. The characteristic phagocytic histiocytes seen in HPS have been confused with malignant histiocytes in the past, but are now known to be reactive. The clinical findings, histologic, and immunohistochemical features of 10 cases of HPS with cutaneous lesions were reviewed. Immunohistochemical markers included KP-1, beta F-1, UCHL-1, L-26, MAC-387, factor XIIIa, and S100 protein. RESULTS: The HPS was associated with T-cell lymphoma and/or viral infection. Most biopsy specimens showed edema and hemorrhage with a lymphohistiocytic infiltrate and prominent histiocytic cells showing erythrophagocytosis and, in some cases, cytophagocytosis. The histiocytic cells showed positive reactions for KP-1 and negative reactions for the lymphoid markers. In all cases the lymphoid cells showed a mixed pattern with most cells positive for beta F-1 and UCHL-1, and a small percentage positive for L-26. CONCLUSION: In HPS, the prominent phagocytic histiocytes are reactive and are stimulated by T-cell lymphocytes, either neoplastic or in response to viral infection. Many of the findings in the HPS may also be due directly or indirectly to cytokines produced by proliferating T-cell lymphocytes and/or reactive phagocytic histiocytes.

Acquired Immunodeficiency Syndrome↗
PubMed1991

In vivo recovery and survival of monoclonal-antibody-purified factor VIII concentrates.

In response to reports of discrepant in vitro assays of high-purity concentrates, a double-blind crossover study of in vivo recovery and half-life of two brands of monoclonal-antibody-purified factor VIII concentrates (Monoclate and Hemofil-M) was performed in 23 patients with hemophilia A. In vivo recoveries were close to values predicted from the labelled unitage when plasma samples were assayed by a one-stage method. When a two-stage assay was used, lower recoveries were calculated and the recovery with Hemofil-M was slightly but significantly lower than that with Monoclate. The concentrates were re-assayed in vitro by the two-stage method. Monoclate (which is assayed by the manufacturer using a two-stage method) contained 97% of the labelled potency and Hemofil-M (which is assayed by the manufacturer using a one-stage method) contained 81% of the labelled potency. Differences in in vitro and in vivo assay methods contribute to disparities between expected and observed factor VIII recovery. Clearance of Hemofil-M was significantly faster than that of Monoclate, but volume of distribution at the steady state, mean residence time, and plasma half-disappearance times of the two concentrates were not significantly different.

Adolescent↗
PubMed1991

Localization of the human tissue factor recognition determinant of human factor VIIa.

Tissue factor is an integral membrane glycoprotein that serves as an essential cofactor for the blood coagulation factor VIIa. Recent studies have attempted to localize the tissue factor recognition determinant of human factor VIIa. While several regions of factor VIIa have been implicated as important for tissue factor binding, the high affinity tissue factor recognition determinant of human factor VIIa is unknown. In order to define the determinant, we constructed a set of six chimeric proteins composed of portions of factor VII and factor IX. We then utilized the chimeras in competition experiments with 125I-labeled factor VIIa for recombinant tissue factor bound to an Immobilon-P membrane. The data indicate that the high affinity tissue factor recognition determinant of human factor VIIa is within the epidermal growth factor domains.

Base Sequence↗
PubMed1991

Expression and characterization of human factor IX. Factor IXthr-397 and factor IXval-397.

Factor IXLong Beach has a single amino acid substitution at 397 (Ile to Thr) in the catalytic domain which results in severe hemophilia B. Recent investigations have shown that the substitution of threonine for isoleucine at 397 may affect a part of the macromolecular substrate binding site. Because threonine has a hydroxyl group in its side chain, it is possible that this hydroxyl group makes new hydrogen bonds and disturbs the substrate binding site. We used three techniques: molecular biology, which includes site-directed mutagenesis and recombinant protein expression in tissue culture; computer-aided kinetic data analysis; and molecular modeling to study this mutation site. We have produced two mutant factor IX molecules that have isoleucine 397 replaced by valine or threonine. Factor IXwild type and the two mutants (factor IXVal and factor IXThr) were expressed in human kidney cells and purified using a conformation-specific monoclonal antibody column. After the activation by factor XIa, these three molecules were able to bind p-aminobenzamidine and increase its fluorescence intensity in a similar manner. Factor IXVal and factor IXwild type had indistinguishable activities in an activated partial thromboplastin time (aPTT) assay and similar kinetic parameters with factor X as a substrate. Factor IXThr had only 5% clotting activity compared with normal factor IX, a slightly lower Km and significantly reduced kcat, using factor X as a substrate. We developed energy-refined (AMBER v.3.1) computer models of the three factor IX molecules based on previous work. Three factor IXa models (Ile, Val, or Thr at 397) with a fragment of the factor X activation site were used to predict the effect of the mutation at 397 and evaluate the significance of the new hydrogen bond thought to form between the side chain hydroxyl group of threonine 397 and the carbonyl oxygen of tryptophan 385. This new hydrogen bond would affect the position of an amide proton of adjacent glycine 386 which has been proposed to make a hydrogen bond with a backbone carbonyl oxygen of the P3 residue of factor X. In addition to the new hydrogen bond, there is significant movement in the side chain of tryptophan 385 between the factor IXawild type-factor X model and the factor IXaThr-factor X model that could interfere with substrate binding. This movement could be caused by the change in the molecular volume, the orientation of the side chain at 397, and the new hydrogen bond.

Base Sequence↗
PubMed1991

Identification of FAP locus genes from chromosome 5q21.

Recent studies suggest that one or more genes on chromosome 5q21 are important for the development of colorectal cancers, particularly those associated with familial adenomatous polyposis (FAP). To facilitate the identification of genes from this locus, a portion of the region that is tightly linked to FAP was cloned. Six contiguous stretches of sequence (contigs) containing approximately 5.5 Mb of DNA were isolated. Subclones from these contigs were used to identify and position six genes, all of which were expressed in normal colonic mucosa. Two of these genes (APC and MCC) are likely to contribute to colorectal tumorigenesis. The MCC gene had previously been identified by virtue of its mutation in human colorectal tumors. The APC gene was identified in a contig initiated from the MCC gene and was found to encode an unusually large protein. These two closely spaced genes encode proteins predicted to contain coiled-coil regions. Both genes were also expressed in a wide variety of tissues. Further studies of MCC and APC and their potential interaction should prove useful for understanding colorectal neoplasia.

Adenomatous Polyposis Coli↗
PubMed1991

Reinnervation of denervated skeletal muscle by central neurons regenerating via ventral roots implanted into the spinal cord.

The reinnervation of denervated skeletal muscle by central axons regenerating via a ventral root implanted into the spinal cord was examined in rats. The 8th thoracic ventral root was severed and its distal end implanted into the ventro-lateral column of the spinal cord via a stab incision. In control animals the root was severed, but was not implanted into the stab incision. After 12-14 months the animals were examined electrophysiologically to determine the presence or absence of motor units in the 8th intercostal muscle which were reinnervated by centrally derived axons regenerating via the implant. Such units were found in implanted animals, but in none of the controls. Evidence that the motor units were reinnervated by central axons included the facts that the units could be activated either, (1) reflexly (i.e. trans-synaptically) by electrical stimulation of the dorsal roots or spinal cord, or (2) pharmacologically by either the intraspinal injection of glutamate or acetycholine, or by the systemic administration of strychnine. Great care was taken to ensure that the only feasible connection between the spinal cord and the 8th intercostal muscle was via the site of implantation. The EMG signals from the motor units were of large amplitude, typical of reinnervated muscle, and their individual activation resulted in discernible contractions of regions of the T8 intercostal muscle. We conclude that regenerating CNS neurons can be guided to innervate denervated skeletal muscle by the implantation of severed ventral roots into the spinal cord. The neuromuscular synapses formed are functional and persistent. The findings may be relevant to the restoration of function after nervous injuries, such as the avulsion of ventral roots.

Animals↗