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Biomedical subjects

C I Smith

Publications and source records attributed to C I Smith.

At least 55 records · Page 3Linked to original sources
PubMed1998

X-ray imaging and microspectroscopy of plants and fungi.

X-ray fluorescence microscopy and microspectroscopy with micrometre spatial resolution and unprecedented capabilities for the study of biological and environmental samples are reported. These new capabilities are a result of both the combination of high-brilliance synchrotron radiation and high-performance X-ray microfocusing optics and the intrinsic advantages of X-rays for elemental mapping and chemical-state imaging. In this paper, these capabilities are illustrated by experimental results on hard X-ray phase-contrast imaging, X-ray fluorescence (XRF) imaging and microspectroscopy of mycorrhizal plant roots and fungi in their natural hydrated state. The XRF microprobe is demonstrated by the simultaneous mapping of the elemental distributions of P, S, K, Ca, Mn, Fe, Ni, Cu and Zn with a spatial resolution of approximately 1 x 3 micron and with an elemental sensitivity of approximately 500 p.p.b. Microspectroscopy with the same spatial resolution is demonstrated by recording near-edge X-ray absorption (XANES) spectra of Mn at a concentration of approximately 3 p.p.m.

Journal Article↗
PubMed1998

Thermal unfolding of small proteins with SH3 domain folding pattern.

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Solution structure of the SH3 domain from Bruton's tyrosine kinase.

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk). Btk belongs to the Tec family of tyrosine kinases. Each member of the family contains five regions and mutations causing XLA have been isolated in all five regions. We have determined the solution structure of the Src homology 3 (SH3) domain of Btk using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on natural abundance and 15N-labeled protein material. The structure determination is complemented by investigation of backbone dynamics based on 15N NMR relaxation. The Btk SH3 forms a well-defined structure and shows the typical SH3 topology of two short antiparallel beta-sheets packed almost perpendicular to each other in a sandwich-like fold. The N- and C-termini are more flexible as are peptide fragments in the RT and n-Src loops. The studied Btk SH3 fragment adopts two slowly interconverting conformations with a relative concentration ratio of 7:1. The overall fold of the minor form is similar to that of the major form, as judged on the basis of observed NOE connectivities and small chemical shift differences. A tryptophan (W251) ring flip is the favored mechanism for interconversion, although other possibilities cannot be excluded. The side chain of Y223, which becomes autophosphorylated upon activation of Btk, is exposed within the potential SH3 ligand binding site. Finally, we compare the present Btk SH3 structure with other SH3 structures.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Interactions between SH2 and SH3 domains.

Src homology 2 (SH2) and SH3 domains are abundant protein and peptide binding modules in signalling molecules. Certain SH2 and SH3 domains have been shown to form functional and physical interactions. The structural basis of dimer formation was studied by docking three dimensional structures of the domains and by analysing structural and functional properties of the dimers. The experimentally verified dimers were noticed to have very large buried surfaces, extensive hydrogen bonding networks, and complementary surfaces, properties which are characteristic for protein-protein interactions. The number of hydrogen bonds between the domains is exceptionally high for interacting protein pairs. Also the buried accessible surface is large, especially when considering the small size of the domains. The dimer results were used to describe mutation information in structural terms and to discuss regulation of protein tyrosine kinases.

Dimerization↗
PubMed1998

BTKbase, mutation database for X-linked agammaglobulinemia (XLA).

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 463 mutation entries from 406 unrelated families showing 303 unique molecular events. In addition to mutations, the database also lists variants or polymorphisms. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites that code for arginine residues. The putative structural implications of all the missense mutations are given in the database. The improved version of the registry having a number of new features is available at http://www. helsinki.fi/science/signal/btkbase.html

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Characterization of the CDR3 region of rearranged alpha heavy chain genes in human fetal liver.

The human fetal liver is an early site for B cell development. Pre B cells are first detectable in human fetal life at 8 weeks of gestation, when the rearrangement of the mu heavy chain genes starts. In this study we characterize the CDR3 region of rearranged alpha heavy chain transcripts from four human fetal livers ranging from 8 to 11 weeks of gestation. Each fetal liver showed a limited number of variations in CDR3 sequences compared with adult peripheral blood mononuclear cells (PBMC). Sequence analysis of 91 clones demonstrated that there was no preference for the usage of a certain JH gene segment, whereas a preference for usage of DH family genes, DXP and DLR, was seen in most cases during early fetal life. This is the first study where rearranged alpha heavy chain genes in fetal liver have been characterized. Our data suggest that the usage of JH genes is random, while there is a preference for DH family genes in human fetal liver.

Adult↗
PubMed1998

Mutation screening of the BTK gene in 56 families with X-linked agammaglobulinemia (XLA): 47 unique mutations without correlation to clinical course.

OBJECTIVES: To determine the utility of single-stranded conformation polymorphism (SSCP) analysis for mutation screening in the BTK (Bruton's tyrosine kinase) gene, we investigated 56 X-linked agammaglobulinemia (XLA) families. To obtain genotype/ phenotype correlations, predicted protein aberrations were correlated with the clinical course of the disease. PATIENTS: This study included 56 patients with XLA, with or without a positive family history, who were diagnosed on the basis of their clinical features, low peripheral B-cell count, and low immunoglobulin levels. Ten patients with isolated hypogammaglobulinemia and 50 healthy males served as controls. METHODS: SSCP analysis was performed for the entire BTK gene, including the exon-intron boundaries and the promoter region. Structural implications of the missense mutations were investigated by molecular modeling, and the functional consequences of some mutations also were evaluated by in vitro kinase assays and Western blot analysis. RESULTS: We report the largest series of patients with XLA to date. All but 5 of the 56 index patients with XLA screened with SSCP analysis showed BTK gene abnormalities, and in 2 of the 5 SSCP-negative patients, no BTK protein was found by Western blot analysis. There were 51 mutations, including 37 novel ones, distributed across the entire gene. This report contains the first promoter mutation as well as 14 novel missense mutations with the first ones described for the Tec homology domain and the glycine-rich motif in the SH1 domain. Each index patient had a different mutation, except for four mutations, each in two unrelated individuals. This result supports the strong tendency for private mutations in this disease. No mutations were found in the controls. CONCLUSIONS: Our results demonstrate that molecular genetic testing by SSCP analysis provides an accurate tool for the definitive diagnosis of XLA and the discrimination of borderline cases, such as certain hypogammaglobulinemia or common variable immunodeficiency patients with overlapping clinical features. Genotype/ phenotype correlations are not currently possible, making prediction of the clinical course based on molecular genetic data infeasible.

Adolescent↗
PubMed1998

Emergency upper gastrointestinal bleeding. Management and outcomes in specialty private practice.

Severe upper gastrointestinal (UGI) bleeding is a common medical emergency associated with significant morbidity and mortality. Recent studies from selected academic medical centers show that emergency UGI endoscopy with therapeutic intervention prevents recurrent hemorrhage, reduces complications, and limits costs. We determined prospective outcomes for patients presenting to 11 hospitals in Minneapolis and treated by 17 gastroenterologists from an independent single-specialty group. All 291 patients with severe UGI bleeding seen from July 1994 to January 1995 were enrolled and treated according to a guideline that the gastroenterologists had previously agreed upon. Chart review after hospital discharge showed that therapeutic endoscopy resulted in substantial reductions in the risk of recurrent bleeding compared with recent historic controls; the reductions were comparable to those seen in randomized studies from academic centers. Low risk of recurrent bleeding was associated with fewer blood transfusions and fewer days in hospital and in ICU. We conclude that 1) committed specialists can develop and adhere to treatment plans that optimize patient benefit and limit costs, and 2) therapeutic endoscopy performed by gastroenterologists in community hospitals may be as effective as endoscopy performed by academicians with a special interest in UGI bleeding.

Electrocoagulation↗
PubMed1997

Missense mutations affecting a conserved cysteine pair in the TH domain of Btk.

Tec family protein tyrosine kinases have in their N-terminus two domains. The PH domain is followed by Tec homology (TH) domain, which consists of two motifs. The first pattern, Btk motif, is also present in some Ras GAP molecules. C-terminal half of the TH domain, a proline-rich region, has been shown to bind to SH3 domains. Mutations in Bruton's tyrosine kinase (Btk) belonging to the Tec family cause X-linked agammaglobulinemia (XLA) due to developmental arrest of B cells. Here we present the first missense mutations in the TH domain. The substitutions affect a conserved pair of cysteines, residues 154 and 155, involved in Zn2+ binding and thereby the mutations alter protein folding and stability.

Adult↗
PubMed1997

Preventive effect of IgG from EBV-seropositive donors on the development of human lympho-proliferative disease in SCID mice.

The effect of weekly treatments with various gammaglobulin preparations on the development of human B-cell tumors was studied in severe combined immunodeficient (SCID) mice. SCID mice were injected i.p. with human peripheral blood mononuclear cells (PBMCs) from an Epstein-Barr virus (EBV)-seropositive healthy blood donor. Repopulated SCID mice were divided into 7 treatment groups receiving either PBS, 2 commercial gammaglobulin preparations, purified IgG prepared from pooled plasma from EBV-seronegative or -seropositive blood donors, a rabbit anti-serum against EBV envelope glycoprotein gp340 or interferon (IFN)-alpha. All treatments started 1 day after injection of PBMC and continued for 8 weeks. In the PBS-treated control group, 85% of mice developed tumors in the abdominal cavity, mostly with liver metastasis within 150 days. Tumor formation was prevented by treatment with the 2 commercial gammaglobulin preparations as well as by purified IgG from EBV-seropositive donors. In contrast, purified IgG from EBV-seronegative donors, rabbit anti-gp340 anti-serum or IFN-alpha had no effect. Our results indicate that the effect of gammaglobulin is due to the presence of specific antibodies against EBV antigens. Further experiments showed that both the time of onset and the duration of treatment, as well as the dose of Ig, are important factors for prevention of tumor formation. Studies aiming at identification of target antigens for antibodies which prevent lymphoma development may be clinically relevant for prevention and possibly treatment of lympho-proliferative disease in severely immuno-compromised patients.

Abdominal Neoplasms↗
PubMed1997

Gap junctions promote the bystander effect of herpes simplex virus thymidine kinase in vivo.

Transfer of the herpes simplex thymidine kinase gene (HSVtk) into tumor cells followed by the administration of ganciclovir (GCV) provides a potential strategy for the treatment of some malignancies. During GCV treatment, not only the cells that express the HSVtk gene are killed but also frequently neighboring tumor cells that are not genetically altered. This has been called the "bystander effect." Although the mechanism of the bystander effect in vivo remains elusive, our results suggest that gap junction formation between neighboring cells is an important contributing factor. The C6 rat glioma cell line, which exhibits a low level of intercellular communication by gap junctions and connexin43 (Cx43)-transfected clones of this cell line forming gap junctions from a moderate level (Cx43-12 and Cx43-14) to a high level (Cx43-13), were transduced with HSVtk. Transduced and nontransduced cells were mixed in various concentrations and then cultured in vitro or injected s.c. into C.B-17/SCID-beige mice followed by i.p. injections of GCV. Cx43-transfected clones showed a significant increase of the bystander effect compared with the less coupled C6 parental cell line. In 11 of 12 mice injected with cells of Cx43-transfected clones, no tumors were seen at the inoculation site when a mixture of 50% HSVtk-negative and HSVtk-positive cells was used. Moreover, in mice injected with cells of clone Cx43-13, which exhibits the highest intercellular communication, tumors were frequently undetectable at the inoculation site when using mixtures of 75% HSVtk-negative and 25% HSVtk-positive cells, and even mixtures containing 5% HSVtk-positive cells of Cx43-transfected clones showed tumor size reduction. All animals in control groups (n = 26) developed large tumors at every injection site. These results demonstrate that gap junctions are an important component in mediating the bystander effect in vivo.

Animals↗
PubMed1997

BTKbase, mutation database for X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 368 entries from 318 unrelated families showing 228 unique molecular events. In addition to mutations the database lists also some polymorphisms and site-directed mutations. Each patient is given a unique patient identity number (PIN). Information is provided regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites forming arginine residues. These hot spots have generally pyrimidines 5'and purines 3'to the mutated cytosine. A decreased frequency of missense mutations was found in the TH, SH3 and the upper lobe of the kinase domain. The putative structural implications of all the missense mutations are given in the database showing 228 unique molecular events, including a novel missense mutation causing an R28C substitution as previously seen in the Xid mouse.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1997

BTK, the tyrosine kinase affected in X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency disorder that is caused by a differentiation block leading to almost complete absence of B lymphocytes and plasma cells. The affected protein is a cytoplasmic protein tyrosine kinase, Bruton's agammaglobulinemia tyrosine kinase (Btk). Btk along with Tec, Itk and Bmx belong to a distinct family of protein kinases. These proteins contain five regions; PH, TH, SH3, SH2 and kinase domains. Mutations causing XLA may affect any of these domains. About 200 unique mutations have been identified and are collected in a mutation database, BTKbase. Here, we describle, the structure, function, and interactions of the affected signaling molecules in atomic detail.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1997

Registries of immunodeficiency patients and mutations.

Immunodeficiencies form a distinct group of human hereditary diseases with several rare disorders. During recent years, information has been collected concerning immunodeficiency patients and mutations causing disorders. The large European (ESID) registry contains clinical data for some 7,000 patients. At present, international mutation databases have information for > 1,000 immunodeficiency patients, including X-linked chronic granulomatous disease (XCGD), Wiskott-Aldrich syndrome (WAS), and X-linked thrombocytopenia (XLT), X-linked hyper-IgM syndrome (XHIM), X-linked agammaglobulinemia (XLA), and X-linked severe combined immunodeficiency (XSCID). The databases are available on Internet. The mutation spectra of patients in these registries were compared. Mutational hotspots were found in CpG dinucleotides with a preference for selected flanking bases.

Allergy and Immunology↗
PubMed1997

Mutation pattern in the Bruton's tyrosine kinase gene in 26 unrelated patients with X-linked agammaglobulinemia.

Mutation pattern was characterized in the Bruton's tyrosine kinase gene (BTK) in 26 patients with X-linked agammaglobulinemia, the first described immunoglobulin deficiency, and was related to BTK expression. A total of 24 different mutations were identified. Most BTK mutations were found to result in premature termination of the translation product. Mutations were detected in most BTK exons with a predominance of frameshift and nonsense mutations in the 5' end of the gene and missense mutations in its 3' part, corresponding to the catalytic domain of the enzyme. Nonsense and frameshift mutations were associated with diminished levels of BTK mRNA expression, except for a frameshift mutation in exon 17 and two nonsense mutations in exon 2, indicating that these cases are not confined to penultimate exons. One amino acid substitution (R28H) was found in the pleckstrin homology domain's residue, which is mutated in mice bearing the X-linked immunodeficiency phenotype; another substitution (R307G) was identified in the src homology domain 2. All remaining amino acid substitutions were found in the catalytic domain of Btk.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1997

Direct and sequential switching from mu to epsilon in patients with Schistosoma mansoni infection and atopic dermatitis.

Immunoglobulin isotype switching to IgE in patients infected with Schistosoma mansoni and patients with atopic dermatitis was studied. Patients with parasitic infections or allergic diseases have a higher production of IgE. We found a four-fold increased production of I epsilon RNA in both patient groups as compared to control donors. The increased expression of germ-line transcripts correlates with higher serum IgE levels. Nested primer polymerase chain reaction was used to generate S mu/S epsilon fragments from DNA of patient peripheral blood mononuclear cells. Twenty-nine out of fourty sequenced switch fragments had undergone direct joining from S mu to S epsilon whereas seven fragments showed mono sequential switching from S mu via either S mu, S gamma2, S gamma4 or S epsilon to S epsilon and four fragments demonstrated double sequential switch: S mu/S mu/S gamma1/S epsilon, S mu/S gamma2/S epsilon/S epsilon or S mu/S gamma1/ S gamma2/S epsilon. The sequential switching had occurred either via deletions or inversions. Mapping of the breakpoints showed hot spots for recombination within S mu, S gamma1 and S epsilon. To our knowledge, this is the first in vivo study in humans demonstrating that switching to IgE can occur from sequential rearrangements via gamma1, gamma2 or gamma4.

Base Sequence↗