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Biomedical subjects

G Gilliland

Publications and source records attributed to G Gilliland.

At least 19 recordsLinked to original sources
PubMed2006

Mutations of the transcription factor PU.1 are not associated with acute lymphoblastic leukaemia.

The transcription factor PU.1 plays a crucial role during normal haematopoiesis in both myeloid cells and B-lymphocytes. Mice with a disruption in both alleles of the PU.1 locus were found to lack macrophages and B cells and had delayed appearance of neutrophils. In addition, critical decrease of PU.1 expression is sufficient to cause acute myeloid leukaemia (AML) and lymphomas in mice. Recently, we reported that heterozygous mutations in the PU.1 gene are present in some patients with AML. Thus, we hypothesised that PU.1 mutations might also contribute to the development of acute leukaemias of the B-cell lineage. Here, we screened 62 patients with B-cell acute lymphoblastic leukaemia (B-ALL) at diagnosis for genomic mutations by direct sequencing of all five exons of the PU.1 gene. We found no genomic alteration of the PU.1 gene suggesting that PU.1 mutations are not likely to be common in B-ALL.

Adolescent↗
PubMed2006

Identification of additional cytogenetic and molecular genetic abnormalities in acute myeloid leukaemia with t(8;21)/AML1-ETO.

AML1-ETO collaborates with further genetic abnormalities to induce acute myeloid leukaemia (AML). We analysed 99 patients with an AML1-ETO rearrangement for additional aberrations. Frequent genetic abnormalities were, loss of a sex chromosome (56/99, 56.5%) and del(9)(q22) (24/99, 24.2%). The most frequent molecular aberrations were mutations of KITD816 (3/23, 13%) and NRAS (8/89, 8.9%). Further molecular abnormalities were FLT3 mutations (3/87, 3.4%), AML1 (1/26, 3.8%) and PU1 (1/14, 7.1%). MLL-PTD, KRAS and CEBPA mutations were not found. These clinical findings support the model that AML1-ETO collaborates with other genetic alterations, such as mutations of receptor tyrosine kinases, to induce AML.

Acute Disease↗
PubMed2001

The PDB data uniformity project.

The Protein Data Bank (PDB; http://www.rcsb.org/pdb/) is the single worldwide archive of structural data of biological macromolecules. This paper describes the data uniformity project that is underway to address the inconsistency in PDB data.

Databases, Factual↗
PubMed2000

The Protein Data Bank.

The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.

Databases, Factual↗
PubMed1998

A comparison of spousal anticipatory grief and conventional grief.

The purpose of the present investigation was to compare the degree of similarity between the grief experienced by spouses of terminally-ill patients prior to (anticipatory grief) and following the death (conventional grief). Responses of this sample were also compared with those of two control groups: spouses of chronically-ill patients and spouses of relatively healthy individuals from the community. The impact of such factors as the quality of the marital relationship, perceived ability to cope, concurrent stressors, previous losses, perceived social support, and perception of spouse's pain and suffering on anticipatory and conventional grief was also systematically explored. Results indicated that these two phenomena are statistically similar with regard to the majority of subscales on the Grief Experience Inventory. Furthermore, when compared with conventional grief, anticipatory grief was unexpectedly associated with higher intensities of anger, loss of emotional control, and atypical grief responses.

Aged↗
PubMed1997

High-dose cyclophosphamide, carmustine, and etoposide with autologous transplantation in Hodgkin's disease: a prognostic model for treatment outcomes.

PURPOSE: To identify clinical factors predictive of treatment outcome after high-dose chemotherapy (HDC) for Hodgkin's disease and to develop a prognostic model for progression-free and overall survival. PATIENTS AND METHODS: 102 patients with relapsed or refractory Hodgkin's disease were treated with high-dose cyclophosphamide, carmustine, and etoposide and autologous marrow and/or peripheral blood progenitor cell support. Median follow-up of survivors is 4.1 years (1.8-7.5 years). Factors potentially important for treatment outcome were examined in univariate analysis, and Cox regression with forward selection was performed. A prognostic model was developed. RESULTS: Poorer progression-free and overall survival were associated with nodular sclerosis histology, abnormal performance status, progressive disease at HDC, more than one extranodal site of disease, and shorter time from initial diagnosis to HDC. These factors and the presence of B symptoms at relapse also predicted for decreased overall survival. Progressive disease immediately prior to HDC, more than one extranodal disease site, and abnormal performance status retained significance for both progression-free and overall survival in multivariate analysis. Progression-free and overall survival are 42% (95% confidence interval, CI, 34 to 53) and 65% (95% CI 54 to 73) at three years. A model based on number of risk factors present divides patients into low, intermediate, and high risk groups with three-year actuarial survival of 82%, 56%, and 19% respectively. Treatment outcome for patients treated with HDC at first chemotherapy relapse was not significantly different from that of the group overall (p > 0.3). CONCLUSIONS: Asymptomatic patients with Hodgkin's disease involving at most one extranodal site whose disease is controlled by conventional dose chemotherapy or radiation therapy at the time of HDC have good outcomes after this therapy. Presence of increasing numbers of risk factors are associated with poorer outcomes. Results of HDC compare favorably to those of standard dose salvage therapy. These data can be used to estimate likely outcomes in patients undergoing HDC for Hodgkin's disease, to identify potential candidates for innovative therapies, and to evaluate strategies for the optimal use of HDC in Hodgkin's disease.

Adult↗
PubMed1996

Positive and negative cooperativities at subsequent steps of oxygenation regulate the allosteric behavior of multistate sebacylhemoglobin.

Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3,5-dibromosalicyl) sebacate. Peptide maps and crystallographic analyses confirm the presence of the 10 carbon atom long sebacyl residue cross-linking the two beta82 lysines of the beta-cleft (DecHb). The Adair's constants, obtained from the oxygen binding isotherms, show that at the first step of oxygenation normal hemoglobin and DecHb have a very similar oxygen affinity. In DecHb negative binding cooperativity is present at the second step of oxygenation, which has an affinity 27 times lower than at the first step. Positive cooperativity is present at the third binding step, whose affinity is 380 times that of the second step. The fourth binding step shows a weak negative cooperativity with an affinity one-half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 angstroms resolution. The resulting atomic coordinates are very similar to those of Fermi et al. [(1984) J. Mol.Biol. 175, 159-174] and Fronticelli et al. [(1994) J. Biol Chem. 269, 23965-23969] for deoxy-HbA. The electron density map of deoxy-DecHb indicates the presence of the 10 carbon bridge between the beta82 lysines. Molecular modeling confirms that insertion of the linker into the T structure requires only slight displacement of the two beta82 lysines. Instead, insertion of the linker into the R and R2 structures [Shaanan (1983) J. Mol. Biol. 171, 31-59; Silva et al. (1992) J. Biol. Chem. 267, 17248-17256] is hindered by serious sterical restrictions. The linker primarily affects the partially and fully liganded states of hemoglobin. The data suggest in DecHb concerted conformational changes at each step of oxygenation.

Allosteric Regulation↗
PubMed1995

Prodomain mutations at the subtilisin interface: correlation of binding energy and the rate of catalyzed folding.

The in vivo folding of subtilisin is dependent on a 77 amino acid prosequence, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme. The recent determination of the structure of a complex of the prodomain and a calcium-free subtilisin mutant has suggested how the prodomain may catalyze subtilisin folding [Bryan, P., Wang, L., Hoskins, J., Ruvinov, S., Strausberg, S., Alexander, P., Almog, O., Gilliland, G., & Gallagher, T. (1995) Biochemistry 34, 10310-10318]. In the complex, the prodomain packs against the two parallel surface helices of subtilisin (residues 104-116 and residues 133-144) and supplies caps to the N-termini of the two helices. The binding site is contained almost entirely in the linear sequence 100-144 of subtilisin. The C-terminus of the prodomain (residues 72-77) extends out from its central part to bind like a substrate in subtilisin's active site cleft. The simplest model of catalyzed folding is one in which the observed binding interaction in the complex accelerates folding by stabilizing an intermediate which includes the 45 amino acid alpha beta alpha substructure in subtilisin. According to our hypothesis, amino acids 100-144 would have a native-like fold in the intermediate which the prodomain stabilizes. Guided by the structure of the bimolecular complex of subtilisin and its prodomain, we have constructed mutations in the C-terminal region of the prodomain. Analysis of five mutants reveals a general correlation between the ability of the prodomain to bind to native subtilisin and its ability to accelerate subtilisin folding.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutation↗
PubMed1995

The prosegment-subtilisin BPN' complex: crystal structure of a specific 'foldase'.

BACKGROUND: The folding of the bacterial protease subtilisin BPN' (SBT) is dependent on its 77-residue prosegment, which is then autocatalytically removed to give the mature enzyme. Mature subtilisin represents a class of proteins that lacks an efficient folding pathway. Refolding of mature SBT is extremely slow unless catalyzed by the independently expressed prosegment, leading to a bimolecular complex. RESULTS: We report the crystal structure at 2.0 A resolution of the prosegment-SBT complex and consider its implications for prosubtilisin BPN' maturation and folding catalysis. The prosegment forms a compact domain that binds SBT through an extensive interface involving the enzyme's two parallel surface helices (residues 104-116 and 133-144), supplying negatively charged caps to the N termini of these helices. The prosegment C terminus binds in the enzyme active site in a product-like manner, with Tyr77 in the P1 binding pocket. CONCLUSIONS: The structure of the complex supports a unimolecular mechanism for prosubtilisin cleavage, involving a 25 A rearrangement of the SBT N terminus in a late folding step. A mechanism of folding catalysis in which the two helices and their connecting beta strand form a prosegment-stabilized folding nucleus is proposed. While this putative nucleus is stabilized by prosegment binding, the N-terminal and C-terminal subdomains of SBT could fold by propagation.

Crystallography, X-Ray↗
PubMed1995

Catalysis of a protein folding reaction: mechanistic implications of the 2.0 A structure of the subtilisin-prodomain complex.

Biosynthesis of subtilisin is dependent on a 77 amino acid, N-terminal prodomain, which is autocatalytically processed to create the mature form of the enzyme [Ikemura, H., Takagi, H., & Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864]. In order to better understand the role of the prodomain in subtilisin folding, we have determined the structure of the processed complex between the prodomain and subtilisin Sbt-70, a mutant engineered for facilitated folding. The prodomain is largely unstructured by itself but folds into a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices when complexed with subtilisin. The Ka of the complex is 2 x 10(8) M-1 at 25 degrees C. The prodomain binds on subtilisin's two parallel surface alpha-helices and supplies caps to the N-termini of the two helices. The C-terminal strand of the prodomain binds in the subtilisin substrate binding cleft. While Sbt-70 is capable of independent folding, the prodomain accelerates the process by a factor of > 10(7) M-1 of prodomain in 30 mM Tris-HCl, pH 7.5, at 25 degrees C. X-ray structures of the mutant subtilisin folded in vitro either with or without the prodomain are compared and show that the identical folded state is achieved in either case. A model of the folding reaction of Sbt-70 and the prodomain is described as the following equilibria: P + Su<-->Pf--SI<-->Pf--Sf, where Su and P are Sbt-70 and prodomain, respectively, which are largely unstructured at the start of the reaction, Pf--SI is a collision complex of a partially folded Sbt-70 and folded prodomain, and Pf--Sf is the complex of folded Sbt-70 and prodomain.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Regions of the JAK2 tyrosine kinase required for coupling to the growth hormone receptor.

Growth hormone (GH) treatment of cells promotes activation of JAK2, a GH receptor (GHR)-associated tyrosine kinase. We now explore JAK2 regions required for GHR-induced signaling. Wild-type (WT) JAK2 and JAK2 molecules with deletions of the amino terminus (JAK2ATD), carboxyl terminus (JAK2CTD), or kinase-like domain (JAK2PKD) were each transiently coexpressed in COS-7 cells with the rabbit GHR. The following responses were assayed: GH-induced transactivation of a luciferase reporter governed by a c-fos enhancer element; GH-induced shift in the molecular mass of a cotransfected epitope-tagged extracellular signal-regulated kinase molecule; and GH-induced antiphosphotyrosine immunoprecipitability of the transfected JAK2 form. In each assay, WTJAK2 and JAK2PKD allowed GH-induced signaling, whereas JAK2ATD and JAK2CTD did not. Anti-GHR serum coimmunoprecipitated WTJAK2, JAK2PKD, and JAK2CTD, but not JAK2ATD. Finally, a chimera in which the JAK2 kinase domain replaced the GHR cytoplasmic domain signaled GH-induced transactivation. We conclude: 1) kinase-like domain deletion eliminates neither physical nor functional interaction between JAK2 and the GHR; 2) kinase domain deletion eliminates functional but not physical coupling of JAK2 to the GHR; 3) interaction with the GHR appears dependent on the NH2-terminal one-fifth of JAK2; and 4) a GH-responsive signaling unit can include as little as the GHR external and transmembrane domains and the JAK2 kinase domain.

Amino Acid Sequence↗
PubMed1994

Orbital aneurysmal bone cyst in a patient with renal cell carcinoma.

A 43-year-old man with presumed renal cell carcinoma who was found to have an asymptomatic hot spot in his right zygoma on a bone scan during a metastatic evaluation. At the time of nephrectomy, an excisional biopsy of the lateral orbital wall was performed to rule out solitary metastasis of his renal cell carcinoma. The lateral wall was reconstructed with a rib autograft and multiple rigid fixation. Histopathologic examination of the specimen showed a fibrous matrix with large, dilated, sinusoidal, vascular channels typical of an aneurysmal bone cyst. In the absence of metastasis, the five-year survival rate is approximately 90% with nephrectomy alone.

Adult↗
PubMed1994

Transorbital penetrating head injury with a hunting arrow: case report.

The development of more complex and diverse hunting weapons may result in an increase of uncommon forms of penetrating injury to the brain. We present a case of nonfatal transorbital arrow injury to the brain. High velocity projectile injuries merit certain management adaptations from gunshot or low velocity stab wounds. This case highlights the necessity for anterograde removal of the arrow in the direction of its line of trajectory. Early assessment of the patient with cerebral angiography to identify surgically correctable vascular injury is recommended.

Adult↗
PubMed1994

Treatment of IM-9 cells with human growth hormone (GH) promotes rapid disulfide linkage of the GH receptor.

Human GH (hGH) is believed to elicit its signal by promoting dimerization of the hGH receptor (hGHR). In this study, we examined a covalent linkage of receptors induced by hGH treatment of IM-9 cells. hGH induced a time- and concentration-dependent appearance of a disulfide-linked species of 215-230 kilodaltons, designated p215-230, that at 37 C was long-lived (> 1 h). p215-230 was confirmed to contain the hGHR (115-140 kilodaltons) as at least one of its constituents by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. hGH induction of p215-230 required intact cells and was inhibitable by pretreatment of cells with N-ethylmaleimide (NEM), a sulfhydryl-reactive alkylating agent. NEM pretreatment did not, however, prevent hGH-dependent formation of a nondisulfide-linked p215-230 form, which was detected in NEM-pretreated hGH-stimulated cells by chemical cross-linking of detergent cell extracts. The disulfide-linked form of the hGHR accounted for a substantial fraction of the receptors that became tyrosine phosphorylated early into hGH treatment. However, formation of the disulfide-linked hGHR was not blocked by attenuation of tyrosine kinase activation, in that pretreatment of cells with staurosporine (1.25 microM) prevented detectable hGH-induced tyrosine phosphorylation without preventing the appearance of p215-230. These findings indicate that hGH induces its receptor to form a noncovalently associated complex, which then undergoes a rapid transition to a disulfide-linked form. These processes may have relevance to hGH signaling and/or hGHR trafficking.

B-Lymphocytes↗
PubMed1994

Interaction of the growth hormone receptor cytoplasmic domain with the JAK2 tyrosine kinase.

An early step in GH action involves tyrosine phosphorylation of various cellular proteins. Recently, it has been shown in murine preadipocytes that GH promotes the association of its receptor (the GHR) with and the activation of the JAK2 tyrosine kinase. In this study, we confirmed the human (h) GH-induced association of JAK2 with hGHR in IM-9 cells by coimmunoprecipitation experiments using anti-hGHR serum. We further examined the interaction of JAK2 with the GHR cytoplasmic domain by two lines of investigation. For in vitro studies, we assayed by immunoblotting the ability of cell-derived JAK2 to interact with glutathione S-transferase fusion proteins containing elements of the hGHR cytoplasmic domain. A fusion protein containing the entire hGHR cytoplasmic domain (residues 271-620) specifically associated with JAK2 independent of prior stimulation of cells with hGH. This interaction was not dependent on tyrosine phosphorylation of either partner. Mutational analysis of the hGHR cytoplasmic domain component of the fusions indicated that a membrane-proximal 20-residue region that includes the proline-rich box 1 was necessary for the interaction. This region appeared to cooperate with another region(s), largely in the N-terminal one third of the cytoplasmic domain, to promote full interaction with JAK2. For in vivo reconstitution experiments, wild-type (WT) and mutant rabbit GHRs (rGHRs) along with murine JAK2 were expressed by transient transfection in COS-7 cells. rGHR mutations were confined to the cytoplasmic domain and included C-terminal truncations as well as internal deletions of residues 297-406 and 278-292 (the latter contains box 1). All mutant rGHRs were expressed at the cell surface and bound hGH to a degree similar to the WT rGHR. Receptors were tested for their ability to mediate the hGH-induced immunoprecipitability of JAK2 with phosphotyrosine (APT) antibodies. A rGHR truncated to residue 275 [rGHR-(1-275)], which contains only five cytoplasmic residues, failed to mediate JAK2 APT precipitability in response to hGH. In contrast, WT rGHR; the C-terminal truncations rGHR-(1-542), rGHR-(1-390), and rGHR-(1-317); and the rGHR-(d297-406) deletion mutant maintained this ability. Deletion of the 278-292 box 1-containing region in the context of either rGHR-(d297-406) or WT rGHR eliminated detectable hGH-induced JAK2 APT precipitability. Interestingly, rGHR-(1-292), which includes box 1, was not able to mediate significant hGH-induced JAK2 APT precipitability.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

An engineered disulfide cross-link accelerates the refolding rate of calcium-free subtilisin by 850-fold.

The mature form of subtilisin is an unusual example of a monomeric protein with a high kinetic barrier to folding and unfolding. Using site-directed mutagenesis of subtilisin BPN', we are attempting to determine the physical and energetic nature of the kinetic barrier. The high-affinity calcium-binding site A has been shown to create a large enthalpic barrier to unfolding. Removing the calcium-binding site A from subtilisin by deleting amino acids 75-83 greatly accelerates both unfolding and refolding reactions. Here a disulfide cross-link is introduced between residues 22 and 87 in delta 75-83 subtilisin. This was done to probe the conformational entropy of the transition state for folding. The 1.8-A X-ray structure of this mutant and the effects of the cross-link on the kinetics of unfolding and refolding are reported. Consistent with an expected loss of entropy of the unfolded protein due to the cross-link, the disulfide accelerates folding relative to the uncross-linked form. The magnitude of the acceleration of folding rate (700-850-fold at 25 degrees C) indicates that residues 22 and 87 are ordered in the transition state such that the disulfide does not affect its total entropy. Although early organization of structure around amino acids 22 and 87 greatly accelerates folding, we do not know whether the early folding of this region is a highly populated folding pathway in the absence of the cross-link. The slow step in the delta 75-83 subtilisin folding reaction may be forming initial structures capable of propagating the folding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗