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Biomedical subjects

A M Buckle

Publications and source records attributed to A M Buckle.

34 records · Page 2Linked to original sources
PubMed1995

Development of a clinically applicable high-speed flow cytometer for the isolation of transplantable human hematopoietic stem cells.

A high-speed cell sorter capable of a throughput speed 4-5-fold greater than commercially available systems was developed and evaluated as a processing tool for isolating purified hematopoietic stem cell grafts. The clinical high-speed sorter (CHSS) serves as a single-pass, multiparameter processing tool that provides the means to isolate a highly purified population of cells from starting cell populations with extremely low frequencies. The sorter incorporates environmental barriers to create a sterile environment for cell processing. Monoclonal antibodies and reagents produced under good manufacturing practices (GMP) are used to isolate hematopoietic stem cells by means of the CHSS. Using this technology, the CD34+Thy-1+Lin- hematopoietic stem cell population has been isolated from normal adult bone marrow and mobilized peripheral blood. The sorted cells have been shown to be sterile and viable and to retain hematopoietic function.

Cell Separation↗
PubMed1994

Stability and function: two constraints in the evolution of barstar and other proteins.

BACKGROUND: Barstar is the intracellular inhibitor of barnase, an extracellular RNAse of Bacillus amyloliquefaciens. The dissociation constant of the barnase-barstar complex is 10(-14) M with an association rate constant between barnase and barstar of 3.7 x 10(8) s-1 M-1. The rapid association arises in part from the clustering of four acidic residues (Asp35, Asp39, Glu76 and Glu80) on the barnase-binding surface of barstar. The negatively charged barnase-binding surface of barstar effectively 'steers' the inhibitor towards the positively charged active site of barnase. RESULTS: Mutating any one of the four acidic side chains of barstar to an alanine results in an approximately two-fold decrease in the association rate constant, while the dissociation rate constant increases from five orders of magnitude for Asp39-->Ala, to no significant change for Glu80-->Ala. The stability of barstar is increased by all four mutations, the increase ranging from 0.3 kcal mol-1 for Asp35-->Ala or Asp39-->Ala, to 2.1 kcal mol-1 for Glu80-->Ala. CONCLUSIONS: The evolutionary pressure on barstar for rapid binding of barnase is so strong that glutamate is preferred over alanine at position 80, even though it does not directly interact with barnase in the complex and significantly destabilizes the inhibitor structure. This, and other examples from the literature, suggest that proteins evolve primarily to optimize their function in vivo, with relatively little evolutionary pressure to increase stability above a certain threshold, thus allowing greater latitude in the evolution of enzyme activity.

Bacillus↗
PubMed1994

Protein-protein recognition: crystal structural analysis of a barnase-barstar complex at 2.0-A resolution.

We have solved, refined, and analyzed the 2.0-å resolution crystal structure of a 1:1 complex between the bacterial ribonuclease, barnase, and a Cys-->Ala(40,82) double mutant of its intracellular polypeptide inhibitor, barstar. Barstar inhibits barnase by sterically blocking the active site with a helix and adjacent loop segment. Almost half of the 14 hydrogen bonds between barnase and barstar involve two charged residues, and a third involve one charged partner. The electrostatic contribution to the overall binding energy is considerably greater than for other protein-protein interactions. Consequently, the very high rate constant for the barnase-barstar association (10(8) s-1 M-1) is most likely due to electrostatic steering effects. The barnase active-site residue His102 is located in a pocket on the surface of barstar, and its hydrogen bonds with Asp39 and Gly31 residues of barstar are directly responsible for the pH dependence of barnase-barstar binding. There is a high degree of complementarity both of the shape and of the charge of the interacting surfaces, but neither is perfect. The surface complementarity is slightly poorer than in protease-inhibitor complexes but a little better than in antibody-antigen interactions. However, since the burial of solvent in the barnase-barstar interface improves the fit significantly by filling in the majority of gaps, as well as stabilizing unfavorable electrostatic interactions, its role seems to be more important than in other protein-protein complexes. The electrostatic interactions between barnase and barstar are very similar to those between barnase and the tetranucleotide d(CGAC). In the barnase-barstar complex, the two phosphate-binding sites in the barnase active site are occupied by Asp39 and Gly43 of barstar. However, barstar has no equivalent for a guanine base of an RNA substrate, resulting in the occupation of the guanine recognition site in the barnase-barstar complex by nine ordered water molecules. Upon barnase-barstar binding, entropy losses resulting from the immobilization of segments of the protein chain and the energetic costs of conformational changes are minimized due to the essentially preformed active site of barnase. However, a certain degree of flexibility within the barnase active site is required to allow for the structural differences between barnase-barstar binding and barnase-RNA binding. A comparison between the bound and the free barstar structure shows that the overall structural response to barnase-binding is significant. This response can be best described as outwardly oriented, rigid-body movements of the four alpha-helices of barstar, resulting in the structure of bound barstar being somewhat expanded.

Bacterial Proteins↗
PubMed1994

Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression.

Resolution of inflammation involves removal of recruited neutrophils from inflamed sites via a noninflammatory mechanism, possibly involving neutrophil apoptosis and engulfment/phagocytosis by macrophages. In this study, we describe the reduction in surface expression (> 90%) of the neutrophil molecule Fc gamma RIII (CD16) during in vitro culture at 37 degrees C, which was found to be temporally associated with the appearance of neutrophils with apoptotic morphology during in vitro culture and inhibitable by granulocyte-macrophage colony-stimulating factor (GM-CSF), which postpones apoptosis in the neutrophil. By using dual fluorescence analysis, CD16 "low" expressing neutrophils showed reduced staining with the DNA-binding dye propidium iodide, suggesting that CD16 low expressing neutrophils were apoptotic. Separation of CD16 "high" and CD16 "low" expressing neutrophils by fluorescence-activated cell sorting revealed that morphologically apoptotic cells exhibited the CD16 low phenotype. We did not observe similar marked changes in expression of other neutrophil surface molecules (including other phosphatidylinositol (PI)-linked molecules), indicating that generalized loss of surface molecules does not occur during apoptosis. We believe this to be the first reported cell type-specific membrane alteration in a surface glycoprotein associated with apoptosis, suggesting that the program of cell death in the neutrophil, in addition to morphologic and nuclear changes, includes alterations in expression of surface receptors.

Apoptosis↗
PubMed1994

Subsite binding in an RNase: structure of a barnase-tetranucleotide complex at 1.76-A resolution.

A set of subsites in barnase has been proposed from kinetic studies. A specific substrate analog, the tetradeoxynucleotide, CGAC, has been designed from this information. We report the crystal structure of its complex with barnase at 1.76-A resolution. The structure was solved by molecular replacement from a model of free barnase and refined to a crystallographic R factor of 19.0%. The stoichiometry of the asymmetric unit dimeric complex is [barnase:d(CGAC)]2, with 2-fold noncrystallographic symmetry. Each barnase molecule binds one oligonucleotide whereby the recognition site is occupied by guanine, and all three phosphate groups of the nucleotide make electrostatic interactions with basic residues in a strongly electropositive region at the bottom of the active site. The active-site His 102 packs against the adenine base of the nucleotide in an almost identical manner to the guanine base in the barnase-d(GpC) complex and defines a possible subsite in the Michaelis complex. The overall protein structure is unchanged on forming the complex with d(CGAC), but there are small differences in the active site and in crystal packing regions. The protein coordinates will be useful for theoretical calculations since some disorder induced by packing constraints in the crystals of the free enzyme are absent in the crystals of the complex. The interface of the dimer is formed by a His 102-adenine-adenine-His 102 face-to-face ring stack directly on the 2-fold axis. The edge of the adenine-adenine stack packs closely onto the face of a 3'-cytosine-3'-cytosine interaction, which has a "base-pair"-like conformation but too great a separation of the bases to form hydrogen bonds. This unusual arrangement is the major stabilizing interaction within the dimeric complex, since there are no direct protein-protein interactions. Using the structure of the complex as a starting point for model building, the nature of the enzyme-substrate and enzyme-transition state complexes is investigated.

Bacterial Proteins↗
PubMed1993

Crystal structural analysis of mutations in the hydrophobic cores of barnase.

We have solved and analysed the crystal structures of five mutants in the hydrophobic core of barnase to investigate the structural basis for the contribution of hydrophobic residues and side-chain packing to the stability of globular proteins. In case ease, an amino acid side-chain has been replaced with one of smaller volume. The overall structures of four Ile-->Val mutants (residues 51, 76, 88 and 96) and one Leu-->Val mutant (residue 89) are all isomorphous with the wild-type structure. The magnitude and nature of structural shifts in the three hydrophobic core regions of barnase depend on the local environment of the substitution site, but have some features in common. (1) Side-chain atoms move to a greater extent than do main-chain atoms. (2) Repacking at the substitution site is achieved by either a rigid body shift of side-chain atoms (for Ile-->Val76 and Ile-->Val96 mutants), or by a combination of a side-chain shift and rotation (for Ile-->Val51 and Ile-->Val88 mutants). The mutated residue moves to the greatest extent, and generally in the direction of the created cavity (the largest atomic shift is 0.9 A, for Ile-->Val51). The space left behind from such shifts is not seen to be filled by neighbouring side-chains. (3) Where a cavity remains after mutation, it does not contain any solvent molecules. (4) There is no correlation between the extent of structural movements and the atomic temperature factors of atoms that have moved. (5) Structural movements are not large enough to disrupt hydrogen bonding. Valine 88, in the Ile-->Val88 mutant, is disordered and the electron density suggests several side-chain conformations. The reduction in the volumes of the cavities introduced upon mutation, due to collapse of the surrounding structure, ranges from 11% (Ile-->Val96) to 90% (Ile-->Val51).

Amino Acid Sequence↗
PubMed1993

Thy-1-expressing CD34+ human cells express multiple hematopoietic potentialities in vitro and in SCID-hu mice.

We have detected a minor subset of fetal liver and bone marrow CD34+ cells that coexpress the Thy-1 antigen at their surface. In immune-deficient SCID mice engrafted with human fetal hematopoietic tissues, CD34+ Thy-1+ reconstitute the full range of thymic lymphocytes and yield a progeny of myeloid and B cells in the human bone marrow. The frequency of cells initiating long-term human myeloid and B lymphocytes in vitro cultures at the contact of the Sys-1 stromal cell line was also found to be 10-15 times higher among CD34+ Thy-1+ cells than in the Thy-1- counterpart population. We propose that CD34+ Thy-1+ cells include candidate human hematopoietic stem cells.

Animals↗
PubMed1992

Isolation of a candidate human hematopoietic stem-cell population.

We have identified a rare (0.05-0.1%) subset of human fetal bone marrow cells that contains multipotent hematopoietic precursors. The population of human precursor cells that express Thy-1 and CD34 but no known lineage markers is enriched for clonogenic activity that establishes long-term, multilineage (myelomonocytic and B lymphoid) cultures on mouse marrow stromal lines. Further, the Thy-1+CD34+ subset that takes up little of the fluorescent mitochondrial dye rhodamine 123 contains virtually all the cells that establish long-term cultures. In human fetal thymus transplanted into SCID (severe combined immunodeficiency) mice, Thy-1+CD34+ fetal bone marrow cells differentiate into T lymphocytes. In two of nine cases, allogeneic Thy-1+CD34+ cells could engraft intact human fetal bone marrow grown in SCID mice, resulting in donor-derived myeloid and B cells. By extrapolation, the rare human Thy-1+Lin-CD34+ cell population contains pluripotent hematopoietic progenitors; we propose that it is highly enriched for candidate hematopoietic stem cells.

Animals↗
PubMed1990

Human memory T cells express intercellular adhesion molecule-1 which can be increased by interleukin 2 and interferon-gamma.

We have shown that low levels of intercellular adhesion molecule-1 (ICAM-1) expression can be detected on approximately 40% of the resting peripheral blood T cell population. The ICAM-1+ T cells have the phenotypic markers of memory cells which can be distinguished functionally from naive T cells by their ability to respond rapidly to previously experienced antigen. These cells appear to be in a state of marginal activation in that they also express low levels of the interleukin 2 receptor (Tac antigen) and have increased cell size as compared to the naive T cells. In addition we have shown that the cytokines interleukin 2 and interferon-gamma, both of which are products of activated T cells, are able to increase the expression of ICAM-1 on T cells. Finally, pretreating T cells with an anti-ICAM-1 monoclonal antibody inhibits their response to recall antigens, strongly suggesting a functional role for this protein on the memory T cell.

Antigens, Differentiation↗
PubMed1990

Colony-stimulating factors and interferon-gamma differentially affect cell surface molecules shared by monocytes and neutrophils.

Monocytes and neutrophils share a common progenitor and perform many similar functions, as reflected in the expression of several shared membrane molecules. We show here that such molecules can be independently regulated by the cytokines interferon-gamma (IFN-gamma) and the colony-stimulating factors (CSF) in the context of the cell type on which they are present. Thus, IFN-gamma causes neutrophils to express the high-affinity receptor for IgG, Fc gamma RI, which has been considered to be a monocyte-specific receptor. Although neutrophil Fc gamma RI never reaches the levels present on monocytes, it is induced more rapidly and by lower amounts of IFN-gamma than monocyte Fc gamma RI. Of the CSF, only macrophage (M) CSF has an effect which is to cause a decrease in expression of monocyte Fc gamma RI. Secondly, after culture monocytes express Fc gamma RIII which is constitutively expressed by neutrophils. Although neutrophil Fc gamma RIII can be readily modulated by IFN-gamma, granulocyte (G) CSF and granulocyte-macrophage (GM) CSF, these cytokines do not alter the low levels of monocyte Fc gamma RIII. Thirdly, expression of the gp55 protein recognized by CD14 monoclonal antibodies is decreased after exposure of monocytes to IFN-gamma. Neutrophils express low levels of CD14 and, in this case, IFN-gamma causes an increase in the CD14 antigen on these cells. Of all the molecules investigated, only HLA class II is confined to monocytes, with increases in expression induced by IFN-gamma but not by the CSF.

Antibodies, Monoclonal↗
PubMed1989

The effect of IFN-gamma and colony-stimulating factors on the expression of neutrophil cell membrane receptors.

The recombinant cytokines IFN-gamma, granulocyte-macrophage (GM)-CSF, and granulocyte (G)-CSF are all known to affect the function of neutrophils. We have investigated their ability to alter the surface expression of neutrophil Fc gamma R and CR. FcRI, formerly considered to be mononuclear phagocyte specific, was found on a variable percentage of neutrophils after 12 h of tissue culture. These levels could be readily enhanced by IFN-gamma at 0.5 U/ml suggesting that neutrophil FcRI is extremely sensitive IFN-gamma. In addition, membrane expression of FcRIII, the most abundant receptor of neutrophils, could also be increased by IFN-gamma and by GM-CSF and G-CSF. However, the opposite effect was observed in samples in which the initial levels of FcRIII were high. In these situations IFN-gamma and GM-CSF caused a decrease in receptor expression. No consistent alterations were found in the levels of FcRII. Cytokine-induced increases in both FcRI and FcRIII were not evident within the first hour of treatment suggesting that these molecules are not available in an intracellular compartment but must be newly synthesized. In contrast, the CR, CR1 and CR3, could be rapidly induced by GM-CSF indicating that this cytokine causes the mobilization of preformed molecules to the membrane. G-CSF and IFN-gamma did not alter the expression of CR1 and CR3. Thus, the very different effects of IFN-gamma, GM-CSF, and G-CSF suggest that the neutrophil functional profile will be distinctively affected by exposure to each of these cytokines.

Antigens, Differentiation↗
PubMed1989

The Fc receptor, FcRI, and other activation molecules on human mononuclear phagocytes after treatment with interferon-gamma.

Human monocytes and the myeloid cell lines U937 and HL60 have been tested with monoclonal antibodies (MoAbs) reactive with 22 different cell surface molecules before and after treatment with interferon-gamma (IFN-gamma). An increase in the expression of the high affinity Fc receptor, FcRI, and the receptor for interleukin 2, IL-2R, were the most consistent alterations which were observed. In addition, expression of the gp55 molecule recognized by CD14 MoAbs was decreased on monocytes. Of the MHC Class II molecules, there was little expression by the myeloid cell lines and no enhancement after IFN-gamma treatment. In contrast monocytes expressed all three MHC Class II subloci with DR much greater than DQ and DP. However there was much variation in IFN-gamma-mediated increase in expression of the individual subregions. In monocytes, the alteration in expression of FcRI, IL-2R, gp55 and MHC Class II molecules took place in a co-ordinate fashion and reached a plateau only after 48 h. In U937 cells, activation proceeded more rapidly and was at maximum levels between 12-16 h. This increase in FcRI appears to be a hallmark of IFN-gamma activation for mononuclear phagocytes (Mph) as the other alterations are either not found on all types of Mph (gp55, MHC Class II) or are induced by other cytokines on Mph and on other cells (IL-2R, MHC Class II). Conversely, other cytokines do not induce FcRI on Mph. These results also suggest that the cell membrane phenotypic changes induced in Mph by IFN-gamma may not be extensive and that FcRI must play a specific role in the IFN-gamma-activated Mph.

Antigens, Surface↗
PubMed1988

Loss of polymorphic A and B locus HLA antigens in colon carcinoma.

In the present study we have confirmed that approximately one third of human colorectal carcinomas fail to express the HLA - A,B,C monomorphic determinant reactive with the W6/32 MAb, and 44% express class II HLA antigens as shown by reactivity with NFK-1 MAb. Reduced staining with the W6/32 MAb was not always associated with the loss of beta 2m. In addition, the expression of HLA-A2 and Bw4 class I specific haplotypes on normal colon epithelium and tumour biopsy tissue was assessed. All normal colonic epithelia stained positively with MAb against A2 and Bw4 antigens, but a loss of these determinants was shown on tumour biopsies from patients tissue typed for the respective specificities. Loss of the A2 haplotype was shown in 4 of 15 tumour tissue samples, and loss of Bw4 specificities in 5 out of 7 tissue samples. The failure to detect specific loci determinants was not necessarily associated with loss of reactivity with W6/32 MAb.

Adenocarcinoma↗
PubMed1987

The effect of the immune status of the TAR mouse on the growth and metastasis of tumour xenografts.

Mice thymectomised at 3-4 weeks of age and subsequently given whole-body irradiation (9 Gy) under cytosine arabinoside cover (TAR mice) provide an alternative model to the athymic nude (nu+/nu+) mouse for studying the biological characteristics of tumour xenografts. In the present study we have evaluated the repopulation events in the bone marrow and spleen following whole body irradiation of TAR mice, and analysed immune competence up to 98 days following irradiation. Repopulation of both bone marrow and spleen was evident in the weeks following whole body irradiation, and an initial increase in the relative proportion of T-lymphocytes present in the spleen was followed by a decrease in the percentage of lymphocytes expressing T-cell markers, which remained below the level observed in control mouse spleen cell preparations. TAR mice exhibited a decreased ability to respond to a non-specific T-cell mitogen and to elicit a T-cell dependent antibody response to influenza viral antigen. Both TAR and control mice possessed macrophages which could be activated to the tumouricidal state, and natural killer activity of TAR mice was enhanced greater than 3-fold above control values. The ability of TAR mice to accept tumour xenografts decreased with the increasing time interval between irradiation and subcutaneous implantation of tumour cells, and (in some instances) spontaneous regression was observed. In addition, a hamster tumour cell line possessing high metastatic potential in its syngeneic host was shown to metastasise to the regional lymph node, lungs, liver, kidneys and spleen of TAR mice from a cell inoculum implanted subcutaneously immediately after irradiation; however, with increasing time between irradiation and inoculation of tumour cells tumour metastasis decreased. The ability of TAR mice to support the growth and metastasis of tumour xenografts would appear to inversely correlate with the increase in natural killer cell activity following irradiation.

Animals↗
PubMed1983

Inhibitor of prostacyclin production in sporadic haemolytic uraemic syndrome.

Prostacyclin (PGI2) production was diminished when rat aortic rings were incubated with plasma from 5 of 6 patients with the sporadic form of haemolytic uraemic syndrome but was normal in the presence of plasma from 7 patients with the epidemic form of haemolytic uraemic syndrome or from patients with other renal diseases. The reduced PGI2 production was caused by an unstable inhibitor, extractable into polar lipid solvents, in sporadic haemolytic uraemic plasma. These results suggest that there may be at least 2 different pathogenetic mechanisms in epidemic and sporadic haemolytic uraemic syndrome and that the reduced PGI2 production observed in the sporadic type is due to an inhibitor of PGI2 production rather than a deficiency of stimulating factors.

Adolescent↗