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Biomedical subjects

M J Stone

Publications and source records attributed to M J Stone.

At least 55 records · Page 3Linked to original sources
PubMed1995

Overexpression of myoglobin and assignment of its amide, C alpha and C beta resonances.

Sperm whale apomyoglobin was expressed to high levels on minimal media and isotopically labeled with 13C and 15N nuclei. The isotopically labeled apoprotein was purified to homogeneity in a single step by reversed-phase chromatography and reconstituted with hemin and carbon monoxide gas for NMR analysis. Sequence-specific backbone 1HN, 15N and 13C alpha as well as side-chain 13C beta resonance assignments have been made for over 90% of the amino acids in the carbon monoxide complex of the protein. Resonance assignments were made by analysis of a series of 3D triple resonance spectra measured on the uniformly labeled sample. These assignments will provide the basis for analyzing the effects of point site mutations on the structure, stability and dynamics of the protein in solution.

Amides↗
PubMed1994

Anti-CD40 antibody binding modulates human multiple myeloma clonogenicity in vitro.

Ligand binding of the B-cell lineage antigen CD40 enhances growth and interleukin-6 (IL-6) secretion in human B cells (the CD40/IL-6 loop). IL-6 has an autocrine and paracrine role in human multiple myeloma (MM) cell growth. With the use of the CD40 monoclonal antibody (MoAb) G28-5, we examined CD40 expression and the effect of CD40 binding on MM clonogenic colony (MCC) formation to characterize the IL-6/CD40 loop activity in MM. CD40 was expressed on plasmacytoid cells in 21 of 28 plasma cell dyscrasia (PCD) bone marrow (BM) biopsies tested (10 of 14 MM, 2 of 2 Waldenstrom's macroglobulinemia [WM], 2 of 2 plasma cell leukemia [PCL], 6 of 8 monoclonal gammopathy of undetermined significance [MGUS], and 1 of 2 primary amyloidosis [AL]). G28-5 binding increased MCCs by 35% to 150% in 11 of 17 CD40+ PCD BM cultures, but did not affect MCC formation in CD40- specimens or normal BM colony forming units (CFU-GEMM, CFU-GM, BFU-E). Responsive cultures originated from BM of patients with MM (2 of 5 cases tested), WM (2 of 2), PCL (2 of 2), and MGUS (5 of 6). CD40-responsiveness was not significantly inhibited by the presence of an anti-IL-6 MoAb (2 of 2 MGUS cultures tested), and did not correlate with the capacity to respond to IL-6 stimulation (n = 17, P > .05) or a detectable level of endogenous IL-6 (n = 15, P > .05). Additional studies were performed with PCD cell lines to characterize the interrelationship of CD40 activation and IL-6 production. Fifty percent to greater than 95% of cells from the RPMI 8226 and ARH77 lines expressed CD40, whereas 6% of U266 cells were CD40+. For RPMI 8226, ARH-77, and U266 cells, the increased MCC formation after anti-CD40 stimulation was not affected by the presence of an anti-IL-6 neutralizing MoAb and was not accompanied by detectable IL-6 secretion. There was no apparent increase in IL-6 mRNA transcription following G28-5 treatment of U266 or RPMI 8226 cells. Our observations indicate that CD40 is expressed in a subset of human myeloma cells present in various PCDs. Cell-line studies suggest that the CD40+ myeloma cell may regulate MM clonogenic colony formation without activating the IL-6 pathway.

Antigen-Antibody Reactions↗
PubMed1994

HLA compatibility and liver transplant outcome. Improved patient survival by HLA and cross-matching.

In liver transplantation (LTx), numerous studies have failed to demonstrate an adverse effect of HLA-A,B,DR incompatibility or of donor-specific positive cross-match on survival of the recipients. In this study, we examined the effect of antidonor cytotoxic antibody and HLA compatibility in 800 LTx recipients with CsA-based immunosuppression. Thirty-four of 482 (7%) recipients were transplanted across a positive donor-specific T cell cross-match. Four-year patient and graft survival was 71% and 67%, respectively, in negative cross-match recipients and 53% and 50%, respectively, in positive cross-match recipients (P = 0.0051 and P = 0.023). Neither B cell-positive cross-match nor the presence of panel reactive antibody (PRA) had an adverse impact on the liver allograft outcome. Interestingly, 21/58 (36.2%) patients with PRA > or = 10% had a positive T cell cross-match, whereas only 7/382 (1.8%) patients with PRA < 10% did (P < 0.0001). This indicates the predictive value of PRA cross-match results. B lymphocyte cross-match results also were strongly correlated with the presence of PRA, as 26/57 (45.6%) of the patients with PRA > or = 10% had a positive cross-match, whereas only 22/394 (5.6%) with PRA < 10% did (P < 0.0001). Analysis of HLA compatibility demonstrated a significant impact on patient's survival, comparing only 0-2 vs. 6 HLA-A+B+DR mismatches and 0 vs. 1 vs. 2 HLA-DR mismatches. Four-year patient survival rate for 0 to 2 antigen mismatches was 86%, whereas for 6 antigen mismatches it was 62% (P = 0.025). Overall actuarial 4-year patient survival rate in HLA-DR-mismatched groups (0 vs. 1 vs. 2) was 84%, 73%, and 64%, respectively (P = 0.033). In no mismatched category was graft survival rate significantly different. Sepsis or rejection was the cause of graft loss in 1/10 (10%), 21/75 (28%), and 34/85 (40%) patients with 0, 1, and 2 HLA-DR mismatches, respectively. The difference between patient and graft survival was accounted for by survival after retransplantation, which was lower in patients with more HLA-DR mismatches in primary transplants. The latter group received intensive immunosuppressive therapy during the first month after primary transplantation, as compared with those with fewer HLA-DR mismatches (P = 0.04). The above data suggest that prospective cross-match should be performed in patients with > or = 10% PRA if it is logistically feasible.(ABSTRACT TRUNCATED AT 250 WORDS)

Actuarial Analysis↗
PubMed1994

Mutational mapping of functional residues in tissue factor: identification of factor VII recognition determinants in both structural modules of the predicted cytokine receptor homology domain.

Alanine scanning mutagenesis of tissue factor, the initiating receptor and cofactor molecule for the coagulation pathways, was used to define residue side chains with functional contributions. Approximately half of the residues were exchanged, and several stretches of functional residues throughout the entire extracellular domain were identified which contributed to overall coagulant function. Mutants were further characterized with respect to their affinity for binding of ligand, providing evidence that identified functional sequence spans are involved in ligand interaction. The tissue factor extracellular domain is suggested to adopt the folding pattern of the cytokine receptor homology unit, which is typically composed of two seven-beta-strand modules. Evaluation of the mutational analysis within this structural context suggests that functionally important residues are spatially proximate and clustered at the boundary of the predicted beta-strand modules. Residues contributing to ligand binding by tissue factor were identified in positions corresponding to ligand interactive residues in the growth hormone receptor and contact residues of other cytokine receptors, consistent with a conserved structural region for ligand interaction throughout the cytokine receptor family.

Alanine↗
PubMed1994

Characterization of skin-infiltrating cells during acute graft-versus-host disease following bone marrow transplantation using unrelated marrow donors.

To characterize skin-infiltrating T lymphocytes during acute GVHD, skin biopsies were obtained from two patients who received unrelated marrow matched for HLA-A, -B, -DR, and -DQ but mismatched for -DP. A total of 120 T-cell clones were generated. Phenotype analysis of the clones showed that the majority of cells were CD4+ and expressed alpha/beta TCR. HLA-DP oligonucleotide genotyping of the clones revealed the presence of lymphoid chimerism. PLT assay showed the lack of HLA specificity, including mismatched HLA-DP. However, mAb to HLA antigens blocked proliferation of the majority of the clones, indicating that the clones recognized HLA-associated molecules. Interestingly, proliferation of two CD4+ T-cell clones was inhibited by class I mAb. A few of the clones revealed augmented proliferation in the presence of CMV antigens and a few revealed cytolytic activity. The above study suggests that (a) CD4+ helper T cells may be primarily responsible for immunopathogenesis of skin manifestations during acute GVHD, (b) there is a mixed lymphoid chimerism in skin during acute GVHD, (c) HLA-DP may not be a factor contributing to the development of acute GVHD, (d) the peptide of the HLA groove or superantigen associated with HLA molecules may be the stimulatory antigen, and (e) CMV antigens appear to stimulate some of the skin-infiltrating T lymphocytes.

Base Sequence↗
PubMed1993

A phase I study of an anti-CD22-deglycosylated ricin A chain immunotoxin in the treatment of B-cell lymphomas resistant to conventional therapy.

Twenty-six patients, whose B-cell lymphoma had relapsed after conventional therapies, were treated in a phase I dose escalation study with an immunotoxin consisting of a mouse CD22 monoclonal antibody (RFB4:IgG1K) coupled to chemically deglycosylated ricin A chain (dgA). Two to 12 doses of the immunotoxin were infused intravenously at 48-hour intervals. The peak serum concentration and half-life (T1/2) did not correlate directly with the dose and averaged 3.8 micrograms/mL and 7.8 hours, respectively. The main dose-limiting toxicity was caused by the vascular leak syndrome (VLS) consisting of weight gain, edema, serum albumin decrease, and critically by pulmonary edema. Myalgia occurred frequently and was only dose limiting in one patient who developed rhabdomyolysis. The presence of lymphoma cells in the blood (> or = 10(10)/L) and clinically detectable splenomegaly were associated with reduced toxicity and a shorter T1/2. Nine of 24 evaluable patients (37.5%) made antibody to either mouse Ig or dgA. There were five partial responses (PR) and one complete response (CR) lasting 30 to 78 days. High peak concentrations of immunotoxin in the serum, a long T1/2, and large areas under the curve (AUC) correlated with both clinical response and toxicity. None of three patients with CD5+ lymphomas (including two CLL patients) had more than mild toxicity or responded to the immunotoxin.

Adult↗
PubMed1993

Molecular basis for methoxyamine-initiated mutagenesis: 1H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-modified cytosine residues.

Methoxyamine, N4-methoxycytidine and its 2'-deoxyribo analogue are transition mutagens. The mechanism by which the latter acts after incorporation into or generation within DNA has been ascribed to the ability of the base analogue to pair effectively with both adenine and guanine. To obtain a detailed understanding of these interactions, the solution structures of the self-complementary octanucleotide d(CGGATCCG) and its analogues d(CGGATTCG), d(CGGATMCG) and d(CGGATPCG) (designated 8mer-GC, -GT, -GM and -GP, respectively) were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2] oxazin-7-one. A variable temperature study showed the order of stability as 8mer GC > GP > GT > GM. Nuclear Overhauser spectroscopy permitted the assignment of the base, anomeric and H2'/H2" protons in these 8mers. All had spectra consistent with regular B-DNA duplex structures. Imino proton spectra showed that the 8mers GC, GP and GM involved Watson-Crick base-pairing but that the G.P and to a greater extent G.M base-pairs were in slow exchange on the nuclear magnetic resonance time-scale with the wobble configuration. Indeed, the G.M pair showed an additional exchange process interpreted in terms of the presence of syn and anti conformers of the methoxy group in the wobble pair. This accounts for the destabilization of M compared with the P-containing duplex. The observations are compared with those made earlier on the corresponding AT, AP and AM octamers. It is evident that M and P can form stable base-pairs with both A and G with essentially Watson-Crick geometry. This confirms the earlier, although unsubstantiated explanation for the transition mutational propenstty of methoxyamine which, in turn, was based on the fact that methoxycytosine bases have tautomeric constants (KT) much nearer to unity than the normal bases. The same general explanation for hydroxylamine and hydrazine-induced mutations is correspondingly rendered more certain.

Base Composition↗
PubMed1993

Disseminated growth of a human multiple myeloma cell line in mice with severe combined immunodeficiency disease.

We have successfully engrafted a human multiple myeloma cell line, ARH-77, into C.B. 17 SCID mice. When ARH-77 cells were injected s.c., tumors grew only at the site of inoculation (five of five). When ARH-77 cells were injected i.v. tumors did not grow in any of the mice (zero of five). However, when mice were given gamma-irradiation with 150 rads and then inoculated i.v. with 10(7) ARH-77 cells, 100% (13 of 13) of the mice developed tumors. Hind leg paralysis was observed in 13 of 16 mice as a result of compression of the spinal cord by tumor. Histological analysis demonstrated that myeloma cells proliferated and formed osteolytic lesions (15 of 16) in the vertebrae and bones of the skull (14 of 16). Tumor cells also invaded the brain and meninges (14 of 16), lung (13 of 15), liver (seven of 15), and kidney (two of 15). Flow cytometric analysis demonstrated that the phenotype of 31% of the bone marrow cells in the vertebrae and 79% of s.c. tumor cells was similar to ARH-77 cells (CD38+, PCA-1+, HLA-Classes 1 and II+). Furthermore, DNA hybridization with a human AluI probe confirmed their human origin. ARH-77-derived human immunoglobulin was detected in the serum of SCID/ARH-77 mice by ELISA. These observations demonstrate systemic involvement of human multiple myeloma following i.v. injection of ARH-77 cells into irradiated mice. This in vivo model should be useful for evaluating new therapeutic modalities for myeloma.

Animals↗
PubMed1993

Comparison of backbone and tryptophan side-chain dynamics of reduced and oxidized Escherichia coli thioredoxin using 15N NMR relaxation measurements.

The backbone and tryptophan side-chain dynamics of both the reduced and oxidized forms of uniformly 15N-labeled Escherichia coli thioredoxin have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured for more than 90% of the protonated backbone nitrogen atoms and for the protonated indole nitrogen atoms of the two tryptophan residues. These data were analyzed by using a model free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The reduced and oxidized forms exhibit almost identical dynamic behavior on the picosecond to nanosecond time scale. The W31 side chain is significantly more mobile than the W28 side chain, consistent with the positions of W31 on the protein surface and W28 buried in the hydrophobic core. Backbone regions which are significantly more mobile than the average include the N-terminus, which is constrained in the crystal structure of oxidized thioredoxin by specific contacts with a Cu2+ ion, the C-terminus, residues 20-22, which constitute a linker region between the first alpha-helix and the second beta-strand, and residues 73-75 and 93-94, which are located adjacent to the active site. In contrast, on the microsecond to millisecond time scale, reduced thioredoxin exhibits considerable dynamic mobility in the residue 73-75 region, while oxidized thioredoxin exhibits no significant mobility in this region. The possible functional implications of the dynamics results are discussed.

Amino Acid Sequence↗
PubMed1993

Neoadjuvant chemotherapy and liver transplantation for hepatocellular carcinoma: a pilot study in 20 patients.

BACKGROUND: Liver transplantation for unresectable hepatocellular carcinoma yields disappointing results. Most cases recur within 2 years, often in the transplanted liver. METHODS: A combination of neoadjuvant doxorubicin and orthotopic liver transplantation was used in 20 patients with unresectable hepatocellular carcinoma confined to the liver. Seventeen patients had tumors > 5 cm in greatest diameter, and 11 cases were stage IVA by the TNM classification. Doxorubicin was administered preoperatively, intraoperatively, and postoperatively at a dose of 10 mg/m2 weekly, totaling 200 mg/m2. RESULTS: Chemotherapy was well tolerated although leukopenia was observed in 70% of patients. Eight patients died, five of recurrent tumor and three of hepatitis B. Three others remain alive 8-22 months after tumor recurrence. One patient had initial tumor recurrence in the allograft. Actuarial survival is 59% and tumor-free survival is 54% at 3 years. For the 17 patients with tumors > 5 cm, overall survival is 63% and tumor-free survival is 49% at 3 years. CONCLUSION: The results of this pilot study suggest that neoadjuvant doxorubicin chemotherapy favorably alters the post-transplant survival of patients with hepatocellular carcinoma.

Adult↗
PubMed1993

Characterization of T-cell clones generated from skin of patients with psoriasis.

BACKGROUND: The infiltrating cells in psoriasis include a subpopulation of autoreactive T cells. OBJECTIVE: The aim of this study was to further characterize skin-infiltrating T lymphocytes in patients with psoriasis. METHODS: Forty-five T-cell clones were generated from skin biopsy specimens from two patients. RESULTS: Phenotypic studies on 25 of 45 clones revealed that 19 (76%) of the clones were CD4+, 5 (20%) were CD8+, and 1 (4%) clone was CD4- and CD8-. Twenty-three clones were stained for identification of T-cell receptors. Twenty-two clones expressed alpha/beta T-cell receptors and one clone (CD4-/CD8-) expressed no T-cell receptor. Nineteen clones (42%) were autoreactive with no restriction to class I or class II HLA antigens. By contrast, proliferation of two of the seven clones was inhibited by class I monoclonal antibody, whereas proliferation of four of seven clones was inhibited by class II monoclonal antibody. CONCLUSION: These data suggest that skin-infiltrating lymphocytes in patients with psoriasis may recognize HLA-associated molecules, perhaps the peptide of the HLA groove. The recognition of the peptide is presumably inhibited when monoclonal antibody is bound to the HLA molecule.

CD4 Antigens↗
PubMed1992

The evolutionary role of secondary metabolites--a review.

It is argued that organisms have evolved the ability to biosynthesise secondary metabolites ('natural products') due to the selectional advantages they obtain as a result of the functions of the compounds. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes in Streptomyces, is interpreted in terms of the defense roles of antibiotics. The clustering together of antibiotic biosynthesis, regulation, and resistance genes, and in particular the staggering complexity shown in the case of the gene cluster for erythromycin A biosynthesis, implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature.

Anti-Bacterial Agents↗
PubMed1992

Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements.

The backbone dynamics of the uniformly 15N-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively; however, a small number of residues exhibited significantly slower relaxation. Similarly, (1H)-15N NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs. The molecular rotational correlation time was 6.24 +/- 0.01 ns; most residues had order parameters in the range 0.75-0.90 and tau e values of less than ca. 25 ps. Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered; the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins; and the region from A146 to S149. All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure.

Amides↗
PubMed1992

On the evolution of functional secondary metabolites (natural products).

It is argued that organisms have evolved the ability to biosynthesize secondary metabolites (natural products) because of the selectional advantages they obtain as a result of the functions of the compounds. The clustering together of antibiotic biosynthesis, regulation, and resistance genes implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes, is interpreted in terms of the defence roles of antibiotics. We suggest a general mechanism for the evolution of secondary metabolite biosynthesis pathways, and argue against the hypothesis that modern antibiotics had prebiotic effector functions, on the basis that it does not account for modern biosynthetic pathways.

Anti-Bacterial Agents↗
PubMed1991

Molecular basis for methoxyamine initiated mutagenesis. 1H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides.

In order to reach a more detailed understanding of the mechanism of the mutagenic action of methoxyamine and of N4-methoxycytidine and its 2'-deoxyribo-analogue, the solution structures of the self-complementary octanucleotide, d(CGAATTCG) and its analogues, d(CGAATCCG), d(CGAATMCG) and d(CGAATPCG) (designated 8mer-AT, 8mer-AC, 8mer-AM, and 8mer-AP, respectively), were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2]oxazin-7-one, in which the N-O bond is held in the anti configuration with respect to N3 of the cytosine ring. Correlated spectroscopy and nuclear Overhauser spectroscopy allowed assignment of the base, anomeric and H2'/H2" protons in 8mers-AT, -AM and -AP, and showed that all three had features consistent with a regular B-DNA duplex structure. Duplex-to-coil transition temperatures were determined to be 52(+/- 2) degrees C (8mer-AT), 51(+/- 2) degrees C (8mer-AP), 32(+/- 2) degrees C (8mer-AM); on the chemical shift timescale, the melting transition was fast for 8mer-AT and 8mer-AP, but slow for 8mer-AM. Imino proton spectra were indicative of Watson-Crick base-pairing in 8mers-AT, -AP and -AM. The 8mer-AP duplex had a structure and melting characteristics virtually identical with those of the 8mer-AT duplex. The preferred syn configuration of the methoxyl group in M had a destabilising effect on the 8mer-AM duplex. At low temperatures, the A.M base-pair was in fast equilibrium between Watson-Crick and wobble configurations, with the methoxyl function anti-oriented, but the melting transition was accompanied by isomerization of the methoxyl group to the syn conformation. This syn-anti isomerization was the rate-determining step in the duplex-to-coil transition. The 8mer-AC oligomer did not form a stable duplex.

Cytidine↗