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Biomedical subjects

A J Barrett

Publications and source records attributed to A J Barrett.

At least 181 records · Page 10Linked to original sources
PubMed1991

Clostripain: characterization of the active site.

In view of the probability that clostripain (EC 3.4.22.8) is fundamentally different in structure from other known cysteine endopeptidases, it was of interest to examine the characteristics of the active site. Z-Phe-Lys-CH2S(CH3)2 irreversibly and rapidly inactivated clostripain, and leupeptin was found to be the most potent reversible inhibitor yet reported for the enzyme. Clostripain was inhibited weakly by some protein inhibitors of serine endopeptidases, and required Ca2+ for stability and activity. Mg2+ and Sr2+ were ineffective. Rapid inactivation by diethylpyrocarbonate, reversed by hydroxylamine, indicated that histidine is essential for catalytic activity. Clostripain was more rapidly inactivated by iodoacetamide than by iodoacetate, with unique pH-dependences of reaction.

Amino Acid Sequence↗
PubMed1991

Human sputum cathepsin B degrades proteoglycan, is inhibited by alpha 2-macroglobulin and is modulated by neutrophil elastase cleavage of cathepsin B precursor and cystatin C.

The high-Mr alkali-stable form of cathepsin B was purified from purulent human sputum. It was shown to solubilize proteoglycan monomer entrapped in polyacrylamide at a rate comparable with that of human lysosomal cathepsin B. Like the enzyme from lysosomes, sputum cathepsin B was bound by human alpha 2-macroglobulin, which inhibited its action on proteoglycan. Cystatin C in purulent sputum was shown to be the N-terminally truncated form generated by neutrophil elastase cleavage, and sputum cathepsin B was only weakly inhibited by recombinant cystatin C that had been cleaved by neutrophil elastase in vitro. Addition of neutrophil elastase to mucoid sputum led to a 5-fold increase in cathepsin B activity concomitant with a lowering in Mr of the cysteine proteinase from 40,000 to 37,000, i.e. the size of the active enzyme purified from purulent sputum. It is concluded that the high-Mr form of cathepsin B present in purulent sputum is a functional proteinase, unlike similar forms of the enzyme secreted by mammary gland in organ culture. The activity of cathepsin B in sputum is modulated by neutrophil elastase, by a combination of inhibitor inactivation and zymogen activation.

Amino Acid Sequence↗
PubMed1991

Homologues of insulinase, a new superfamily of metalloendopeptidases.

On the basis of a statistical analysis of an alignment of the amino acid sequences, a new superfamily of metalloendopeptidases is proposed, consisting of human insulinase, Escherichia coli protease III and mitochondrial processing endopeptidases from Saccharomyces and Neurospora. These enzymes do not contain the 'HEXXH' consensus sequence found in all previously recognized zinc metalloendopeptidases.

Amino Acid Sequence↗
PubMed1991

Immunosuppressive therapy in bone marrow transplantation.

Immunosuppressive and immunomodulatory techniques have a central role in bone marrow transplantation (BMT). A wide range of agents with different mechanisms of action, including steroids, cyclosporin, cytotoxic agents, irradiation and monoclonal antibodies are used to achieve the following treatment aims: (1) Suppression of the recipient's immunity to permit donor marrow engraftment and prevent graft rejection; (2) Prevention of graft versus host disease (GvHD); (3) Treatment of established acute or chronic GvHD.

Bone Marrow Transplantation↗
PubMed1991

Acute myeloid leukaemia relapsing following interleukin-2 treatment expresses the alpha chain of the interleukin-2 receptor.

Immunotherapy with recombinant human Interleukin-2 (rhIL-2) was given to nine patients in first complete remission from acute myeloid leukaemia (AML). Five patients relapsed. The median time to relapse after commencing rhIL-2 was 26 weeks (range 2-44). Four patients were studied at relapse. The morphological and cytochemical features at relapse and presentation were similar. Cytogenetic analysis at relapse in patients 1 and 3 showed a normal karyotype. At relapse, patient 4 had the abnormality 46,XY, t(2;3). Patient 2 had the chromosomal abnormality t(8;21) at presentation and relapse. Patients 3 and 4 with M5 AML relapsed rapidly at 2 and 9 weeks after starting rhIL-2 treatment. Relapse leukaemia cells had features normally associated with lymphoid development. Patient 3 was TdT positive, with rearranged immunoglobulin genes, and a proportion of cells expressing the CD7 antigen; patient 4 also expressed the CD7 antigen. Relapse leukaemic cells from three of four patients expressed the alpha chain of the IL-2 receptor as assessed by flow cytometry. After overnight incubation and removal of T-lymphocytes the proportion of cells from these patients expressing the alpha chain increased from 15% to 61% (P less than 0.01). Using tritiated thymidine uptake to assess cell proliferation, two of three patients who expressed the IL-2 receptor alpha chain proliferated in response to 1000 u/ml of rhIL-2 in vitro, with a stimulation index greater than 1.95 (P less than 0.05). Following rhIL-2 immunotherapy for AML, relapse cells may express an inducible form of the alpha chain of the IL-2 receptor, which can mediate a proliferative response. It is possible that rhIL-2 when administered to AML patients in remission, may induce relapse. This may be a particular risk in patients with the M5 subtype.

Adult↗
PubMed1991

Second transplants for leukaemic relapse after bone marrow transplantation: high early mortality but favourable effect of chronic GVHD on continued remission. A report by the EBMT Leukaemia Working Party.

Ninety second bone marrow transplants (BMT) for relapsed leukaemia were carried out in 30 European BMT centres. At second BMT, after further treatment in 64 cases, 43 patients were in complete remission or in chronic phase of CML, and 47 were in continuing relapse, accelerated phase or blast crisis of CML. Seventy patients died, 37 from early transplant-related toxicity and relapse or failure to eradicate leukaemia which occurred in 23. There were 20 survivors. The actuarial disease-free survival was 11% with a relapse probability of 69% at 3 years. Associated with reduced graft-versus-host disease (GVHD) prophylaxis during second BMT, the incidence and severity of acute and chronic GVHD, was increased when compared with the first BMT (P = 0.02, and 0.002 respectively for acute and chronic GVHD). In multivariate analysis survival was shown to be favoured by a prolonged interval between first and second BMT (relative risk 1.3/year, P = 0.02), and no or mild chronic GVHD following first BMT (relative risk 2.3, P = 0.02). Continuing remission was favoured by chronic GVHD occurring after second BMT (relative risk 8.1, P = 0.004). These results confirm the high treatment-related mortality following second BMT, but identify superior survival in selected patients. Improved results might be achieved by further reduction in preparative regimen intensity, and increasing graft-versus-leukaemia reactivity.

Acute Disease↗
PubMed1991

Graft-versus-leukaemia following allogeneic bone marrow transplantation: emergence of cytotoxic T lymphocytes reacting to host leukaemia cells.

Cytotoxic T lymphocyte precursor (CTLp) frequency assays were examined in patients with chronic myeloid leukaemia (CML) following bone marrow transplantation (BMT) using recipient lymphocytes or CML cells as targets in a 51Cr release cytotoxicity assay. Eighteen patients were studied; 11 received marrow from a fully HLA A, B and DR matched sibling donor, and six from matched unrelated donors or a partially matched sibling (one patient). Two of the unrelated donor transplant recipients received marrow depleted of T lymphocytes, and the remainder received unmanipulated marrow and cyclosporin with or without methotrexate as prophylaxis against graft-versus-host disease (GVHD). Donor cells tested before BMT did not generate CTL against the patients' leukaemia, but up to 9 months after BMT a low frequency of CTLp directed against the patients' CML cells (Lk-CTLp) was detected in all patients. The Lk-CTLp frequency was significantly lower than the frequency of CTLp directed against the recipients' PHA transformed pretransplant lymphocytes (Ly-CTLp) (p less than 0.05). Lk-CTLp showed MHC restricted cytotoxicity and did not demonstrate cytotoxicity in an NK assay. The Lk-CTLp frequency correlated with both GVHD severity and relapse: severe GVHD was only seen with Lk-CTLp frequencies greater than 1:400,000, while leukaemic relapse was only observed in two patients with Lk-CTLp frequencies less than 1:400,000. These results show that a low frequency of alloreactive cells of presumed donor origin with cytotoxic potential against residual leukaemia normally circulate after BMT. Their relationship with the graft-versus-leukaemia phenomenon and their cross-reaction with GVHD reacting cells remain to be determined.

Adolescent↗
PubMed1990

Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase.

Pz-peptidase was purified from chicken liver as a protein of Mr 80,000 and pI 5.2. The purified enzyme hydrolysed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys. 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-(2,4-dinitropheny l)Lys, benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate, Ac-Ala4 (at the Ala-1-Ala-2 bond) and bradykinin (at the Phe-5-Ser-6 bond). No hydrolysis of proteins was detected. Loss of activity in the presence of EDTA or 1,10-phenanthroline was time-dependent. Metal ions found to restore activity after treatment with EDTA were Zn2+, Mn2+, Ca2+, Co2+ and Cd2+, in decreasing order of effectiveness. Ni2+, Fe2+ and higher concentrations of Zn2+ were inhibitory. Inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate and related compounds showed Ki values (down to 5 nM) somewhat lower than those for the rat enzyme. Pz-peptidase was activated by low concentrations of 2-mercaptoethanol and dithiothreitol, but inhibited by higher concentrations. p-Chloromercuribenzoate and some other thiol-blocking reagents were inhibitory. Inactivation by diethyl pyrocarbonate that was reversible by hydroxylamine showed the presence of essential histidine residue(s). We conclude that chicken Pz-peptidase is a metallo-endopeptidase with thiol-dependence. Moreover, the properties of chicken Pz-peptidase agree with those described for mammalian soluble metallo-endopeptidase and endo-oligopeptidase A. consistent with the view that these three types of activity are all attributable to the single enzyme for which the name thimet peptidase has been proposed.

Amino Acid Sequence↗
PubMed1990

Inhibition of cysteine proteinases by a protein inhibitor from potato.

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.

Cystatins↗
PubMed1990

The amino acid sequence of a novel inhibitor of cathepsin D from potato.

The amino acid sequence of a cathepsin D inhibitor isolated from potato is described. It was determined by analysis of peptides generated by use of the glycine-specific proteinase PPIV. The order of the peptides was established by examination of tryptic peptides derived from the two cyanogen bromide peptides. The inhibitor comprises 187 amino acid residues, and has a calculated Mr of 20,450.

Amino Acid Sequence↗
PubMed1990

A distinct thimet peptidase from rat liver mitochondria.

Thimet peptidase has been purified from rat liver mitochondria and found to share the characteristics of a thiol-dependent metallo-endopeptidase previously described for an enzyme in the cytosolic fraction from rat testis: inhibition by EDTA, reactivation by Zn2+, requirement of dithiothreitol for maximal and stable activity, and inhibition by N-ethylmaleimide. The Ki for inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoic acid is 2.6 microM, 100-fold higher than the value for the cytosolic form. The mitochondrial form is not inhibited by antisera against the cytosolic form, and the two forms of the enzyme show quantitative differences in substrate specificity. The name thimet peptidase II is suggested for the enzyme from rat mitochondria.

Amino Acid Sequence↗
PubMed1990

Thiol-dependent metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit.

Pz-peptidase was purified from rat testis and rabbit muscle. Zinc was detectable in the rat enzyme. The activity of the enzyme from both species was slowly but completely abolished by EDTA and restored by Zn2+. Free thiol groups were also important for the catalytic activity of rat Pz-peptidase, as previously reported for the rabbit enzyme. We conclude that in both species Pz-peptidase has the characteristics of a thiol-dependent metallo-endopeptidase.

Amino Acid Sequence↗
PubMed1990

The cysteine proteinases of the pineapple plant.

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

Amino Acid Sequence↗