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Biomedical subjects

P Carter

Publications and source records attributed to P Carter.

At least 109 records · Page 6Linked to original sources
PubMed1992

Weyers' ulnar ray/oligodactyly syndrome and the association of midline malformations with ulnar ray defects.

We describe a two generation family with variable ulnar and radial ray reduction and midline craniofacial abnormalities. The features suggest a diagnosis of Weyers' ulnar ray/oligodactyly syndrome originally described in two isolated cases. Syndromes of ulnar ray reduction are briefly reviewed and the relationship between limb bud and midline development discussed.

Abnormalities, Multiple↗
PubMed1992

Organising a continence advisory service.

Modern surgical techniques have greatly improved the treatment of incontinence, but there remains a large number of patients for whom conservative management will be necessary. This paper reports the appointment of a continence adviser 10 years ago, and describes the subsequent development of a District Continence Advisory Service. Experience has shown that this appointment is essential, not only in terms of facilitating the service, but also as a means of ensuring cost efficiency.

Community Health Nursing↗
PubMed1992

Engineering a humanized bispecific F(ab')2 fragment for improved binding to T cells.

We recently constructed a humanized bispecific antibody (BsF(ab')2v1) by separate E. coli expression of each Fab' arm followed by directed chemical coupling in vitro. BsF(ab')2 v1 (anti-CD3/anti-p185HER2) was demonstrated to retarget the cytotoxic activity of human CD3+ CTL in vitro against the human breast-tumor cell line, SK-BR-3, which over-expresses the p185HER2 product of the proto-oncogene HER2. Our minimalistic humanization strategy is to install as few murine residues as possible into a human antibody in order to recruit antigen-binding affinity and biological properties comparable to that of the murine parent antibody. This strategy proved very successful for the anti-p185HER2 arm of BsF(ab')2 v1. In contrast BsF(ab')2 v1 binds to T cells via its anti-CD3 arm much less efficiently than does the chimeric BsF(ab')2 which contains the variable domains of the murine parent anti-CD3 antibody. Here we have constructed additional BsF(ab')2 fragments containing variant anti-CD3 arms with selected amino acid replacements in an attempt to improve antibody binding to T cells. One such variant, BsF(ab')2 v9, was created by replacing 6 residues in the second hypervariable loop of the anti-CD3 heavy chain variable domain of BsF(ab')2 v1 with their counterparts from the murine parent anti-CD3 antibody. BsF(ab')2 v9 binds to T cells (Jurkat) much more efficiently than does BsF(ab')2 v1 and almost as efficiently as the chimeric BsF(ab')2. This improvement in the efficiency of T-cell binding of the humanized BsF(ab')2 is an important step in its development as a potential therapeutic agent for the treatment of p185HER2 over-expressing cancers.

Amino Acid Sequence↗
PubMed1991

Probing the mechanism and improving the rate of substrate-assisted catalysis in subtilisin BPN'.

A mutant of the serine protease, subtilisin BPN', in which the catalytic His64 is replaced by Ala (H64A), is very specific for substrates containing a histidine, presumably by the substrate-bound histidine assisting in catalysis [Carter, P., & Wells, J.A. (1987) Science (Washington, D.C.) 237, 394-399]. Here we probe the catalytic mechanism of H64A subtilisin for cleaving His and non-His substrates. We show that the ratio of aminolysis to hydrolysis is the same for ester and amide substrates as catalyzed by the H64A subtilisin. This is consistent with formation of a common acyl-enzyme intermediate for H64A subtilisin, analogous to the mechanism of the wild-type enzyme. However, the catalytic efficiencies (kcat/KM) for amidase and esterase activities with His-containing substrates are reduced by 5000-fold and 14-fold, respectively, relative to wild-type subtilisin BPN, suggesting that acylation is more compromised than deacylation in the H64A mutant. High concentrations of imidazole are much less effective than His substrates in promoting hydrolysis by the H64A variant, suggesting that the His residue on the bound (not free) substrate is involved in catalysis. The reduction in catalytic efficiency kcat/KM for hydrolysis of the amide substrate upon replacement of the oxyanion stabilizing asparagine (N155G) is only 7-fold greater for wild-type than H64A subtilisin. In contrast, the reductions in kcat/KM upon replacement of the catalytic serine (S221A) or aspartate (D32A) are about 3000-fold greater for wild-type than H64A subtilisin, suggesting that the functional interactions between the Asp32 and Ser221 with the substrate histidine are more compromised in substrate-assisted catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1990

Functional interaction among catalytic residues in subtilisin BPN'.

Variants of the serine protease, subtilisin BPN', in which the catalytic triad residues (Ser-221, His-64, and Asp-32) are replaced singly or in combination by alanine retain activities with the substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPF-pna) that are at least 10(3) to 10(4) above the non-enzymatic rate [Carter, P., Wells, J.A. Nature (London) 322:564-568, 1988]. A possible source of the residual activity was the hydrogen bond with the N delta 2 of Asn-155 that helps to stabilize the oxyanion generated in the tetrahedral transition state during amide bond hydrolysis by the wild-type enzyme. Replacing Asn-155 by Gly (N155G) lowers the turnover number (kcat) for sAAPF-pna by 150-fold with virtually no change in the Michaelis constant (KM). However, upon combining the N155G and S221A mutations to give N155G:S221A, kcat is actually 5-fold greater than for the S221A enzyme. Thus, the catalytic role of Asn-155 is dependent upon the presence of Ser-221. The residual activity of the N155G:S221A enzyme (approximately 10(4)-fold above the uncatalyzed rate) is not an artifact because it can be completely inhibited by the third domain of the turkey ovomucoid inhibitor (OMTKY3), which forms a strong 1:1 complex with the active site. The mutations N155G and S221A individually weaken the interaction between subtilisin and OMTKY3 by 1.8 and 2.0 kcal/mol, respectively, and in combination by 2.1 kcal/mol. This is consistent with disruption of stabilizing interactions around the reactive site carbonyl of the OMTKY3 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1990

RO 23-6457 prolongs survival of vascularized allografts in rodents and primates.

The ability of RO 23-6457, a retinoid compound with marked in vitro immunosuppressive properties, to prolong vascularized allografts was examined in several in vivo transplantation models. In the murine heterotopic heart model, efficacy was shown in two H-2 incompatible strain combinations, with indefinite graft survival at some doses. In the rat heterotopic heart model, oral administration prolonged Wistar-Furth grafts in Lewis hosts an average of 1 week, with no long-term survivors at a variety of doses. Given subcutaneously, grafts were further prolonged, but the compound proved toxic. In a bilaterally nephrectomized renal transplant model in cynomolgus monkeys treated intravenously at a dose of 2 mg/kg/day, host survival was prolonged to 18, 32, 33, and 74 days, compared with 11, 11, 12, 13, and 26 days in untreated controls (P less than 0.05 by rank-sum testing). The three shorter surviving recipients died from anorexia and weight loss with normal renal function, while the longest survivor rejected its kidney when exhaustion of iv sites precluded further treatment. The toxic effects of the compound resemble the syndrome of hypervitaminosis A. RO 23-6457 will prolong graft survival as a single agent, justifying further preclinical testing and efforts to reduce toxicity.

Animals↗
PubMed1989

Engineering subtilisin BPN' for site-specific proteolysis.

A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.

Amino Acid Sequence↗
PubMed1989

Plunging ranula: literature review and report of three cases.

Three cases of plunging ranula are described and the literature is reviewed. In many cases, a plunging ranula is iatrogenic and follows surgery to an oral ranula. In the cases presented, the cervical swelling was associated with prolongations of sublingual gland into or through the mylohyoid muscle. All patients were cured by partial or total excision of the sublingual gland.

Adult↗
PubMed1989

The structures of human C1r and C1s and their relationship to other serine proteases.

The recent sequencing of the C1 subcomponents has allowed comparison with other molecules of homologous primary structure. Where tertiary structures are available for at least one member of the family it is possible to make further progress by modelling the amino acid sequence of the complement protein into the three-dimensional coordinates of the directly determined structure, thereby obtaining an approximation of the structure of the complement protein. Molecular modelling allows structure-function relationships to be explored and suggests further experiments that may be amenable to techniques such as site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1989

Molecular defects of the C1-inhibitor gene in hereditary angioedema.

Family studies of hereditary angioedema are discussed with particular emphasis on the molecular basis of the more common form of the disease (type I), characterized by quantitative C1-Inhibitor deficiencies. Results obtained in several laboratories can be interpreted in the light of the restriction maps and of the exon-intron structure of the normal C1-Inhibitor gene that have been published recently. Several partial deletions of either exon 4 or exon 7 or of larger portions of the 5' end of the gene and a duplication of exon 4 have been described. These gene alterations are essentially family-specific and they account for at least fifteen-percent of the defects in type I hereditary angioedema. Detailed sequences analyses of cloned defective genes indicate that clusters of repetitive sequences of the Alu family, located within several introns, are responsible for the relatively high frequency of gross gene alterations observed at the C1-Inhibitor locus. In contrast, most of the defects that lead to production of dysfunctional proteins appear to be point mutations within the P1 residue of the reactive site, whose codon features a hypermutable CpG dinucleotide.

Angioedema↗
PubMed1989

Antigenic expression of B-cell chronic lymphocytic leukemic lymphocytes.

A flow cytometric analysis of lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL) samples and in monocyte-depleted and T-cell-depleted normal peripheral blood (B-PBL) samples was undertaken using 129 reagents from the blind panel (BP) and 72 reagents from the cluster designation (CD) panel obtained from the Fourth International Leucocyte Differentiation Conference and Workshop, B-Cell Section. After determining the average mean channel fluorescence and the average percentage of positive cells for the B-CLL and the normal B-PBL preparations, a combined ratio and difference analysis was performed for each monoclonal antibody reactivity. This analysis confirmed the intense expression of class II antigens on B-CLL and the preferential expression of CD19, CD20, CD23 and CD24 antigens. In addition, three new clustered and three new unclustered antigens were also preferentially expressed on B-CLL lymphocytes. Cluster analysis of these differences suggests the existence of at least three overlapping immunophenotypic subpopulations, composed of CD19, CD20, CD21, CD22, CD23, CD24, CD75, CD76 and CDw78.

Antigens, CD↗
PubMed1988

Dissecting the catalytic triad of a serine protease.

Serine proteases are present in virtually all organisms and function both inside and outside the cell; they exist as two families, the 'trypsin-like' and the 'subtilisin-like', that have independently evolved a similar catalytic device characterized by the Ser, His, Asp triad, an oxyanion binding site, and possibly other determinants that stabilize the transition state (Fig. 1). For Bacillus amyloliquefaciens subtilisin, these functional elements impart a total rate enhancement of at least 10(9) to 10(10) times the non-enzymatic hydrolysis of amide bonds. We have examined the catalytic importance and interplay between residues within the catalytic triad by individual or multiple replacement with alanine(s), using site-directed mutagenesis of the cloned B. amyloliquefaciens subtilisin gene. Alanine substitutions were chosen to minimize unfavourable steric contacts and to avoid imposing new charge interactions or hydrogen bonds from the substituted side chains. In contrast to the effect of mutations in residues involved in substrate binding, the mutations in the catalytic triad greatly reduce the turnover number and cause only minor effects on the Michaelis constant. Kinetic analyses of the multiple mutants demonstrate that the residues within the triad interact synergistically to accelerate amide bond hydrolysis by a factor of approximately 2 X 10(6).

Amino Acid Sequence↗
PubMed1987

Renal brush border glutamine transport: comparison between in situ and isolate membrane vesicle uptake.

Glutamine uptake by renal cortical brush-border vesicles was compared to transport expressed by the functioning isolated kidney. Comparisons were made with regard to sodium dependency and the adaptive increase induced by chronic metabolic acidosis in the rat. The results show an absolute dependency upon a sodium gradient; sodium-independent glutamine uptake has no counterpart in situ. In addition, acidosis-induced adaptive increase in vesicle glutamine uptake has no counterpart in situ. Rather, the apparent adaptation reflects extravesicular gamma-glutamyltransferase-mediated conversion to glutamate and subsequent accumulation; acidosis-induced adaptation of this enzyme largely explains the apparent adaptation in glutamine uptake. Consequently the role of membrane transport in glutamine flux regulation can be assessed providing metabolic conversion is controlled.

Acidosis↗
PubMed1987

Engineering enzyme specificity by "substrate-assisted catalysis".

A novel approach to engineering enzyme specificity is presented in which a catalytic group from an enzyme is first removed by site-directed mutagenesis causing inactivation. Activity is then partially restored by substrates containing the missing catalytic functional group. Replacement of the catalytic His with Ala in the Bacillus amyloliquefaciens subtilisin gene (the mutant is designated His64Ala) by site-directed mutagenesis reduces the catalytic efficiency (kcat/Km) by a factor of a million when assayed with N-succinyl-L-Phe-L-Ala-L-Ala-L-Phe-p-nitroanilide (sFAAF-pNA). Model building studies showed that a His side chain at the P2 position of a substrate bound at the active site of subtilisin could be virtually superimposed on the catalytic His side chain of this serine protease. Accordingly, the His64Ala mutant hydrolyzes a His P2 substrate (sFAHF-pNA) up to 400 times faster than a homologous Ala P2 or Gln P2 substrate (sFAAF-pNA or sFAQF-pNA) at pH 8.0. In contrast, the wild-type enzyme hydrolyzes these three substrates with similar catalytic efficiencies. Additional data from substrate-dependent pH profiles and hydrolysis of large polypeptides indicate that the His64Ala mutant enzyme can recover partially the function of the lost catalytic histidine from a His P2 side chain on the substrate. Such "substrate-assisted catalysis" provides a new basis for engineering enzymes with very narrow and potentially useful substrate specificities. These studies also suggest a possible functional intermediate in the evolution of the catalytic triad of serine proteases.

Amino Acid Sequence↗