M. James C. Crabbe

The emzymatic ring expansion of penicillins to cephalosporins : side chain specificity

Abstract Structural variants of the acylamino side chain of penicillins have been tested as substrates for Deacetoxy/Deacetyl Cephalosporin C Synthetase from C.acremonium CO 728. A six carbon chain terminating in a carboxyl group was found to permit efficient ring expansion to cephems, with the exception of δ-( L -α-aminoadipoyl).1

A spectrophotometric assay for deacetoxycephalosporin C synthase

A continuous direct spectrophotometric assay for deacetoxycephalosporin C synthase was developed, based on the absorption at 260 nm characteristic of the dihydrothiazine moiety of cephalosporins. K m values of 0.18 mM for penicillin N and 0.16 mM for α‐ketoglutarate were determined. A coupled assay using succinate thiokinase, pyruvate kinase and lactate dehydrogenase showed that succinate was a product of both deacetoxycephalosporin C synthase and hydroxylase reactions. The expandase reaction exhibited a 1:1.06 stoichiometry for deacetoxycephalosporin C and succinate.

Distribution-free computer methods for analysing ligand binding and enzyme mechanisms

BASIC computer programs have been designed to calculate enzyme kinetic and ligand binding parameters using distribution-free methods. Statistical estimates for the parameters can be calculated, and models distinguished by the F-test. Integrated rate equations are derived for complex enzyme mechanisms.

Enzymatic ring expansion of penicillins to cephalosporins: side chain specificity

Structural variants on the acylamino-side chains of penicillins as substrates for the ring expansion enzyme from Cephalosporium acremonium CO728 show that a six carbon chain terminating in a carboxy group permits efficient conversion into cephems with the exception of δ-(L-α-aminoadipoly)[5-(5S)-amino-5-carboxypentanoyl].

Microcomputer analysis of hyperbolic and non-hyperbolic steady-state kinetics

A BASIC computer program has been developed which has been used to show that bovine lens aldose reductase with NADPH as substrate follows a 1:1 function, while rabbit lens hexokinase has a rate equation of minimum degree 2:2, and bovine lens polyol dehydrogenase has a rate equation of minimum degree 1:2 with xylitol as substrate. The parameter estimates obtained are very close to those from the BMDP3R curvefitting program on an ICL 2980 mainframe computer, with identical conclusions as to the minimum degree of the rate equation. The computer program can be run on any microcomputer with high resolution graphics in less than 48 K of random access memory.

The penetration of Sorbinil, an aldose reductase inhibitor, into lens, aqueous humour and erythrocytes of patients undergoing cataract extraction

Two studies to investigate the penetration of the aldose reductase inhibitor, Sorbinil, were conducted. In the first study, 24 diabetic patients undergoing intracapsular extraction were randomised into three groups on a double masked basis. In the week immediately preceding the operation, all patients were requested to take two capsules daily before breakfast. Each capsule contained 200 mg Sorbinil, 100 mg Sorbinil, or a placebo. On measuring Sorbinil levels in lens, plasma and erythrocytes using HPLC, three clearly defined groups of patients were obtained. In one group no Sorbinil was detected, in the second group there were moderate levels of Sorbinil, while the third group had significantly higher levels of Sorbinil. The ratio of erythrocyte/plasma Sorbinil was 0.225, while the ratio for lens/plasma was 0.7, for both groups where Sorbinil was detected. In a second study, 20 patients were treated topically with a single dose of 0.5 mg ophthalmic Sorbinil at times ranging from 0-14 hr preoperatively. Sorbinil was detected in both lens and aqueous. Transport into the lens was complete within about 2 hr, and although aqueous levels were negligible after 6 hr, Sorbinil persisted up to 14 hr in the lens. Three out of 16 patients taking Sorbinil orally developed a maculopapular rash with pyrexia approximately 8 days after commencing the drug. No side effects were noted in any patients given the topical ophthalmic preparation.

Collagen crosslinking: Isolation of a dimeric crosslinked peptide of α1-CB6 from bovine corneal and scleral collagens

The pattern of CNBr fragments of various collagens on SDS-polyacrylamide electrophoresis gels includes a major band with mobility intermediate between a2-CB(3-5) and a2-CB4 which has been called (Al-CB(3-7) and cxl-CB(S-8-3) on the basis of molecular weights from the gels [ 1,2]. We have noted a similar component which we will call component X, in gel patterns of CNBr fragments from unreduced corneal collagen [3], reduced cornea1 collagen and reduced scleral collagen. Our CNBr digestion conditions are harsher than those most used and we find no methionine in the digest; also we have not isolated any uncleaved fragments [3], so its seems unlikely that this major component could be an uncleaved peptide as has been suggested. Furthermore oil-CB3, part of both the proposed uncleaved peptides, is readily isolated in high yield (<60%) from CNBr digests of both cornea1 and scleral collagen (J .J.H., N. A. Panjwani, M.J.C.C., unpublished results). In the present work we have isolated component X from CNBr digests of cornea1 and scleral collagen and identified it as a dimer of al-CB6.

Partial sequence homology of human myc oncogene protein to beta and gamma crystallins

The human cellular myc gene is one of about 20 cellular oncogenes which code for a variety of proteins including protein kinases and growth factors [1]. The human gene is related to the avian myelocytomatosis leukaemia virus MC29 [2] and produces a binding protein which may be involved in regulation of gene expression [3] and cellular differentiation and proliferation [4]. The crystallins are proteins in the eye lens synthesised at different stages of cell differentiation and proliferation, and whose short range order is necessary for lens transparency [5,6]. Computer‐based sequence comparisons show that beta Bp and gamma II crystallins, which show partial sequence homology and conservation of ‘Greek Key’ motives [7,8] are also partially homologous to two regions on the human myc protein, though this protein probably does not conserve the ‘Greek Key’ structural motives.

Collagen crosslinking: isolation of two crosslinked peptides involving α2-CB(3–5) from bovine scleral collagen

Collagen, the major structural protein m the eye, as elsewhere, 1s responsible for givmg the eye its unique and functional shape. Collagenous tissues are strengthened by the covalent crosslinks between the collagen molecules. The approximate positions of several crosslinks are known [ 11. In degenerative myopia, the sclera appears thmned with loss of organisatron m the scleral fibres. This change m fibrillar structure may be due to changes m collagen crosshnkmg and we have found two crosslinked peptrdes m mature bovine scleral collagen after reduction with trrtiated potassmm borohydride and cleavage with cyanogen bromide. These crosslmked peptides both involve the peptrde a2CB (3-5) the largest cyanogen bromide fragment, and are mtermolecular. The first IS a drmer of fragment a2CB (3-5) while the second consists of fragment (w2CB (3-S) lamed to one or more small peptides, similar to the crosslink found m bovme cornea [I].

Enrichment analysis of Alu elements with different spatial chromatin proximity in the human genome

ABSTRACTTransposable elements (TEs) have no longer been totally considered as “junk DNA” for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C (chromosome conformation capture) have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: rxa0=xa00.9, P < 2.2 × 1016; IMR90 fibroblasts: rxa0=xa00.94, P < 2.2 × 1016) and also have a significant positive correlation with some remote functional DNA elements like enhancers and promoters (Enhancer: hESC: rxa0=xa00.997, Pxa0=xa02.3 × 10−4; IMR90: rxa0=xa00.934, Pxa0=xa02 × 10−2; Promoter: hESC: rxa0=xa00.995, Pxa0=xa03.8 × 10−4; IMR90: rxa0=xa00.996, Pxa0=xa03.2 × 10−4). Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue-specific genes.