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Biomedical subjects

M C Wilkinson

Publications and source records attributed to M C Wilkinson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Human diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase is a member of the MutT family of nucleotide pyrophosphatases.

The cDNA and derived amino acid sequence of human diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase have been determined with the aid of the GenBank Expressed Sequence Tag database. This enzyme possesses a modification of the MutT sequence motif found in certain nucleotide pyrophosphatases. It is unrelated to the enzymes of diadenosine tetraphosphate catabolism found in prokaryotes and fungi.

Amino Acid Sequence↗
PubMed1995

Characterisation of a purified phospholipase A2 from the venom of the Papuan black snake (Pseudechis papuanus).

A neutral phospholipase A2 (PLA2) was separated from Pseudechis papuanus venom by a two-stage FPLC procedure of cation exhange and phenyl-Superose chromatography. It had a molecular mass of 15 kDa and a lower LD50 value than a co-separated haemorrhagic fraction, indicating a higher lethal potency. In vitro tests confirmed the powerful inhibition of platelet aggregation by the PLA2 and strong anticoagulant activity initially observed with whole venom. Ultrastructural studies showed that platelets lost their discoid shape and developed membranous projections with a general decrease in electron-density of the cytosol and disruption of the microfilaments following incubation with the enzyme. Amino acid sequence analysis of the N-terminus and some internal peptides demonstrated a high degree of homology with PLA2s from other Pseudechis venoms. Our results indicate that this fraction is the main agent responsible for the haemostatic disorders in envenomed patients.

Amino Acid Sequence↗
PubMed1995

Urinary tract infection and hip fracture.

A routine audit revealed that in 25 per cent of patients with proximal femoral fracture, hospital stay was complicated by urinary tract infection (UTI). A prospective study was undertaken to investigate the relationship of UTI to fracture type, timing of surgery and the effect of perioperative antibiotics. Eighty-eight patients were investigated over a 4-month period with urine specimens obtained at time of operation and 48 h from operation. Of the patients, all female, 12.5 per cent had positive urine cultures at the time of operation. Of all patients, 42 per cent had positive urine cultures 48 h after operation. Females with intra-capsular fractures were more likely to have positive cultures both pre- and post-operatively (P < 0.005). Age (P < 0.05) and operative delay beyond 48 h (P < 0.05) were also found to predispose to infection. All patients except one who had urinary infection at the time of surgery had post-operative urine infection with the same organism. Present audit methods have significantly under estimated the presence of UTI in these patients. Fracture type and operative delay would appear to be the most significant determinants of a positive urine culture 48 h after operation. Prophylactic antibiotics appear to be ineffective in eradicating pre-existing or preventing early post-operative infection.

Adult↗
PubMed1995

Immunocytochemical distribution of the calcium-binding protein p9Ka in normal rat tissues: variation in the cellular location in different tissues.

The family of S-100-related proteins consists of a number of small potential calcium-binding proteins of unknown function. Elevated expression of one of these proteins, p9Ka, or of its mRNA, correlates with the metastatic potential of cultured mammary epithelial cells from rat or mouse. Over-expression of p9Ka by transfection of benign rat mammary epithelial tumor cells with the gene for p9Ka induces the metastatic phenotype. At present there is little information on the occurrence of p9Ka in normal rat tissues. A specific antiserum immunocytochemically detects p9Ka intracellularly in most normal adult rat tissues studied, including smooth muscle, brown adipose tissue, and liver. In other tissues, p9Ka is localized specifically to some absorptive and keratinized epithelia, the acid-secreting parietal cells of the stomach, the neuronal cells within plexuses of the autonomic nervous system, and a proportion of cells of the immune system in spleen, lymph nodes, bone marrow, and blood. p9Ka is found widely in both arteries and veins, particularly in the smooth muscle and in the endothelium of smaller veins. In mammary gland, the pattern of staining suggests that p9Ka is extracellularly located in a region surrounding the ducts.

Adipose Tissue, Brown↗
PubMed1995

Repair of the common peroneal nerve.

Elective repair of lesions of the common peroneal nerve was carried out in 27 patients between 1982 and 1992. Twenty-three have been reviewed of whom 11 recovered power sufficient to prevent foot drop and 13 recovered protective sensation or better.

Adolescent↗
PubMed1994

Interactions in vitro of p9Ka, the rat S-100-related, metastasis-inducing, calcium-binding protein.

The S-100 proteins are a structurally related family displaying diverse intracellular and extracellular interactions. One such protein p9Ka (also known as calvasculin), or its mRNA (also known as CAPL, 42A, 18A2, mts, pEL 98), becomes elevated upon changes in the growth and differentiation of cells. Overexpression of p9Ka in benign rat mammary cells induces the metastatic phenotype. In order to help understand the role of p9Ka in these processes, the molecular properties of recombinant rat p9Ka have been studied. Recombinant p9Ka forms multimers in vitro, which are not due to intermolecular disulfide bridges, it binds 2 mol of calcium ions/mol of protein, and the binding of calcium ions is strongly antagonized by monovalent and divalent cations tested. Immunofluorescence studies indicate that p9Ka is located on cytoskeletal elements in a pattern which is identical to actin filaments stained with phalloidin. In vitro, it is shown that recombinant p9Ka binds to sites on at least two intracellular polypeptides. These sites display the same binding capacity for p9Ka in extracts of cultured rat mammary cells which show widely differing levels of expression of natural p9Ka. The results suggest that the production of p9Ka, and not of its target molecules, may be associated with the changes seen in cultured cells.

Amino Acid Sequence↗
PubMed1993

Ectopic production of heparin-binding growth factors and receptors for basic fibroblast growth factor by rat mammary epithelial cell lines derived from malignant metastatic tumours.

A rat mammary (Rama) epithelial cell line, Rama 704, derived from normal rat mammary gland does not possess any detectable cell-surface receptors for basic fibroblast growth factor (bFGF), produces a barely detectable level of bFGF mRNA and does not contain detectable levels of bFGF-like activity. Similar results have been obtained with the Rama 37 epithelial cells derived from benign tumours. However, 4 independently isolated epithelial cell lines derived from malignant rat mammary tumours and their metastases possess receptors for bFGF and contain between 2 ng and 9 ng heparin-binding, growth-stimulatory activity per 10(6) cells. The weakly metastatic Rama 600 cells possess high- and low-affinity receptors for bFGF, (KD 20 pM and 8 nM, respectively), while the moderately metastatic Rama 800 cells possess only high-affinity receptors (KD 40 pM). The moderately metastatic C18PLN and 267LU cells, derived from metastases arising from benign Rama 37 cells which had been transfected with DNA from the malignant Rama 800 cells, also possess only high-affinity receptors (KD 36 pM and 80 pM, respectively). Our results show that within the Rama system there is a correlation between the appearance of heparin-binding growth factors and of high-affinity but not low-affinity receptors for bFGF with the malignant phenotype.

Animals↗
PubMed1993

Brachial plexus injury: when to amputate?

Amputation of the upper limb was performed in 20 out of 750 patients with traction lesions of the brachial plexus between August 1969 and June 1991. Urgent amputation was necessary in three patients with irreparable vascular injury and in two more because of overwhelming sepsis. Thirteen patients chose amputation of their flail and useless arm. Two patients have been lost to follow-up. Urgent amputation is indicated when perfusion of the limb cannot be restored, for sepsis, and for a limb which is so severely injured that there can be no prospect for a return of any function. Elective amputation is performed at the patient's request and may be considered as an element of rehabilitation. The pain of preganglionic injury of the brachial plexus is not relieved by amputation.

Accidents, Traffic↗
PubMed1993

Immunocytochemical identification of basic fibroblast growth factor in the developing rat mammary gland: variations in location are dependent on glandular structure and differentiation.

We raised antiserum to human recombinant basic fibroblast growth factor (rbFGF) in rabbits. With this affinity-purified antiserum, other antisera to rbFGF, and commercial antiserum to bovine pituitary bFGF, we undertook immunocytochemical detection of bFGF in histological sections of rat mammary glands at different developmental stages. In non-growing ducts, anti-bFGF serum stains the basement membrane/myoepithelial cells, whereas in serial sections most of this stain is observed to be associated with anti-laminin-staining basement membranes rather than with anti-callus-keratin-staining myoepithelial cells. The weak staining of the myoepithelial cells is enhanced when NiCl2 is included in the detection system, but little staining for bFGF is observed in the epithelial cells. In growing neonatal ducts from 1-day-old rats, in growing terminal end buds (TEBs) and, to a lesser extent, in growing alveolar buds (ABs) in prepubescent (21-day) and pubescent (50-day) rats, both their inner and outer cells are stained moderately by anti-bFGF sera. In non-growing ducts from rats aged 6 days, in non-growing ABs of rats aged 60 days and more, and in alveoli from pregnant and lactating rats, only the basement membrane/myoepithelial cell area is stained by anti-bFGF sera; the epithelial cells are unstained. Staining of the myoepithelial cells is enhanced by mixtures of rbFGF and anti-bFGF sera in non-growing ducts, but there is little change in the staining of growing TEBs. All staining by anti-bFGF sera is abolished with heparin in the reactions. We suggest that the immunoreactive bFGF is present mainly bound to heparan sulfate glycosaminoglycans in the basement membrane of resting structures, but that immunoreactive bFGF becomes associated with proliferating cells, particularly those intermediate in characteristics between epithelial and myoepithelial cells in growing structures such as TEBs.

Animals↗
PubMed1993

The expression of basic fibroblast growth factor and its receptor in cell lines derived from normal human mammary gland and a benign mammary lesion.

mRNA for basic Fibroblast Growth Factor (bFGF) was expressed in a series of SV40-transformed human mammary cell lines as molecules of 7.1, 3.6, 2.0 and 1.2 kb. This expression was much weaker in those lines of epithelial morphology than in myoepithelial-like cell lines derived from them. It was confirmed, using northern hybridization to single-stranded RNA probes, that the multiple mRNAs were transcribed from the coding strand for bFGF. bFGF activity was detected in extracts of the cells and the relative amounts of activity corresponded in general to the amounts of mRNA found. Similar results were obtained from spontaneously transformed cell lines derived from a human benign breast lesion. The presence of bFGF protein in the extracts was confirmed by western blotting, which showed a band of 18-19 kDa, migrating in the same position as authentic bFGF; in addition, the myoepithelial-like cells showed prominent bands of bFGF at 24 and 26 kDa. No FGF receptor was detectable by the binding of 125I-bFGF to the SV40-transformed cell lines or to the epithelial cell lines from the benign breast lesion, but both high- and low-affinity receptors were found on myoepithelial-like cells derived from the latter. The results indicate that differentiation to the human myoepithelial-like phenotype in culture is associated with the enhanced expression of bFGF, and it is suggested that bFGF, immunocytochemically detected in the basement membrane of the human breast, may arise, at least in part, from the myoepithelial cells of the mammary parenchyma.

Breast↗
PubMed1992

Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate.

The structural properties of fibroblast heparan sulfate (HS) that are necessary for it to bind strongly to basic fibroblast growth factor (bFGF) have been investigated using bFGF affinity chromatography. Specific enzymic and chemical scission of HS, together with chemical N-desulfation, revealed that N-sulfate groups and iduronate-2-sulfates (IdoA(2-OSO3)) were essential for the interaction. bFGF-affinity chromatography of sulfated oligosaccharides released from HS by treatment with heparitinase led to the identification of an oligosaccharide component (oligo-H), seven disaccharides in length, with a similar affinity for bFGF as the parent molecule. Heparinase treatment of this fraction abolished the high affinity binding to bFGF. Analysis of oligo-H indicated that 74% of the disaccharide units had the structure IdoA(2-OSO3)alpha 1,4GlcNSO3; the remainder comprised N-acetylated and N-sulfated units, the majority of which were devoid of O-sulfate groups. Oligo-H was fully degraded to disaccharides by treatment with nitrous acid. These results indicate that the sequence of oligo-H is as shown below. delta GlcA beta 1,4GlcNSO3 alpha 1,4[IdoA(2-OSO3)alpha 1,4GlcNSO3]5 alpha 1, 4IdoA alpha 1,4GlcNAc Sulfated oligosaccharides of similar size but with a lower affinity for bFGF had a reduced concentration of IdoA(2-OSO3) but significant quantities of GlcNSO3(6-OSO3) and GlcNAc(6-OSO3). The data indicate a primary role for contiguous sequences of IdoA(2-OSO3)alpha 1,4GlcNSO3 in mediating the high affinity binding between fibroblast HS and bFGF.

Binding Sites↗
PubMed1992

Cement burn of the sciatic nerve.

We present the case of a 63-year-old woman who sustained an acrylic cement burn of the sciatic nerve at hip replacement. She was treated by resection of the damaged segment and grafting. Electron microscopy showed that the nerve was nearly normal 1 cm from the cement margin indicating that this is a safe level for resection.

Amputation, Surgical↗
PubMed1991

Isolation and cDNA cloning of a rat O6-alkylguanine-DNA-alkyltransferase gene, molecular analysis of expression in rat liver.

A rat O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA has been isolated from a rat liver cDNA library by hybridization with the human homologue. The candidate 806 bp cDNA was sequenced and shown to contain a 630 bp open reading frame that could encode a protein of 22.2 kd. Fluorography of labelled ATase indicates a 24 kd protein which is consistent with several previous reports. The derived amino acid sequence demonstrated 81% similarity with the human ATase and 94% identity over a 67 residue region encompassing the putative alkyl acceptor site. Peptide sequences derived from cleaved homogeneous rat ATase have been located in the predicted protein providing additional confirmation of the identity of the cDNA. A 1.05 kb mRNA has been detected in rat liver by Northern analysis; treatment of adult rats with 2-acetylaminofluorene causes an approximately 10-fold induction of this message in liver. Following site directed mutagenesis of the 806 bp cDNA, the 630 bp protein coding sequence has been ligated into an Escherichia coli expression vector to achieve ATase levels of greater than 3% of total protein in bacterial extracts.

2-Acetylaminofluorene↗
PubMed1990

High-level production of human acidic fibroblast growth factor in E. coli cells: inhibition of DNA synthesis in rat mammary fibroblasts at high concentrations of growth factor.

Recombinant human acidic fibroblast growth factor has been produced in E. coli cells at a level of at least 50 mg/l culture. The recombinant and natural acidic fibroblast growth factors are almost identical to one another when tested on rat mammary fibroblasts for their ability to stimulate DNA synthesis, to bind to the high-affinity surface receptors of the cells and to inhibit DNA synthesis when present in the culture medium at high concentrations. The recombinant acidic fibroblast growth factor binds to two cell-surface polypeptides of molecular masses 160 kDa and 140 kDa, which are the same size as the receptors for basic fibroblast growth factor, and it binds preferentially to the smaller polypeptide.

Animals↗
PubMed1990

Purification to apparent homogeneity and partial amino acid sequence of rat liver O6-alkylguanine-DNA-alkyltransferase.

O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.

Amino Acid Sequence↗
PubMed1989

Purification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides.

The E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.

Alkylating Agents↗