Surgical management of an angiomyolipoma extending into the inferior vena cava.
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Angiomyolipoma↗
Biomedical subjects
P Jackson
Publications and source records attributed to P Jackson.
Comparison of labelling by bromodeoxyuridine, MIB-1, and proliferating cell nuclear antigen in gastric mucosal biopsy specimens.
AIMS: To compare proliferating cell nuclear antigen (PCNA) and MIB-1 with bromodeoxyuridine (BrdU) pulse labelling, a specific marker of cell proliferation, in endoscopic gastric biopsy specimens. METHODS: Twenty four biopsy specimens were obtained from 12 patients: 10 antral and eight body specimens were suitable. Each specimen was routinely processed and stained with haematoxylin and eosin. A modified Giemsa stain was used to detect the presence of Helicobacter pylori. Sections of the specimens were labelled with BrdU, MIB-1, and PC10. Gastric mucosa specimens were divided into three zones. The numbers of positively staining nuclei for 500 epithelial cell nuclei were counted in each zone and expressed as a percentage. RESULTS: The proportion of PCNA positive cells (range 0-90%) was much greater in all specimens (10 antrum, eight body). BrdU positive cells were virtually all confined to zone 2 (0-17% cells in this zone were positive) (zone 1 = surface and gastric pit, zone 2 = isthmus, zone 3 = gland base), while PCNA positive cells were present in all three zones (1 = 23-90%, 2 = 43-90%, 3 = 0-74%). Spearman's rank coefficient correlation of 0.57 confirmed that the percentage of positively staining cells varied in the same direction for both PCNA and BrdU (p < 0.001). PCNA, however, was overexpressed in all zones of the gastric epithelium compared with BrdU. In 38 biopsy specimens from 19 patients, of which 14 antrum and 11 body were suitable, the proportion of MIB-1 positive cells (0-59%) was greater than BrdU in most. As with BrdU labelling, the MIB-1 positive cells were confined to zone 2 (zone 1 = 1-11%); zone 2 = 21-59%; zone 3 = 0-13%) and the coefficient correlation for MIB-1 and BrdU was 0.63 (p < 0.001). CONCLUSIONS: MIB-1 accurately reflects the S-phase fraction in gastric mucosa, determined by BrdU labelling in conventionally processed gastric biopsy material. Caution is needed in the interpretation of PCNA labelling detected by PC10, which should not be accepted uncritically as a marker of cell proliferation in paraffin wax embedded material.
Antibodies, Monoclonal↗
Ketoconazole blocks organic osmolyte efflux independently of its effect on arachidonic acid conversion.
Ketoconazole, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, and gossypol are reported inhibitors of the lipoxygenase (LO) and cytochrome P-450 enzyme systems and are potent blockers of swelling-activated efflux of organic osmolytes and volume-sensitive anion channels in C6 glioma cells. To directly test the hypothesis that LO- or cytochrome P-450-derived products of arachidonic acid (AA) participate in the regulation of these volume-sensitive transport pathways, we incubated C6 cells with [1-14C]AA and observed the extent and profile of its conversion under basal conditions and after acute swelling. High-performance liquid chromatographic analysis revealed that most (70-80%) of the labeled AA remained unchanged with only 6-8% and 10-20% of label converted to LO- [12(S)- and 15(S)-hydroxyeicosatetraenoic acid (12- and 15-HETE)] and cyclooxygenase- [prostaglandin (PG) E2 and PGF2a] derived products, respectively. Leukotrienes and epoxyeicosatrienoic acid compounds were not produced. The conversion profile of [1-14C]AA was not altered substantially by cell swelling. Treatment of cells with the LO-derived products 5-, 12-, and 15-HETE or their immediate metabolic precursors, 5(S)-, 12(S)-, and 15(S)-hydroxyperoxyeicosatetraenoic acid, at 5 microM concentrations did not stimulate efflux of [3H]inositol. In addition, treatment with HETEs did not override the inhibition of efflux observed with the LO-cytochrome P-450 blocker ketoconazole. Whole cell patch-clamp experiments demonstrated that volume-sensitive anion channels, the postulated pathway for organic osmolyte efflux in C6 cells, are rapidly and reversibly blocked by ketoconazole in a fashion suggestive of direct inhibition rather than via interruption of a second messenger pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin.
Paxillin and vinculin are cytoskeletal proteins that colocalise to focal adhesions, specialised regions of the cell involved in attachment to the extracellular matrix. These two molecules form part of a complex of proteins that link the actin network to the plasma membrane. Paxillin has been shown to bind directly in vitro to the C-terminal region of vinculin (Turner et al. (1990). J. Cell Biol. 111, 1059-1068), which also contains a focal adhesion targeting sequence (Bendori et al. (1989). J. Cell Biol. 108, 2383-2393). In the present study, we have used a series of vinculin deletion mutants to map more precisely the sites in vinculin responsible for paxillin binding and focal adhesion localisation. A glutathione-S-transferase fusion protein spanning vinculin residues 881-1000 was sufficient to support 125I-paxillin binding in a gel-blot assay while no detectable binding was observed to a fusion protein spanning residues 881-978. Transfection experiments using cDNAs encoding chick vinculin residues 398-1066 and 398-1028 demonstrated that amino acids C-terminal to residue 1028 were not necessary for targeting to focal adhesions. In contrast, a vinculin polypeptide expressed from a cDNA encoding residues 398-1000 failed to localise to focal adhesions in stably transfected NIH3T3 cells. We have therefore identified a region of 50 amino acids (residues 979-1028) within the C-terminal region of vinculin that contains both the paxillin-binding site and the focal adhesion targeting sequence. This region is highly conserved in human and chicken vinculin and is likely to be important in regulation of the assembly of focal adhesions.
3T3 Cells↗
Onchocerciasis in two dogs.
Although widely reported in cattle, horses, and other ungulates, onchocerciasis has only recently been reported in 1 dog. We report 2 additional dogs with onchocerciasis involving the palpebral conjunctiva, third palpebra, and sclera. Both dogs were evaluated because of ocular or periocular masses. Histologically, viable adult parasites were surrounded by minimal fibrosis and few macrophages. Pyogranulomas were often centered around degenerating or mineralized Onchocerca organisms. Onchocerciasis should be considered in the differential diagnosis of ocular or periocular nodules in dogs, particularly dogs from western states.
Animals↗
Two-dimensional polyacrylamide gel electrophoresis of proteins labeled with the fluorophore monobromobimane prior to first-dimensional isoelectric focusing: imaging of the fluorescent protein spot patterns using a cooled charge-coupled device.
A new method for visualizing 2D protein spot patterns is described whereby proteins containing sulfhydryl groups are labeled with the fluorophore monobromobimane prior to the isoelectric focusing step of 2D polyacrylamide gel electrophoresis. The method requires the addition of a single reagent and a delay of only 15 min during sample preparation. High resolution spot patterns were obtained without the need for any post-electrophoretic gel handling. The gel patterns were imaged digitally immediately post-electrophoresis while the gels were still within their glass electrophoresis cassettes. The imaging system was based on a cooled charge-coupled device. The fluorescent spot patterns of proteins from the lymphoid cell-line IM9 were similar but not identical to silver-stained gel spot patterns of the same but underivatized sample. There was an overall increase (approx. 10%) in the number of spots visible on the fluorescent gels compared to those visible on the silver-stained gels.
Bridged Bicyclo Compounds↗
Hormone-conditional transformation by fusion proteins of c-Abl and its transforming variants.
Fusion of the hormone binding domain (HBD) of steroid receptors to transcription factors renders them hormone-dependent. We show here that an SH3-deleted, oncogenic variant of the Abl tyrosine kinase becomes hormone-dependent for transformation by fusion to the estrogen receptor (ER) HBD, extending the phenomenon to tyrosine kinases. Surprisingly, fusion of the HBD to the normal, non-transforming c-Abl (IV) protein activated transforming activity in a hormone-dependent fashion. In the presence of hormone, the c-Abl:ER fusion protein was transforming, cytoplasmic and tyrosine phosphorylated, whereas it was non-transforming, nuclear and hypophosphorylated without hormone. We have examined the kinetics of activation of the c-Abl:ER protein and found that protein synthesis is required both for kinase activation and for redistribution of the c-Abl:ER protein from the nucleus to the cytoplasm. We suggest that the activation of c-Abl could be due to HBD-mediated dimerization and/or to the ability to overexpress conditionally the normally toxic c-Abl protein. This novel approach may be applicable to a wide variety of proteins, particularly when activating mutations or physiological inducers are unknown or when the protein is toxic to cells.
3T3 Cells↗
Kawasaki disease--the Northern Ireland experience.
In the period 1985-1991 inclusive, forty five cases of Kawasaki Disease were identified in Northern Ireland. The number of cases increased each year until 1988 when the incidence was 19 per million population under 16 years, the highest reported for any region in the United Kingdom (data from British Paediatric Surveillance Unit). A high incidence of cardiac involvement was found, with 18(40%) having proximal coronary artery dilatation or aneurysm. and 8(17.7%) with pericardial effusion detected by 2D Echocardiography during or shortly after the acute illness. One year after disease onset, persistent coronary artery changes were detectable by 2D Echocardiography in 53% of those initially affected. There have been no deaths and no patient has so far developed myocardial infarction. One patient has required coronary artery bypass grafting. A protocol for the acute management and long term follow-up of Kawasaki Disease is suggested.
Child↗
Fluorophore-assisted carbohydrate electrophoresis: a new technology for the analysis of glycans.
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Aminoacridines↗
The cytoskeletal protein talin contains at least two distinct vinculin binding domains.
We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2-6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin.
3T3 Cells↗
How to recruit and retain radiologic technologists.
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Adult↗
Wild-type mouse p53 down-regulates transcription from different virus enhancer/promoters.
The protein encoded by the tumour-suppressor gene p53 can complex with SV40 virus large T antigen, the adenovirus E1B 58-kDa protein and the E6 protein of human papillomavirus type 16. The functions of these complexes are unclear, but there is some evidence to suggest that binding of p53 to these viral proteins may inactivate p53 function. Recent reports have shown that p53 is involved in regulation of transcription. We have considered the possibility that p53 may regulate transcription of viral genes important for virus replication and/or transformation. Inactivation of p53 function by formation of such complexes might then permit correct expression of these viral genes. Since p53 can bind to the SV40 virus enhancer/promoter, we have investigated the effect of p53 on transcription from this promoter and report here that mouse p53 is a potent repressor of the SV40 enhancer/promoter. Mutations within p53 severely inhibited this activity and provided some evidence to show that the N-terminus of p53 contains residues essential for this function. We also show that mouse p53 represses transcription from the promoters of viruses that do not express proteins that complex with p53: the human cytomegalovirus early promoter and the Rous sarcoma virus long terminal repeat. By studying the effect of p53 on transcription in different cell lines, we show that the effects of p53 on promoters may be cell type specific.
Animals↗
Mutually exclusive splicing of calcium-binding domain exons in chick alpha-actinin.
We have determined the complete sequence of chick brain alpha-actinin (892 amino acids; 107,644 Da). The sequence differs from that of smooth muscle alpha-actinin only in the region of the first EF-hand calcium-binding motif, where 27 residues in brain alpha-actinin are replaced by just 22 residues in the smooth muscle isoform. This probably accounts for the different calcium sensitivities of the two isoforms with respect to actin binding. Analysis of the gene structure showed that this region of sequence divergence is encoded by two separate exons whose incorporation is mutually exclusive. We have determined the proportion of the two transcripts in various tissues and cell lines using poly(A)+ RNA and a quantitative assay based on the polymerase chain reaction. MRC-5 fibroblasts and HeLa cells express mRNAs encoding both isoforms, whereas Namalwa lymphoblastoid cells, which lack actin stress fibers, express only the non-muscle mRNA. Both isoforms of alpha-actinin became incorporated into stress fibers and cell-matrix junctions when full-length chick alpha-actinin cDNAs were expressed in monkey COS cells. The levels of chick alpha-actinin mRNAs were found to be serum-inducible, suggesting that alpha-actinin may be an early response gene.
Actinin↗
Cytochrome c aided resolution of Lupinus albus isoperoxidases in a cathodal polyacrylamide gel electrophoresis system.
In a cathodal polyacrylamide gel electrophoresis system, three distinct groups of isoperoxidases from Lupinus albus were found to achieve retention factors (rf) dependent on the quantity of sample applied onto the gel. The possibility of extract-derived substances weakly associating with peroxidase samples was investigated. Association of the putative agents survived dialysis against electrophoresis buffer with and without 2 M CaCl2 and freeze-thaw treatments. The addition of polyvinylpolypyrrolidone and polyethylene glycol to the homogenization buffer also proved ineffective in eliminating the variation in isoperoxidase rf although differences in the zymogram profiles of these samples were evident. The addition of spermine and cytochrome c to samples was found to increase the rf of some peroxidase bands. Electrophoresis of samples with cytochrome c resulted in the resolution of peroxidase groups to distinct bands at rf independent of the quantity of peroxidase applied. Control experiments indicate that this treatment did not introduce any detectable artifacts.
Cell Wall↗
Management of patients with severe hypertriglyceridaemia during pregnancy: report of two cases with familial lipoprotein lipase deficiency.
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Adult↗
Further characterisation of the talin-binding site in the cytoskeletal protein vinculin.
The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and alpha-actinin. In this study, we further characterise the talin-binding domain in vinculin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gel-blot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207. Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin. However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathione-agarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1-258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5' portion of the chicken vinculin gene, and by analysing vinculin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.
Amino Acid Sequence↗
Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone: subpicomolar detection using an imaging system based on a cooled charge-coupled device.
Numerous monosaccharides and oligosaccharides were derivatized at their reducing end groups with the fluorophore 2-aminoacridone. The resulting fluorescent compounds were separated by PAGE using two different buffer systems. One of these, a Tris borate buffer, enabled all of the fluorescent saccharide derivatives tested to be electrophoresed and various positional isomers, anomers, and epimers could be separated. The other system consisted of a discontinuous Tris-HCl/Tris-glycine buffer and enabled the electrophoresis of acidic, but not neutral, saccharide derivatives. The acidic and neutral saccharides could thus be distinguished unequivocally. The fluorescent labeling procedure was virtually quantitative and as little as 0.63 pmol could be detected photographically when gels were illuminated by uv light. When gels were viewed using an imaging system based on a cooled charge-coupled device, as little as 0.2 pmol was detected. The method may be useful for the structural analysis of the carbohydrates of glycoconjugates and other naturally occurring oligosaccharides.
Aminoacridines↗