Biomedical subjects
Publications and source records attributed to J Sharpe.
A modified caval preservation technique with the potential for decreased incidence of venous outflow obstruction and haemorrhage.
The selection of patients for resection of colorectal liver metastasis (CRLM) is based around a set of established rules and principles, some of which date back to and have changed little since the mid 1980's. In this paper the authors challenge this accepted doctrine and describe the criteria used for selection of patients for surgery in their own centre, criteria which permit the inclusion of many more patients for potentially curative surgery. They go on to describe methods used to increase resectability and discuss their own results achieved for the resection of CRLM.
In the present study, we evaluated proapoptotic protein Bax on mitochondria and Ca2+ homeostasis in primary cultured astrocytes. We found that recombinant Bax (rBax, 10 and 100 ng/ml) induces a loss in mitochondrial membrane potential (Delta Psi m). This effect might be related to the inhibition of respiratory rates and a partial release of cytochrome c, which may change mitochondrial morphology. The loss of Delta Psi m and a selective permeabilization of mitochondrial membranes contribute to the release of Ca2+ from the mitochondria. This was inhibited by cyclosporin A (5 microM) and Ruthenium Red (1 microg/ml), indicating the involvement of mitochondrial Ca2+ transport mechanisms. Bax-induced mitochondrial Ca2+ release evokes Ca2+ waves and wave propagation between cells. Our results show that Bax induces mitochondrial alteration that affects Ca2+ homeostasis and signaling. These changes show that Ca2+ signals might be correlated with the proapoptotic activities of Bax.
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Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.
The mouse mutants of the hemimelia-luxate group (lx, lu, lst, Dh, Xt, and the more recently identified Hx, Xpl and Rim4; [1] [2] [3] [4] [5]) have in common preaxial polydactyly and longbone abnormalities. Associated with the duplication of digits are changes in the regulation of development of the anterior limb bud resulting in ectopic expression of signalling components such as Sonic hedgehog (Shh) and fibroblast growth factor-4 (Fgf4), but little is known about the molecular causes of this misregulation. We generated, by a transgene insertion event, a new member of this group of mutants, Sasquatch (Ssq), which disrupted aspects of both anteroposterior (AP) and dorsoventral (DV) patterning. The mutant displayed preaxial polydactyly in the hindlimbs of heterozygous embryos, and in both hindlimbs and forelimbs of homozygotes. The Shh, Fgf4, Fgf8, Hoxd12 and Hoxd13 genes were all ectopically expressed in the anterior region of affected limb buds. The insertion site was found to lie close to the Shh locus. Furthermore, expression from the transgene reporter has come under the control of a regulatory element that directs a pattern mirroring the endogenous expression pattern of Shh in limbs. In abnormal limbs, both Shh and the reporter were ectopically induced in the anterior region, whereas in normal limbs the reporter and Shh were restricted to the zone of polarising activity (ZPA). These data strongly suggest that Ssq is caused by direct interference with the cis regulation of the Shh gene.
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The clustered organisation of Hox complexes is highly conserved in vertebrates and the reasons for this are believed to be linked with the regulatory mechanisms governing their expression. In analysis of the Hoxb4-Hoxb6 region of the HoxB complex we identified enhancers which lie in the intergenic region between Hoxb4 and Hoxb5, and which are capable of mediating the correct boundaries of neural and mesodermal expression for Hoxb5. We examined their regulatory properties in the context of the local genomic region spanning the two genes by transgenic analysis, in which each promoter was independently marked with a different reporter, to monitor simultaneously the relative transcriptional read-outs from each gene. Our analysis revealed that within this intergenic region: (i) a limb and a neural enhancer selectively activate Hoxb4 as opposed to Hoxb5; (ii) a separate neural enhancer is able to activate both genes, but expression is dependent upon competition between the two promoters for the enhancer and is influenced by the local genomic context; (iii) mesodermal enhancer activities can be shared between the genes. We found similar types of regulatory interactions between Hoxb5 and Hoxb6. Together these results provide evidence for three separate general mechanisms: selectivity, competition and sharing, that control the balance of cis-regulatory interactions necessary for generating the proper spatial and temporal patterns of Hox gene expression. We suggest that these mechanisms are part of a regulatory basis for maintenance of Hox organisation.
Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.
Thrombospondin is a multifunctional 450 kD glycoprotein which may be secreted into the extracellular matrix by a wide variety of cells. Occasional foci of immunoreactive thrombospondin have previously been demonstrated within normal human glomeruli. A specific polyclonal antibody directed against thrombospondin 1 was used to examine the distribution of this regulatory glycoprotein in renal biopsies from patients with a variety of renal diseases, including rapidly progressive glomerulonephritis associated with circulating antibodies to neutrophils, active or quiescent systemic lupus erythematosus, and membranous nephropathy, together with normal renal tissue. The results demonstrated the marked up-regulation of thrombospondin expression in acutely inflamed renal tissue with strongly positive, predominantly extracellular staining of glomerular crescents, although cytoplasmic staining of epithelial cells was also seen, indicating that these cells may contribute to thrombospondin accumulation at these sites. Occasional segmental mesangial staining was seen in cases of active lupus and rapidly progressive glomerulonephritis, while some focal interstitial staining around peritubular capillaries was seen in all renal tissue examined. These results suggest that thrombospondin may play an important role in the regulation of cellular recruitment, proliferation, and function in crescentic glomerulonephritis.
A comprehensive database has been maintained on patients attending the St. Paul's Hospital Diabetes Teaching and Treatment Centre (DTTC) since 1984. In November 1995, four sets of patients, all of whom had returned to the Centre, were identified for an outcome study. The sets were: insulin-dependent diabetes mellitus (IDDM), diet-treated non-insulin-dependent diabetes mellitus (NIDDM), oral agent-treated NIDDM, insulin-treated NIDDM. Data on glycosylated hemoglobin (A1c) values, percent ideal body weight (%IBW), home blood glucose monitoring (HBGM/week were analysed for all sets; data on hypoglycemic events/month were analysed only for the group with IDDM. Results demonstrated that patients in all groups performed significantly more HBGM over time. Downward change in %IBW in the diet-treated and oral agent groups was significant. Upward change in %IBW was significant in the IDDM group. Hypoglycemic events did not significantly increase in IDDM patients even though A1c improved. Most notably, the A1c values improved in all four groups up to 8 years after the first DTTC visit. Implications for practice are suggested.
We report a case of renal vein occlusion in a transplant kidney that occurred secondary to extrinsic compression from a large kidney being placed extraperitoneally in a small iliac fossa. Prompt reexploration in the immediate postoperative period resulted in salvage of the graft. The abdominal wall was reconstructed using prosthetic mesh, which decreased the compartmental pressure within the iliac fossa sufficiently to allow the kidney to perfuse and the renal vein to remain patent. The patient was eventually discharged home with a functioning graft and normal flow in the vessels, as demonstrated by duplex Doppler studies.
The developing vertebrate hindbrain consists of segments known as rhombomeres, which express combinations of Hox genes implicated in specifying segmental identity. Using chick-chick and chick-transgenic mouse graftings, we show that anterior to posterior rhombomere transpositions result in a progressive posterior transformation and coordinate induction of new Hox expression. This shows that hindbrain plasticity is evolutionarily conserved and implies rhombomeres may be undergoing continual assessment of their identities. The nature of the changes is dependent on both the anteroposterior position of the graft and its origin. Transposed somites from specific axial levels and developmental stages have a graded ability to induce changes in Hox expression, indicating that paraxial mesoderm is a source of the environmental signal responsible for the plasticity.
Four patients are presented, who were initially diagnosed and treated for differentiated thyroid carcinoma, but subsequently discovered to have medullary carcinoma. We suggest that tumour histopathology needs to be carefully reviewed in all cases of thyroid cancer, especially those having atypical clinical or pathological features. This should be completed prior to further therapeutic intervention, such as the administration of ablative radioactive iodine.
The adsorption of serum proteins to calcium phosphate bone substitute materials, positively-charged dextrose and negatively-charged polystyrene microcarrier beads was compared by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Protein adsorption to hydroxyapatite (HA)-based materials was influenced by chemical composition. Surface charge sign, distribution and/or functional group affected protein adsorption to microcarrier beads. Enhanced chemiluminescence was used to quantify adsorption of fibronectin and vitronectin following Western blotting, and to monitor the kinetics of adsorption of these two proteins to HA and Biosilon. Relative to total protein, fluctuating levels of fibronectin were detected on both materials. In contrast, vitronectin adsorption increased over the course of the incubation period with maximal relative adsorption detected after 30 min on Biosilon and 60 min on HA.