Comparison of a long spinal board and vacuum mattress for spinal immobilisation.

OBJECTIVES: This study was designed to compare the stability and comfort afforded by the long spinal board (backboard) and the vacuum mattress. METHODS: Nine volunteers wearing standardised clothing and rigid neck collars were secured on to a backboard and vacuum mattress using a standard strapping arrangement. An operating department table was used to tilt the volunteers from 45 degrees head up to 45 degrees head down, and additionally 45 degrees laterally. Movements of the head, sternum, and pubic symphysis (pelvis) from a fixed position were then recorded. The comfort level during the procedure was assessed using a 10 point numerical rating scale (NRS) where 1=no pain and 10=worst pain imaginable. RESULTS: The mean body movements in the head up position (23.3 v 6.66 mm), head down (40.89 v 8.33mm), and lateral tilt (18.33 v 4.26mm) were significantly greater on the backboard than on the vacuum mattress (p<0.01 for all planes of movement). Using the NRS the vacuum mattress (mean score=1.88) was significantly more comfortable than the backboard (mean score=5.22) (p<0.01). CONCLUSIONS: In the measured planes the vacuum mattress provides significantly superior stability and comfort than a backboard.

Reversal of P-glycoprotein-mediated multidrug resistance by XR9051, a novel diketopiperazine derivative.

XR9051 (N-(4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydro-2-isoquinolyl)ethyl)phe nyl)-3-((3Z,6Z)-6-benzylidene-1-methyl-2,5-dioxo-3-pipera zinylidene) methylbenzamide) was identified as a potent modulator of P-glycoprotein-mediated multidrug resistance (MDR) following a synthetic chemistry programme based on a natural product lead compound. The activity of XR9051 was determined using a panel of human and murine drug-resistant cell lines (H69/LX4, 2780AD, EMT6/AR 1.0, MC26 and P388/DX Johnson). XR9051 was able to reverse resistance to a variety of cytotoxic drugs, including doxorubicin, etoposide and vincristine, which are associated with classical MDR. At a concentration of 0.3-0.5 microM, XR9051 was able to fully sensitize resistant cells to cytotoxics, whereas little or no effect was observed on the corresponding parental cell lines. No effect of XR9051 was observed on the response of cells to non-MDR cytotoxics such as methotrexate and 5-fluorouracil. XR9051 was consistently more potent than cyclosporin A (CsA) and verapamil (Vpm) in all assays used. XR9051 inhibited the efflux of [3H]daunorubicin from preloaded cells and, unlike CsA and Vpm, remained active for several hours after removal of resistance-modifying agent. In photoaffinity labelling experiments employing [3H]azidopine, XR9051 was able to displace binding to P-glycoprotein. In binding studies using [3H]vinblastine, XR9051 was shown to be a potent inhibitor of the binding of the cytotoxic to P-glycoprotein (EC50 = 1.4 +/- 0.5 nM). Taken together, the results indicate that XR9051 reverses the MDR phenotype through direct interaction with P-glycoprotein.

Structure and methylation-based silencing of a gene (DBCCR1) within a candidate bladder cancer tumor suppressor region at 9q32-q33.

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, indicating the presence of one or more relevant tumor suppressor genes. We previously mapped one of these putative tumor suppressor loci to 9q32-q33 and localized the candidate region within a single YAC 840 kb in size. This locus has been designated DBC1 (for deleted in bladder cancer gene 1). We have identified a novel gene, DBCCR1, in this candidate region by searching for expressed sequence tags (ESTs) that map to YACs spanning the region. Database searching using the entire DBCCR1 cDNA sequence identified several human ESTs and a few homologous mouse. ESTs. However, the predicted 761-amino-acid sequence had no significant homology to known protein sequences. Mutation analysis of the coding region and Southern blot analysis detected neither somatic mutations nor gross genetic alterations in primary TCCs. Although DBCCR1 was expressed in multiple normal human tissues including urothelium, mRNA expression was absent in 5 of 10 (50%) bladder cancer cell lines. Methylation analysis of the CpG island at the 5' region of the gene and the induction of de novo expression by a demethylating agent indicated that this island might be a frequent target for hypermethylation and that hypermethylation-based silencing of the gene occurs in TCC. These findings make DBCCR1 a good candidate for DBC1.

Allelic loss on chromosomes 8p, 22q and 18q (DCC) in human prostate cancer.

Previous studies have suggested the involvement of tumour-suppressor genes on chromosomes 8p, 22q and 18q (DCC) in prostate cancer. The aim of this study was to further characterize these regions. We investigated 20 polymorphic regions on the 3 chromosome arms in 43 cancers and 10 cases of benign prostatic hyperplasia (BPH). Allelic loss was observed in 72% of cancers on 8p, 16% on 22q and 24% at DCC. For BPH, loss was observed in 20% on 8p and in 12% at DCC. The low incidence of LOH on 22q implies that this locus has no significant role in prostate carcinogenesis. At DCC, although the overall incidence was low, tumours with LOH were mostly of high grade or had metastases, suggesting a role for this gene in prostate cancer progression. On chromosome 8p, 29% of cancers had deletions at the LPL locus on 8p22 and 60% had deletions within a region flanked by the markers D8S339 and ANKI on 8p 11.1-p21.1. Within this region, 2 distinct areas of allelic loss were observed, at one or both ANKI and D8S255, and in the region defined by the markers D8S259-D8S505. For the regions 8p22 and ANKI-D8S255, tumours with metastases had a greater frequency of LOH compared to non-metastasizing tumours, suggesting the presence of putative metastasis-suppressor genes in these regions.

Regulation of c-fgr proto-oncogene expression in Burkitt's lymphoma cells: effect of interferon treatment and relationship to EBV status and c-myc mRNA levels.

The proto-oncogene c-fgr is expressed in transformed human B lymphoid cell lines and has been reported to be induced in cells infected with the Epstein-Barr virus (EBV). We have compared the levels of c-fgr mRNA in several B cell lines and have examined the effects of interferon-induced changes in growth rate of Daudi cells on the concentration of this mRNA. High levels were found in exponentially growing EBV-positive Raji and Daudi cells but the amounts in B95-8 and P3HR-1 cells (also EBV-positive) were lower than in the EBV-negative cell line Ramos. Growth inhibition of Daudi cells by interferon-alpha is preceded by a reduction in c-fgr expression, with a 56% decrease observed within 6 h. The differences in the amounts of c-fgr mRNA in the various cell lines and in control versus interferon-treated cells are similar to the differences in the c-myc mRNA contents of these cells. These results indicate that c-fgr expression bears little relationship to the EBV status of B lymphoid cell lines but may play a role, in conjunction with c-myc expression, in growth regulation by interferons. Other conditions which influence Daudi cell proliferation, such as treatment with a phorbol ester or growth to high cell density, also inhibit c-fgr expression but to a lesser extent than interferon treatment.

A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity.

We have studied various factors involved in the optimal use of a tetrazolium (MTT) based colorimetric assay for cell growth and chemosensitivity. The assay is dependent on the ability of viable cells to metabolise a water-soluble tetrazolium salt into a water-insoluble formazan product. We have found that DMSO is the best solvent for dissolving the formazan product, especially where a significant amount of residual medium is left in the wells of the microtitre tray used for the assay. A reaction occurs between medium and a solution of MTT formazan in DMSO which changes the shape of the absorbance spectrum of the solution. The resulting optical density is not however greatly dependent upon the volume of added medium in the range 1-10 microliters. Between 10 and 40 microliters of added medium results in a gradually lower optical density than that produced by the smaller volumes. Above 40 microliters, the optical density increases again due to turbidity as protein precipitation occurs. When cells are incubated with MTT, the resulting optical density of the formazan product is dependent upon both the concentration of MTT and the incubation time. The optical density is stable for several hours after solution of the formazan in DMSO. A linear relationship is seen between optical density and cell number for incubation times of 2, 4, 6 or 24 h with 20 microliters of MTT (5 mg ml-1) added to 200 microliters medium. We have adopted 4 h as the standard incubation time for the assay. Only a small amount of MTT formazan product can be detected in the growth medium of wells in which cells have been exposed to MTT. Comparative chemosensitivity data for EMT6 mouse tumour cells show good agreement between results obtained using the MTT assay and results based on total cell count after a fixed period of growth.

Multiple complexes of thrombin and heparin.

Fluorescence polarization has been used to study the interaction of thrombin and heparin, and the catalysis by heparin of the combination of thrombin and antithrombin. At low ionic strength (20 mM Tris, pH 7.4), the addition of heparins of known molecular weights to thrombin led to the formation of large complexes (defined as 'complex 1'). Further addition of heparin led to a rearrangement of these large complexes to form smaller complexes (defined as 'complex 2'). The molar ratio of thrombin to heparin in complex 1 increased with increasing heparin molecular weight, and corresponded to one thrombin molecule for every heparin segment of Mr 3000. The stoichiometry of complex 2 was 1 heparin to 1 thrombin, irrespective of the heparin molecular weight. At higher ionic strength (150 mM NaCl) some complex 1 was still formed. However, by reversing the titration and adding thrombin to fluorescein-heparin the dissociation constant for complex 2 was estimated to be 1-3 microM and independent of the heparin molecular weight. The complex formed between thrombin and heparin, to which antithrombin was attached, has a dissociation constant of 1-2 microM, again irrespective of the heparin molecular weight. In the heparin-catalysed thrombin-antithrombin reaction, an increase in the size of heparin leads to a lowering of the observed Km for thrombin. A possible explanation is that thrombin, after initial binding to the heparin, moves rapidly to the site where it combines with antithrombin.

Low-resolution structure of the complex of human blood platelet factor 4 with heparin determined by small-angle neutron scattering.

Small-angle neutron scattering was used to confirm that human platelet factor 4 was a compact tetrameric globular protein of radius of gyration 1.74 nm and indistinguishable from a sphere. The same technique, when applied to the 1:1 mol/mol complex of platelet factor and heparin of Mr 14000, revealed that the radius of gyration of the particle varied, depending on the relative proportion of 2H2O to H2O in the solvent. Analysis of this variation by the method of Ibel and Stuhrmann (Ibel, K. and Stuhrmann, H.B. (1975) J. Mol. Biol. 93, 255-266) revealed that in the complex the material of greatest neutron-scattering length (the highly sulphated polysaccharide heparin) was furthest from the centre of the particle. This confirms the postulate of Luscombe and Holbrook (Luscombe, M. and Holbrook, J.J. (1983) in Glycoconjugates (Chester, A.M., Heinegård, D., Lundblad, A. and Svensson, S., eds.), pp. 818-819, Secretariat, Lund) that the exact 1:1 mole ratio of heparin (Mr greater than 10 000) to platelet factor in this stable complex arises from the heparin winding around the outside of a globular protein core.

The catalysis by heparin of the reaction between thrombin and antithrombin.

Fluorescence polarization has been used to study the kinetics of the combination of thrombin with antithrombin and its catalysis by the polysaccharide heparin. The heparin-catalysed combination of thrombin and antithrombin is saturable with respect to both thrombin and antithrombin. The rate-determining step of the reaction is approximately 1.7 s-1. The kinetics observed can be explained by proposing that the catalyst of the reaction is not heparin alone but a complex of heparin and antithrombin (bound at the high-affinity site). The temperature dependence of the heparin-catalysed reaction is indistinguishable from that of the uncatalysed reaction. This coincidence is consistent with the rate-limiting step being the same in both cases.

Immunohistological and biochemical evidence for a role for hyaluronic acid in the growth and development of the placenta.

A monoclonal antibody, designated NDOG1, has been used to stain a series of human and monkey placentae as well as several adult human tissues using immunoperoxidase techniques. In early placentae, NDOG1 was found to stain extracellular material associated with proliferating, extravillous cytotrophoblast cell columns and with the cytotrophoblast shell at the feto-maternal junction. The immunohistology suggests that NDOG1 antigen may be secreted by the anchoring cytotrophoblast into the immediately adjacent maternal tissues. NDOG1 antibody also shows extracellular staining in the stroma of early human placentae and reacted with the apical villous syncytiotrophoblast plasma membrane throughout pregnancy. Biochemical experiments demonstrated that extracts of this latter membrane contained NDOG1 antigenic activity which was susceptible to digestion with bovine testicular hyaluronidase. Hyaluronic acid was the only glycosaminoglycan found in this membrane, thereby implying a reaction between NDOG1 antibody and hyaluronic acid. Whilst no such direct interaction could be demonstrated in vitro, NDOG1 was shown to compete with two other antibodies which themselves demonstrated specificity for hyaluronic acid. The proposed identity between the NDOG1 antigen and hyaluronic acid is discussed particularly in terms of placentation where the distribution of NDOG1 staining may confirm the role of hyaluronic acid in providing an open matrix structure during stages of cell proliferation, migration and invasion.

The interaction of platelet factor 4 with heparins of different chain length.

The interaction of platelet factor 4 with heparin of varying chain lengths has been investigated by labelling the heparin with fluorescein isothiocyanate and monitoring the change in anisotropy of fluorescence when the protein is added to a solution of the polysaccharide. The shape of the titration curve depends on the Mr of the heparin and chains of Mr greater than 10 000 showed a definite break when the concentration of polysaccharide and protein became equimolar. Evidence is presented to show that most of the fluorescein label is linked to residual serines on the heparin. Similar break-points were observed if total fluorescence or light-scattering was used to monitor the interaction. Unlabelled heparin was used for the latter method. These results together with those obtained in buffer of high ionic strength lead us to propose a model where the heparin is wrapped around the tetrameric protein.

The effect of the chain length of heparin on its interaction with lipoprotein lipase.

The interactions of bovine milk lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34) with the glycosaminoglycans heparin and heparan sulphate were investigated using the technique of fluorescence polarization spectroscopy. The type of complex formed with the enzyme depends on the chain length of the heparin. In 0.05 M NaCl and when the heparin was in molar excess, one heparin chain of Mr 10000-18400 formed a very stable complex with the dimeric protein molecule (the 1:1 complex). With excess protein, weaker interactions produced complexes with higher molecular weights. These two classes of complex were also detected with shorter heparins (Mr 6600-8000), although in these circumstances the more stable complex possessed a heparin:protein dimer ratio of 2:1. In higher salt (0.2 M NaCl) and lower heparin concentrations (less than 6 . 10(-8) M) the weaker class of compound was undetectable and Kd values of 4 . 10(-8) M and 6 . 10(-9) M were assigned to the 2:1 and 1:1 complexes, respectively. Heparan sulphate of Mr 17000 could only form one class of complex. This had a 1:1 stoichiometry and with Kd values of 3 . 10(-8) M and 1.6 . 10(-7) M at 0.05 and 0.2 M NaCl, respectively. The results could be explained if there is a distinct binding region for glycosaminoglycans on each subunit of the dimeric enzyme and a single heparin chain of Mr greater than 10000 can satisfy both sites to form a 1:1 complex. Smaller heparin chains are unable to span the sites and, in order to occupy them, two chains must interact with each enzyme molecule.

The effect of bromhexine on experimentally induced diabetic nephropathy.

Diabetes was induced in male Wistar rats by a single i.v. injection of streptozotocin (40 mg/kg). Animals were treated with bromhexine at 2 dose levels (2.5 mg/kg/day and 25 mg/kg/day) for 13 months thereafter and compared to non-diabetic controls and untreated diabetic animals. Renal pathology showed a significant increase in glomerular volume and basement membrane thickening in untreated diabetic animals. The higher dose bromhexine treated diabetic animals showed a significant decrease in glomerular volume as compared with diabetic animals not given bromhexine.

The transfer of platelet factor 4 from its proteoglycan carrier to natural and synthetic polymers.

1. Transfer of dansyl-platelet factor 4 complexed with a series of glycosaminoglycans to heparin has been detected and studied by measuring changes in the anisotropy of the dansyl fluorescence. The protein was most easily transferred from chondroitin sulphate and least easily from heparan sulphate. 2. The transfer of the dye-labelled protein from its biological chondroitin 4-sulphate proteoglycan carrier to natural and synthetic anionic polymers was similarily followed. The transfer to heparin and dermatan sulphate was shown to be the same whether 3 mM Ca2+ or 8 mM EGTA was present in the solution. 3. The shapes of the binding curves of the dansyl-factor to the polymers have been compared at I = 0.4M. 4. The observed changes in anisotropy of dye fluorescence have been correlated with the charge density and the stereochemistry of the charged groups of the natural polymers. Large complexes are observed with polymers of high negative charge/weight ratios. Less charged polymers containing disaccharide units of iduronic acid and glucosamine N-sulphate will also from large complexes at I = 0.15 M. 5. It is demonstrated that the release of a platelet factor 4 proteoglycan complex in vivo would result in the transfer of the protein to heparin, moderate quantities of either dermatan or heparan sulphates would not prevent this transfer.

The multiple complexes formed by the interaction of platelet factor 4 with heparin.

The anisotropy of the fluorescence of dansyl (5-dimethylaminonaphthalene-1- sulphonyl) groups covalently attached to human platelet factor 4 was used to detect the macromolecular compounds formed when the factor was mixed with heparin. At low heparin/protein ratios a very-high-molecular-weight compound (1) was formed that dissociated to give a smaller compound (2) when excess heparin was added. 2. A large complex was also detected as a precipitate that formed at high protein concentrations in chloride buffer. It contained 15.7% (w/w) polysaccharide, equivalent to four or five heparin tetrasaccharide units per protein tetramer. In this complex, more than one molecule of protein binds to each heparin molecule of molecular weight greater than about 6 X 10(3).3. The stability of these complexes varied with pH, salt concentration and the chain length of the heparin. The limit complexes found in excess of the larger heparins consisted of only one heparin molecule per protein tetramer, and the failure to observe complexes with four heparin molecules/protein tetramer is discussed.

Action of degradative enzymes on the light fraction of bovine septa protein polysaccharide.

1. Fragments from enzymic degradation of protein-polysaccharide light fraction (PPL) have been analysed. 2. The time-course of action of some proteolytic enzymes and of hyaluronidase on PPL has been followed by viscometric techniques. 3. It is suggested that papain acts to produce single polysaccharide chains, whereas other proteolytic enzymes tried give evidence of twin-chain residues. 4. The molecular weight of the fragments derived from complete enzyme action on PPL supports this postulate. 5. A structure of the PPL complex is suggested.

The composition and physicochemical properties of bovine nasal-septa protein-polysaccharide complex.

1. Protein-polysaccharide complexes were prepared in three different ways and the gross stoicheiometry of the complexes compared. 2. The neutral sugar content was ascertained and the possibility of a glycoprotein occurring with chondroitin sulphate and keratosulphate is discussed. 3. Physical data support a molecule of molecular weight 3.2x10(6)-5.8x10(6) with a roughly spherical domain and an average radius of gyration of 1390A. Such a particle is highly solvated. The complex is heavily charged with the sulphate groups on the outside. 4. These findings are discussed in the light of the physiological role of protein-polysaccharide light fraction (PPL) in cartilage.