Growth and morphology control of carbon nanotubes at the apexes of pyramidal silicon tips.

We describe the development of catalysed chemical vapour deposition (cCVD) growth schemes suitable for the production of carbon nanotube atomic force microscopy (CNT-AFM) probes. Growth and sample processing conditions are utilized that both incorporate safety in the process, e.g. the use of ethanol (EtOH) vapour as a carbon feedstock and hydrogen at only 4% (flow proportion), and simplicity, e.g. no catalyst patterning is required. Cobalt is employed as the growth catalyst and thin films of aluminium on silicon as the substrate material. Purpose-fabricated silicon substrates containing large numbers of tip structures are used as models of AFM probes. This enables growth to be carried out on many tips at once, facilitating a thorough investigation of the effect of different growth schemes on yields. cCVD growth schemes are chosen which produce stabilizing high density networks of carbon nanotubes on the sidewalls of the pyramidal tips to aid in anchoring the apex protruding carbon nanotube(s) in place. This results in long-lasting AFM imaging tips. We demonstrate that through rational tailoring of cCVD conditions it is possible to tune the growth conditions such that CNTs which protrude straight from tip apexes can be obtained at yields of greater than or equal to 78%. Application of suitable growth schemes to CNT growth on commercially available AFM probes resulted in CNT-AFM probes which were found to be extremely useful for extended lifetime metrological profiling of complex structures.

Preparation and characterization of a magnetic and optical dual-modality molecular probe.

Multi-modality imaging probes combine the advantages of individual imaging techniques to yield highly detailed anatomic and molecular information in living organisms. Herein, we report the synthesis and characterization of a dual-modality nanoprobe that couples the magnetic properties of ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) with the near infrared fluorescence of Cy5.5. The fluorophore is encapsulated in a biocompatible shell of silica surrounding the iron oxide core for a final diameter of approximately 17 nm. This silica-coated iron oxide nanoparticle (SCION) has been analyzed by transmission electron microscopy, dynamic light scattering, and superconducting quantum interference device (SQUID). The particle demonstrates a strong negative surface charge and maintains colloidal stability in the physiological pH range. Magnetic hysteresis analysis confirms superparamagnetic properties that could be manipulated for thermotherapy. The viability of primary human monocytes, T cells, and B cells incubated with the particle has been examined in vitro. In vivo analysis of agent leakage into subcutaneous A431 tumors in mice was also conducted. This particle has been designed for diagnostic application with magnetic resonance and fluorescence imaging, and has future potential to serve as a heat-sensitive targeted drug delivery platform.

Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide.

The N-terminal sequence of a novel sheep-derived peptide with growth inhibitory activity has been obtained. The N-terminal fragment was chemically synthesised and designated EPL001. The kidney was chosen as the first mammalian system in which to study EPL001 since kidney growth can be accurately quantified following a surgical reduction in renal mass. Cell proliferation was measured in mouse collecting duct kidney (MCDK) cells stimulated with insulin-like growth factor I (IGF-I). Compensatory renal growth (CRG) was induced in Wistar rats and either EPL001 or an EPL001 antibody delivered by continuous renal tissue infusion. Mouse monoclonal antibodies to EPL001 were generated for immunoneutralisation, rabbit polyclonal antibodies were generated for immunohistochemistry. EPL001 had no apparent effect on IGF-I stimulated cell proliferation in MCDK cells in vitro, yet provoked a dose-dependent inhibition of CRG in vivo. An EPL001 antibody potentiated CRG, in the absence of exogenous EPL001, consistent with an inhibitory role in kidney growth for an endogenous peptide containing the EPL001 sequence. Tubular staining for epitopes to the EPL001 sequence was detected in normal human kidney sections and enhanced in renal cell carcinoma. Results support the presence of growth inhibitory activity in the N-terminus of a sheep-derived peptide with evidence for both its presence and endogenous activity in the kidney. Attempts to further characterise its structure and activity are ongoing.

Mass spectrometry tools and metabolite-specific databases for molecular identification in metabolomics.

The chemical identification of mass spectrometric signals in metabolomic applications is important to provide conversion of analytical data to biological knowledge about metabolic pathways. The complexity of electrospray mass spectrometric data acquired from a range of samples (serum, urine, yeast intracellular extracts, yeast metabolic footprints, placental tissue metabolic footprints) has been investigated and has defined the frequency of different ion types routinely detected. Although some ion types were expected (protonated and deprotonated peaks, isotope peaks, multiply charged peaks) others were not expected (sodium formate adduct ions). In parallel, the Manchester Metabolomics Database (MMD) has been constructed with data from genome scale metabolic reconstructions, HMDB, KEGG, Lipid Maps, BioCyc and DrugBank to provide knowledge on 42,687 endogenous and exogenous metabolite species. The combination of accurate mass data for a large collection of metabolites, theoretical isotope abundance data and knowledge of the different ion types detected provided a greater number of electrospray mass spectrometric signals which were putatively identified and with greater confidence in the samples studied. To provide definitive identification metabolite-specific mass spectral libraries for UPLC-MS and GC-MS have been constructed for 1,065 commercially available authentic standards. The MMD data are available at http://dbkgroup.org/MMD/.

Constitutive and inducible expression of HLA class II determinants by human osteoblast-like cells in vitro.

Activated immune cells release cytokines which modulate the activity of bone cells in vitro. Expression of major histocompatibility complex (HLA in humans) class II determinants on bone surface cells may be important in local immune cell activation. In this study, expression of HLA-DR and DQ by cultured human bone cells (HBC) derived from normal trabecular bone surfaces was assessed by fluorescence-activated cell sorter (FACS) analysis and immunoperoxidase techniques using monoclonal antibodies. A subset of HBC (10-30%) expressed DR constitutively while 5-15% displayed DQ during long-term culture. HBC lacked a number of monocyte and lymphocyte markers. In addition, both DR+ and DR- HBC (FACS separated) produced osteocalcin stimulated by 1,25-dihydroxyvitamin D2 (1,25(OH)2D3). This suggests that both phenotypes belong to the osteoblast lineage. The number of DR+ HBC was increased by interferon-gamma (IFN gamma; 40-95% DR+ cells) whereas DQ+ HBC remained unchanged or was slightly increased (5-20% DQ+ cells). Moreover, 1,25(OH)2D3 enhanced IFN gamma-induced DR expression and at high concentration (10(-7) M) augmented DR expression by itself. Other major osteotropic factors, parathyroid hormone, interleukin 1, and calcitonin, did not affect HBC DR expression. The findings suggest that HBC may participate in activation of the immune system and that some osteotropic factors may regulate this function.

Intraoperative red blood-cell salvage in revision hip surgery. A case-matched study.

BACKGROUND: Revision hip arthroplasty is commonly associated with substantial blood loss and the subsequent need for transfusion. This leads to an increased risk of blood-borne infection and hemolytic reactions. The purpose of this study was to demonstrate whether the use of intraoperative red blood-cell salvage in revision hip arthroplasty reduces the overall rate of allogeneic transfusion. METHODS: Forty-seven patients who had undergone revision hip arthroplasty with the use of intraoperative cell salvage were identified. A computer database was used to individually match these patients, for age, sex, and eleven operative variables, to control patients who had undergone revision hip arthroplasty in the same unit without intraoperative cell salvage. Data gathered included the total allogeneic transfusion requirement for each patient, preoperative and postoperative hemoglobin levels, and operative time. RESULTS: The total allogeneic transfusion requirement was significantly lower in the group that had intraoperative cell salvage than in the control group (median, 2 compared with 6 U of packed red blood cells, p = 0.0006), with a median reduction in allogeneic transfusion of 4 U. There was no significant difference in preoperative or postoperative hemoglobin levels between the groups. CONCLUSIONS: The use of intraoperative cell salvage significantly lowered the allogeneic transfusion requirement, which can lead to substantial cost savings. To our knowledge, this is the first study in which the use of intraoperative red blood-cell salvage in revision hip arthroplasty was evaluated by matching patients on the basis of age, sex, and operative variables.

National comparative audit of the use of platelet transfusions in the UK.

The objective of this national audit was to examine the use of platelet transfusions against audit standards developed from national guidelines. Hospitals were asked to provide data on 40 consecutive patients receiving platelet transfusions (15 haematology patients, 10 cardiac, 10 critical care and five in other clinical specialties). One hundred and eighty-seven UK hospitals participated, including 168/263 (64%) hospitals in England. A total of 4421 patients receiving platelet transfusions were audited. The reason for transfusion was documented in the medical records for 93% of transfusions and 57% were used for prophylaxis (in the absence of bleeding). Overall 3726/4421 (84%) of the transfusions were evaluable and 43% (1601/3726) were found to be non-compliant with the audit standards. A major non-compliance was failure to measure the platelet count before transfusion (29% of transfusions). Other non-compliances included the use of platelet transfusion in the absence of bleeding in 11% of cardiac surgery patients receiving platelet transfusions, the use of a threshold platelet count more than 10 x 10(9)/L for 60% of prophylactic platelet transfusions in haematology patients without risk factors indicating the need for a higher threshold, and a threshold platelet count more than 30 x 10(9)/L for 59% of prophylactic platelet transfusions in critical care. The reasons for the high rate of non-compliance were not explored in this audit, but this is a topic worthy of further study. The main recommendations were that hospitals should ensure there are written local guidelines for platelet transfusions, clinicians must be provided with training about their appropriate use, and hospitals should carry out regular audits of practice. More research should be carried out to develop the evidence base for the use of platelet transfusions, more detailed guidelines should be developed for platelet transfusions in critical care and cardiac surgery, and the audit should be repeated in about three years.

Repair of ionizing radiation DNA base damage in ataxia-telangiectasia cells.

Micrococcus luteus endonuclease sensitive sites were measured by alkaline elution in normal human and ataxia-telangiectasia (AT) fibroblasts after ionizing radiation. Due to the sensitivity of this assay, repair of base damage after 3 to 6 kilorads has been measured after oxic or hypoxic radiation. With 5.5 kilorads of oxic radiation, more than 50% of the base damage was removed after 1.5 h of repair incubation in all cells, including exr+ and exr- AT cells, and approximately 75% was removed by 4 h. After 3 or 4.5 kilorads of hypoxic X-irradiation, repair was equivalent in normal and exr- AT cells. This study included three exr- AT strains which have been reported to be deficient in the removal of gamma-ray base damage at higher doses. Since these strains repaired ionizing radiation base damage normally at lower doses, which are more relevant to survival, it is concluded that the X-ray hypersensitivity of AT cells is probably not related to the repair of base damage.

Inhibition of acyltransferase in lymphocytes by concanavalin A.

The effects of concanavalin A and succinylated concanavalin A on the transformation of mouse splenic lymphocytes, and on early biochemical events in the transformation, were compared. 1. The transformation of lymphocytes is biphasic with respect to concanavalin A concentration with optimal activation at about 1 microgram/ml. Activation by succinyl concanavalin A is not biphasic over a range of lectin concentration of 1--16 microgram/ml. 2. In intact lymphocytes cultured for 4 h, the enzyme Acyl-CoA:1-acylglycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23) was not activated by concanavalin A but was inhibited at all concentrations tested, and was about 60% inhibited at 16 micrograms concanavalin A per ml. Succinyl concanavalin A gave little or no inhibition at similar concentrations. 3. Lymphocytes become committed to divide while their acyltransferase activities are markedly inhibited by concanavalin A. 4. The inhibition of acyltransferase by concanavalin A can be lifted by displacing the lectin from the cells by alpha-methylmannoside. Lowered enzyme activity is not caused by cell agglutination or by direct cross-linking of lectin receptors. It is unlikely that the inhibition of acyltransferase is due to indirect cross-linking via the cytoskeleton since colchicine did not reverse the inhibition. 5. The inhibition of acyltransferase and the reduced stimulation of transformation by higher levels of concanavalin A appear to be due to hydrophobic interaction of the lectin with the plasma membrane, as shown by liposome aggregation studies.

The mitogenic action of recombinant basic FGF in Swiss 3T3 cells is independent of early diradylglycerol production and downregulatable protein kinase C activity.

In this study we have investigated the requirement for phosphoinositide metabolism, diradylglycerol (DG) production and protein kinase C (PKC) activation in recombinant basic fibroblast growth factor (rbFGF)-mediated reinitiation of DNA synthesis in Swiss 3T3 cells. We have assessed the involvement of PKC activation in rbFGF-induced DNA synthesis by two approaches; enzymic inhibition by H7 and down-regulation by prolonged phorbol-ester treatment. In both conditions we observed that rbFGF was able to sustain a significant component of its mitogenic response, therefore denying an exclusive role for the activation of downregulatable and H7-sensitive PKC isoforms in rbFGF-induced reinitiation of DNA synthesis. Moreover, we have found no evidence for diacylglycerol accumulation in response to rbFGF by 3T3 cells. In previous studies, we observed that rbFGF caused a moderate and slow accumulation of total inositol phosphates. This effect was significant only after a 60 min incubation. It is our contention that rbFGF, in our culture system, does not exert a direct effect on phosphoinositide metabolism.

Stimulation of arachidonic-acid release from Swiss 3T3 cells by recombinant basic fibroblast growth factor: independence from phosphoinositide turnover.

In this study we have attempted to characterize the mechanism of recombinant bovine basic fibroblast growth factor (rbFGF)-induced release of arachidonic acid from prelabelled Swiss 3T3 fibroblasts. Recombinant bFGF caused the release of [3H]arachidonic acid from metabolically labelled cells in a dose- and time-dependent manner. This effect was maximal with 10 ng rbFGF/ml and became significant after a 30-min incubation. Although rbFGF was able to cause a modest increase in total inositol phosphate accumulation, an examination of the time-course of the latter effect revealed that enhanced [3H]arachidonic-acid release could not have been derived from phosphoinositide metabolism. Evidence suggesting that rbFGF-induced release of [3H]arachidonic acid was being mediated via a PLA2 pathway was obtained by pharmacological antagonism using mepacrine, a putative PLA2 inhibitor. Moreover, treatment of cells with neomycin failed to attenuate rbFGF-mediated release of [3H]arachidonic acid. Chelation of extracellular calcium by EGTA was found to abrogate rbFGF-induced liberation of [3H]arachidonic add. Down-regulation of protein kinase C (PKC) by prolonged treatment of cells with the phorbol ester, PMA, was observed to have no effect on the action of rbFGF on [3H]arachidonic add release from Swiss 3T3 fibroblasts. While rbFGF was found to cause the indomethacin-sensitive production of prostaglandin E2 (PGE2) in a dose-dependent manner, this effect was independent of rbFGF-induced reinitiation of DNA synthesis. Clearly, the effect of rbFGF on cellular DNA synthesis was being mediated independently of PGE2 biosynthesis. We discuss the potential importance of the PLA2-signalling pathway in the mechanism of action of fibroblast growth factors.

Muscarinic acetylcholine receptor activation causes inhibition of cyclic AMP accumulation, prolactin and growth hormone secretion in GH3 rat anterior pituitary tumour cells.

Acetylcholine, oxotremorine and carbachol, compounds that exhibit muscarinic agonist activity, maximally inhibited basal prolactin secretion from GH3 cells by approx. 50% and intracellular cyclic AMP levels by approx. 20%. Both parameters were inhibited with similar potencies by each agonist. These inhibitory effects were blocked by a muscarinic but not by a nicotinic receptor antagonist. In the presence of VIP or IBMX, which raise intracellular cyclic AMP levels and stimulate hormone release, the degree of muscarinic inhibition was increased, but the potency remained unchanged. Similar changes in the secretory rate of prolactin and growth hormone occurred in these and in cell perifusion experiments. These results suggest that the inhibition of hormone secretion from GH3 cells by muscarinic agonists is mediated by a decrease in intracellular cyclic AMP levels.

Interactive regulation of signalling pathways in bone cells: possible modulation of PGE2-stimulated adenylyl cyclase activity by protein kinase C.

We have reported previously that tumour-promoting phorbol esters modulate both basal and vasoactive intestinal polypeptide (VIP)-stimulated adenylyl cyclase activity in GH3 (an established pituitary cell line). Here, we probe the receptor and cell specificity of this response. Experiments were performed in the presence of isobutylmethylxanthine. Unlike the response in GH3 cells, the tumour-promoting phorbol ester (tetradecanoyl phorbol acetate (TPA] did not affect either basal adenylyl cyclase activity nor VIP-stimulated activity in the rat osteosarcoma subclones UMR 106-01 and UMR 106-06. In addition, the cyclase responses to parathyroid hormone (PTH), and, in the case of UMR 106-06, to calcitonin were unaffected by tumour-promoting phorbol ester. However, prostaglandin E2-stimulated cyclase activity in both of these subclones was attenuated in a dose-dependent manner.

Bradykinin stimulates the production of prostaglandin E2 and interleukin-6 in human osteoblast-like cells.

The effect of bradykinin (BK) on proteinase activity, prostaglandin synthesis, and the production of interleukin-6 (IL-6) was investigated in cultures of human osteoblast-like cells. Bradykinin had no effect on stromelysin activity and plasminogen activator activity produced by human osteoblast-like cells. However, BK stimulated the production of prostaglandin E2, an effect that was markedly enhanced by pre-incubation with recombinant interleukin-1 alpha (rhIL-1 alpha), but was apparently unaffected by BK receptor antagonists types 1 and 2. Bradykinin stimulated the intracellular accumulation of total inositol phosphates suggesting that its effects were mediated by stimulation of phosphoinositide metabolism. Bradykinin within the dose range of 10(-11)-10(-5) M also significantly stimulated the production of IL-6. Bradykinin may, therefore, mediate a variety of responses in bone under both physiological and pathological conditions.

Anthocyanins from red wine--their stability under simulated gastrointestinal digestion.

The stability of anthocyanins from red wine was assessed using an in vitro digestion system that simulated the physiochemical changes that occur in the upper gastrointestinal tract. Anthocyanins in red wine were stable to gastric conditions whereas there was a small loss in total phenol content. After pancreatic digestion, the total anthocyanins were very poorly recovered compared to the bulk phenols in the IN sample, which was previously described as the "serum-available" fraction, and the majority of the anthocyanins and phenols were recovered in the OUT fraction, previously described as the "colon-available" fraction. Removing alcohol from the wine samples prior to the procedure did not markedly affect this pattern. The composition of anthocyanins in the post gastric, IN and OUT samples was analysed using liquid chromatography mass-spectrometry. The red wine used contained over 20 identifiable anthocyanins of which the main components were 3-O-glucosides of malvidin, peonidin, petundin, delphidin and cyanidin. Coumaroylated-glucoside derivatives of malvidin, petundin, peonidin, and delphinidin were observed and acetylated glucosides of peonidin, petundin and malvidin were also identified. Anthocyanins with modified aglycones similar to vitisin A derivatives of delphinidin, peonidin, petunidin and malvidin were also identified. After the in vitro digestion procedure, only five anthocyanins could be detected in the IN (serum-available) and the OUT (colon-available) fractions, which were confirmed as malvidin-3-O-glucoside and the vitisin A adducts of malvidin-3-O-glucoside, malvidin-3-O-acetylglucoside, malvidin-3-O-coumaroylglucoside and peonidin-3-O-glucoside. Malvidin-3-O-glucoside was recovered at 0.2% in the IN fraction and 0.9% in the OUT fraction. However, the vitisin derivatives were much more stable to pancreatic digestion. Assuming that the vitisin A derivatives display similar biological properties to their parent anthocyanins, their enhanced gastrointestinal stability could lead to enhanced bioavailability and bio-effectiveness in vivo.

Caspase-3-like activity is necessary but not sufficient for daunorubicin-induced apoptosis in Jurkat human lymphoblastic leukemia cells.

In the present study we have shown that the cancer therapeutic drug, daunorubicin, induces apoptosis in the human lymphoblastic leukemia cell line Jurkat E6.1. This effect was both dose-and time-dependent with nuclear fragmentation detectable by 8 h. Caspases have been implicated in pro-apoptotic events. By utilizing synthetic fluorochrome-linked substrates of the caspases, we observed that a caspase-3-like enzyme had dramatically increased activity (3340 130% with respect to basal levels) in response to daunorubicin treatment. Furthermore, by using an inhibitor to caspase-3, Ac-DEVD-CHO, we have shown that activation of a caspase-3-like enzyme appears to be necessary for nuclear fragmentation and apoptotic body formation, but is not required for chromatin condensation. In contrast, a general caspase inhibitor, Z-VAD-fmk, inhibited all apoptotic parameters measured. Ceramide has been implicated in daunorubicin-induced apoptosis in human myeloid leukemia cells. However, in Jurkat cells, caspase activation does not appear to be a consequence of ceramide generation since, although ceramide levels were elevated through the action of ceramide synthase in response to daunorubicin treatment, this occurred with slower kinetics than either nuclear fragmentation or caspase activation. In contrast, caspase inhibitors abrogated ceramide elevation induced by DNR treatment, suggesting that ceramide synthase may be a downstream target for caspase action. Therefore, daunorubicin-induced apoptosis does not appear to be mediated by ceramide in the lymphoblastic leukemia cell line, Jurkat E6.1. Instead, caspase 3 activity appears to be necessary, but not sufficient for this process.

A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein.

The GH receptor (GHR) is a member of the cytokine receptor family. Short isoforms resulting from alternative splicing have been reported for a number of proteins in this family. RT-PCR experiments, in human liver and cultured IM-9 cells, using primers in exon 7 and 10 of the GHR, revealed three bands reflecting alternative splicing of GHR mRNA: the predicted product at 453 bp and two other products at 427 and 383 bp. The 427-bp product (GHR1-279) utilized an alternative 3'-acceptor splice site 26 bp downstream in exon 9; the predicted C-terminal residues are six frameshifted exon 9 codons ending in an inframe stop codon. The 383-bp product (GHR1-277) resulted from skipping of exon 9; the predicted C-terminal residues are three frame-shifted exon 10 codons ending in an in-frame stop codon. RNase protection experiments confirmed the presence of the GHR1-279 variant in IM-9 cells and human liver. The proportion of alternative splice to full length was 1-10% for GHR1-279 and less than 1% for GHR1-277. The function of GHR1-279 was examined after subcloning in an expression vector and transient transfection in 293 cells. Scatchard analysis of competition curves for [125l]-hGH bound to cells transfected either with GHR full length (GHRfl) or GHR1-279 revealed a 2-fold reduced affinity and 6-fold increased number of binding sites for GHR1-279. The increased expression of GHR1-279 was confirmed by cross-linking studies. The media of cells transfected with GHR1-279 contained 20-fold more GH-binding protein (GHBP) than that found in the media of cells transfected with the full-length receptor. Immunoprecipitation and Western blotting experiments, using a combination of antibodies directed against extracellular and intracellular GHR epitopes, demonstrated that GHRfl and GHR1-279 can form heterodimers and that the two forms also generate a 60-kDa GHBP similar in size to the GHBP in human serum. Functional tests using a reporter gene, containing Stat5-binding elements, confirmed that while the variant form was inactive by itself, it could inhibit the function of the full-length receptor. We have demonstrated the presence of a splice variant of the GHR in human liver encoding a short form of the receptor similar in size to a protein previously identified in human liver and choroid plexus. Expression studies in 293 cells support the hypothesis that while the expression of the splice variant accounts for only a small proportion of the total GHR transcript, it produces a short isoform that modulates the function of the full-length receptor, inhibits signaling, and generates large amounts of GHBP. The differential expression of GHR receptor short forms may regulate the production of GHBP, and truncated receptors may act as transport proteins or negative regulators of GHR signaling.

An isolated osteochondroma with underlying non-Hodgkin's lymphoma of bone.

Osteochondroma is the most common benign bone tumour. The risk of sarcomatous change in an isolated lesion is approximately 1%. We report a case of an isolated osteochondroma which appeared benign on clinical and plain radiographic examination but routine histological analysis revealed non-Hodgkin's lymphoma in the underlying bone. This association has not previously been reported and the case emphasises the importance of routine histological analysis, even if a lesion appears benign.