Biomechanical analysis of practitioner's gesture for peripheral venous catheter insertion.

Peripheral venous catheter insertion (PVCI) is one of the most common procedures performed by healthcare professionals but remains technically difficult. To develop new medical simulators with better representativeness of the human forearm, an experimental study was performed to collect data related to the puncturing of human skin and a vein in the antebrachial area. A total of 31 volunteers participated in this study. Force sensors and digital image correlation were used to measure the force during the palpation and puncturing of the vein and to retrieve the kinematics of the practitioner's gesture. The in vivo skin rupture load, vein rupture load, and friction loads for skin only and for both the skin and vein were (mean ± standard deviation) 0.85 ± 0.34 N, 1.25 ± 0.37 N, -0.49 ± 0.19 N, and -0.51 ± 0.16 N, respectively. The results of this study can be used to develop realistic skin and vein substitutes and mechanically assess them by reproducing the practitioner's gesture in a controlled fashion.

Nasal immunization with subunit proteosome influenza vaccines induces serum HAI, mucosal IgA and protection against influenza challenge.

The immunogenicity of a mucosally delivered subunit influenza vaccine was assessed in mice. Split influenza virus vaccine (sFlu) was formulated with proteosomes (Pr-sFlu), administered intranasally, and the induced immunity was compared with the responses elicited by sFlu alone given either intramuscularly or intranasally. Intranasal (i.n.) immunization with Pr-sFlu induced specific serum IgG and hemagglutination inhibition (HAI) titers comparable to or better than those induced by intramuscular (i.m.) sFlu, and in contrast to sFlu alone, i.n. Pr-sFlu also induced high levels of influenza-specific IgA in lung and nasal washes. Mice receiving i.n. Pr-sFlu were completely protected against live virus challenge, as were mice immunized by injection with sFlu alone. The i.n. Pr-sFlu elicited cytokine responses polarized towards a type 1 phenotype whereas those elicited by sFlu alone were of a mixed type 1/type 2 phenotype. The data strongly suggest that i.n. proteosome-formulated influenza antigens are highly effective and are excellent candidates for a non-invasive human vaccine.

The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.

2,2'-Dithiobis(N-ethyl-spermine-5-carboxamide) is a high affinity, membrane-impermeant antagonist of the mammalian polyamine transport system.

We have synthesized 2,2'-dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), its thiol monomer (MESC), and the mixed MESC-cysteamine disulfide (DEASC) as potential inhibitors of polyamine transport in mammalian cells. DESC was the most potent antagonist of spermine transport in ZR-75-1 human breast cancer cells, with Ki values of 5. 0 +/- 0.7, 80 +/- 31, and 16 +/- 3 microM for DESC, MESC, and DEASC, respectively. DESC also strongly blocked putrescine and spermidine uptake in ZR-75-1 cells (Ki = 1.6 +/- 0.5 and 2.7 +/- 1.1 microM, respectively). While DESC and MESC were purely competitive inhibitors of putrescine transport, DEASC was a mixed competitive/noncompetitive antagonist. Remarkably, DESC was virtually impermeant in ZR-75-1 cells despite its low Ki toward polyamine transport. The marked difference in affinity between DESC and MESC was essentially due to the tail-to-tail juxtaposition of two spermine-like structures, suggesting that dimeric ligands of the polyamine transporter might simultaneously interact with more than one binding site. While DESC strongly decreased the initial rate of [3H]spermidine transport, even a 40-fold molar excess of antagonist could not completely abolish intracellular spermidine accumulation. Moreover, as little as 0.3 microM spermidine fully restored growth in ZR-75-1 cells treated with an inhibitor of polyamine biosynthesis in the presence of 50 microM DESC, thus emphasizing the importance of uptake of trace amounts of exogenous polyamines. Thus, reducing the exogenous supply of polyamines with a potent competitive inhibitor may be kinetically inadequate to block replenishment of the polyamine pool in polyamine-depleted tumor cells that display high transport capacity. These results demonstrate that polyamine analogues cross-linked into a dimeric structure such as DESC interact with high affinity with the mammalian polyamine carrier without being used as substrates. These novel properties provide a framework for the design of specific irreversible inhibitors of the polyamine transporter, which should present advantages over competitive antagonists for an efficient blockade of polyamine transport in tumor cells.

Substrate protection against inactivation of the mammalian polyamine-transport system by 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide.

Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11 +/- 4 and 96 +/- 16 microM after 30 min at 22 degrees C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and K(m) for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 degrees C respectively. Spermine (10 microM) almost fully protected against spermidine-transport inactivation by EDC at 22 degrees C, and decreased the rate of inactivation at 37 degrees C by about 80%. Putrescine, spermidine and spermine were all effective in protecting against EDC-mediated inactivation of [3H]spermidine and [3H]putrescine uptake at 22 degrees C with EC50 values estimated at 10, 1 and less than 1 microM respectively. The nucleophile glycine ethyl ester (up to 50 mM) prevented the inhibition brought about by 1 mM EDC. Inhibition by 1 mM EDC was greater at pH 7.2 than at pH 5.8 (89 +/- 3 compared with 44 +/- 5%), whereas the converse was true for 100 microM DCC (81 +/- 3 compared with 92 +/- 5%). On the other hand, spermine did not protect against inactivation of spermidine uptake by DCC. Moreover, DCC, but not EDC, inhibited Na(+)-dependent amino acid uptake. The present data indicate that (i) EDC and DCC inhibit polyamine transport through distinct mechanisms, (ii) substrate binding occludes one or several carboxy groups lying in a polar environment of the carrier and (iii) these carboxyl residues might be activated by EDC to crosslink a neighbouring nucleophile side group, resulting in a conformation of the polyamine carrier which is inactive for transport.

Tyrosine 1356 in the carboxyl-terminal tail of the HGF/SF receptor is essential for the transduction of signals for cell motility and morphogenesis.

The met proto-oncogene is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF is a multifunctional cytokine that stimulates mitogenesis, motility, invasion, and tubulogenesis of a spectrum of epithelial and endothelial cells in culture. Using a chimeric receptor (CSF-MET), containing the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor fused to the transmembrane and intracellular domain of the Met receptor, we have previously demonstrated that activation of the Met kinase domain is sufficient to mediate the motility, invasion and morphogenic signals of HGF/SF in Madin-Darby canine kidney epithelial cells (MDCK). In this study we have analyzed the role of tyrosine phosphorylation of the Met receptor in the transmission of these signals by site-directed mutagenesis of specific tyrosine residues. Mutation of two tyrosine residues (tyrosine 1234 and tyrosine 1235), involved in activation of the catalytic activity of the kinase, abrogates the biological activity of the chimera. In addition, we have identified a single noncatalytic tyrosine residue (tyrosine 1356) in the carboxyl terminus of the Met receptor, that is essential for the biological activity of the chimeric receptor. Mutation of tyrosine 1356 to a nonphosphorylatable phenylalanine residue does not affect the exogenous kinase activity of the receptor toward enolase, but it impairs the ability of the mutant protein to associate with the adaptor protein Grb2, and MDCK cells expressing this mutant fail to scatter, invade, and form branching tubules in response to CSF-1. These results support a crucial role for tyrosine 1356 in activation of signaling pathways involved in the biological activity of the Met receptor in response to HGF/SF.

Phorbol esters inhibit insulin-induced receptor down-regulation in cultured human lymphocytes: association with diminished insulin receptor autophosphorylation.

Exposure of cells to phorbol 12-myristate 13-acetate (PMA) has been reported to result in resistance to the acute biological effects of insulin and an associated reduction in insulin-receptor tyrosine kinase activity. To investigate the relationship of insulin receptor autophosphorylation with a longer-term action of insulin the effect of PMA on insulin-stimulated receptor down-regulation was examined in cultured human lymphocytes (IM-9). Lymphocytes bound [3H]phorbol dibutyrate specifically with characteristics typical of binding to protein kinase C (PKC). Acute exposure (30 min) to PMA resulted in a transient decrease of insulin binding which is consistent with a decrease in receptor number. Chronic (18 h) exposure to PMA (5 nM) resulted in inhibition of insulin-induced down-regulation of its cognate receptor. Sphingosine, an inhibitor of PKC, or chronic pre-exposure to a high concentration of PMA (1 microM), which is known to inactivate PKC, blocked the effect of PMA. PMA inhibited insulin-stimulated receptor internalization by 26% and receptor degradation by 82%. Exposure of intact cells to PMA followed by insulin treatment inhibited insulin-receptor autophosphorylation subsequently assayed in vitro, as well as beta-subunit tyrosine phosphorylation in situ. In summary, PMA inhibited insulin-stimulated receptor down-regulation via activation of PKC. This was associated with an inhibition of both receptor internalization and receptor degradation. There was a concomitant inhibition of receptor tyrosine autophosphorylation consistent with a requirement of receptor kinase activation for both short-term and long-term biological effects of insulin.

The penetration of ciprofloxacin into bronchial mucosa, lung parenchyma, and pleural tissue after intravenous administration.

We have studied the concentrations of ciprofloxacin in serum, bronchial mucosa, lung parenchyma, and pleural tissue after a single intravenous dose of 200 mg in 20 patients subjected to lung surgery. The concentrations of ciprofloxacin in the tissues exceeded that in the serum by 3-fold to 7-fold: serum 0.6 micrograms.ml-1, bronchial mucosa 1.9 micrograms.g-1, lung parenchyma 3.4 micrograms.g-, and pleural tissue 1.7 micrograms.g-1. The achievable concentrations of ciprofloxacin in the tissues of the lower respiratory tract are above the MICs for most lung pathogens.

Baculovirus expression of mammalian G protein alpha subunits.

Complementary DNAs encoding three subtypes of the alpha subunit (alpha i-1, alpha o and alpha s) of rat guanyl nucleotide regulatory proteins were used to construct recombinant baculoviruses which direct high-level expression of the corresponding proteins in cultured Sf9 insect cells. The expressed proteins were recognized by polyclonal antisera specific for the different alpha chains, and co-migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive alpha chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [35S]methionine or [3H]myristate showed that both soluble and particulate forms of alpha i-1 and alpha o were myristoylated; in contrast, alpha s did not incorporate myristate. The soluble fractions from cells expressing alpha chains showed high levels of GTP-binding activity over that observed in uninfected cells, or in cells infected with wild-type virus. The peak expression levels observed at 72 h post-infection were highest for alpha o at ca. 400 pmol of GTP-gamma-35S/mg protein, or roughly 2% of the total soluble protein. The results of this work show that the baculovirus system can be employed for high-level production of mammalian G protein alpha chains which retain GTP-binding activity and are appropriately modified by myristoylation.

Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes.

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.

Glycosyltransfer by pea membranes from sugar nucleotides to added prenyl phosphates.

Pea membranes supplied with GDP-[14C]mannose, UDP-N-[14C]acetylglucosamine or UDP-[14C]glucose catalyze the transfer of 14C-labeled sugars or sugar phosphates to endogenous lipid acceptors as well as to exogenously added dolichyl phosphates. Fully unsaturated polyprenyl phosphates were not used as effective acceptors by this system. Mannosyl-P-dolichol was formed most rapidly in the presence of long-chained dolichyl-P while mannosyl-PP-, glucosyl-PP- and GlcNAc-PP-dolichol were preferentially formed from relatively short-chained dolichyl phosphate acceptors. Glucosyl-PP- and mannosyl-PP-dolichol accumulated in the preparation without further metabolism, but GlcNAc-PP-dolichol was lengthened by addition of a second GlcNAc plus several [14C]mannose units to form an oligosaccharide fraction susceptible to the action of endoglycosidase H. This lipid-linked oligosaccharide could then be glycosylated in the presence of UDP-[14C]glucose to form a longer oligosaccharide. It is concluded that levels of endogenous dolichyl phosphates in pea membranes are rate-limiting for several of the key glycosyltransferases required for oligosaccharide assembly.

Long-term results of surgical treatment for alkaline reflux gastritis in gastrectomized patients.

Sixteen gastrectomized patients underwent surgical treatment for alkaline reflux gastritis by means of a Roux-en-Y loop duodenal diversion. Long-term evaluation of results was performed 5-9 years later. Ten patients (62.5%) showed good results, with absence of digestive symptoms and with an increase in body weight. Two patients (12.5%) had moderate results, with presence of sporadic and mild epigastric pain. Four patients (25%) had unsatisfactory results, with persistence of epigastric pain and absence of body weight increase. No patient had recurrent biliary vomiting or endoscopic evidence of endogastric biliary reflux. Among the six patients with moderate and unsatisfactory results, two had a significant alcoholic intake, two showed a high degree of anxiety on psychological assessment, and two had both factors. Alcoholism and psychological disturbances should be considered exclusion criteria when evaluating a gastrectomized patient for surgical cure of alkaline reflux gastritis.