Surgical planning and microvascular reconstruction of the mandible with a fibular flap using computer-aided design, rapid prototype modelling, and precontoured titanium reconstruction plates: a prospective study.

The standard of mandibular reconstruction has increased since the introduction of computer-assisted design (CAD) and rapid prototype modelling (RPM) for surgical planning. Between 2008 and 2013, a prospective pilot study of 20 patients was planned to compare the outcomes of patients treated by mandibular reconstruction who had CAD and RPM-guided operations using a precontoured titanium plate, with the outcomes of patients treated conventionally. We recorded the time taken for reconstruction, total operating time, and whether this type of planning could improve the results of mandibular reconstruction. We found significant differences in the incidence of dental malocclusion (p=0.03) and exposure of the titanium plate (p=0.009). The mean operating time for reconstruction in the preoperative planning group was 135 (37)min compared with 176 (58)min in the conventional group (p=0.04). Preoperative planning using CAD and RPM can increase the accuracy of microvascular mandibular reconstruction and reduce the operating time for reconstruction.

Simultaneous determination of Δ9-tetrahydrocannabinol, cannabidiol, cannabinol and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in hair using liquid chromatography-tandem mass spectrometry.

For several years, hair analyses have become a powerful tool to investigate past exposure towards xenobiotics. In the case of illicit drugs and more precisely of cannabis exposure, four compounds are usually investigated: Δ(9)-tetrahydrocannabinol (THC), the main active compound of cannabis, one of its metabolites [11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)] and two cannabinoids (cannabinol and cannabidiol). Up until now, the hair determination of the carboxylic metabolite of THC, which has been described as the only marker allowing distinguishing consumption and passive exposure, has been performed using a gas chromatography-tandem mass spectrometry method. The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of the four markers. The sample preparation was based on an alkaline hydrolysis of hair samples followed by a liquid-liquid extraction of compounds in acidic conditions using a hexane/ethyl acetate mixture. The method was validated and the results were satisfactory: intra- and inter-assay accuracies below 9% and relative standard deviation below 15% for the four compounds. Moreover, the limit of quantification for THC-COOH, the most challenging compound, was validated at 0.2 pg/mg. This concentration is in accordance with the recommendations made by a scientific society which specializes in hair testing. It makes it possible to distinguish the kind of exposure to cannabis.

Cell adhesion and signaling on the fibronectin 1st type III repeat; requisite roles for cell surface proteoglycans and integrins.

BACKGROUND: The first type III repeat of fibronectin is known to be involved in fibronectin matrix assembly, and recombinant proteins from this type III repeat can inhibit cell proliferation, tumor metastasis and angiogenesis. We have analyzed the way rat aortic smooth muscle cells (RASMCs) interact with a recombinant protein encompassing a C-terminal portion of the first type III repeat of fibronectin (protein III1-C). RESULTS: Cells are able to adhere to and spread on III1-C coated on a dish. Both beta1 integrins and cell surface heparan sulfate proteoglycans serve as receptors for III1-C. For example, cell attachment to III1-C is partially inhibited by agents that block beta1 integrins or by heparin. Complete inhibition of cell attachment is seen only when integrin blocking agents are combined with heparin. Affinity chromatography revealed the binding of proteins that likely represent the integrin beta1 and alpha5 submits to a III1-C column. Cell adhesion to III1-C results in robust ERK1/2 activation that is blocked by integrin-blocking agents. In addition, cell adhesion to III1-C and ERK1/2 activation by III1-C are both inhibited by heparan sulfate but not by chondroitin sulfate. Moreover, heparitinase treatment, but not chondroitinase treatment of RASMCs results in reduced cell adhesion and ERK1/2 activation. Affinity chromatography experiments demonstrated that 35SO4-labeled cell surface heparan sulfate proteoglycans bound specifically to III1-C. CONCLUSIONS: The results suggest that the 1st type III repeat of fibronectin contains a previously unrecognized cell adhesion domain that stimulates robust ERK1/2 activation in RASMCs. Cells interact with this domain through cell surface heparan sulfate proteoglycans and integrins, and both classes of receptors are required for optimal cell adhesion and ERK1/2 activation.

Direct cell adhesion to the angiopoietins mediated by integrins.

Genetic ablation of angiopoietin-1 (Ang-1) or of its cognate receptor, Tie2, disrupts angiogenesis in mouse embryos. The endothelial cells in growing blood vessels of Ang-1 knockout mice have a rounded appearance and are poorly associated with one another and their underlying basement membranes (Dumont, D. J., Gradwohl, G., Fong, G. H., Puri, M. C., Gertsenstein, M., Auerbach, A., and Breitman, M. L. (1994) Genes Dev. 8, 1897--1909; Sato, T. N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fujiwara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W., and Qin, Y. (1995) Nature 376, 70--74; Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato, T. N., and Yancopoulos, G. D. (1996) Cell 87, 1171--1180). It is therefore possible that Ang-1 regulates endothelial cell adhesion. In this study we asked whether Ang-1 might act as a direct substrate for cell adhesion. Human umbilical vein endothelial cells (HUVECs) plated for a brief period on different substrates were found to adhere and spread well on Ang-1. Similar results were seen on angiopoietin-2 (Ang-2)-coated surfaces, although cells did not spread well on Ang-2. Ang-1, but not Ang-2, supported HUVEC migration, and this was independent of growth factor activity. When the same experiments were done with fibroblasts that either lacked, or stably expressed, Tie2, results similar to those with HUVECs were seen, suggesting that adhesion to the angiopoietins was independent of Tie2 and not limited to endothelial cells. Interestingly, when integrin-blocking agents were included in these assays, adhesion to either angiopoietin was significantly reduced. Moreover, Chinese hamster ovary-B2 cells lacking the alpha(5) integrin subunit did not adhere to Ang-1, but they did adhere to Ang-2. Stable expression of the human alpha(5) integrin subunit in these cells rescued adhesion to Ang-1 and promoted an increase in adhesion to Ang-2. We also found that Ang-1 and Ang-2 bind rather selectively to vitronectin. These results suggest that, beyond their role in modulating Tie2 signaling, Ang-1 and Ang-2 can directly support cell adhesion mediated by integrins.

Control of smooth muscle cell proliferation and phenotype by integrin signaling through focal adhesion kinase.

Extracellular matrix proteins such as fibronectin (FN) and laminin (LM) are known to help control the growth and phenotype of vascular smooth muscle cells (VSMCs). Here we have analyzed the relationship between growth factor and integrin signaling pathways in VSMCs. Culturing porcine coronary artery smooth muscle cells (PCASMCs) on FN and LM leads to distinct effects on cell proliferation and contractile protein expression. PCASMCs cultured on FN proliferate at a higher rate than cells cultured on LM, regardless of the growth factor used to support proliferation. Moreover, cells cultured on LM show higher levels of expression of smooth muscle myosin heavy chain (a marker of smooth muscle cell differentiation) than cells cultured on FN. In contrast to the effects on proliferation and contractile protein expression, both FN and LM supported cell migration in response to PDGF. Also, both FN and LM supported activation of ERK1 and ERK2 in response to PDGF and bFGF. However, FN and LM did show a difference in their ability to support signaling through the focal adhesion kinase (FAK). PCASMCs cultured on FN show robust activation of FAK in response to either PDGF or bFGF, however, cells cultured on LM show little-to-no activation of FAK in response to the growth factors. The results show that integrin signaling pathways have a profound effect on VSMC proliferation and phenotype, and that FAK is an important intermediate in these signaling pathways. The implications of our findings on the mechanisms controlling VSMC proliferation and phenotype in pathological states such as atherosclerosis and restenosis are discussed.

[Multicenter study of resistance in enterococci. The role of teicoplanin. Spanish Group for Study and Surveillance of Resistance]. / Estudio multicéntrico de resistencias en enterococos. Papel de la teicoplanina. Grupo Espãnol de Estudio y Vigilancia de Resistencias.

Enterococci are a frequent cause of nosocomial and community infections, especially the E. faecalis and E. faecium species. They often show intrinsic resistance to cephalosporins and quinolones, and acquired resistance to other antimicrobials, such as glycopeptides, has also been described. In order to test the impact of antibiotic resistance in enterococci isolated from infections, we carried out a multicenter study in 19 hospitals in Spain. We verified whether resistance to a high concentration of aminopenicillins and aminoglycosides was high (30.86% for ampicillin, 32.32% for gentamicin at a 500 micrograms dose, 55.93% for streptomycin at a 1000 micrograms dose) while the resistance to glycopeptides was low (1.8% for vancomycin and 1% for teicoplanin). It was also shown that teicoplanin had greater intrinsic activity than vancomycin, with teicoplanin 0.5 mg/l inhibiting 86.1% of the strains studied, whereas only 12.8% were inhibited with the same concentration of vancomycin.

Inhibition of vascular smooth muscle cell growth by inhibition of fibronectin matrix assembly.

The regulation of vascular smooth muscle cell (VSMC) proliferation by the fibronectin matrix was tested by treating human umbilical artery smooth muscle cells (HUASMCs) with a recombinant fragment of fibronectin (protein III1-C) that has previously been shown to modulate fibronectin matrix assembly. III1-C inhibited HUASMC proliferation by 75% to 90%. The inhibition of growth was time dependent; III1-C had no effect on DNA synthesis after 0 to 5 hours of treatment but did have an effect at 24 hours and beyond. III1-C did not stimulate apoptosis in these cells, indicating that the inhibition of proliferation was not due to an induction of programmed cell death. The effects of III1-C on cell growth were only specific for normal diploid smooth muscle cells. III1-C had no effect on the proliferation of IMR-90 fibroblasts, endothelial cells, NIH 3T3 cells, or the rat aortic smooth muscle cell line A7r5. However, III1-C did inhibit proliferation by primary rat aortic smooth muscle cells. An analysis of HUASMC fibronectin receptor (integrin alpha5beta1) distribution revealed that III1-C did not inhibit alpha5beta1 localization to focal contacts. Moreover, III1-C had no effect on the relative expression levels of seven different integrin subunits on HUASMCs. However, III1-C did inhibit fibronectin matrix assembly by rat aortic smooth muscle cells, HUASMCs, A7r5 cells, IMR-90 cells, and endothelial cells. An analysis of fibronectin synthesis indicated that the inhibition of fibronectin matrix assembly by III1-C was not due solely to a decrease in fibronectin synthesis. Finally, treatment of HUASMCs with anti-fibronectin monoclonal antibody L8 (which is known to inhibit fibronectin matrix assembly) also decreased the rate of HUASMC DNA synthesis. These results demonstrate that III1-C inhibits VSMC proliferation and suggest that this effect may be mediated by the inhibition of fibronectin matrix assembly.

In vitro activity of biapenem against beta-lactamase producing Enterobacteriaceae.

The activity of biapenem was compared with that of imipenem and cefotaxime against 108 strains of beta-lactamase producing Enterobacteriaceae. Biapenem and imipenem were very active, inhibiting 90% of the strains at a concentration of 0.5 microgram/ml. Both carbapenems were very active against plasmidic beta-lactamase producers, with MIC90s below 1 microgram/ml. However, the MIC90 of biapenem for cephalosporinase producers was 1 microgram/ml. Against strains producing extended-spectrum beta-lactamases, biapenem exhibited better activity against TEM-type producers (MIC90 0.25 microgram/ml) than against SHV-type producers (MIC90 0.5 microgram/ml). Overall, the in vitro antibacterial activity of biapenem is similar to that of imipenem.

Superfibronectin is a functionally distinct form of fibronectin.

Fibronectin is an extracellular matrix protein that is important in development, wound healing and tumorigenesis. In the blood it is dimeric, but in tissues forms disulphide crosslinked fibrils. Here we show that a fragment from the first type-III repeat of fibronectin binds to fibronectin and induces spontaneous disulphide crosslinking of the molecule into multimers of high relative molecular mass which resemble matrix fibrils. Treatment of fibronectin with this inducing fragment also converts fibronectin into a form that has greatly enhanced adhesive properties (hence the term superfibronectin) and which suppresses cell migration. Whereas cells attach to fibronectin through integrins, cell attachment to superfibronectin is mediated both by integrins and by receptors with properties distinct from those of integrins. Superfibronectin may be closely related to the natural matrix form of fibronectin.

The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin.

The fibronectin receptor, alpha 5 beta 1, has been shown to be required for fibronectin matrix assembly and plays an important role in cell migration on fibronectin. However, it is not clear whether other fibronectin binding integrins can take the place of alpha 5 beta 1 during matrix assembly and cell migration. To test this, we expressed the human alpha v subunit in the CHO cell line CHO-B2 that lacks the alpha 5 subunit. We found that the human alpha v combined with CHO cell beta 1 to form the integrin alpha v beta 1. Cells that expressed alpha v beta 1 attached to and spread well on fibronectin-coated dishes, but did so less well on vitronectin-coated dishes. This, along with other data, indicated that alpha v beta 1 functions as a fibronectin receptor in CHO-B2 cells. The alpha v beta 1-expressing cells failed to produce a fibronectin matrix or to migrate on fibronectin, although the same cells transfected with alpha 5 do produce a matrix and migrate on fibronectin. The affinity of the alpha v beta 1-expressing cells for fibronectin was fourfold lower than that of the alpha 5 beta 1-expressing cells. In addition, alpha v beta 1 was distributed diffusely throughout the cell surface, whereas alpha 5 beta 1 was localized to focal adhesions when cells were seeded onto fibronectin-coated surfaces. Thus, of the two fibronectin receptors, alpha v beta 1 and alpha 5 beta 1, only alpha 5 beta 1 supports fibronectin matrix assembly and promotes cell migration on fibronectin in the CHO-B2 cells. Possible reasons for this difference in the activities of alpha v beta 1 and alpha 5 beta 1 include the lower affinity of alpha v beta 1 for fibronectin and the failure of this integrin to localize in adhesion plaques on a fibronectin substrate. These results show that two integrins with similar ligand specificities and cell attachment functions may be quite different in their ability to support fibronectin matrix assembly and cell motility on fibronectin.

[Molecular study of ampicillin resistance in clinical isolates of Salmonella]. / Estudio molecular de la resistencia a ampicilina en aislamientos clínicos de Salmonella.

BACKGROUND: The purpose of this work was to study the molecular basis of beta-lactamase production in ampicillin-resistant strains of Salmonella spp. METHODS: It was performed analytical isoelectric focusing of beta-lactamases produced by a group of 33 strains selected in basis of their resistance phenotype. Plasmid profile analysis and assays of transferable drug resistance were developed. The study was completed by hybridization experiments with an intragenic TEM probe which allowed the location of the bla-TEM gene. RESULTS: By analytical isoelectrofocusing we found that 26 out of the 27 ampicillin-resistant strains produced beta-lactamases with pl 5.4 and/or 5.6 corresponding to TEM-1 and/or TEM-2 type. Analysis of plasmid DNA revealed in almost all strains plasmids ranging in size from 1.1 to 125 Mdal. This plasmids were responsible of the resistance and, moreover, were able to transfer the resistance by conjugation mechanisms. Southern blot analysis detected the gene that code the TEM beta-lactamase at the 125, 8 and 5.8 Mdal plasmids. CONCLUSIONS: Resistance to ampicillin in the strains of Salmonella studied was due to the presence of TEM type beta-lactamases coded by conjugative plasmids. These plasmids coded also resistance to other antimicrobial agents. Our results showed that the use of a DNA probe to the detect TEM-type beta-lactamases using a non radioactive probe, could be a suitable alternative to isoelectric focusing.

A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide.

The active form of fibronectin is its extracellular matrix form, which allows for the attachment of cells and influences both the growth and migration of cells. The matrix form is assembled by cells; however, many cells are defective in this regard. Several regions within fibronectin have been shown to play a role in matrix assembly by cells. One such region has been localized into the first type III repeat of fibronectin (Chernousov, M. A., F. J. Fogerty, V. E. Koteliansky, and D. F. Mosher. J. Biol. Chem. 266:10851-10858). We have identified this site as a fibronectin-fibronectin binding site and reproduced it as a synthetic peptide. This site is contained in a 14-kD fragment that corresponds to portions of the first two type III repeats. The 14-kD fragment was found to bind to cell monolayers and to inhibit fibronectin matrix assembly. The 14-kD fragment only slightly reduced the binding of fibronectin to cell surfaces but it significantly inhibited the subsequent incorporation of fibronectin into the extracellular matrix. The 14-kD fragment also bound to purified fibronectin and inhibited fibronectin-fibronectin binding. A synthetic 31-amino acid peptide (P1) representing a segment of the 14-kD fragment retained the ability to inhibit fibronectin-fibronectin binding. Peptide P1 specifically bound fibronectin from plasma in affinity chromatography, whereas a column containing another peptide from the 14-kD fragment did not. These results define a fibronectin-fibronectin binding site that appears to promote matrix assembly by allowing the assembly of fibronectin molecules into nascent fibrils. The 14-kD fragment and the P1 peptide that contain this site inhibit matrix assembly by competing for the fibronectin-fibronectin binding.

Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase.

The retinoblastoma gene product (RB) is a nuclear protein which has been shown to function as a tumor suppressor. It is phosphorylated from S to M phase of the cell cycle and dephosphorylated in G1. This suggests that the function of RB is regulated by its phosphorylation in the cell cycle. Ten phosphotryptic peptides are found in human RB proteins. The pattern of RB phosphorylation does not change from S to M phases of the cell cycle. Hypophosphorylated RB prepared from insect cells infected with an RB-recombinant baculovirus is used as a substrate for in vitro phosphorylation reactions. Of several protein kinases tested, only cdc2 kinase phosphorylates RB efficiently and all 10 peptides can be phosphorylated by cdc2 in vitro. Removal of cdc2 from mitotic cell extracts by immunoprecipitation causes a concomitant depletion of RB kinase activity. These results indicate that cdc2 or a kinase with similar substrate specificity is involved in the cell cycle-dependent phosphorylation of the RB protein.

Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis.

Tyrosine phosphorylation of cdc2 is regulated in the cell cycle of mouse 3T3 fibroblasts. Phosphotyrosine in cdc2 is detectable at the onset of DNA synthesis and becomes maximal in the G2 phase of the cell cycle. Quantitative tyrosine dephosphorylation of cdc2 occurs during entry into mitosis and no phosphotyrosine is detected during the G1 phase of the cell cycle. While increasing tyrosine phosphorylation of cdc2 correlates with the formation of a cdc2/p62 complex, the tyrosine phosphorylated cdc2 is inactive as a histone H1 kinase. cdc2 is fully dephosphorylated in its most active mitotic form, yet specific tyrosine dephosphorylation of interphase cdc2 in vitro is insufficient to activate the kinase. In vivo inhibition of tyrosine dephosphorylation by exposure of cells to a phosphatase inhibitor is associated with G2 arrest, which is reversible upon the removal of the phosphatase inhibitor. Tyrosine dephosphorylation of cdc2 may be one of a number of obligatory steps in the mitotic activation of the kinase.

Hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in interleukin-3-dependent murine cell lines.

By immunoblotting with antibodies for phosphotyrosine, we have demonstrated that the hematopoietic growth factors interleukin-2, interleukin-3, interleukin-4, and granulocyte-macrophage colony-stimulating factor stimulate the tyrosine phosphorylation of specific sets of proteins in murine hematopoietic progenitor cell lines. The stimulation of tyrosine phosphorylation is a receptor-dependent transient event. The effect of these hematopoietic growth factors on protein tyrosine phosphorylation was not mediated through protein kinase C.

Reduction in protein tyrosine phosphorylation during differentiation of human leukemia cell line K-562.

Human chronic myelogenous leukemia cell line K-562 expresses the bcr/c-abl fusion protein which is an active protein tyrosine kinase. Multiple tyrosine-phosphorylated proteins were detected in K-562 cells by immunoblotting with a high-affinity anti-phosphotyrosine antibody. When K-562 cells were induced with hemin to progress through the erythroid differentiation pathway, reduction in the levels of these tyrosine-phosphorylated proteins was observed. This reduction in tyrosine-phosphorylated proteins was not found in another chronic myelogenous leukemia cell line which could not be induced to differentiate by hemin. This and other observations established that the reduction in protein tyrosine phosphorylation is a specific differentiation response. The bcr/c-abl protein synthesis was reduced in hemin-treated K-562 cells. Thus, erythroid differentiation of K-562 cells reduces the level of the bcr/c-abl tyrosine kinase and the phosphotyrosine content of its substrate proteins.

Protein tyrosine phosphorylation in the cell cycle of BALB/c 3T3 fibroblasts.

Cell cycle-dependent regulation of protein tyrosine phosphorylation in normal BALB/c 3T3 fibroblasts was examined by immunoblotting with a high-affinity antibody specific for phosphotyrosine. At least 15 different tyrosine-phosphorylated proteins are found in normal 3T3 cells. The level of tyrosine phosphorylation is higher in growing cells than in quiescent cells. However, a prominent tyrosine-phosphorylated protein of Mr 150,000 is present in quiescent cells, and its level is inversely proportional to the growth rate of these fibroblasts. Stimulation of quiescent cells with serum causes a major, yet transient, increase in tyrosine phosphorylation. The immediate tyrosine phosphorylation reactions in response to serum stimulation are independent of protein synthesis, but tyrosine phosphorylation reactions occurring later in the G1 phase of the cell cycle are inhibited by cycloheximide. Thus, tyrosine phosphorylation of proteins in normal 3T3 cells occurs predominantly at the G0 to G1 transition of the cell cycle. Maintenance of steady-state tyrosine phosphorylation is dependent on the presence of serum, but at least one tyrosine phosphorylation reaction occurs in the absence of cell growth.