Fibronectin coating of collagen modules increases in vivo HUVEC survival and vessel formation in SCID mice.

Modular tissue engineering is a novel approach to creating scalable, self-assembling, three-dimensional tissue constructs with inherent vascularization. Under initial methods, the subcutaneous implantation of human umbilical vein endothelial cell (HUVEC)-covered collagen modules in immunocompromised mice resulted in significant host inflammation and limited HUVEC survival. A minimally invasive injection technique was used to minimize surgery-related inflammation, and cell death was attributed to extensive apoptosis within 72 h of implantation. Coating collagen modules with fibronectin (Fn) was shown in vivo to reduce short-term HUVEC TUNEL staining by nearly 40%, while increasing long-term HUVEC survival by 30-45%, relative to collagen modules without fibronectin. Consequently, a ∼100% increase in the number of HUVEC-lined vessels was observed with Fn-coated modules, as compared to collagen-only modules, at 7 and 14 days post-implantation. Furthermore, vessels appeared to be perfused with host erythrocytes by day 7, and vessel maturation and stabilization was evident by day 14.

Near infrared spectroscopy as a rapid tool to measure volatile aroma compounds in Riesling wine: possibilities and limits.

Volatile chemical compounds responsible for the aroma of wine are derived from a number of different biochemical and chemical pathways. These chemical compounds are formed during grape berry metabolism, crushing of the berries, fermentation processes (i.e. yeast and malolactic bacteria) and also from the ageing and storage of wine. Not surprisingly, there are a large number of chemical classes of compounds found in wine which are present at varying concentrations (ng L(-1) to mg L(-1)), exhibit differing potencies, and have a broad range of volatilities and boiling points. The aim of this work was to investigate the potential use of near infrared (NIR) spectroscopy combined with chemometrics as a rapid and low-cost technique to measure volatile compounds in Riesling wines. Samples of commercial Riesling wine were analyzed using an NIR instrument and volatile compounds by gas chromatography (GC) coupled with selected ion monitoring mass spectrometry. Correlation between the NIR and GC data were developed using partial least-squares (PLS) regression with full cross validation (leave one out). Coefficients of determination in cross validation (R(2)) and the standard error in cross validation (SECV) were 0.74 (SECV: 313.6 microg L(-1)) for esters, 0.90 (SECV: 20.9 microg L(-1)) for monoterpenes and 0.80 (SECV: 1658 microg L(-1)) for short-chain fatty acids. This study has shown that volatile chemical compounds present in wine can be measured by NIR spectroscopy. Further development with larger data sets will be required to test the predictive ability of the NIR calibration models developed.

Complement inhibition reduces material-induced leukocyte activation with PEG modified polystyrene beads (Tentagel) but not polystyrene beads.

With isolated leukocytes, inhibiting complement reduced material-induced leukocyte activation (CD11b) with polyethylene glycol modified polystyrene beads (PS-PEG), but not with polystyrene beads (PS). The PS-PEG beads (TentaGel) were complement activating as measured by SC5b-9 levels consistent with the sensitivity of these beads to leukocyte inhibition with complement inhibitors. Following contact with PS and PS-PEG beads, isolated leukocytes in plasma and in the absence in platelets were found to significantly upregulate CD11b, while TF expression and exposure of phosphatidylserine remained at background levels. Complement inhibition by means of sCR1 partially reduced CD11b upregulation on PS-PEG beads, but had no effect with PS beads. Pyridoxal-5-phosphate (P5P) was able to significantly reduce both CD11b upregulation and exposure of phosphatidylserine with PS-PEG beads, although it did not appear to inhibit SC5b-9 production. Pentamidine and NAAGA inhibited complement and were effective in reducing CD11b upregulation with both PS and PS-PEG. However, they also had an inhibitory effect on leukocyte signaling mechanisms, precluding their utility for further study in this context. Leukocyte adhesion occurred to similar extents on both PS and PS-PEG beads. While sCR1 and P5P blocked adhesion and activation (for adherent leukocytes) on PS-PEG beads, they had no effect on leukocytes adherent to PS beads. The role of complement in leukocyte activation and adhesion was found to be material-dependent. Thus, leukocyte-material compatibility may be resolved by complement inhibition in some but not all cases. For these other materials (example here was PS), other mechanisms, such as fibrinogen adsorption and direct leukocyte release, may need exploitation to minimize leukocyte activation and adhesion.

Viability of hydroxyethyl methacrylate-methyl methacrylate-microencapsulated PC12 cells after omental pouch implantation within agarose gels.

Hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA, 75 mol% HEMA). Microcapsules containing viable PC12 cells (as an allogeneic transplant model) were implanted into omental pouches in Wistar rats. Two different capsule preparations were tested, based on differences in polymer solutions during extrusion: 10% HEMA-MMA in TEG, and 9% HEMA-MMA in TEG with 30% poly(vinyl pyrrolidone) (PVP). The omental pouch proved to be an ideal transplant site in terms of implantation, recovery, and blood vessel proximity (nutrient supply). To minimize the fibrous overgrowth and damaged capsules previously seen on implantation of individual capsules, agarose gels were used to embed the capsules before implantation. Cells proliferated within the microcapsule-agarose device during the first 7 days of implantation, but overall cell viability declined over the 3-week period, when compared with similar capsules maintained in vitro. Nonetheless, approximately 50% of the initial encapsulated cells were still viable after 3 weeks in vivo. This approach to HEMA-MMA microcapsule implantation improved cell viability and capsule integrity after 3 weeks in vivo, compared with capsules implanted without agarose.

Material-induced tissue factor expression but not CD11b upregulation depends on the presence of platelets.

Biomaterials activate leukocytes as well as platelets when exposed to blood. One feature of leukocyte activation at least at times beyond a few hours is tissue factor expression, contributing to a procoagulant state. We show here that platelet activation and specifically platelet-monocyte aggregate formation appears to be a precondition for tissue factor expression. Material-induced Tissue Factor (TF) expression by isolated leukocytes (6 x 10(6) cells/mL) resuspended in increasing concentrations of platelets in plasma was elevated when the platelet concentration was 50 x 10(6) platelets/mL or more; at lower platelet concentrations (1-25 x 10(6). cells/mL) the TF expression remained at background levels. On the other hand, significant CD11b upregulation was observed on leukocytes, in bulk and adherent to beads, at all platelet concentrations. This platelet effect on material-induced TF expression appeared to be mediated by the formation of platelet-monocyte aggregates. Anti-P-selectin, which blocked the association between platelets and leukocytes, reduced monocyte adhesion and material-induced TF expression for bulk monocytes. Anti-GPIIb/IIIa, a GPIIb/IIIa platelet antagonist, also reduced monocyte adhesion and material-induced TF expression in the bulk, most likely due to its inhibiting effect on the formation of platelet-monocyte aggregates, secondary to platelet activation. However, the antibody-associated reductions for bulk leukocytes (mainly neutrophils) were small and incomplete. Similar levels of TF expression, in the bulk, were observed with both polystyrene (PS), a strong platelet activator, and polyethylene glycol-modified PEG (PS-PEG), a mild platelet activator. The role of platelets in material-induced TF expression appears to be mediated in part via the formation of platelet-monocyte aggregates, although other mechanisms are likely also involved.

Tissue engineering as a platform for controlled release of therapeutic agents: implantation of microencapsulated dopamine producing cells in the brains of rats.

Tissue engineering can lead to novel controlled release devices and controlled release strategies (e.g., of growth factors) can enhance the performance of tissue engineered constructs. There are however a number of technical challenges that must be overcome before these goals can be realized. The apparently 'simple' challenge of implanting the device (e.g., capsules) in the optimal site must be met. In addition, adequate nutrient supply to the capsules is required to maintain cell viability. To illustrate this problem we describe a guide and delivery cannula technique to provide reliable and reproducible delivery of up to 120 PC12 cell containing capsules into the caudate putamen (CPu). Microencapsulation of mammalian cells is potentially a powerful means of delivering therapeutically important molecules such as insulin. It can also have numerous applications as a platform for gene therapy. However, realizing this potential has been more difficult than first anticipated.

Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations.

The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

Leukocyte activation and leukocyte procoagulant activities after blood contact with polystyrene and polyethylene glycol-immobilized polystyrene beads.

Beads (45 microm) of polystyrene (PS) and polyethylene glycol modified PS (TentaGel) with an amino or hydroxyl terminal group were incubated with blood to assess the effect of surface area and material chemistry on leukocyte activation. After a 2-hour incubation, blood contact with beads activated leukocytes in the bulk (tissue factor expression, CD11b up-regulation, and association with platelets) independently of material surface chemistry. On the other hand, activation of adherent leukocytes was material dependent. After blood contact with PS, polyethylene glycol-immobilized PS (PS-PEG) and PS-PEG-NH2 beads, CD11b up-regulation in the bulk, platelet-leukocyte aggregates, and leukocyte adhesion were all dependent on surface area, whereas tissue factor (TF) expression was not. Material-induced leukocyte activation in the bulk was also independent of the beads' capacity to activate platelets. However, monocyte adhesion and TF expression on beads appeared to be related to the presence of platelets on the surface. Material-induced TF expression was able to initiate the extrinsic pathway of coagulation, resulting in significant fibrin formation. Although not all of our markers of leukocyte activation varied with material area or chemistry, it was clear that these materials activated leukocytes in a way that resulted in increased procoagulant activity. During blood-material interactions, material-induced leukocyte activation may then contribute to thrombogenesis.

Does surface chemistry affect thrombogenicity of surface modified polymers?

With some exceptions, surface chemistry had little effect on platelet and leukocyte activation, and cell deposition, by scanning electron microscopy after blood exposure and clotting times among a group of 12 unmodified and plasma modified tubings. All materials activated platelets and leukocytes to detectable levels, although some materials increased the value of one activation parameter but not another. Unmodified materials [polyethylene (PE), Pellethane (PEU), latex, nylon, and Silastic] and modified materials (H(2)O plasma treated PE and PEU, CF(4) plasma treated PE, fluorinated PEU, NH(4) plasma treated PEU, polyethylene imine treated PEU, and heparin treated PEU) were characterised by XPS and contact angle. The objective of this project was to define a series of assays for the evaluation of hemocompatibility of cardiovascular devices with a view to clarify the specific requirements of ISO-10993-4, and to define an appropriate screening program for new blood contacting biomaterials. PE, PE--CF(4), PE--H(2)0, PEU--F, latex, and PEU-heparin were the exceptions to the general observations, although each behaved differently. PE proved to be least reactive, whereas PE-CF(4) was most reactive by several assays. Platelet microparticle formation (determined by flow cytometry), PTT, postblood exposure SEM, total SC5b-9, C3a, and platelet and leukocyte loss (cell counts) were able to distinguish differences among these materials, and often, but not always, showed expected correlations.

Quantitative analysis of polymeric procyanidins (Tannins) from grape (Vitis vinifera) seeds by reverse phase high-performance liquid chromatography.

A reverse phase C(18) HPLC method with potential for high automated throughput has been developed for the quantitative analysis of polymeric procyanidins (tannins) in grape seed extracts. Chromatography gave rise to 13 distinct UV-absorbing peaks with good baseline separation. The UV-absorbing peak eluting last is distinct and therefore easily quantified. Biochemical analyses including ultrafiltration, protein precipitation, and Sephadex LH20 chromatography combined with electrospray mass spectrometric analyses establish that this peak predominantly contains polymeric procyanidins. The polymers, which appear to be galloylated to various degrees and seem to fragment in a characteristic manner during electrospray mass spectrometry, are well separated from catechins and procyanidin oligomers of up to 4 units. The recovery of polymeric grape seed tannins with this HPLC method was 86%, which is similar to the 89% recovery achieved with commercial quebracho tannins. The concentration of tannins in seeds from ripe Vitis vinifera cv. Shiraz grapes ranged from 1360 to 2830 mg/kg of berries.

Complement activation by PVA as measured by ELIFA (enzyme-linked immunoflow assay) for SC5b-9.

Polyvinyl alcohol (PVA) coated onto polyethylene (PE) tubes exposed to human serum for 1 hour at 37 degrees C resulted in the production of 1.03 +/- 0.04 microg/cm2 of the soluble form of the terminal membrane attack complex, SC5b-9. This was approximately 20 x that produced by the polyethylene. About one quarter of this total was found associated with the surface of PVA. SC5b-9 concentrations were determined by enzyme-linked immunoflow assay (ELIFA) a variant on ELISA that involved drawing the test sample, the antibodies and the chromogenic reagent through a nitrocellulose membrane filter. ELIFA enabled analysis of protein concentrations in the presence of SDS, so that SDS (0.05%) was used to desorb adsorbed SC5b-9 prior to analysis together with SC5b-9 in the bulk to get a more complete picture of PVA-associated complement activation.

Expression of the G protein-coupled receptor kinase 2 during early mouse embryogenesis.

Whilst several G protein-coupled receptors (GPCRs) have been shown to play important roles during development, little study has been carried out on the G protein-coupled receptor kinases (GRKs) that modulate their activity in embryos. Here, we have analyzed the expression of GRK2, the predominant GRK expressed during embryogenesis. We show that at early stages, the expression of GRK2 is restricted to populations of cells that are undifferentiated, multipotent and in many cases, migratory. As such, GRK2 transcripts were found in the early mesoderm and neural crest as they migrate from the primitive streak and the neural tube, respectively. In the limb bud, GRK2 transcripts were observed in cells of the progress zone and in the interdigital areas. At later stages, the expression in the heart is compatible with the phenotype observed in the GRK2 deficient mice.

Microencapsulation of normal and transfected L929 fibroblasts in a HEMA-MMA copolymer.

Mouse L929 fibroblasts transfected to express a secreted form of human alkaline phosphatase (SEAP) were encapsulated in approximately 400-microm poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA) microcapsules as a baseline for the use of genetically engineered cells in encapsulation therapy. Although incubation of microcapsules with serum-containing medium resulted in maintaining the number of live encapsulated cells with the passage of time, incubation in a serum-free medium resulted in a three-fold proliferation of the encapsulated cells within a 3-week observation period. Similar to the results for incubation with serum-containing medium, co-encapsulation with a bovine dermal type I collagen, i.e., the inclusion of a matrix in the core of the capsules, resulted in maintenance of the initial number of live cells with the passage of time. SEAP measurements indicated that the transfected cells not only continued to express the transgene product after encapsulation, but also adapted to the capsule microenvironment to secrete SEAP at progressively larger amounts with the passage of time. However, SEAP expression only occurred when the transfected cells (encapsulated or non-encapsulated) were cultivated in serum-containing medium.

Viability of HEMA-MMA microencapsulated model hepatoma cells in rats and the host response.

Small diameter hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA; 75% HEMA) microcapsules containing an aggregate of viable rat hepatoma H4IIEC3 cells, after implantation into an omental pouch in Wistar rats, contained viable cells at 7 days but not 14 days. A similar transplantation of microencapsulated aggregates of human hepatoma HepG2 cells did not result in viable cells even at 7 days. The loss of viability was attributed to the tissue reaction, because both encapsulated cell types remained viable in vitro. However, it is not clear if the cells lost their viability in vivo, leading to the aggressive tissue reaction or if the latter caused the cells to starve or otherwise die. The tissue reactions to microcapsules containing rat or human hepatoma cells at day 1 was one cell layer thick and avascular. At later times, tissue reactions were comprised of three regions: macrophages, fibroblasts, and some foreign body giant cells apposed to the polymer membrane, a dense region of fibroblasts and collagen, and a region of vascularized granulation tissue. Prompt vascularization of the tissue reactions occurred after 4 days and was maintained for up to 14 days. Even at 14 days, immune cells were observed, suggesting a continued immune response toward antigens shed from the encapsulated cells.

Exploring redundancy in the yeast genome: an improved strategy for use of the cre-loxP system.

Gene families having more than three members are a common phenomenon in the Saccharomyces cerevisiae genome. As yeast research enters the post-genome era, the development of existing deletion strategies is crucial for tackling this apparent redundancy, hence a method for performing rapid multiple gene disruptions in this organism has been developed. We constructed three replacement cassettes in which different selectable markers were placed between two loxP loci. Multiple deletions (of members of a gene family) were generated, in one strain, using sequential integration of different replacement markers (kanMX, LYS2, KlURA3 and SpHIS5). Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinase vector, which carries the cycloheximide-resistance gene from Candida maltosa as a selectable marker. Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particularly useful for studying gene families in either laboratory or industrial yeast strains.

Hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymers for cell microencapsulation: effect of HEMA purity.

Thermoplastic copolymers of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) (molar ratio: 75/25 HEMA-MMA) were synthesized using HEMA containing different amounts of ethylene glycol dimethacrylate (EGDMA) to investigate their suitability for cell microencapsulation. Pure HEMA (0.0% EGDMA) was obtained with preparative chromatography to prepare a linear copolymer. Microcapsules (with a diameter of 300-400 microm) were readily made with the copolymers by interfacial precipitation. Smaller and more transparent capsules were obtained using the copolymer prepared from purer HEMA. Chinese hamster ovary (CHO) fibroblasts, as model cells, were microencapsulated in the linear copolymer. The CHO cells survived the microencapsulation process and the metabolic activity of the encapsulated cells increased within the 14 days observation period.

Acid-catalyzed hydrolysis of alcohols and their beta-D-glucopyranosides.

The hydrolysis, in model wine at pH 3, of the allylic, homoallylic, and propargylic glycosides, geranyl-beta-D-glucopyranoside, [3'-(1' '-cyclohexenyl)-1'-methyl-2'-propynyl]-beta-D-glucopyranoside, (3'RS, 9'SR)-(3'-hydroxy-5'-megastigmen-7-yn-9-yl)-beta-D-glucopyra noside, (3',5',5'-trimethyl-3'-cyclohexenyl)-beta-D-glucopyranoside, E-(7'-oxo-5',8'-megastigmadien-3'-yl)-beta-D-glucopyranoside (3-hydroxy-beta-damascone-beta-D-glucopyranoside), and their corresponding aglycons has been studied. In general, aglycons were more rapidly converted to transformation products than were the corresponding glucosides. Glycoconjugation of geraniol in grapes is a process that reduces the flavor impact of this compound in wine, not only because geraniol is an important flavor component of some wines but also because the rate of formation of other flavor compounds from geraniol during bottle-aging is reduced. However, when flavor compounds such as beta-damascenone are formed in competition with flavorless byproducts, such as 3-hydroxy-beta-damascone, by acid-catalyzed hydrolytic reactions of polyols, then glycoconjugation is a process that could enhance as well as suppress the formation of flavor, depending on the position of glycosylation. (3'RS, 9'SR)-(3'-Hydroxy-5'-megastigmen-7'-yn-9'-yl)-beta-D-glucopy ranoside hydrolyzed more slowly but gave a higher proportion of beta-damascenone in the products than did the aglycon at 50 degrees C. Reaction temperature also effected the relative proportion of the hydrolysis products. Accelerated studies do not parallel natural processes precisely but only approximate them.

An abnormal response of retinoblastoma cells (Y-79) to neurotrophins.

PURPOSE: To clarify the expression of neurotrophins and their receptors in retinoblastoma (Rb) cells, to elucidate their potential role in the proliferation of neuroectodermal tumor cells, and to establish conditions for Rb cell differentiation. METHODS: The Rb-derived cell line Y-79 was grown in serum-free suspension or monolayer culture. Proliferating and differentiated cells were isolated and submitted to semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, immunostaining, and flow cytometry. The proliferation rate of the cells was estimated by 5-bromo-2'-deoxyuridine (BrdU) incorporation, and the effects of neurotrophins and laminin on BrdU-incorporation, process outgrowth, or immunostaining were determined. RESULTS: In contrast to previously studied normal retinal precursor cells, Y-79 cells not only express nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and p75, but also the corresponding high affinity receptors TrkA, TrkB, and TrkC. Proliferation was stimulated by exogenous and endogenous neurotrophin receptor ligands. Inhibition of protein kinase phosphorylation with K252a blocked proliferation and promoted differentiation. The effect of K252a on differentiation was enhanced by the addition of soluble laminin. After 9 days of combined treatment, the fraction of differentiated cells amounted to 30%, differentiation being characterized by improved attachment, neurite outgrowth, expression of NF-68, and a loss of glial fibrillary acidic protein (GFAP) and parvalbumin immunoreactivity. These changes were accompanied by a downregulation of TrkB and TrkC, but not TrkA or p75. Differentiated cells were isolated and further grown in the absence of K252a. However, despite the high level of TrkA expression in differentiated cells, the addition of NGF had no effect on their survival. CONCLUSIONS: A mitogenic action of neurotrophins could contribute to retinal tumor growth.

Quantitative analysis of 4-ethylphenol and 4-ethylguaiacol in red wine.

2,3,5,6-[2H4]-4-Ethylphenol (d4-4-ethylphenol) was synthesised for use as an internal standard in a new, rapid and accurate analytical method, employing gas chromatography-mass spectrometry to determine the concentration of the important aroma compounds 4-ethylphenol and 4-ethylguaiacol in red wine. The concentrations of both compounds in wine stored in 44 American and 47 French new and used oak barrels from several suppliers were measured. Wine stored in shaved and refired oak barrels contained up to 85% less 4-ethylphenol and 4-ethylguaiacol than wine stored in normal barrels of the same age that were not shaved. The concentration of 4-ethylphenol found in 61 bottled commercial Australian red wines of various ages ranged from 2 microg/l in a Merlot up to 2660 microg/l in a Shiraz, with a mean concentration of 795 microg/l. 4-Ethylguaiacol was also detected in every red wine analysed, ranging in concentration from 1 microg/l (in a Pinot Noir) up to 437 microg/l (in a Merlot) with a mean concentration of 99 microg/l.