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PubMed2003

Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous.

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.

Basidiomycota↗
PubMed2003

Nanocapillary array interconnects for gated analyte injections and electrophoretic separations in multilayer microfluidic architectures.

An electrokinetic injection technique is described which uses a nuclear track-etched nanocapillary array to inject sample plugs from one layer of a microfluidic device into another vertically separated layer for electrophoretic separations. Gated injection protocols for analyte separations, reported here, establish nanocapillary array interconnects as a route to multilevel microfluidic analytical designs. The hybrid nanofluidic/microfluidic gated injection protocol allows sample preparation and separation to be implemented in separate horizontal planes, thereby achieving multilayer integration. Repeated injections and separations of FITC-labeled arginine and tryptophan, using 200-nm pore-diameter capillary array injectors in place of traditional cross injectors are used to demonstrate gated injection with a bias configuration that uses relay switching of a single high-voltage source. Injection times as rapid as 0.3 s along with separation reproducibilities as low as 1% for FITC-labeled arginine exemplify the capability for fast, serial separations and analyses. Impedance analysis of the micro-/nanofluidic network is used to gain further insight into the mechanism by which this actively controlled nanofluidic-interconnect injection method works. Gated sample introduction via a nanocapillary array interconnect allows the injection and separation protocols to be optimized independently, thus realizing the versatility needed for real-world implementation of rapid, serial microchip analyses.

Arginine↗
PubMed2003

Indium microrod tags for electrochemical detection of DNA hybridization.

The preparation and advantages of indium microrod tracers for solid-state electrochemical detection of DNA hybridization are described. The cylindrical metal particles were prepared by a template-directed electrochemical synthetic route involving plating of indium into the pores of a host membrane. The linear relationship between the charge passed during the preparation and the resulting particle size allows tailoring of the sensitivity of the electrical DNA assay. The resulting micrometer-long rods thus offer a greatly lower detection limit (250 zmol), as compared to common bioassays' spherical nanoparticle tags. Indium offers a very attractive electrochemical stripping behavior and is not normally present in biological samples or reagents. Solid-state derivative-chronopotentiometric measurements of the indium tracer have been realized through a "magnetic" collection of the DNA-linked particle assembly onto a thick-film electrode transducer. Factors affecting the performance, including the preparation of the microrods and pretreatment of the transducer surface, were evaluated and optimized. The resulting protocol offers great promise for other affinity bioassays, as well as for electrical coding and identification (through the plating of different metal markers and of multimetal redox-encoded tags).

Base Pairing↗
PubMedDate not supplied

Co-aggregation of penicillin g acylase and polyionic polymers: an easy methodology to prepare enzyme biocatalysts stable in organic media.

A novel type of biocatalyst that combines the good properties of cross-linked enzyme aggregates (CLEAs) and hydrophilic microenvironments has been developed. Dextran sulfate- and polyethyleneimine-coated CLEAs of penicillin acylase (CLEA-GDP) were prepared by adding the polymers of different sizes before the precipitation stage of the enzyme. This study presents the development and optimization of a protocol to produce such a biocatalyst using penicillin acylase as a model. Experiments show that CLEA-GDPs have a highly increased stability in organic media. The average half-life of the preparations was much higher than standard CLEA without a microenvironment (CLEA-G), (e.g., more than 25-fold) in the presence of dioxane. However, their thermal stability was not increased, which leads to the conclusion that the stability of CLEA-GDPs in organic media is due to the hydrophilic microenvironment that surrounds the protein enzyme more than to a conformational stiffening effect. This is further supported by solvation experiments that show a preferential hydration of CLEA when polymers are used to coat the enzyme. CLEA-GDPs are clearly better than other biocatalysts in terms of solvent stability.

Catalysis↗
PubMed2003

On the nature of the multivalency effect: a thermodynamic model.

A quantitative model is proposed for the analysis of the thermodynamic parameters of multivalent interactions in dilute solutions or with immobilized multimeric receptor. The model takes into account all bound species and describes multivalent binding via two microscopic binding energies corresponding to inter- and intramolecular interactions (Delta G(o)inter and Delta G(o)intra), the relative contributions of which depend on the distribution of complexes with different numbers of occupied binding sites. The third component of the overall free energy, which we call the "avidity entropy" term, is a function of the degeneracy of bound states, Omega(i), which is calculated on the basis of the topology of interaction and the distribution of all bound species. This term grows rapidly with the number of receptor sites and ligand multivalency, it always favors binding, and explains why multivalency can overcome the loss of conformational entropy when ligands displayed at the ends of long tethers are bound. The microscopic parameters and may be determined from the observed binding energies for a set of oligovalent ligands by nonlinear fitting with the theoretical model. Here binding data obtained from two series of oligovalent carbohydrate inhibitors for Shiga-like toxins were used to verify the theory. The decavalent and octavalent inhibitors exhibit subnanomolar activity and are the most active soluble inhibitors yet seen that block Shiga-like toxin binding to its native receptor. The theory developed here in conjunction with our protocol for the optimization of tether length provides a predictive approach to design and maximize the avidity of multivalent ligands.

Kinetics↗
PubMed2006

Optimization, standardization, and testing of a new NMR method for the determination of zeolite host-organic guest crystal structures.

An optimized and automated protocol for determining the location of guest sorbate molecules in highly siliceous zeolites from (29)Si INADEQUATE and (1)H/(29)Si cross polarization (CP) magic-angle spinning (MAS) NMR experiments is described. With the peaks in the (29)Si MAS NMR spectrum assigned to the unique Si sites in the zeolite framework by a 2D (29)Si INADEQUATE experiment, the location of the sorbate molecule is found by systematically searching for sorbate locations for which the measured rates of (1)H/(29)Si cross polarization of the different Si sites correlate linearly with (1)H/(29)Si second moments calculated from H-Si distances. Due to the (1)H/(29)Si cross polarization being in the "slow CP regime" for many zeolite-sorbate complexes, it is proposed that the CP rate constants are best measured by (1)H/(29)Si cross polarization drain experiments, if possible, to avoid complications that may arise from fast (1)H and (29)Si T(1)rho relaxations. An algorithm for determining the sorbate molecule location is described in detail. A number of ways to effectively summarize and display the large number of solutions which typically result from a prediction of the structure from the CP MAS NMR data are presented, including estimates of the errors involved in the structure determinations. As a working example throughout this paper, the structure of the low loaded p-dichlorobenzene/ZSM-5 complex is determined under different conditions from solid-state (1)H/(29)Si CP MAS NMR data, and the solutions are shown to be in excellent agreement with the known single-crystal X-ray diffraction structure. This structure determination approach is shown to be quite insensitive to the use of relative rate constants rather than absolute values, to the detailed structure of the zeolite framework, and relatively insensitive to temperature and motions.

Journal Article↗
PubMed2003

Patulin in Italian commercial apple products.

Patulin is a mycotoxin produced by microscopic fungi belonging to the Penicillium and Aspergillus genera. The natural occurrence of patulin in four apple products marketed in Italy and purchased from the supermarket, herbalist, and retail shops was studied. Thirty-three samples of the four products had no detectable patulin contamination. The 11 positive samples had a concentration ranging between 1.4 and 74.2 microg/L with a mean of 26.7 microg/L. All vinegar samples were negative for patulin; of 10 apple-based baby foods, two samples were contaminated with 17.7 and 13.1 microg/L and both were labeled as "organic food". Comparing organic and conventional agricultural practices, no significant differences were found. Finally, optimization of extraction protocol more general and useful for juices, clarified juices, baby foods, vinegars, and purees was performed. The low incidence of the patulin level in Italian apple products is a clear parameter to judge the quality of the fruit, and the process is of a high standard.

Acetic Acid↗
PubMed2004

Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA.

An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.

Animal Feed↗
PubMed2005

Direct atom-efficient esterification between carboxylic acids and alcohols catalyzed by amphoteric, water-tolerant TiO(acac)2.

A diverse array of oxometallic species were examined as catalysts for a test direct condensation of benzoic acid and 2-phenylethanol in 1:1 stoichiometry. Besides group IVB MOCl2-xH2O and TiOX2-xH2O, group VB VOCl2-xTHF and group IVB TiO(acac)2 were found to be the most efficient and water-tolerant catalysts for the test reaction. The new neutral catalytic protocol with the optimal TiO(acac)2 tolerates many stereo/electronic structural variations in both (di)acid (1 degree -3 degrees alkyl and aryl) and (di)alcohol (1 degree, 2 degrees alkyl, and aryl) components with high chemoselectivity.

Journal Article↗
PubMed2006

Bioactive galactose-branched polyelectrolyte multilayers and microcapsules: self-assembly, characterization, and biospecific lectin adsorption.

We describe the fabrication of multilayers and microcapsules with biologically designed targeting activity using chemoenzymatic synthesized carbohydrate-branched polyelectrolytes. A novel cationic d-galactose-branched copolymer [poly(vinyl galactose ester-co-methacryloxyethyl trimethylammonium chloride), PGEDMC] is alternated with poly(styrene sulfonate) (PSS) to form thin multifilms by the layer-by-layer (LbL) technique on such different solid surfaces as quartz slides, poly(ethylene terephthalate) (PET) films, silicon wafers, and polystyrene (PS) microparticles. The experimental protocols were first optimized on flat, smooth silica substrates using UV-vis, contact angle, and atomic force microscopy (AFM) measurements. The film properties of PGEDMC/PSS multilayers are modified by varying polyelectrolyte concentration, ionic strength, and counteranion types. Hollow capsules were formed after the removal of colloidal templates; transmission (TEM) and scanning (SEM) electron microscopy were used to verify the LbL process integrity. PGEDMC/PSS planar films and capsules carrying beta-galactose as recognition signals have specific recognition abilities with peanut agglutinin (PNA) lectin rather than concanavalin A (Con A) lectin observed by fluorescence spectroscopy.

Adsorption↗
PubMedDate not supplied

Analyzing alkaline proteins in human colon crypt proteome.

Normal human colon crypt protein extract was resolved by two-dimensional gel electrophoresis using pH 6-11 immobilized pH gradient strips in the first dimension. The optimized isoelectric focusing protocol includes cup-loading sample application at the anode and 1.2% hydroxyethyl disulfide (DeStreak), 15% 2-propanol and 5% glycerol in the rehydration buffer. Spots were well resolved across the entire pH range up to 11. A total of 311 protein spots were identified by mass spectrometry and peptide mass mapping. After combining isoforms, 231 nonredundant proteins were grouped into 16 categories according to their subcellular locations, and 17 categories according to their physiological functions. Histone proteins, ribosomal proteins and mitochondrial proteins were among the well-resolved highest p/ proteins. Application of this protocol to the analysis of normal and neoplastic colon crypts will contribute to the proteomic study of colorectal tumorigenesis since a significant portion of the human proteins is in basic pH range.

Cell Extracts↗
PubMed2007

Immunoaffinity purification of plasma membrane with secondary antibody superparamagnetic beads for proteomic analysis.

Plasma membrane (PM) has very important roles in cell-cell interaction and signal transduction, and it has been extensively targeted for drug design. A major prerequisite for the analysis of PM proteome is the preparation of PM with high purity. Density gradient centrifugation has been commonly employed to isolate PM, but it often occurred with contamination of internal membrane. Here we describe a method for plasma membrane purification using second antibody superparamagnetic beads that combines subcellular fractionation and immunoisolation strategies. Four methods of immunoaffinity were compared, and the variation of crude plasma membrane (CPM), superparamagnetic beads, and antibodies was studied. The optimized method and the number of CPM, beads, and antibodies suitable for proteome analysis were obtained. The PM of mouse liver was enriched 3-fold in comparison with the density gradient centrifugation method, and contamination from mitochondria was reduced 2-fold. The PM protein bands were extracted and trypsin-digested, and the resulting peptides were resolved and characterized by MALDI-TOF-TOF and ESI-Q-TOF, respectively. Mascot software was used to analyze the data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which 67% are PM or PM-related proteins. No endoplasmic reticulum (ER) or nuclear proteins were identified according to the GO annotation in the optimized method. Our protocol represents a simple, economic, and reproducible tool for the proteomic characterization of liver plasma membrane.

Animals↗
PubMed1998

Assessment of vascularity in histological sections: effects of methodology and value as an index of angiogenesis in breast tumours.

The aims of this study were to (a) determine how the quantification of blood vessels in histological sections (vascularity) is affected by the methodology used and (b) assess the value of vascularity as an index of angiogenesis by comparing tumour and normal breast tissue. Archival specimens of breast, lung and oral carcinoma, oral dysplasia and normal breast tissue were used to test the effects of the following experimental variables on vascularity: pretreatment of the sections (enzymatic digestion, heating), endothelial markers (von Willebrand factor and CD31 antibodies), method of quantification (highest microvascular density, average microvascular density and microvascular volume) and interobserver variations. All the variables examined significantly affected the estimated vascularity; this depended on the type of tissue and method used. The pretreatment of the sections before staining was the most important variable, altering the vascularity ranking of the tumours. Vascularity in breast tumours was similar to that of the normal breast intralobular stroma, suggesting that an area of high microvascular density in the tumour does not necessarily represent tumour-induced angiogenesis. Contradictory results have been published regarding the value of vascularity as a tumour prognostic factor. Our results suggest that statistically significant differences in vascularity values are most likely to arise from failure to optimize the staining protocol and from the method used to assess vascularity.

Blood Vessels↗
PubMed1999

Effect of thermal variables on human breast cancer in cryosurgery.

There is a growing interest in the use of cryosurgery to treat breast cancer, following recent breakthroughs in noninvasive imaging and in cryotechnology, as well as the recent success of cryosurgery in treating various types of cancer. However, since haphazard freezing does not guarantee tissue destruction, in order to apply this technique effectively it is essential to determine the thermal parameters that produce complete destruction of malignant tissue. This study seeks to quantitatively identify the relationship between thermal variables and the degree of freezing damage to human breast cancer cells. In order to do this, human breast cancer and normal cells were frozen with controlled thermal parameters using a directional solidification apparatus. Cell viability was determined after thawing using trypan blue, and correlated to the thermal variables used during freezing. Cellular damage is observed to increase with increasing cooling rates, due to the higher probability of intracellular ice formation. A double freeze thaw cycle significantly increases the extent of cell damage, and is sufficient to ensure complete cell destruction at final freezing temperatures of -40 degrees C for a 25 degrees C/min cooling rate, and -20 degrees C for a 50 degrees C/min cooling rate. The correlations between cell death and thermal parameters are qualitatively identical for all the cell types in this study, although there is some variation from one cell type to another in the overall susceptibility to freezing damage. The correlations established in this study can be used to design systematic and optimal breast cryosurgery protocols.

Breast Neoplasms↗
PubMed2003

Amyloid-beta immunization in Alzheimer's disease transgenic mouse models and wildtype mice.

Alzheimer's disease is the most prevalent form of dementia worldwide. Therapies are desperately needed to prevent and cure the disease. Mouse models of amyloid-beta deposition [APP and PSAPP transgenic (tg) mice] have been useful in determining the role of amyloid-beta (A beta) in both the pathogenesis and cognitive changes in AD. In addition, they have allowed scientists to investigate potential AD therapies in living animals. Active and passive A beta immunizations have been employed successfully in APP and PSAPP tg mice to lower cerebral A beta levels and improve cognition. Optimization of immunization protocols and characterization of immune responses in wildtype mice have been reported. Based on the promising results of A beta immunization studies in mice, a clinical trial was initiated for A beta vaccination in humans with AD. Although no adverse effects were reported in the Phase I safety trials, about 5% of AD patients in the phase II clinical trial developed meningoencephalitis, ending the trial prematurely in March 2002. Studies in AD mouse models and wildtype mice may help elucidate the mechanism for these unwanted side effects and will be useful for testing newer, safer vaccines for future use in human clinical trials.

Alzheimer Disease↗
PubMed2002

Islet/pancreas transplantation: challenges for pediatrics.

Beta cell replacement is a valid alternative to exogenous insulin injections to treat type 1 diabetic patients. The rate of success obtained after whole-pancreas transplantation, performed alone or in combination with kidney, and, as shown recently, by islet transplantation, justifies optimism and sets the stage for a larger clinical application of these approaches. Lifetime immunosuppression, however, required to protect the graft against recurrent autoimmune destruction and allorejection, raises serious doubts about the safety of its employment in children. While it is evident that children may be helped even more than adults by the possibility to correct diabetic metabolic disorders without exogenous insulin, and to lower in a more effective way the chance to develop secondary complications, the drawbacks of the currently used immunosuppressive drugs largely overcome the potential benefits. A great step forward for immediate applicability of transplantation to children involves the optimization of tolerogenic protocols and a better understanding of the concept of immune ignorance. Functional tolerance should be sufficient to entail the absence of immune reactivity against self- and graft antigens, while maintaining immune reactivity against other non-self, non-donor antigens. In addition, novel strategies aimed at utilizing surrogate beta cells obtained from non-islet cells, or by genetic manipulation of beta-cell precursors merit consideration as the use of xenogeneic donors. However, much work is still needed for their safe clinical implementation.

Journal Article↗
PubMed2000

Dynamic evaluation of fluid shifts during normothermic and hypothermic cardiopulmonary bypass in piglets.

BACKGROUND: Edema, generalized overhydration and organ dysfunction commonly occur in patients undergoing open-heart surgery using cardiopulmonary bypass (CPB) and induced hypothermia. Activation of inflammatory reactions induced by contact between blood and foreign surfaces are commonly held responsible for the disturbances of fluid balance ("capillary leak syndrome"). We used an online technique to determine fluid shifts between the intravascular and the interstitial space during normothermic and hypothermic CPB. METHODS: Piglets were placed on CPB (fixed pump flow) via thoracotomy in general anesthesia. In the normothermic group (n=7), the core temperature was kept at 38 degrees C prior to and during 2 h on CPB, whereas in the hypothermic group (n=7) temperature was lowered to 28 degrees C during bypass. The CPB circuit was primed with acetated Ringer's solution. The blood level in the CPB circuit reservoir was held constant during bypass. Ringer's solution was added when fluid substitution was needed (falling blood level in the reservoir). In addition to invasive hemodynamic monitoring, fluid input and losses were accurately recorded. Inflammatory mediators or markers were not measured in this study. RESULTS: Cardiac output, s-electrolytes and arterial blood gases were similar in the two groups in the pre-bypass period. At start of CPB the blood level in the machine reservoir fell markedly in both groups, necessitating fluid supplementation and leading to a markedly reduced hematocrit. This extra fluid need was transient in the normothermic group, but persisted in the hypothermic animals. After 2 h of CPB the hypothermic animals had received 7 times more fluid as compared to the normothermic pigs. CONCLUSION: We found strong indications for a greater fluid extravasation during hypothermic CPB compared with normothermic CPB. The experimental model using the CPB-circuit reservoir as a fluid gauge gives us the opportunity to study further fluid volume shifts, its causes and potential ways to optimize fluid therapy protocols.

Animals↗
PubMed1991

Compartmental analysis of [11C]flumazenil kinetics for the estimation of ligand transport rate and receptor distribution using positron emission tomography.

The in vivo kinetic behavior of [11C]flumazenil ([11C]FMZ), a non-subtype-specific central benzodiazepine antagonist, is characterized using compartmental analysis with the aim of producing an optimized data acquisition protocol and tracer kinetic model configuration for the assessment of [11C]FMZ binding to benzodiazepine receptors (BZRs) in human brain. The approach presented is simple, requiring only a single radioligand injection. Dynamic positron emission tomography data were acquired on 18 normal volunteers using a 60- to 90-min sequence of scans and were analyzed with model configurations that included a three-compartment, four-parameter model, a three-compartment, three-parameter model, with a fixed value for free plus nonspecific binding; and a two-compartment, two-parameter model. Statistical analysis indicated that a four-parameter model did not yield significantly better fits than a three-parameter model. Goodness of fit was improved for three- versus two-parameter configurations in regions with low receptor density, but not in regions with moderate to high receptor density. Thus, a two-compartment, two-parameter configuration was found to adequately describe the kinetic behavior of [11C]FMZ in human brain, with stable estimates of the model parameters obtainable from as little as 20-30 min of data. Pixel-by-pixel analysis yields functional images of transport rate (K1) and ligand distribution volume (DV"), and thus provides independent estimates of ligand delivery and BZR binding.

Adult↗