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Electrophoretic identification and quantitation of compounds in the polyphenolic fraction of extra-virgin olive oil.

A capillary zone electrophoresis method has been carried out to determine and quantitate some compounds of the polyphenolic fraction of virgin olive oil which have never previously been determined before using capillary electrophoresis, such as elenolic acid, ligstroside aglycon, oleuropein aglycon, and (+)-pinoresinol. The compounds were identified using standards obtained by semipreparative high-performance liquid chromatography (HPLC). A detailed method optimization was performed to separate the phenolic compounds present in olive oil using a methanol-water extract of Picual extra-virgin olive oil, and different extraction systems were compared (C18-solid phase extraction (SPE), Diol-SPE, Sax-SPE and liquid-liquid extraction). The optimized parameters were 30 mM sodium tetraborate buffer (pH 9.3) at 25 kV with 8 s hydrodynamic injection, and the quantitation was carried out by the use of two reference compounds at two different wavelengths.

Chromatography, High Pressure Liquid↗

Preparation of porcine carotid arteries for vascular tissue engineering applications.

Biomaterials derived from tissue continue to offer viable alternatives to synthetic materials when autologous materials are unavailable for transplantation due to their unique chemical and mechanical properties. Tissue processing aims to stabilize the material against host degradation and render it immunologically inert by removing cellular material and crosslinking the structural proteins. It is clear that different approaches taken to achieve these goals have very different chemical and mechanical effects on the material. We describe herein the development of a tissue processing methodology to generate acellular scaffolds for tissue engineering small-diameter vascular grafts. Carotid arteries were isolated from Great White pigs and exposed to various solvent treatments, xylene, butanol, and ethanol to determine optimal parameters for the extraction of host lipids. The tissue was then exposed to a limited proteolysis with trypsin to disrupt cellular protein. This resulted in a controlled digestion that disrupted porcine nuclear DNA and cleared bulk cellular protein, leaving the more resistant structural proteins largely intact and retaining the bulk mechanical properties of the matrix. Histological analysis and scanning electron microscopy illustrated the complete removal of intact cells and nuclear material. The decellularized graft was stabilized by crosslinking with the photooxidative dye methylene green in the presence of 30,000 LUX of broad-band light energy. High-performance liquid chromatography analysis showed that the crosslinked tissue yielded 78.6% less hydroxyproline, compared with control tissue, after 20 h incubation with pepsin. Analysis of the crosslinked vessels' burst-pressure and stress-strain characteristics have shown comparable mechanical properties to those of control vessels. Assessment of in vitro cell adhesion and compatibility was conducted by seeding primary human umbilical vein endothelial cells and adult human vascular smooth muscle cells onto the lumenal and ablumenal surfaces, respectively; these cells were shown to adhere and proliferate under traditional static culture conditions.

Animals↗

Adhesion molecule expression by osteogenic cells cultured on various biodegradable scaffolds.

Design of tissue-engineered cell-loaded device involves cells seeding onto scaffolds in vitro, allowing them to settle and grow before in vivo transplantation. Interaction between scaffold and cells is important in the development of desired tissues. The present study aimed to investigate the effect of cell-polymer interactions on cell morphology and expression of surface markers of osteogenic MBA-15 cells cultured on various bioresorbable polymers. In this study, we used various polymers: poly(L-lactic acid) (PLLA), poly(DL-lactic acid) (PDLLA), poly(L-lactic-glycolic acid) (PLGA), and poly(DL-lactide-glycolide acid) PDLGA1 and PDLGA2. Expression of integrinalpha-M (CD11b), selectin-E (CD62E), and PECAM-1 (CD31), important in cell-cell and cell-matrix interactions, were quantified by flow-cytometry analysis. Cells grown on PDLGA1 films demonstrated fivefold increase in CD62E expression and two-folds increase in CD11b expression. None of the polymers affected the levels of CD31. Identified differential effect of polymers on the expression of cell-adhesion molecules by osteoprogenitors in vitro might help to choose optimal parameters for successful engraftment of cell-loaded constructs.

Animals↗

Abrasive water jet cutting as a new procedure for cutting cancellous bone--in vitro testing in comparison with the oscillating saw.

The quality of bone cuts is assessed by the accuracy and biological potency of the cut surfaces. Conventional tools (such as saws and milling machines) can cause thermal damage to bone tissue. Water jet cutting is nonthermal; that is, it does not generate heat. This study investigates whether the abrasive jet cutting quality in cancellous bone with a biocompatible abrasive is sufficient for the implantation of endoprostheses or for osteotomies. Sixty porcine femoral condyles were cut with an abrasive water jet and with an oscillating saw. alpha-lactose-monohydrate was used as a biocompatible abrasive. Water pressure (pW = 35 and 70 MPa) and abrasive feed rate (m = 0.5, 1, and 2 g/s) were varied. As a measure of the quality of the cut surface the cutting gap angle (delta) and the surface roughness (Ra) were determined. The surface roughness was lowest for an abrasive feed rate of m = 2 g/s (jet direction: 39 +/- 16 microm, advance direction: 54 +/- 22 microm). However, this was still significantly higher than the surface roughness for the saw group (jet direction: 28 +/- 12 microm, advance direction: 36 +/- 19 microm) (p < 0.001 for both directions). At both pressure levels the greatest cutting gap angle was observed for a mass flow rate of m = 1 g/s (pW = 35 MPa: delta = 2.40 +/- 4.67 degrees ; pW = 70 MPa: delta = 4.13 +/- 4.65 degrees), which was greater than for m = 0.5 g/s (pW = 35 MPa: delta = 1.63 +/- 3.89 degrees ; pW = 70 MPa: delta = 0.36 +/- 1.70 degrees) and m = 2 g/s (pW =70 MPa: delta = 0.06 +/- 2.40 degrees). Abrasive water jets are suitable for cutting cancellous bone. The large variation of the cutting gap angle is, however, unfavorable, as the jet direction cannot be adjusted by a predefined value. If it is possible to improve the cutting quality by a further parameter optimization, the abrasive water jet may be the cutting technique of the future for robotic usage.

Animals↗

A review of fetal organ measurements obtained with ultrasound: normal growth.

This review article presents fetal organ measurements obtained with ultrasound. Data derived from measurements of the brain, heart, lung, thymus, liver, spleen, pancreas, stomach, gallbladder, kidney, adrenal glands, intestine, and bladder are summarized, compared and evaluated critically. The selection of optimal parameters, development of valid measurement procedures, and use of mathematical modeling and descriptive statistics are necessary for quantitative studies by ultrasound of fetal organ growth.

Abdomen↗

Regional myocardial blood volume and flow: first-pass MR imaging with polylysine-Gd-DTPA.

The authors investigated the utility of an intravascular magnetic resonance (MR) contrast agent, poly-L-lysine-gadolinium diethylenetriaminepentaacetic acid (DTPA), for differentiating acutely ischemic from normally perfused myocardium with first-pass MR imaging. Hypoperfused regions, identified with microspheres, on the first-pass images displayed significantly decreased signal intensities compared with normally perfused myocardium (P < .0007). Estimates of regional myocardial blood content, obtained by measuring the ratio of areas under the signal intensity-versus-time curves in tissue regions and the left ventricular chamber, averaged 0.12 mL/g +/- 0.04 (n = 35), compared with a value of 0.11 mL/g +/- 0.05 measured with radiolabeled albumin in the same tissue regions. To obtain MR estimates of regional myocardial blood flow, in situ calibration curves were used to transform first-pass intensity-time curves into content-time curves for analysis with a multiple-pathway, axially distributed model. Flow estimates, obtained by automated parameter optimization, averaged 1.2 mL/min/g +/- 0.5 (n = 29), compared with 1.3 mL/min/g +/- 0.3 obtained with tracer microspheres in the same tissue specimens at the same time. The results represent a combination of T1-weighted first-pass imaging, intravascular relaxation agents, and a spatially distributed perfusion model to obtain absolute regional myocardial blood flow and volume.

Animals↗

Combined tag tracking and strain reconstruction from tagged cardiac MR images without user-defined myocardial contours.

PURPOSE: To develop an unsupervised method for measuring quantitative three-dimensional regional strain in the left ventricular wall from tagged cardiac MR images. MATERIALS AND METHODS: A total of 10 normal human volunteers and eight patients with myocardial infarction were imaged using a parallel tagged imaging protocol. Each study was analyzed using the combined tag tracking and strain reconstruction (COTTER) algorithm. In contrast to existing techniques, which first track tag lines independently in each slice, then reconstruct myocardial deformation, the COTTER algorithm fits a three-dimensional cardiac deformation model directly to the image data. This approach ensures that tag line positions identified in the image data are consistent from slice to slice. A total of 10 imaging studies (six normals, four patients) were used to optimize parameters of the COTTER algorithm. RESULTS: In the remaining eight imaging studies, the root-mean-square difference between tags tracked by COTTER and user-supervised analysis was 0.66 pixels at end-systole. The correlation coefficient between circumferential shortening strains at end-systole computed by COTTER and user-supervised analysis was 0.84 (P < 0.005) at the midwall. CONCLUSION: The COTTER algorithm can compute accurate measurements of three-dimensional regional strain without user supervision.

Algorithms↗

Magnetic resonance imaging artifacts caused by aneurysm clips and shunt valves: dependence on field strength (1.5 and 3 T) and imaging parameters.

PURPOSE: To evaluate artifact sizes at 3 T compared to at 1.5 T, and to evaluate the influence of scanning parameters with respect to artifact size on a 3-T magnetic resonance imaging (MRI) system. MATERIALS AND METHODS: Two aneurysm clips and five shunt valves were imaged in a water phantom at 1.5 and 3 T. At 3 T the influence of bandwidth (spin echo (SE) images) and echo time (gradient echo (GRE) images) on artifact size (area and extension in two orthogonal directions) was investigated. RESULTS: Artifact sizes increased substantially (typically 5-10 mm) at 3 T, compared to at 1.5 T, for implants entirely made of metallic materials, whereas the increase was the size less prominent (0-5 mm) for implants only partly containing metal. Artifact areas could be altered by changing the bandwidth or the echo time to about the same extent as it was affected by the increased field strength. CONCLUSION: Artifact sizes increase at 3 T, compared to at 1.5 T, depending on the type and composition of the implant, but can be substantially reduced by altering the imaging parameters. Optimization of imaging protocols to minimize artifacts is therefore important at higher field strengths.

Artifacts↗

In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3-year experience.

We report our experience in development of the in situ hybridization (ISH) procedure to detect messenger RNAs (mRNAs) coding for various molecules involved in endocrine glands and central nervous system activity, including mRNAs coding for endorphin precursors [preproenkephalin A (PPA), pro-opiocortin (POMC)], vasopressin, and transferrin. Various conditions of fixation and handling of the tissues were tested to establish optimal parameters for mRNA detection. Double-stranded DNA probes labeled by nick translation, synthetic oligonucleotides labeled at their 5' end, as well as single-stranded RNA probes were used, after incorporation of 32P- or 35S-labeled nucleotides. Specific requirements for efficient and reproducible ISH investigations are discussed. Cells expressing the PPA gene in the adrenal medulla and in the brain were detected by ISH. The results show that ISH is as sensitive as immunohistochemistry in detecting peptide-producing cells in the adrenal and that it allows detection of PPA cell bodies in brain in conditions in which they are inconstantly detected by immunohistochemistry. Unilateral destruction of substantia nigra provokes a dramatic decrease in the number of neurons expressing the PPA gene in the contralateral striatum. Cells expressing the POMC gene were detected in the pituitary of various species including man and in the rat arcuate nucleus. Neurons containing vasopressin mRNA were visualized in the supraoptic paraventricular and suprachiasmatic nucleus of the adult rat by using a synthetic oligonucleotide probe. Transferrin gene expression was shown in the central nervous system of the rat brain in two cell populations, the oligodendrocytes and the epithelial cells of the choroid plexus, by demonstration of simultaneous presence in them of transferrin immunoreactivity together with transferrin mRNA. These results show that the ISH procedure is a technique that can be routinely used to investigate gene transcription anatomically in complex heterocellular tissues such as the endocrine glands and the nervous system.

Adrenal Medulla↗

Optimization of near ultraviolet irradiation conditions for isolation of relaxed mutants of Escherichia coli.

We describe optimized conditions for isolation of relaxed mutants of bacteria with 4-thiouridine-containing tRNAs. The results presented here imply that besides the knowledge of the action spectra for near UV-induced growth inhibition of stringent and relaxed cells the fluence--fluence rate dependence of growth inhibition is an essential factor for optimizing the enrichment of relaxed mutants. We investigated systematically the dependence of growth inhibition of both stringent and relaxed strains of E. coli on the wavelength lambda, on the fluence F and on the fluence rate I within the ranges lambda = 301-365 nm, F = 0-48 kJ X m-2 and I = 0-60 W X m-2. The optimized conditions for selection of relaxed mutants of E. coli are lambda = 334 nm, F = 48 kJ X m-2 and I = 60 W X m-2. These optimized parameters determined for E. coli may be used in general to select relaxed mutants of other bacterial species by combining the turbidostat technique with near UV irradiation (Riesenberg et al. 1983).

Bacteriological Techniques↗

An assay to measure adriamycin binding in osteosarcoma cells.

Adjuvant chemotherapy is currently employed in the treatment of patients with osteosarcoma, but the drug regimens, although effective in improving disease-free survival, are unsuccessful in 20-40% of patients and very toxic. It would be useful to know whether tumor cells are sensitive to a given drug prior to its use. To this end, we developed a method of assessing Adriamycin (doxorubicin) binding to tumor nuclei as a possible means of detecting sensitivity to the drug. Adriamycin-sensitive murine osteosarcoma cells were used to develop the assay. The in vitro conditions (drug concentration, duration of incubation, and temperature) were optimized with use of the murine osteosarcoma cells in culture. After the cells had been incubated with Adriamycin, cell viability was determined and Adriamycin fluorescence intensity was measured with a cytofluorometer. The optimal parameters for Adriamycin binding were found to be a 30-minute incubation in a 10 micrograms/ml concentration of Adriamycin at 37 degrees C; the frequency of cells that emitted Adriamycin fluorescence from the nucleus compared with the total number of living cells reached 100% under these conditions. In a murine leukemia cell line with known sensitivity to Adriamycin, the cells emitted red fluorescence from the nucleus and cytoplasm, whereas in a resistant line the cells emitted Adriamycin fluorescence from only the cytoplasm. We demonstrated that it is possible to differentiate nuclear from cytoplasmic concentration of Adriamycin in a tumor cell with use of a fluorescent microscope and that resistant cell lines can be distinguished from sensitive cell lines by this method.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

A kinetic analysis of substance P trafficking.

The potential for administering substance P (SP) nocitoxins for the treatment of chronic pain has been identified. To characterize treatment protocols for the spinal cord or elsewhere, binding/internalization of these compounds at the cellular targets must be understood quantitatively. Thus, a kinetic model of SP binding and intracellular trafficking has been developed from data. The eight differential equation model describes surface binding between SP and neurokinin 1 receptor, clathrin-mediated endocytosis followed by spatial translation to a perinuclear endosome where SP is sorted from its receptor, SP degradation in late endosomes/early lysosomes, and return of sorted receptor to plasma membrane via recycling endosomes. With suitably optimized parameters, the model accounts for the kinetics of total, membrane-associated, and internalized SP in cells continuously exposed to SP, as well as the fractions of internalized SP remaining intact at 30 and 60 min. Simultaneously, the model accounts for the kinetics of internalization and receptor recycling after SP preloading of membrane and subsequent exposure to SP-free media. Rate constants (min(-1)) are: 0.034 +/- 0.004 (receptor off-rate), 0.15 +/- 0.03 (internalization), 0.048 +/- 0.003 (exit from sorting endosome), 0.062 +/- 0.008 (exit of labeled SP amino acids from prelysosome), and 0.029 +/- 0.004 (receptor return from recycling endosome to plasma membrane). The SP kinetics resemble those of transferrin and its receptor at the internalization step, but are several-fold slower in the sorting and recycling steps.

Algorithms↗

The determination of sulfonamides in honey by high performance liquid chromatography--mass spectrometry method (LC/MS).

The sulfonamides (SAs) are stable chemotherapeutics used against the bacterial disease affecting bees, known as American foulbrood (Bacillus larvae), so their residues could appear in the honey of treated bees. Their presence at a concentration above the limit value could be a potential danger to human health. Therefore, a simple, rapid, and reliable method for determination of 11 available SAs in honey was optimized. The samples were homogenized and cleaned with extraction on solid phase by means of Chromabond C18 end-capped cartridge followed by LC/MS analyses. A detection limit of 25 microg/kg was achieved for all analytes. The repeatability of the method was proven and the optimal parameters for temperature and pH of the mobile phase and acetic buffer, respectively, were determined. In this study, 20 samples of domestic honey were included. Six of the analyzed samples were positive, but all results were below the Croatian permissible limit value (100 microg/kg).

Anti-Infective Agents↗

Pulsed Ho:YAG laser meniscectomy: effect of pulsewidth on tissue penetration rate and lateral thermal damage.

BACKGROUND AND OBJECTIVE: Studies need to define the optimal parameters under which the holmium laser should operate for arthroscopic meniscectomy. This study was designed to analyze the effect of various Holmium wavelength pulsewidths on human meniscal tissue penetration rates and lateral thermal injury. STUDY DESIGN/MATERIALS AND METHODS: Using a pulsed Holmium: YAG laser at a wavelength of 2.1 microns, the effect of various pulse-widths on tissue penetration rates as well as the degree of accompanying thermal damage in human meniscal tissue was evaluated in a specially designed jig. Holding the energy constant at 500 mJ per pulse, the pulsewidth was varied between 100 and 600 microseconds. RESULTS: Fiber penetration of meniscal tissue was found to be fastest at a pulsewidth of 250 microseconds. As the pulsewidth was increased or decreased around this number, the observed penetration time decreased, although no statistical difference was found. The size of the hole created was inversely related to the penetration time. Microscopic examination revealed zones of lateral thermal effect extending 800 microns from the ablation site. CONCLUSION: No relationship between the pulsewidth and the lateral thermal effect could be found.

Adult↗

Electroconvulsive therapy in Parkinson's disease and other movement disorders.

Early case reports note marked improvements in the signs of Parkinson's disease (PD) in several patients with coexisting psychiatric disorders after treatment with electroconvulsive therapy (ECT). Studies since 1959 reveal improvement of parkinsonism in over half of PD patients receiving ECT, regardless of the presence or absence of psychiatric comorbidity. Drug-induced parkinsonism, tardive dystonia, and tardive dyskinesia have also been shown to improve with ECT administration; tic syndromes have achieved mixed results. In animals, ECT enhances dopamine-mediated effects and increases GABA concentrations in the CNS. Optimal parameters relevant to the antiparkinsonism effects of ECT require further study.

Depressive Disorder↗

Protein fold recognition without Boltzmann statistics or explicit physical basis.

We present a fast method for finding optimal parameters for a low-resolution (threading) force field intended to distinguish correct from incorrect folds for a given protein sequence. In contrast to other methods, the parameterization uses information from >10(7) misfolded structures as well as a set of native sequence-structure pairs. In addition to testing the resulting force field's performance on the protein sequence threading problem, results are shown that characterize the number of parameters necessary for effective structure recognition.

Algorithms↗

Folding of a small helical protein using hydrogen bonds and hydrophobicity forces.

A reduced protein model with five to six atoms per amino acid and five amino acid types is developed and tested on a three-helix-bundle protein, a 46-amino acid fragment from staphylococcal protein A. The model does not rely on the widely used Go approximation, which ignores non-native interactions. We find that the collapse transition is considerably more abrupt for the protein A sequence than for random sequences with the same composition. The chain collapse is found to be at least as fast as helix formation. Energy minimization restricted to the thermodynamically favored topology gives a structure that has a root-mean-square deviation of 1.8 A from the native structure. The sequence-dependent part of our potential is pairwise additive. Our calculations suggest that fine-tuning this potential by parameter optimization is of limited use.

Amino Acid Sequence↗

Molecular modeling of an antigenic complex between a viral peptide and a class I major histocompatibility glycoprotein.

Computer simulation of the conformations of short antigenic peptides (5-10 residues) either free or bound to their receptor, the major histocompatibility complex (MHC)-encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energy-minimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall alpha-helical conformation through regular i,i + 4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i + 3 hydrogen bonds at their N-terminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H-2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energy-minimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template-derived optimal parameters. The bound peptide retains mainly its alpha-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts alpha-helical conformation for the bound peptide.

Computer Simulation↗