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Agarose Gel (agarose + gel)
Terms modified by Agarose Gel Selected AbstractsPolyamines interact with DNA as molecular aggregatesFEBS JOURNAL, Issue 17 2002Luciano D'Agostino New compounds, named nuclear aggregates of polyamines, having a molecular mass of 8000, 4800 and <,1000 Da, were found in the nuclear extracts of several replicating cells. Their molecular structure is based on the formation of ionic bonds between polyamine ammonium and phosphate groups. The production of the 4800 Da compound, resulting from the aggregation of five or more <,1000 Da units, was increased in Caco-2 cells treated with the mitogen gastrin. Dissolving single polyamines in phosphate buffer resulted in the in vitro aggregation of polyamines with the formation of compounds with molecular masses identical to those of natural aggregates. After the interaction of the 4800 Da molecular aggregate with the genomic DNA at 37 °C, both the absorbance of DNA in phosphate buffer and the DNA mobility in agarose gel increased greatly. Furthermore, these compounds were able to protect the genomic DNA from digestion by DNase I, a phosphodiesterasic endonuclease. Our data indicate that the nuclear aggregate of polyamines interacts with DNA phosphate groups and influence, more efficaciously than single polyamines, both the conformation and the protection of the DNA. [source] A case of mucosal leishmaniasis: beneficial usage of polymerase chain reaction for diagnosisINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 12 2001Hironori Onuma MD A 36-year-old woman, who had emigrated from Japan to Paraguay as a 4-year-old child before returning to Japan in 1991, visited our clinic on November 10, 1997. She had suffered from a persistent ulcer on her forearm as a 6-year-old child and received intravenous injections for a few months, although she did not remember the details of therapy. Since May 1997, she had been aware of redness and swelling on her nose and had been treated with topical corticosteroid, but no improvement had been noted. Physical examination revealed erythematous plaque with crust from the left internal naris to nasolabial region (Fig. 1a). The atrophic plaque that had resulted from prolonged ulceration was found on the right forearm (Fig. 1b). In a biopsy specimen from the erythematous plaque on the nasolabial region, mononuclear dermal infiltrate, consisting of lymphocytes and histiocytes, was seen (Fig. 2a). The histiocytes were filled with Leishman-Donovan (L-D) bodies on a Giemsa staining sample (Fig. 2b). Fiberscopic examination revealed white plaque in the pharynx. The biopsy from the affected mucosa showed the same histopathological finding as with the skin. Figure 1. (a) Erythematous plaque with crust from the left internal naris to nasolabial region. (b) Atrophic plaque on the right forearm Figure 2. (a) In the biopsy specimen from the erythematous plaque on the nasolabial region, a mononuclear dermal infiltrate consisting of lymphocytes and histiocytes was seen. (Hematoxylin-Eosin stain, × 100) (b) The histiocytes were filled with Leishman-Donovan bodies. (Giemsa staining, × 400) Total DNA was purified from the skin biopsy specimen for polymerase chain reaction (PCR) analysis using a specific primer for L (V) braziliensis.1,2 A 70-bp product was amplified (Fig. 3a); furthermore, the specificity of the PCR product was confirmed by Southern hybridization with the probe for L (V) braziliensis (Fig. 3b) and DNA sequence analysis (data not shown). From December 2, 1997, the patient received 20 mg/kg/day sodium stibogluconate (PentostamTM) intravenously for 20 days. After 5 days of treatment, the redness and swelling of the skin lesion was improved, and faint erythema remained at the end of 20 days' treatment. After a 2-week interval, since the erythema remained, another 20-day treatment was performed. All of the skin lesion became scar tissue and L-D bodies could not be found in a skin biopsy specimen. However, L-D bodies were still found in a biopsy from the pharyngeal mucosa that had a normal appearance. Though another additional treatment was planned, the patient refused it. Figure 3. (a) The results of PCR. 70-bps bands appear in lanes 2 and 6. Lane 1, a size marker (pUC19/HapII); lane 2, DNA extracted from the formalin-fixed patient's sample; lane 3, DNA extracted from a formalin-fixed control sample; lane 4, DNA (,); lane 5, DNA extracted from L (V) tropica; lane 6, DNA extracted from L (V) braziliensis. (b) Results of Southern blotting using the PCR products. The PCR products were transferred from agarose gel as shown in Fig. 3 (a). Specific probes were hybridized with 70-bps bands on lanes 2 and 6 [source] Preparation of monomethyl poly(ethylene glycol)- g -chitosan copolymers with various degrees of substitution: Their ability to encapsulate and condense plasmid DNAJOURNAL OF APPLIED POLYMER SCIENCE, Issue 5 2008Wei Zhang Abstract Chitosan (CS) has great potential as a nonvirus gene delivery vector, but its application is limited because of poor water solubility. Monomethyl poly(ethylene glycol) (mPEG)- graft -CS copolymers were synthesized by the reaction of mPEG,aldehyde (oxidized mPEG) with amino groups on CS chains; they showed enhanced solubility in water. Copolymers with various mPEG degrees of substitution (DS) and CS molecular weights were obtained, and their capabilities of DNA encapsulation were compared through gel retardation assay and particle size and , potential measurements. The effects of different ratios of primary amines on CS to the phosphate groups on DNA (N/P ratios), DS, and molecular weights on particle size and encapsulation efficiency were investigated. The results show that high N/P ratios and proper DS were necessary for the formation of well-distributed complex particles. Among all of these samples, mPEG (3.55),CS (50 kDa)/DNA complexes [where the parentheses following mPEG indicate DS (%), and the parentheses following CS indicate the molecular weight of CS] raised the , potential from negative to positive most quickly, yielded the smallest particle size, and were retarded in agarose gel at the lowest N/P ratio; this indicated the best efficiency of DNA encapsulation. On the contrary, mPEG (0.80),CS (50 kDa)/DNA complexes raised the , potential to positive most slowly, fluctuated around the value 0 from N/P ratios of 15 : 1 to 30 : 1, and were retarded in agarose gel at the highest N/P ratio; this indicated the lowest efficiency of encapsulating plasmids. Copolymers with desirable efficiencies of DNA encapsulation could be promising gene carriers. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] Fabrication by three-phase emulsification of pellicular adsorbents customised for liquid fluidised bed adsorption of bioproductsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2003Mohsen Jahanshahi Abstract A novel dense pellicular adsorbent, custom-designed for liquid fluidised bed adsorption of protein bioproducts, has been fabricated by coating zirconia,silica particles with agarose gel in a three-phase emulsification process. A slurry feedstock comprising solid zirconia,silica particles (120 µm average diameter) suspended in an aqueous solution of agarose was emulsified in an oil,surfactant mixture in a stirred vessel to yield composite droplets. These were subsequently stabilised by cooling to form spherical pellicular particles characterised by a porous, pellicular coat cast upon a solid core. The impact of agitation speed, surfactant concentration, oil viscosity and slurry composition upon the pellicle depth and overall particle diameter was investigated. Pellicle depth decreased with increasing impeller speed and decreased oil viscosity, whilst increased slurry viscosity enhanced that parameter. Initial increases from low concentrations of Span 80 surfactant (0.1% w/v oil) reduced the depth of the agarose pellicle, but the highest values investigated (1.5% w/v oil) promoted particle aggregation. The fluidisation behaviour of particles fabricated under various conditions was characterised by the measurement of expansion coefficients and axial dispersion coefficients for the liquid phase when operated in a standard fluidised bed contactor. Both parameters were found to be comparable or superior to those reported for conventional, composite fluidised bed adsorbents. The controlled coating of porous agarose upon a solid core to yield specific pellicular geometries is discussed in the context of the fabrication of adsorbents customised for the recovery of a variety of bioproducts (macromolecules, nanoparticulates) from complex particulate feedstocks (whole broths, cell disruptates and unclarified bio-extracts). Given the agreement between the size of the pellicular particles and the trends expected from theory, the large-scale manufacture of such particles for customised industrial use is recommended. Copyright © 2003 Society of Chemical Industry [source] WHEN POSITIVELY CHARGED MILK PROTEINS CAN BIND TO DNAJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2002MAHMOUD SITOHY ABSTRACT The binding of three esterified milk proteins (,-lactoglobutin, ,-lactalbumin and ,-casein) to plasmid DNAs at pH 7.1 was followed by agarose-gel electrophoresis. Highly esterified ,-lactoglobulin and ,-lactalbumin samples showed DNA-binding capacities comparable to those exhibited by native basic proteins such as lysozymes and histones. All the studied esterified ,-casein samples failed to bind to DNA at the applied pH. Complete retardation of DNA migration on agarose gel was observed at a 1:1 ratio of protein basic groups (Lys + Arg) to DNA phosphate add groups in the case of highly esterified ,-lactoglobulin, esterified ,-lactalbumin and native basic proteins (lysozyme and histone). Binding capacity was dependent on the degree of esterification of the milk proteins. Hydrolysis of esterified milk proteins either suppressed or reduced their DNA-binding capacities according to the degree of hydrolysis and consequently to the average size of the resulting peptides. A prolonged peptic hydrolysis (25% degree of hydrolysis) completely suppressed DNA-binding capacity probably because of the small sizes of the resulting peptic peptides (< 1 kDa). Treatment with trypsin, which hydrolyzed the esterified proteins into relatively large peptide fragments, reduced the DNA-binding capacity to levels inversely proportional to the degree of hydrolysis. In the range of 2.7,12.3 kb, there was no influence of the DNA size on the binding of esterified milk proteins. The interactions DNA-esterified milk proteins did not depend on the DNA shape (circular or linear). Circular dichroism spectra of DNA in complex with methylated ,-lactoglobulin were markedly altered as compared to those obtained when DNA was in complex with native ,-lactoghbutin. [source] AFM measurement of the stiffness of layers of agarose gel patterned with polylysineMICROSCOPY RESEARCH AND TECHNIQUE, Issue 10 2010Marco Salerno Abstract Films of agarose gel microspotted with polylysine aqueous solution have been characterized by atomic force microscopy carried out in deionized water. Thickness and surface morphology of the layers have been checked, and the effect of polylysine impregnation on the local elasticity has been investigated. An increase in contact stiffness of the organic layer at the spotted areas has been observed, correlated with the polylysine concentration. For the considered agarose layer thickness of ,0.9 ,m in dry condition, the concentration threshold at which stiffening appears is ,0.1 mg/mL. Above this threshold, the stiffening coefficient becomes approximately twofold and seems not to increase significantly with concentration in the range 0.3,0.7 mg/mL. For concentrations above the stiffening threshold, this effect is also accompanied by a locally lower film thickness. For quantitative determination of the stiffness, force,distance curves extracted from the regions of interest of spots and agarose substrate have been selected and processed. These curves were fitted to the Hertz model of purely elastic tip-surface interaction, under appropriate assumptions on both tip shape and optimum indentation depth. In this way, we could determine the Young's modulus of the agarose layer to be ,50 kPa and quantitatively confirm the stiffening due to polylysine. Microsc. Res. Tech. 73:982,990, 2010. © 2010 Wiley-Liss, Inc. [source] Identification of the endangered small red brocket deer (Mazama bororo) using noninvasive genetic techniques (Mammalia; Cervidae)MOLECULAR ECOLOGY RESOURCES, Issue 3 2009SUSANA GONZÁLEZ Abstract The small red brocket deer Mazama bororo is one of the most endangered deer in the Neotropics. The great morphological similarities with three other sympatric brocket deer species, coupled with the fact that they inhabit densely forested habitats complicate detection and prevent the use of traditional methodologies for accurate identification of species. The ability to determine the presence of this endangered species in an area is crucial for estimating its distribution range, and is critical for establishing conservation management strategies. Here we describe a fast and reliable noninvasive genetic method for species identification of Mazama species from faeces. We designed a primer set that amplifies a short 224-bp fragment of the cytochrome b and demonstrate its effectiveness in successful amplification of DNA isolated from both tissue and faecal samples. This fragment contains a BSTNI/ECORII digestion site that is unique to the endangered M. bororo. The digested polymerase chain reaction products yielded a 160-bp fragment that is clearly visible in a 2% agarose gel. Two other diagnostic sites were identified to differentiate the other three sympatric species, SspI (M. gouazoubira) and AflIII (M. americana, and M. nana). [source] Analysis of von Willebrand factor structure by multimer analysis,AMERICAN JOURNAL OF HEMATOLOGY, Issue 7 2010Marlies R. Ledford-Kraemer Analysis of von Willebrand factor (VWF) structure is achieved by performing a highly specialized procedure, VWF multimer analysis. The test is reserved for the reference or specialized laboratory environment. The assay is qualitative (though under some circumstances multimers may be quantified) in that it assesses the overall size distribution of VWF multimers as well as their individual internal structure. The test is used predominantly to type or subtype von Willebrand disease. The analysis of VWF multimers generally consists of four steps: (1) electrophoresis of plasma in an agarose gel, (2) either gel fixation or transfer of the electrophoretic protein product to a membrane, (3) immunodetection of the protein, and (4) evaluation of the protein in the gel or membrane. The assay is complex, time consuming, requires specialized equipment and technical expertise, and is not standardized. Am. J. Hematol., 2010. © 2010 Wiley-Liss, Inc. [source] A quick method for the assessment of activity and inhibition of fish amylasesAQUACULTURE NUTRITION, Issue 1 2001I. Fernández A sensitive and quick method was developed to determine the presence of ,-amylase in the gut of aquatic organisms, as well as its sensitivity to inhibitors. The assay is based on the utilization of Petri dishes filled with starch,agarose gel as a substrate for the enzyme solution, which is placed in small wells punched in the surface. Circular zones produced by the action of amylase remain colourless after staining with lugol. Pure commercial porcine amylase was used to fit the better conditions for developing the assay (1 g L,1 starch in the gels, 4 h of incubation). The diameter of the cleared zones were related to the activity of enzyme and the method detected linearly amylase activity in a range of 2,20 U well,1, so it was used to reveal the presence of amylase in digestive extracts obtained from different sparid fish. The method was also used to evaluate the effect produced by a specific inhibitor on fish amylases, showing a linear response when the ratio inhibitor:enzyme (in units) changed from 20:1 to 2:1. Comparison of the cleared zones produced by amylases of sparid fish in the presence or absence of inhibitor, revealed differences in their sensitivity to inhibition, which ranged from 15 to 50% of total activity. The assay is proposed for a preliminary evaluation of possible inhibitors contained in feedstuffs used in fish feeding. [source] Crystal quality and differential crystal-growth behaviour of three proteins crystallized in gel at high hydrostatic pressureACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2005A. Kadri Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75,100,MPa (lysozymes) and 250,MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with ,20% less ordered water molecules in crystals grown at 150,MPa when compared with those grown at atmospheric pressure (0.1,MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered. [source] Effects of macromolecular impurities and of crystallization method on the quality of eubacterial aspartyl-tRNA synthetase crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2005A. Moreno Although macromolecular purity is thought to be essential for the growth of flawless protein crystals, only a few studies have investigated how contaminants alter the crystallization process and crystal quality. Likewise, the outcome of a crystallization process may vary with the crystallization method. Here, it is reported how these two variables affect the crystallogenesis of aspartyl-tRNA synthetase from the eubacterium Thermus thermophilus. This homodimeric enzyme (Mr = 130,000) possesses a multi-domain architecture and crystallizes either in a monoclinic or an orthorhombic habit. Minute amounts of protein impurities alter to a different extent the growth of each crystal form. The best synthetase crystals are only obtained when the crystallizing solution is either enclosed in capillaries or immobilized in agarose gel. In these two environments convection is reduced with regard to that existing in an unconstrained solution. [source] Accurate rocking-curve measurements on protein crystals grown in a homogeneous magnetic field of 2.4,TACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2004Daniel Lübbert Differences in mosaicity between lysozyme crystals grown inside and outside a homogeneous magnetic field of 2.4,T and with and without agarose gel were investigated by X-ray diffraction rocking-curve measurements. High angular resolution was achieved using an Si(113) four-reflection Bartels monochromator. The results show that (i) all crystals were highly perfect, (ii) the mosaicities were clearly anisotropic and (iii) the mosaicities varied more strongly within each group of crystals (grown under identical conditions) than the average values across groups. In particular, the effect of the magnetic field on crystal mosaicity was found to be very small. Finally, the spatial distribution of mosaic blocks inside a protein crystal was visualized with a novel diffraction technique using a high spatial resolution two-dimensional CCD detector. [source] Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 10-1 2002Crystals of the collagen-like polypeptide (PPG)10 were obtained within the Advanced Protein Crystallization Facility on board the International Space Station, during the STS-105/STS-108 mission. The duration of this mission was such to ensure that the crystallization process had reached its end. Crystals were grown both in the presence and in the absence of agarose gel, to compare the quality of the crystals obtained from these different environments. As a result, crystals grown in the absence of agarose on Earth as well as in microgravity showed X-ray diffraction up to 1.15 Ĺ. The intensity/sigma ratio was slightly higher for microgravity grown crystals. Crystals grown in agarose gel, both in microgravity and on ground, showed a comparable diffraction power, with a resolution limit of 1.45 Ĺ. [source] Crystallization and preliminary crystallographic studies of a new crystal form of Escherichia colil -asparaginase II (Ser58Ala mutant)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2000Maciej Kozak Periplasmic Escherichia colil -asparaginase II with an Ser58Ala mutation in the active-site cavity has been crystallized in a new orthorhombic form (space group P21212). Crystals of this polymorph suitable for X-ray diffraction have been obtained by vapour diffusion using two sets of conditions: (i) 1% agarose gel using MPD as precipitant (pH 4.8) and (ii) liquid droplets using PEG-MME 550 (pH 9.0). The crystals grown in agarose gel are characterized by unit-cell parameters a = 226.9, b = 128.4, c = 61.9,Ĺ and diffract to 2.3,Ĺ resolution. The asymmetric unit contains six protein molecules arranged into one pseudo-222-symmetric homotetramer and an active-site competent dimer from which another homotetramer is generated by crystallographic symmetry. [source] Electronic Speckle Pattern Interferometry: A Tool for Determining Diffusion and Partition Coefficients for Proteins in GelsBIOTECHNOLOGY PROGRESS, Issue 6 2002David Karlsson The aim of this study was to demonstrate electronic speckle pattern interferometry (ESPI) as a powerful tool in determining diffusion coefficients and partition coefficients for proteins in gels. ESPI employs a CCD camera instead of a holographic plate as in conventional holographic interferometry. This gives the advantage of being able to choose the reference state freely. If a hologram at the reference state is taken and compared to a hologram during the diffusion process, an interferometric picture can be generated that describes the refraction index gradients and thus the concentration gradients in the gel as well as in the liquid. MATLAB is then used to fit Fick's law to the experimental data to obtain the diffusion coefficients in gel and liquid. The partition coefficient is obtained from the same experiment from the flux condition at the interface between gel and liquid. This makes the comparison between the different diffusants more reliable than when the measurements are performed in separate experiments. The diffusion and partitioning coefficients of lysozyme, BSA, and IgG in 4% agarose gel at pH 5.6 and in 0.1 M NaCl have been determined. In the gel the diffusion coefficients were 11.2 ± 1.6, 4.8 ± 0.6, and 3.0 ± 0.3 m2/s for lysozyme, BSA, and IgG, respectively. The partition coefficients were determined to be 0.65 ± 0.04, 0.44 ± 0.06, and 0.51 ± 0.04 for lysozyme, BSA, and IgG, respectively. The current study shows that ESPI is easy to use and gives diffusion coefficients and partition coefficients for proteins with sufficient accuracy from the same experiment. [source] Microfabricated Polymer Chip for Capillary Gel ElectrophoresisBIOTECHNOLOGY PROGRESS, Issue 5 2001Jong Wook Hong A polymer (PDMS: poly(dimethylsiloxane)) microchip for capillary gel electrophoresis that can separate different sizes of DNA molecules in a small experimental scale is presented. This microchip can be easily produced by a simple PDMS molding method against a microfabricated master without the use of elaborate bonding processes. This PDMS microchip could be used as a single use device unlike conventional microchips made of glass, quartz or silicon. The capillary channel on the chip was partially filled with agarose gel that can enhance separation resolution of different sizes of DNA molecules and can shorten the channel length required for the separation of the sample compared to capillary electrophoresis in free-flow or polymer solution format. We discuss the optimal conditions for the gel preparation that could be used in the microchannel. DNA molecules were successfully driven by an electric field and separated to form bands in the range of 100 bp to 1 kbp in a 2.0% agarose-filled microchannel with 8 mm of effective separation length. [source] Size-Tunable Highly Luminescent SiO2 Particles Impregnated with Number-Adjusted CdTe NanocrystalsCHEMPHYSCHEM, Issue 4 2010Ping Yang Dr. Abstract Highly luminescent SiO2 particles impregnated with CdTe nanocrystals (NCs) are prepared by a sol,gel procedure. Partial ligand exchange from thioglycolic acid to 3-mercaptopropyltrimethoxysilane (MPS) on the NCs enables retention of the initial photoluminescence (PL) efficiency of the NCs in water, while the simultaneous addition of a poor solvent (ethanol) results in regulated assembly of the NCs through condensation of hydrolyzed MPS. The SiO2 particles thus prepared have, for example, a diameter of 16 nm and contain three NCs each. The PL efficiency of these particles is 40,%, while the initial efficiency is 46,% in a colloidal solution. The redshift and narrowed spectral width in PL observed after impregnation indicate that the concentration of NCs in these nearly reaches the ultimate value (on the order of 1021 particles per liter). The porosity of these particles is investigated by means of N2 adsorption,desorption isotherms. Due to the SiO2 shell, these particles have higher stability in phosphate-buffered saline buffer solution than the initial NCs. Their potential use for labeling in bio-applications is investigated by conjugating biotinylated immunoglobulin G to them by using streptavidin maleimide as linker. Successful conjugation is confirmed by electrophoresis in agarose gel. This preparation method is an important step towards fabricating intensely emitting biocompatible SiO2 particles impregnated with semiconductor NCs. [source] Subtracted restriction fingerprinting , a new typing technique using magnetic capture of tagged restriction fragmentsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2004Valeri Terletski Abstract Molecular typing of bacterial pathogens is an important issue in the epidemiological analysis of emerging infections in humans and animals. Numerous methods have been developed for and applied to a wide variety of bacteria of medical, veterinary and zoonotic importance. The present minireview provides a description of a new typing approach designated subtracted restriction fingerprinting (SRF), its use for typing of Salmonella isolates and a comparison with the most widely used typing techniques for these bacteria. SRF is based on double restriction endonuclease digestion of whole cell DNA, followed by a fill-in reaction with specifically tagged nucleotides and subtractive capture of selected restriction fragments. This results in a reduced number of fragments optimal for separation in standard agarose gels. [source] Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing,HUMAN MUTATION, Issue 3 2008Edgar A. Otto Abstract Nephronophthisis (NPHP), an autosomal recessive kidney disease, is the most frequent genetic cause of chronic renal failure in the first three decades of life. Mutations in eight genes (NPHP1,8) have been identified. We here describe a combined approach for mutation screening of NPHP1, NPHP2, NPHP3, NPHP4, and NPHP5 in a worldwide cohort of 470 unrelated patients with NPHP. First, homozygous NPHP1 deletions were detected in 97 patients (21%) by multiplex PCR. Second, 25 patients with infantile NPHP were screened for mutations in inversin (NPHP2/INVS). We detected a novel compound heterozygous frameshift mutation (p.[Q485fs]+[R687fs]), and a homozygous nonsense mutation (p.R899X). Third, 37 patients presenting with NPHP and retinitis pigmentosa (Senior-Lřken syndrome [SLS]) were screened for NPHP5/IQCB1 mutations by direct sequencing. We discovered five different (three novel) homozygous premature termination codon (PTC) mutations (p.F142fsX; p.R461X; p.R489X; p.W444X; and c.488,1G>A). The remaining 366 patients were further investigated for mutations in NPHP1, NPHP3, and NPHP4. We applied a "homozygosity only" strategy and typed three highly polymorphic microsatellite markers at the respective loci. A total of 32, eight, and 14 patients showed homozygosity, and were screened by heteroduplex crude celery extract (CEL I) endonuclease digests. The sensitivity of CEL I was established as 92%, as it detected 73 out of 79 different known mutations simply on agarose gels. A total of 10 novel PTC mutations were found in NPHP1 (p.P186fs, p.R347X, p.V492fs, p.Y509X, and c.1884+1G>A), in NPHP3 (c.3812+2T>C and p.R1259X), and in NPHP4 (p.R59X, p.T1004fs, and p.V1091fs). The combined homozygosity mapping and CEL I endonuclease mutation analysis approach allowed us to identify rare mutations in a large cohort of patients at low cost. Hum Mutat 29(3), 418,426, 2008. © 2007 Wiley-Liss, Inc. [source] Aging Increases the Interleukin-1,,Induced INOS Gene Expression and Nitric Oxide (NO) Production in Vascular Smooth Muscle CellsJOURNAL OF CARDIAC SURGERY, Issue 6 2002Gabriel HH Chan Objectives: Inducible form of nitric oxide synthase (iNOS) is induced by cytokines (e.g. interleukin-1, (IL-1,)) during pathological conditions, such as sepsis. Excessive NO synthesis in blood vessels during sepsis can result in massive vasodilation and life-threatening hypotension. In addition, chronic expression of iNOS contributes to onset of diabetes, autoimmune diseases, arthritis, renal toxicity, and neurodegenerative disorders. The purpose of the present study was to examine the effect of aging on the levels of expression of iNOS induced by a low concentration (5 ng/ml) of IL-1, in VSMCs. Methods: Gene expression of iNOS was determined by RT-PCR and analysis of the PCR products by both agarose gel electrophoresis and capillary electrophoresis with laser-induced fluorescence detector (CE-LIF). This new CE-LIF technique, just developed in our laboratory, provides greater than 1,000 fold better sensitivity compared to agarose gels. The production of nitrite, the stable metabolite of NO, was measured (by a modified Griess reaction) in the media of cultured VSMCs isolated from young and elderly rats (3-month and 20-months old, respectively) of both genders following the exposure to IL-1, (5 ng/ml). VSMCs were used in their 1st passage to avoid phenotypic changes that typically occur in cultures of VSMCs after 3-10 passages. Results: IL-1, (5 ng/ml) caused a much larger increase in iNOS mRNA in VSMCs of elderly rats as compared to young rats. Furthermore, IL-1, (5 ng/ml) had no significant effect on nitrite levels in VSMCs of young, but significantly increased nitrite levels by 7.9 fold in VSMCs from elderly male rats and by 2.6 fold in VSMCs from elderly female rats, as compared to young rats. A report had previously shown that the neuropeptide CGRP could synergistically enhance the expression of iNOS caused by IL-1, in later passages (10-15 passages) of rat aortic VSMCs (i.e. phenotypically modulated VSMCs). We found that IL-1, and CGRP together did not act synergistically to increase production of nitrite in our phenotypically normal (1st passage) VSMCs. Conclusion: IL-1,, at a low concentration (5 ng/ml), preferentially induces iNOS expression and increases production of NO in VSMCs of elderly rats as compared to young rats. The data suggest that aging enhances the responsiveness of VSMCs to the iNOS-inducing actions of the cytokine IL-1,. This may be a contributing factor in the increased risk of developing severe hypotension in elderly patients with sepsis. (Supported by a Direct Grant for Research). [source] Mapping of intrinsic bent DNA sites in the upstream region of DNA puff BhC4-1 amplified geneJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2001Adriana Fiorini Abstract We have identified bent DNA sites in the distal and proximal DNA puff BhC4-1 amplified gene promoter region of Bradysia hygida. The 2D modeling of the 3D DNA path and the ENDS ratio values calculated in this promoter region resulted in the identification of ten pronounced bent sites named BhC4B ,,9 to +,1. The 1847 bp fragment (,,3697 to ,,1850) in relation to the transcription start site shows multiple bending sites, BhC4B ,,9 to BhC4B ,,4, with periodicity ,300 bp. The analysis of the other identified bent region, starting at position ,,957, reveals that the BhC4B +,1 bent site colocalizes with the putative BhC4-1 minimal promoter. The sequence analysis of bent site BhC4B ,,4 shows a distribution of dA,dT at ,10 bp intervals between the middle of each tract, but intervals with more than one turn, ,20 bp, two helix turns, were detected in the other bent sites described here. The bent sites BhC4B ,,6 and BhC4B ,,4, contain two consensus sequences, with 60 bp each. The apparent molecular weight of fragments in the BhC4-1 promoter region were estimated in agarose gels and compared with the data obtained in polyacrylamide gels without and with ethidium bromide. The mobility reduction ratios (R -values) were determined, and a high R -value, 1.80, for a 1215 bp fragment in the distal promoter region and a 1.23 significant R -value for a 662 bp fragment in the proximal segment were found. To further analyze the predicted bent DNA sites in these fragments, the 2D trajectories of the 3D DNA path and other parameters, AT percentage, roll angle, ENDS ratio and ,G, were determined. The role of these bent sites in the BhC4-1 transcription regulation is discussed. © 2001 Wiley-Liss, Inc. [source] Protein adsorption kinetics in charged agarose gels: Effect of agarose content and modelingAICHE JOURNAL, Issue 2 2009Emily B. Schirmer Abstract The adsorption kinetics of myoglobin in charged gels of varying agarose content have been measured macroscopically, through batch uptake experiments, and microscopically, using light microscopy with gels supported in microfluidics chips. The apparent effective pore diffusivities, determined by fitting either set of rate data to the shrinking core model, were greater than the free solution diffusivity and concentration-dependent. Moreover, the microscopically derived concentration profiles were qualitatively different from the predicted ones. Therefore, a new model taking into account an assumed favorable partitioning of the protein in the pore liquid is proposed to describe the adsorption kinetics. The new model yields effective pore diffusivities that are in approximate agreement with the values determined chromatographically under nonbinding conditions and with hindered diffusion theory. In addition, it predicts concentration profiles in the gel that are consistent with those observed microscopically. The overall increase in mass transfer is attributed to the favorable partitioning of the protein in the pores at low ionic strength, which results in a greater diffusional driving force. © 2008 American Institute of Chemical Engineers AIChE J, 2009 [source] Effect of dynamic loading on solute transport in soft gels implication for drug deliveryAICHE JOURNAL, Issue 3 2008F. Urciuolo Abstract Solute transport through soft gels and tissues is intimately coupled to mechanical stress and deformation of the macromolecular network. The aim of this study was to investigate the effect of periodic mechanical stimuli upon solute transport through agarose gels at different concentrations. For this purpose it was experimentally evaluated the materials parameters that govern the coupling between elasto-dynamic and solute transport: hydraulic conductivity (K), elastic modulus (HA), and macromolecular diffusivity (Dg) along with their strain dependence behavior. Mechanical activated solute transport simulation was carried out in order to elucidate the role of amplitude and frequency of soliciting mechanical stimuli on mass kinetics release. Results show that mechanical loading affects the release of macromolecules from a gel in a frequency and strain dependent manner. These findings pave the way for novel strategies for the design and engineering of smart drug delivery devices with transport mechanisms triggered by mechanical stimuli. © 2008 American Institute of Chemical Engineers AIChE J, 2008 [source] Expression of metalloproteinases and their tissue inhibitors in inflamed gingival biopsiesJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2008L. D. R. Gonçalves Objectives:, Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are known to be involved in the periodontal disease process. Results of in vivo MMPs and TIMPs gene expressions in the gingiva, though, are still controversial. In the present study, we compared the gene expression of MMP-1, -2, -9, -13 and TIMP-1, -2 in healthy and inflamed gingiva. Methods:, 38 gingival samples were collected from gingivitis (n = 10), advanced chronic periodontitis (n = 10), generalized aggressive periodontitis (n = 8) and periodontally healthy individuals (n = 10). Total RNA isolated from those samples was subjected to reverse transcription followed by amplification by polymerase chain reaction (RT-PCR). Products were visualized in agarose gels and quantified by optical densitometry. Samples were also processed for gelatin zymography and Western blotting for MMP-2 and MMP-9 in order to assess for post-transcriptional MMP regulation at the protein level. Results:, The frequencies and levels of transcripts encoding MMPs and TIMPs were found to be not significantly different among groups (p > 0.05, Fisher's Exact and Kruskall-Wallis tests). There is a trend towards higher MMP-2 and -9 gelatinase activities in the inflamed samples, although not statistically significant. In contrast, zymography and Western blotting studies show that MMP-2 is virtually absent in the chronic periodontitis group. Conclusion:, These results could reflect a complex regulation of MMPs and TIMPs' gene expression in the course of gingival inflammation. They also reveal a great biological diversity even among individuals with similar periodontal status. [source] Isolation and characterization of microsatellite loci in SenecioMOLECULAR ECOLOGY RESOURCES, Issue 4 2004GUOQING LIU Abstract Interspecific hybridization has resulted in the recent origin of several hybrid Senecio taxa at diploid, tetraploid and hexaploid levels. As part of research aimed at constructing and comparing genomic maps of each of these taxa and their parents, we have isolated microsatellite loci from genomic DNA libraries of S. vulgaris and S. squalidus. Primers of 35 loci amplified microsatellites resolved in agarose gels from one or more of S. vulgaris, S. squalidus, S. aethnensis and S. chrysanthemifolius. Approximately 71% of primers amplified a product in all four species. A survey of microsatellite variation in S. chrysanthemifolius over a subset of 14 loci resolved 2,11 alleles per locus in polyacrylamide gels with expected heterozygosity (HE) ranging from 0.26 to 0.87. [source] Calycopterin, an immunoinhibitory compound from the extract of Dracocephalum kotschyiPHYTOTHERAPY RESEARCH, Issue 9 2008Najme Faham Abstract Medicinal plants have been widely investigated for their various effects. Dracocephalum kotschyi Boiss (Labiatae) is used in Iranian traditional medicine for the treatment of rheumatoid diseases. The inhibitory effect of D. kotschyi on the lectin-induced cellular immune response has been demonstrated previously. In this study, mitogen-treated lymphocytes were exposed to the extract of D. kotschyi and analysed for the induction of apoptosis using flow cytometry and gel electrophoresis. The data obtained indicated a dose-dependent increase of cells in the sub-G1 phase of cell cycle. Study of internucleosomal DNA fragmentation showed a typical DNA laddering in agarose gels. A bioactivity-guided fractionation assay to find the active components responsible for the inhibitory effect of D. kotschyi on mitogen-induced lymphocyte proliferation led to the isolation of calycopterin from the ethyl acetate extract of D. kotschyi. Its structure was identified by spectroscopic methods including 1H-NMR, 13C-NMR, MS and UV spectra. Calycopterin inhibited lymphocyte proliferation in a dose-dependent manner with an IC50 value of 1.7 µg/mL. In conclusion, the results of this study suggest that D. kotschyi extract has the capacity to induce apoptosis in the lymphocytes and that isolated calycopterin is responsible for the inhibitory effect of D. kotschyi on lymphocyte proliferation. Copyright © 2008 John Wiley & Sons, Ltd. [source] Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEFPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2007Jukka Hellman Dr. Abstract Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20,50,ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS. [source] Type II collagen levels correlate with mineralization by articular cartilage vesiclesARTHRITIS & RHEUMATISM, Issue 9 2009Brian Jubeck Objective Pathologic mineralization is common in osteoarthritic (OA) cartilage and may be mediated by extracellular organelles known as articular cartilage vesicles (ACVs). Paradoxically, ACVs isolated from OA human cartilage mineralize poorly in vitro compared with those isolated from normal porcine cartilage. We recently showed that collagens regulate ACV mineralization. We sought to determine differences between collagens and collagen receptors on human and porcine ACVs as a potential explanation of their different mineralization behaviors. Methods ACVs were enzymatically released from old and young human and porcine hyaline articular cartilage. Western blotting was used to determine the presence of types I, II, VI, and X collagen and various collagen receptors on ACVs. Type II collagen was quantified by enzyme-linked immunosorbent assay. Biomineralization was assessed by measuring the uptake of 45Ca by isolated ACVs in agarose gels and by ACVs in situ in freeze-thawed cartilage. Results As previously shown, isolated human ACVs mineralized poorly in response to ATP compared with porcine ACVs, but human and porcine ACVs mineralized similarly in situ in freeze-thawed cartilage. Type II collagen levels were 100-fold higher in isolated human ACVs than in porcine ACVs. Type II collagen in human ACVs was of high molecular weight. Transglutaminase-crosslinked type II collagen showed increased resistance to collagenase, suggesting a possible explanation for residual collagen on human ACVs. Expression of other collagens and collagen receptors was similar on human and porcine ACVs. Conclusion Higher levels of type II collagen in human ACV preparations, perhaps mediated by increased transglutaminase crosslinking, may contribute to the decreased mineralization observed in isolated human ACVs in vitro. [source] Introducing human population biology through an easy laboratory exercise on mitochondrial DNABIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 2 2010Antonio F. Pardińas Abstract This article describes an easy and cheap laboratory exercise for students to discover their own mitochondrial haplogroup. Students use buccal swabs to obtain mucosa cells as noninvasive tissue samples, extract DNA, and with a simple polymerase chain reaction-restriction fragment length polymorphism analysis they can obtain DNA fragments of different sizes that can be visualized in agarose gels. The analysis of these fragments can reveal the mitochondrial haplogroup of each student. The results of the exercise can be used to provide additional insights into the genetic variation of human populations. [source] Review modeling the free solution and gel electrophoresis of biopolymers: The bead array-effective medium modelBIOPOLYMERS, Issue 2-3 2007Stuart A. Allison Abstract Free solution and gel electrophoresis is an extremely useful tool in the separation of biopolymers. The complex nature of biopolymers, coupled with the usefulness of electrophoretic methods, has stimulated the development of theoretical modeling over the last 30 years. In this work, these developments are first reviewed with emphasis on Boundary Element and bead methodologies that enable the investigator to design realistic models of biopolymers. In the present work, the bead methodology is generalized to include the presence of a gel through the Effective Medium model. The biopolymer is represented as a bead array. A peptide, for example, made up of N amino acids is modeled as 2N beads. Duplex DNA is modeled as a discrete wormlike chain consisting of touching beads. The technical details of the method are placed in three Appendices. To illustrate the accuracy and effectiveness of the approach, two applications are considered. Model studies on both the free solution mobility of 73 peptides ranging in size from 2 to 42 amino acids, and the mobility of short duplex DNA in dilute agarose gels are discussed. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 102,114, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] | |||||||||||||||||||||||||||||||