The adsorption of methylene blue (MB) in plasma on cross-linked agarose beads entrapped activated charcoal (CAAC-I), cross-linked agarose coated activated charcoal (CAAC-II) and cross-linked agar beads entrapped attapulgite (CAA) was tested in this study. These adsorbents are intended to be applied to blood purification for eliminating MB from virucidal MB-phototrated plasma units. The experimental results indicated that the adsorption of MB in plasma on CAAC-I and CAA was quick and efficacious, but it was slower on CAAC-II. The relationship between the adsorption time and the adsorption rate was examined with reference to the plasma with a specific concentration of MB flowing over a certain volume of CAA. A flow rate of about 1 ml.min-1 through 10 ml CAA at 25 degrees C could eliminate more than 95% of the MB in 200 ml human plasma (1 mumol.L-1).
Adsorption ; Agar ; chemistry ; Animals ; Charcoal ; chemistry ; Hemofiltration ; instrumentation ; Humans ; In Vitro Techniques ; Methylene Blue ; isolation & purification ; Rabbits

The principal purpose of this study is to set up efficient purification techniques of small DNAs which are suitable for isolation of from tens to three hundred bases of genes. On the bases of the technique, purification methods for big DNA fragments are established. In the experiment, the DNA bands were cut after agarose gel electrophoresis and put into 0.5 mL of tubes with silica wool, glass wood, absorbent cotton and cotton at the bottom. And then 10 000 r/min for 2 min, the liquid was collected. The results indicated that silica wool was the best of the materials. The recovery rate for DNAs below 200bp was over 90%, 85% to approximately 90% for 300bp. And the technique can be applied to purify bigger DNA fragments. The kits for DNA purification hardly recovered DNA below 150bp. The recovery rate for 150bp of DNA was 5%, 60% even for 300bp. The efficiencies of enzymic digestion and enzymic connection for the DNAs purified by the technique were the same as those for the DNAs isolated by the kits. So, the technique is obviously superior to kit purification methods.
DNA ; genetics ; isolation & purification ; Electrophoresis, Agar Gel ; methods ; Silicon Dioxide ; chemistry

OBJECTIVESTo assess the effect of freezing on the sperm DNA.

METHODSTo assess the sperm DNA preserved at -80 degrees C by using the single cell gel electrophoresis (SCGE).

RESULTSThere was no statistical difference on the time factor by analysis of variance (ANOVA).

CONCLUSIONSIntegrity of sperm DNA could not be devastated in frozen states.

Adult ; Cryopreservation ; DNA ; analysis ; Electrophoresis, Agar Gel ; Freezing ; Humans ; Male ; Semen Preservation ; Spermatozoa ; chemistry

A method for preparing the double cross-linked agar beads entrapped attapulgite clay for hemopurification is reported. Attapulgite clay was coated with agar and cross-linked by epichlorohydrin. After the process of "drying-out", the cross-linked agar beads entrapped attapulgite clay (CAA) was cross-linked again by 10% toluene 2,4,-diisocyanate in acetone at 35 degrees C for 3 h and 30 min. The products withstood autoclaving at 121 degrees C for 30 min. The performance tests showed that the adsorption of the double cross-linked agar beads entrapped attapulgite clay (DCAA) on methylene blue was about 4 times the adsorption of CAA on methylene blue. The intensity of DCAA was raised 6 times, and the appearance of DCAA was denser. Investigation on the blood being in contact with DCAA showed: at 1 h of contact, the loss of leucocyte was <1%, of erythrocyte <5%, and of blood platelets <8%; at 2h of contact, the loss of leucocyte was <2%, of erythrocyte <10%, and of blood platelets <20%.
Adsorption ; Agar ; Animals ; Biocompatible Materials ; Epichlorohydrin ; chemistry ; Hemoperfusion ; instrumentation ; Magnesium Compounds ; chemical synthesis ; chemistry ; Materials Testing ; Rats ; Silicon Compounds ; chemical synthesis ; chemistry

We report a case of type III hyperlipoproteinemia which is called a broad-beta disease. A 53 year old female patient visited our clinic for the evaluation of multiple yellowish papules on extremities and eyelids. The patient showed various types of xanthoma includiiig eruptive, tuberous, tendinous xanthomas and xanthelasma palpebrarum, xanthoma striatum palmare. The blood chemistry revealed a marked elevstion of cholesterol and triglycerides and agarose gel electrophoresis showed a single peak at prebeta and beta portion without separation. On histopathologic studies, typical foam cells were showen.
Chemistry ; Cholesterol ; Electrophoresis, Agar Gel ; Extremities ; Eyelids ; Female ; Foam Cells ; Humans ; Hyperlipoproteinemia Type III* ; Hyperlipoproteinemias ; Middle Aged ; Triglycerides ; Xanthomatosis

OBJECTIVETo analyse clinical feasibility of two electrophoresis procedures of seminal plasma proteins, agarose gel electrophoresis and SDS-agarose gel electrophoresis.

METHODSSixty-nine semen samples were examined and classified into three groups: the asthenozoospermia (n = 22), the asthenoteratozoospermia (n = 19), and the relative normal group (n = 28) with normal routine and special test results, according to WHO routine and special test criterion. Then, the seminal plasma protenis were separated by two different electrophoresis, with SDS-agarose and agarose support medium, the buffer pH 7.0 and 9.2 respectively. The agarose gel electrophoresis was done under various sample loading time, motion power and staining modules. The completed gels were scanned and compared the each other statistically.

RESULTSSeminal plasma proteins can be separated into 4 strips by SDS-agarose gel electrophoresis with acid crystal violet, and the strips were diffusion and with dark background. However, 6 clear strips named A, B, C, D, E, and F can be obtained by agarose gel electrophoresis with 6 min. After samples were loaded and stained by amidoblack, there showed appropriate spaces among strips, and it was very easy to scan the drying gel by a densitometer. Using agarose gel electrophoresis, the statistical difference in strip C and E was shown between the asthenozoospermia and the relative normal group, and between the asthenozoospermia and the asthenoteratozoospermia, however, not between the relative normal and the asthenoteratozoospermia group. Moreover, the samples in the relative normal group with normal routine and special test results were in different electrophoresis maps.

CONCLUSIONAgarose gel electrophoresis of seminal plasma proteins with buffer pH 9.2, 6 min. sample loading and amidoblack stain was a simple, fast and fit technique for clinic.

Adult ; Electrophoresis, Agar Gel ; methods ; Electrophoresis, Polyacrylamide Gel ; methods ; Humans ; Male ; Proteins ; analysis ; Semen ; chemistry ; Staining and Labeling

OBJECTIVETo investigate single-molecule detection and characterization of DNA replication.

METHODSSingle-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication.

RESULTSThe designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis.

CONCLUSIONSThe combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

Biotinylation ; DNA ; chemistry ; DNA Replication ; DNA, Single-Stranded ; chemistry ; DNA-Directed DNA Polymerase ; Electrophoresis, Agar Gel ; Microscopy, Atomic Force ; Nucleic Acid Hybridization ; Streptavidin

OBJECTIVETo prepare chitosan nanoparticles (NPs) as gene carriers and study its pharmaceutical characteristics and gene transfection efficiency in vitro.

METHODSThe plasmid expressing green fluorescent protein (pGFP) was used as the reporter gene, and the chitosan-pGFP NPs were prepared using a complex coacervation process. The binding of pDNA was evaluated by agarose gel electrophoresis analysis, and the encapsulation rate was determined with colorimetry. The size distribution and polydispersity of the NPs were measured by nanoparticle size analyzer, and their morphologies observed by atomic force microscope. The transfection studies were performed with LoVo cells in vitro.

RESULTSThe results of gel electrophoresis demonstrated full binding of chitosan with the pDNA by electrostatic interaction. The encapsulation rates for these NPs all exceeded 90%. The morphology of the chitosan NPs was mostly spherical and well distributed, with a mean diameter of about 209 nm and polydispersity of 0.32. The in vitro transfection of chitosan-pGFP NP was efficient in LoVo cells and the expression of green fluorescent proteins was observed under fluorescent microscope.

CONCLUSIONSChitosan NP prepared by complex coacervation can bind to the pDNA efficiently with high encapsulation rate and diameter distribution of 100 to 500 nm. These NPs allow efficient delivery of the reporter genes into cells in vitro for their expression. The chitosan-pDNA NPs may serve as an effective non-viral carrier for delivery of nucleotides into cells.

Cell Line, Tumor ; Chitosan ; chemistry ; DNA ; chemistry ; genetics ; Electrophoresis, Agar Gel ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Atomic Force ; Microscopy, Fluorescence ; Nanoparticles ; chemistry ; Transfection ; methods

We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMerieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80degrees C, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.
Agar/chemistry ; Bacterial Proteins/genetics ; Bacteriological Techniques/*methods ; Chromogenic Compounds/chemistry/metabolism ; Clostridium difficile/genetics/*isolation & purification ; Culture Media/chemistry ; DNA, Bacterial/analysis/metabolism ; Diarrhea/microbiology/pathology ; Feces/*microbiology ; Humans ; Polymerase Chain Reaction ; Time Factors

Virus inactivation of plasma can be achieved by phototreatment with methylene blue (MB). Subsequently, elimination of MB may reduce the adverse effects of MB. This study examined the effects of adsorbing MB with the use of cross-linked agar bead entrapped attapulgite clay (CAA) on normal ingredients in MB-treated plasma units. The biomedical characteristics of CAA were assessed by determination of partial biochemical indexes, coagulation potency and some cationic concentration in a control sample and the MB-treated plasma eluted from CAA column. The biochemistry indexes or K+, Na+ in plasma were almost unaltered before and after CAA adsorption. In contrast, the concentrations of CA2+ and Mg2+ increased and the blood ammonium decreased obviously. The activated partial thromboplastin time (APTT) was prolonged from 42 s to 53 s, and prothrombin time (PT) from 13 s to 14 s. The result indicates that CAA as an adsorbent for hemopurification retains the most important characters of human plasma. CAA can be useful for the elimination of MB in MB-treated plasma and does not bring on harmful alteration in clinical significance.
Adsorption ; Agar ; Blood Chemical Analysis ; Blood Coagulation Tests ; Hemofiltration ; instrumentation ; Humans ; Magnesium Compounds ; Methylene Blue ; analysis ; Plasma ; chemistry ; cytology ; Silicon Compounds