Detection of DNA in agarose gels using berberine and Mordant Yellow 3R.

A nontoxic and simple staining method for the detection of DNA in agarose gels is described. After eletrophoretic separation, the gels were stained with 5 microg/ml of berberine (BB) prepared in distilled water and then the gels were soaked in 20 microg/ml of aqueous Mordant Yellow 3R (MY3R) solution. Employment of MY3R as a counterion dye efficiently quenched unwanted background fluorescence of BB. This method can detect as little as 10 ng of plasma membrane H(+)-ATPase cDNA obtained from Arabidopsis thaliana L. (AHA1, 3.2 kb) under a long wavelength of UV irradiation (366 nm) within 1 h.     

Filtration performance of microporous ceramic supports

The use of inorganic membranes in pollution treatment is actually limited by the cost of such membranes. Advantages of inorganic membranes are their chemical, thermal and pH properties. The purpose of this work was the development of microporous ceramic materials based on clay for liquid waste processing. The supports or ceramic filters having various compositions were prepared and thermally treated at 1100 °C. The results show that, at the temperature studied, porosity varied according to the support composition from 12% for the double-layered (ceramic) support to 47% for the activated carbon- filled support with a mean pore diameter between 0.8 and 1.3 μm, respectively. Volumes of 5 l of distilled water were filtered tangentially for 3 h under an applied pressure of 3.5 and 5.5 bar. The retention of tubular supports prepared was tested with molecules of varying size (Evans blue, NaCl and Sacharose). The study of the liquid filtration and flow through these supports showed that the retention rate depends on support composition and pore diameter, and solute molecular weight. The S1 support (mixture of barbotine and 1% (w/w) activated carbon) gave a flux for distilled water of 68 L/m² h while the double-layered support resulted in a flux of 8 L/m² h for the same solution at the pressure of 3.5 bar. At a pressure of 5.5 bar an increase in the distilled water flux through the various supports was observed. It was significant for the S1 support (230 L/m h).     

氟骨症不同动物模型的建立与评价

目的 为选择合适的实验动物并建立氟骨症动物模型,研究不同动物对氟化物致骨骼损伤的敏感性.方法 分别选取离乳新西兰兔、Hartley豚鼠、Wistar大鼠、KM小鼠各16只,每种动物随机分为两组,每组8只,即对照组(C):饲喂标准饲料,饮用去离子水;模型组(M):饲喂标准饲料,饮用150 mg/L氟化钠(NaF)去离子水.在饲喂60 d后取股骨制作组织切片观察组织形态变化,并测定股骨骨氟、骨钙、骨磷的含量及血清碱性磷酸酶(ALP)活性.结果 与对照组相比,各种实验动物模型组生长发育受到显著的抑制,股骨形态破坏明显,骨矿化显著减少,骨氟含量显著升高,血清ALP活性显著增强,但股骨钙和磷含量差异无显著性(P＞0.05).结论 高氟可不同程度的损害豚鼠、家兔、小鼠、大鼠的生长发育及骨骼系统,导致产生氟骨症,但综合考察,以豚鼠、家兔较为敏感,其次为小鼠、大鼠.     

Rapid transfer of DNA from agarose gels to nylon membranes.

The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in sensitivity of subsequent hybridization analyses. In addition, we have found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon membrane, reduces its ability to hybridize.     

Transformation in Naegleria gruberi: patterns of RNA synthesis examined by polyacrylamide gel electrophoresis

Naegleria gruberi were grown on bacteria and methods were devised to free the cellular RNA from bacterial RNA contamination. Use of actinomycin D and cycloheximide showed that the transformation of Naegleria from amoeba to flagellate required RNA synthesis for 30 min and protein synthesis for 40 min after the initial stimulus of distilled water. Comparison of the patterns of RNA synthesized during transformation with those during growth indicated a considerable amount of new RNA produced during the phenotypic change. Most marked was the increase in RNA co-migrating on polyacrylamide gels with the small ribosomal sub-unit RNA, together with RNAs between the latter and transfer RNA. These results were compared with other published results using axenically-grown cells cells and sucrose density gradient centrifugation. Cells placed in 80 mM NaCl instead of distilled water fail to transform but the pattern of newly-synthesized RNAs was not significantly different from that seen in transforming cells. This suggested that high salt concentrations inhibit transformation by inhibiting synthesis and/or assembly of certain proteins rather than RNA synthesis. Eluted material from various regions of polyacrylamide gels containing RNA extracted from transforming cells was used in a cell-free system. Incorporation of 3H-glutamic acid but not 3H-tryptophan was stimulated by material extracted from the 18S regions of the gels.     

Activity of pancreatic antioxidative enzymes and malondialdehyde concentrations in rats with hyperglycemia caused by fluoride intoxication.

The aim of this work was to examine the effect of fluoride ions on antioxidative enzyme activity in the pancreas of rats exposed during 4 months to NaF in drinking water. The study was carried out in 30 four-week-old male Wistar FL rats, that were randomly assigned to three equal groups and given distilled water ad libitum for three weeks. Subsequently, two examined groups of animals were exposed to NaF in drinking water: group 1 (10 rats) at 50 mg F(-)/L (2.63 mmol/L), group 2 (10 rats) at 100 mg F(-)/L (5.26 mmol/L). The control group (10 rats) received distilled water. After 4 months the animals were anesthetized with ether prior to collection of pancreas and cardiac blood. Serum concentrations of glucose and fluoride, as well as activities of the cytoplasmic (CuZn-SOD) and the mitochondrial (Mn-SOD) superoxide dismutase, glutathione peroxidase (GSH-Px) and concentrations of malondialdehyde (MDA) in the homogenized pancreas were measured. The activity of CuZn-SOD was reduced by 50% and a tendency to lower activities of Mn-SOD was observed. No changes were noted in the activity of GSH-Px or concentrations of MDA. We conclude that: 1) the fluoride caused hyperglycemia in rats in this study is not accompanied by an activation of the free radical production in the pancreas; 2) the hyperglycemia in the exposed rats cannot be attributed to pancreatic damage caused by fluoride ions (the cause in this case appears to be extrapancreatic); 3) the inhibition of pancreatic CuZn-SOD is probably due to the direct action of fluoride on the enzyme.     

Carbopol-Based Gels for Nasal Delivery of Progesterone

The purpose of this study was to investigate the nasal absorption of progesterone from carbopol-based nasal gels in rabbits. Progesterone nasal gels were prepared by dispersing carbopol 974 (1%, 1.5%, and 2%) in distilled water followed by addition of progesterone/progesterone–β cyclodextrin complex dissolved in propylene glycol then neutralization. The potential use of β cyclodextrin (CD) as nasal absorption enhancer by simple addition, as a physical mixture and as a complex with progesterone was investigated. The absolute bioavailability of progesterone from nasal gels in rabbits was studied by estimating the serum progesterone level by competitive solid-phase enzyme immunoassay in comparison to intravenous injection. The carbopol gel formulations produced a significant increase in bioavailability. CD complex promotes the nasal absorption of progesterone from carbopol gels as compared with gels where the CD is added by simple addition and gels which do not contain CD. This method of addition of CD as an inclusion complex in the gels could be considered as a preferred platform in nasal drug administration.     

Legionnaires' disease caused by Legionella dumoffii in distilled water.

Five cases of Legionnaires'' disease caused by Legionella dumoffii were identified within an 11-month period in a hospital in the Quebec City area. In four cases bacterial isolates were obtained from clinical specimens, and in one case seroconversion was demonstrated. All the patients had been admitted to hospital within 10 days before diagnosis. Two of the patients were immunosuppressed children. Only 1 of the 40 hot water samples from the hospital yielded L. dumoffii; however, 6 of 11 distilled water samples contained the bacterium. All the patients had been exposed to distilled water, four through respiratory therapy equipment and one through a room humidifier. Following the use of sterile distilled water in the apparatus, no further cases were identified. This is the first reported outbreak of Legionnaires'' disease caused by L. dumoffii, and it is the first time that nosocomial legionellosis has been linked to contaminated distilled water in Canada.     

Effect of the removal of the COOH-terminal region of bacteriorhodopsin on its light-induced H+ changes.

Removal of the COOH-terminal region of bacteriorhodopsin by digestion with trypsin or papain reduces the yield of light-induced H+ release by 50-70%. The rate of H+ release is not affected significantly, but the half time of H+ uptake increases almost twofold. However, there is no effect on the photocycle of bacteriorhodopsin as judged by the yield and decay kinetics of the M412 photointermediate. The H+:M ratio in enzyme-digested membranes is approximately 0.4-0.8, whereas untreated membranes have a H+:M ratio of approximately 2. Purple membrane sheets stored in distilled water at 4 degrees C for prolonged periods also have a low H+:M ratio, probably due to protease activity associated with bacterial contamination. Electrophoresis on sodium dodecylsulfate-polyacrylamide gels showed that both the enzyme-treated and the stored purple membrane samples have a higher electrophoretic mobility compared to the fresh preparation. The reduction in molecular weight can be accounted for by the loss of several residues from the COOH-terminal portion of the bacteriorhodopsin. We propose that the COOH-terminal region is partially responsible for the high yield of H+ release by the purple membrane.     

拟黑多刺蚁提取液可缓解小鼠体力疲劳

研究拟黑多刺蚁Polyrhachis vicina Roger提取液的缓解体力疲劳作用。采用清洁级雄性昆明种小鼠,拟黑多刺蚁水和乙醇双重提取液按8.3mL/kg.bw,16.7mL/kg.bw,25.0mL/kg.bw连续灌胃30d,测定小鼠负重游泳时间、血乳酸、血清尿素氮和肝糖原含量。结果:3个实验组及乙醇对照组小鼠负重游泳时间均显著长于蒸馏水对照组(P<0.05,P<0.01);8.3mL/kg.bw剂量组小鼠负重游泳时间显著长于乙醇对照组(P<0.01)。8.3mL/kg.bw剂量组小鼠3个时点血乳酸曲线下面积显著低于蒸馏水对照组和乙醇对照组(P<0.05、P<0.01),16.7mL/kg.bw剂量组小鼠3个时点血乳酸曲线下面积明显低于蒸馏水对照组(P<0.01)。乙醇对照组及拟黑多刺蚁提取液3个剂量组小鼠血清尿素氮含量均显著低于蒸馏水对照组(P<0.05、P<0.01),16.7mL/kg.bw剂量组小鼠血清尿素氮含量低于乙醇对照组(P<0.05)。拟黑多刺蚁提取液具有缓解体力疲劳的作用。     

Direct immunodetection of antigens within the precast polyacrylamide gel

Detection of specific proteins separated by SDS-PAGE is the basis for studying specific antigens. Immunodetection of antigens is commonly performed using Western blotting technique. In this paper we have shown that it is possible to eliminate Western blotting and to detect the antigens directly within the precast polyacrylamide gels by pretreating the gels with 50% isopropanol followed by distilled water treatment. This method would be valuable for large or difficult to transfer proteins.     

Artifacts associated with quick-freezing and freeze-drying

We have studied the structures produced when nonbiological samples were subjected to quick-freezing and freeze-drying with a liquid helium cooled freeze-slamming device. Samples examined in this way included sodium chloride, sucrose, and Tris buffer. A variety of filamentlike and trabeculumlike structures were formed in these preparations. These structures may represent eutectic mixtures formed during the growth of small ice crystals during the freezing process, and exposed during the rapid sublimation of pure ice during the etching process. Samples of biological membranes (isolated chloroplast membranes) were prepared in various buffers by means of this technique. In distilled water, excellent replicas of membrane surfaces were obtained. In salt solutions, however, the membranes appeared to be embedded in a network of thin filaments appearing very much like a cytoskeletal lattice. It is concluded that extreme caution must be used when employing this preparation technique for studies of cell architecture, and that extensive washing of cell components in distilled water may be necessary to obtain faithful representations of cell structure.     

Separation of both the Bbeta- and the gamma-polypeptide chains of human fibrinogen into two main types which differ in sialic acid content

The gamma- and Bbeta-polypeptide chains of purified human fibrinogen have each been resolved into two major species: gammaL and gammaR and BbetaL and BbetaR. These molecular variants, separable on CM-cellulose, differ from each other in sialic acid content: approximately 2 residues of sialic acid per molecule of polypeptide chain for the L species to 1 residue of sialic acid per molecule for the R species. The two types of each polypeptide are demonstrable in preparations of fibrinogen from single donors as well as in pooled fibrinogen. The L and R forms of the gamma chains or the Bbeta chains do not differ in their electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels, suggesting that they are similar in molecular weight. They are also indistinguishable in polyacrylamide gels in the presence of urea at pH 2.7. Maps of ninhydrin-positive tryptic peptides of the L and R forms of the gamma chain displayed differences within a small group of peptides which have been shown to contain the sialic acid residues present in the gamma-polypeptides. No differences between the peptide maps of BbetaL and BbetaR chains were obvious. A larger ratio of L/R in the gamma and Bbeta chains of dysfibrinogenemia fibrinogen "Zürich II" than in those of normal fibrinogen explains the higher content of sialic acid measured in the native Zürich II fibrinogen molecule.     

Comigration of Two Autophagosome-Associated Dehydrogenases on Two-Dimensional Polyacrylamide Gels

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (~37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (~35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-asssisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-a-hydroxysteroid dehydrogenase (3aHSD). Silver staining indicated that this 3aHSD form would selectively bind to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3aHSD demonstrated immunostaining of four 3aHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3aHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3aHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.     

A glutathione-based hydrogel and its site-selective interactions with water

Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is ubiquitous biological tripeptide with multiple functions and possible therapeutic uses. The oxidized disulfide form (GSSG) self-assembles into fibrillar aggregates and gels in organic solvents, but not in solvent mixtures with high water content. Here, the disulfide bond has been replaced with a pyrenyl moiety in order to test the ability of GSH to direct noncovalent self-assembly in H2O, when combined with a hydrophobic driving force for aggregation. The resulting GSH-pyrene forms gels in 95% H2O:5% DMSO. The gamma-glutamyl group is critical for gelation, as it is with GSSG organo-gels, inasmuch as neither S-(pyrenyl)-cysteinyl-glycine nor the iodo-acetamido-pyrene precursor gels under any conditions studied. Circular dichroism and fluorescence spectroscopy indicate that the pyrene moieties cluster within the gels. Scanning and transmission electron microscopy reveal fibrous networks with individual strands of approximately 50-100 nm diameter. Saturation transfer difference (STD) NMR studies demonstrate that water interacts strongly with GSH-borne protons in both solution and gel states, but only the gels include water-pyrenyl interactions with significant residence times.     

A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis

We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.     

Radiation Processed Amniotic Membranes in the Treatment of Non-Healing Ulcers of Different Etiologies

The amniotic membranes were collected from the placentae of selected and screened donors. Processing was done by washing the fresh amniotic membrane successively in sterile saline, 0.05% sodium hypochlorite solution and sterile distilled water until it was completely cleared of blood particles. The membranes were sterilized by gamma irradiation at 25 kGy. The processed amniotic membranes were applied to 50 open wounds comprising of 42 full thickness defects and eight partial thickness defects. These included leprotic, diabetic, traumatic, gravitational ulcers and superficial burn in the form of scald and corrosive burn. The radiation processed amniotic membranes favoured healing of unresponsive and non-healing ulcers of different etiologies. Ulcers with duration of minimum 3 weeks to maximum 12 months were found to heal in 2-6 weeks by the application of amniotic membranes.     

Comigration of two autophagosome-associated dehydrogenases on two-dimensional polyacrylamide gels

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.     

Effect of ultrasonically nebulized distilled water on airway epithelial cell swelling in guinea pigs.

To investigate the pathogenesis of ultrasonically nebulized distilled water-induced airway narrowing, we studied the role of airway epithelial cells during a distilled water-inhalation challenge in an animal model of airway inflammation. Guinea pigs were divided into four groups: 1) a sham/saline (S/S) group: sham ozone followed by saline inhalation; 2) a sham/water (S/W) group: sham ozone followed by water inhalation; 3) an ozone/saline (O/S) group: ozone followed by saline inhalation; and 4) an ozone/water (O/W) group: ozone followed by water inhalation. After exposure to either 3.0 parts/million ozone or air at the same flow rate for 2 h, guinea pigs were anesthetized and tracheostomized, and then lung resistance (RL) was measured. For morphometric assessment, tissues were fixed with formaldehyde, stained with hematoxylin and eosin, and cut into transverse sections. Airway dimensions were either measured directly or calculated from the internal perimeter, the external perimeter, and airway wall area. There were no statistical differences in the values of RL before distilled water inhalation between the sham groups and the ozone groups. RL increased significantly after 10 min of distilled water inhalation in both the S/W group and the O/W group. In the S/W group, epithelial cells were swollen, and intercellular spaces were wider, resulting in significant increase in epithelial wall thickness, but there was no significant infiltration by inflammatory cells. In the O/S group, the epithelium showed infiltration by inflammatory cells without change in cell volume. In the O/W group, the epithelium showed both infiltration and a greater increase in epithelial wall thickness compared with the S/W group. These results suggest that airway epithelial cell swelling, induced by inhaled distilled water, increases with RL in guinea pigs and that this reaction may be accelerated by airway inflammation.     

In Vitro Evaluation of Proniosomes as a Drug Carrier for Flurbiprofen

The purpose of the present investigation is to formulate and evaluate proniosomal transdermal carrier systems for flurbiprofen. Proniosomes were prepared using various non-ionic surfactants, namely span 20 (Sp 20), span 40 (Sp 40), span 60 (Sp 60) and span 80 (Sp 80) without and with cholesterol at percentages ranging from 0% to 50%. The effect of surfactant type and cholesterol content on drug release was investigated. Drug release was tested by diffusion through cellophane membrane and rabbit skin. Drug release from the prepared systems was compared to that from flurbiprofen suspensions in distilled water and HPMC (hydroxypropylmethylcellulose) gels. In case of Sp 20 and Sp 80, the added amount of cholesterol affected the preparation type to be either proniosomal alcoholic solutions or liquid crystalline gel systems. On the other hand, both Sp 40 and Sp 60 produced gel systems in presence or absence of cholesterol. Microscopic observations showed that either proniosomal solutions or gel formulations immediately converted to niosomal dispersions upon hydration. Due to the skin permeation barrier, rabbit skin showed lower drug diffusion rates compared to cellophane membrane. The proniosomal composition controlled drug diffusion rates to be either faster or slower than the prepared flurbiprofen suspensions in HPMC gels or distilled water, respectively. In conclusion, this study demonstrated the possibility of using proniosomal formulations for transdermal drug delivery.