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Controlled and Reproducible Fixation of the Lung for Correlated Studies
Published in Joan Gil, Models of Lung Disease, 2020
Regarding fixative, the most commonly used solution is 1 % glutaraldehyde in isotonic buffer (phosphate or cacodilate) (Gil, 1977; Hayat, 1981). The lung is then immersed into the same fixative and diced. Fragments of lung must then be washed in isotonic buffer (1 h, or 3 × 10 min) if possible while shaking, and this must be followed by osmification for 1-2 h (1-1.5 % osmium tetroxide in isotonic buffer, collidine, or similar).
Reactivities of Amino Acids and Proteins with Iodine
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
The method has already been introduced in Chapter 2, Section III, and it may be used for iodoamino acids under the same general conditions as for radioiodide and its oxidative products. In addition to butanol-acetic acid and propanol-ammonia-water (see Chapter 2, Section III.B.2), pyridine-2M-acetic acid and collidine-2M-acetic acid are useful solvents in this field. Their Rf values are summarized in Table 20. It is seen that none of the solvents gives a perfect resolution of all the compounds listed. Thus, monoiodotyrosine and diio- dohistidine chromatograph too close to each other in butanol-acetic acid, and, similarly, the resolution of the iodotyrosines in pyridine-acetic acid is inadequate. However, this problem can be overcome by running samples of the same preparation simultaneously in two different solvents as one-dimensional chromatograms. (This obviates the problem of location as it arises in two-dimensional chromatograms.)
Chemical Synthesis of Core Structures
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
Chemical syntheses of oligosaccharides comprising α-(l→3)- and α-(l→7)-linked disaccharides of l-glycero-d-manno-heptopyrmosy, residues (l,d-Hepp), which are common subunits of enterobacterial LPS, were first accomplished using a heptopyranosyl trichloroacetimidate donor activated by benzyl ether-protecting groups (56). Under catalysis with anhydrous toluene sulfonic acid, the α-(l→3)-linked disaccharide was obtained in 50% yield, whereas the α-(l→7)-linked isomer was prepared in 91% yield as a 3.5:1 ato-(3 mixture. Further extension of the synthetic scheme under similar conditions gave, after deblocking, the trisaccharide l-α-d-Hepp-(1 →7)-l-α-d-Hepp-(1 →3)-l,d-Hep. In an alternative preparation, the disaccharide structures were elaborated using 2-(9-acetyl-3,4,6,7-tetra-O-benzyl-heptopyranosyl chloride 34 as a donor and silver trifiate as promoter to give the α-(1→3)- and the α-(1→7)-linked disaccharide derivatives in good yields (43). The heptopyranosyl chloride 34 was also employed for the assembly of trisaccharides occurring in the inner core of Citrobacter PCM 1487 LPS (57). Simultaneous formation of two glycosidic linkages by silver trifiate promoted condensation of 34 with methyl 2,4,6-tri-O-benzyl l-glycero-α-d-manno-heptopymno-side as acceptor afforded the protected branched trisaccharide methyl glycoside l-α-d-Hepp-(l→3)-[l-α-DHepp-(l→7)]-l-α-d-Hepp-0Me in excellent yield (71%). Furthermore, a heptopyranoside monosaccha-ride acceptor containing the versatile dimethyl-(phenyl)silyl function as a hydroxy-masking group at C-7 was compatible with a coupling reaction using tetra-O-benzyl-glucopyranosyl chloride as donor in the presence of silver trifiate giving the α-(l→3)-linked disaccharide in 90% yield without formation of the p anomer. Subsequent transformation of the carbon-silicon bond afforded a free 7-OH group, which in turn was glycosylated with the heptopyranosyl chloride 34 in 89% yield. Conventional deblocking afforded the branched trisaccharide methyl glycoside α-d-Glcp-(1 →3)-[l-α-d-Hepp-(1 →7)]-l-α-d-Hepp-0Me (57). The same trisaccharide had previously been obtained in comparable overall yield using acetylated heptopyranosyl bromide as the glycosyl donor in the presence of silver trifiate and collidine (58).
Induction of Dbp by a histone deacetylase inhibitor is involved in amelioration of insulin sensitivity via adipocyte differentiation in ob/ob mice
Published in Chronobiology International, 2019
Chisato Suzuki, Kentaro Ushijima, Hitoshi Ando, Hiroko Kitamura, Michiko Horiguchi, Tomomi Akita, Chikamasa Yamashita, Akio Fujimura
Measurement of the cell size distribution of adipocytes was performed as described previously, albeit with minor modification (Kaplan et al. 1980; Oh and Kaplan 1995). Fractionated adipocytes were fixed in 2% osmium tetroxide (OsO4) (VIII) solution containing 50 mM γ-collidine at room temperature for 2 weeks. Fixed adipocytes were washed twice with Isoton solution (Beckman Coulter, Brea, CA, USA). Cell size distribution of adipocytes was measured using a Z1D Coulter counter with an aperture tube (diameter: 200 µm; Beckman Coulter). One sample was applied to six different threshold values: 40, 50, 60, 80, 100, and 120 µm. Since the number of cells counted was bigger than the setting threshold value, the 40 µm threshold value count was used as the total cell number. Finally, cell number percentages in each segment were calculated.
Methemoglobin Forming Effect of Dimethyl Trisulfide in Mice
Published in Hemoglobin, 2018
Márton Kiss, Ilona Petrikovics, David E. Thompson
Unless otherwise stated, all solutions were prepared using type I deionized (DI) water that was produced by a Millipore Milli-Q Direct 8 water purification system (MilliporeSigma, Burlington, MA, USA). A pH 7.0 collidine buffer [0.675% (V/V)] was prepared by mixing 337.5 μL of 2,4,6-trimethylpyridine, 40 mL DI water, and 1.75 mL of 1 M HCl; confirming a pH of 7.0 using a benchtop pH-meter (Orion star A111, Thermo Scientific, Waltham, MA, USA); transferring the buffer solution to a 50 mL volumetric flask and diluting to the mark. A 10,000 U/mL heparin solution was prepared by dissolving 277 mg of heparin sodium salt in 5 mL DI water. This solution was subsequently diluted to yield a 500 U/mL solution. The DMTS-AF formulation (50 mg/mL) was prepared by diluting 500 mg of DMTS to 10.0 mL with 15.0% aqueous P80 solution [11]. The DMTS-NAF formulation (originally developed by Southwest Research Institute, San Antonio, TX, USA) was prepared by mixing 2.00 g of DMTS into a surfactant blend consisting of 0.75 g Span 80 and 2.25 g P80. Following mixing, the DMTS-NAF formulation was homogenized by vortexing. A 5.0% (w/v) KCN solution was made by diluting 250 mg of KCN salt to the mark with DI water in a 5 mL volumetric flask.