Mouse model of dermal fibrosis induced by one-time injection of bleomycin-poly(L-lactic acid) microspheres.

OBJECTIVE: Animal models are useful tools to study various aspects of human diseases. Bleomycin (BLM)-induced scleroderma mouse has been widely investigated as an animal model of scleroderma. Repeated injections of BLM, either daily or every other day, for 3-4 weeks are required to induce scleroderma in mice. Poly(L-lactic acid) (PLA) is a biodegradable, biocompatible and bioabsorbable device that has been widely investigated for controlled drug release. In this study, we fabricated BLM-containing PLA microspheres and subcutaneously injected them into C3H mice for only one time. METHODS: Treated skins were harvested at days 7 and 21. Then, histological examination and collagen content measurement assay were performed. The mRNA expression of alpha1(I) collagen (COL1A1), monocyte chemoattractant protein-1 (MCP-1), TGF-beta(1) and connective tissue growth factor (CTGF) were quantified by real-time PCR. RESULTS: Dermal fibrosis was histologically observed at day 7 after injection and remained present at day 21. Tissue responses against BLM-PLA microspheres alone were mild. Soluble collagen content and expression level of alpha1(I) collagen mRNA were significantly elevated at day 21. Expression levels of MCP-1 mRNA and TGF-beta(1) mRNA at day 7 and CTGF mRNA at day 21 were also elevated. CONCLUSION: The present study demonstrated for the first time that one-time injection of BLM-PLA microspheres can induce dermal fibrosis in C3H mice. BLM-PLA microspheres thus offer a labour-saving, simple and powerful tool to establish an animal model of BLM-induced dermal fibrosis.

Compound heterozygosity in sibling patients with recessive dystrophic epidermolysis bullosa associated with a mild phenotype.

We describe two cases of a 3-year-old Japanese boy and his 1-year-old sister presenting recessive dystrophic epidermolysis bullosa; a relatively mild phenotype. Blistering and scarring were limited to the acral region, and some fingernails and toenails were lost. PCR-RFLP and DNA sequencing analyses revealed compound heterozygotes for a splice-site mutation (6573 +1GtoC) and a nonsense mutation (E2857X) in the type VII collagen gene (COL7A1). Both mutations caused a premature termination codon (PTC). The mutation E2857X was located behind the candidate cleavage site within the NC-2 domain required for the assembly of anchoring fibrils. This PTC position may explain their mild phenotype.

c-kit Mutation in generalized lentigines associated with gastrointestinal stromal tumor.

We describe the case of a 59-year-old Japanese woman presenting with generalized lentigines without systemic anomalies. She had a medical history of gastrointestinal stromal tumors (GISTs), in which gain-of-function mutations of the c-kit gene had recently been found. We detected a point mutation at codon 557 in exon 11 of leukocyte DNA from the patient. The stem cell factor-type III receptor tyrosine kinase pathway plays important roles in the regulation of melanocyte proliferation and differentiation. We speculate that the generalized lentigines of the patient may be caused by melanocyte proliferation due to the c-kit gene mutation.

Separation and retention of human blood cells by surface-affinity chromatography.

This review describes the surface-affinity chromatography of human peripheral blood cells on polyethylene glycol (PEG) and polypropylene glycol (PPG) chemically bonded columns. The affinity of surface blood cells to bonded PEG and PPG stationary phases is apparently selective, and granulocytes and lymphocytes are more strongly retained on the column than erythrocytes and platelets. The retention factors of granulocytes and lymphocytes increased with increase in the hydrophobicities (Delta log K values) of PPG-agarose column packing beads. Hydrophobic interactions contributed to the retention of the blood cells on the PPG-bonded agarose columns.

Sunlight induces N epsilon-(carboxymethyl)lysine formation from glycated polylysine-iron(III) complex.

Sunlight was found to strongly induce the formation of N epsilon-(carboxymethyl)lysine (CML) from glycated polylysine in the presence of Fe(III) ion. The initial step of this Fe(III)-catalyzed CML formation was noted to be similar to that of blueprint photography as was confirmed by the production of Turnbull's blue in sunlight-exposed glycated human serum albumin ferricyanide solution in the presence of Fe(III). Based on this, photoinduced oxidative C-C bond cleavage of the Amadori compound was assumed to be initiated by photochemical single electron transfer front ligand to Fe(III) in the Fe(III)-Amadori compound complex affording the Fe(II)-Amadori compound radical intermediate, which eventually yields either CML or active oxygen species. CML is thus a useful oxidative stress marker. The mechanism proposed here would explain the high accumulation of CML in lens protein and skin actinic elastosis.

High-speed counter-current chromatography of apple procyanidins.

Apple procyanidins were separated by high-speed counter-current chromatography using a type-J multilayer coil planet centrifuge. Several two-phase solvent systems with a wide range of hydrophobicities from a non-polar hexane system to polar n-butanol systems were evaluated their performance in terms of the partition coefficient and the retention of the phase. The best separation of procyanidins B and C was achieved with a two-phase solvent system composed of n-butanol-methyl tert.-butyl ether-acetonitrile-0.1% trifluoroacetic acid (2:4:3:8) using the lower phase as a mobile at a flow-rate of 1.0 ml/min.

Surface affinity chromatography of human peripheral blood cells.

Column packings with chemically bonded polyethylene glycol (PEG) and polypropylene glycol (PPG) were prepared and the separation of human peripheral blood cells by an aqueous polymeric two phase system could be performed in a partition chromatography. In this chromatographic system, the interaction of blood cells with bonded PEG, PPG stationary phases is apparently selective, and granulocytes and lymphocytes are more strongly retained on the column than erythrocytes and platelets. The hydrophobic interactions between the hydrophobic region on the cell surface membrane and the bonded PEG, PPG phases is probably the main factor affecting the retention of lymphocytes and granulocytes. The selective separation of human peripheral platelets, granulocytes and lymphocytes was investigated using another type of column packing bonded with methoxyethoxymethyl (MEM) groups. The isolation of platelets and lymphocytes from human leukocyte-rich plasma was performed with a MEM-Sephadex column and elution with 0.27 M sucrose solution. Platelets could be selectively collected from the same column by elution with 0.31 M methyl-alpha-D-mannoside at the high recovery of 100%.

One-step purification of proteins from chicken egg white using counter-current chromatography.

Proteins present in chicken egg white are separated by counter-current chromatography (CCC) in one step using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% (w/w) poly(ethylene glycol) 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 1.0 ml/min. From about 20 g of the crude egg white solution, lysozyme, ovalbumin, and ovotransferrin were resolved within 5.5 h. Each component was identified by 12% SDS gel electrophoresis with Coomassie brilliant blue staining.

Lipoproteins: comparison of different separation strategies.

This review describes two chromatographic techniques for the separation of three main classes of lipoproteins (HDLs, LDLs and VLDLs) from human serum: hydroxyapatite chromatography and counter-current chromatography. The HDLs, LDLs and VLDLs were purified by the combined use of the two chromatographic techniques without prior ultracentrifugation.

Purification of lactic acid dehydrogenase from bovine heart crude extract by counter-current chromatography.

To test the utility of counter-current chromatography in purifying proteins, lactic acid dehydrogenase (LDH) was extracted from a crude bovine heart filtrate using a cross-axis coil planet centrifuge. The purification was performed with several polymer phase systems composed of 16% (w/w) poly(ethylene glycol) (PEG) 1000-12.5% (w/w) potassium phosphate buffers and 4.4% (w/w) PEG 8000-7.0 (w/w) dextran T500 at pH values ranging from 6.5 to 11.0. The best purification was achieved using PEG 1000-potassium phosphate buffer system at pH 7.3 by eluting the upper phase at 1.0 ml/min. Fractions were analyzed by LDH enzymatic activity and sodium dodecyl sulfate slab gel electrophoresis (SDS-PAGE). The LDH was purified directly from bovine heart crude extract within 3 h.

Selective separation of human peripheral platelets, granulocytes and lymphocytes by surface affinity chromatography.

Selective separation of human peripheral platelets, granulocytes and lymphocytes was investigated by column liquid chromatography using methoxyethoxymethyl (MEM) bonded-phase columns (25 x 0.9 cm I.D.). Isotonic solutions containing mono- and disaccharides, methyl-alpha-D-pyranosides and a physiological saline at pH 7.4 were used as the mobile phase. Granulocytes and lymphocytes were separated on the MEM-Cellulofine GH-25 column by elution with 0.3 M D-mannose solution. The isolation of platelets and lymphocytes from human leukocyte-rich plasma was performed with a MEM-Sephadex G25 column and elution with 0.27 M sucrose solution. On the same column platelets could also be collected selectively by elution with 0.31 M methyl-alpha-D-mannoside at the high recovery of 100%. The isolated cells were viable for more than 90%.

Complementary use of counter-current chromatography and hydroxyapatite chromatography for the separation of three main classes of lipoproteins from human serum.

High-density, low-density and very-low-density lipoproteins (HDLs, LDLs and VLDLs) were purified from human serum by the combined use of counter-current chromatography (CCC) and hydroxyapatite chromatography. Polymer-phase CCC of human serum using the cross-axis coil planet centrifuge yielded two lipoprotein fractions, one containing HDLs and LDLs and the other VLDLs and serum proteins. Each fraction was concentrated and subjected to hydroxyapatite chromatography to obtain three lipoprotein fractions, all free from serum proteins. Each lipoprotein was confirmed by agarose gel electrophoresis.

Studies on an abnormally sharpened elution peak observed in counter-current chromatography.

Counter-current chromatography (CCC) of the bromoacetylation product of 3,3',5-triiodo-L-thyronine (T3) produced an unusually sharp peak for the desired product, N-bromoacetyl T3 (BrAcT3). A series of experiments revealed that bromoacetic acid, probably present as a side reaction product in the sample solution, was responsible. This compound repressed the ionization of the carboxyl group of BrAcT3 forcing it into the less polar stationary phase until the bromoacetic acid had eluted completely from the apparatus. At this point, the sudden increase of pH and consequent ionization of the BrAcT3 allowed the ammonium salt of the latter to enter the more polar moving phase where it eluted rapidly from the column as a sharp peak. The same phenomenon was observed in the CCC fractionation of a series of indole auxins where addition of trifluoroacetic acid to the sample caused peak sharpening by the same process. The phenomenon recalls pH gradient elution and isoelectric focussing except that the substance responsible for the pH range here is added along with the sample in one bolus forming a sharp pH gradient at its trailing edge. As with gradient elution, the technique is of practical interest since it permits collection of the eluting compounds with increased detectability in fewer fractions. The technique can also enhance separation of compounds whose partition coefficients differ with a change in pH.

Complementary use of counter-current chromatography and preparative reversed-phase high-performance liquid chromatography in the separation of a synthetic mixture of brominated tetrachlorofluoresceins.

A synthetically prepared mixture of brominated 4,5,6,7-tetrachlorofloresceins was separated by a combination of preparative reversed-phase high-performance liquid chromatography and high-speed counter-current chromatography. Two new lower-brominated subsidiary colors of D&C Red Nos. 27 and 28 (phloxine B), 4',5'-dibromo-4, 5,6,7-tetrachlorofluorescein and 2',4',5'-tribromo-4,5,6, 7-tetrachlorofluorescein, were isolated and characterized by 1H NMR and chemical ionization mass spectrometry.

Counter-current chromatography of lipoproteins with a polymer phase system using the cross-axis synchronous coil planet centrifuge.

Lipoproteins were separated by counter-current chromatography using the type-XLL coil planet centrifuge. The separation was performed with a polymer phase system composed of 16% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 0.5 ml/min. About 5 ml of the sample solution containing approximately 150 mg of a lipoprotein mixture were loaded. High- and low-density lipoproteins were resolved within 12 h. Each component was detected by gel electrophoresis with oil red staining.

Protein separation with aqueous-aqueous polymer systems by two types of counter-current chromatographs.

Two different types of counter-current chromatographs, the cross-axis coil planet centrifuge (X-axis CPC) and horizontal flow-through coil planet centrifuge (horizontal CPC), were evaluated for protein separation with an aqueous-aqueous two-phase polymer system. The sample solution, containing 10-200 mg each of cytochrome c, myoglobin, ovalbumin and hemoglobin in 2 ml of each phase was eluted with the lower phase. In both instruments, the effects of the flow-rate, revolution speed, and parameter beta (helical diameters of the multilayer coil) on the protein separation were investigated. The best results were obtained from the X-axis CPC operated at 750 rpm and a flow-rate of 2.0 ml/min using a multilayer coil with a small helical diameter (beta = 0.25-0.60). Four protein samples were well resolved in less than 5 h.

Separation of human serum lipoproteins into three major classes by hydroxyapatite chromatography.

The separation of normolipidemic male serum lipoprotein fraction, prepared by ultracentrifugal flotation, was studied on hydroxyapatite columns. Potassium phosphate buffers in the pH range 5.6-7.4 were evaluated as eluents. The three main classes of the lipoproteins (high density, low density and very low density) can be separated on the Tiselius-type hydroxyapatite (Bio-Gel HTP DNA grade) column by elution with 75, 250 and 300 mM potassium phosphate buffer (pH 7.4), respectively.

Surface affinity chromatographic separation of blood cells. VII. Relationship between capacity factors of human peripheral blood cells and the rate of penetration of liquids into xerogel column packings.

Eleven kinds of column packing gels which bonded poly(ethylene glycol) (PEG) to Sepharose 6B (PEG-C10-Sepharose) were prepared. Human peripheral blood cells were chromatographed on these gel columns by eluting with 0.09 M phosphate-buffered 2% (w/w) dextran T40 solution at the pH of the respective isoelectric points of the blood cells. The rate of penetration of water or the mobile phases into the PEG-C10-Sepharose xerogels as a measure of the hydrophobicity of the gels depended on both the oxyethylene residue content and the number of oxyethylene units of the packing gels. The capacity factors of granulocytes and lymphocytes were increased on the columns packed with gels having a slower rate of penetration of the liquids into the xerogels.

Surface affinity chromatographic separation of blood cells. VI. Capacity factors of human peripheral blood cells on poly(propylene glycol) bonded chromagel columns and their correlation with surface hydrophobicities of the gel beads and the cells.

The chromatographic behaviour of human peripheral blood cells was investigated on poly(propylene glycol) (PPG)-bonded Chromagel. The mobile phase was 0.09 M phosphate-buffered 2% (w/w) dextran T40 solution at the pH of the respective isoelectric points of human blood cells. The elution order of four kinds of blood cells from PPG-C3-Chromagel columns coincided with that of the delta log K values of these cells determined by the hydrophobic affinity partition method using Pluronic P84 as a hydrophobic ligand. The capacity factors of granulocytes and lymphocytes increased with increase in the delta log K values of PPG-C3-Chromagel beads. Hydrophobic interactions contributed to the retention of the four kinds of blood cells on PPG-bonded Chromagel columns.