BACKGROUND: Though elastic fibers are as important as collagen fibers in interpretation of the histopathologic findings, it is impossible to observe them on the hematoxylin & eosin (H&E) stained specimen. OBJECTIVE: Characterizing eosin fluorescence emitted by elastic fibers in H&E stained specimens. METHODS: Normal skin tissue sections were stained in 4 different ways (unstained, hematoxylin only, eosin only, H&E) and observed under a fluorescence microscope using a FITC filter set. Fluorescent findings of 30 H&E-stained specimens showing abnormal dermal findings were compared with bright field findings of Miller's elastic stained specimen. RESULTS: Strong eosin fluorescence was related to the differential binding property of eosin with elastic fibers. Hematoxylin stain quenched excessive eosin fluorescence from other tissue components and contributed to better contrast. Fluorescence microscopy of H&E-stained sections was found to be especially useful in observing mature elastic fibers in the reticular dermis. In 74% of the specimens, eosin fluorescence findings of elastic fibers in reticular dermis matched well with that of specimens with elastic fiber special stain. CONCLUSION: Analysis of skin elastic fibers by fluorescence microscopy is a useful and complementary method to reveal hidden elastic fibers in H&E-stained specimens.
Collagen ; Dermis ; Elastic Tissue ; Enzyme Multiplied Immunoassay Technique ; Eosine Yellowish-(YS) ; Fluorescein-5-isothiocyanate ; Fluorescence ; Hematoxylin ; Microscopy, Fluorescence ; Skin

OBJECTIVETo establish an HPLC method for the determination of four alkaloids, i.e., 2-hydroxy-1-methoxyaporphine, pronuciferine, nuciferine and roemerine, in Nelumbo nucifera and its alkaloid fraction.

METHODThe determination was carried out at 35 degrees C on a Hypersil C18 column (4.6 mm x 250 mm, 5 microm), eluting with acetonitrile-water containing 0.1% triethylamine as mobile phases in gradient mode. The flow rate was 1.0 mL x min(-1) and detection at the wavelength was set at 270 nm.

RESULTThe linear ranges of 2-hydroxy-1-methoxyaporphine, pronuciferine, nuciferine and roemerine were 0.110-0.658 microg (r = 0.9995), 0.0210-0.126 microg (r = 0.9995), 0.103-0.618 microg (r = 0.9998), 0.085 6-0.514 microg (r = 0.9995), with the average recoveries (n=6) were 101.5%, 99.14%, 99.21% and 98.41% for the alkaloid fraction of N. nucifera and 99.53%, 100.5%, 97.51% and 100.1% for N. nucifera respectively.

CONCLUSIONThe determination results of the three batches of samples showed that the method was easy and accurate which could be used to determine the contents of four components in N. nucifera and its alkaloid fraction.

Alkaloids ; chemistry ; Aporphines ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Nelumbo ; chemistry ; Spiro Compounds ; chemistry

The present study aimed at identifying cell cycle inhibitors from the fermentation broth of Streptomyces pseudoverticillus YN17707. Activity-guided isolation was performed on tsFT210 cells. Compounds were isolated through various chromatographic methods and elucidated by spectroscopic analyses. Flow cytometry was used to evaluate the cell cycle inhibitory activities of the fractions and compounds. Two compounds were obtained and identified as pteridic acid hydrate (1) and pteridic acid C (2), which arrested the tsFT210 cells at the G0/G1 phase with the MIC values being 32.8 and 68.9 μmol·L(-1), respectively. These results provide a basis for future development of Compounds 1 and 2 as novel cell cycle inhibitors for cancer therapy.
Cell Cycle Checkpoints ; drug effects ; Cell Line ; Heptanoic Acids ; chemistry ; isolation & purification ; pharmacology ; Humans ; Molecular Structure ; Spiro Compounds ; chemistry ; isolation & purification ; pharmacology ; Streptomyces ; chemistry

BACKGROUNDDiabetic nephropathy (DN) is the leading cause of end-stage renal disease. Various treatment regimens and combinations of therapies provide only partial renoprotection. Therefore new approaches are needed to retard the progression of DN. The aim of the present study was to evaluate the role of a novel spiroalkaloid from Acorus tatarinowii named acortatarin A (AcorA) in inhibiting high glucose-induced extracellular matrix accumulation in mesangial cells (MCs).

METHODSThe cytotoxity of AcorA on MCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay. The expression of fibronectin and collagen IV was examined by real time PCR and western blotting. The expression of p22(phox) and p47(phox) was detected by western blot. The interaction between p22(phox) and p47(phox) was examined by co-immunoprecipitation. The phosphorylation of p47(phox) was examined by immunoprecipitation. The phosphorylation of protein kinase C (PKC) α, PKCβ, phospholiase C gamma (PLCγ1), and the p85 subunit of PI3K was determined by Western blotting.

RESULTSAcorA significantly inhibited high glucose-induced activation of NADPH oxidase, a ROS-generating enzyme, by increasing phosphorylation of p47(phox) and enhancing interaction between p22(phox) and p47(phox). Preincubation of AcorA with MCs inhibited high glucose-induced collagen IV and fibronectin production in a dose-dependent manner. Moreover, AcorA attenuated high glucose enhanced phosphorylation of PKCα, PKCβ, PLCγ1, and the p85 subunit of PI3K.

CONCLUSIONAcorA inhibits high glucose-induced extracellular matrix production via blocking NADPH oxidase activation.

Animals ; Blotting, Western ; Cell Line ; Extracellular Matrix ; metabolism ; Glucose ; metabolism ; Immunoprecipitation ; Mesangial Cells ; drug effects ; metabolism ; Morpholines ; metabolism ; Rats ; Real-Time Polymerase Chain Reaction ; Spiro Compounds ; metabolism

OBJECTIVETo study the chemical constituents of Costus speciosus and C. tonkinensis (Zingiberaceae) distributed in Yunnan province.

METHODChromatography and spectral analyses were used to isolate the constituents and elucidate their structure.

RESULTSix compounds were isolated from the rhizome of C. speciosus and elucidated as diosgenin(1), prosapogenin B of dioscin(2), diosgenone(3), cycloartanol(4), 25-en-cycloartenol(5) and octacosanoic acid(6). Four compounds were isolated from the rhizome of Costus tonkinensis and elucidated as tetracosanoic acid(7), succinic acid(8), beta-sitosterol(9) and daucosterin(10).

CONCLUSIONCompounds of 3-6 were obtained from C. speciosus for the first time and compounds of 7-10 were obtained from C. tonkinensis for the first time too.

Costus ; chemistry ; classification ; Fatty Acids ; chemistry ; isolation & purification ; Plants, Medicinal ; chemistry ; Rhizome ; chemistry ; Spiro Compounds ; chemistry ; isolation & purification ; Triterpenes ; chemistry ; isolation & purification

Author investigated the deposition of immunoglobulins and complements in thc skin of 56 patients with 19 various dermatoses by direct immunofluorescent (DIF) staining. The biopsied specimens were quick-frozen(by dry ice-acetone) and stained with FITC conjugated antihuman immunoglobulin and complement after cutting in a cryostat at -20C~30C. (countinued...)
Complement System Proteins ; Dronabinol ; Fluorescein-5-isothiocyanate ; Humans ; Immunoglobulins ; Skin ; Skin Diseases*

Natural cytotoxicity mediated by natural killer (NK) cells is believed to play an important role in host anticancer defense mechanisms. The aim of this study is to compare the number of NK cells in patients with colorectal cancer and hemorrhoids, and before and after surgery in patients with colorectal cancer. Twenty colorectal cancer patients and twenty hemorrhoid ones were studied. Venous blood samples were obtained preoperatively, and on the 7th, and 14th postoperative days. Mononuclear cells were isolated over Ficoll-Hypaque gradients, and T cells, B cells, and NK cells were measured with CD3 FITC (T cell), CD 19 PE (B cell), and CD56 FITC (NK cell) antibody, The number of T cell (/mm3) was 1224, 1280, and 1125 at preoperative, 7th, and 14th postoperative day in hemorrhoid patients and 1195, 901, and 1060 in colorectal cancer patients respectively. The number of B cell (/mm3) was 243, 160, and 250 in hemorrhoid patients and 147, 78, and 113 in colorectal cancer patients. The number NK cell (/mm3) was 148, 156, and 143 in hemorrhoid patients and 129, 85, and 128 in colorectal cancer patients. There was no difference among Dukes stages in the number of NK cells. In conclusion, the number of NK cells was not changed in colorectal cancer patients compared with hemorrhoid ones. Major operation changed the number of NK cells in colorectal cancer patients.
B-Lymphocytes ; Colorectal Neoplasms* ; Defense Mechanisms ; Fluorescein-5-isothiocyanate ; Hemorrhoids ; Humans ; Killer Cells, Natural* ; T-Lymphocytes

AIMTo establish a simple and effective method for RBCs labeling and survival assays, and the qualities of rabbit RBCs preserved in GMA solution at 4 degrees C were verified.

METHODSThe bloods were taken through the ear arteries of the rabbits. The RBCs were labeled by fluorescein isothiocyanate (FITC), and were reinjected to the same rabbit through ear veins. The percentage of FITC labeled RBCs was assayed by FACS at a series of times after injection. The SAS software was employed to analyze the data and establish the regression equations. The 24-hour recovery and the half-life span of the labeled RBCs were calculated according to the equations.

RESULTSThe 24-hour recovery and the half-life span of the labeled RBCs in the control group were 93.76% +/- 5.40% and 22.50% +/- 4.37 days respectively, which was in agreement with the previous papers. The 24-hour recovery and the half-life span of the labeled RBCs in the GMA group were 89.13% +/- 7.10% and 11.41% +/- 1.63 days respectively, which was coincident with the infusion conditions.

CONCLUSIONCompared with other methods of RBCs labeling in vivo, FITC labeling was thought to be easier and cheaper to use, which could facilitate the analysis of the biological character of the labeled cells, and could be used to trace the fate of labeled cells.

Animals ; Blood Preservation ; methods ; Erythrocyte Aging ; physiology ; Erythrocyte Count ; Erythrocytes ; physiology ; Fluorescein-5-isothiocyanate ; Rabbits ; Software

In order to prepare angiopep-2 modified fluorescein isothiocyanate-labeled neurotoxin nanoparticles( ANG-NPs/FITCNT),emulsion/solvent evaporation method was used with m PEG-PLA and ANG-PEG-PLA( in proper proportions) as carriers and with FITC-NT as drug. With particle size and encapsulation efficiency as comprehensive indexes,the effects of different ultrasound power and ultrasound time combinations on the process were investigated. The in vitro release characteristics of nanoparticles in PBS buffer at p H 7. 4 and p H 6. 5 were investigated by dialysis method. The results indicated that the optimum process for preparing ANG-NPs/FITC-NT was as follows: ultrasonic power 90 W,ultrasonic time 30 s. In such optimal process,ANG-NPs/FITC-NT were well-shaped under the transmission electron microscope,with an average particle size of( 123. 9±0. 5) nm,Zeta potential of(-10. 5±0. 5) m V,encapsulation efficiency of( 68. 1±0. 4) %,and the drug loading of( 0. 82±0. 01) %. The in vitro drug release profiles of the nanoparticles in PBS buffer at p H 7. 4 and p H 6. 5 were both consistent with Ritger-Peppas equation,ln Q = 0. 508 8 lnt-2. 285 0,r = 0. 961 5( p H 7. 4) and ln Q= 0. 449 9 lnt-1. 855 3,r = 0. 970 3( p H 6. 5),respectively. The experiment results proved that the nanoparticles prepared by emulsion/solvent evaporation method had uniform particle size,high encapsulation efficiency and in vitro sustained release characteristic,which might be a potential carrier for NT intracerebral drug delivery.
Drug Carriers ; Fluorescein-5-isothiocyanate ; Nanoparticles ; Particle Size ; Peptides ; Polyethylene Glycols

The objective of this work is to evaluate the effect of polyamidoamine (PAMAM) dendrimers on electroosmotic flow (EOF) through skin. The effect of size and concentration of dendrimer was studied, using generation 1, 4 and 7 dendrimer (G1, G4 and G7, respectively). As a marker molecule for the direction and magnitude of EOF, a neutral molecule, acetoaminophen (AAP) was used. The visualization of dendrimer permeation into the current conducting pore (CCP) of skin was made using G4–fluorescein isothiocyanate (FITC) conjugate and confocal microscopy. Without dendrimer, anodal flux of AAP was much higher than cathodal or passive flux. When G1 dendrimer was added, anodal flux decreased, presumably due to the decrease in EOF by the association of G1 dendrimer with net negative charge in CCP. As the generation increased, larger decrease in anodal flux was observed, and the direction of EOF was reversed. Small amount of methanol used for the preparation of dendrimer solution also contributed to the decrease in anodal flux of AAP. Cross-sectional view perpendicular to the skin surface by confocal laser scanning microscope (CLSM) study showed that G4 dendrimer-FITC conjugate (G4-FITC) can penetrate into the viable epidermis and dermis under anodal current. The permeation route seemed to be localized on hair follicle region. These results suggest that PAMAM dendrimers can permeate into CCP and change the magnitude and direction of EOF. Overall, we obtained a better understanding on the mechanistic insights into the electroosmosis phenomena and its role on flux during iontophoresis.
Acetaminophen ; Dendrimers* ; Dermis ; Electroosmosis* ; Epidermis ; Fluorescein-5-isothiocyanate ; Hair Follicle ; Iontophoresis ; Methanol ; Microscopy, Confocal ; Skin*