The purpose of this study is to investigate the penetration effects and mechanism of N-arginine chitosan (ACS). This novel transdermal enhancer with a mimetic structure of cell-penetration peptides was synthesized by introducing hydrophilic arginine groups to the amino-group on chitosan's side chain. The structure of ACS was confirmed by FT-IR, 1H NMR and element analysis. In addition, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the protein conformation and the water content of stratum corneum, and the result suggested that ACS can change the orderly arrangement of the molecules in the stratum corneum, making the stack structure of keratin become loose. And ACS can increase the water content of the stratum corneurn. Inverted fluorescence microscope and flow cytometry were used to examine penetration effect of ACS on Hacat cell. The result confirmed that the uptake of ACS was enhanced with increased substitution degree of arginine by 4-8 folds compared to chitosan. In vitro penetration studies on three electrical types of drugs were carried out using three model drugs of negatively charged aspirin, positively charged terazosin and neutral drug isosorbide mononitrate by the method of Franz diffusion cells. The results showed that ACS has obviously penetration of the negatively charged drug aspirin, and certain penetration of neutral drug issorbide mononitrate, but inhibition of positively charged terazosin. In vivo imaging technology research results show that the ACS can significantly enhance the fluorescence intensity of morin, which is the auto-fluorescence anionic drug. These obtained results suggested that ACS, as a promising transdermal enhancer, can change the structure of the keratinocytes and analog penetrating peptides promote absorption, but have certain selectivity for the drug.
Administration, Cutaneous ; Animals ; Arginine ; chemical synthesis ; chemistry ; pharmacology ; Aspirin ; administration & dosage ; pharmacokinetics ; Cell Line ; Cell Survival ; drug effects ; Cell-Penetrating Peptides ; chemical synthesis ; chemistry ; pharmacology ; Chitosan ; chemical synthesis ; chemistry ; pharmacology ; Drug Carriers ; Humans ; Isosorbide Dinitrate ; administration & dosage ; analogs & derivatives ; pharmacokinetics ; Keratinocytes ; cytology ; Male ; Mice ; Prazosin ; administration & dosage ; analogs & derivatives ; pharmacokinetics ; Skin Absorption ; drug effects

Our previous work has indicated that mycelium growth and exopolysaccharide accumulation in submerged fermentation by Pholiota squarrosa (Pers. ex Fr.) Quel. AS 5.245 are strongly affected by many internal and external factors, including medium constituents and fermentation conditions. In this study, we use an effective two-phase statistical approach to enhance exopolysaccharide production. In the first phase, Plackett-Burman design was undertaken to evaluate the effects of the twenty factors, i.e., glucose, fructose, maltose, yeast extract, tryptone, K2HPO4, KH2PO4, (NH4)2SO4, NaNO3, FeSO4, MgSO4, MnCl2, ZnCl2, FeCl3, CuSO4.5H2O, vitamin B1, initial pH, the temperature, the medium volume and the duration, to the fermentation. By regression analysis, yeast extract, tryptone, fructose, MgSO4, MnCl2, initial pH and temperature were found to be important for exopolysaccharide production, while glucose, maltose, NaNO3, ZnCl2, vitamin B1, the duration and the volume are important to the mycelium biomass. In the second phase of the optimization process, a response surface methodology (RSM) was used to optimize the above critical internal factors, and to find out the optimal concentration levels and the relationships between these factors. Based on the results of the first phase, a five-level six-factor (yeast extract, fructose, MgSO4, maltose, ZnCl2 and initial pH) central composite rotatable design (CCRD) was employed. By solving the quadratic regression model equation using appropriate statistic methods, the optimal concentrations for obtaining 876.32 microg exopolysaccharide per milliliter of fermentation liquor were calculated as: 6.0g/L yeast extract, 11.5g/L fructose, 0.5g/L MgSO4, 9.6g/L maltose, 38.6mg/L ZnCl2 and with the initial pH 5.3. The experimental data under various conditions have validated the theoretical values.
Culture Media ; Fermentation ; Fructose ; metabolism ; Hydrogen-Ion Concentration ; Maltose ; metabolism ; Pholiota ; growth & development ; metabolism ; Polysaccharides ; analysis ; biosynthesis ; Temperature

BACKGROUNDWWOX and FHIT are two candidate tumor suppressor genes located in active fragile sites, the damage of which has been associated with the development of breast cancer. The association of the expression of these genes and the development of breast cancer has not been fully explored. We evaluated mRNA and protein expression of WWOX and FHIT in breast tissue with normal histological appearances, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive cancer to see if a progressive decline in expression was present.

METHODSReverse transcription-polymerase chain reaction and Western blotting were used to evaluate the specimens for mRNA and protein expression, including 28 specimens with normal tissue, 28 specimens with atypical ductal hyperplasia, 33 specimens with ductal carcinoma in situ, and 51 specimens with invasive ductal carcinoma.

RESULTSCompared with in situ and invasive cancer specimens, both normal and atypical hyperplasia specimens had greater rates of detectable mRNA (WWOX rate ratio = 2.95, 95% CI 1.24 - 7.08; FHIT rate ratio = 4.58, 95% CI 1.82 - 11.81) and Western blotting detectable protein (WWOX rate ratio = 4.12, 95% CI 1.63 - 10.73; FHIT rate ratio = 3.76, 95% CI 1.44 - 10.06). For both proteins, differences between normal and atypical hyperplasia specimens and between in situ and invasive carcinoma specimens were explainable by chance (P > 0.05 for each analysis). Within each histological category, differences among fractions of specimens showed that FHIT and WWOX mRNA and protein expression were explainable by chance (P > 0.05 for each analysis).

CONCLUSIONExpression of FHIT and WWOX decreases along with breast tissue progress from a normal histological appearance to atypical ductal hyperplasia, in situ cancer, and the final invasive cancer.

Acid Anhydride Hydrolases ; analysis ; genetics ; Breast ; pathology ; Breast Neoplasms ; genetics ; Chromosome Fragile Sites ; Female ; Genes, Tumor Suppressor ; Humans ; Hyperplasia ; Neoplasm Proteins ; analysis ; genetics ; Oxidoreductases ; analysis ; genetics ; Tumor Suppressor Proteins ; analysis ; genetics ; WW Domain-Containing Oxidoreductase

OBJECTIVETo prepare microbubble, made of N-carboxymethyl chitosan, as ultrasound contrast agent and evaluate its characteristics and acoustic effects in vivo.

METHODSOil-Water-Oil multiple emulsion/solvent evaporation method was used to prepare the microbubble contrast agent. Both optical micrography and scanning electron micrography were performed to determine the bubble size and morphology. The acoustic effect of the N-carboxymethyl chitosan echo contrast agent was evaluated in vivo in rabbit. Liver echo images were recorded with ultrasound machine before and after intravenous bolus injecting 0.5 ml of the agent.

RESULTSThe novel N-carboxymethyl chitosan echo contrast agent was formulated as lyophilized product, with a mean diameter of 2-3 microm and a shell thickness of 250-300 nm. Its size is relatively uniform. The imaging effect was remarkably enhanced with the ultrasonic contrast agent when applied in rabbit livers.

CONCLUSIONIt is feasible to prepare excellent microbubble ultrasound contrast agent with N-carboxymethyl chitosan as membrane components.

Animals ; Chitin ; analogs & derivatives ; chemical synthesis ; chemistry ; Contrast Media ; Liver ; diagnostic imaging ; Microbubbles ; Rabbits ; Ultrasonics ; Ultrasonography

BACKGROUNDThe successful end-point of in vitro fertilization (IVF) treatment is for a woman to give live birth. This outcome is based on various factors including adequate number of retrieved eggs. Failure to recruit adequate follicles, from which the eggs are retrieved, is called a "poor response". How to improve the clinical pregnancy rates of poor responders was one of the tough problems for IVF.

METHODSThe study involved 51 patients who responded poorly to high dose gonadotropin treatment in their previous cycles at our reproductive center, between April 2010 and February 2012. The previous cycle (group A) received routine long protocol; the subsequent cycle (group B) received modified super-long down-regulation protocol. The primary outcome of the study was the number of oocytes fertilized. The increase in the pregnancy rate was the secondary outcome. Differences between the groups were assessed by using Student's t test and c(2) test where appropriate.

RESULTSThe patients' average age was (36.64 ± 3.85) years. The mean duration of ovarian stimulation cycles of the group A patients was longer than those of the group B patients. The total dose of follicle-stimulating hormone (FSH) was significantly lower in the subsequent cycle. The peak value of serum estradiol on human chorionic gonadotrophin (hCG) day was lower in group A as compared with group B. The number of metaphase II oocytes recovered was significantly higher in group B. The cleavage rate in group A was significantly lower than in group B, 49 patients in group B reached embryo transfer stage, while 46 patients in group A reached this stage. Patients in group B received significantly more embryos per transfer as compared with group A. More pregnancies and more clinical pregnancies with fetal heart activity were achieved in group B.

CONCLUSIONSThis comparative trial shows that poor responder women undergoing repeated assisted reproduction treatment using modified super-long down-regulation protocol achieve more oocytes, leading to higher fertilization rate, compared to women receiving routine long protocol. Our study also showed that clinical pregnancy rate was significantly improved.

Adult ; Chorionic Gonadotropin ; therapeutic use ; Embryo Transfer ; Estradiol ; blood ; Female ; Fertilization in Vitro ; methods ; Follicle Stimulating Hormone ; therapeutic use ; Humans ; Male ; Ovulation Induction ; methods ; Pregnancy

Isolation and idenfication of lipopeptides from Bacillus subtilis fmbJ was carried out in this paper. With HPLC method, it was determined that the antimicrobial substance was composed of many components, and one of them had the similar retention time similar to surfactin. In addition, the antimicrobial substance was proved to include the closed cycle peptide bind by TLC, and one of them had the migrating rate similar to surfactin. Furthermore, ESI-MS analysis showed that the antimicrobial substance contained five homologues of fengycin, such as m/z1449.9, m/zl1463.8, m/zl1477.8, m/z1491.9 and m/z1505.9, and three homologues of surfactin, such as m/z1008.8, m/z1022.8 and m/z1036.8.
Anti-Infective Agents ; chemistry ; isolation & purification ; Bacillus subtilis ; metabolism ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Lipopeptides ; chemistry ; isolation & purification ; Spectrometry, Mass, Electrospray Ionization

The resistance effect on Newcastle disease virus (NDV) and Infectious Bursal Disease Virus(IBDV) in vitro of a new antimicrobial substance (AS), which produced by a Bacillus subtilis strain named B. subtilis fmbJ. Results showed that the TD50 and TD0 value of this AS on Chicken Embryo Fibroblasts cell (CEF) were 128.95mg/L and 25.79mg/L, respectively. This AS could strongly inhibit the cytopathic effects of cell induced by NDV as well as IBDV, and increase the survival rate of cell remarkably. This AS could inhibit the function of NDV and IBDV, and it could defend against the infection and inhibit multiplication of NDV and IBDV, and the effect was the same as the antiviral medicine Ribavirin. It had lower toxicity to CEF cell, therefore we would study it further that it was as antiviral medicine.
Animals ; Antiviral Agents ; metabolism ; toxicity ; Bacillus subtilis ; metabolism ; Chick Embryo ; cytology ; Fibroblasts ; cytology ; drug effects ; Infectious bursal disease virus ; drug effects ; Newcastle disease virus ; drug effects

The novel antimicrobial peptide in submerged fermentation by Bacillus sp. fmbJ224 is strongly influenced by many internal and external factors, namely medium constituents and fermentation conditions. In this study, Plackett-Burman design was undertaken to evaluate the effects of the seventeen factors. By the statistical regression analysis, the significant factors affecting the novel antimicrobial peptide in submerged fermentation by Bacillus sp. fmbJ224 were determined as follows: glucose, NH4NO3, glutamic acid, CaCl2, MnSO4. In the second phase of the optimization process, a response surface methodology (RSM) was used to optimize the above critical internal factors, and to find out the optimization concentraction levels and the relationships between these factors. By solving the quadratic regression model equation using appropriate statistic methods, the optimal concentration of the variables were determined as: 8.13 g/L glucose, 6.14 g/L NH4NO3, 4.2 g/L glutamic acid, 3.98 mg/L CaCl2, 4.87 mg/L MnSO4. The content of the novel antimicrobial peptide was increased from 1304.21 microg/mL to 1487.58 microg/mL. The experimental data under various conditions have validated the theoretical values.
Anti-Infective Agents ; metabolism ; Bacillus ; growth & development ; metabolism ; Cell Culture Techniques ; methods ; Culture Media ; Fermentation ; Peptides ; metabolism ; Regression Analysis

China has richly and inexpensive fat and oils from animal and plants, but these resources could not get effectively utilization. In order to make the best of these resources, lipase-catalyzed acidolysis of lard with caprylic acid to produce functional lipid in solvent free system was investigated. Of the five lipases that were tested in the initial screening, immobilized lipase TL IM fromca T. languginosa resulted in the highest incorporation of capry lic acid into lard. This enzyme was further studied for the effect of enzyme load, substrate ratib, reaction time, reaction temperature and added water content on the incorporation of caprylic acid into lard. HPLC analyzed the products from the acidolysis reaction. The highest incorporation was attained at 20% enzyme load. Time course studied suggest that the incorporation of caprylic acid into lard was increased up to 38.77 mol% after 24h. Desirable mole ratio of substrates was 1:2 (lard: caprylic acid), caprylic acid incorporation up to 30.95 mol%. In the range of 45 - 60 degrees C , temperature had no significant effect on enzyme activity and caprylic acid incorporation changed little. When temperature was above 60 degrees C, incorporation of caprylic acid into lard was decreased. The highest incorporation of caprylic acid into lard 35.76 mol% was attained when added water content was 2.5%.
Caprylates ; chemistry ; Catalysis ; Chromatography, High Pressure Liquid ; Dietary Fats ; metabolism ; Enzymes, Immobilized ; metabolism ; Lipase ; metabolism ; Lipids ; chemical synthesis ; Solvents

OBJECTIVETo evaluate the expression of CD117 in human testicular germ cell tumors and its value in the differential diagnosis of seminoma and nonseminoma.

METHODSSeventy-four human testicular germ cell tumor specimens were studied by ABC kit immunohistochemical staining detection using CD117 monoclonal antibodies. The immunoreaction scores (IRS) of all the specimens were calculated and analysed for their clinical significance.

RESULTSAmong the 74 germ cell tumors, 31 out of 32 (9 6.9%) seminomas showed positive staining of the CD117 mostly on the cell membrane. Four of 31 (12.9%) nonseminomas displayed a weak positive staining of CD117 only in the cytoplasm of a few cells. In 10 of 11 mixed germ cell tumors, a relatively weak expression of CD117 was shown only in the seminoma component. The CD117 expression was diagnostically decreased from seminoma to mixed seminoma and to nonseminoma successively, with IRS of 6.82 +/- 2.76, 1.25 +/- 0.42 and 0.60 +/- 0.16, respectively. There was a significant difference in the CD117 expression between seminoma and nonseminoma (P < 0.05). CD 117 proteins were positively expressed in all the 20 specimens of the normal testis.

CONCLUSIONThe detection of CD117 by immunohistochemical staining using CD117 monoclonal antibodies is a newly developed useful method for differentiating seminoma and nonseminoma.

Adolescent ; Adult ; Diagnosis, Differential ; Humans ; Immunohistochemistry ; Male ; Neoplasms, Germ Cell and Embryonal ; diagnosis ; metabolism ; Predictive Value of Tests ; Proto-Oncogene Proteins c-kit ; biosynthesis ; Seminoma ; diagnosis ; metabolism ; Testicular Neoplasms ; diagnosis ; metabolism