This article reviews the resultsof the basic research about epidermal growth factor and its receptor,and the development of the novel drug, EGF eyedrop, that containing chemically synthesised EGF gene,the construction of EGF expression vector, the transformation of the host cells , the purification of the recombinant protein EGF, the preparation of three batches of the EGF product and identification, the preclinical and clinical trials. Relevant studies show that recombinant EGF consisting of 51 amino acids can be secreted into the medium under the control of the α factor leading sequence in the yeast cells.The EGF can accelerate the growth of corneal-limbal epithelial cells and the healing of an alkali burned corneal. The EGF can be used in curing oral cavity ulcer and skin burned wound. And it has the preventive effects on experimental duodenal ulcer of rat. The antiserum was made for test of the con- centration of blood EGF and urine EGF by RIA. Data from studies demonstrate the inhibition effect of EGF on the growth of tumor cells, such as A431 and BT325 cells in the presence of high EGF concentration (＞10 ng/ml). The expression of EGFR and DNA ploidy in renal carcinoma has clinical significance. Crystallization and preliminary x-ray diffraction studies of the EGF has been made. The MW of the EGF product is 6000, and the pI is about 4. 6 and it has correct N-terminal amino acids sequences , immunogenicity and biological activity. There is no vestige of the DNA of the yeast cells. Animal experiments reveal that there is no cumulation of the EGF in the body, and EGF can promote corneal epithelial healing. There is no toxicological effect during cornea wound healing of rabbit. A randomized, double-blind, placebo-controlled, multi-center clinical trial was conducted in four hospitals to assess safety,ocular tolerance and efficacy of an ophthalmic solution of EGF for 200 cases of cornea transplantation and 247 cases of nebulae. Unequivocal results were obtained as the eyedrop really accelerate the wounded cornea healing. So, the EGF eyedrop as a novel drug of class I is approved by the National Drug Administration, and this is the first gene engineering drug that come from yeast expression system in China.

AIMTo study the effects of rhBNP and milrinone on the cardiac hemodynamics and renal function in anesthetized dogs.

METHODSThe actions of rhBNP given cumulatively i.v. 10, 30 and 100 ng.kg-1 for 30 min and milrinone of single dose (100 micrograms.kg-1, i.v.) on cardiac hemodynamics and renal function were studied in anesthetized open-chest dogs.

RESULTSIn anesthetized dogs (n = 7) intravenous infusion of rhBNP at 10-100 ng.kg-1, caused decreases in mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), LVdp/dtmax, pulmonary arterial pressure (PAP), left ventricular end diastolic pressure (LVEDP), total peripheral resistance (TPR) and renal vascular resistance (RVR) dose-dependently, without significant changes in cardiac output (CO), LV(dp/dt)/P, renal blood flow (RBF) and heart rate (HR), increases in urinary volume and sodium excretion. In anesthetized dogs (n = 6), there were remarkable decreases in MAP, LVEDP, PAP, TPR, RBF, RVR and urinary volume following the MIL (100 micrograms.kg-1, i.v.), with significant increases of LVSP, +/- LVdp/dtmax, HR and CO, but no marked changes in urinary volume and sodium excretion.

CONCLUSIONrhBNP reduces the pre-load and after-load in the anaesthetized dogs but showed no distinct effect on the contractility of the heart. Positive inotropic and chronotropic actions have been demonstrated after intravenous injection of milrinone 100 micrograms.kg-1 in anesthetized dogs.

Anesthesia ; Animals ; Cardiotonic Agents ; pharmacology ; Dogs ; Drug Interactions ; Female ; Heart ; drug effects ; physiology ; Hemodynamics ; Kidney ; drug effects ; physiology ; Male ; Milrinone ; pharmacology ; Natriuretic Peptide, Brain ; pharmacology ; Recombinant Proteins ; pharmacology ; Sodium ; blood ; urine ; Urine

By electrophysiological methods, cultured Drosophila embryonic and larval central neurons have been widely used to study ion channels, neurotransmitter release and intracellular message regulation. Voltage-activated K(+) channels play a crucial role in repolarizing the membrane following action potentials, stabilizing membrane potentials and shaping firing patterns of cells. In this study, a mechanical vibration-isolation system was used to produce a sufficient number of acutely dissociated larval central neurons, of which the majority were type II neurons (2~5 microm in diameter). Using patch clamp technique, the whole-cell K(+) currents in type II neurons were characterized by containing a transient 4-AP-sensitive current (I(A)) and a more slowly inactivating, TEA-sensitive component (I(K)). According to their kinetic properties, five types of whole-cell K(+) currents were identified. Type A current exhibited primarily fast transient K(+) currents that activated and inactivated rapidly. The majority of the neurons, however, slowly inactivated K(+) currents with variable inactivation time course (type B current). Type C current, being present in a small number of the cells, was mainly composed of noninactivating components. Some of the neurons expressed both transient and slow inactivating components, but the slowly inactivating components could reach more than 50% of the peak current (type D current). Type E current showed distinct voltage-dependent activation properties, characterized by its bell-shaped activation curve. Type E current was inhibited by application of Ca(2+)-free solution or 0.1 mmol/L Cd(2+). Moreover, this novel current ran down much more rapidly than other types. These results indicate that different K(+) channels, which have different kinetic and pharmacological properties, underlie the whole-cell K(+) currents in type II neurons of Drosophila larval central nervous system.
Action Potentials ; Animals ; Cell Separation ; methods ; Drosophila ; metabolism ; physiology ; Larva ; cytology ; Membrane Potentials ; Neurons ; metabolism ; physiology ; Patch-Clamp Techniques ; Potassium ; physiology

The present study was to investigate the mRNA, protein expression and the activity of calcineurin in the hypertrophic heart, and to determine the effect of calcineurin inhibitor--cyclosporine A (CsA) on the regression of cardiac hypertrophy in renovascular hypertensive rats. Renovascular hypertension was induced by two kidney-one clip methods. Two months after the operation, cardiac hypertrophy was determined by histological analysis performed in some rats (2K1C-2M), then the rats were subdivided into 2 groups: (1) 3-month old two kidney-one clip group (2K1C-3M) with rats receiving 0.9% NaCl per day for one month, and (2) CsA-treated group with rats treated with CsA for one month. Sham-operated rats were used as control. The ratio of the left ventricular weight to tibial length (LVW/TL), the area of cardiac myocyte, mRNA and protein expression and the activity of calcineurin were determined. Both the LVW/TL and the cardiomyocyte area were significantly larger in 2K1C-2M and 2K1C-3M rats than in age-matched sham-operated rats. Treatment with CsA significantly attenuated the increase in the LVW/TL as well as the cardiomyocyte area. The mRNA, protein expression and the activity of calcineurin were significantly higher in 2K1C-2M and 2K1C-3M rats than those in the age-matched sham-operated rats, while the elevation of mRNA, protein expression and activity of calcineurin were significantly suppressed in the CsA-treated rats. In conclusion, calcineurin plays a role in the progression of cardiac hypertrophy in renovascular hypertensive rats. The inhibition of calcineurin can reverse cardiac hypertrophy.
Animals ; Calcineurin ; biosynthesis ; genetics ; metabolism ; Cyclosporine ; pharmacology ; Hypertension, Renovascular ; complications ; metabolism ; physiopathology ; Hypertrophy, Left Ventricular ; etiology ; metabolism ; physiopathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVESix1 and Six4 are expressed in several tumors, and associated with tumor progress and poor prognosis. The aim of this study was to investigate the expression of Six1 and Six4 in esophageal squamous cell carcinoma (ESCC), and to evaluate their correlation with the clinicopathological factors and prognosis.

METHODSTissue microarray technology and immunohistochemical method (EnVision) were used to detect the expression of Six1 and Six4 in the tumor tissues and corresponding adjacent normal epithelium of esophagus from 292 ESCC patients.

RESULTSAmong the 292 ESCC patients, the positive rates of Six1 and Six4 protein expression in tumor tissues were 72.9% (213/292) and 56.2% (164/292), respectively, significantly higher than the expression rate of 33.2% (97/292) and 32.5% (95/292) in adjacent normal epithelium of esophagus (P < 0.05). Chi square test showed that the expression of Six1 protein was related to tumor size, depth of tumor invasion and patient survival status; higher Six4 protein expression level was related to poor differentiation and increased depth of invasion. Single factor Log-rank analysis revealed that gender, TNM stage, Six1 protein expression level were related to the overall survival of ESCC patients (P < 0.05), while the five-year survival rate was significantly higher in the Six1-negative group than the Six1-positive group [51.9% (41/79) vs. 43.7% (93/213)]. Multi-factor Cox proportional risk model analysis showed that TNM stage and positive expression of Six1 were independent prognostic factors for ESCC patients (P < 0.05).

CONCLUSIONSSix1 and Six4 are highly expressed in ESCC. Their expression levels are closely related to the progress and prognosis of ESCC. Over-expression of Six1 is related to poor prognosis in ESCC patients. Thus, Six1 could be used as an important prognostic indicator for ESCC patients.

Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; surgery ; Esophageal Neoplasms ; metabolism ; pathology ; surgery ; Female ; Follow-Up Studies ; Homeodomain Proteins ; metabolism ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; Prognosis ; Proportional Hazards Models ; Risk Factors ; Survival Rate ; Trans-Activators ; metabolism ; Tumor Burden

Objective To obtain recombinant human epidermal growth factor(hEGF) that can be used in animal experiments and clinical trial. Method Chemically synthesized hEGF gene was expressed in Yeast Pichia pastoris and the secretory hEGF was purified by Phenysepharose 6 Fast Flow(high sub), Q-sepharose High Performance, and Superdex 30 chromatography, and its characters were studied by respective methods. Results The purified hEGF doesn' t contain pyrogen, endotoxin, or yeast chromosome DNA and the purity reached 98%. The recombinant human EGF has correct molecular weight, pI, N-terminal amino acids se quences, peptide map, ultraviolet spectrum and well-biological activity. Conclusion The purified hEGF is in accord with the requirements for animal experiments and clinical trial which provids the basis of prepar ing EGF agents for clinical test.

Objective: To realize the classification and identification of Cynomorii Herba from different producing areas based on fourier transform infrared spectroscopy (FTIR) and chemometrics. Method: FTIR spectrum data of 106 batches of Cynomorii Herba from 12 cities in 5 provinces were collected by transmission method and preprocessed. The FTIR fingerprints of Cynomorii Herba were established, and spectrum analysis was performed. The FTIR similarities of Cynomorii Herba from different producing areas were calculated by correlation coefficient method. The first derivative (1D) spectrum of average FTIR of Cynomorii Herba from different producing areas were obtained. The soft independent modeling of class analog (SIMCA) model based on principal component analysis (PCA) was established by the preprocessed 1D spectrum data. The orthogonal partial least squares (OPLS) model was established by top 6 principal components. Result: The FTIR fingerprint trend and main absorption peaks of Cynomorii Herba from different producing areas were basically the same,and 16 common characteristic absorption peaks were recognized. Similarity and 1D spectrum of FTIR fingerprint of Cynomorii Herba from different producing areas showed significant and unique characteristics. The established SIMCA model can realize the classification and identification of Cynomorii Herba from different provinces,while OPLS model can realize accurate classification and identification of Cynomorii Herba in different cities. The classification and identification of Cynomorii Herba from 12 city producing areas showed obvious geographical clustering characteristics. Conclusion: The established method based on FTIR and chemometrics can realize the classification and identification of Cynomorii Herba from 12 cities.