[Preliminary study on the relationship between different blood glucose levels and periodontitis in patients with diabetes mellitus type 2].

Objective: To determine the correlation between the diabetes mellitus control and periodontitis. Methods: This study was a cross-sectional survey using stratified system sampling model design. The target population was the patients with diabetes investigated from May to July 2018 in Huangpu District of Shanghai. In the present study, severe periodontitis was defined as at least at two sites in different quadrants with probing depth (PD)≥6 mm and clinical attachment loss (CAL)≥ 5 mm. Edentulous induced by periodontitis were also classified as severe periodontitis and the others were classified as non-severe periodontitis subjects. Diabetes control levels were divided into the following three groups: poorly controlled group [glycated hemoglobin (HbA1c)>7.5% and fasting blood glucose (FPG)>7.0 mmol/L], well controlled group (6.5%≤HbA1c≤7.5％ or 6.1 mmol/L≤FPG≤7.0 mmol/L) and ideally controlled group (HbA1c<6.5% and FPG<6.1 mmol/L). SPSS 25.0 was used for statistical analysis. Chi square test was used for demographic data and frequency distribution, α=0.05, two-sided test. Ordinal regression model was used for PD and diabetes control status to balance confounding factors (including age, gender, education and smoking status). After matching the propensity scores between severe periodontitis group and non-severe periodontitis group, logistic regression analysis was used to analyze the level of diabetes control and periodontitis. Results: A total of 5 220 adults over the age of 18 with a medical history of diabetes participated in the survey, of which 3 064 subjects with diabetes mellitus type 2 (T2DM) who were given both oral and laboratory examinations and were included in this study. Statistics showed that the prevalence of moderate and severe periodontitis was 10.57% (324/3 064). In the severe periodontitis group, 79.01% (256/324) of the subjects were over 65 years old, 55.56% (180/324) were male, 58.33% (189/324) had lower education level than high school level, and 21.91% (71/324) were smokers, which were significantly higher than those in the non-severe periodontitis group (P<0.01). In different T2DM status groups, the percentage of severe periodontitis increased with the aggravation of T2DM status. In severe periodontitis group, the proportion of patients with poor glycemic control was higher. T2DM patients with poor glycemic control accounted for 68.52% (222/324) in severe periodontitis group, which was significantly higher than the proportion of non-severe periodontitis group of 60.99% (1 671/2 740) (P<0.05). The regression coefficient of PD was 0.191, and PD had a significant negative effect on the level of blood glucose (P<0.01). There was a significant positive correlation between diabetes glycemic control and severe periodontitis (OR=2.800, P<0.05). Conclusions: In Huangpu District of Shanghai, among T2DM patients, the age of severe periodontitis group was higher than that of non-severe periodontitis group, most of them were male, with lower education level and higher proportion of smoking. The severity of diabetes was related to periodontitis and the proportion of severe periodontitis was higher in patients with poor glycemic control.

[CD38 dictates cardiac damage through mitochondrial apoptotic pathway under hypoxic-ischemic conditions].

Objective: To explore the mechanism of CD38-mediated cardiac damage under hypoxic-ischemic (H/I) conditions. Methods: Twenty CD38(-/-) male mice (8-week-old) and 20 wild-type (WT) male C57BL/6J mice (8-week-old) were randomly selected to construct the model of approximately 25% of the total body surface area (TBSA) burn injury. The cardiomyocytes (CMs) were separated from neonatal mice (1day) to construct the H/I injury model. Ad-CD38 adenovirus was transfected into CD38(-)/- primary CMs to callback CD38 expression. Animal experiments were grouped into WT-control group, CD38(-/-)-control group, WT-burn group, and CD38(-/-)-burn group (10 mice in each group). Primary CMs were divided into 6 groups: WT-normoxia group, CD38(-/-)-normoxia group, CD38(-/-)+Ad-CD38-normoxia group, WT-H/I group, CD38(-/-)-H/I group, CD38(-/-)+Ad-CD38-H/I group. The release of lactic dehydrogenase (LDH) from CMs and the cell viability were measured to estimate the level of myocardial injury. Ultrastructure of cardiomyocytes was examined by electron microscope. CD38 protein level and mitochondrial apoptosis-related proteins were detected by Western blot. Flow cytometry was used to detect mitochondrial reactive oxygen species (MitoSOX) of CMs under H/I condition. Cardiac function of mice was detected by ultrasonic apparatus. Results: (1) Animal experiments: The expression level of cardiac CD38 in WT-burn group was significantly higher than that in sham group (P<0.001). The heart function of CD38(-/-)-burn group was obviously better than WT-burn group [ejection fraction (EF)%: (84.70±2.31)% vs (76.10±2.96)%, shortening fraction (FS)%: (48.90±5.00)% vs (38.10±2.80)%] (both P<0.001). (2) Cell experiments: The expression level of cardiac CD38 in WT CMs under H/I condition was significantly higher than that in WT CMs under normoxia condition (P<0.05). The level of LDH, apoptotic cell and MitoSOX in CD38(-/-)-H/I group were fewer than WT-H/I group and CD38(-/-)+Ad-CD38(-)H/I group [(11.2±3.0)% vs (18.2±3.4)% and (17.6±4.0)%, (13.0±2.8)% vs (23.1±4.9)% and (23.3±6.0)%, (162±11)% vs (228±18)% and (220±18)%] (all P<0.001). The levels of cleaved-caspase3, Cytochrome-C in CD38(-/-)-H/I group were significantly lower than those in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group (P<0.001). The cell viability in CD38(-/-)-H/I group was higher than that in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group (0.355±0.043 vs 0.280±0.051 and 0.291±0.024) (all P<0.05). Electron microscopy results showed that structure of mitochondria in CD38(-/-)-H/I group was better than in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group. Conclusion: Overexpression of CD38 contributes to cardiac damage by stimulating mitochondrial apoptotic pathway.

High-glucose promotes proliferation of human bladder cancer T24 cells by activating Wnt/ß-catenin signaling pathway.

OBJECTIVE: Bladder cancer is the most prevalent genitourinary malignant disorder worldwide. We aimed to observe effects of high-glucose on bladder cancer proliferation and explore the associated mechanisms. MATERIALS AND METHODS: Human bladder cancer cell line, T24, was divided into Blank, Control (Ctrl), 10 mmol/l, 20 mmol/l and 30 mmol/l group. T24 cell proliferation was evaluated by using multiple table tournament (MTT) assay and colony formation analysis, respectively. Quantitative Real-time PCR (qRT-PCR) assay was employed to examine mRNA expression of Wnt-5a and ß-catenin. Meanwhile, Western blot assay was used to evaluate expression of Wnt-5a and ß-catenin protein. The linear regression analysis was utilized to analyze correlation between Wnt-5a/ß-catenin expression and T24 cell proliferation. RESULTS: High-glucose significantly enhanced proliferation of T24 cells compared to that of Blank and Ctrl group (p < 0.05). High-glucose significantly promoted colony formation of T24 cells compared to that of Blank and Ctrl group (p < 0.05). High-glucose significantly up-regulated Wnt-5a mRNA and protein expression compared to that of Blank and Ctrl group (p < 0.01). High-glucose significantly increased ß-catenin mRNA and protein expression compared to that of Blank and Ctrl group (p < 0.01). Effects of high-glucose on T24 cell proliferation were increased following with the enhanced glucose concentration. Wnt/ß-catenin signaling pathway molecules were correlated with colony formation of T24 cells (p < 0.05). CONCLUSIONS: High-glucose promoted the proliferation of T24 cells by activating the Wnt/ß-catenin signaling pathway. This study would provide the novel targets for bladder cancer therapy.

[Experimental research on mesenchymal stem cells (MSCs) therapy for autoimmune uveitis].

Autoimmune uveitis is a major cause of visual disability. Treatment of chronic and recurrent uveitis can be extremely difficult, as various complications of it could impede the long-term usage of corticosteroids and immunosuppressants. Mesenchymal stem cells are of both immunosuppressive and neurotrophic effect, and can enhance the antimicrobial ability of the body, thereby hold great promise in clinical application for treating uveitis. (Chin J Ophthalmol, 2018, 54: 712-715).

Influence of Hydrostatic Pressure on the Corrosion Behavior of Superhydrophobic Surfaces on Bare and Oxidized Aluminum Substrates.

It is generally recognized that superhydrophobic surfaces in water may be used for corrosion resistance due to the entrapped air in the solid/liquid interface and could find potential applications in the protection of ship hull. For a superhydrophobic surface, as its immersion depth into water increases, the resultant hydrostatic pressure is also increased, and the entrapped air can be squeezed out much more easily. It is therefore predicted that high hydrostatic pressure would cause an unexpected decrease in corrosion resistance for the vessels in deep water (e.g., submarines) because of the unstable entrapped air. In this work, in order to clarify the role of hydrostatic pressure in the corrosion behavior of superhydrophobic surfaces, two typical superhydrophobic surfaces (SHSs) were prepared on bare and oxidized aluminum substrates, respectively, and then were immersed into the NaCl aqueous solutions with different depths of ∼0 cm (hydrostatic pressure ∼0 kPa), 10 cm (1 kPa), and 150 cm (15 kPa). It was found out for the SHSs on the oxidized Al, as the hydrostatic pressure increased, the corrosion behavior became severe. However, for the SHSs on the bare Al, their corrosion behavior was complex due to hydrostatic pressure. It was found that the corrosion resistance under 1 kPa was the highest. Further mechanism analysis revealed that this alleviated corrosion behavior under 1 kPa resulted from suppressing the oxygen diffusion through the liquid and reducing the subsequent corrosion rate as compared with 0 kPa, whereas the relatively low hydrostatic pressure (HP) could stabilize the entrapped air and hence enhance the corrosion resistance, compared with 15 kPa. The present study therefore provided a fundamental understanding for the applications of SHSs to prevent the corrosion, especially for various vessels in deep water.

Cofilin is correlated with sperm quality and influences sperm fertilizing capacity in humans.

Spermatozoa should undergo a series of biochemical modifications in female reproduction tract, which is collectively called sperm capacitation. The capacitated spermatozoa can bind to the egg zona pellucida, resulting in the occurrence of acrosome reaction which enabled spermatozoa penetrate into the egg. The formation of actin plays an important role in these processes. Actin polymerized during sperm capacitation, but the polymers dispersed before acrosome reaction. In this study, we take our focus on actin-binding protein, cofilin. Our results showed that the % and intensity of sperm expressing cofilin in normal sperm were significantly higher than in abnormal sperm, and the sperm expressing cofilin was correlated with sperm quality. Furthermore, treatment with anti-cofilin antibody increased the percentage of sperm capacitation and inhibited progesterone- or A23187- induced acrosome reaction in a dose-dependent manner. The presence of 100 ng/mL anti-cofilin antibodies markedly blocked the sperm penetration of zona-free hamster eggs. Besides, immunofluorescence results revealed that cofilin was colocalized with F-actin in the midpiece of spermatozoa; however, phospho-cofilin was expressed in the tail rather than in the midpiece of spermatozoa, which was not colocalized with F-actin in spermatozoa. Moreover, western blot revealed that phospho-cofilin increased in sperm capacitation, and the total cofilin and cofilin in insoluble fraction increased in acrosome reaction; immunofluorescence results showed that the amount of cofilin in acrosome increased in sperm capacitation. In conclusion, our study revealed that cofilin expression in human sperm is correlated with sperm quality and the alterations of cofilin and phospho-cofilin in fertilization affects sperm capacitation, acrosome reaction, and spermatozoa-oocyte fusion.

Expression of ß-glucuronidase on the surface of bacteria enhances activation of glucuronide prodrugs.

Extracellular activation of hydrophilic glucuronide prodrugs by ß-glucuronidase (ßG) was examined to increase the therapeutic efficacy of bacteria-directed enzyme prodrug therapy (BDEPT). ßG was expressed on the surface of Escherichia coli by fusion to either the bacterial autotransporter protein Adhesin (membrane ßG (mßG)/AIDA) or the lipoprotein (lpp) outermembrane protein A (mßG/lpp). Both mßG/AIDA and mßG/lpp were expressed on the bacterial surface, but only mßG/AIDA displayed enzymatic activity. The rate of substrate hydrolysis by mßG/AIDA-BL21cells was 2.6-fold greater than by pßG-BL21 cells, which express periplasmic ßG. Human colon cancer HCT116 cells that were incubated with mßG/AIDA-BL21 bacteria were sensitive to a glucuronide prodrug (p-hydroxy aniline mustard ß-D-glucuronide, HAMG) with an half maximal inhibitory concentration (IC50) value of 226.53±45.4 µM, similar to the IC50 value of the active drug (p-hydroxy aniline mustard, pHAM; 70.6±6.75 µM), indicating that mßG/AIDA on BL21 bacteria could rapidly and efficiently convert HAMG to an active anticancer agent. These results suggest that surface display of functional ßG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.

A novel intronic mutation and a missense mutation of MEN1 identified in two Chinese families with multiple endocrine neoplasia type 1.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) caused by MEN1 mutation is widely recognized. To date, 14 novel mutations were reported in Chinese and intronic mutations are getting more attention. AIM: To explore clinical features and MEN1 mutations in two Chinese families suffering from MEN1. METHODS: Nineteen individuals (10 males and 9 females) from two unrelated families with MEN1 were studied. Mutations of MEN1 were analyzed by direct sequencing of PCR products. In vitro splicing analysis was also performed with minigenes containing both wildtype and novel mutant fragments. Through the RNAstructure program, we analyzed the secondary structure of the wild type MEN1 pre-mRNA and then introduced T>G mutation at +2 donor splice site of intron 7. RESULTS: Clinical features of 3 patients in two families were described, and 5 individuals were proven to be carriers of MEN1 mutation without apparent symptoms. A novel splicing site mutation of the intron 7 (IVS7+2 T→G) was identified in the first family. In vitro analysis also verified this mutation caused the aberrant splicing of MEN1 mRNA. With the RNAstructure program, we could figure out that the global secondary structure as well as the number of stems and loops of pre-mRNA greatly changed after this mutation. The mutation c. 1227 C>A (C409X) was identified in another family, which also caused the truncation of menin. CONCLUSION: We reported a novel intronic mutation and a missense mutations in two Chinese families suffering from MEN1.

Rosiglitazone protects diabetic rats from liver destruction.

AIMS: To investigate whether rosiglitazone (ROS) protects diabetic rats from destructive changes in the liver. METHODS: Twenty-four Sprague Dawley rats were randomly divided into 3 groups: control (NC) group (no.=8), streptozocin (STZ)-treated diabetic (DM) group (no.=8), and STZ+ROStreated diabetic (RSG) group (no.=8). After 8 weeks, the liver structure was observed by light microscopy and transmission electron microscopy. Apoptosis was detected by TUNEL, and apoptosis index was calculated. The Fas ligand (FasL) mRNA expression of apoptosis-promoting gene and cyclooxygenase- 2 (COX-2) mRNA in the liver were detected by RTPCR. COX-2 protein in the liver was tested via immunohistochemical staining. RESULTS: Compared to NC group, DM group showed a visible fatty degeneration and inflammatory cell infiltration in the liver under microscopy. Obvious hepatocyte swelling with atrophic mitochondria was observed, and the central zone of cholangiole was severely outstretched. Meanwhile, in RSG group, the hepatocyte steatosis and inflammatory cell infiltration decreased, and the hepatic ultra-structure was markedly improved. Hepatocyte apoptosis (p<0.05) and the expression levels for hepatic COX-2 mRNA (p<0.05), FasL mRNA (p<0.01), and COX-2 protein (p<0.05) were higher in DM group compared to the NC group, while the expression level of hepatic COX-2 mRNA (p<0.05), FasL mRNA (p<0.01), COX-2 protein (p<0.05), and hepatocyte apoptosis (p<0.05) in RSG group were decreased compared to DM group. CONCLUSION: Diabetes causes severe liver injury and ROS can protect diabetic rats from liver destruction, which may be related to inhibition of the expression of COX-2 and the hepatocyte apoptosis induced by FasL gene over expression.

Mitochondrial apoptotic pathways: a mechanism for low androgen-induced vascular endothelial injury in male rats.

The aim of the study was to investigate the role of mitochondrial apoptotic pathways in vascular endothelial injury in male rats with low androgen. 8 week-old adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups (n=6/each group): control group, castrated group (low androgen), and replacement group (given androgen after castration). After 10 weeks, endothelial structure was observed by general light microscope and transmission electron microscope (TEM) respectively. Isolated mitochondria and mitochondrial membrane potential (MMP) were detected by fluorescence to access mitochondrial function. Chromatin degradation was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine-biotin nick end labeling (TUNEL) staining method. The mRNA and protein of bcl-2, cytochrome C (Cyt C), caspase-9, and caspase-3 were analyzed for apoptosis. Cell shrinkage and condensed chromatin, less mitochondria and a fall in MMP levels were observed in the castrated group, along with more apoptotic endothelial cells. Bcl-2 level was reduced and the expression of caspase-9, caspase-3 and Cyt C were elevated in the castrated group (p<0.05). But there was no significant difference between the replacement group and the control group (p>0.05). It was concluded that low androgen caused vascular endothelial damage. It may be, at least in part, related with the activating mitochondrial apoptotic pathways.

Changes of pituitary and penile structure in male adult rats following castration and high-fat diet.

AIM: To investigate the influence of low androgen levels and high-fat diet on the structure of pituitary and penis in male rats. METHODS: Ten-week-old adult male Sprague-Dawley rats were randomly divided into 2 groups, one fed a high-fat diet the other fed a normal diet; each group consisted of 3 subgroups: controls, castrated rats (with low androgen), and castrated rats given undecanoate replenishment. After 11 weeks, the structure of pituitary and penis were observed under light microscopy. Immunohistochemistry was used to assess the expression of FSH in pituitary and cyclooxygenase-2 (COX-2) in corpora cavernosa penis. RESULTS: The structures of pituitary and penis in castrated rats were injured, and were more damaged in castration together with high-fat diet. Immunohistochemistry showed FSH expression in castrated rats pituitary while castrated rats on a high-fat diet had less positive staining than those on a normal diet. Vascular structure of corpora cavernosa penis, showed a strongly positive COX-2 expression in high-fat diet rats. CONCLUSIONS: Castration and high-fat diet could induce structural damages of pituitary and penis in male rats. Replacement with testosterone could partially restore the impaired structure. The positive expression of COX-2 implied inflammatory pathway existence on vascular structure of penis in high-fat diet and low-androgen male rats.

Telbivudine preserves T-helper 1 cytokine production and downregulates programmed death ligand 1 in a mouse model of viral hepatitis.

Telbivudine is an orally bioavailable L-nucleoside with potent and specific anti-hepatitis B virus activity. The higher rate of hepatitis B e antigen (HBeAg) seroconversion during telbivudine treatment than other potent anti-HBV agents suggests a potential immunomodulatory effect. We sought to determine the effects of telbivudine on the immune system, particularly on cytokine production and T-cell response, using an animal model with mouse hepatitis virus strain 3 (MHV-3)-induced hepatitis. The effects of telbivudine on virus replication and cytokine production were investigated in vitro using MHV-3-infected macrophages, and the effects on T-cell response were investigated in vivo in an MHV-3-induced viral hepatitis model. Telbivudine had no effect on MHV-3 replication in macrophages. However, the production of tumour necrosis factor-alpha and interleukin-12 was increased significantly in MHV-3-induced macrophages treated with telbivudine. In vivo survival was enhanced in telbivudine-treated mice, with marked normalization in clinical conditions and histological lesions. Serum levels of interferon-gamma were elevated significantly after telbivudine treatment in MHV-3-infected C3H mice. In contrast, serum interleukin-4 levels were decreased significantly. Furthermore, telbivudine treatment enhanced the ability of T cells to undergo proliferation and secrete cytokines but did not affect cytotoxicity of infected hepatocytes. Of note, we found that telbivudine treatment suppressed programmed death ligand 1 expression on T cells. The results demonstrate the immunomodulatory properties of telbivudine, independent of its antiviral activity, in a mouse model of MHV-3-induced hepatitis.

Tumor-targeting prodrug-activating bacteria for cancer therapy.

Increasing the specificity of chemotherapy may improve the efficacy of cancer treatment. Toward this aim, we developed a strain of bacteria to express enzymes for selective prodrug activation and non-invasive imaging in tumors. beta-glucuronidase and the luxCDABE gene cluster were expressed in the DH5alpha strain of Escherichia coli to generate DH5alpha-lux/betaG. These bacteria emitted light for imaging and hydrolyzed the glucuronide prodrug 9ACG to the topoisomerase I inhibitor 9-aminocamptothecin (9AC). By optical imaging, colony-forming units (CFUs) and staining for betaG activity, we found that DH5alpha-lux/betaG preferentially localized and replicated within CL1-5 human lung tumors in mice. The intensity of luminescence, CFU and betaG activity increased with time, indicating bacterial replication occurred in tumors. In comparison with DH5alpha-lux/betaG, 9AC or 9ACG treatment, combined systemic administration of DH5alpha-lux/betaG followed by 9ACG prodrug treatment significantly (P<0.005) delayed the growth of CL1-5 tumors. Our results demonstrate that prodrug-activating bacteria may be useful for selective cancer chemotherapy.

A membrane antibody receptor for noninvasive imaging of gene expression.

Monitoring gene expression is important to optimize gene therapy protocols and ensure that the proper tissue distribution is achieved in clinical practice. We developed a noninvasive imaging system based on the expression of artificial antibody receptors to trap hapten-labeled imaging probes. Functional membrane-bound anti-dansyl antibodies (DNS receptor) were stably expressed on melanoma cells in vitro and in vivo. A bivalent (DNS)2-diethylenetriaminepentaacetic 111Indium probe specifically bound to cells that expressed DNS receptors but not control scFv receptors. Importantly, the 111In probe preferentially localized to DNS receptors but not control receptors on tumors in mice as assessed by gamma camera imaging. By 48 h after intravenous injection, the uptake of the probe in tumors expressing DNS receptors was 72 times greater than the amount of probe in the blood. This targeting strategy may allow noninvasive assessment of the location, extent and persistence of gene expression in living animals and in the clinic.

Ex vivo gene therapy in autologous bone marrow stromal stem cells for tissue-engineered maxillofacial bone regeneration.

This study examines the clinical relevance of tissue engineering integrating gene therapy and polymer science to bone regeneration. Bilateral maxillary defects (3 x 1.2 cm(2)) in 20 miniature swine were bridged with a bioresorbable internal splint. Constructs were created using ex vivo adenovirus bone morphogenetic protein (BMP)-2-mediated gene transfer to the expanded bone marrow mesenchymal stem cells (MSCs) 7 days before implantation. Controls were performed using adenovirus beta-galactosidase. The BMP-2 cell/construct displayed white solid bone formation after 3 months. Meanwhile, the hematoxylin and eosin and Von Kossa stains demonstrated exhibited mature woven bone with good mineralization. Additionally, three-dimensional computer tomography imaging revealed a nearly complete infraorbital rim repair. Quantitative analysis demonstrated a significant difference (P<0.001) in bone formation. Finally, biomechanical testing revealed no statistically significant difference in the maximal compressive strength of new bone formed by BMP-2 cell constructs and the normal maxilla. The data evidenced de novo bone formation capable of sustaining axial compressive loads. The measurement results showed that ex vivo replication defective adenovirus-mediated human BMP-2 gene transfer to MSCs enhances autologous bone formation in the repair of maxillary defects.

Effects of androgen and ageing on gene expression of vasoactive intestinal polypeptide in rat corpus cavernosum.

OBJECTIVE: To clarify the effects of androgens (testosterone and dihydrotestosterone, DHT) and ageing on gene expression of vasoactive intestinal polypeptide (VIP), assessed as the expression of VIP mRNA, in rat corpus cavernosum. Materials and methods The study comprised 160 male Sprague Dawley rats divided into group A (56 rats, 5 weeks old), group B (50 rats, 10 weeks old) and group C (54 rats, 58 weeks old). Groups A-C were subdivided, respectively, into subgroups 1 (intact controls), 2 (castrated), 3 (castrated but given testosterone undecanoate 25 mg/kg per month by intramuscular injection), 4 (castrated with testosterone undecanoate 50 mg/kg per month) and 5 (treated with finasteride 4.5 mg/kg per day, orally). At 4 and 10 weeks after these treatments half the rats were killed. Serum samples were taken for the measurement of total and free testosterone and DHT, using a radioimmunoassay. Penile samples (corpus cavernosum) were frozen in liquid nitrogen and stored at -80 degrees C. VIP mRNA was estimated using a semi-quantitative reverse-transcription polymerase chain reaction. RESULTS: There was no significant difference in VIP mRNA in rat corpus cavernosum between intact control and castrated subgroups, or subgroups treated with finasteride, in groups A-C, including both the 4- and 10-week old animals (P > 0.05). Penile VIP mRNA was unchanged at any dose of testosterone in the castrated subgroups in all groups (P > 0.05). There was no significant relationship between penile VIP mRNA and ageing (P > 0.05). CONCLUSIONS: The gene expression of VIP in rat corpus cavernosum is independent of androgens (testosterone and DHT) and ageing. Androgens probably induce penile erection by pathways other than VIP; ageing may have little effect on penile VIP mRNA.

EGFR antisense RNA blocks expression of the epidermal growth factor receptor and partially reverse the malignant phenotype of human breast cancer MDA-MB-231 cells.

The effects of human EGFR to the malignant phenotype of human breast cancer cell line MDA-MB-231 were investigated experimentally. A retroviral vector containing a 5'1350bp fragment of the human EGFR cDNA in the antisense orientation was transfected into targeted cells by lipofectamine. The effects on cell proliferation, cell cycle and adherent ability to extracellular matrix (ECM) components were studied after the expression of antisense transcripts to EGFR 5'1350bp fragment in target cells. In vitro studies showed that the growth ability of the transfected cells was partially inhibited in comparison to parental cells and to cells transfected with the plasmid containing the neomycin resistance gene only. It was found that EGF (10 ng/ml) had an argumenation effect on the growth of transfected MDA-AS10 cells but not MDA-MB-231 cells. Flow cytometric analysis showed that the cell cycle of the transfected cells was abnormal with a decrease of cells in G2/M and S phases and an increase of cells in G1 phase, indicating a blockage in phase G1. Immunofluorescence of EGFR expression in transfectants stained with an anti-EGFR antibody was decreased and their growth in soft agarose was also severely impaired. The transfected cells showed less adherence to laminin (LN) and fibronectin (FN). In short, EGFR antisense RNA decreases the expression of EGFR on MDA-MB-231 cells and partially reverses their malignant phenotype as well.

Clinical study on the termination of 388 high-risk early pregnancies with mifepristone and misoprostol.

PIP: The safety, efficacy, and acceptability of a mifepristone-misoprostol combination for the termination of high-risk early pregnancies were documented in a study conducted at International Peace Maternity and Child Hospital in Shanghai, China, in 1993-96. The 388 study participants required pregnancy termination for reasons including scarred uterus, reproductive tract malformations, uterine fibroids, history of repeated abortions, or pregnancy during lactation. All pregnancies were under 70 days of gestation (mean, 46.7 days). Women received 150 mg of oral mifepristone followed, on the 3rd day, by 600 mcg of misoprostol. The complete abortion rate was 92.3%, the incomplete abortion rate was 6.2%, and the pregnancy rate was 1.5%. The complete abortion rate was significantly higher in women with amenorrhea of 49 days or less (95.5%) than in those with amenorrhea of 50-69 days of duration (83.3%). In 11 of the 12 cases in which heavy bleeding necessitated emergency management, the pregnancy exceeded 49 days of gestation. 92% of study participants were satisfied with this regimen. In cases of method failure, the softening and dilatation of the cervix induced by the drugs made vacuum aspiration easier and less painful.^ieng

Differentiation induced by the c-Mpl cytokine receptor is blocked by mutant Shc adaptor protein.

c-Mpl, a member of the cytokine receptor superfamily, induces both proliferative and differentiation responses when stimulated with its ligand thrombopoietin (TPO). To examine signal transduction pathways associated with differentiation versus proliferation, 32D clone 3 cells, a murine interleukin 3-(IL-3)-dependent cell line capable of granulocytic differentiation, were engineered to express human c-Mpl (designated 32DM.2). Human TPO-containing medium was produced by transient transfection of 293 cells. Treatment of 32DM.2 cells with human TPO induced cellular aggregates within 12 h of exposure to ligand. 32DM.2 cells maintained in the presence of TPO did not change in cell number over a 72-h period and acquired characteristics of granulocytic differentiation as evidenced by metamyelocytic cellular morphology. The differentiation effect of TPO was observed in the absence and presence of the mitogen IL-3. Evaluation of protein tyrosine phosphorylation following exposure to ligand revealed that TPO stimulation induced an elevated level of tyrosine phosphorylation of the adaptor protein Shc when compared with IL-3. However, treatment of 32DM.2 cells with TPO did not result in the phosphorylation of mitogen-activated protein kinase (MAPK). To evaluate the potential role of Shc in c-Mpl differentiation, we transfected 32DM.2 cells with a mutant Shc gene that lacked the region coding for the phosphotyrosine interaction domain (delta PI-Shc). Expression of the delta PI-Shc protein in 32DM.2 cells blocked the TPO differentiation response with no effect on IL-3-stimulated proliferation. These studies demonstrate that c-Mpl-induced differentiation results from the activation of signal transduction pathways that are dominant to the IL-3 proliferative response and independent of the Ras/MAPK signal transduction pathway. The ability of the delta PI-Shc protein to block TPO-induced differentiation implicates Shc as a mediator of signal transduction pathways leading to differentiation, which is distinct from its role as a mediator in activating the Ras/MAPK pathway.