Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system / 中国生物制品学杂志

@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

Aberrant signal transduction in Indian triple-negative breast cancer patients

Aim of Study: The aim of this study is to correlate the prominin-1 or CD133 association with functional pathway markers of cancer stemness in Indian triple-negative breast cancer (TNBC) patient samples. Materials and Methods: TNBC samples were confirmed for the absence of hormone receptors (estrogen receptor–ER/progesterone receptor) and human epidermal growth factor receptor-2 or proto-oncogene neu or erbB2 or CD340 by immunohistochemical analysis. Formalin-fixed paraffin-embedded samples of patients were used to collect the total RNA. Then, one-step reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the cancer stemness-related transcript levels in the different samples. The RT-PCR products were analyzed semi-quantitatively on agarose gels. The band intensities of respective samples for different transcripts were analyzed by densitometry. Results: TNBC-confirmed samples had shown increased levels of CD133 transcript than control tissues. Further, elevated CD133 transcripts are correlated with higher transcript levels of NOTCH1/FZD7/transforming growth factor-beta receptor Type III R/patched-1 pathway mediators. Conclusions: This work has clearly indicated that there is a correlation between CD133 and functional pathways that control cancer stem cells in TNBC. These observations may indicate the possible association between cancer stemness and TNBC malignancy

Changes of vasoactive substances and adrenergic beta receptors in rabbit models of neurogenic pulmonary edema / 中国小儿急救医学

Objective To establish an animal model of neurogenic pulmonary edema (NPE),and to study the role of catecholamine and beta receptors in the occurrence of NPE. Methods The NPE model was established by injecting fibrinogen and thrombin into the cerebellum medullary pool of the rabbits. Twenty-four rabbits were divided into the control group,the saline group and the experimental group by random num-ber table. In the control group,only cerebellar medullary cistern puncture was carried out,and no drug was in-jected. Cerebrospinal fluid was drew out and the same amount of saline was injected into the cerebellum me-dullary pool in the saline group. Fibrinogen and thrombin were injected into the cerebellum medullary pool in the experimental group. The animals were intubated by tracheotomy,the femoral artery and the internal jugu-lar vein were dissected and connected with the PiCCO instrument to detect the blood pressure,heart rate,and respiratory rate before puncture and at 1 min,10 min,30 min after puncture. Serum samples were collected for the determination of epinephrine,norepinephrine,acetylcholine,endothelin-1,cardiac troponin I,brain natri-uretic peptide and neuropeptide Y levels before puncture and at 1 min,10 min after puncture. The rabbits weresacrificed at 3 hours after successful modeling,the pathological examination of lung was performed. Myocar-dial samples were taken to detect adrenergic beta receptors mRNA. Results (1)The heart rate,respiratory rate and mean arterial pressure at 1 min and 10 min after puncture in experimental group were significantly higher than those in control group and saline group. (2) The pathological examination of the rabbits′ lungs in experimental group found that the lung tissue was swollen and dark red in appearance with large areas of con-gestion. Under the microscope,the lung tissue was edema,bleeding,and inflammatory cells were infiltrated in the alveolar cavity,which was consistent with characteristics of NPE. (3)There was no difference in epineph-rine and norepinephrine concentration in all groups before the cerebellar medullary pool puncture. The concen-tration of epinephrine and norepinephrine at 1 min after puncture time were (200. 0 ± 251. 7)μg/ L,(448. 9 ± 356. 7)μg/ L in the experimental group,whcih were significantly higher than those of control group[(15. 4 ± 3. 4)μg/ L,(15. 9 ± 9. 7)μg/ L] and saline group[(17. 1 ± 3. 8) μg/ L,(29. 6 ± 18. 4) μg/ L] (P < 0. 05). The concentration of epinephrine and norepinephrine at 10 min after puncture were (397. 0 ± 797. 7)μg/ L, (221. 4 ± 173. 7)μg/ L in the experimental group,whcih were significantly higher than those of control group [(23. 3 ± 6. 4) μg/ L,(18. 8 ± 3. 9) μg/ L] and saline group[(16. 7 ± 9. 1) μg/ L,(20. 3 ± 6. 5) μg/ L] (P < 0. 05). (4)There was no significant difference in the levels of serum neuropeptide Y,acetylcholine and endothelin-1 among the three groups. (5)The mRNA of adrenergic beta-1 receptor in the experimental group was 0. 37 ± 0. 12,which was significantly lower than those in the control group (0. 54 ± 0. 13) and saline group (0. 56 ± 0. 14) (P < 0. 05). There was no significant difference in adrenergic beta-3 receptor mRNA among the three groups. Conclusion The NPE animal model was constructed by injecting fibrinogen and thrombin into the cerebellum medullary pool of the rabbits. Catecholamine and beta-1 receptor play a role in the occurrence of NPE rabbit model. There is no significant correlation between neuropeptide Y,acetylcholine,en-dothelin-1 and the occurrence of NPE in rabbits.

Función de los confórmeros de ataque cercano en la acilación enantioselectiva del (R,S)-propranolol catalizada por lipasa B de Candida antárctica / Rol of the near attack conformers during enantioselective acylation of (R,S)-propranolol catalysed by Candida antarctica lipase B

RESUMEN La lipasa B de Candida antárctica (CalB) se ha utilizado en la acilación quimio- y enantioselectiva del racemato (R,S)-propranolol. CalB tiene enant¡oselect¡v¡dad moderada (£=63) por el R-propranolol. La enantioselectividad, se origina en la reacción de transferencia del grupo acilo desde la serina catalítica, acilada, al propranolol. La fase inicial de esta reacción involucra la formación de complejos de Michaelis y posteriormente conformaciones de ataque cercano. El análisis de las conformaciones de ataque cercano ha permitido en varios casos explicar el origen de la catálisis o reproducir el efecto catalítico. En este trabajo se profundiza en la comprensión la función de las conformaciones de ataque cercano en la enantioselectividad de la acilación del (R,S)-propranolol catalizada por CalB. Para lo anterior se realizó un estudio detallado de los complejos de Michaelis y de las conformaciones de ataque cercano del paso enantioselectivo de la reacción de acilación del (R,S)-propranolol utilizando un protocolo de dinámica molecular QM/MM (SCCDFTB/CHARMM) utilizando 6 distribuciones de velocidades iniciales y simulaciones de 2,5 ns. Se estudiaron 7 complejos CalB-propranolol. Los enlaces de hidrógeno del sitio activo de CalB acilada relevantes para la actividad catalítica fueron estables en todas las simulaciones. Las poblaciones de los complejos de Michaelis y de las conformaciones de ataque cercano son dependientes de la distribución de las velocidades iniciales de la dinámica molecular. La enantioselectividad moderada de CalB acilada, encontrada experimentalmente, puede ser parcialmente atribuida a la alta población de conformaciones de ataque cercano observada para el S-propranolol.

ABSTRACT Candida antarctica lipase B (CalB) has been used for chemo- and enantioselective acylation of racemic (R,S)-propranolol, with moderate enantioselectivity (£=63) for R-propranolol. The enantioselective step in this reaction is the transfer of an acyl group from the catalytic acylated serine to propranolol. The initial phase of this reaction involves the formation of Michaelis complexes, followed by the formation of near-attack complexes. The analysis of the near-attack complexes has in several cases permitted to explain the origin of the catalysis or to reproduce the catalytic effect. The aim of this study was improve the understanding of the role of the near-attack complexes for the enantioselectivity of the acylation of (R,S)-propranolol, catalyzed by CalB. To this purpose a detailed investigation of the Michaelis and near-attack complexes of the enantioselective step of the acylation of (R,S)-propranolol using QM/MM molecular dynamics was performed. Several simulations (each 2,5 ns) with different initial velocity distributions were performed. In total seven CalB-propranolol complexes were studied. The hydrogen bonds in the active site of CalB, which are relevant for the catalytic activity, are stable in all simulations. The lifetime of the Michaelis complexes is considerably shorter than the simulation time. Conclusions: The populations of the Michaelis and near-attack complexes depend on the initial velocity distribution in the molecular dynamics simulations. The experimentally observed moderate enantioselectivity may be partially attributed to the high population of near-attack conformations of S-propranolol.

10-year Trend of Early β-blocker Use in Acute Myocardial Infarction Patients in Eastern Urban China / 中国循环杂志

Objective: To assess trends of β-blocker use within 24h of admission in ideal candidates with acute myocardial infarction (AMI) in eastern urban China from 2001 to 2011. Method: A 2-stage random sampling design was performed. In the first stage, a simple random-sampling was used to identify participating hospitals. In the second stage, a systematic sampling was conducted in 2001, 2006 and 2011 to select cases from the participating hospitals. Data was obtained by central medical record abstraction. 10-year trend and predictors of early β-blocker application were assessed with weighted calculation for each year to represent the overall situation of eastern urban China. Results: 35 hospitals were sampled and 32 of them were finally participated. With necessary exclusion, 1399 ideal candidates were included in this analysis. The early weighted β-blocker application rates in 2001, 2006 and 2011 were 64.7%, 69.7%, and 60.9% respectively, P=0.0447 for trend. Patients with chest pain at admission (OR=2.22, 95% CI 1.19-4.13), higher systolic blood pressure (OR=1.40, 95% CI 1.11-1.77) or faster heart rate (OR=2.01, 95% CI 1.58-2.55) were more likely to use β-blocker; in contrast, compared with NSTEMI patients, STEMI patients seemed less likely to receive such treatment (OR=0.55, 95% CI 0.37-0.81). Conclusion: The early β-blocker therapy in eastern urban China is suboptimal for ideal AMI patients who could benefit from it. The application pattern has not been changed from 2001 to 2011 which might be related to physicians' misunderstanding of relevant evidence or guidelines. Our study may help to create an important target to improve the quality of AMI care.

10-year Trend of Early Beta Receptor Blocker Application for Acute Myocardial Infarction Patients in Western Rural China / 中国循环杂志

Objective: To assess the trend of early beta receptor blocker (β-blocker) application (with 24h of admission) for acute myocardial infarction (AMI) patients in western rural China from 2001 to 2011. Methods: A 2-stage random sampling design was performed. The 1st stage: a simple random sampling was used to identify participating hospitals and the 2nd stage: a systematic random sampling approach was conducted in 3 specific years of 2001, 2006 and 2011 to take case study for central medical information abstraction. The changing trends and impact factors of early β-blocker application for AMI patients in western rural area were assessed by multivariate model analysis. Results: 35 hospitals were sampled and 33 of them were finally participated. With necessary exclusion, a total of 486 AMI patients without β-blocker contraindication were enrolled for 2 groups: Suitable group, the patients were suitable for early β-blocker application, n=247 and High risk group, the patients with the high risk for shock occurrence, n=239. The application rates for β-blocker within 24h of admission at 2001, 2006 and 2011 in Suitable group were 19.06%, 54.30% and 56.20%, Ptrend=0.0020; in High risk group were 31.53%, 59.49% and 69.62%, Ptrend=0.0001. In Suitable group, the patients with history of hypertension (OR=1.87, 95% CI 1.06-3.29), smoking (OR=1.97, 95% CI 1.11-3.48) or admitted in 2006 (OR=2.93, 95% CI 1.22-7.03) and 2011(OR=4.67, 95% CI 2.06-10.59) had the higher chance to use β-blocker within 24h of admission. Conclusion: Application of β-blocker within 24h of admission in AMI patients presented the increasing trend in western rural China from 2001 to 2011, while there was still difference from the guideline recommendation. Improved normative application of β-blocker is helpful to enhance the quality of care and prognosis in AMI patients.

Effect of Total Flavonoids from Litchi chinensis Sonn on Expression of TGF-β1 Receptor and Collagen in Rats with Liver Fibrosis / 医药导报

Objective To observe the prevention and treatment of the total flavonoids from Litchi chinensis Sonn( TFL) on hepatic fibrosis induced by dimethylnitrosamine(DMN)in rats, and to explore its mechanism. Methods Ninety SD rats were randomly divided into six groups, normal control group, model control group, colchicine group, high-, medium- and low-dose TFL group(n=15).Expect for normal control group, the other groups were given intraperitoneal injection of 2 mL.kg-1 of 5% dimethylnitrosamine for 4 weeks as the model group. The rats in the normal control group and model control group were given 5 mL.kg-1of 0.9% sodium chloride solution, colchicine group was treated with 0.1 mg.kg-1 colchicine.High-, medium-and low-dose TFL groups were given 200, 100 and 50 mg.kg-1 of TFL.The rats were sacrificed and the livers were harvested and stained with HE and Masson staining to observe pathological changes and liver fibrosis in the same part 6 weeks after all the medicine was given to the rats each day. Immunohistochemistry and Western blotting were used to detect the expression of the transforming growth factor β-Ⅰ/type Ⅱ receptor ( TβRⅠ/Ⅱ) , collagen Ⅰ( Col Ⅰ) and Ⅲ collagen ( Col Ⅲ) . Results Compared with the normal control group, the semiquantitative score of liver fiber and the protein expression of TβRⅠ, TβRⅡ, ColⅠ and Col Ⅲ in the model control group were significantly increased(P<0.01).Compared with the model control group, the protein expression levels of TβR, TβRⅡ, ColⅠand ColⅢwere significantly decreased( P<0.01) in the high-,medium-and low-dose TFL group.The semiquantitative score of liver fiber was significantly decreased( P<0.01) with a dose-effect relationship. Conclusion TFL can inhibit formation of DMN-induced liver fibrosis in rats, which may be related with reduction of expression of TβRⅠ/Ⅱ of hepatic fibrosis promoting factor TGF-β1 , inhibition of the activation and increase of hepatic stellate cells, reduction of the collagen content.

Neurovascular Manifestation of Loeys-Dietz Syndrome: A Case Report

Loeys-Dietz syndrome (LDS) is an autosomal dominant disorder caused by heterozygous mutations in the genes encoding transforming growth factor-beta receptor type 1 or 2. It is typically characterized by a triad of hypertelorism, cleft palate or bifid uvula, and arterial tortuosity with aneurysm or dissection. Characteristic vascular abnormalities such as tortuosity, aneurysms, dissections, and stenosis are the most severe complications of LDS and can occur in the neurovascular system. We report a 5-year-old boy who presented with headaches and neurovascular abnormalities and was diagnosed with LDS with a novel mutation of the TGFBR1 gene. It is the first Korean report of neurovascular abnormalities in LDS.

An experimental research on effect of LTβR and P65 in progression of bladder cancer / 中华检验医学杂志

ObjectiveTo investigate the relationship of lymphotoxin β receptor (LTβR) and classical nuclear factor-κB (NF-κB) activation pathway in the pathogenesis and progress of cystitis and bladder cancer.MethodsThe LTβR and P65 mRNA expression were detected by Real-time quantitative PCR in 108 cases of fresh bladder tissue specimens (75 cases of bladder cancer,10 cases of inflammation and 23 normal bladder mucosa cases grouped by the tissue classification ),and protein expression were analyzed by immunohistochemistry assay in 118 cases of paraffin-embedded biopsy specimens (73 cases of bladder cancer,30 cases of cysitis and 15 normal bladder mucosa cases).The correlation analysis between the expressions of LTβR and P65 with clinical pathological data was then performed.Differences between LTβR and P65 mRNA and protein expression level were compared in different groups of bladder tissues using Kruskal-Wallis H test and the Chi-square test.Results( 1 )The mRNA expressions of LTβR and NF-κB/P65were higher in bladder cancer than those in normal group ( LTβR:29.4 ( 14.2 - 46.7 ) × 10 - 3/1.2 ( 0.3 -7.0) ×10-3,Z=-5.508; P65:9.7 (2.7 -21.1) ×10-3/1.0(0.8 ～1.8) ×10-3,Z=-5.030,P＜0.05 ).There were significantly differences between bladder cancer with different histological grades ( LTβR:18.2(2.1-31.3) × 10-3/ 28.4(16.6-36.2) × 10-3/47.9(34.3 -70.5) ×10-3,x2K-W=20.378;P65:4.9(1.3 - 12.0) × 10-3/7.4(3.0-21.9) × 10-3/17.0(10.0 ～28.3)× 10-3 ,x2K-W2 =15.219,P all ＜0.05) and lymph node metastasis (LTβR:27.2(9.7-40.1) ×10-3/39.4(26.7 -52.6) ×10-3,Z=-2.552; P65:7.4(2.3-15.6) ×10-3/13.4(6.7-23.3) ×10-3,Z=-2.026,P＜0.05).(2)The positive rates of LTβR and phosphorylated P65 ( p-P65 ) protein in cancer were higber than those of normal group (LTβR:69.8％/13.3％,x2 =16.600 ; p-P65:56.2％/6.7％,x2 =12.220,P ＜ 0.05 ).Upregulation of LTβR and p-P65 were associated with the histological grade (LTβR:56.3％/70.0％/90.4％,x2 =7.055; p-P65:40.6％ /60.0％/76.2％,x2 =6.679,P ＜0.05) and with lymph node metastasis (LTβR:58.3％/92.0％,x2 =8.849; p-P65:52.1％/64.0％,x2 =5.088,P ＜0.05).(3)There was a positive correlation between LTβR and P65 expression ( mRNA:r =0.654,P ＜ 0.05,protein:r =0.399,P ＜ 0.05 )in the bladder cancer and cystitis (r =0.521,P＜0.05).ConclusionsThe activation of LTβR and P65 was associated with progression and metastasis of bladder cancer.The activation of classical NF-κB pathway by LTβR may be achieved by P65.

Síndrome del QT largo / Syndrome of long Q-T interval

El síndrome del QT largo congénito de tipo Romano-Ward es una canalopatía arritmogénica poco frecuente, caracterizada por una grave alteración en la repolarización ventricular y traducida en el electrocardiograma por un alargamiento del intervalo QTc. Se documenta el caso de una paciente con síndrome del QT largo congénito de tipo 1, asintomßtica, con antecedentes familiares de muerte súbita y síndrome del QT largo, a quien se le realizó estudio ecocardiográfico, prueba ergométrica, detección de potenciales tardíos y dispersión del QT como complementos diagnósticos y estratificadores de riesgo. Se prescribió tratamiento farmacológico y semanas después se valoró su efectividad

The syndrome of congenital long Q-T interval (Q-T-i) of Romano-Ward type is an uncommon arrthymic channel disease, characterized by a severe alteration in ventricular repolarization and translated in the electrocardiogram by a lengthening of QTc interval. This is the case of a female patient presenting with type 1 congenital long Q-T, asymptomatic, with family history of sudden death and syndrome of long Q-T; an echocardiography study, ergometer test, late potentials detection and Q-T dispersion as diagnostic complements and risk stratification. Drug therapy was prescribed and several weeks later its effectiveness was assessed

Mechanism of dermal fibroblasts transdifferentiation into myofibroblasts induced by estrogen / 中华医学美学美容杂志

Objective To explore the possible pathway and regulatory mechanism of dermal fibroblasts' transdifferentiation into myofibroblasts induced by estrogen. Methods Human dermal fibroblasts were divided into six groups (A: control; B: estrogen; C: estrogen + ICI-182780; D: estrogen + SB203580; E: estrogen + PD98059; F: estrogen + SP600125). The cells were collected for RNA extraction and the expression of α-SMA was detected by real time quantitative RT-PCR. Some cells were analyzed by single cell RT-PCR to detect positive expression percentage of α-SMA.Results The expression and positive rate of α-SMA in estrogen group were significantly increased (Group B vs. Group A, 7. 385±0. 246 vs 1. 367±0. 034, P＜0.01) and those in ICI-182780 group and SP600125 group were significantly inhibited (Groups C and F vs. Group B, 4. 619 ±0. 164,2. 409±0. 091 vs 7. 385±0. 246, P＜0. 05). Conclusions In the process of fibroblast transdifferentiation into myofibroblasts induced by estrogen, estrogen β receptor and JNK-MAPK signal transduction pathway may play an important role.

Expression of TβRⅡRH mRNA in PBMCs of patients with lupus nephritis / 中华风湿病学杂志

Objective To investigate the association of TGF-β receptor typeⅡ(TβRⅡ)mRNA with lupus nephritis (LN) and disease activity by testing its expression levelin peripheral blood mononuclear cells (PBMCs).Methotis Forty-four patients with LN were included in this study.They were all had active LN.Twepty-eight LN patients were taking glueocorticoids and/or immunosuppressive agents and sixteen had never taken steroids or immunosuppressive agents.The expression levels of T13R H mRNA were semi-quantitativelydetermined by reverse transcription-polymerase chain reaction(RT-PCR).Resuits The expression levels of TβRⅡ mRNA in PBMCs from LN patients(1.7±1.0)were lower than those of non-lupus nephritis(4.0±3.1) and healthy subiects(4.1±2.5),(P<0.01).The difference of the expression levels between patients who took and had never taken glucocorticoids and/or immunosuppressive drugs was significantly statistically(P<0.05).The expression levels of TβRⅡ mRNA in PBMCs of patients with LN were correlated significantly with the systemic lupus erythematosus disease activity index (SLEDAI)scores(r-0.309.P<0.05),titers of anti-dsDNA antibody(r=-0.401,P<0.01)and serum complement C3 level(r=0.621,P<0.01).Conclusion This study suggests that TβRⅡ may be involved in the development of LN,and the TβRⅡ mRNA expression levels in PBMCs from patients with SLE are significantly correlated with LN activity.Glucocortieoids or immunosuppressive drugs can increase the expression levels of TβRⅡ mRNA and ameliorate renal damage.

The Clinical Significance of Transforming Growth Factor (TGF) beta1, TGF beta Receptor beta II, p53 Protein and K-ras Point Mutation in Pancreatic Cancer

PURPOSE: Many cancers, including pancreatic cancer, harbor defects in TGF beta signaling and are resistant to TGF beta mediated growth inhibition. In addition, the expression of the p53 gene and mutations in K-ras might play an important role in the multistep carcinogenesis of pancreatic cancer. This study examined the expression level of TGF beta 1, TGF beta receptorII (T beta RII), p53 protein and K-ras mutation in pancreatic cancer, along with their role and clinical significance. METHODS: The overexpression of TGF beta 1, T beta RII and p53 protein was evaluated using an immunohistochemical assay. The K-ras mutation was analyzed by PCR-RFLP in the surgical resected pancreatic tissue from 26 pancreatic ductal adenocarcinomas and 5 normal pancreases. RESULTS: Immunohistochemical analysis of TGF beta 1 and T beta RII revealed positive immunostaining in 73.1% and 76.9% of the tumors, respectively, which were significantly higher than the normal pancreas (P=0.008). The p53 protein was positive in none of the 5 normal ducts and 16 out of 26 (61.5%) pancreatic carcinoma specimens. The K-ras mutation was positive in none of the 5 normal ducts, and in 20 of the 26 pancreatic carcinoma specimens (76.9%). The presence of TGF beta1 and T beta RII in the cancer samples was significantly associated with node metastasis, advanced tumor stage (P<0.01), and a short survival time (P<0.05). The p53-positive pancreatic cancers showed a significantly lower survival rate than those with p53-negative tumors (P<0.05). There was no correlation between K-ras mutations and the survival rates. CONCLUSION: The detection of K-ras mutations and TGF beta 1, T beta RII and p53 protein overexpression can predict the prognosis of pancreatic carcinoma patients.

Nebivolol in the Inhibition of eNOS Activity in Cadiocytes with or without Heart Failure / 中国药房

0.1), but which were inhibited significantly in the left ventricular cardiac myocytes of the subjects with heart failure(P≤0.05). Both carvedilol and metoprolol exhibited no effect on eNOS activity in all the investigated cardiac myocytes. CONCLUSIONS: Nebivolol does no effect on eNOS activity of left ventricular cadiocytes in subjects or rats without hear failure but it can inhibit eNOS activity of cadiocytes in subjects or rats with heart failure so as to exert its beneficial clinical effect.

The Prognostic Significance of Chromosome 18 Monosomy in the Colon Cancer: Correlations with the Expressions of Smad4 and TGF-beta Receptor II Proteins

PURPOSE: Chromosomal instability of chromosome 18 and inhibition of the transforming growth factor beta (TGF-beta) signaling pathway, which is mediated through Smad4, play important roles in the tumorigenesis of colon cancer. This study evaluated the value of the expression of chromosome 18 monosomy in colon cancer as a prognostic factor and its correlations with the expressions of Smad4 and TGF-beta receptor II proteins. METHODS: We analyzed the rate of the expression of chromosome 18 monosomy in 66 colon cancers with using chromogenic in situ hybridization (CISH) and we evaluated its value as a prognostic factor by determining its correlation with the pathologic factors and immunohistochemical expressions of Smad4 and TGF-beta receptor II proteins. RESULTS: Of the 66 colon cancers, monosomy of chromosome 18, as determined by CISH, was observed in 18 cases (27.3%), and the decreased expression of Smad4 and TGF-beta receptor II proteins was observed in 30 cases (45.5%) and 25 cases (37.9%), respectively. The monosomy of chromosome 18 and the decreased expression of Smad4 proteins showed statistically significant correlations with the histologic differentiation, the presence of tumor emboli, the nodal status and the stage. The decreased expression of TGF-beta receptor II proteins had statistically significant correlations with the histologic differentiation, the T-stage, the nodal status and the stage. The monosomy of chromosome 18 showed a statistically significant correlation with the decreased expression of Smad4 and TGF-beta receptor II proteins. CONCLUSION: These results suggested that chromosome 18 monosomy may have prognostic value for colon cancer.

Effects of Transforming Growth Factor-beta1 and Its Receptor on the Development, Recurrence and Progression of Human Bladder Cancer / 대한비뇨기과학회지

PURPOSE: We investigated whether the expression levels of Transforming growth factor beta1 (TGF-beta1) and its receptors were related to the development, recurrence, progression and disease-free survival in the patients with bladder cancer. MATERIALS AND METHODS: The mRNA levels of TGF-beta1 and its receptors were examined in 102 tumor specimens from patients with primary bladder cancer, 29 corresponding normal bladder mucosae specimens surrounding these tumors and 15 normal bladder mucosae specimens by performing quantitative competitive PCR (QC-PCR). The protein levels of TGF-beta1 and its receptors were investigated by performing immunohistochemical staining on sections cut from 86 archival bladder tissue paraffin blocks. RESULTS: QC-PCR analysis showed that expressions of TGF-beta1, TGF-beta receptor I (TGF-betaRI) and receptor II (TGF-betaRII) in the superficial and low-grade bladder cancers were significantly higher than those in both the corresponding normal bladder mucosae surrounding the cancer (p= 0.0069, 0.0022 and 0.0046, respectively) and the control's normal bladder mucosae (p=0.0014, 0.0125 and 0.0089, respectively). Expressions of TGF-beta1 and its receptors were enhanced in the non-recurred and non-progressed patients compared to the recurred cases (p=0.0022, 0.0003 and 0.0001, respectively) and the progressed cases (p=0.0002, <0.0001 and <0.0001, respectively). Patients with high expression of TGF-beta and its receptors had a significantly higher disease-free survival rate than those patients with low expressions (p=0.0129, 0.0121 and 0.0132, respectively). CONCLUSIONS: The enhanced expression of TGF-beta1 and its receptors was correlated not only with superficial and low-grade bladder cancer, but also with enhanced patient survival. In conclusion, our findings suggest that the expressions of TGF-beta1 and its receptors are useful prognostic markers for a patient's resistance to disease recurrence and/or progression.

EXPRESSIONS OF TRANSFORMING GROWTH FACTOR-BETA 1 AND ITS TYPE Ⅱ RECEPTOR IN EXPERIMENTAL RAT ASCENDING AORTIC ANEURYSM / 解剖学报

Objective To investigate the expressions and significance of transforming growth factor-beta 1(TGF?1) and its typeⅡ receptor(TGF?RⅡ) in experimental rat ascending aortic aneurysm of rat model.Methods The rat ascending aortic aneurysm models were made by banding ascending aorta of young Wistar rats.The ascending aortas were taken 4 months after operation.Immunohistochemistry staining and Western blotting were used to investigate the expressions of TGF?1 and TGF?RⅡ.Result Immunohistochemistry staining results showed that TGF?1 expressed in all layers of the aortic aneurysm and the control.TGF?RⅡ was extensively located in the hyperplastic intima and tunica media smooth muscle cells in the aortic aneurysm,while there was only a little positive staining in the control group.Western blotting results indicated that the expression levels of TGF?1 and TGF?RⅡ in the aortic aneurysm were stronger than the control,P

Expression of Transforming Growth Factor-beta Receptors in Food Protein-Induced Enterocolitis Syndrome in Infancy / 소아알레르기및호흡기학회지

PURPOSE: Food protein-induced enterocolitis syndrome (FPIES) is a symptom complex of vomiting and diarrhea caused by non-IgE mediated allergy to cow's milk and/or soy in young infants. Transforming growth factor (TGF)-beta has been reported to protect the epithelial barrier of the gut from foreign antigens. We studied the expression of type 1 and 2 TGF-beta receptors in the mucosa of small intestine to investigate their roles in the pathogenesis of FPIES. METHODS: Twenty-eight patients, aged 7 to 120 days (mean 49 days) who were diagnosed with FPIES by clinical criteria and challenge tests were included. Immunohistochemical stainings for type 1 and 2 TGF-beta receptors were performed on endoscopic duodenal biopsy specimens. RESULTS: Type 1 and 2 TGF-beta receptors were expressed in the villous and crypt epithelial cells but nearly absent in the lamina propria in both patients and controls. Type 1 TGF-beta receptor expression was significantly lower in the patients who had villous atrophy than in the patients who had not and in controls. The expression of type 1 TGF-beta receptor was negatively correlated with the severity of villous atrophy. Type 2 TGF-beta receptor expression showed no significant difference between the patients and controls. CONCLUSION: Our results suggests that the decreased activity of type 1 TGF-beta receptor is implicated in the pathogenesis of FPIES in young infants.

Role of TGF-beta signaling and Ski/SnoN mRNA expression in cervical carcinomas / 대한산부인과학회잡지

OBJECTIVE: TGF-beta signaling is dependent on the heterodimerization of the type II TGF-beta receptor (TbetaR-II) with the type I TGF-beta receptor (TbetaR-I). which mediate intracellular signals through Smad proteins. Whereas physiologic concentrations of SnoN and Ski allow a feedback regulation of TGF-beta signaling, deregulation of SnoN or Ski expression leads to total inhibition of TGF-beta signaling and of the tumor suppressors Smad2 and Smad4, which can explain the role of SnoN and Ski as oncogenes. In order to identify possible molecular mechanisms responsible for TGF-beta resistance, the author investigated the mutation and expression of TGF-beta1, its receptors, Ski/SnoN in cervical carcinomas. METHODS: From December 1995 to December 1999, 45 carcinomas and 7 normal cervical tissue specimens were obtained by surgical resection in the Kyung Hee University Medical Center. Tissue specimens were snap-forzen in liquid N2 and stored at -70 degrees C until used. Total RNA was extracted from specimens and evaluated the expression levels using densitometric analysis of quantitative RT-PCR products (TGF-beta1, Tbeta1R-I, Tbeta1R-II, Ski/SnoN), and the mutations were investigated by quantitative genomic-PCR followed by nonisotopic RT-PCR-SSCP analysis (Tbeta1R-II, Tbeta1R-I, Ski/SnoN). The abnorally expressed levels of RT-PCR products (TGF-beta1, Tbeta1R-II) were analysed for the clinicopathologic characteristics. RESULTS: Quantitative RT-PCR analysis demonstrated variable expression of TGF-beta1 mRNA (0.05-0.89) in tumors and significantly increased TGF-beta1 expression level (>0.48) in 15 of 45 samples (33.3%). There is no significant reduction of Tbeta1R-I expression (1.36) in 2 of 45 samples (4.4%), and there is no amplification of Ski/SnoN gene by quantitative genomic-PCR analysis. CONCLUSIONS: The overexpression of TGF-beta1 mRNA and the reduced or absent expression of Tbeta1R-II may be an important contributing factors, and the abnormally low genomic levels and no mutational alterations of Tbeta1R-II is caused by monoallelic deletion suggesting that Tbeta1R-II might play as a tumor suppressor of haloinsufficiency in cervical carcinomas. We could not show that high levels of Ski/SnoN expression could produce a disruption of TGF-beta signaling in cervical carcinomas.

Correlation between the Expression Levels of Transforming Growth Factor-beta (TGF-beta) Receptors and Responsiveness to TGF-beta1-Induced Growth Inhibitory Effects in Leukemic Cell Lines / 대한혈액학회지

BACKGROUND: Defects in TGF-beta receptors have been found in a variety of malignant cells, we therefore investigated whether these defects could be also demonstrated in leukemic cells. In addition, we analyzed the relation between TGF-beta receptor expression and responsiveness to TGF-beta-induced growth inhibitory effects. METHODS: Eleven human leukemic cell lines and two normal cell lines were recruited for the study. To evaluate the expression of TGF-beta receptor type I (RI) and type II (RII), Western blotting analysis was conducted utilizing two kinds of primary antibodies against both RI and RII. Band strength was quantitated with densitometry. Moreover, specific peptides against primary antibodies were employed for competitive inhibition assay. Responsiveness to TGF-beta1 was assessed by [3H] thymidine uptake. RESULTS: Bands of same molecular size were demonstrated by two different primary antibodies. Peptides against RI or RII antibodies successfully blocked the emergence of RI or RII message, respectively, verifying that former bands represented specific RI or RII. Relative RI levels in leukemic cells except HL-60 compared with CCL-64, normal lung epithelial cells, were in the range of 0.48~1.14. In contrast, relative RII levels, although variable between individual cells, were less than 0.25 in all the leukemic cells. Percents [3H]thymidine uptake of leukemic cells at 10 ng/mL of TGF-beta1 compared with untreated control were widely distributed in the range of 7.7~62.9%. Positive correlation between RII levels and TGF-beta responsiveness was observed (P=0.025). CONCLUSION: Defective RI expression seems to be rare, however, defective RII expression appears to be rather common in leukemic cells. Positive correlation between RII levels and TGF-beta responsiveness suggests role of defective RII expression in the acquisition of resistance to TGF-beta in leukemic cells.