[Analysis of strong invasive clinical features and poor prognosis in macrotrabecular-massive hepatocellular carcinoma].

Objective: To analyze the correlation between the histological classification of hepatocellular carcinoma (HCC), especially macrotrabecular-massive (MTM), tumor invasiveness, and prognosis. Methods: The clinical and follow-up data of 246 consecutive HCC cases who met the inclusion criteria from 2015 to 2018 were retrospectively analyzed. They were divided into trabecular/pseudoglandular, trabecular/patchy, and MTM types according to the histological classification. The relationship between each type and related clinicopathological features was analyzed. The Kaplan-Meier method was used to plot tumor-free survival (DFS) and overall survival (OS) curves. Log rank tests, COX univariate, and multivariate regression analyses were conducted to analyze the relationship between clinical features, including histological classification, DFS, and OS. Results: Trabecular/pseudoglandular, trabecular/nodular, and MTM type proportions were 44.7% (110 cases), 32.9% (81 cases), and 22.4% (55 cases), respectively. The results of the clinicopathological features showed that MTM-type HCC was significantly more invasive than the other two types, with alpha-fetoprotein (AFP) ≥400 ng/ml, tumor diameter≥8 cm, no tumor capsule, poor differentiation, and MVI positivity proportions, and the differences were statistically significant (P < 0.05).The proportion of MTM-type HCC patients with American Joint Committee on Cancer TNM Stage III to IV and Chinese Liver Cancer Staging (CNLC) II to II was significantly higher than that of the first two types, and the differences were statistically significant (P < 0.05). In addition, the proportion of MTM subtypes undergoing transcatheter arterial chemoembolization was also raised (P < 0.05). The DFS and OS were significantly lower for MTM-type HCC compared to trabecular/pseudoductal-type HCC at 1-, 3-, and 5-years, and the differences were statistically significant (P < 0.05). Univariate analysis indicated that strongly invasive clinical pathological features such as alpha fetoprotein (AFP) ≥400 ng/ml, tumor diameter ≥ 8 cm, no tumor capsule, poor differentiation, positive microvascular invasion, tumor stage, and MTM staging were all risk factors affecting DFS and OS (P < 0.05). Multivariate COX analysis showed that MTM histological staging, AFP ≥ 400 ng/ml, tumor non-encapsulation, satellite nodules, CNLC stages II~III, and TNM stages III~IV were independent risk factors for DFS (P < 0.05), while AFP ≥ 400 ng/ml, tumor non-encapsulation, and CNLC stage II~III were independent risk factors for OS ( P < 0.05). Conclusion: Histological classification is highly correlated with tumor invasiveness and HCC prognosis. Trabecular/pseudoglandular types have lower malignancy and a better prognosis, while MTM types exhibit strong invasive features and a poor prognosis.

[Impact of regional positive lymph node ratio of gallbladder carcinoma on prognosis].

Objective: To study the impact of regional positive lymph node ratio (LNR) on prognosis of patients with gallbladder carcinoma. Methods: The clinicopathological and survival data of 53 patients with gallbladder carcinoma who underwent radical resection with regional lymph node metastasis in Ningbo University Affiliated Lihuili Hospital from May 2012 to December 2020 were collected, and receiver operating characteristic curve (ROC) was used to determine the optimal cut-off value of LNR for predicting postoperative survival status in patients with gallbladder carcinoma. According to the critical value, the patients were divided into low LNR group and high LNR group. The clinicopathological features and prognosis of the two groups were compared. Log rank test was used for univariate analysis of prognostic factors in patients with gallbladder carcinoma, and Cox proportional hazards model was used for multivariate analysis. Results: A total of 417 regional lymph nodes were dissected in 53 patients, of which 144 lymph nodes were positive, with a positive rate of 34.5%. The optimal cut-off value of LNR for predicting postoperative survival status of patients with gallbladder carcinoma was 0.33. According to this cut-off value, patients were divided into low LNR group (LNR≤0.33, 28 cases) and high LNR group (LNR>0.33, 25 cases). The recurrence rates were 64.3% (18/28) and 88.0 % (22/25) in low LNR group and high LNR group, respectively. The median recurrence-free survival (RFS) was 8 and 7 months, respectively (P=0.032). In the low LNR group, the 1-, 3-, and 5-year survival rates were 56.2%, 38.4%, and 32.0%, respectively, and the median overall survival (OS) was 16 months. In the high LNR group, the 1-, 3-, and 5-year survival rates were 37.9%, 5.4%, and 0, respectively, and the median OS was 9 months. The postoperative survival rate of patients in the low LNR group was better than that in the high LNR group (P=0.008). Univariate analysis showed that LNR was even associated with RFS and OS in patients with gallbladder carcinoma (P<0.05). Multivariate analysis showed that LNR>0.33 was an independent risk factor for postoperative RFS (HR=1.977, 95% CI: 1.045-3.740), but not for OS (HR=1.561, 95% CI: 0.685-3.553). Conclusion: On the basis of clearing a sufficient number of regional lymph nodes, patients with gallbladder carcinoma with regional LNR>0.33 are more likely to relapse after operation, but the predictive value of LNR>0.33 OS is insufficient.

[Efficacy of in-situ full-left/full-right split liver transplantation for adult recipients using the living donor liver transplantation technique:a single-center report of 25 cases].

Objective: To evaluate the efficacy of in-situ full size split liver transplantation(fSLT) for adult recipients using the living donor liver transplantation(LDLT) technique and to compare the characteristics of the left hemiliver graft (LHG) and the right hemiliver graft(RHG)transplantation. Methods: Deceased donor and recipient data of 25 consecutive cases of fSLT at Department of Hepatopancreatobiliary Surgery, Ningbo Medical Center Lihuili Hospital from March to December 2021 was retrieved and the patients divided into two groups:LHG group and RHG group. Among the 13 donors,11 were male and 2 were female,aged (M(IQR))38(19) years(range: 25 to 56 years),with height of 168(5) cm(range:160 to 175 cm) and weight of 65(9) kg(range: 50 to 75 kg). The median age of the 25 recipients was 52(14) years(range:35 to 71 years),17 were male and 8 were female,15 had primary liver cancer and 10 had benign end-stage liver disease,model for end-stage liver disease score was 10(9) points(range:7 to 23 points). Of the 25 recipients,10 recipients had previously undergone hepatobiliary surgery. The follow-up period was to January 2022. Demographic,clinicopathological,surgical outcomes and postoperative complications were evaluated and compared between the two groups. Continuous quantitative data were compared using Mann-Whitney U test. Classification data were expressed as frequencies,and were compared between groups using χ2 test or Fisher exact probability method. Results: Using LDLT technique,in-situ full-left/full-right liver splitting was performed and 13 viable pairs of hemiliver grafts were harvested with acquisition time of 230(53) minutes(range:125 to 352 minutes) and blood loss of 250(100) ml(range:150 to 1 000 ml). A total of 25 hemiliver grafts(13 LHG and 12 RHG) were allocated to patients listed for liver transplantation in our center by China Organ Transplant Response System. In the LHG group(13 cases),there were more females and more patients with benign end-stage liver disease than in the RHG group(12 cases)(P<0.05). The body weight and graft weight of recipients in the LHG group were lower than that in RHG group(both P<0.05). There were no significant differences in other baseline data between the two groups(all P>0.05). The graft to recipient weight ratio(GRWR) was 1.2(0.4)%(range:0.7% to 1.9%) for 25 recipients,1.1(0.5)%(range:0.7% to 1.6%)for the LHG group and 1.3(0.5)%(range:0.9% to 1.9%)for the RHG group. There was no significant difference between the two groups (P>0.05). Sharing patterns of hepatic vessels and the common bile duct are as follows:all the trunk of middle hepatic vein were allocated to the LHG group. The proportion of celiac trunk,main portal vein and common bile duct assigned to LHG and RHG was 10∶3 (P=0.009), 9∶4 (P>0.05) and 4∶9 (P=0.027),respectively. The vena cava of 12 donors in early stage retained in LHG and that of last one was shared between LHG and RHG (P<0.01). The median cold ischemia time of 25 hemiliver grafts was 240(90) minutes(range:138 to 420 minutes). For the total of 25 fSLT,the median anhepatic phase was 50(16) minutes(range:31 to 98 minutes) and the operation time was 474(138)minutes(range:294 to 680 minutes) with blood loss of 800(640) ml(range:200 to 5 000 ml). There were no significant differences in all of operation data between two groups. In the LHG group,3 patients with GRWR≤0.8% had postoperative small-for-size syndrome which improved after treatment. Postoperative Clavien-Dindo grade≥Ⅲ complications were observed in 6 cases(24.0%),4 cases(4/13) in the LHG group and 2 cases(2/12) in the RHG group,respectively. The difference was not statistically significant. Among them,5 cases improved after re-operation and intervention,1 case in LHG group died of secondary infection 2 weeks after operation,and the mortality was 4.0%. Analysis of serious postoperative complications and death has suggested that conventional caval interposition should not be used for LHG transplantation. Conclusion: Relying on accurate donor-recipient evaluation and the apply of LDLT technique,the morbidity and mortality of in-situ fSLT in adults is acceptable.

[Evaluations of total hilar en bloc resection and reconstruction in comparison with portal vein resection and reconstruction in treatment of perihilar cholangiocarcinoma].

Objective: To evaluate the feasibility and efficacy of total hilar en bloc resection and reconstruction(THERR) and portal vein resection and reconstruction(PVRR) in treatment of perihilar cholangiocarcinoma(PHC). Methods: Data of a total of 101 consecutive patients with PHC who underwent bile duct resection with various types of hepatectomies from June 2013 to December 2019 at Department of Hepatopancreatobiliary,Lihuili Hospital were retrospectively analyzed. Patients who underwent PHC resection combined with THERR or PVRR were identified and grouped accordingly. Fourteen patients(6 males, 8 females, aged (64.3±9.7)years old) underwent hepatectomy combined with THERR, 19 patients(11 males, 8 females, aged (63.8±8.6)years old) underwent hepatectomy combined with PVRR. Indications and surgical procedures of THERR and PVRR were reported. The clinicopathological characteristics and operation data, as well as the short and long-term outcomes of patients of the two groups were compared by Student's t-test and the χ2 test or Fisher exact test, respectively. The actual survivals rates were calculated by using the Kaplan-Meier method, and compared using the Log-rank test. Results: There were no statistically significant differences between the two groups in respect to age,sex and whether they had preoperative biliary drainage or not. The types of combined hepatectomy carried out predominately between the two groups were statistically different with the left side being predominant in the THERR group(10/14,P=0.010) and right side in PVRR group(12/19,P=0.001). There were no significant differences between the two groups in respect to whether they received preoperative portal vein embolization,intraoperative blood loss,curative degree,number of lymph node dissections, and whether there was lymphatic metastasis or not. However, both the times of operation and continuous Pringle maneuver were statistically longer in the THERR group((586±158)minutes and (32.5±7.3)minutes)than those in the PVRR group((453±88)minutes and (12.4±3.8)minutes),respectively(t=3.087,P=0.004;t=10.325,P<0.01). One patient in the THERR group died of liver failure 9 days postoperative, the cumulative 1-, 3- and 5-year survival rates were 84.9%, 57.1% and 37.0% for the THERR group and 81.9%, 37.8% and 30.2% for the PVRR group, respectively. There was no statistically significant differences between the two groups(χ²=0.150,P=0.698). Conclusions: Compared to the role of PVRR in the treatment of PHC, THERR is a novel and technically demanding procedure that is feasible in selected patients for the treatment of advanced PHC with invasion of both the hepatic artery and portal vein. However,due to the small size of this primary study,the value of THERR needs further evaluation.

Prognostic model for identifying candidates for hepatectomy among patients with hepatocellular carcinoma and hepatic vein invasion.

BACKGROUND: Hepatic vein tumour thrombus (HVTT) is a major determinant of survival outcomes for patients with hepatocellular carcinoma (HCC). An Eastern Hepatobiliary Surgery Hospital (EHBH)-HVTT model was established to predict the prognosis of patients with HCC and HVTT after liver resection, in order to identify optimal candidates for liver resection. METHODS: Patients with HCC and HVTT from 15 hospitals in China were included. The EHBH-HVTT model with contour plot was developed using a non-linear model in the training cohort, and subsequently validated in internal and external cohorts. RESULTS: Of 850 patients who met the inclusion criteria, there were 292 patients who had liver resection and 198 who did not in the training cohort, and 124 and 236 in the internal and external validation cohorts respectively. Contour plots for the EHBH-HVTT model were established to predict overall survival (OS) rates of patients visually, based on tumour diameter, number of tumours and portal vein tumour thrombus. This differentiated patients into low- and high-risk groups with distinct long-term prognoses in the liver resection cohort (median OS 34·7 versus 12·0 months; P < 0·001), internal validation cohort (32·8 versus 10·4 months; P = 0·002) and external validation cohort (15·2 versus 6·5 months; P = 0·006). On subgroup analysis, the model showed the same efficacy in differentiating patients with HVTT in peripheral and major hepatic veins, the inferior vena cava, or in patients with coexisting portal vein tumour thrombus. CONCLUSION: The EHBH-HVTT model was accurate in predicting prognosis in patients with HCC and HVTT after liver resection. It identified optimal candidates for liver resection among patients with HCC and HVTT, including tumour thrombus in the inferior vena cava, or coexisting portal vein tumour thrombus.

ANTECEDENTES: La trombosis tumoral de la vena hepática (hepatic vein tumour thrombus, HVTT) es un determinante importante de los resultados de supervivencia en pacientes con carcinoma hepatocelular (hepatocellular carcinoma, HCC). Se desarrolló el modelo llamado Eastern Hepatobiliary Surgery Hospital (EHBH)-HVTT para predecir el pronóstico de los pacientes con HCC y HVTT después de la resección hepática (liver resection, LR), con el fin de identificar los candidatos óptimos para LR entre estos pacientes. MÉTODOS: Se incluyeron pacientes con HCC y HVTT de 15 hospitales en China. El modelo EHBH-HVTT con gráfico de contorno se desarrolló utilizando un modelo no lineal en la cohorte de entrenamiento, siendo posteriormente validado en cohortes internas y externas. RESULTADOS: De 850 pacientes que cumplieron con los criterios de inclusión, hubo 292 pacientes en el grupo LR y 198 pacientes en el grupo no LR en la cohorte de entrenamiento, y 124 y 236 en las cohortes de validación interna y externa. Los gráficos de contorno del modelo EHBH-HVTT se establecieron para predecir visualmente las tasas de supervivencia global (overall survival, OS) de los pacientes, en función del diámetro del tumor, número de tumores y del trombo tumoral de la vena porta (portal vein tumour thrombus, PVTT). Esto diferenciaba a los pacientes en los grupos de alto y bajo riesgo, con distinto pronóstico a largo plazo en las 3 cohortes (34,7 versus 12,0 meses, 32,8 versus 10,4 meses y 15,2 versus 6,5 meses, P < 0,001). En el análisis de subgrupos, el modelo mostró la misma eficacia en la diferenciación de pacientes con HVTT, con trombo tumoral en la vena cava inferior (inferior vena cava tumour thrombus, IVCTT) o en pacientes con PVTT coexistente. CONCLUSIÓN: El modelo EHBH-HVTT fue preciso para la predicción del pronóstico en pacientes con HCC y HVTT después de la LR. Identificó candidatos óptimos para LR en pacientes con HCC y HVTT, incluyendo IVCTT o PVTT coexistente.

[PAHs exposure and occupational health risk assessment of workers in coal tar pitch factory].

Objective: To Investigation of industrial sites involving coal tar pitch to detect PAHs in the occupational environment and to assess their occupational health risks to workers. Methods: Taking coal tar pitch enterprises as the research object, and making the occupational health field investigation and inspection. Detecting the 16 PAHs in the samples by high performance liquid chromatography (HPLC) , analyze the PAHs exposed dose of the workers in the place, and using cumulative toxic equivalent quantity method, loss of life expectancy method and carcinogenic risk factor method to assessment worker's occupational health risk. Results: In the 15 posts involved, Some workers' exposed to the total concentration of PAHs is higher than others, the maximum exposure concentration is 1931.45ng/m3. There are different hazard risk levels in different working post due to different processes.The lifetime risk of workers is significantly higher than the acceptable range. Some workers has higher carcinogenic risk and workers' life expectancy loss is up to 1033.95 hours. Conclusion: Part of the coal tar pitch workers exposed to higher concentrations of PAHs, and beyond the occupational exposure limits.And there is a high occupational health risk due to high exposure to PAHs.

[Investigation and analysis on occupational hazards in a carbon enterprise].

Objective: To explore occupational-disease-inductive in a carbon enterprise workplace and personnel occupational health examination, providing the basis for occupational disease prevention and control of the industry. Methods: Field occupational health survey and inspection law were used to study the the situation and degree of occupational disease hazards in carbon enterprise from 2013 to 2015.Occupational health monitoring was used for workers, physical examination, detection of occupational hazard factors and physical examination results were analyzed comprehensive. Results: Dust, coal tar pitch volatiles, and noise in carbon enterprise were more serious than others. Among them, the over standard rate of coal tar pitch volatiles was 76.67%, the maximum point detection was 1.06 mg/m(3), and the maximum of the individual detection was 0.67 mg/m(3). There was no statistical difference among the 3 years (P>0.05) . There were no significant differences in the incidence of occupation health examination, chest X-ray, skin audiometry, blood routine, blood pressure, electrocardiogram between 3 years (P>0.05) , in which the skin and audiometry abnormal rate was higher than 10% per year. Conclusion: Dust, coal tar, and noise are the main occupational hazard factors of carbon enterprise, should strengthen the corresponding protection.

[A association of genetic polymorphisms of XRCC1 and damage levels in workers exposed to coal tar pitch].

Objective: To study the relationship between XRCC1 gene polymorphism and DNA damage in peripheral blood lymphocytes of workers exposed to coal tar pitch. Methods: 203 coal tar asphalt device operation area workers (exposure group) and 76 logistics management personnel (control group) as the research ob-ject, determination of 1-hydroxypyrene concentrations in the urine as polycyclic aromatic hydrocarbons expo-sure dose, using the alkaline comet assay evaluation a peripheral blood lymphocyte DNA damage degree, using TaqMan MGB real time PCR method to detect XRCC1 gene 3 loci (XRCC1-194, XRCC1-280 and XRCC1-399) single nucleotide polymorphism. Results: No significant differences was observed in age, sex, smoking and alco-hol consumption between the two groups (P>0.05). The level of 1-OHP in the exposed group was significantly higher than that in the control group (1.27±0.93 µg/g creatinine) (P<0.05). The comet Olive tail moment level (3.21±0.93) in the peripheral blood lymphocytes of the exposed group was significantly higher than that in the control group (0.94 ± 0.39) (P<0.05). There was no significant difference in genotype distribution of XRCC1-194, XRCC1-280 and XRCC1-399 between the two groups (P>0.05). There was a significant correlation be-tween the XRCC1-280 locus gene polymorphism and comet Olive tail moment in the exposure group (P<0.05) af-ter adjustment for sex, age, smoking rate, drinking rate, length of service and urinary 1-OHP concentration. The comet Olive tail moment level of GG individuals carrying wild homozygous genotype was significantly lower than that of individuals carrying heterozygous genotype GA and carrying mutant homozygous genotype AA (P< 0.05) , and the difference was statistically significant (P<0.05) The comet Olive tail moment level of heterozy-gous genotype GA was significantly lower than that of genotype AA with mutational homozygous genotype AA (P<0.05) , and the difference was statistically significant (P<0.05). Conclusion: Arg280His locus polymor-phism of XRCC1 gene may influence the DNA damage level of peripheral blood lymphocytes induced by occupational exposure to coal tar pitch.

[The effect of occupational exposure to coal tar pitch workers' health].

Objective: To investigate the effect of occupational exposure to coal tar pitch on workers' health and metabolism. Methods: 805 workers exposed to coal tar pitch were selected as exposure group from the produce and em-ploy factory. Other people handle administrative and logistical affairs who not exposed to coal tar pitch were selected as control group. Fix-point sample of air were collected to detect the concentration of coal tar pitch. Do physical examination and questionnaire to collect workers' basic and healthy information. To detect the metabolic product of urine samples in laboratory. Results: Anomaly detection rate of the skin in exposure group is 10.61. The lung function indices (FEV1.0%) in exposure group were significantly lower than control group (P<0.05) . The monocyte count and monocyte rate in expo-sure group were significantly higher than control group (P<0.05) . The metabolic product content of PAHS in urine sam-ples is significantly higher in exposed group than control group (P<0.05) . Conclusion: The metabolic product content of polycyclic aromatic hydrocarbon is higher in exposed workers. Coal tar pitch damage workers' skin and lung function. It can cause pruritus chromatodermatosis and so on.

Structure of Bacillus subtilis superoxide dismutase.

The sodA gene of Bacillus subtilis was expressed in Escherichia coli, purified and crystallized. The crystal structure of MnSOD was solved by molecular replacement with four dimers per asymmetric unit and refined to an R factor of 21.1% at 1.8 A resolution. The dimer structure is very similar to that of the related enzyme from B. anthracis. Larger structural differences were observed with the human MnSOD, which has one less helix in the helical domain and a longer loop between two beta-strands and also showed differences in three amino acids at the intersubunit interface in the dimer compared with the two bacterial MnSODs. These structural differences can be exploited in the design of drugs that selectively target the Bacillus enzymes.

Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas.

A novel inhibitor of apoptosis designated survivin has recently been found in many common human cancers but not in normal tissues. A potential distribution of survivin in gastric cancer and its implication for apoptosis inhibition have been investigated. Recombinant survivin expressed in Escherichia coli as a glutathione S-transferase fusion protein was used to raise a novel panel of mouse monoclonal antibodies. In an immunohistochemical analysis of 174 cases of gastric carcinomas (stages I-III), anti-survivin monoclonal antibody 8E2 (IgG1) reacted with 34.5% of cases (60 of 174 cases) with a variable number of tumor cells stained (20-100%). In contrast, no expression of survivin in neighboring normal tissues was observed. When stratified for p53 and bcl-2 expression and apoptotic index, the expression of survivin significantly segregated with p53- and bcl-2-positive cases [56.1 versus 15.2% (P = 0.001) and 69.2 versus 31.6% (P = 0.006), respectively] and with a decreased apoptotic index as compared with that of survivin-negative tumors (0.97 +/- 0.64 versus 0.62 +/- 0.39%, P < 0.001). These data identify a role for survivin in promoting aberrantly increased cell viability in gastric cancer and suggest a potential correlation between accumulated p53 and survivin expression in neoplasia.

Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer.

Deregulated inhibition of apoptosis (programmed cell death) may facilitate the insurgence of neoplasia, but whether it also influences the outcome of common cancers has remained controversial. In this study, we investigated the expression of a novel inhibitor of apoptosis, survivin, in colorectal cancer and its relationship with tumor cell apoptosis and overall prognosis. By immunohistochemistry, survivin was expressed in 91 of 171 (53.2%) cases of colorectal carcinomas of histological stages 0 to IV. In contrast, normal colon epithelium did not express survivin. Although survivin expression did not correlate with p53 abnormalities (46.5% versus 58.0%; P = 0.18), survivin-positive cases were strongly associated with bcl-2 expression (72.5% versus 27.4%; P < 0.0001) and reduced apoptotic index (0.76% +/- 0.39% versus 1.17% +/- 0.62%; P < 0.0001). Expression of survivin alone in bcl-2-negative (discordant) cases also resulted in reduced apoptotic index (0.82% +/- 0.57% versus 1.16% +/- 0.66%; P = 0.0046). When analyzed for prognostic significance, patients with low apoptotic index (< 0.97%) had worse survival rates than the group with high apoptosis (P < 0.001), and a multivariate Cox proportional hazard model identified reduced apoptosis as an independent predictive factor for overall survival (P < 0.0001). These data demonstrate that apoptosis inhibition by survivin, alone or in cooperation with bcl-2, is an important predictive/prognostic parameter of poor outcome in colorectal carcinoma and identify survivin as a new diagnostic/therapeutic target in cancer.

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.

Changes of angiogenesis and tumor cell apoptosis during colorectal carcinogenesis.

Activation of the angiogenic process occurs during tumorigenesis, as does disturbance of cell proliferation and apoptosis. Seeking a potential correlation, we investigated tumor cell apoptosis, proliferation, and angiogenesis in the adenoma-carcinoma sequence of colorectal carcinogenesis using an in situ apoptosis detection kit and MIB-1 and anti-CD34 antibodies in 27 adenomas with low dysplasia, 17 adenomas with high dysplasia, and 26 carcinomas in adenoma, as well as assessed p53 and bcl-2 expressions. The results showed that the potential for apoptosis was augmented, paralleling the increment of proliferation, in adenomas with low dysplasia but diminished when adenomas progressed from low dysplasia to high dysplasia and cancer. A gradual increment of microvessel density was observed during the progression with an increase during transition from low dysplasia to high dysplasia and cancer. Correlation coefficient test showed an inverse correlation between apoptotic index and microvessel density when all of the lesions were taken into account. No apparent impact of aberrant p53 on angiogenesis or bcl-2 on apoptosis was observed in this study. These results suggest that the angiogenesis initiates during transition from low dysplasia to high dysplasia and cancer, which may, in turn, contribute to the reduction of tumor cell apoptosis during colorectal carcinogenesis.

DNA damage regulates ARID1A stability via SCF ubiquitin ligase in gastric cancer cells.

OBJECTIVE: The gene product of the AT-rich interactive domain 1A (SWI-like) gene (ARID1A) is a member of the SWI/SNF adenosine triphosphate-dependent chromatin-remodeling complexes, which plays an essential role in controlling gene expression and is also involved in cancer development. ARID1A is frequently mutated in a wild variety of cancers and function as a tumor suppressor in several kinds of cancers. ARID1A was down-regulated in gastric cancer, and associated poor patient prognosis. However, how ARID1A protein is regulated in gastric cancer remains largely unknown. MATERIALS AND METHODS: Here, we show that ARID1A protein is rapidly ubiquitinated and degradated in gastric cancer cells in response to DNA damage treatment. RESULTS: Using genetic and pharmacologic Cullin inactivation coupled with in vitro ubiquitination assay, we demonstrate that ARID1A is a substrate of the Cullin-SKP1-F-box protein (SCF) complexes. Moreover, gastric cancer cells with forced expression of ARID1A showed an increased sensitivity to DNA damage reagents. Thus, our data uncovered a previous unknown posttranscriptional regulation of ARID1A by SCF E3 ligase in gastric cancer cells in DNA damage response. CONCLUSIONS: These findings suggest ARID1A might be a promising drug target in gastric cancer treatment.

Complete sequence of the Salmonella typhimurium gene encoding malate dehydrogenase.

The nucleotide (nt) sequence of mdh (encoding malate dehydrogenase) in Salmonella typhimurium is presented. The relative positions of argR (encoding arginine repressor) and mdh on the chromosome of S. typhimurium were determined from analysis of the nt sequence. The homology of the 3'-terminal end with only one of two published mdh sequences in Escherichia coli identifies the correct sequence in this organism.

In vitro cleavage of HPV16 E6 and E7 RNA fragments by synthetic ribozymes and transcribed ribozymes from RNA-trimming plasmids.

A RNA-trimming plasmid pRG523 is constructed, in which three Rz genes, GR5(5'-cis-Rz gene), HR2G(trans-Rz gene) and GR3(3'-cis-Rz gene), are arranged in the order from 5' to 3' downstream from the T7 promoter. In vitro transcription of this plasmid shows that the trans-Rz can be trimmed to definite lengths by the cis-Rz on both sides of the trans-Rz. In vitro cleavage of HPV16 E6 and E7 RNA fragments of different lengths by synthetic Rz and that of E7 RNA with a length of 171 nt by synthetic Rz and transcribed Rzs with different lengths of flanking sequences is studied. The results show that the non-base-pairing flanking sequences on both Rz and target RNA can affect the cleavage reaction.

Conformation of DNA-DNA polymerase I complex observed by scanning tunneling microscopy.

The conformation of a complex of a 41 mer/31 mer DNA fragment and the Klenow fragment of DNA polymerase I of Escherichia coli was studied by scanning tunnelling microscopy (STM). The results shows that near two turns of double helix of this DNA fragment was outside of enzyme while another part containing more than one turn of helix and 10 nucleotides single strand was combined with enzyme. The dimension and shape of DNA polymerase I (KF) in complex were different from that of free enzyme. The conformation of DNA-DNA polymerase I (KF) complex and the application of STM in studying structure of complex of DNA polymerase with DNA were discussed.

Feed intake and weight gain of growing goats fed diets of various energy and protein levels.

Growing goats, 45 Alpine and 45 Nubian, were used in a 3 x 3 factorial arrangement to quantify the influence of dietary energy and protein levels on daily DM intake and nutrient utilization for growth. Goats had ad libitum access to complete mixed diets containing either 2.46, 2.77 or 3.05 Mcal/kg ME plus 11.2, 12.7 or 15.1% CP for 16 wk. Dry matter intake decreased curvilinearly as dietary ME density increased (P less than .001). Dry matter intake increased linearly (P less than .05) as dietary CP level increased during all growth intervals except wk 25 to 28 of age. Average daily gain was 115, 113 and 99 g/d for goats fed diets containing 2.46, 2.77 and 3.05 Mcal/kg ME, respectively. Average daily gain was 104, 106 and 117 g/d for goats fed diets with 11.2, 12.7 and 15.1% CP, respectively. Dry matter intake was higher (P less than .01) for Alpine than for Nubian goats, whereas ADG was similar between breeds. Intake of ME was 248, 260 and 198 kcal/(kg.75.d) for goats fed the low- medium- and high-energy diets, respectively. Intake of CP was 9.1, 10.7 and 13.2 g/(kg.75.d) for goats fed low-, medium- and high-protein diets, respectively. Average requirements for growth derived from regression analysis of all data points were 4.6 kcal ME and .26 g CP/g ADG. The prediction equation for intake of growing goats of 4 to 8 mo of age was: DMI, g/d = 1,749 - 496 DE, kcal/g + 18 live weight, kg + 3 ADG, g/d; r2 = .73 (Sy.x = 127, P less than .0001, n = 90). The requirement of ME for growth was 33% lower than the value recommended in 1981 by the National Research Council.

Sulfate supplementation of alpine goats: effects on milk yield and composition, metabolites, nutrient digestibilities, and acid-base balance.

Effects of sulfate supplementation on milk yield and composition, ruminal and blood metabolites, acid-base status, and nutrient digestibilities were determined using 30 multiparous, lactating Alpine does. Goats were fed isonitrogenous diets containing .16 (basal), .26, or .36% S (DM basis) during a 13-wk lactation trial that coincided with wk 3 to 15 of lactation. During wk 16 to 17, four does from each treatment were placed in a metabolism crate to measure nutrient digestibility and balance. Feed intake, yield of 4% fat-corrected milk, and milk S content were not affected by added S, but the goats fed the .26% S diet had a trend of higher persistency of lactation (P < .20). During wk 10 and 15 of lactation, milk solids-not-fat percentage was higher (P < .10) for does fed the .26% S diet. Sulfur supplementation resulted in quadratic decreases in ruminal ammonia N (P < .05) in wk 15 and in plasma urea N in wk 10 and 15 (P < .05) but in linear increases (P < .05) in ruminal protein S concentrations throughout the experiment. Added S had little effect on blood acid-base status. Apparent digestibilities of DM, OM, ash, ADF, and GE were increased linearly (P < .10) by added S. The milk N:S ratio remained constant. Increasing S from .16 to .26% of diet DM was beneficial to lactating Alpine goats during early lactation.