[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.

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.

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.

Silk gland specific secretory expression of egfp gene in silkworm Bombyx mori with rAcMNPV system.

To evaluate the possibility of establishing an in vivo baculovirus expression system in a silk gland specific secretory way, the recombinant Autographa californica nucleopolyhedrovirus (AcserpegfpDeltaEGT) carrying the reporter gene egfp downstream of silkworm ser1 promoter and signal peptide coding sequence was generated. The purified recombinant baculovirus AcserpegfpDeltaEGT was injected into the haemocoel of newly ecdysed 5thHendekl) instar silkworm larvae at the amount of 10(6) pfu per larva. At 5 days post injection, green fluorescence derived from EGFP could be observed with fluorescent microscope in only the silk gland but not other tissues after dissection of the silkworm. By making an opening on the silk gland wall, green fluorescence could be observed in the outflow of silk gland indicating the secretion of EGFP and the effectiveness of ser1 signal peptide. Western blotting assay confirmed that EGFP exists in the water-soluble part of cocoon silk too. We also established a simple protocol to purify EGFP from the secreted silk proteins.

Transient in vivo gene delivery to the silkworm Bombyx mori by EGT-null recombinant AcNPV using EGFP as a reporter.

Several strains of silkworm Bombyx mori were tested for the gene delivery feasibility of Autographa californica nucleopolyhedrovirus (AcNPV) in vivo. In contrast to the general belief that silkworms were non-permissive to AcNPV, we found that 3 of 7 tested strains were AcNPV permissive. To dispel the physiological influence of the ecdysteroid UDP-glucosyltransferase (EGT) on the silkworm, we modified the AcNPV bacmid by disruption of that gene. Expression pattern of EGFP in tissues of silkworm larvae after injection of EGT-null AcNPV vector carrying EGFP cassette was revealed by green fluorescence and Western blot analysis. Viral DNA was detected and semi-quantified in various kinds of tissues by dot blot assay. Active recombinant virus from larval hemolymph was detectable by TCID(50). Our results indicate that some strains of silkworm were permissive to AcNPV, which could serve as a novel gene deliver tool to silkworm in vivo.

Molecular characterization and regulation of the aguBA operon, responsible for agmatine utilization in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB). The aguBA and adjacent aguR genes were cloned and characterized. The predicted AguB protein (M(r) 32,759; 292 amino acids) displayed sequence similarity (< or =60% identity) to enzymes of the beta-alanine synthase/nitrilase family. While the deduced AguA protein (M(r) 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (M(r) 24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon. The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function.

Nutritional evaluation of solar dried sardines as a ruminant protein supplement.

Solar dried sardines of various qualities were analyzed for nutrient content and for nutrient digestibility and nitrogen balance in sheep. Additionally, key serum enzymes and metabolites were examined to identify potential toxic effects. Dried sardine protein, ether extract and crude ash content ranged from 65 to 39%, 5.7 to 5.1%, and 22 to 51%, respectively, for high to low quality dried sardines. Visual appraisal of the dried sardines did not appear to be very reliable for determining dried sardine quality. Crude protein content was highly correlated (r(2)=0.962) with crude ash content based on the following equation (dry matter basis): crude protein%=86.0 - (0.961xcrude ash%). Therefore, crude ash could be used to estimate crude protein content and dried sardine quality. Digestibility in Omani sheep was determined on diets composed of a composite of dried sardines collected from Al-Batinah region of the Sultanate (the average crude protein content was 51%) and compared to that of a similar diet using soybean meal (also 51% crude protein) as the major protein source. The digestibility of crude protein, ether extract, total carbohydrates, digestible energy and metabolizable energy in diets utilizing dried sardines versus the soybean meal diet were 74 versus 76%, 69 versus 68%, 67 versus 69%, 64 versus 66%, and 52 versus 56%, respectively. Although, protein digestibility was similar, nitrogen balance data suggested that nitrogen in the dried sardine diet was not as efficiently utilized as nitrogen in the soybean meal diet. Key serum enzymes and metabolites did not suggest that dried sardines at 10% of the total dietary intake would cause metabolic disturbances in sheep. The study suggests that solar dried sardines may be effectively used in ruminant diets but may not have the protein by-pass value of commercial, processed fish meals.

The gdhB gene of Pseudomonas aeruginosa encodes an arginine-inducible NAD(+)-dependent glutamate dehydrogenase which is subject to allosteric regulation.

The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.

Amino acid-mediated induction of the basic amino acid-specific outer membrane porin OprD from Pseudomonas aeruginosa.

Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction of oprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the oprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression of oprD is linked to both carbon and nitrogen metabolism of Pseudomonas aeruginosa.

The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.

Loss of p27Kip1 expression independently predicts poor prognosis for patients with resectable pancreatic adenocarcinoma.

BACKGROUND: As a universal inhibitor of cyclin-dependent kinases in mammalian cells, p27Kip1 expression has been implicated in tumor progression and has proved to be a prognostic predictor for several human cancers. In this study, the authors investigated the expression of p27Kip1 and its potential prognostic significance in patients with resectable pancreatic adenocarcinoma. METHODS: The correlation between p27Kip1 expression and prognosis was investigated retrospectively in 38 patients who had undergone resection of pancreatic adenocarcinoma including 2 cystadenocarcinomas and 4 mucin-producing tumors. Immunohistochemical staining using an anti-p27Kip1 antibody, along with an assessment of tumor cell Ki-67 labeling index, was performed on resected specimens. RESULTS: p27Kip1 expression in pancreatic adenocarcinoma was decreased dramatically when compared with that in normal pancreatic tissue. Based on 35 cases available for survival analysis, loss of p27Kip1 expression in 16 cases defined as having no or <1% p27Kip1 positive tumor cells showed 1-, 1.5-, and 2-year survival rates of 37.5%, 15.6%, and 0%, respectively, in comparison with 1-, 2-, and 3-year survival rates of 68.4%, 62.2%, and 49.8%, respectively, in the other cases (P = 0.001). After excluding cases of cystadenocarcinoma and mucin-producing tumors, the survival advantage for patients with p27Kip1 positive tumors remained apparent (P = 0.024). In each Cox regression model, both those including cystadenocarcinoma and mucin-producing tumors and those that did not, p27Kip1 expression proved to be an independent predictor for overall survival by multivariate analysis. CONCLUSIONS: These results reveal the significance of p27Kip1 immunostaining in predicting the outcome of patients with resectable pancreatic adenocarcinoma, adding a novel predictor in the evaluation of prognosis for this lethal disease.

Role of ArgR in activation of the ast operon, encoding enzymes of the arginine succinyltransferase pathway in Salmonella typhimurium.

The ast operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from Salmonella typhimurium, and the nucleotide sequence for the upstream flanking region was determined. The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR. The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites. Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an argR derivative. AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR. The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of the ast operon in response to nitrogen or carbon limitation.