[Clinical application of complete laparoscopic gastrectomy with function preservation of cardia in gastric carcinoma].

To investigate the feasibility and safety of total laparoscopic cardia function preserving gastrectomy for gastric carcinoma. Clinical data of 10 patients undergoing total laparoscopic cardia function preserving gastrectomy for gastric carcinoma from November 2020 to December 2021 were retrospectively collected. There were 7 males and 3 females. The mean age was (66.1±12.9) years (ranged from 38 to 86 years). All of the 10 patients were successfully performed total laparoscopic cardia function preserving gastrectomy without conversion to laparotomy. The time of digestive tract reconstruction was (24.8±3.3) min (20-30 min), and the intraoperative blood loss was (35±24) ml(20-100 ml). The time of postoperative exhaust was (2.5±0.9) days(2-3 d), the time of postoperative liquid diet was (2.25±0.87) days(2-3 d), postoperative hospital stay was (9.5±2.1) days(6-13 d). No surgical complications such as bleeding, anastomotic fistula or anastomotic stenosis occurred. Postoperative pathology showed that the proximal and distal margins of resected specimens were negative. Patients were followed up for 2 to 15 months, respectively. No death or tumor recurrence and metastasis occurred during the follow-up period. There were no symptoms of reflux after operation. Compared with total gastrectomy and proximal gastrectomy, total laparoscopic cardia function preserving gastrectomy can theoretically reduce the incidence of reflux esophagitis. We used manual suture method for digestive tract reconstruction, which can reduce the application of 2-3 stapling studs and reduce the cost of surgical materials. Compared with subtotal gastrectomy, total laparoscopic cardia function preserving gastrectomy has the advantages of more thorough lymph node dissection, with little residual gastric tissue; therefore, the blood supply is relatively better. The incidence of reflux esophagitis of total laparoscopic cardia function preserving gastrectomy for gastric cancer may was lower than total gastrectomy.

[Clinical effect of minimally invasive duodenum preserving pancreatic head resection for benign and pre-malignant lesions of pancreatic head].

Objective: To examine the clinical effect of minimally invasive duodenum preserving pancreatic head resection(DPPHR) for benign and pre-malignant lesions of pancreatic head. Methods: The clinical data of patients with diagnosis of benign or pre-malignant pancreatic head tumor were retrospectively collected and analyzed,all of them underwent laparoscopic or robotic DPPHR between October 2015 and September 2021 at Division of Gastrointestinal and Pancreatic surgery,Zhejiang Provincial People's Hospital. Thirty-three patients were enrolled with 10 males and 23 females. The age(M(IQR)) was 54(32) years old(range: 11 to 77 years old) and the body mass index was 21.9(2.9)kg/m2(range: 18.1 to 30.1 kg/m2). The presenting symptoms included abdominal pain(n=12), Whipple triad(n=2), and asymptomatic(n=19). There were 7 patients with hypertension and 1 patient with diabetes mellitus. There were 19 patients who were diagnosed as American Society of Anesthesiologists class Ⅰ and 14 patients who were diagnosed as class Ⅱ. The student t test,U test, χ2 test or Fisher exact test was used to compare continuous data or categorized data,respectively. All the perioperative data and metabolic morbidity were analyzed and experiences on minimally invasive DPPHR were concluded. Results: Fourteen patients underwent laparoscopic DPPHR,while the rest of 19 patients received robotic DPPHR. Indocyanine green fluorescence imaging was used in 19 patients to guide operation. Five patients were performed pancreatico-gastrostomy and the rest 28 patients underwent pancreaticojejunostomy. Pathological outcomes confirmed 9 solid pseudo-papillary neoplasms, 9 intraductal papillary mucinous neoplasms, 7 serous cystic neoplasms, 6 pancreatic neuroendocrine tumors, 1 mucous cystic neoplasm, 1 chronic pancreatitis. The operative time was (309.4±50.3) minutes(range:180 to 420 minutes),and the blood loss was (97.9±48.3)ml(range:20 to 200 ml). Eighteen patients suffered from postoperative complications,including 3 patients experienced severe complications(Clavien-Dindo Grade ≥Ⅲ). Pancreatic fistula occurred in 16 patients,including 8 patients with biochemical leak,7 patients with grade B pancreatic fistula and 1 patient with grade C pancreatic fistula. No one suffered from the duodenal necrosis and none perioperative death was occurred. The length of hospital stay was 14(7) days (range:6 to 87 days). The follow-up was 22.6(24.5)months(range:2 to 74 months). None suffered from recurrence or metastasis. During the follow-up,all the patients were free of refractory cholangitis. Moreover,in the term of endocrine dysfunction,no postoperative new onset of diabetes mellitus were observed in the long-term follow-up. However,in the view of exocrine insufficiency,pancreatic exocrine insufficiency and non-alcoholic fatty liver disease (NAFLD) was complicated in 2 and 1 patient,respectively,with the supplement of pancreatic enzyme,steatorrhea and weight loss relieved,but NAFLD was awaited to be seen. Conclusions: Minimally invasive DPPHR is feasible and safe for benign or pre-malignant lesions of pancreatic head. Moreover,it is oncological equivalent to pancreaticoduodenectomy with preservation of metabolic function without refractory cholangitis.

[Clinical analysis of minimal invasive surgical treatment for pancreatic neuroendocrine tumors].

Objective: To analyze the safety and effectiveness of minimal invasive surgery treating pancreatic neuroendocrine tumors (pNETs), and to summarize surgical characteristics and share experience. Methods: The clinical data of 80 pNETs treated by a single hospital from January 2015 to December 2019 were retrospectively analyzed. The patients were divided into laparoscopic group and robot group. And surgical procedures included pancreaticoduodenectomy (PD), distal pancreatectomy (DP), central pancreatectomy (CP), and tumor enucleation. Results: Of 80 patients, 76 cases (95%) underwent minimal invasive surgery and 4 cases (5%) changed to open surgery. There were 38 females, with median age of 54.4 (20-80) years and median BMI (17.0-38.0) kg/m(2). Among them, 24 patients (31.6%) underwent PD, 36 patients (47.4%) underwent DP, 8 patients (10.5%) underwent CP and 8 patients (10.5%) received tumor enucleation. The postoperative incidence of grade B/C pancreatic fistula was 35.5%, the incidence of abdominal infection was 10.5%, the postoperative bleeding was 7.9%, and the reoperation rate was 6.6%, without perioperative deaths. There was no significant difference in postoperative complications among different surgical methods, including postoperative pancreatic fistula (P=0.396), postoperative bleeding (P=0.297), postoperative abdominal infection (P=0.339) and reoperation (P=0.396). Conclusions: Surgical resection is an effective treatment for pNETs. pNETs are suitable for minimally invasive surgery with earlier stage and smaller tumor diameter. Minimally invasive surgery for pNETs is safe and feasible, and functional preserving surgery could take into consideration.

[Early experience of minimal invasive surgery for adolescent with pancreatic head tumor: a report of 15 cases].

Objective: To summarize the characteristics and difficulties of minimal invasive surgery for adolescent with pancreatic head tumor. Methods: The data of adolescent younger than 28 years old with pancreatic head tumor treated at Department of Gastroenterology and Pancreatic Surgery, Zhejiang Province People's Hospital from January 2014 to December 2019 were analyzed retrospectively. Fifteen cases were enrolled, included 5 males and 10 females. The median age was 20 years(range: 11 to 27 years) and the body mass index was (19.8±2.3)kg/m(2)(range: 17.3 to 21.6 kg/m(2)). Results: Standard pancreaticoduodenectomy was performed in 12 cases, including 9 cases of laparoscopic surgery and 3 cases of robotic-assisted surgery, and laparoscopic pancreaticoduodenectomy with resection of hepatic flexure of colon was performed on 1 case, and robotic-assisted duodenum-preserving pancreatic head resection with pancreatogastrostomy was performed on other 2 cases. The operative time was (269.0±65.1)minutes(range: 150 to 410 minutes), and the blood loss was (135.6±52.7)ml(range: 50 to 400 ml). Six patients got postoperative complications with gastrointestinal bleeding biochemical leakage and intestinal obstruction(n=1), pancreatic biochemical leakage(n=1), bile leakage(n=1), chylous leakage(n=1), wound infection(n=1), hepatic injury(n=1). The median postoperative hospital stay was 13 days(range: 9 to 22 days).The pathologic findings were solid pseudopapillary neoplasms(n=8), neuroendocrine neoplasms(n=3), introductal papillary mucinous neoplasm(n=1), cystic fibroma(n=1), serous cystadenoma(n=1), Ewing sarcoma(n=1).The median follow-up was 37 months(range: 2 to 75 months).The patient with Ewing sarcoma was diagnosed as liver metastasis at 41 months after surgery and died at 63 months after surgery.All the other patients survived without tumor.Three patients got the long-term complication of bile duct. Conclusions: Most of pancreatic head tumors for adolescent are benign or low malignant. Minimally invasive surgery be the first choice, and function-preserving surgery should be taken into account as much as possible.Perioperative management and communication is essential as the parents pay much attention to the quality of life after surgery.

[Laparoscopic gastroduodenostomy with manual suture for Billroth â anastomosis: a report of 36 cases].

Objective: To examine the clinical efficiency of laparoscopic gastroduodenostomy with BillrothⅠanastomosis with manual suture. Methods: The clinic data of 36 patients with gastric cancer who underwent laparoscopic gastroduodenostomy with Billroth Ⅰ anastomosis from November 2017 to September 2019 in Department of Gastrointestinal and Pancreatic Surgery, Zhejiang Provincial People's Hospital were analyzed retrospectively.There were 22 males and 14 females, aged (64.3±9.3) years(range: 43 to 80 years), underwent complete laparoscopic gastroduodenostomy. The laparoscopic manual suture was used for Billroth Ⅰ anastomosis. Results: All the laparoscopic radical gastrectomy and manual suturing gastroduodenostomy were successfully performed. The operation time was (226.7±40.4) minutes (range: 180 to 320 minutes), including (24.8±7.1) minutes (range: 15 to 48 minutes) for gastroduodenostomy.There was (3.8±0.9) days (range: 2 to 6 days) for anal exhaust, (5.7±2.0) days (range: 3 to 13 days) for extubation of gastric tube, and (10.3±3.1) days (range: 7 to 19 days) for hospitalization. There was no death in perioperative period. Postoperative pathological report showed 3 cases of highly differentiated adenocarcinoma, 5 cases of moderately differentiated adenocarcinoma, 22 cases of poorly differentiated adenocarcinoma and 6 cases of signet ring cell carcinoma, including 27 cases in T1 stage and 9 cases in T2 stage. The number of lymph nodes harvested was 36.4±8.9 (range: 23 to 60). Lymph node metastasis was positive in 7 cases and negative in 29 cases. TNM stage included 24 cases in ⅠA stage, 8 cases in ⅠB stage and 4 cases in Ⅱ stage. After the operation, the upper digestive tract radiography showed that the anastomosis opening was unobstructed without complications such as anastomotic stenosis. Conclusion: Laparoscopic gastroduodenostomy with Billroth Ⅰ anastomosis with manual suture is safe and feasible, has a good short-term effect.

[The current status and future of laparoscopic pancreaticoduodenectomy].

This review focused on the progress in laparoscopic pancreaticoduodenectomy(LPD) in the past six years.With the appropriate approaches under laparoscopy, including the resection and reconstruction, LPD has been proved to be safe and feasible. In some centers, LPD has been routine with rapid growth of numbers, it not only benefit the patients with fast recovery, but also benefit the trainees with similar sights as the primary surgeon and good videos of the procedures. However, LPD is still controversial as the more complications in some centers and inconclusive oncologic outcomes. Thus, in the further, a long-time outcome monitoring of LPD is essential. A registry of a prospectively maintained database may be a need for LPD to evaluate its outcomes by multicenter randomized control trials, and real world research may be of value. Structured LPD training programs are valuable for the new surgeons.

[Preliminary application of endoscopic titanium clip localization combined with three-dimensional CT reconstruction in the determination of resection margin of gastric central cancer under laparoscopy].

Objective: To evaluate the accuracy of endoscopic titanium clip localization combined with CT three-dimensional reconstruction for the control of incision margin in early gastric cancer under laparoscopy. Methods: A prospective analysis was made for gastric cancer whose lesions were located in the middle of the stomach and T stage was 1 to 2 from October 2017 to January 2019 at Department of Gastrointestinal and Pancreatic Surgery, Zhejiang Provincial People's Hospital. Totally 25 patients were eventually enrolled in the study. There were 17 males and 8 females aging of (63.6± 7.2) years (range: 48 to 77 years). All cases were treated with titanium clip localization under endoscope combined with CT three-dimensional (3D) reconstruction to construct a virtual panorama of gastric cavity and lesions, and to design surgical margins. Laparoscopic surgical resection was performed according to the surgical margins designed before operation. The distance from the gastric angle to the origin of the minor curvature of the incisional margin, the distance from the gastric angle to the the center of lesion and the distance of the upper incision margin were measured under three-dimensional CT reconstruction and under actual specimen. Paired t test was used to compare the three distances measured by two methods. Results: The measured distances from the gastric angle to the center of the lesion and the proximal incisional margin under 3D reconstruction CT were according to the measured values of actual specimens ((2.67±1.38) cm vs. (2.83±1.56) cm, t=1.51, P=0.14; (5.23±0.60) cm vs. 5 cm, t=1.93, P=0.07); the measured distances from the gastric angle to the origin of the minor curvature of the incisional margin under CT 3D reconstruction were different with the measured values of solid specimens ((5.94±0.94) cm vs. (6.37±0.90) cm, t=3.52, P=0.00). Conclusion: The method of titanium clip localization combined with CT 3D reconstruction can provide a feasible laparoscopic localization method and incision edge solution for T1 to 2 gastric central cancer.

[Surgical treatment of port-site metastases after laparoscopic radical resection of gastric cancer].

Objective: To investigate the feasibility of surgical treatment of port-site metastasis after laparoscopic radical resection of gastric cancer. Methods: The clinical and follow-up data of five patients with port-site metastases after laparoscopic radical resection of gastric cancer at Zhejiang Provincial People's Hospital between January 2014 and January 2018 were retrospectively analyzed. Results: Port-site metastases occurred within 6 months after gastrointestinal tumor resection in three patients, 10 months after the operation in one patient, and 30 months after the operation in one patient, respectively. Metastasis to the abdominal cavity or distant metastasis was excluded before the surgical treatment of the port-site metastases, and all patients recovered well after the operation. No incisional infection or hernia occurred. By December 2018, two patients died (they survived for 13 and 24 months, respectively) and three patients survived. The follow-up duration ranged from 7 to 19 months. Conclusions: Surgical resection of port-site metastases is not difficult due to their superficial location. Surgical treatment can improve the prognosis of patients without abdominal or distant metastasis/recurrence.

[Experience on postoperative complications of laparoscopic pancreaticoduodenectomy].

Objective: To summarize the incidence and characteristics of postoperative complications after laparoscopic pancreaticoduodenectomy(LPD), and to share our experience on management of complications. Methods: The clinical data of 320 LPD performed by a single team in Sir Run Run Shaw Hospital and Zhejiang Provincial People's Hospital between September 2012 and September 2017 were retrospectively analyzed, among which there were 196 males and 124 females with age of (60.2±11.6) years old.There were 306 patients who underwent standard LPD, and 14 patients who underwent extended LPD. The patients were divided into 2 groups of former 160 LPD and later 160 LPD according to the time order. By analyzing the differences of clinical outcomes between the two groups, especially focusing on the incidence of postoperative complications.The experience on management of complications was concluded. The prior surgical history of latter group was significantly higher than the former group(30.0%(48/160)vs. 18.8%(30/160), χ(2)=5.49, P=0.019), and the rest of baseline characteristics remained the comparable (P>0.05). For resectable lesions, LPD was performed by "No back" approach, following the principle of "From distal to cephalad, from ventral to dorsal, and from left to right" . As for the borderline resectable patients, LPD was performed by "Easy first" strategy. Student t test, χ(2) test or Fisher test was used to analyzed the data between the two groups respectively. Results: Of 320 LPD patients, 306 cases underwent standard LPD, 14 cases underwent LPD with resection of other organs.There were 278 LPD cases who followed "No back" approach, and 42 cases who followed "Easy first" strategy because of difficulty in creating the retro-pancreatic tunnel. And the overall morbidity was 32.2%(103/320) with reoperation rate of 5.3%(17/320). The perioperative mortality was 0.6%(2/320). The operation time of latter group was ((346.6±48.8)minutes), which was shorter than that of former group((358.0±54.4)minutes)(t=1.97, P=0.048). The blood loss of former and latter group remained comparable((207.9±135.8)ml vs.(189.6±121.4)ml, P=0.205). However, in subgroup analysis, the patients with blood loss less <200 ml of latter group decreased significantly from 59.4%(95/160) to 47.5%(76/160)(χ(2)=4.53, P=0.033). The overall morbidity of latter group was 28.8%(46/160), indicated a decrease from 35.6%(57/160) of former group without significant difference(P=0.188). Moreover, Grade A/B/C pancreatic fistula rate, Grade A/C bile leakage rate, Grade B/C postoperative hemorrhage rate of the later group tended to decrease, although they also didn't reach a significant difference. However, the abdominal infection rate decreased significantly(χ(2)=3.93, P=0.047). The length of hospital stay remained comparable(P=0.156). Conclusions: The most common complications after LPD were postoperative hemorrhage and pancreatic fistula. With specialized team and accumulated experience, the morbidity can decrease progressively by analyzing the leading cause and improving the technical skills.

[Strategy in management of adjacent organ lesion during laparoscopic pancreaticoduodenectomy].

Objective: To evaluate the safety and feasible of adjacent organ resection during laparoscopic pancreaticoduodenectomy(LPD), and summary the surgical strategies. Methods: Clinical data of 15 adjacent organ resections combined with LPD from March 2013 to September 2017 were reviewed.There were 10 male and 5 female patients aging from 20 to 86 years, and the body mass index ranged from 19.6 to 34.5 kg/m(2).Two patients had previous abdominal surgical history.Two patients underwent preoperative chemotherapy. Results: The resected adjacent organs included liver(n=4), stomach(n=3), colon(n=6), right kidney with embolectomy and vasoplastic of inferior vena cava(n=1), and spleen artery aneurysms(n=1). The operative time ranged from 280 to 450 minutes, and the blood loss ranged from 100 to 450 ml.The total complication rate was 5/15 and no one died in 90 days after surgery.The postoperative hospital stay ranged from 10 to 42 days with medium 18 days.The pathology included adenocarcinoma of stomach and duodenum(n=1), gastric cancer invading pancreas or duodenum(n=2), ampullary adenocarcinoma with left hepatolithiasis(n=1), ampullary adenocarcinoma with a benign lesion in left liver(n=1), ampullary adenocarcinoma with single liver metastasis(n=1), ampullary adenocarcinoma(n=1), pancreatic intraductal papillary mucinous neoplasm with splenic artery aneurysms(n=1), pancreatic neuroendocrine neoplasm with colon cancer(n=1), distal common bile duct adenocarcinoma involving righ hepatic duct(n=1), pancreatic neuroendocrine neoplasm invading inferior vena cava and right renal vein(n=1), duodenal adnocarcinoma(n=1), duodenal ewing's sarcoma(n=1), duodenal intesititialoma(n=2). The follow-up was from 3 to 40 months with the medium survival of 17.5 months. Conclusions: The oncological outcomes of PD combined with adjacent organ resection is acceptable.Surgical treatment for those patients with periampullary neoplasma and adjacent organ lesions should be aggressive.

[Laparoscopic radical antegrade modular pancreatosplenectomy for left-sided pancreatic adenocarcinoma: analysis of 12 cases].

Objective: To evaluate the safety and feasibility of laparoscopic radical antegrade modular pancreatosplenectomy(Lap-RAMPS) for left-sided pancreatic adenocarcinoma. Methods: Clinical data of total 12 patients underwent Lap-RAMPS for left-sided pancreatic adenocarcinoma at Department of Gastrointestinal and Pancreatic Surgery, Zhejiang Provincial People's Hospital from March 2016 to August 2017 were reviewed retrospectively.There were 7 male patients and 5 female patients, with median age of 60.5 years old(47-68 years old). Abdominal enhanced CT, pancreatic MRI, PET-CT were performed on all patients to evaluate the lesion and exclude metastasis.Follow-up were done with out-patient clinic or telephone consultancy until October 2017. Results: All patients underwent pure Lap-RAMPS.The medium operative time was 250 minutes(180-445 minutes), and the blood loss was 150 ml(50-500 ml). The medium first flatus time and diet resumption time were 3.0 days(1-5 days) and 3.5 days(1-7 days) respectively.The medium postoperative hospital stay was 9 days(4-18 days). Morbidity occurred in 8 patients with gastric empty delay(n=1), bleeding(n=1), fluid collection(n=3). There was no mortality.The medium overall number of retrived lymph nodes was 15.6 and the positive rate was 41.7%. The R0 rate was 100%.The medium follow-up was 10 months.One patient was diagnosed as liver metastasis after 8 months and accepted chemotherapy.One patient died after 14 months for tumor recurrence and metastasis.Others survived without tumor recurrence or metasitasis. Conclusion: Lap-RAMPS is safe and feasible with accepted oncological outcomes for selected left side pancreatic adenocarcinoma under skilled hands.

[Laparoscopic pancreaticoduodenectomy: a report of 233 cases by a single team].

Objective: To summary the experience of 233 cases of laparoscopic pancreaticoduodenectomy (LPD) performed by a single surgical team. Methods: Data of patients undergoing LPD from September 2012 to October 2016 were reviewed. There were 145 males and 88 females with the mean age of(60.3±13.0)years old, ranging from 19 to 92 years old, and the mean body mass index of (22.8±3.5)kg/m(2,) ranging from 16.3 to 36.8 kg/m(2). There were 195 patients with clinical manifestation and 54 patients who had the history of abdominal surgery. Results: LPD were performed on 233 patients by same surgical team consecutively. The mean operative time was(368.0±57.4)minutes. Mean blood loss was(203.8±138.6)ml. The postoperative morbidity rate was 33.5%, with 6.9% of grade B or C pancreatic fistula and 9.9% of bleeding. The reoperation rate was 5.6%. The mortality during 30 days after operation was 0.9%. Mean postoperative hospital stay was (18.1±11.2)days. Mean tumor size was (3.9±2.4)cm, and the mean number of lymph nodes harvested was 21.3±11.9.One hundred and sixty-three patients were diagnosed as malignant tumor, including pancreatic adenocarcinoma(n=84), cholangiocarcinoma(n=17), ampullary adenocarcinoma(n=55), duodenal adenocarcinoma(n=5), gastric cancer(n=1)and duel cancer (n=1) located in distal stomach and duodenum. Conclusion: The key point to make laparoscopic pancreaticduodenectomy a routine safe procedure is to operate the procedure under skilled hands in selected patients via suitable surgical approaches.

c-Met and RON expression levels in endometrial adenocarcinoma tissue and their relationship with prognosis.

OBJECTIVE: To investigate the potential relevance of c-Met and RON gene expression in patients with adenocarcinoma of the endometrium and analyze the relationships among the c-Met and RON expression, clinicopathological characteristics, and patient survival. MATERIALS AND METHODS: The study included 60 cases diagnosed with endometrial adenocarcinoma with more than five-years follow-up. Total RNA from formalin-fixed paraffin-embedded tissues of 60 adenocarcinomas of the endometrium and normal endometrium tissues were isolated for c-Met and RON quantitative analysis by real-time real-time polymerase chain reaction (RT-PCR). RESULTS: The c-Met and RON expression in endometrial adenocarcinoma was significantly higher than that in normal endometrial tissues (p < 0.01), with average up-regulated levels of 3.94 ± 1.88 and 2.74 ± 0.88, respectively. Moreover, high c-Met expression was significantly correlated with the histological stage (p = 0.017), and high RON expression was related to histological stage (p = 0.035), muscle invasion (p = 0.006), and lymph node metastasis (p = 0.018). Multivariate Cox regression analysis revealed that the co-expression of c-Met and RON was an independent prognostic factor for adenocarcinoma of the endometrium and was significantly associated with decreased overall survival (HR = 3.571, p = 0.014). CONCLUSION: The co-expression of c-Met and RON is associated with a poor prognosis in endometrial adenocarcinoma and is an independent prognostic marker for endometrioid adenocarcinoma.

Distribution of the tandem repeat sequences and karyotyping in cucumber (Cucumis sativus L.) by fluorescence in situ hybridization.

We analyzed repeat sequences composition in the genome of cucumber inbred line 9930 using whole-genome shotgun reads. The analysis showed that satellite DNA sequences are the most dominant components in the cucumber genome. The distribution pattern of several tandem repeat sequences (Type I/II, Type III and Type IV) on cucumber chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of the Type III and 45S rDNA provide useful cytogenetic markers, whose position and fluorescence intensity allow for easy identification of all somatic metaphase chromosomes. A karyotype showing the position and fluorescence intensity of several tandem repeat sequences is constructed. The establishment of this FISH-based karyotype has created the basis for the integration of molecular, genetic and cytogenetic maps in Cucumis sativus and for the ultimate genome sequencing project as well.

[Detection and analysis of alien genes in transgenic rice by fluorescence in situ hybridization].

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a transferred barnase-psl DNA sequence onto chromosomes in 8 rice transgenic plants. All the tested rice transgenic lines showed hybridization signals on the middle and terminal regions of chromosome arms except for those close to centromeres. In two lines, two different integration sites were identified, and the other lines showed only one integration site. With the aid of Southern analysis and expression detection, we found that the barnase tended to show a higher level expression in the lines whose integration sites near the distal regions of chromosomes, while the expression level became lower in the lines whose integration sites near the centromeres. This result suggested a possible relationship between chromosomal location of transgenes and the expression level. However it showed no obvious relationship between copy numbers and expression levels. In most cases, the results of multi-color FISH showed that barnase-ps1 always integrated at the same position on the chrmosome as the reporter genes(pHctinG).

Ca(2+)-dependent activation of c-jun NH(2)-terminal kinase in primary rabbit proximal tubule epithelial cells.

Previous work from this laboratory demonstrated that arachidonic acid activates c-jun NH(2)-terminal kinase (JNK) through oxidative intermediates in a Ca(2+)-independent manner (Cui X and Douglas JG. Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells. Proc Natl Acad Sci USA 94: 3771-3776, 1997.). We now report that JNK can also be activated via a Ca(2+)-dependent mechanism by agents that increase the cytosolic Ca(2+) concentration (Ca(2+) ionophore A(23187), Ca(2+)-ATPase inhibitor thapsigargin) or deplete intracellular Ca(2+) stores [intracellular Ca(2+) chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM]. The activation of JNK by BAPTA-AM occurs despite a decrease in cytosolic Ca(2+) concentration as detected by the indicator dye fura 2, but appears to be related to Ca(2+) metabolism, because modification of BAPTA with two methyl groups increases not only the chelation affinity for Ca(2+), but also the potency for JNK activation. BAPTA-AM stimulates Ca(2+) influx across the plasma membrane, and the resulting local Ca(2+) increases are probably involved in activation of JNK because Ca(2+) influx inhibitors (SKF-96365, nifedipine) and lowering of the free extracellular Ca(2+) concentration with EGTA reduce the BAPTA-induced JNK activation.

Plasmid DNA encoding transforming growth factor-beta1 suppresses chronic disease in a streptococcal cell wall-induced arthritis model.

Transforming growth factor beta is a potent immunomodulator with both pro- and antiinflammatory activities. Based on its immunosuppressive actions, exogenous TGF-beta has been shown to inhibit autoimmune and chronic inflammatory diseases. To further explore the potential therapeutic role of TGF-beta, we administered a plasmid DNA encoding human TGF-beta1 intramuscularly to rats with streptococcal cell wall-induced arthritis. A single dose of 300 microg plasmid DNA encoding TGF-beta1, but not vector DNA, administered at the peak of the acute phase profoundly suppressed the subsequent evolution of chronic erosive disease typified by disabling joint swelling and deformity (articular index = 8.17+/-0. 17 vs. 1.25+/-0.76, n = 6, day 26, P < 0.01). Moreover, delivery of the TGF-beta1 DNA even as the chronic phase commenced virtually eliminated subsequent inflammation and arthritis. Both radiologic and histopathologic as well as molecular evidence supported the marked inhibitory effect of TGF-beta1 DNA on synovial pathology, with decreases in the inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and the expression of proinflammatory cytokines that characterize this model. Increases in TGF-beta1 protein were detected in the circulation of TGF-beta1 DNA-treated animals, consistent with the observed therapeutic effects being TGF-beta1 dependent. These observations provide the first evidence that gene transfer of plasmid DNA encoding TGF-beta1 provides a mechanism to deliver this potent cytokine that effectively suppresses ongoing inflammatory pathology in arthritis.

Cytokine regulation of schistosome-induced granuloma and fibrosis.

Schistosomiasis mansoni, a major cause of hepatic fibrosis in many developing countries, triggers a granulomatous inflammatory reaction in response to its eggs that lodge in the liver. The egg antigens are eliminated slowly, and the persistent granulomatous response leads to prolonged matrix synthesis and hepatic fibrosis. In mice, soluble egg antigens (SEA) induce interleukin 4 synthesis, promoting a dominant T helper type 2 lymphocyte accumulation with the release of additional cytokines (IL-5, IL-10), which not only suppress Th1 lymphocyte subset cytokines, but mediate the characteristic pathophysiology. Manipulation of the cytokine profile with antagonists or exogenous cytokine delivery alters the course of the hepatic inflammation and fibrosis. In the evolution of the granulomatous response to the S. mansoni eggs, transforming growth factor beta (TGF-beta) is also produced that may modulate inflammation and regulate fibrogenesis. In TGF-beta 1-gene targeted mutant mice that over-express TGF-beta 1 (TGF-beta 1 transgenics) or in which TGF-beta 1 has been inactivated (TGF-beta 1-/-; null mutation) or partially inactivated (TGF-beta 1+/-; null mutation heterozygotes), the altered production of TGF-beta 1 impacts on S. mansoni granuloma and hepatic fibrosis. In addition to the Th1/Th2 cytokine balance, modulation of TGF-beta 1 may change the outcome of chronic inflammatory fibrotic disease.

Beta 2 integrin-dependent tyrosine phosphorylation of paxillin in human neutrophils treated with tumor necrosis factor.

The focal adhesion protein paxillin undergoes tyrosine phosphorylation in response to signals mediated by integrins, neuropeptides and oncogene products, possibly via activation of the focal adhesion-associated kinase, p125FAK. In the present work, tumor necrosis factor-alpha (TNF) stimulated tyrosine phosphorylation of paxillin in human neutrophils. Cell adhesion and participation of the beta 2 integrin CD18 were necessary, but not sufficient, for the response. Adherent neutrophils also tyrosine phosphorylated paxillin in response to phorbol ester, formylmethionyl-leucyl-phenylalanine and opsonized bacteria. In contrast, p125FAK was constitutively tyrosine phosphorylated in a manner unaffected by adherence and/or TNF. Thus, cytokines and microbial products are among the stimuli that can induce the tyrosine phosphorylation of paxillin, and kinases other than p125FAK may be responsible. This is the first identification of paxillin and p125FAK in human cells and neutrophils, and one of the few identifications of a specific protein that undergoes tyrosine phosphorylation in response to any agonist in neutrophils or in response to TNF in any cell.