Massage of the Hepatoduodenal Ligament Recovers Portal Vein Flow Immediately After the Pringle Maneuver in Hepatectomy.

BACKGROUND: The Pringle maneuver is often used in liver surgery to minimize bleeding during liver transection. Many authors have demonstrated that intermittent use of the Pringle maneuver is safe and effective when performed appropriately. However, some studies have reported that the Pringle maneuver is a significant risk factor for portal vein thrombosis. In this study, we evaluated the effectiveness of portal vein flow after the Pringle maneuver and the impact that massaging the hepatoduodenal ligament after the Pringle maneuver has on portal vein flow. MATERIALS AND METHODS: Patients treated with the Pringle maneuver for hepatectomies performed to treat hepatic disease at our hospital between August 2014 and March 2019 were included in the study (N = 101). We divided these patients into two groups, a massage group and nonmassage group. We measured portal vein blood flow with ultrasonography before and after clamping of the hepatoduodenal ligament. We also evaluated laboratory data after the hepatectomy. RESULTS: Portal vein flow was significantly lower after the Pringle maneuver than before clamping of the hepatoduodenal ligament. The portal vein flow after the Pringle maneuver was improved following massage of the hepatoduodenal ligament. After hepatectomy, serum prothrombin time was significantly higher and serum C-reactive protein was significantly lower in the massage group than in the nonmassage group. CONCLUSION: Massaging the hepatoduodenal ligament after the Pringle maneuver is recommended in order to quickly recover portal vein flow during hepatectomy and to improve coagulability.

Crystal structures of ß-primeverosidase in complex with disaccharide amidine inhibitors.

ß-Primeverosidase (PD) is a disaccharide-specific ß-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes ß-primeveroside (6-O-ß-d-xylopyranosyl-ß-d-glucopyranoside) at the ß-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones. PD only accepts primeverose as the glycone substrate, but broadly accepts various aglycones, including 2-phenylethanol, benzyl alcohol, linalool, and geraniol. We determined the crystal structure of PD complexes using highly specific disaccharide amidine inhibitors, N-ß-primeverosylamidines, and revealed the architecture of the active site responsible for substrate specificity. We identified three subsites in the active site: subsite -2 specific for 6-O-ß-d-xylopyranosyl, subsite -1 well conserved among ß-glucosidases and specific for ß-d-glucopyranosyl, and wide subsite +1 for hydrophobic aglycone. Glu-470, Ser-473, and Gln-477 act as the specific hydrogen bond donors for 6-O-ß-d-xylopyranosyl in subsite -2. On the other hand, subsite +1 was a large hydrophobic cavity that accommodates various aromatic aglycones. Compared with aglycone-specific ß-glucosidases of the glycoside hydrolase family 1, PD lacks the Trp crucial for aglycone recognition, and the resultant large cavity accepts aglycone and 6-O-ß-d-xylopyranosyl together. PD recognizes the ß-primeverosides in subsites -1 and -2 by hydrogen bonds, whereas the large subsite +1 loosely accommodates various aglycones. The glycone-specific activity of PD for broad aglycone substrates results in selective and multiple release of temporally stored alcoholic volatile aglycones of ß-primeveroside.

Induction of apoptosis by intrapleural perfusion hyperthermo-chemotherapy for malignant pleural mesothelioma.

PURPOSE: Despite extensive clinical research, no effective therapy for advanced malignant pleural mesothelioma has been established. In this study, we induced apoptosis in patients with this disease, using intrapleural perfusion hyperthermo-chemotherapy, a new procedure developed in our surgical department. We then measured the tumorcidal effect. MATERIAL AND METHODS: Our study included 6 consecutive patients with malignant pleural mesothelioma (stage III: 5; stage IV: 1). Because of the advanced stage of the disease, none of the patients underwent tumor resection or pleurectomy. All patients, however, received perfusion treatment. Tumor cells collected from pleural effusions pre-and at 0, 24, and 48 h postperfusion were examined using an immunocytochemical stain to determine apoptosis. The percentage of positively stained cells was expressed as the apoptotic index. RESULTS: Preperfusion, the apoptotic index was 3.8%+/-2.0%, indicating spontaneous apoptosis of untreated tumor cells. Postperfusion, the apoptotic index at 0, 24, and 48 h was 22.8%+/-5.15%, 63.8%+/-8.2%, and 47.8%+/-6.9%, respectively. The patients had a median survival time of 30 months. No patient morbidity was associated with the perfusion treatment. CONCLUSION: In patients with malignant pleural mesothelioma, intrapleural perfusion hyperthermo-chemotherapy induced potent apoptosis of tumor cells, increasing immediately postperfusion and peaking at 24 h.

Profiling of fluorouracil-related genes by microdissection technique in hepatocellular carcinoma.

BACKGROUND/AIMS: 5-fluorouracil (5-FU)-related metabolic enzymes, including dihydropyrimidine dehydrogenase (DPD), thymidylate synthase (TS), thymidylate phosphorylase (TP), and orotate phosphoribosyl transferase (OPRT) are initial, rate-limiting enzymes in the metabolism of 5-FU. The therapeutic implications of these enzymes in hepatocellular carcinoma (HCC) remain poorly understood. We used a newly developed laser-captured microdissection technique combined with RNA extraction to examine the mRNA levels of 5-FU-related metabolic enzymes in HCC and adjacent liver tissue. METHODOLOGY: The study material comprised 43 paired specimens of HCC and adjacent liver tissue. The mRNA levels of 5-FU-related metabolic enzymes were quantified by real-time reverse-transcriptase polymerase chain reaction combined with laser-captured microdissection. RESULTS: The DPD mRNA level in HCC (4.31 +/- 4.21) was lower than that in adjacent liver (6.53 +/- 2.93) (p < 0.001). The TS mRNA level in HCC (3.55 +/- 2.54) was higher than that in adjacent liver (1.90 +/- 0.11) p < 0.001). The TP and the OPRT mRNA levels did not differ significantly between HCC and adjacent liver. The TS mRNA level of HCC with portal invasion (4.47 +/- 2.76) was higher than that of HCC without portal invasion (2.71 +/- 1.96) (p = 0.015). The DPD mRNA level of HCC with septum formation (4.89 +/- 4.82) was significantly higher than that of HCC without septum formation (2.12 +/- 0.61) (p < 0.027). The OPRT mRNA level of poorly differentiated HCC (1.18 +/- 0.49) was lower than that of moderately or well-differentiated HCC (2.42 +/- 1.82) (p = 0.037). CONCLUSIONS: The DPD mRNA level was lower and the TS mRNA level was higher in HCC than in adjacent liver. Our results will hopefully stimulate further investigations designed to optimize the use of 5-FU in patients with HCC.

Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli.

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.

Intrapleural hyperthermic perfusion with chemotherapy increases apoptosis in malignant pleuritis.

BACKGROUND: Previously, we reported on the effectiveness of intrapleural hyperthermic perfusion with chemotherapy, a new treatment we developed for patients with malignant pleuritis. The present study analyzes the mechanism of the effectiveness of this therapy by examining the induction ratio of apoptosis among tumor cells following the perfusion treatment. METHODS: This study included 11 consecutive patients with primary pulmonary adenocarcinoma and accompanying pleural seedlings and pleural effusions containing tumor cells but without distant metastasis. All patients underwent surgical resection of the primary lesion and then received sequential perfusion treatment. Tumor cells collected from the effusion both before and again at 24 hours following the perfusion treatment were subsequently examined using an immunocytochemical stain to determine apoptosis among tumor cells. The percentage of positively stained cells was expressed as the apoptotic index. We compared the survival rate of these 11 patients with the survival rate of a second group of 11 patients with malignant pleuritis who underwent surgical resection of the primary lesion but who did not receive the perfusion treatment (control group). RESULTS: The ratio of spontaneous apoptosis of untreated tumor cells was 2.8% +/- 2.0%. Following the perfusion, apoptosis among tumor cells was 25.2% +/- 4.6%, clearly a significant increase. While the median survival time for patients receiving the perfusion treatment was 20 months, the median survival time for the control group was 6 months. CONCLUSIONS: In patients with malignant pleuritis, intrapleural hyperthermic perfusion with chemotherapy induced potent apoptosis of tumor cells in the pleural cavity and also improved the survival rate of these patients as compared with patients who did not receive the perfusion treatment.