Robotic versus laparoscopic hepatectomy: A single-center, propensity score- matched study.

BACKGROUND: Although the effectiveness of robotic hepatectomy (RH) has been evaluated in several studies, the superiority of RH over other approaches has not been definitely established. Therefore, in the present propensity score-matched cohort study, we compared RH and laparoscopic hepatectomy (LH) in terms of perioperative and oncologic outcomes. METHODS: This retrospective study included patients who underwent RH or LH for benign and malignant liver lesions at a single center in Taiwan at any time between 2014 and 2020. Confounding factors, specifically age, sex, body mass index, American Society of Anesthesiologists score, IWATE criteria, and Charlson comorbidity index, were adjusted through propensity score matching (PSM). RESULTS: A total of 329 patients were finally included in this study. Two homogeneous groups (RH and LH; n, 72 each) were formed using PSM. The RH group had a longer operative time (median: 231 vs.180 min, respectively; P = .001) and lower conversion (to open surgery) rate (9.7% vs.0.0%, respectively; P = .013) than did the LH group. However, the two groups did not differ in terms of other perioperative outcomes, specifically blood loss, hospital stay, intensive care unit admission, mortality, morbidity, or tumor margin status. CONCLUSIONS: The rate of conversion to open surgery is lower in RH than in LH. Although operative time is longer in RH than in LH, RH is feasible and safe for patients with benign or malignant liver lesion. Our study also demonstrated comparable oncological results in patients with hepatocellular carcinoma between LH and RH group.

Galectin-1-mediated MET/AXL signaling enhances sorafenib resistance in hepatocellular carcinoma by escaping ferroptosis.

Sorafenib, a small-molecule inhibitor targeting several tyrosine kinase pathways, is the standard treatment for advanced hepatocellular carcinoma (HCC). However, not all patients with HCC respond well to sorafenib, and 30% of patients develop resistance to sorafenib after short-term treatment. Galectin-1 modulates cell-cell and cell-matrix interactions and plays a crucial role in HCC progression. However, whether Galectin-1 regulates receptor tyrosine kinases by sensitizing HCC to sorafenib remains unclear. Herein, we established a sorafenib-resistant HCC cell line (Huh-7/SR) and determined that Galectin-1 expression was significantly higher in Huh-7/SR cells than in parent cells. Galectin-1 knockdown reduced sorafenib resistance in Huh-7/SR cells, whereas Galectin-1 overexpression in Huh-7 cells increased sorafenib resistance. Galectin-1 regulated ferroptosis by inhibiting excessive lipid peroxidation, protecting sorafenib-resistant HCC cells from sorafenib-mediated ferroptosis. Galectin-1 expression was positively correlated with poor prognostic outcomes for HCC patients. Galectin-1 overexpression promoted the phosphorylation of AXL receptor tyrosine kinase (AXL) and MET proto-oncogene, receptor tyrosine kinase (MET) signaling, which increased sorafenib resistance. MET and AXL were highly expressed in patients with HCC, and AXL expression was positively correlated with Galectin-1 expression. These findings indicate that Galectin-1 regulates sorafenib resistance in HCC cells through AXL and MET signaling. Consequently, Galectin-1 is a promising therapeutic target for reducing sorafenib resistance and sorafenib-mediated ferroptosis in patients with HCC.

Chaperone-mediated autophagy degrade Dicer to promote breast cancer metastasis.

Metastasis in breast cancer usually lead to the majority of deaths on clinical patients. Accordingly, diagnosis of metastasis at the early stage in breast cancer is important to improve the prognosis. We observed that Dicer protein levels are significant decrease in highly invasive breast cancer cells and usually correlated with poor clinical outcomes. Following, we aim to clarify the molecular regulatory mechanism of this phenomenon in breast cancer to provide a new therapeutic target. In this study, we obtained that Dicer expression correlated with metastasis and invasion without affect cell stability in breast cancer cells. Importantly, we identified the regulatory mechanism of Dicer protein degradation, the chaperone-mediated autophagy (CMA)-mediated degradation that is major mechanism to decrease Dicer protein expression and lead to cancer metastasis. We discovered that heat shock cognate 71-kDa protein (Hsc70) which as a CMA-related factor interacts with the CMA-targeting motif I333A/K334A on Dicer to promote degradation through CMA. Taken together, our findings hint that Dicer highly correlated with cancer metastasis, we reveal the tumor-promoting effect of CMA-mediated Dicer degradation in breast cancer.

Comparison of surgical outcomes for laparoscopic liver resection of large hepatocellular carcinomas: A retrospective observation from single-center experience.

BACKGROUND/PURPOSE: Although laparoscopic liver resection (LLR) is a common surgical procedure for hepatocellular carcinoma (HCC), its suitability for large HCCs (≥5 cm) remains controversial. This study compared surgical outcomes of open hepatectomy with LLR for large HCCs. METHODS: A total of 313 patients with HCC who underwent hepatectomy between January 2010 and June 2017 were analyzed retrospectively. Demographic data, short-term outcomes, and long-term survivals were analyzed. RESULTS: Among patients with large HCCs (n = 122), the open group (n = 85) had larger tumor sizes (10.91 ± 4.72 vs. 7.45 ± 2.95 cm; p < 0.001) and more advanced stages (stages 3/4: 71.8% vs. 45.9%; p = 0.029) than the LLR group (n = 37), while LLR group achieved less blood loss (623.24 ± 841.75 mL vs. 1091.76 ± 1004.72 mL, p = 0.014) and shorter LOS (9.00 ± 5.13 d vs. 12.82 ± 8.51 d, p = 0.013). There were no significant differences in complication and mortality rates between groups. The 5-year overall and recurrence-free survival rates between the two groups were not significantly different (p = 0.408 and 0.644 respectively). The surgical outcomes showed equal benefit of the two operation types. CONCLUSION: With sufficient surgeon experience and appropriate patient selection, LLR is a feasible treatment choice for large HCCs.

Long-term and short-term surgical outcomes of single-incision laparoscopic hepatectomy on anterolateral liver segments.

BACKGROUND: Laparoscopic liver resection yields improved short-term surgical outcomes, whereas the reports about clinical benefits of single-incision laparoscopic hepatectomy (SILH) are scarce. This retrospective study is to compare the surgical outcomes of SILH with those of multi-incision laparoscopic hepatectomy (MILH). METHODS: The study included 54 patients who had undergone SILH and 184 patients who had undergone MILH between January 2010 and December 2017. Short-term outcomes were compared in those of patients who underwent left lateral sectionectomy and partial hepatectomy of segment 5-6. A subgroup analysis of hepatocellular carcinoma (HCC) was also performed for long-term outcome comparisons. RESULTS: In those of patients who underwent left lateral sectionectomy, SILH group had less chronic hepatitis B (15.2 vs. 45.8%; p = 0.004), less liver cirrhosis (12.1 vs. 50.0%; p = 0.002), less tumor proximal to major vessel (6.1 vs. 29.2%; p = 0.018), shorter surgical time (113.2 ± 37.9 vs. 146.0 ± 50.5 min; p = 0.007), and shorter postoperative hospital stays (4.4 ± 1.1 vs. 5.4 ± 1.3 days; p = 0.002) compared with MILH group. In those of patients with tumor located at segment 5-6, no significant differences were observed in surgical time, blood loss, complications, and mortality. Single-incision laparoscopic partial hepatectomy was only associated with wider surgical margins (11.8 ± 7.0 vs. 5.3 ± 5.2 mm; p = 0.003). In the HCC subgroup, SILH had similar 1-, 3-, and 5-year overall survival and 1-, 3-, and 5-year recurrence-free survival rates compared with patients who had undergone MILH. CONCLUSIONS: The study demonstrates the safety and feasibility of single-incision laparoscopic liver resection for left lateral sectionectomy and partial hepatectomy for segment 5-6. In selected patients within the group and by experienced surgical team, the SILH technique results in comparable short-term surgical outcomes and long-term oncological outcomes.

Long-term and short-term surgical outcomes of laparoscopic versus open liver resection for hepatocellular carcinoma: might laparoscopic approach be better in early HCC?

BACKGROUND: This retrospective study compared the short- and long-term outcomes of laparoscopic liver resection (LLR) and open liver resection (OLR) and identified patients who might gain more benefits from LLR. METHODS: The demographic and perioperative data, short-term surgical outcomes, and long-term oncological results of all 313 patients who received elective liver resection for hepatocellular carcinoma (HCC) between January 2010 and June 2017 were analyzed. The patients were then divided into stage-specific subgroups according to the TNM staging system for comparison. RESULTS: LLR was performed in 153 patients and OLR in 160 patients. LLR is associated with less blood loss (p < 0.001), shorter surgical time (p = 0.001), shorter length of hospital stay (p < 0.001), and lower morbidity rate (p = 0.034). The 5-year overall survival (OS) rates in the LLR group were higher than those in the OLR group (78.1 vs. 57.6%; p = 0.002). Stage-specific subgroup analysis revealed similar 5-year OS in the two groups (stage I: 82.8 vs. 82.6%, p = 0.845; stage II: 80.3 vs. 69.2%, p = 0.638; stage III: 55.6 vs. 34.8%, p = 0.681), as did the 5-year recurrence-free survival. Moreover, the short-term outcomes were better in the LLR group in terms of surgical time, blood loss, and length of hospital stay, and these benefits attenuated with advancing tumor stage. CONCLUSIONS: LLR for HCC is a safe and feasible procedure that does not compromise long-term oncological outcomes. In early tumor stages, LLR might be better in terms of short-term surgical outcomes.

Risk Factors Analysis of Postoperative Pleural Effusion after Liver Resection.

BACKGROUND: Pulmonary complications remain relatively high in morbidities that arise after liver surgery and are associated with increased length of hospital stay and higher cost. Identification of possible risk factors in this retrospective analysis may help reduce operative morbidity and achieve better outcomes. METHODS: In all, 363 consecutive patients underwent elective hepatectomies between July 2008 and November 2013 and these were identified and analyzed retrospectively. Patient demographics and perioperative variables were collected. The main outcome was an analysis of risk factors associated with postoperative pleural effusion (PPE). RESULTS: Of 363 patients receiving hepatectomies, 80 patients (22.0%) developed pulmonary complications. The predominant pulmonary complication in this series is pleural effusion (76 patients, 95%). Univariate analysis found that older age, higher body mass index (BMI), chronic obstructive lung disease, asthma, heart disease, hepatitis C infection, heavy smoking, American Society of Anesthesiology class III and IV, hepatectomy site, combined surgeries, perioperative blood transfusion, and cirrhosis of liver were associated with PPE. Only older age, higher BMI, asthma, heavy smoker, combined gastrointestinal surgeries, and perioperative blood transfusion were identified as independent risk factors in multivariate analysis. CONCLUSION: This study identifies 6 risk factors for PPE. Identification and management of some of these factors could possibly reduce morbidity and improve short-term surgical outcomes.

Galectin-3 Inhibits Galectin-8/Parkin-Mediated Ubiquitination of Group A Streptococcus.

Group A streptococcus (GAS) is an important human pathogen that causes a wide variety of cutaneous and systemic infections. Although originally thought to be an extracellular bacterium, numerous studies have demonstrated that GAS can trigger internalization into nonimmune cells to escape from immune surveillance or antibiotic-mediated killing. Epithelial cells possess a defense mechanism involving autophagy-mediated targeting and killing of GAS within lysosome-fused autophagosomes. In endothelial cells, in contrast, we previously showed that autophagy is not sufficient for GAS killing. In the present study, we showed higher galectin-3 (Gal-3) expression and lower Gal-8 expression in endothelial cells than in epithelial cells. The recruitment of Gal-3 to GAS is higher and the recruitment of Gal-8 to GAS is lower in endothelial cells than in epithelial cells. We further showed that Gal-3 promotes GAS replication and diminishes the recruitment of Gal-8 and ubiquitin, the latter of which is a critical protein for autophagy sequestration. After knockdown of Gal-3 in endothelial cells, the colocalization of Gal-8, parkin, and ubiquitin-decorated GAS is significantly increased, as is the interaction of Gal-8 and parkin, an E3 ligase. Furthermore, inhibition of Gal-8 in epithelial cells attenuates recruitment of parkin; both Gal-8 and parkin contribute to ubiquitin recruitment and GAS elimination. Animal studies confirmed that Gal-3-knockout mice develop less-severe skin damage and that GAS replication can be detected only in the air pouch and not in organs and endothelial cells. These results demonstrate that Gal-3 inhibits ubiquitin recruitment by blocking Gal-8 and parkin recruitment, resulting in GAS replication in endothelial cells.IMPORTANCE In epithelial cells, GAS can be efficiently killed within the lysosome-fused autophaosome compartment. However, we previously showed that, in spite of LC-3 recruitment, the autophagic machinery is not sufficient for GAS killing in endothelial cells. In this report, we provide the first evidence that Gal-3, highly expressed in endothelial cells, blocks the tagging of ubiquitin to GAS by inhibiting recruitment of Gal-8 and parkin, leading to an enhancement of GAS replication. We also provide the first demonstration that Gal-8 can interact with parkin, the critical E3 ligase, for resistance to intracellular bacteria by facilitating the decoration of bacteria with ubiquitin chains. Our findings reveal that differential levels of Gal-3 and Gal-8 expression and recruitment to GAS between epithelial cells and endothelial cells may contribute to the different outcomes of GAS elimination or survival and growth of GAS in these two types of cells.

Effect of wound infiltration with ropivacaine or bupivacaine analgesia in breast cancer surgery: A meta-analysis of randomized controlled trials.

BACKGROUND: Although not completely painless, breast-conserving surgery is considerably less painful than modified radical mastectomy. Local anesthetics are speculated to reduce postoperative pain when placed at the surgical site. Thus, we conducted a systematic review of randomized controlled trials to evaluate the efficacy of bupivacaine or ropivacaine analgesia for pain relief in breast cancer surgery. METHODS: PubMed, Embase, the Cochrane Library, Scopus, and the ClinicalTrials.gov registry were searched for studies published up to July 2015. Individual effect sizes were standardized, and a meta-analysis was performed to calculate a pooled effect size by using random effects models. Pain was assessed using a visual analog scale at 1, 2, 12, and 24 h postoperatively. The secondary outcomes included complications and analgesic consumption. RESULTS: We reviewed 13 trials with 1150 patients. We found no difference in postoperative pain reduction at 1, 12, and 24 h after breast cancer surgery between the experimental and control groups. The severity of pain was significantly reduced in the experimental group (weighted mean difference -0.19; 95% confidence interval: -0.39-0.00) at 2 h postoperatively. Moreover, postoperative analgesic consumption did not differ significantly between the groups. No major drug-related complication was observed in any study. CONCLUSION: Administration of the local anesthetics bupivacaine or ropivacaine during breast cancer surgery decreased pain significantly at only 2 h but did not reduce pain at 12, and 24 h postoperatively.

Mutational analysis of the terminal protein Tpg of Streptomyces chromosomes: identification of the deoxynucleotidylation site.

The linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins (TPs) covalently bound to the 5' ends of the DNA. The TPs serve as primers for DNA synthesis that patches in the single-stranded gaps at the telomeres resulting from the bi-directional replication ('end patching'). Typical Streptomyces TPs, designated Tpgs, are conserved in sequence and size (about 185 amino acids), and contain a predicted helix-turn-helix domain and a functional nuclear localization signal. The Tpg-encoding gene (tpg) is often accompanied by an upstream gene tap that encodes an essential telomere-associating protein. Five lone tpg variants (not accompanied by tap) from various Streptomyces species were tested, and three were found to be pseudogenes. The lone tpg variant on the SLP2 plasmid, although functional, still requires the presence of tap on the chromosome for end patching. Using a combination of in vitro deoxynucleotidylation, physical localization, and genetic analysis, we identified the threonine at position 114 (T114) in Tpg of Streptomyces lividans chromosome as the deoxynucleotidylated site. Interestingly, T114 could be substituted by a serine without destroying the priming activity of Tpg in vitro and in vivo. Such T114S substitution is seen in and a number of pseudogenes as well as functional Tpgs. T114 lies in a predicted coil flanked by two short helixes in a highly hydrophilic region. The location and structural arrangement of the deoxynucleotidylated site in Tpg is similar to those in the TPs of phage ø 29 and adenoviruses. However, these TPs are distinct in their sequences and sizes, indicating that they have evolved independently during evolution. Using naturally occurring and artificially created tpg variants, we further identified several amino acid residues in the N-terminus and the helix-turn-helix domain that were important for functionality.

[Effect of Yunnan-cobra venom factor in overcoming acute humoral rejection after allograft cardiac transplantation in presensitized recipients: experiment with rats].

OBJECTIVE: To investigate the effect of Yunnan-cobra venom factor (Y-CVF) in overcoming acute humoral rejection after allograft cardiac transplantation in presensitized recipients. METHODS: Fifteen Lewis rats received the transplantation of full-thickness skin graft of BN rats three times so as to be presensitized. Fifteen pairs of Lewis rat, as recipients of heart, and BN rat, as heart donors, were randomly divided into 2 groups: experimental group (n = 8), and control group (n = 7). The Lewis rats in the experimental group received heart transplantation of the heart of the BN rat 7 approximately 10 days after the third skin transplantation, and were injected with Y-CVF 80 microg/kg 24 hours before the heart transplantation. The Lewis rat in the control group received only the heart transplantation without Y-CVF injection. Blood samples were collected from all rats before pre-sensitization and 7 days after the third skin transplantation so as to determine the titer of anti-BN rat lymphocyte antibody. 0 and 24 hours, and 6 and 8 days after Y-CVF injection blood samples were collected from the Lewis rats to determine the total complement activity with the complement activity before Y-CVF injection defined as 100%. The survival time of the transplanted heart was observed. After the transplanted hearts stopped to beat, they were resected and underwent HE staining and microscopy. Immunohistochemistry was used to examine the deposition of IgG and complement 3 (C3). RESULTS: The titer of anti-BN rat lymphocyte antibody was 0 before the pre-sensitization, and increased to 1:1028 - 1:2056 7 days after the third skin pre-sensitization. The serum total complement activity of the Lewis rats decreased to 0 twenty-four hours after the Y-CVF injection, recovered to 2.01% - 15.41% 6 days after, and returned to the normal level (89.61% - 109.46%) 8 days after. The mean survival time of the transplanted hearts of the control group was 12.71 +/- 13.94 hours (with a range of 1.5 - 15 hours), significantly shorter than that of the experimental group (99.50 +/- 38.72 hours, with a range of 23 - 153 hours, P < 0.01). Pathological examination showed that the acute humoral rejection in the control group was mainly complement-dependent antibody-mediated humoral rejection characterized by intravascular thrombosis, and that in the experimental group was mainly cellular rejection, characterized by extensive infiltration of mononuclear cells. Immunohistochemistry showed that remarkable IgG deposition was seen in the cardiac muscle cells and vascular endothelial cells in both groups; however, C3 deposition in vascular endothelial cells could be seen only in the control group. CONCLUSION: Administration of CVF is effective in overcoming the acute humoral rejection after allograft cardiac transplantation in presensitized recipients.

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom.

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent molecular weights of 15.0 kDa and 14.5 kDa under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for dabocetin alpha and beta subunits were isolated and sequenced. The deduced protein sequences of both subunits were confirmed by N-terminal amino acid sequencing and trypsin-digested peptide mass fingerprinting. Dabocetin did not induce platelet aggregation in platelet-rich plasma. It also had little effect on the platelet aggregation induced by ADP, TMVA or stejnulxin. Whereas, dabocetin inhibited ristocetin-induced platelet agglutination in platelet-rich plasma in a dose-dependent manner with an IC50 value of 0.35 microM. Flow cytometry analysis showed that dabocetin significantly inhibited mAb SZ2 binding to platelet membrane glycoprotein Ib alpha, indicating that platelet membrane glycoprotein Ib is involved in the inhibitory effect of dabocetin on ristocetin-induced platelet agglutination.

A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding.

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization.

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin.

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded alpha-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin.

Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom.

A novel disintegrin, jerdonin, was purified from the Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. Its coding cDNA was also isolated from the venom gland. The jerdonin coding cDNA is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. From the deduced amino acid sequence, jerdonin is composed of 71 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonin was determined to be 7483Da by matrix-assisted laser desorption ionization time of flight mass spectrometry. Jerdonin inhibited ADP- and collagen-induced human platelet aggregation with IC(50) of 220 and 240 nM, respectively. In vivo, jerdonin inhibited the growth of subcutaneously inoculated B16 solid tumor in C57BL/6 mice and improved the survival time of the tumor-bearing mice.

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom.

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors.

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival.

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 microg/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 microg/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB(2) and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 microg/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB(2) in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.

A new protein structure of P-II class snake venom metalloproteinases: it comprises metalloproteinase and disintegrin domains.

A new metalloproteinase-disintegrin, named Jerdonitin, was purified from Trimeresurus jerdonii venom with a molecular weight of 36 kDa on SDS-PAGE. It dose-dependently inhibited ADP-induced human platelet aggregation with IC(50) of 120nM. cDNA cloning and sequencing revealed that Jerdonitin belonged to the class II of snake venom metalloproteinases (SVMPs) (P-II class). Different from other P-II class SVMPs, metalloproteinase and disintegrin domains of its natural protein were not separated, confirmed by internal peptide sequencing. Compared to other P-II class SVMPs, Jerdonitin has two additional cysteines (Cys219 and Cys238) located in the spacer domain and disintegrin domain, respectively. They probably form a disulfide bond and therefore the metalloproteinase and disintegrin domains cannot be separated by posttranslationally processing. In summary, comparison of the amino acid sequences of Jerdonitin with those of other P-II class SVMPs by sequence alignment and phylogenetic analysis, in conjunction with natural protein structure data, suggested that it was a new type of P-II class SVMPs.

Purification and cloning of a novel C-type lectin-like protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom.

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15536 Da) and beta (14873 Da). The mature amino acid sequences of the alpha (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers of (alpha beta)(2) and (alpha beta)(4) with molecular weights of 63680 and 128518 Da, respectively. The (alpha beta)(2) complex is stabilized by an interchain disulfide bridge between the two alpha beta-heterodimers, whereas the stabilization of the (alpha beta)(4) complex seems to involve non-covalent interactions between the (alpha beta)(2) complexes.

A Highly Active Anticomplement Factor from the Venom of Naja kaouthia.

A highly active anticomplement factor (cobra venom factor) from the venom of Naja kaouthia in South Yunnan, China was isolated by sequential column chromatography (SP-Sephadex-C-25, Q Sepharose HP and Sephadex G-150). It displays strong anticomplement activities in vitro and in vivo, and has anticomplement activity of 1 515 u per mg. The purified anticomplement factor was homogeneous on SDS-PAGE with a molecular weight of 149 kD. It is composed of three polypeptide chains which are connected by disulfide bonds. The molecular weights of the three chains were determined to be 65.4 kD (alpha-chain), 52.1 kD (beta-chain), and 35.5 kD (gamma-chain). The gamma-chain displayed size heterogeneity, and two bands could be clearly detected in gels. All polypeptide chains were stained with periodate-Schiff reagent, suggesting the presence of carbohydrate. Neutral sugar and sialicacid were determined to be 1.78% and 0.38%, respectively. The isoelectric point of this anticomplement factor was 6.2. Its amino acid composition was analyzed and N-terminal amino acid sequence of each polypeptide chain was determined.