Molecular cloning of putative chloroplastic cysteine synthase in Leucaena leucocephala.

Cysteine biosynthesis is directed by the successive commitments of serine acetyltransferase, and O-acetylserine (thiol) lyase (OASTL) compounds, which subsequently frame the decameric cysteine synthase complex. The isoforms of OASTL are found in three compartments of the cell: the cytosol, plastid, and mitochondria. In this investigation, we first isolated putative chloroplastic OASTL (Ch-OASTL) from Leucaena leucocephala, and the Ch-OASTL was then expressed in BL21-competent Escherichia coli. The putative Ch-OASTL cDNA clone had 1,543 base pairs with 391 amino acids in its open reading frame and a molecular weight of 41.54 kDa. The purified protein product exhibited cysteine synthesis ability, but not mimosine synthesis activity. However, they both make the common α-aminoacrylate intermediate in their first half reaction scheme with the conventional substrate O-acetyl serine (OAS). Hence, we considered putative Ch-OASTL a cysteine-specific enzyme. Kinetic studies demonstrated that the optimum pH for cysteine synthesis was 7.0, and the optimum temperature was 40 °C. In the cysteine synthesis assay, the Km and kcat values were 838 ± 26 µM and 72.83 s-1 for OAS, respectively, and 60 ± 2 µM and 2.43 s-1 for Na2S, respectively. We can infer that putative Ch-OASTL regulatory role is considered a sensor for sulfur constraint conditions, and it acts as a forerunner of various metabolic compound molecules.

Molecular characterization of cytosolic cysteine synthase in Mimosa pudica.

In the cysteine and mimosine biosynthesis process, O-acetyl-L-serine (OAS) is the common substrate. In the presence of O-acetylserine (thiol) lyase (OASTL, cysteine synthase) the reaction of OAS with sulfide produces cysteine, while with 3-hydroxy-4-pyridone (3H4P) produces mimosine. The enzyme OASTL can either catalyze Cys synthesis or both Cys and mimosine. A cDNA for cytosolic OASTL was cloned from M. pudica for the first time containing 1,410 bp nucleotides. The purified protein product from overexpressed bacterial cells produced Cys only, but not mimosine, indicating it is Cys specific. Kinetic studies revealed that pH and temperature optima for Cys production were 6.5 and 50 °C, respectively. The measured Km, Kcat, and Kcat Km-1 values were 159 ± 21 µM, 33.56 s-1, and 211.07 mM-1s-1 for OAS and 252 ± 25 µM, 32.99 s-1, and 130.91 mM-1s-1 for Na2S according to the in vitro Cys assay. The Cy-OASTL of Mimosa pudica is specific to Cys production, although it contains sensory roles in sulfur assimilation and the reduction network in the intracellular environment of M. pudica.

Temperature controls on the basal emission rate of isoprene in a tropical tree Ficus septica: exploring molecular regulatory mechanisms.

Isoprene emission from plants is very sensitive to environmental temperature both at short-term and long-term scales. Our previous study demonstrated suppression of isoprene emission by cold temperatures in a high emitting tropical tree Ficus septica and revealed a strong correlation of emission to isoprene synthase (IspS) protein levels. When challenged with decreasing daily temperatures from 30 to 12 °C, F. septica completely stopped isoprene emission at 12 °C, only to recover on the second day after re-exposure to 30 °C. Here, we explored this regulation of isoprene emission in response to environmental temperature by a comprehensive analysis of transcriptome data, gene expressions and metabolite pools of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. MEP pathway genes and metabolites dynamics did not support substrate-level limitations as major control over observed basal emission, but transcriptome data, network inferences and putative regulatory elements on IspS promoter suggested transcriptional regulation of IspS gene through circadian rhythm and phytohormone signalling processes. Expression levels of 29 genes involved in these pathways were examined by quantitative real-time PCR. We propose that temperature controls over basal isoprene emission at a time-scale of hours to few days are regulated by phytohormone-mediated transcriptional modulation of IspS gene under synchronization by the circadian clock.

Reoxygenation from chronic hypoxia promotes metastatic processes in pancreatic cancer through the Hedgehog signaling.

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly types of malignancies because of its high ability to metastasize. PDAC is thought to be under hypoxic condition. Therefore, to investigate the mechanism of metastatic processes, chronic-hypoxia-resistant PDAC cells were newly generated under hypoxic condition for 3-6 months and reoxygenation experiments were performed using these chronic-hypoxia-resistant PDAC cells in in vivo-mimicking conditions. Proliferation, invasiveness and tumorigenicity in PDAC cells were significantly increased by reoxygenation. A Hedgehog (Hh) signaling component, Gli1, was significantly increased by reoxygenation. Gli1 knockdown inhibited reoxygenation-induced increases in proliferation and tumorigenicity and decreased invasiveness through suppression of matrix metalloproteinase (MMP) 2 and MMP9. Moreover, inhibition of Sonic Hh and Smoothened abrogated reoxygenation induced increases in proliferation and invasiveness. These results suggest that metastatic processes in PDAC are induced through activation of the Hh signaling pathway. Therefore, the Hh signaling pathway may be a therapeutic target for refractory PDAC in metastatic processes induced by reoxygenation.

Hedgehog Gli3 activator signal augments tumorigenicity of colorectal cancer via upregulation of adherence-related genes.

Hedgehog signal is re-activated in several cancers. In this study, we examined the role of Gli3 on malignant phenotype of tumorigenicity for colorectal cancer and its relationship with p53, WNT and ERK/AKT signals. Gli3 expression was detected in HT29 and SW480 (p53-mutant) cells, but not in DLD-1 (p53-mutant) or HCT116 (p53-wild type) cells by reverse transcription-polymerase chain reaction and immunocytochemistry. Full-length Gli3 transfection increased anchor-independent growth for all cells regardless of p53 status, with upregulation of adhesion-related genes. Exogenous Sonic-Hedgehog increased activator-type of Gli3 and colony formation in Gli3-positive HT29 and SW480 cells. After implantation of Gli3-FL or mock-transfectant DLD-1 cells into SCID mice, tumor formation was highly observed in only Gli3-FL-transfectant group. In clinical specimens, Gli3 expression was detected in subsets of colorectal cancer and related with poorly-differentiated histological type, while Sonic-Hedgehog was present with high incidence. In conclusion, activator Gli3 signal augments tumorigenicity of colorectal cancer irrespective of p53 status.

Risk Factor of Sarcopenia After Gastrectomy in Elderly Patients With Gastric Cancer.

BACKGROUND/AIM: Sarcopenia is a progressive and generalized muscle disorder correlated with an increased risk of adverse outcomes, including falls, fractures, physical disability and mortality. Moreover, sarcopenia is associated with short- and long-term outcomes after surgery in patients with gastrointestinal malignancies. Additionally, severe skeletal muscle loss after surgery reduces quality of life. In this study, we analyzed the perioperative risk factors for skeletal muscle loss after gastrectomy in elderly patients undergoing radical gastrectomy for gastric cancer. PATIENTS AND METHODS: In this case-control study, we enrolled patients aged ≥75 years who underwent radical gastrectomy for gastric cancer between January 2014 and December 2020 at our Institution. The psoas muscle index was used to assess skeletal muscle mass. They were divided into two groups-muscle depletion (D group) and no depletion (ND group)-depending on the ratio of skeletal muscle loss before and after gastrectomy. RESULTS: The D and ND groups comprised 34 and 41 patients, respectively. Univariate analysis showed that open gastrectomy was a potential risk factor for postoperative skeletal muscle loss in elderly gastric cancer patients (p=0.017). In multiple logistic regression analysis using the following variables: sex, operation and approach, the D group had a significantly higher proportion of patients who underwent open surgery than the ND group (p=0.032). CONCLUSION: Open gastrectomy is an independent risk factor for the progression of sarcopenia after gastrectomy in elderly patients with gastric cancer. Laparoscopic surgery is an eligible method for preserving skeletal muscle mass in elderly patients with gastric cancer.

Hedgehog inhibitor decreases chemosensitivity to 5-fluorouracil and gemcitabine under hypoxic conditions in pancreatic cancer.

Pancreatic cancer is one of the deadliest types of cancer. Previously, we showed that hypoxia increases invasiveness through upregulation of Smoothened (Smo) transcription in pancreatic ductal adenocarcinoma (PDAC) cells. Here, we first evaluated whether hypoxia-induced increase in Smo contributes to the proliferation of PDAC cells. We showed that Smo, but not Gli1, inhibition decreases proliferation significantly under hypoxic conditions. To further investigate the effects of Smo on PDAC growth, cell cycle analysis was carried out. Inhibition of Smo under hypoxia led to G(0) /G(1) arrest and decreased S phase. As 5-fluorouracil (5-FU) and gemcitabine, which are first-line drugs for pancreatic cancer, are sensitive to S phase, we then evaluated whether cyclopamine-induced decreased S phase under hypoxia affected the chemosensitivity of 5-FU and gemcitabine in PDAC cells. Cyclopamine treatment under hypoxia significantly decreased chemosensitivity to 5-FU and gemcitabine under hypoxia in both in vitro and in vivo models. In contrast, cis-diamminedichloroplatinum, which is cell cycle-independent, showed significant synergistic effects. These results suggest that hypoxia-induced increase of Smo directly contributes to the proliferation of PDAC cells through a hedgehog/Gli1-independent pathway, and that decreased S phase due to the use of Smo inhibitor under hypoxia leads to chemoresistance in S phase-sensitive anticancer drugs. Our results could be very important clinically because a clinical trial using Smo inhibitors and chemotherapy drugs will begin in the near future.

Cloning and characterization of a small family 19 chitinase from moss (Bryum coronatum).

Chitinase-A (BcChi-A) was purified from a moss, Bryum coronatum, by several steps of column chromatography. The purified BcChi-A was found to be a molecular mass of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.5. A cDNA encoding BcChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1012 nucleotides and encoded an open reading frame of 228 amino acid residues. The predicted mature BcChi-A consists of 205 amino acid residues and has a molecular weight of 22,654. Sequence analysis indicated that BcChi-A is glycoside hydrolase family-19 (GH19) chitinase lacking loops I, II, IV and V, and a C-terminal loop, which are present in the catalytic domain of plant class I and II chitinases. BcChi-A is a compact chitinase that has the fewest loop regions of the GH19 chitinases. Enzymatic experiments using chitooligosaccharides showed that BcChi-A has higher activity toward shorter substrates than class II enzymes. This characteristic is likely due to the loss of the loop regions that are located at the end of the substrate-binding cleft and would be involved in substrate binding of class II enzymes. This is the first report of a chitinase from mosses, nonvascular plants.

Hypoxia activates the hedgehog signaling pathway in a ligand-independent manner by upregulation of Smo transcription in pancreatic cancer.

The hedgehog (Hh) signaling pathway is activated in various types of cancer including pancreatic ductal adenocarcinoma. It has been shown that extremely low oxygen tension (below 1% O2) is found in tumor tissue including pancreatic ductal adenocarcinoma cells (PDAC) and increases the invasiveness of PDAC. To investigate the contribution of the Hh pathway to hypoxia-induced invasiveness, we examined how hypoxia affects Hh pathway activation and the invasiveness of PDAC. In the present study, three human PDAC lines were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions. Hypoxia upregulated the transcription of Sonic hedgehog (Shh), Smoothened (Smo), Gli1 and matrix metalloproteinase9 (MMP9) and increased the invasiveness of PDAC. Significantly, neither the addition of recombinant Shh (rhShh) nor the silencing of Shh affected the transcription of these genes and the invasiveness of PDAC. On the other hand, silencing of Smo decreased the transcription of Gli1 and MMP9 and PDAC invasiveness. Silencing of Gli1 or MMP9 decreased PDAC invasiveness. These results suggest that hypoxia activates the Hh pathway of PDAC by increasing the transcription of Smo in a ligand-independent manner and increases PDAC invasiveness.

An additional suture secures against pitfalls in delta-shaped gastroduodenostomy after laparoscopic distal gastrectomy.

Laparoscopic gastrectomy is widely used as a minimally invasive surgery for gastric cancer. Laparoscopic distal gastrectomy must be followed by either a gastroduodenostomy or gastrojejunostomy to restore continuity of the alimentary tract. The intraabdominal delta-shaped gastroduodenostomy using endoscopic linear staplers, which was developed by Kanaya et al., is one of the feasible reconstructive procedures. However, the clinical results still remain uncertain. In 71 patients treated between February 2008 and February 2009, we found that anastomotic failure occurred in six patients and there was an intraabdominal abscess around the anastomosis in two patients, findings which might be associated with technical pitfalls in the procedure. After considering the mechanisms underlying these unfavorable complications, we developed a modification of the procedure to successfully overcome these complications by reinforcement of the anastomosis using simple suturing at the closed common channel on the greater curvature. This modified Kanaya's procedure will be safer and should provide a better intracorporeal gastroduodenostomy after laparoscopic distal gastrectomy.

The delta-shaped anastomosis in laparoscopic distal gastrectomy: analysis of the initial 100 consecutive procedures of intracorporeal gastroduodenostomy.

BACKGROUND: We developed a new method of intracorporeal gastroduodenostomy, the delta-shaped anastomosis, in which only endoscopic linear staplers are used. In this report, we present the short- and long-term outcomes of our initial experience with this procedure. METHODS: We retrospectively analyzed 100 consecutive gastric cancer patients who underwent the delta-shaped anastomosis procedure from May 2001 to November 2006. All of them underwent a laparoscopic distal gastrectomy with regional lymph node dissection. Quality of life was assessed with a questionnaire 6 months or more postoperatively, and the gastric remnant was evaluated by endoscopy one year following the surgery. RESULTS: Eight surgeons successfully performed the delta-shaped anastomosis without any conversion to laparotomy. The learning curve for all surgeons was steep and the mean time for the anastomosis was 13 min. Only one patient developed an anastomotic leak, and the leak was minor. Sixty-five patients tolerated a 1500 kcal/day soft diet at the time of discharge. The mean follow-up period was 54.9 months. Only one patient reported symptoms indicative of dumping. Two patients were diagnosed with reflux esophagitis, and approximately 70% had evidence of bile reflux, but severe gastritis of the remnant stomach on endoscopy was uncommon. CONCLUSIONS: The wide lumen of the delta-shaped anastomosis led to early, adequate postoperative oral intake without a significant incidence of dumping syndrome. The delta-shaped anastomosis is safe and simple and provides satisfactory postoperative results.

Peucedanum japonicum Thunb inhibits high-fat diet induced obesity in mice.

This study examined the antiobese activity of Peucedanum japonicum Thunb (PJT) in mice. In the first experiment, 4-week-old C57BL/6 mice were fed seven different diets containing 15% corn oil and 0-20% PJT powder for 4 weeks. Feeding the 10% and 20% PJT diet suppressed the body weight gain and the accumulation of abdominal and subcutaneous fats. PJT reduced serum and liver levels of triglyceride and serum levels of leptin in a dose-dependent manner. PJT intake decreased the proportion of saturated fatty acids and increased polyunsaturated and n-3 fatty acids in the liver. To obtain more insight into the antiobese activity of PJT, its effect on lipid absorption and enzyme activities related to lipid metabolism was studied in the second experiment. There was an increased faecal excretion of triglyceride in mice fed 5% and 10% PJT diets. Fatty acid synthase activity was decreased while carnitine palmitoyltransferase activity was increased by 10% PJT intake. These findings pointed to the usefulness of PJT for the development of a safe natural agent to reduce obesity or body weight for the first time. The rationale for the lipid lowering mechanism of PJT and the candidate compound responsible for the observations have also been discussed.

Lymphadenectomy along the left recurrent laryngeal nerve by a minimally invasive esophagectomy in the prone position for thoracic esophageal cancer.

BACKGROUND: A thoracoabdominal esophagectomy for esophageal cancer is a severely invasive procedure. A thoracoscopic esophagectomy may minimize injury to the chest wall and reduce surgical invasiveness. Conventional thoracoscopic procedures are performed in the left lateral-decubitus position. Recently, procedures performed in the prone position have received more attention because of improvements in operative exposure or surgeon ergonomics. However, the efficacy of the prone position in an aggressive thoracoscopic esophagectomy with an extensive lymphadenectomy has not been fully documented. METHODS: We successfully performed a thoracoscopic esophagectomy with a three-field extensive lymphadenectomy in 43 esophageal carcinoma patients in the prone position from December 2007 to December 2009. We describe our procedures with the patients in the prone position, focusing especially on a lymphadenectomy along the left recurrent laryngeal nerve where the nodes are frequently involved and precise dissection is technically challenging. To determine further the advantages of this position, we retrospectively compared surgical outcomes in 43 patients to those of 34 patients who underwent a thoracoscopic esophagectomy in the left lateral decubitus position as a historical control from January 2006 to November 2007. RESULTS: It was easier to explore the operative field around the left recurrent laryngeal nerve during a thoracoscopic esophagectomy in the prone position. The mean duration of the aggressive thoracoscopic procedure in the prone position was 307 min, which was significantly longer than in the left lateral decubitus position, but the total estimated blood loss in the prone position was significantly lower. There was no difference in the incidence of postoperative complications between the two procedures. CONCLUSIONS: A thoracoscopic esophagectomy in the prone position is technically safe and feasible and provides better surgeon ergonomics and better operative exposure around the left recurrent laryngeal nerve during an aggressive esophagectomy.

Postoperative hiatal hernia after minimally invasive esophagectomy for esophageal cancer.

BACKGROUND: Minimally invasive esophagectomy (MIE) can reduce various complications compared with conventional thoracotomic esophagectomy. However, several reports suggested that MIE promoted incidence of post-operative hiatal hernia (HH). In current reports, we retrospectively analyzed incidence and risk factors of HH development after MIE. METHODS: A total of 113 patients undergoing MIE (McKeown esophagectomy) at our institute from April 2009 to December 2015 were included in this study. Patients with clinical stage II and III received neoadjuvant chemotherapy (NAC). RESULTS: Eleven of 113 patients (9.7%) undergoing MIE developed HH. Four of them were female and the ratio of female among the patient with HH was higher than that among the patient without HH after MIE (36.4% vs. 13.7%, P=0.05). Sixty-six patients (58.4%) during the study period were administered NAC and 10 of 11 patients with HH (90.9%) received NAC according to the clinical stage, which was significantly more than in the non-HH group (P=0.02). Type and route of graft organ were not related to HH development. Moreover, the fixation of the conduit organ at the hiatus does not contribute to post-operative HH. CONCLUSIONS: In the current study, we showed that NAC was a major risk factor of HH development after MIE.

A plant class V chitinase from a cycad (Cycas revoluta): biochemical characterization, cDNA isolation, and posttranslational modification.

Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)(3) from the substrate (GlcNAc)(6) through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

Incidence of postoperative complications in transabdominal preperitoneal repair for groin hernia is influenced by poor performance status rather than by old age.

AIM: The present study was designed to evaluate the safety and feasibility of transabdominal preperitoneal (TAPP) repair for very old patients with groin hernia and to identify the risk factors predicting perioperative complications. METHODS: A total of 140 patients treated by TAPP were reviewed retrospectively. They were divided into two groups: patients ≥80 years of age (≥80 years group; n = 26) and those <80 years of age (<80 years group; n = 114). Patient characteristics and surgical outcomes were then statistically compared between the two groups. RESULTS: Number of patients with any comorbidities was significantly higher in the ≥80 years group than in the <80 years group (96.2% vs 61.4%, P = 0.003). There were no significant differences in surgical outcomes between the two groups. In the univariate analysis of perioperative complications, poor performance status (PS) (P = 0.014), lower hemoglobin level (P = 0.038) and lower albumin level (P = 0.016) were significantly associated with the occurrence of postoperative complications, and multivariate analysis showed that only poor PS was an independent factor (PS 0-2 vs 3-4: P = 0.034, OR 5.192 [95% CI; 1.137 to 23.71]). CONCLUSIONS: This is the first report to show that the incidence of postoperative complications in TAPP repair for groin hernia is influenced by poor PS rather than old age. TAPP can be a safe surgical procedure for very old patients with a good PS, with benefits that are equal to those in young patients.

Molecular regulatory mechanism of isoprene emission under short-term drought stress in the tropical tree Ficus septica.

Isoprene is emitted by many plants and is thought to function as an antioxidant under stressful conditions. However, the detailed regulatory mechanism of isoprene emission in relation to the antioxidant system remains unclear. Therefore, in this study, we explored the molecular regulatory mechanism of isoprene emission under short-term drought stress in the tropical tree Ficus septica Burm.f. We found that the soil moisture content gradually decreased from 55% on Day 1 (D1) to 23% (wilting point) on D5 after withholding water for 4 days and then returning to the initial level following re-watering on D6. On D5, drought-stressed plants had more than twofold higher isoprene emission and 90.6% lower photosynthesis rates, 99.5% lower stomatal conductance and 82.3% lower transpiration rates than well-watered control plants. It was also estimated that the isoprene concentration inside the leaf greatly increased on D5 due to the increased isoprene emission rate and reduced stomatal conductance. Among the traits related to the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which is responsible for isoprene biosynthesis, the isoprene synthase (IspS) protein level was positively correlated with the isoprene emission rate in stressed plants. The transcripts of the antioxidant genes peroxidase 2 (POD2), POD4, copper-zinc superoxide dismutase 2 (Cu-ZnSOD2) and manganese superoxide dismutase 1 (Mn-SOD1) also increased during the drying period, while those of ascorbate peroxidase 1 (APX1) decreased. However, there was only a weak correlation between isoprene emission and antioxidant enzyme gene expression, indicating that the regulation of isoprene biosynthesis is not directly linked to the antioxidant defense network in drought-stressed F. septica. These findings suggest that the post-transcriptional regulation of IspS led to the observed change in isoprene emission rate, which enhanced the quenching of reactive oxygen species (ROS) and, in combination with the increased antioxidant enzyme activity, conferred tolerance to drought stress in this species.

Formalin fixation on HER-2 and PD-L1 expression in gastric cancer: A pilot analysis using the same surgical specimens with different fixation times.

BACKGROUND: The needs for human epidermal growth factor receptor 2 (HER-2) and/or programmed death-ligand 1 (PD-L1) evaluations in gastric cancer are dramatically increasing. Although the importance of standardization of sample fixation has been widely recognized, most of the evidence regarding the fixation duration or type of fixing solution are based on breast cancer. AIM: To investigate the real effects of fixation conditions on HER-2 testing or PD-L1 testing for gastric cancer using gastrectomy specimens. METHODS: Thirty-two patients who underwent gastrectomy for gastric cancer were enrolled. Their resected specimens were each divided into four pieces and fixed in four strictly controlled different durations (6 h, 24 h, and 48 h, and 1 wk) by 10% formalin (n = 22) or 10% neutral buffered formalin (NBF) (n = 10). Immunohistochemistry (IHC) of HER-2 and PD-1 was performed, and a pathology examination was conducted. In the HER-2-immunoreactive cases, all four specimens were subjected to dual-color in situ hybridization (DISH). Five cases were assessed as HER-2-positive by IHC and DISH. We used the cut-off values of 1%, 10%, and 50% to assess the IHC findings of PD-L1. RESULTS: No significant difference was observed in comparisons between the shorter fixation period groups (6 h, 24 h, and 48 h) and the prolonged fixation period (1 wk) group in the HER-2 and PD-L1 analyses. Although no significant difference was observed between 10% formalin and 10% NBF within 1 wk of fixation, the superiority of 10% NBF was confirmed in a long-term (> 3 mo) fixation in both the HER-2 and PD-L1 analyses. CONCLUSION: In this small-numbered pilot study, prolonged fixation within 1 wk showed no inferiority in HER-2 or PD-L1 testing. However, a large-numbered prospective study is needed to obtain conclusive results.

The occurrence of HR1b in the venom of the snake Okinawa habu (Protobothrops flavoviridis).

This study describes the first isolation of hemorrhagic metalloproteinase HR1b from the venom of Okinawa habu, and its cDNA cloning. The substrate specificity of isolated HR1b definitely differed from that of HR1a, further supporting the presence of a metalloproteinase distinguishable from HR1a in the venom of Okinawa habu. The deduced amino acid sequence of HR1b showed 99.67% identity with HR1b of Amami habu, with only two amino acid residue replacements.

Antioxidative flavan-3-ol glycosides from stems of Rhizophora stylosa.

Two flavan-3-ol glycosides together with seven flavan-3-ols were isolated from stems of the mangrove plant, Rhizophora stylosa. Their structures were established as glabraoside A (7) and glabraoside B (8) by spectroscopic and chemical methods. The antioxidative activity of these isolated compounds evaluated by the diphenylpicrylhydrazyl (DPPH) radical scavenging reaction was higher than that of L-ascorbic acid.