[Clinical application of laparoscopic sentinel lymph node mapping in early staged cervical cancer].

Objective: To investigate the application of sentinel lymph node biopsy (SLNB) in early-staged cervical cancer by laparoscopy. Methods: It was a prospective, single-arm, single-center clinical study. Seventy-eight cases of cervical cancer patients were collected from July 2015 to December 2018 at the Fourth Hospital of Hebei Medical University. All the patients were injected with tracer into the disease-free block of cervical tissue after anesthesia by the same surgeon who learned sentinel lymph node (SLN) mapping technique in Memorial Sloan-Kettering Cancer Center, and underwent SLN mapping followed by complete pelvic lymphadenectomy. Moreover, all the dissected lymph nodes were stained with hematoxylin eosin staining (HE) pathological examination. Besides, the negative SLN on hematoxylin-eosin staining were detected by immunohistochemistry cytokeratin staining micro-metastasis. To analyze the distribution, detection rate, false negative rate the sensitivity and negative predictive value of the SLN in early-staged cervical cancer by laparoscopy, and explore the value of SLN mapping in predicting the lymph nodes metastasis in early-staged cervical cancer. Results: The overall detection rate of SLN in cervical cancer was 99% (77/78), bilateral detection rate was 87% (68/78). The average of 12.4 lymph node (LN) and 3.6 SLN were dissected for each patients each side. SLN of cervical cancer were mainly distributed in the obturator space (61.5%, 343/558), followed by external iliac (23.5%, 131/558), common iliac (7.3%, 41/558), para-uterine (3.8%, 21/558), internal iliac (2.2%, 12/558), para abdominal aorta (1.1%, 6/558), and anterior sacral lymphatic drainage area (0.7%, 4/558). Fourteen cases of LN metastasis were found among all 78 cases. There were a total of 38 positive LN, including 26 SLN metastasis and 12 none sentinel LN metastasis. Through immunohistochemical staining and pathological ultra-staging, 1 SLN was found to be isolated tumor cells (ITC), and 5 SLNs were found to be micro-metastases (MIC), accounting for 23% (6/26) of positive SLN. SLN mapping with pathological ultra-staging improved the prediction of LN metastasis in cervical cancer (2/14). Metastatic SLN mainly distributed in the obturator space (65%, 17/26), peri-uterine region (12%, 3/26), common iliac region (15%, 4/26), and external iliac region (8%, 2/26). The consistency of the diagnosis of lymph node metastasis by SLN biopsy and postoperative retroperitoneal lymph node metastasis showed that the Kappa value was 1.000 (P<0.001), indicated that the metastasis status of SLN and retroperitoneal lymph node were completely consistent. The sensitivity, specificity, accuracy, false-negative rate, and negative predictive value of SLN biopsy in the diagnosis of lymph node metastasis were 100%, 100%, 100%, 0, and 100%, respectively. Conclusions: SLN in early-staged cervical cancer patients were mainly distributed in the obturator and external iliac space, pathalogical ultra-staging of SLN could improve the prediction of LN metastasis. Intraoperative SLN mapping is safe, feasible and could predict the state of retroperitoneal LN metastasis in early-staged cervical cancer. SLNB may replace systemic pelvic lymphadenectomy.

A Retrospective Study of a Chinese Traditional Medicine YIKEER in the Treatment of Verruca Patients in Liaoning District.

BACKGROUND: There are many possible ways to treat verruca, but no one is the single perfect treatment. YIKEER is a kind of compound preparation of Chinese traditional medicine, which has been used in the treatment of verruca for several years. AIM: To confirm the effects of YIKEER for verruca. METHOD: Patients with verruca vulgaris, verruca plantaris, or verruca plana were instructed to apply YIKEER stock solution or diluent to the lesions once or twice daily for 5-7 days. Then, the YIKEER was ceased for 3-4 days, and sea buckthorn oil was used for wound repairing. The total procession was defined as one session. RESULT: Respective 88.05% verruca vulgaris patients, 86.03% verruca plantaris patients, and 82.42% verruca plana patients achieved complete response. Most patients gained complete or partial responses after 4 treatment sessions. The percentage of patients who achieved at least 50% improvement was 90.34% for verruca vulgaris, 90.60% for verruca plantaris, and 80.91% for verruca plana after 4-session treatment. The efficacy of verruca vulgaris or verruca plantaris was better than that of verruca plana. CONCLUSION: YIKEER is an effective, safe, and well-tolerated agent for treating verruca including verruca vulgaris, verruca plantaris, and verruca plana.

Isolation and characterization of a Kunitz-type trypsin inhibitor with antiproliferative activity from Gymnocladus chinensis (Yunnan bean) seeds.

A 20-kDa Kunitz-type trypsin inhibitor was isolated from Gymnocladus chinensis (Yunnan bean) seeds. The isolation procedure involved ion exchange chromatography on diethylaminoethyl cellulose (DEAE-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on sulfopropyl sepharose (SP-sepharose), and gel filtration by FPLC on Superdex 75. The trypsin inhibitor was adsorbed on DEAE-cellulose, unadsorbed on Affi-gel blue gel, and adsorbed on SP-Sepharose. It dose-dependently inhibited trypsin with an IC(50) value of 0.4 µM. Dithiothreitol reduced its trypsin inhibitory activity, suggesting that an intact disulfide bond is indispensable to the activity. It suppressed [methyl-(3)H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC(50) value of 4.7 and 9.4 µM, respectively. There was no effect on human immunodeficiency virus(4)-1 reverse transcriptase activity and fungal growth when the trypsin inhibitor was tested up to 100 µM.

A laccase with antiproliferative activity against tumor cells from an edible mushroom, white common Agrocybe cylindracea.

A laccase, with HIV-1 reverse transcriptase inhibitory activity (IC(50)=12.7 µM) and antiproliferative activity against HepG2 cells (IC(50)=5.6 µM) and MCF7 cells (IC(50)=6.5 µM), was purified from fresh fruiting bodies of the edible white common Agrocybe cylindracea mushroom. The laccase, which had a novel N-terminal sequence, displayed a molecular mass of 58 kDa within the range reported for most other mushroom laccases. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, SP-Sepharose, and Q-Sepharose and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but unadsorbed on SP-Sepharose. Its optimum pH was pH 3-4 and its optimum temperature was 50°C. The activity of the isolated laccase differed from one substrate to another. The ranking was ABTS>N,N-dimethyl-1,4-phenylenediamine>hydroquinone>catechol>2-methylcatechol>pyrogallol.

Purification, characterization, and cloning of a novel phytase with low pH optimum and strong proteolysis resistance from Aspergillus ficuum NTG-23.

A novel phytase was isolated from Aspergillus ficuum NTG-23 with a procedure involving ion-exchange chromatography on DEAE-cellulose, CM-cellulose and FPLC-gel filtration on Superdex 75. The protein exhibited a molecular mass of 65.5kDa in gel filtration and SDS-PAGE. It possessed an optimal pH of 1.3 and an optimal temperature of 67 degrees C, and manifested a K(m) of 0.295mM and a V(max) of 55.9nmol (phosphate)/min. Phytase activity was not significantly affected by metal ions such as Ca(2+), Mg(2+), Mn(2+), Zn(2+), but was slightly stimulated in the presence of EDTA. The phytase was stable at 60 degrees C with no obvious loss of activity upon its incubation at 70 degrees C for 20min. The enzyme exhibited a broad substrate selectivity and showed strong resistance toward pepsin and trypsin. The unique properties suggest that the phytase has the potential to be useful as an animal feed supplement.

Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis.

Random amplified polymorphic DNA (RAPD) and high performance liquid chromatography (HPLC) were applied to investigate genetic and chemical variations of 2 natural C. sinensis, 16 fungal strains isolated from C. sinensis, and 2 fungal strains of C. militaris. Five of the 68 arbitrary decamer primers were available for discrimination of the investigated samples. As a result, 20 investigated samples were divided into three main clusters according to the genetic distance, and some fungal strains isolated from natural C. sinensis were obviously different. But according to the contents of nucleosides, including uracil, uridine, hypoxanthine, inosine, guanosine, adenosine, adenine, and cordycepin, natural and cultured Cordyceps were in two individual sub-groups, which suggested that chemical characteristics among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one.

Methyl palmitate, an acaricidal compound occurring in green walnut husks.

Walnut, Juglans regia L., is known for its insecticidal activities to a range of herbivores and microbes. Isolation and identification of bioactive compounds from walnut is a potential approach for the development of new pesticides. Laboratory experiments were carried out to investigate the acaricidal activities of green husk extracts of walnut. Bioassay-guided fractionation of petroleum-ether extracts of walnut led to the identification of a common plant-borne fatty acid ester, methyl palmitate (MP), which produced strong acaricidal activity (62.8% mortality) at 1 mg/ml at 24 h. The structure of MP was characterized with infrared spectrum and NMR, and the identification of MP confirmed using an authentic standard on high-performance liquid chromatography. Based on a slide dip bioassay, 10 mg/ml MP provided 97.9% mortality against adults of Tetranychus cinnabarinus (Boisduval) (Acari: Tetranychidae), whereas mortality against eggs was much lower (57.2%).

An antifungal protein from the pea Pisum sativum var. arvense Poir.

An antifungal protein with a molecular mass of 11 kDa and a lysine-rich N-terminal sequence was isolated from the seeds of the pea Pisum sativum var. arvense Poir. The antifungal protein was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-cellulose. It exerted antifungal activity against Physalospora piricola with an IC50 of 0.62 microM, and also antifungal activity against Fusarium oxysporum and Mycosphaerella arachidicola. It inhibited human immunodeficiency virus type 1 reverse transcriptase with an IC50 of 4.7 microM.

Isolation of a ribonuclease from sanchi ginseng (Panax pseudoginseng) flowers distinct from other ginseng ribonucleases.

A single-chained ribonuclease was isolated from the aqueous extract of sanchi ginseng (Panax pseudoginseng) flowers. It exhibited a molecular mass of 23 kDa, an N-terminal sequence with some similarity to other enzymes involved in RNA metabolism but different from known ribonucleases, and considerably higher activity toward poly U than poly C and only slight activity toward poly A and poly G. The purification protocol entailed ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on carboxymethyl (CM)-cellulose, and gel filtration on Superdex 75. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-cellulose. Maximal activity of the ribonuclease was attained at pH 7. On either side of this pH the enzyme activity underwent a drastic decline. The enzyme activity was at its highest at 50 degrees C and dropped to about 20% of the maximal activity when the temperature was decreased to 20 degrees C or elevated to 80 degrees C. The characteristics of sanchi ginseng flower ribonuclease were different from those of the ribonucleases previously purified from sanchi ginseng and Chinese ginseng roots including ribonuclease from Chinese ginseng flowers which are morphologically very similar to sanchi ginseng flowers.

Pharmacological actions of Cordyceps, a prized folk medicine.

Cordyceps species, including C. sinensis, C. militaris, C. pruinosa and C. ophioglossoides, are prized traditional medicinal materials. The aim of this article is to review the chemical constituents and pharmacological actions of Cordyceps species. The chemical constituents include cordycepin (3'-de-oxyadenosine) and its derivatives, ergosterol, polysaccharides, a glycoprotein and peptides containing alpha-aminoisobutyric acid. They include anti-tumour, anti-metastatic, immunomodulatory, antioxidant, anti-inflammatory, insecticidal, antimicrobial, hypolipidaemic, hypoglycaemic, anti-ageing, neuroprotective and renoprotective effects. Polysaccharide accounts for the anti-inflammatory, antioxidant, anti-tumour, anti-metastatic, immunomodulatory, hypoglycaemic, steroidogenic and hypolipidaemic effects. Cordycepin contributes to the anti-tumour, insecticidal and antibacterial activity. Ergosterol exhibits anti-tumour and immunomodulatory activity. A DNase has been characterized.

The antioxidant effects of aqueous and organic extracts of Panax quinquefolium, Panax notoginseng, Codonopsis pilosula, Pseudostellaria heterophylla and Glehnia littoralis.

The roots of Panax quinquefolium, Panax notoginseng, Glehnia littoralis, Codonopsis pilosula and Pseudostellaria heterophylla were extracted with an aqueous extraction method and also with an organic extraction method. The aqueous extracts of Glehnia littoralis and Codonopsis pilosula were the most potent in inhibiting erythrocyte hemolysis. The aqueous extracts of Panax quinquefolium and Panax notoginseng had lower potencies while the aqueous extract of Pseudostellaria heterophylla and the organic extract of Panax quinquefolium were only weakly active. The organic extracts of Glehnia littoralis, Panax heterophylla and Panax quinquefolium were potent in inhibiting lipid peroxidation while the organic extracts of Codonopsis pilosula and Panax notoginseng had weaker potencies. The aqueous extracts possessed much lower potencies the corresponding organic extracts. However, the Glehnia littoralis extract was the most potent aqueous extract. The results suggest that Glehnia littoralis, Codonopsis pilosula, Panax notoginseng and Panax heterophylla are cheaper substitutes of Panax quinquefolium with regard to antioxidant activity.

Isolation of allicepin, a novel antifungal peptide from onion (Allium cepa) bulbs.

From the bulbs of the onion Allium cepa, a novel antifungal peptide distinct from the antimicrobial peptide previously reported from onion seeds was isolated. The antifungal peptide, designated allicepin, was purified with a procedure that involved aqueous extraction, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and FPLC-gel filtration on Superdex 75. Allicepin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel. The molecular weight of allicepin was estimated to be 10 K by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. Allicepin exerted an inhibitory activity on mycelial growth in several fungal species including Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola.

Purification of a novel low-molecular-mass laccase with HIV-1 reverse transcriptase inhibitory activity from the mushroom Tricholoma giganteum.

A laccase with a novel N-terminal sequence, a low molecular mass of 43 kDa smaller than those of previously reported laccases, a pH optimum of 4, and a temperature optimum at 70 degrees C was isolated from fresh fruiting bodies of the mushroom Tricholoma giganteum. The activity of the enzyme rose steadily from 20 to 50 degrees C, increased very slowly from 50 to 70 degrees C, and fell slightly when the temperature was further increased to 80 degrees C. The activity of the laccase underwent little changes over the pH range 3.0-5.0. However, the enzyme activity dwindled to nothing after exposure to 100 degrees C for 10 min and when the ambient pH was 7 or above. The procedure used for purifying the enzyme included ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and FPLC-gel filtration on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-cellulose. It inhibited HIV-1 reverse transcriptase with an IC(50) of 2.2 microM.

A ribonuclease from Chinese ginseng (Panax ginseng) flowers.

A ribonuclease, with a molecular mass of 23kDa, and much higher activity toward poly(U) than poly(C) and only negligible activity toward poly(A) and poly(G), was isolated from the aqueous extract of Chinese ginseng (Panax ginseng) flowers. The ribonuclease was unadsorbed on diethylaminoethyl-cellulose and adsorbed on Affi-gel blue gel and carboxymethyl-cellulose. High activity of the ribonuclease was maintained at pH 6-7. On either side of this pH range, there was a precipitous drop in enzyme activity. The activity of the enzyme peaked at 50 degrees C and fell to about 20% of the maximal activity when the temperature was lowered to 20 degrees C or raised to 80 degrees C. The characteristics of this ribonuclease were different from those of ribonuclease previously purified from ginseng roots.

A distinctive ribonuclease from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum.

A ribonuclease with an N-terminal sequence distinct from other mushroom ribonucleases was isolated from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum. The ribonuclease was adsorbed on DEAE-cellulose and Q-Sepharose, and unadsorbed on CM-Sepharose. It possessed a molecular mass of 42 kDa as judged by gel filtration by fast protein liquid chromatography on Superdex 75 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molecular mass was similar to that of straw mushroom ribonuclease but much higher compared with those of other mushroom ribonucleases. The ribonuclease was unique among mushroom ribonucleases in that it exhibited the highest potency toward poly(U), followed by poly(A). Its activity toward poly(G) and poly(C) was about one-half of that toward poly(A) and one-quarter of that toward poly(U). A pH of 4.0 and a temperature of 60 degrees C were required for optimal activity of the enzyme. The optimum pH was low compared with those reported for other mushroom ribonucleases.

A ubiquitin-like peptide with ribonuclease activity against various polyhomoribonucleotides from the yellow mushroom Cantharellus cibarius.

A peptide, with a molecular weight of 9K and an N-terminal sequence identical to that of ubiquitin, was isolated from fresh fruiting bodies of the yellow mushroom Cantharellus cibarius. The peptide manifested ribonucleolytic activity toward poly A, poly C, poly G, and poly U, with the activity toward the first two polyhomoribonucleotides being higher. Maximal activity toward yeast tRNA was observed at a temperature of 70 degrees C and a pH of 7. The peptide was adsorbed on DEAE-cellulose, CM-Sepharose, and Q-Sepharose. The yield of the peptide was 7mg from 1.8kg mushroom.

Alocasin, an anti-fungal protein from rhizomes of the giant taro Alocasia macrorrhiza.

An anti-fungal protein designated alocasin was isolated from the rhizomes of the giant taro Alocasia macrorrhiza. The isolation protocol involved ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, ion exchange chromatography on sulfopropyl (SP)-Sepharose, and gel filtration on Superdex 75. Alocasin, which was unadsorbed on DEAE-cellulose and SP-Sepharose, possessed the N-terminal sequence APEGEV, which exhibited some similarity to that of the miraculin-like anti-fungal protein from Pisum sativum legumes. It demonstrated a molecular mass of 11kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, and displayed anti-fungal activity against Botrytis cinerea. Alocasin reduced the activity of HIV-1 reverse transcriptase. It exhibited weak hemagglutinating activity, only at a concentration of 1mg/ml.

Ascalin, a new anti-fungal peptide with human immunodeficiency virus type 1 reverse transcriptase-inhibiting activity from shallot bulbs.

An isolation procedure comprising ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and gel filtration on Superdex 75 was used to isolate an anti-fungal peptide from the bulbs of the shallot Allium ascalonicum. The peptide demonstrated a molecular weight of 9.5kDa, and possessed an N-terminal sequence YQCGQGG somewhat similar to chitinases from other Allium species which are however much larger in molecular weight. The peptide designated ascalin manifested a unique specific anti-fungal activity. It inhibited mycelial growth in the fungus Botrytis cinerea but not in the fungi Mycosphaerella arachidicola and Fusarium oxysporum. Ascalin inhibited HIV-1 reverse transcriptase with an IC(50) of 10 microM, much more potently than Allium tuberosum anti-fungal protein and other anti-fungal proteins.

Examination of lectins, polysaccharopeptide, polysaccharide, alkaloid, coumarin and trypsin inhibitors for inhibitory activity against human immunodeficiency virus reverse transcriptase and glycohydrolases.

A variety of lectins were tested in vitro for inhibitory action against the activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the N-glycohydrolases (alpha-glucosidase, beta-glucosidase and beta-glucuronidase). Lectins from Phaseolus vulgaris, Momordica charantia, Ricinus communis and its constituent chains, and Agaricus bisporus were able to inhibit HIV-1 reverse transcriptase. P. vulgaris lectin and A. bisporus lectin were the most potent. The aforementioned lectins had only weak or no inhibitory effects on the glycohydrolases. The inhibitory effect of polysaccharopeptide from the mushroom Coriolus versicolor on HIV-1 reverse transcriptase and alpha-glucosidase was enhanced after chemical modification with chlorosulfonic acid. However, the inhibitory effect of the algal polysaccharide fucoidan on HIV-1 reverse transcriptase and alpha-glucosidase was not augmented by sulfation. Trypsin inhibitors from Phaseolus lunatus and Glycine max, gossypol and alkaloids from Corydalis yanhusuo were able to inhibit HIV-1 reverse transcriptase. Dicoumarol was capable of inhibiting HIV-1 reverse transcriptase, alpha-glucosidase, beta-glucosidase and beta-glucuronidase.

A novel lectin from Pseudostellaria heterophylla roots with sequence simularity to Kunitz-type soybean trypsin inhibitor.

From the roots of the Chinese medicinal herb Pseudostellaria heterophylla a single-chained lectin with a molecular weight of 36 kDa and high hemagglutinating activity was isolated. The lectin was adsorbed on DEAE-cellulose in 10 mM Tris-HCI buffer (pH 7.4) and was eluted by the same buffer containing 50 mM NaCl. It was adsorbed on SP-Sepharose in 10mM NH4OAc (pH 4.5) and eluted by approximately 0.5 M NaCl in the same buffer. The hemagglutinating activity of the lectin could not be inhibited by a large variety of monosaccharides, but was largely abrogated by exposure to 0.05 M HCl, 0.05M NaOH or 80 degrees C. However, about 50% of the activity remained after exposure to 0.025M NaOH or 40 degrees C. Despite possession of an N-terminal sequence exhibiting some similarity to thaumatin-like proteins with antifungal activity, the lectin was devoid of antifungal activity. The lectin exerted some inhibitory effect on the glycohydrolases alpha-glucosidase, beta-glucosidase and beta-glucuronidase which are involved in HIV infection but had no suppressive action on human immunodeficiency virus-type 1 reverse transcriptase.