The use of medicinal leech(Hirudo medicinalis) has been increased during the past years and is a very common procedure for treatment of venous congestion in flaps and replanted part in plastic surgery. One of the main complication in using leeches is infection. We used the medicinal leeches in venous congestion after total ear reconstruction(8 cases) and replanted finger(1 case). A bacteriologic study of the leeches is undertaken before applying it to the patient and an antibiotic susceptibility test was also performed. In 6 patients. 6 pathogenic organisms were identified, and in 2 cases 2 different organisms were identified simultaneously.
Ear ; Humans ; Hyperemia ; Leeches ; Surgery, Plastic

OBJECTIVETo study the anticoagulant constituents in dried leech (Whitmania pigra Whitman).

METHODThe plasma recalcification time (PRT) as index, the constituents with anticoagulant activity were isolated and purified by anion-exchange chromatography on Sephadex DEAE A-50, gel permeation chromatography on Sephadex G-25 and Sephadex LH-20 columns, and then reversed phase high-performance liquid chromatography successively.

RESULTThree anticoagulant polypeptides were isolated and purified. Compounds 1 and 2 can be translated each other in natural conditions, and their molecular weights are 7100 and 5531, respectively. Compound 3 was identified as a pure polypeptide by HPLC and SDS-PAGE, and its molecular weight was determined as 8 608 by MALDI-TOF-MS. The amino acid composition of compound 3 was also determined.

CONCLUSIONCompound 3 was inferred to be a novel anticoagulant, and named whitmanin.

Animals ; Anticoagulants ; analysis ; chemistry ; isolation & purification ; Leeches ; chemistry ; Molecular Weight

Animals ; Bronchi ; Child ; Child, Preschool ; Foreign Bodies ; Humans ; Leeches ; Male

Whitmania pigra is the most widely distributed species of leeches in the market. In this study, the effect of heavy metal lead pollution on the anticoagulant activity of Wh. pigra was studied and the potential mechanism was explored. Pb(NO_3)_2 was used to contaminate the breeding soil which was then used to rear Wh. pigra for 50 days(lead-contaminated group, LC group), and meanwhile the blank control group(CG group) was set. Proteins were extracted from the obtained leech samples, and the differentially expressed proteins between LC and CG groups were analyzed by label-free proteomics technology. In this study, a total of 152 differentially expressed proteins were screened out, of which 93 proteins were up-regulated and 59 proteins were down-regulated in LC group. Bioinformatics analysis showed that the biological processes enriched with the differentially expressed proteins were mainly vesicle-mediated transport and transport positive regulation; the enriched cell components were mainly endocytosis vesicles and apical plasma membrane; the enriched molecular functions mainly included carbohydrate binding. The differentially expressed proteins were enriched in 76 KEGG pathways, which mainly involved metabolic pathways, biosynthesis of secondary metabolites, and bacterial invasion of epithelial cells. In this study, two differentially expressed proteins with Antistasin domain were presumed, which provides reference for further exploring the regulatory mechanism and signal transduction underlying the effect of lead pollution on the anticoagulant activity of leech.
Animals ; Anticoagulants/pharmacology* ; Environmental Pollution ; Leeches ; Metals, Heavy ; Proteomics

Protein kinase C(PKC) is a kind of kinase which is widely involved in cell proliferation and development. PKC(Wp-PKC) in Whitmania pigra body belongs to classic PKC. In order to investigate the effect of Wp-PKC on the development of Wh. pigra germ cells, 17β-estradiol(17β-E2)(100 ng·mL~(-1)) and methyltestosterone(MT)(150 μg·L~(-1)), 150 μg·L~(-1)(MT)+0.5 mg·L~(-1) PKC, 0.5 mg·L~(-1) PKC inhibitor were added to Wh. pigra culture water, and no addition group(control group) was added, and the effects on the development of Wh. pigra germ cells and the expression of Wp-PKC were observed. The results showed that: Wp-PKC in male gonads was always higher than that in female gonads; MT promoted the development of male gonads in Wh. pigra, while the expression of Wp-PKC was significantly higher than that in the control; 17β-E2 promoted the development of female gonads in Wh. pigra and Wp-PKC expression significantly lower than that of the control; while the development of the female and male gonads in the PKC inhibitor group was inhibited, the expression of Wp-PKC was significantly lower than that of the control. In summary, Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads.
Animals ; Estradiol ; Female ; Gonads ; Leeches ; Male ; Methyltestosterone ; Ovary

The objectives of study were to explore the effects of exogenous methyltestosterone( MT) on the growth and gonadal development of overwintering Whitmania pigra. Before overwintering,0. 1,1. 0,10. 0,100. 0,150. 0 μg·L^-1 of MT were added to the aquaculture water for 6 weeks. The changes of growth performance,gonad index,endogenous steroid hormones level and internal quality were measured after hibernate for 60 days. Then the tissue slice technique was used to observe the spermary( ovary) of Wh. pigra.The results showed that the body weight,survival rate and gonadal index increased first and then decreased with the increase of exogenous MT concentration; the male gland index was found the highest at the concentration of MT 10. 0 μg·L^-1 and the female gland index was the highest at the concentration of MT 1. 0 μg·L^-1. The survival rate of Wh. pigra peaked at the concentration of MT 10. 0 μg·L^-1.The weight reaches a peak at a concentration of MT 100. 0 μg·L^-1( P<0. 05). The number of primary spermatocytes in the testis was negatively correlated with the concentration of exogenous MT. The number of secondary spermatocytes and sperm cells increased first and then decreased. The concentration of secondary spermatocytes was the highest when the concentration of MT was 100. 0 μg·L^-1.The number and volume of oocytes in the ovary and the yolk granules increased first and then decreased with the increase of exogenous MT concentration,and the highest was observed at the MT concentration of 100. 0 μg·L^-1. The endogenous steroid hormone of Wh.pigra increased first and then decreased with the increase of exogenous MT concentration. The concentration of androgen and progesterone was the highest in MT 100. 0 μg·L^-1 treatment( P<0. 05),and the concentration of estrogen was found the highest in MT 10. 0 μg·L^-1 treatment( P<0. 05). After adding exogenous MT,Wh. pigra moisture content,acid-insoluble ash content,p H and anti-thrombin activity met the quality criterion of medicinal Wh. pigra in Chinese Pharmacopoeia( 2015 edition). In conclusion,the short-term addition of 1. 0-100. 0 μg·L^-1 exogenous MT before hibernation can promote the growth,the development of sperm cells and the antithrombin activity of Wh. pigra.
Animals ; Estrogens ; Female ; Gonads ; Leeches/physiology* ; Male ; Methyltestosterone ; Ovary ; Progesterone

HSP90 is a widely distributed molecular chaperone that participates in a variety of cellular processes and plays an important role in the meiosis of germ cells. However, its role in the gonadal development of hermaphroditic Whitmania pigra is not yet clear. To explore the effect of HSP90 on the germ cell development of Wh. Pigra, this study cloned the wpHSP90 gene, performed bioinformatics analysis, and measured its expression levels. The results showed that the cloned wpHSP90 was 2 592 bp in length, with an open reading frame(ORF) of 2 373 bp, encoding 790 amino acids. Prediction analysis revealed 85 phosphorylation modification sites on serine, threonine, and tyrosine residues of the wpHSP90 protein. Structural domain prediction and multiple sequence alignment results showed that wpHSP90 contained two conserved domains of HSP90 and exhibited the highest homology with Helobdella robusta, with a sequence similarity of 80.72%. RT-qPCR results showed that the relative expression level of wpHSP90 in the gonads of 5-month-old Wh. pigra was positively correlated with temperature within the range of 12 ℃ to 28 ℃. The expression level in the female gonads was significantly higher than in the male gonads and correlated with the trend of germ cell development in the ovaries and testes. In conclusion, wpHSP90 may be involved in regulating the development of germ cells, particularly oocytes, in Wh. pigra. This study provides a reference for further research on the gonadal development mechanism in Wh. pigra.
Animals ; Female ; Male ; Temperature ; Ovary ; Gonads ; Testis ; Leeches ; Cloning, Molecular

Studies on the variation of amylase, lipase and lrotease activity of Whitmania pigra in 0-6 months old using 3, 5-dinitro- salicylic acid colorimetry, right-nitrophenyl palmitate ester (ρ-NPP) colorimetry and folin-phenol method. The results showed that pro- tease activity remained low before 1.5 months old and with the highest activity in 2 months old, but after showing a small peak in 4 months, alkaline protease rapid declined. Amylase was low at born, then gradually increased the activity of the highest in 2.5 months old. Lipase with a strong vitality at birth, then 1 month with minimum and 2 months peaked, but appeared a small peak in 4 months old. In summary, only lipase exhibits strong activity at birth, lipase with the strongest activity in the digestive tract during develop- ment. Protease, lipase and amylase with the strongest activity at 2-3 months old, but were decreased after 4 months old.
Age Factors ; Amylases ; metabolism ; Animals ; Leeches ; enzymology ; Lipase ; metabolism ; Peptide Hydrolases ; metabolism

OBJECTIVETo study the extraction system of hirudin emulsion liquid membrane with the Poecilobdella manillensis as raw material, di-(2-ethylhexyl) phosphate (D2EHPA) as carrier, Span 80 as emulsifier, octane and D2EHPA mixed to constitute membrane solution, diluted HCl solutions as internal aqueous phase.

METHODUsing the orthogonal experiment to optimize the extraction conditions of hirudin reference substance such as membrane phase, internal aqueous phase volume ratio (MIPVR), external aqueous phase pH, internal aqueous phase pH, mobile carrier concentration and so on, and then using hirudin crude extracts to do purifying experiment, and gaining experimental samples.

RESULTThe optimal conditions of hirudin extraction were as follows: MIPVR 10: 3, internal aqueous phase pH 2.6, external aqueous phase pH 3.4, the mass fraction of carrier D2EHPA 2%. In the optimal extraction conditions, when the initial concentration of hirudin was one anti-thrombin activity units (ATU) x mL(-1), ATU recovery rate of the reference substance was 83.06%. In the purifying experiment of crude extracts, ATU recovery rate was 82.99%, and the specific activity of sample was 3 289.48 the ATU x mg(-1). Discontinuous polyacrylamide gel electrophoresis and spectral scanning, the results showed that the purity and reference substance were considerable.

CONCLUSIONThe method of preparation hirudin was relatively simple, the purity of the experimental samples and ATU recovery were both high.

Animals ; Emulsions ; chemistry ; Hirudins ; analysis ; isolation & purification ; Leeches ; chemistry ; Membranes, Artificial ; Solid Phase Extraction ; instrumentation ; methods

The dried Whitmania pigra is used for the treatment of cardiovascular and cerebrovascular diseases in traditional Chinese medicine. Bellamya purificata is widely distributed in the Chang Jiang River basin, it is natural diets of W. pigra. Current study was conducted to compare and analyze the nutritional ingredient in W. pigra, body fluid and flesh of B. purificata. Results showed that the contents of protein, crude fat and total sugar in W. pigra, body fluid and flesh of B. purificata were significantly different (P < 0.05). Protein content in W. pigra accounts up to 65.01%. The contents of inorganic elements and amino acid were abundant in W. pigra, body fluid and flesh of B. purificata. The content of essential amino acids in them were 32.6, 221.59, 40.78 mg x g(-1), respectively. The content of flavor amino acid in them were 27.51, 14.5, 32.03 mg x g(-1), while the coresponding content of antioxidant amino acid were 8.81, 5.91, 9.73 mg x g(-1), respectively. The individual amino acids of high content in them were Glu, Asp and Leu. Macro elements Ca, P, Mg and trace elements Zn, Si, Fe were abundant. It could be speculated that W. pigra may be a promising novel food, and the present results provide a foundation to develop artificial feed for W. Pigra.
Amino Acids ; analysis ; Animals ; Gastropoda ; chemistry ; Leeches ; chemistry ; Medicine, Chinese Traditional