Protein kinase C(PKC) is a type of protein kinase widely involved in cell proliferation and development, but the developmental mechanism in the gonads of androgynous animals is still unclear. In order to explore the role of protein kinase C in the development of Whitmania pigra germ cells, the Wh. pigra PKC(Wp-PKC) gene was cloned, bioinformatics analysis was conducted, and fluorescent quantitative PCR was used to analyze the expression of female and male gonads. The results showed that:(1)The cloned Wp-PKC had a full length of 2 580 bp, a relative molecular weight of 76 555.19, and contains an open reading frame encoding 670 amino acids, Wp-PKC was closely related to Danio rerio PKC-α and rat PKC-γ. The similarity of amino acid sequence was 55% and 58%.(2)The protein encoded by Wp-PKC had no signal peptide and was a hydrophilic protein. The secondary structure is mainly composed of random coils, α-helices, extended chains, folds and folds, with the largest proportion of random coils and α-helices. Wp-PKC protein does not contain a transmembrane domain. Multiple sequence alignment and domain prediction analysis show that Wp-PKC contains 4 conserved domains of classical protein kinase C.(3)Fluorescence quantitative results showed that the expression of Wp-PKC in Wh. pigra gonads was positively correlated with the development of germ cells, and the expression in male gonads was significantly higher than that in female gonads. In summary, Wp-PKC is a classic PKC, and Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads, and provide references for further research on the developmental mechanisms of Wh. pigra.
Animals ; Cloning, Molecular ; Female ; Gonads ; Leeches/genetics* ; Male ; Ovary ; Protein Kinase C/genetics* ; Rats

OBJECTIVETo evaluate the genetic differentiation and phylogenetic relationship between Whitmania pigra Whitman and Hirudo nipponia Whitman.

METHODBy the sequences of the rDNA internal transcribed spacer (ITS) the molecular phylogenetic tree was constructed by MP method using software MEGA 4.0.

RESULTThe average length of ITS was 857.2-861.2 bp. The A, T, G and C contents in this fragment were 25.12%, 28.28%, 17.34%, 29.29%, respectively. The GC content was higher than the AT content. Little sequence variation was observed in ITS gene fragments with in species, and transition in only 45 loci was revealed in 14 populations. 14 W. pigra and H. nipponia populations were clustered into 2 groups by MP phylogenetic tree.

CONCLUSIONThe results also showed that the intraspecific variation was dominated in variation types of W. pigra and H. nipponia. The classification result of W. pigra and H. nipponia and its adulterants based on DNA sequences are not totally consistent with those based on classification. It showed that a little of mutation of base in ITS sequences had occurred in the process of evolution, such as transition cites, transvertion cites among base or base gap.

Animals ; Base Sequence ; DNA, Ribosomal Spacer ; chemistry ; Leeches ; classification ; genetics ; Molecular Sequence Data ; Phylogeny

There are 60 species of blood-feeding land leeches, 50 species belonging to the family Haemadipsidae and 10 species belonging to the family Xerobdellidae. Despite recent papers on the land leeches, their taxonomic identification is not fully understood, especially at a species level. In Korea, there have been no historical records of the terrestrial leeches, but recently an unrecorded blood-feeding land leech was discovered at Gageo-do (Island), Korea. Molecular analysis was used to identify the species of 29 leeches collected from Mt. Dock-Sil in Gageo-do. Conventional PCR was conducted using nuclear 18S rRNA and mitochondrial cytochrome c oxidase subunit 1 (CO1) genetic marker. The 18S rRNA sequences revealed that the leeches share 99.9% identity with Haemadipsa rjukjuana (inhabiting Taiwan), and the CO1 sequences revealed that the leeches are very close to H. rjukjuana (inhabiting Taiwan). The CO1 sequences were separated into 2 categories, 1 with 94.6% and the other with 94.3% similarity to the H. rjukjuana L00115A (inhabiting Taiwan). This new finding of the land leech is the first record in Korea. In addition, the north range of the distribution of the blood-feeding leech (Hirudiniformes: Haemadipisidae) should be reconsidered including Korea.
Animals ; Base Sequence ; DNA, Mitochondrial/genetics ; Electron Transport Complex IV/*genetics ; Genetic Markers/genetics ; Leeches/*classification/*genetics ; Mitochondria/enzymology/genetics ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 18S/*genetics ; Republic of Korea ; Sequence Alignment ; Sequence Analysis, DNA

OBJECTIVETo study the effect of Shuxuetong injection on cerebral tissue and brain microvascular endothelial cell(BMEC) in secreting tissue-type plasminogen activator (tPA).

METHODThe experiment in vivo was designed for three groups: sham-operation group, focal cerebral ischemia model group, Shuxuetong injection-treated group. The gene expression and the activity of tPA on brains were detected by RT-PCR and chromogenic substrate analysis respectively after treatment for five days; The experiment in vitro: The cultured BMEC were divided into experimental group and control group. The Shuxuetong injection were added into experimental group and the BMECs in the experimatal groups were treated for 24 h. The gene expression and the activity of tPA on cultured BMEC were detected by RT-PCR and chromogenic substrate analysis respectively.

RESULTThe gene expression and the content of tPA on both cerebral tissue and BMEC in the experimatal groups were significantly higher than that in the control groups.

CONCLUSIONShuxuetong injection could accelerate secretion of tPA, and therefore enhance fibrinolysis.

Animals ; Brain ; blood supply ; Brain Ischemia ; metabolism ; pathology ; Cells, Cultured ; Drug Combinations ; Endothelial Cells ; cytology ; metabolism ; Female ; Fibrinolytic Agents ; pharmacology ; Injections ; Leeches ; chemistry ; Male ; Materia Medica ; administration & dosage ; isolation & purification ; pharmacology ; Microcirculation ; cytology ; Oligochaeta ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Tissue Plasminogen Activator ; biosynthesis ; genetics

The present study was designed to identify immunomodulatory components from the leech salivary gland of Haemadipsa sylvestris. The Sephadex G-50, Resource(TM) S column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC) were used to isolate and purify the salivary gland extracts (SGE). Structural analysis of isolated compounds was based on Edman degradation and matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The cDNA encoding the precursor of the compound was cloned from the cDNA library of the salivary gland of H. sylvestris. The levels of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1) were assayed using an enzyme-linked immunosorbent assay (ELISA). The effects on cell proliferation and cell viability were observed using MTT assay. A novel neuropeptide Y (Neuropeptide Y-HS) from the leech salivary gland of H. sylvestris was purified and characterized. It was composed of 36 amino acid residues and the amino acid sequence was determined to be FLEPPERPAVFTSVEQMKSYIKALNDYYLLLGRPRF-NH2, containing an amidated C-terminus. It showed significant inhibitory effects on the production of inflammatory cytokines including TNF-α, IFN-γ, IL-6, and MCP-1. Neuropeptide Y was identified from leeches for the first time. The presence of neuropeptide Y-HS in leech salivary gland may help get blood meal from hosts and inhibit inflammation.
Amino Acid Sequence ; Animals ; Immunologic Factors ; administration & dosage ; chemistry ; genetics ; Inflammation ; drug therapy ; immunology ; Interferon-gamma ; immunology ; Interleukin-6 ; immunology ; Leeches ; chemistry ; Mass Spectrometry ; Mice ; Molecular Sequence Data ; Neuropeptide Y ; administration & dosage ; chemistry ; genetics ; Peptide Mapping ; Salivary Glands ; chemistry ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo explore curative machanism of Shenle capsule on the 5/6 nephrectomy rats.

METHODFibrin plate method was applied to examine activity of urinary plasminogen activator(PA). Semi-quantitative analysis was used to observe stained intensity and area of tissue-type plasminogen activator, urokinas-type plasminogen activator/ plasminogen activator inhibitor(tPA, uPA/PAI-1)in remnant renal tissue. Northern blot was employed to analyze the expression of transforming growth factor (TGF-beta) mRNA.

RESULTIn model control group, the urinary PA activity and protein expression of tPA, uPA were down-regulated, but protein expression of PAI-1, TGF-beta mRNA was up-regulated in remnant renal tissue. In each treated group, the urinary PA activity and protein expression of tPA/uPA were enhanced,but the protein expression of PAI-1, TGF-beta mRNA decreased simultaneously.

CONCLUSIONShenle capsule can delay glomerulosclersis and tubulointerstitial fibrotic lesions of remnant kidney by improving the activity of urinary PA and modulating the expression of tPA, uPA/PAI-1 and TGF-beta mRNA.

Animals ; Capsules ; Codonopsis ; chemistry ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Kidney ; metabolism ; Kidney Failure, Chronic ; drug therapy ; metabolism ; Leeches ; chemistry ; Male ; Materia Medica ; isolation & purification ; pharmacology ; Nephrectomy ; Plasminogen Inactivators ; metabolism ; Polyporales ; chemistry ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Tissue Plasminogen Activator ; metabolism ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Urokinase-Type Plasminogen Activator ; metabolism