SsdchA is a novel secretory cellobiohydrolase driving pathogenicity in Sclerotinia sclerotiorum

The necrotrophic fungus, Sclerotinia sclerotiorum, employs an array of cell wall-degrading enzymes(CWDEs), including cellulase, to dismantle host cell walls. However, the molecular mechanisms through which S. sclerotiorum degrades cellulose remain elusive. Here, we unveil a novel secretory cellobiohydrolase, SsdchA, characterized by a signal peptide and a Glyco_hydro_7(GH7) domain. SsdchA exhibits a robust expression of during early infection stages. Interestingly, colony morphology and growth rates remain unaffected across the wild-type, SsdchA deletion strains and SsdchA overexpression strains on potato dextrose agar(PDA) medium. Nevertheless, the pathogenicity and cellobiohydrolase activity decreased in the SsdchA deletion strains, but enhanced in the SsdchA overexpression strains. Moreover,the heterologous expression of SsdchA in Arabidopsis thaliana leads to reduced cellulose content and heightened susceptibility to S. sclerotiorum. Collectively, our data underscore the pivotal role of the novel cellobiohydrolase SsdchA in the pathogenicity of S. sclerotiorum.

Calcium/calcimimetic via calcium-sensing receptor ameliorates cholera toxin-induced secretory diarrhea in mice

BACKGROUND Enterotoxins produce diarrhea through direct epithelial action and indirectly by activating the enteric nervous system.Calcium-sensing receptor(CaSR)inhibits both actions.The latter has been well documented in vitro but not in vivo.The hypothesis to be tested was that activating CaSR inhibits diarrhea in vivo.AIM To determine whether CaSR agonists ameliorate secretory diarrhea evoked by cholera toxin(CTX)in mice.METHODS CTX was given orally to C57BL/6 mice to induce diarrhea.Calcium and calci-mimetic R568 were used to activate CaSR.To maximize their local intestinal actions,calcium was administered luminally via oral rehydration solution(ORS),whereas R568 was applied serosally using an intraperitoneal route.To verify that their actions resulted from the intestine,effects were also examined on Cre-lox intestine-specific CaSR knockouts.Diarrhea outcome was measured biochemically by monitoring changes in fecal Cl-or clinically by assessing stool consistency and weight loss.RESULTS CTX induced secretory diarrhea,as evidenced by increases in fecal Cl-,stool consistency,and weight loss following CTX exposure,but did not alter CaSR,neither in content nor in function.Accordingly,calcium and R568 were each able to ameliorate diarrhea when applied to diseased intestines.Intestinal CaSR involvement is suggested by gene knockout experiments where the anti-diarrheal actions of R568 were lost in intestinal epithelial CaSR knockouts(villinCre/Casrflox/flox)and neuronal CaSR knockouts(nestinCre/Casrflox/flox).CONCLUSION Treatment of acute secretory diarrheas remains a global challenge.Despite advances in diarrhea research,few have been made in the realm of diarrhea therapeutics.ORS therapy has remained the standard of care,although it does not halt the losses of intestinal fluid and ions caused by pathogens.There is no cost-effective therapeutic for diarrhea.This and other studies suggest that adding calcium to ORS or using calcimimetics to activate intestinal CaSR might represent a novel approach for treating secretory diarrheal diseases.

THE SECRETORY STIGMAS IN POPULUS: DEVELOPMENT, CYTOCHEMISTRY, ULTRASTRUCTURE AND THEIR RELATION TO INTERSECTIONAL INCOMPATIBILITY *

The stigmas of five species, Populus euphratica Oliv., P. alba L., P. simonii Carr., P. lasiocarpa Oliv. and P. nigra L. have been studied. Scanning electron microscopy re veals that exudates are present in the intercellular spaces, in the clefts between the multicel lular papillae and on the receptive surface. Release and movement of exudates can be visual ized when the fresh stigmas are stained with sudan Ⅲ and auramine O. Paraffin and semi thin resin sections of stigmas after glutaraldehyde osmium fixation evidence the lipidic nature of the exudates. Transmission electron microscopy reveals the glandular features of the stigmatic papillae cells, such as abundance of rough and smooth endoplasmic reticulum, polyribosomes, and well developed dictyosomes with secretory vesicles. Pellicle and epicuticular lamellate layers which have been considered as typical features of the dry type stigmas are also present in the species where stigmas appear extremely wet. It is concluded that stigmas in all of the five species are secretory at the receptive stage. Well developed generative and sperm cells were observed in the pollen tubes penetrating through the deep layers of the stigmatic tissue in the reciprocal crosses between P. euphratica and P. simonii, which indicated that there is no significant barrier in the stigma.

Paracrine and endocrine actions of bone——the functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts

The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin（SOST） that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influencea A osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or ＂osteokines＂from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin（OCN） secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2（LCN2） is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain.We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.

Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation

Rat protein DE is an androgen-dependent cysteine-rich secretory protein （CRISP） synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target. Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 （S2）. The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx- 1 （CRISP-2） was capable of binding to rodent eggs, human epididymal AEG-related protein （ARP） and helothermine （from lizard saliva） were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx- 1 and anti-Tpx- 1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx- 1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods. （Asian J Androl 2007 July; 9： 528-532）

Clinicopathologic and molecular characteristics of 44 patients with pure secretory breast carcinoma

Objective: Secretory breast carcinoma(SBC) is a rare type of breast malignancy, accounting for less than 0.02% of all infiltrating breast malignancies. The pure SBC, a type of SBC without another type of breast malignant neoplasm, is particularly rare. This study aimed to investigate the clinicopathologic and molecular features of pure SBC.Methods: The main pathological parameters such as estrogen receptor(ER), progesterone receptor(PR), and human epithelial growth factor receptor 2(C-erbB-2) were detected by immunohistochemistry(IHC), and the clinicopathologic and prognostic difference were compared with invasive ductal carcinoma(IDC). Fluorescent in situ hybridization(FISH) and reverse transcription polymerase chain reaction(RT-PCR) was performed to identify the ETV6-NTRK3 rearrangement of SBC.Results: We found that the positivity rates of ER, PR, C-erbB-2, p53, and S-100 were 47.7%(21/44), 52.3%(23/44), 36.4%(16/44), 27.3%(12/44), and 95.5%(42/44), respectively, which were higher than those reported in previous studies. Special periodic acid-Schiff analysis was performed in 36 patients, and the value of the Ki-67 index ranged from 1% to 50%(mean value:10%). Interestingly, most patients with pure SBC harbored an ETV6-NTRK3 rearrangement with an 88.6%(39/44) expression rate. Compared with IDC, the tumor size of most patients with SBC was larger than 2 cm(P = 0.024). Ultrasound showed benign lesions, and the total misdiagnosis rate was higher(P = 0.020). Although the pathological classification was mostly triple-negative breast cancers(P = 0.036), there was less metastasis(P = 0.029), and the overall prognosis was better than that of the IDC group.Conclusions: Although axillary lymph node metastasis, local recurrence, or distant metastasis may occur, SBC is also considered an indolent neoplasm with a good prognosis. Once diagnosed, surgical treatment should be performed as soon as possible,followed by appropriate adjuvant chemotherapy, irradiation, and endocrine therapies.

Balloon dilation of Eustachian tube combined with tympanostomy tube insertion and middle ear pressure equalization therapy for recurrent secretory otitis media

Objective: To report outcomes of balloon dilation Eustachian tuboplasty combined with tympanostomy tube insertion and middle ear pressure equalization therapy in treatment of recurrent secretory otitis media. Methods: Fifty one patients with recurrent secretory otitis media (62 ears) underwent balloon dilation of Eustachian tube and tympanic tube insertion under general anesthesia, followed by long term middle ear pressure equalization therapies. The Eustachian tube score (ETS) and Eustachian tube function questionnaire (ETDQ-7) were used for pre- and postoperative (up to 12 months) evaluation of Eustachian tube functions. Results: The mean ETS score was 2.34 ± 0.97 preoperatively, and 6.17 ± 1.54, 7.23 ± 1.62, 8.24 ± 1.97, and 7.63 ± 1.86 at 1, 3, 6 and 12 months postoperatively, respectively (P < 0.05). The ETDQ-7 score was 4.82 ± 1.07 preoperatively, and 2.20 ± 0.54, 2.32 ± 0.68, 2.53 ± 0.79, and 2.67 ± 0.76 at 1, 3, 6 and 12 months postoperatively, respectively (P < 0.05). Conclusion: Balloon dilation of Eustachian tube combined with tympanostomy and catheterization resulted in significant improvement of subjective symptoms and objective evaluation of Eustachian tube functions in most patients with recurrent secretory otitis media, as indicated by the ETS and ETDQ-7 scores, demonstrating high levels of efficacy and patient satisfaction.

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory /secretory product

AIM： To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory （ES） product. METHODS： NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA （cDNA） array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semiquantitative SYBR-based real-time RT-PCR. RESULTS： Among a total of 15 000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serinethreonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O.viverrini ES product, The expression level of signal transduction genes, pkC, pdgfra, jak 1, eps 8, tgfβ 1/4,strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC,eps 8 and tgfβ3 1/4 which are the downstream signaling molecules of either epidermal growth factor （EGF） or transforming growth factor-β （TGF-β） showed statistical significance （P 〈 0.05）.CONCLUSION： O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-β and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

Expression of serum human epididymal secretory protein E4 at low grade and high grade serous carcinomas

Objective:To investigate the value of serum human epididymis protein 4(HE4) in differential diagnosis of patients with low-grade serous(LGSC) and high-grade serous carcinoma(HGSC) serous ovarian cancer.Methods:LGSC and HGSC serous ovarian cancer were diagnosed by the two-tier grade system,serum levels of HE4 and carbohydrate antigen 12S(CA125) were measured by ELBA and radioisotope method,respectively in 60 serous ovarian cancer patients. HE4 and TPS3 protein in cancer tissue were measured by immunohistochemical method. Results:The difference in density of HE4 and TP53 protein was significant between LGSC and HGSC tissue,while serum CA12S did not show significant difference between different serum samples.There was significant difference in serum HE4 levels between LGSC and HGSC and the result was different within FIGO(Ⅰ+Ⅱ) stage,suggesting HE4 was not a reliable biomarker for the discrimination between LGSC and HCSC.HE4 had potential as a biomarker for the discrimination between LGSC and HGSC but the role in early diagnosis was limited.Conclusions:HE4 may be a reliable marker for differential diagnosis of LGSC and HGSC.But its role in early diagnosis of LGSC and HGSC need to be confirmed from the perspective of two-tier grade system.

Construction and Secretory Expression of β-Galactosidase Gene from Lactobacillus Bulgaricus in Lactococcus Lactis

Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.

Structure and function of epididymal protein cysteine-rich secretory protein-1

Cysteine-rich secretory protein-1 （CRISP-1） is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP- 1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract. （Asian J Androl 2007 July; 9： 508-514）

Animal secretory endolysosome channel discovery

Secretory pore-forming proteins(PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs(af-PFPs) and trefoil factors(TFFs). As a toad af-PFP, Bm ALP1 can be reversibly regulated between active and inactive forms, with its paralog Bm ALP3 acting as a negative regulator. Bm ALP1 interacts with Bm TFF3 to form a cellular active complex called βγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of βγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence,we hereby propose a secretory endolysosome channel(SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with βγ-CAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms.

Secretory Expression of E2 Main Antigen Domain of CSFV C Strain and the Establishment of Indirect ELISA Assay

The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21–CodonPlus (DE3)–RIL using the pGEX-4T-1 expression vector and the soluble recombinant product was purified with Glutathione Sepharose TM4B by centrifugation. The soluble recombinant protein showed good immune reactions and was confirmed by Western blot using anti-CSFV-specific antibodies. Then an indirect ELISA with the purified E2 protein as the coating antigen was established to detect antibody against CSFV. The result revealed that the optimal concentration of coated antigen was 0.6 μg/well and the optimal dilution of serum was 1:80. The positive cut-off value of this ELISA assay was OD tested serum / OD negative serum≥2.1. The E2-ELISA method was evaluated by comparison with the indirect hemagglutination test (IHAT). When a total of 100 field serum samples were tested the sensitivity and specificity were 90.3% and 94.7% respectively. Specificity analysis showed that there were no cross-reactions between BVD serum and the purified E2 protein in the E2-ELISA.

Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa

Aim： To determine the cellular distribution of secretory phospholipase A2 （sPLA2） in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. Methods： Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA2 and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used. Results： Although sPLA2 was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA2 was associated with enhanced binding of annexin V-fluoroscein isothiocyanate （FITC） to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA2, disturbance of acrosome structure, and loss of vitality. Conclusion： The ability of spermatozoa to release secretory phospholipase A2 is related to the acrosomal state. Premature destabi- lization of the acrosome and loss of sPLA2 can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA2 in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM:To determine whether SP-TAT-apoptin induces apoptosis and also maintains its tumor cell specificity. METHODS:In this study,we designed a secretory protein by adding a secretory signal peptide(SP) to the N terminus of Transactivating Transcription(TAT)-apoptin(SP-TAT-apoptin),to test the hypothesis that it gains an additive bystander effect as an anti-cancer therapy. We used an artificial human secretory SP whose amino acid sequence and corresponding cDNA sequence were generated by the SP hidden Markov model. RESULTS:In human liver carcinoma HepG2 cells,SP-TAT-apoptin expression showed a diffuse pattern in the early phase after transfection. After 48 h,however,it translocated into the nuclear compartment and caused massive apoptotic cell death,as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and annexin-V binding assay. SP-TAT-apoptin did not,however,cause any cell death in non-malignant human umbilical vein endothelial cells(HUVECs). Most importantly,the conditioned medium from Chinese hamster ovary(CHO) cells transfected with SP-TAT-apoptin also induced significant cell deathin HepG2 cells,but not in HUVECs. CONCLUSION:The data demonstrated that SP-TAT-apoptin induces apoptosis only in malignant cells,and its secretory property might greatly increase its potency once it is delivered in vivo for cancer therapy.

Changes in secretory pathway Ca^(2+)-ATPase 2 following focal cerebral ischemia/reperfusion injury

This study aimed to investigate changes in secretory pathway Ca2＋-ATPase 2 expression following cerebral ischemia/reperfusion injury, and to define the role of Ca2＋-ATPases in oxidative stress. A rat model of cerebral ischemia/reperfusion injury was established using the unilateral middle cerebral artery occlusion method. Immunohistochemistry and reverse transcription-PCR assay results showed that compared with the control group, the expression of secretory pathway Ca2＋-ATPase 2 protein and mRNA in the cerebral cortex and hippocampus of male rats did not significantly change during the ischemic period. However, secretory pathway Ca2＋-ATPase 2 protein and mRNA expression reduced gradually at 1, 3, and 24 hours during the reperfusion period. Our experimental findings indicate that levels of secretory pathway Ca2＋-ATPase 2 protein and mRNA expression in brain tissue change in response to cerebral ischemia/reperfusion injury.

Research advances of secretory proteins in malignant tumors

Secretory proteins in tumor tissues are important components of the tumor microenvironment.Secretory proteins act on tumor cells or stromal cells or mediate interactions between tumor cells and stromal cells,thereby affecting tumor progression and clinical treatment efficacy.In this paper,recent research advances in secretory proteins in malignant tumors are reviewed.

Evaluation of the Clinical Efficacy of Qingqiao Capsule (清窍胶囊) in Treating Patients with Secretory Otitis Media

Objective： To observe the clinical efficacy of Qingqiao Capsule （清窍胶囊, QQC） in treating patients with secretory otitis media （SOM）. Methods： A total of 90 patients were randomly assigned into the treated group （n：45） and the control group （n=45）. Patients in the treated group were administrated with QQC, 5 capsules each time, 3 times a day for totally 10-14 days, and those in the control group were given per os cefaclor capsules 0.5g each time for adult, 3 times a day, or 20mg/（kg·d） for children, for 10-14 days. The therapeutic efficacy of treatment on the patients was observed and compared after treatment and followed up for 3-6 months. Results： （1） The clinical efficacy in the treated group was superior to that in the control group with significant statistical difference （P〈0.01）; （2） Comparison of the efficacies in patients of three different TCM syndrome types （the external pathogenic wind invasion caused auditory orifice stuffiness type, the Gan-Dan damp-heat steaming up auditory orifice type and the Pi-deficiency dysfunction induced dirty dampness blocking ear type） showed no statistically significant difference（P〉0.05）; （3） The vanishing rate and time needed of the main symptoms and signs in the treated group were superior to those in the control group on ear muffle, tinnitus, hearing impairment, hydrotypanum, pure tone threshold and abnormal tongue figure, and the difference was statistically significant （P〈0.05 or P〈0.01）, only those of earache, otopiesis and abnornal pulse figure were insignificantly different between the two groups （P〉0.05）. Conclusion： QQC is an effective Chinese composite medicine on patients with SOM, and shows no obvious adverse reaction.

Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris

AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coilyeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GSl15 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Hut ＋ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS： The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g （DCW）/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION： The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

The key target of neuroprotection after the onset of ischemic stroke: secretory pathway Ca^(2+)-ATPase 1

The regulatory mechanisms of cytoplasmic Ca2＋ after myocardial infarction-induced Ca2＋ overload involve secretory pathway Ca2＋-ATPase 1 and the Golgi apparatus and are well understood. However, the effect of Golgi apparatus on Ca2＋ overload after cerebral ischemia and reperfusion remains unclear. Four-vessel occlusion rats were used as animal models of cerebral ischemia. The expression of secretory pathway Ca2＋-ATPase 1 in the cortex and hippocampus was detected by immunoblotting, and Ca2＋ concentrations in the cytoplasm and Golgi vesicles were determined. Results showed an overload of cytoplasmic Ca2＋ during ischemia and reperfusion that reached a peak after reperfusion. Levels of Golgi Ca2＋ showed an opposite effect. The expression of Golgi-specific secretory pathway Ca2＋-ATPase 1 in the cortex and hippocampus decreased before ischemia and reperfusion, and increased after reperfusion for 6 hours. This variation was similar to the alteration of calcium in separated Golgi vesicles. These results indicate that the Golgi apparatus participates in the formation and alleviation of calcium overload, and that secretory pathway Ca2＋-ATPase 1 tightly responds to ischemia and reperfusion in nerve cells. Thus, we concluded that secretory pathway Ca2＋-ATPase 1 plays an essential role in cytosolic calcium regulation and its expression can be used as a marker of Golgi stress, responding to cerebral ischemia and reperfusion. The secretory pathway Ca2＋-ATPase 1 can be an important neuroprotective target of ischemic stroke.