食神鞘氨醇杆菌(Sphingo spiritivorum)肝素酶的制备和性质研究

筛选新型肝素酶产生菌,制备新型肝素酶并对其降解肝素能力进行测试。采用定向富集方法对土壤中的菌群进行培养,筛选分离产肝素酶的新种菌株,利用微生物发酵和蛋白纯化技术制备肝素酶,考察新型肝素酶的理化性质和酶切位点,之后使用新型肝素酶降解肝素并测试产物特征。筛选分离得到1株新的肝素酶产生菌,属食神鞘氨醇杆菌(Sphingo spiritivorum)。发酵后得到的粗酶,经多步柱层析纯化后得到的新型肝素酶SShep I和SShep II,用得到的酶降解肝素得到两种新型酶制低分子肝素。利用食神鞘氨醇杆菌中分离纯化得到的两种新型肝素酶SShep I和SShep II制备的新型酶制低分子肝素,有望开发成抗凝血效果更佳或具有新临床功能的低分子肝素药物。

刀额新对虾脑部胶质细胞和神经鞘

本文用透射电镜观察了刀额新对虾（Ｍｅｔａｐｅｎａｅｕｓ ｅｎｓｉｓ）脑部神经胶质细胞和神经纤维髓鞘；结果表明。这些胶质细胞呈圆形或椭圆形，直径约６４００ｎｍ。在胞质中，没有观察到有线粒体，高尔基体等细胞结构，细胞核大电子致密的异染色质位于核边缘且连成块状，髓鞘直径约６４００—９３００ｎｍ。鞘层厚约５００—６７０ｎｍ。在髓鞘层中存在着微管结构，微管直径约８３ｎｍ。

Ex vivo non-viral vector-mediated neurotrophin-3 gene transfer to olfactory ensheathing glia: effects on axonal regeneration and functional recovery after implantation in rats with spinal cord injury

Objective Combine olfactory ensheathing glia （OEG） implantation with ex vivo non-viral vector-based neurotrophin- 3 （NT-3） gene therapy in attempting to enhance regeneration after thoracic spinal cord injury （SCI）. Methods Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1 （＋）-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord （T9） contusion by the New York University impactor. The animals in 3 different groups received 4x 1050EG transfected with pcDNA3.1 （＋）-NT3 or pcDNA3.1 （＋） plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. Results NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1 （＋）-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. Conclusion Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT- 3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.

Expression of vascular endothelial growth factor and its receptors VEGFR-1 and 2 in gastrointestinal stromal tumors,leiomyomas and schwannomas

AIM： To investigate the role of vascular endothelial growth factor （VEGF/and its receptors VEGFR-1 and 2 in the growth and differentiation of gastrointestinal strornal tumors （GISTs）. METHODS： Thirty-three GISTs, 15 leiomyomas and 6 schwannomas were examined by immunohistochemistry in this study. RESULTS： VEGF protein was expressed in the cytoplasm of tumor cells, and VEGFRol and 2 were expressed both in the cytoplasm and on the membrane of all tumors. Irnrnunohistochernical staining revealed that 26 GISTs （78.8%）, 9 leiornyornas （60.0%） and 3 schwannornas （50.0%/were positive for VEGF; 24 GISTs （72.7%/, 12 leiornyornas （80.0%） and 4 schwannornas （66.7%） were positive for VEGFR-1; 30 GISTs （90.9%/, 5 leiornyornas （33.3%/and 4 schwannornas （66.7%） were positive for VEGFR-2. VEGFR-2 expression was statistically different between GISTs and leiomyomas （P 〈 0.0001）. However, there was no correlation between the expression of VEGF pathway componenets and the clinical risk categories. CONCLUSION： Our results suggest that the VEGF pathway may play an important role in the differentiation of GISTs, leiomyomas and schwannomas.

Recent Advances in Researches on Physiologically Active Substances in Holothurians

In this report, we reviewed recent literature on physiologically active substances from sea cucumbers (SCs) andtheir activities together with results obtained from our study. Preventive properties against lipid metabolism were reported inrats using a whole SC preparation with no particular constituent specified. Administration of the preparation lowered serumand hepatic cholesterol levels and improved the HDL/LDL ratio. These functions may be attributed to the stimulatory effectof the extract on the secretion of cholesterol in feces. Novel fucosylated chondroitin sulfates (FCSs) from Ludwigothureagrisea significantly induced fibroblast growth factor 2-dependent angiogenesis in human umbilical vein endothelial cells (HU-VECs). The proangiogenetic activity seemed attributable to the action of the sulfated fucose branches on the polysaccharide.SCs contain mycosporine-like amino acids (MAAs) that are capable of absorbing UV. A biogenetic precursor of MAAs wasfirst reported in SCs. The anti-proliferative effects of a branched chain fatty acid from a sea cucumber on prostate cancercells was reported with the activity of 5-lipoxygenase. Glycosphingolipid constituents in SCs have been systematically ana-lyzed over the past ten years. The results showed that the gangliosides in several SCs differed from those of mammals in thata sialic acid of SC gangliosides directly binded to glucose of cerebroside. Neuritogenic activity of the glycosphingolipids wasdemonstrated in vitro experiments and may lead to the development of therapeutic products for neurological disorders. Ourstudy also showed that sphingoid bases, the hydrolyzed products of glycosphingolipids from SCs, induced significant apoptosisin several tumor cell lines.

Sigmoid schwannoma: A rare case

Schwannomas are rare tumors derived from the cells of Schwann that form the neural sheath. When located in the gastrointestinal tract, they constitute together with leiomyoma, leiomyoblastoma, and leiomyosarcoma, the gastrointestinal stromal tumors (GIST). Peripheral nerve sheath tumors represent 2-6% GIST with most common location, the stomach and the small intestine. Schwannomas of the colon and rectum are extremely rare and radical excision with wide margins is mandatory, due to their tendency to recur locally and become malignant, if left untreated. In the present study, we report a rare case of a sigmoid schwannoma, which was successfully treated in our department and reviewed the literature.

Acute inflammatory demyelinating polyneuropathy associated with pegylated interferon α 2a therapy for chronic hepatitis C virus infection

The combination of pegylated interferon (Peg-IFN) and ribavirin is the standard of care for chronic hepatitis C virus (HCV) infection treatment. In general, common side effects related to this combination therapy are mild and are very well tolerated. However, peripheral neuropathy including demyelinating polyneuropathy related to Peg-IFN is extremely rare. We present the first case of an acute inflammatory demyelinating polyneuropathy (AIDP) associated with Peg-IFN-α 2a (Pegasys) after 16 wk of a combination therapy with Pegasys and ribavirin in a 65-year-old woman with chronic HCV infection. She developed tingling, numbness, and weakness of her upper and lower extremities and was hospitalized for acute neurological deficits. Her clinical course, neurological findings, an electromyogram (EMG), nerve conductions studies (NCS), muscle biopsy, and a sural nerve biopsy were all consistent with AIDP likely related to Pegasys use. The patient recovered completely with the use of intravenous immunoglobulin (IVIG) including physical therapy and neurological rehabilitation. It is very important that gastroenterologists and/or hepatologists recognize this rare neurological complication related to Peg-IFN treatment very early, since it requires a prompt discontinuation of therapy including an immediate referral to a neurologist for the confirmation of diagnosis, management, and the prevention of long-term neurological deficits.

Expression of Ets-1 proto-oncoprotein in gastrointestinal stromal tumors, leiomyomas and schwannomas

AIM： Gastrointestinal stromal tumors （GISTs） are rare. GISTs differ from other mesenchymal tumors of the gastrointestinal tract （e.g. leiornyomas and schwannomas）. The purpose of this study was to investigate the role of Ets-1 in the growth and differentiation of GISTs. METHODS： Twenty-eight GISTs, nine leiomyomas and six schwannomas were examined by immunohistochemical staining method for Ets-1 in this study. Specimens were selected from surgical pathology archival tissues at Nagasaki University Hospital. RESULTS： Ets-1 protein was expressed in the cytoplasm of cells in all of these tumors. Immunohistochemical staining revealed that 27 GISTs （96.4%）, six leiomyomas （66.7%）, and five schwannomas （83.3%） were positive for Ets-1. Ets-1 expression was statistically different between GISTs and leiomyomas （P〈0.005）. However, there was no correlation between Ets-1 expression and clinical risk categories. CONCLUSION： Ets-1 plays an important role in the growth and differentiation of GISTs, leiomyomas and schwannomas.

Mucosal Schwann cell “Hamartoma”:A new entity?

Schwannoma is a well-described,benign nerve sheath tumor of the soft tissue,but is rare in the gastrointestinal tract.Gastrointestinal schwannomas are often incidentally discovered as small polypoid intraluminal lesions.In this report,we describe the clinicopathologic and immunohistochemical features of a distinctive neural mucosal polyp composed of a diffuse cellular proliferation of uniform bland spindled cells in the lamina propria that entraps the colonic crypts.Immunohistochemical analysis revealed strong and diffuse positivity for the S-100 protein.To avoid confusion of these solitary colorectal polyps containing pure spindled Schwann cell proliferation in the lamina propria with neural lesions that have significant association with inherited syndromes,it is better to use the designation "mucosal Schwann hamartoma".

Research advancements of axonal remyelination in spinal cord injury

Spinal cord injuries(SCI)usually result in impairment of axonal conduction and sensorimotor function.There are no effective therapy to completely repair SCI.Axonal demyelination is very common as a pathologic change in SCI,and demyelination partly contributes to neural function impairment.So,it may be reasonable that remyelination of demyelinated axons become one of effective therapeutic targets for SCI treatment. Demyelination involves myelin breakdown and loss of myelin-forming cells(oligodendrocytes).The death of oligodendrocytes plays a key role in axonal demyelination in SCI.Recently a number of studies demonstrate that cell replacements could facilitate axonal remyelination and restore axonal conductive func- tion.Thus,it is expected that myelinogenetic cell transplantation(oligodendroglial lineage)will have good prospect as an effective therapy to improve axonal remyelination and restore neural function for SCI treat- ment in the near future.

Common bile duct schwannoma:A case report and review of literature

Schwannoma is a myelin sheath tumor complicated with neurofibroma, neurofibromatosis and neurogenic sarcoma. Peripheral nerve sheath tumors represent 2%-6% of gastrointestinal tract stromal tumors (GIST), but there are deficient data about location of neurogenic tumors in the biliary system and only nine cases of schwannoma of the extrahepatic biliary tract have been reported. These tumors are clinically non-specific. They are usually symptomatic by compressing the close or adjacent structures when being retroperitoneal, and their preoperative diagnosis is extremely difficult. This paper reviews the literature data and describes a case of schwannoma of the common bile duct associated with cholestasis in a healthy young woman, diagnosed and treated in our department. This case is of interest on account of the complexity of its diagnosis and the atypical macroscopic growth pattern of the tumor.

Association between HER-2 Over-Expression and Prognosis in Human Osteosarcoma:a Meta-Analysis

OBJECTIVE Various studies examining the relationship be-tween HER-2 over-expression and the response to chemotherapy and clinical outcome in patients with osteosarcoma have yielded inconclusive results.The purpose of the current study was to evaluate the relation of HER-2 status with the response to chemo-therapy and clinical outcome in osteosarcoma.METHODS We conducted a meta-analysis of 6 studies that evaluated the correlation between HER-2 status and histologic response to chemotherapy and 2-year survival.Data were syn-thesized in summary receiver operating characteristic curves and with summary likelihood ratios(LRs) and relative risk.RESULTS The quantitative synthesis showed that HER-2 status is not a prognostic factor for the response to chemotherapy.The positive LR was 1.27(95% conf idence interval,0.91～1.77),and the negative LR was 0.68(95% confidence interval,0.38～1.22).There was no significant between-study heterogeneity.HER2-positive status tended to be associated with a worse 2-year survival,but the overall results were not formally statistically signif icant.CONCLUSION HER-2 status is not associated with the histo-logic response to chemotherapy in patients with osteosarcoma,whereas HER-2 positive patients may be associated with decreased survival.

Comparative analysis of CT and pathological findings of peripheral nerve sheath tumors

Objective: To improve the qualitative diagnosis of peripheral nerve sheath tumors by computed tomography (CT). Methods: CT findings of 64 cases of pathologically confirmed nerve sheath tumors were compared with the pathological findings of the tumors. Results: Low density of the tumors shown in plain CT images was related to dominating reticular structure in the tumor as found pathologically. Tumors with intact capsule found by pathological findings were shown with smooth margin in CT images. Inhomogeneous density and enhancement of the tumors in CT images was related to tumor necrosis, liquefaction and cystic degeneration, and inhomogeneous enhancement also involved the reticular structure. Conclusion: Nerve sheath tumors are characterized by distribution along the nerves, lower density than that of muscles in plain CT images, and inhomogeneous enhancement in enhanced CT, which can help differentiate nerve sheath tumors from other soft tissue tumors. When nerve sheath tumors lack distinctive CT features, the diagnoses have to depend on their pathological findings.

The yins and yangs of ceramide

Since their discovery over 100 years ago, sphingolipids have caught the eyes and the imagination of scientists.Modern science has made many new insights on the cell biology and day-to-day functions of many integral sphingolipids, especially those of ceramide. Ceramide is recognized as a vital second messenger in the signal transduction process mediated by receptors of many cytokines and growth factors.A great part of our current understanding of ceramide has been achieved from apoptosis-related studies, however recent data in the fields of immunology, endocrinology and neurobiology, also suggest a fundamental involvement of ceramide in the onset of diseases. Therefore, understanding the biology of ceramide could be a key to unraveling many biological mechanisms and provide information for the treatment of some common diseases.

THE ANALYSIS OF NF2 GENE MUTATION IN SPORADIC SCHWANNOMAS

Objective To analyze the mutation of NF2 gene (exon 2,4,6 and 13) in schwannomas. Methods The NF2 gene mutation in 36 schwannomas were observed by PCR-SSCP and DNA sequence. The proliferative index of schwannoma was detected by immunohistochemistry. Results We found 13 mutations in 36 schwannomas, including 6 deletion or insertion resulting in a frameshift, 2 nonsense mutations, 2 missense mutations, and 3 alterations affecting acceptor or donor of splicing sites in E4,E6,E13. The proliferative index of schwannomas with mutation were significantly higher than those without mutation(P<0.05). Conclusion NF2 gene mutation is the frequent event in the tumorigenesis of schwannomas, and there is some correlation between the mutation and clinical behavior(tumor proliferation).

P2X4 receptor and brain-derived neurotrophic factor in neuropathic pain

Objective:To investigate whether the activation of p38MAPK is involved in the neuropathic pain induced by P2X4 receptor,and the effects of activated P2X4 receptor and p38MAPK on expression of brain-derived neurotrophic factor (BDNF) in the chronic neuropathic pain.Methods:Lumbar intrathecal catheters were chronically implanted in male Sprague-Dawley rats.The right sciatic nerve was loosely ligated proximal to the sciatica's trifurcation at approximately 1.0 mm intervals with 4-0 silk sutures.The microglia inhibitor minocycline,P2X4 antagonist (TNP-ATP) and p38MAPK inhibitor (SB203580) were intrathecally administered every 12 h,3 d post-chronic constriction injury (CCI).Mechanical nociceptive thresholds were assessed with the paw withdrawal threshold (PWT) to von Frey filaments.The expression of P2X4 and BDNF were assessed by both immunohistochemical analysis and RT-PCR.Results:Intrathecal injection of minocycline or TNP-ATP or SB203580 significantly attenuated CCI-induced mechanical allodynia.The time courses of P2X4 receptor and BDNF expression were increased at all points after CCI and reached a peak level on postoperative d 7.Intrathecal injection of minocycline or TNP-ATP or SB203580 markedly suppressed the increase of CCI-induced P2X4 receptor and BDNF expression in the spinal cord.Conclusion:The activation of P2X4 receptor BDNF pathways contributes to neuropathic pain in CCI rats,and the activation of p38MAPK is involved in the neuropathic pain induced by P2X4 receptor.

Intrathecal injection with methotrexate plus dexamethasone in the treatment of central nervous system involvement in systemic lupus erythematosus

Abstract:Objective To investigate the effect of intrathecal injection (IT) with methotrexate (MTX) plus dexamethasone (DXM) in treating central nervous system involvement in systemic lupus erythematosus (CNS lupus). Methods Twenty-four CNS lupus patients that were refractory to conventional steroid therapy were selected for IT with MTX 10-20?mg plus DXM 10-20?mg. The effects and side effects of IT were closely observed. Results The symptoms and signs of 22/24 (91.7%) CNS lupus patients receiving IT improved considerably. Cerebrospinal fluid pressure,protein and WBC levels declined from 201.5±155.4?mm?H2O, 145.2±87.6?mg/dl and 25.1±14.3/mm3 to 128.7±108.1?mm?H2O, 60.8±38.3?mg/dl and 6.8±2.1/mm3 respectively. Transient side effects were observed in 4 patients: 1 with itching sensation of lower limbs, 2 with headache and 1 with incontinence.Conclusion IT with MTX plus DXM is a promising method for treating CNS lupus and deserves further investigation.

Sphingomyelin synthase activity measurement by the fluorescent product in cell culture medium or animal plasma

Sphingolipids, a new class of lipid mediators, are involved in a variety of important physiological and pathological processes. Sphingomyelin synthase （SMS） is an enzyme to convert the ceramide （Cer.） and phosphatidylcholine into sphingomyelin （SM） and diacylglycerol, which plays a key role in sphingolipid biosynthesis. Two SMS isoforms, SMS1 and SMS2, have been identified with different subceUular localizations and expression level in tissues. Previous studies have shown that SMS may serve as a potential therapeutic target for the treatment of various diseases, such as cardiovascular and metabolic diseases. Thus, there is an urgent need for a rapid and sensitive method for SMS activity analysis. In our study, we developed a novel method for SMS activity by monitoring the appearance of the product, NBD-SM, in the tissue culture medium or blood and applied this method in cells and mice. In Huh7 cells, the interassay coefficient of variation of the SMS activity assay was （3.60±0.07）% . In wild type （WT） mice, we observed accumulation of NBD-SM in blood in a time dependent fashion. In SMS2 KO mice, NBD-SM in plasma collected at 5- （0%, P〈0.01）, 30- （16%, P〈0.01）, and 60 min （21%, P〈0.01） after injection of fluorescence liposome solution was significantly decreased compared with WT mice. However, in SMS1 KO mice, NBD-SM in plasma collected 5- and 30 min is similar to that in WT mice. Our results suggest that this method could be used for SMS activity measurement in vitro and in vivo.

Repairing peripheral nerve defects with tissue engineered artificial nerves in rats

Objective： To observe the effect of tissue engineered nerves in repairing peripheral nerve defects （ about 1. 5 cm in length） in rats to provide data for clinical application. Methods： Glycerinated sciatic nerves （2 cm in length） from 10 Sprague Dawiey （SD） rats （aged 4 months） were used to prepare homologous dermal acellular matrix. Other 10 neonate SD rats （aged 5-7 days） were killed by neck dislocation. After removing the epineurium, the separated sciatic nerve tracts were cut into small pieces, then digested by 2.5 g/L trypsin and 625 U/nd collagenase and cultured in Dulbecco＇ s modified Eagle＇ s medium （DMEM） for 3 weeks. After proliferation, the Schwann cells （SCs） were identified and prepared for use. And other 40 female adult SD rats （ weighing 200 g and aged 3 months） with sciatic nerve defects of 1.5 cm in length were randomly divided into four groups： the defects of 10 rats bridged with proliferated SCs and homologous dermal acellular matrix （the tissue engineered nerve group, Group A）, 10 rats with no SCs but homologous dermal acellular matrix with internal scaffolds （Group B ）, 10 with autologous nerves （Group C ）, and the other 10 with nothing （the blank control group, Group D）. The general status of the rats was observed, the wet weight of triceps muscle of calf was monitored, and the histological observation of the regenerated nerves were made at 12 weeks after operation. Results： The wounds of all 40 rats healed after operation and no death was found. No foot ulceration was found in Groups A, B and C, but 7 rats suffered from foot ulceration in Group D. The triceps muscles of calf were depauperated in the experimental sides in all the groups compared with the uninjured sides, which was much more obvious in Group D. The wet weight of triceps muscle of calf and nerve electrophysiologic monitoring showed no statistical difference between Group A and Group C, but statistical difference was found between Groups A and B and Groups B and D. And significant statistical difference was found between Group B and Group D. Obvious compound muscle （ or motor） action potential （CMAP） could be evoked in Group A and Group C, but the evoked amplitude was very low in Group B and Group D. The axons of regenerated nerves penetrated through the whole graft in Group A and Group C, and partly penetrated through the graft in Group B, but did not penetrate in Group D. The two tips of the separated sciatic nerves of Groups A , B , and C were connected together, without formation of neuroma. But those of Group D were not connected together and neuroma formed in 6 rats. Conclusions： Tissue engineered nerves can be used for repairing long defects of the peripheral nerves of rats and ideal repairing effects can be obtained.