Wuxistatin, a novel and potent statin, is converted from lovastatin by Amycolatopsis sp. CGMCC1149. In the bioconversion, lovastatin is firstly hydroxylated by a hydroxylase. To obtain the critical hydroxylase, a novel hydroxylase gene was isolated from Amycolatopsis sp. CGMCC1149 by Degenerate PCR and Self-Formed Adaptor PCR and expressed in Escherichia coli. BLAST sequence analysis revealed that the gene belonged to cytochrome P450 gene superfamily and could encode a 403-amino-acid protein with a molecular weight of 44.8 kDa. The secondary structure prediction result showed that this protein contained many typical functional regions of P450, such as oxygen binding site, ion-pair region and heme binding region. Meanwhile, a catalytic function verification system was constructed by NADH, ferredoxin and ferredoxin reductase which could catalyze lovastatin hydroxylation into the target product. These would be helpful for further studies in large-scale production of wuxistatin.
Actinomycetales ; enzymology ; genetics ; Amino Acid Sequence ; Butyrates ; metabolism ; Cloning, Molecular ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Hydroxylation ; Industrial Microbiology ; Lovastatin ; metabolism ; Molecular Sequence Data

BACKGROUNDNeuropathic pain results from a lesion or disease affecting the somatosensory system at either the peripheral or central level. The transmission of nociception within the central nervous system is subject to modulation by release and reuptake of neurotransmitters, which maintain a dynamic balance through the assembly and disassembly of the SNARE complex as well as a series of neurotransmitter transporters (inhibitory GABA transporters GAT and excitatory glutamate transporters GT). Neuronal hyper-excitability or defected inhibition involved in neuropathic pain is one of the outcomes caused by imbalanced neurotransmission. SNAP-25, which is one of the SNARE complexes, can modulate the release of neurotransmitters. Glia glutamate transporter (GLT) is one of the two glutamate transporters which account for most synaptic glutamate uptake in the CNS. The role of SNAP-25 and GLT as well as GAT is not clearly understood.

METHODSWe used the rat chronic constriction injury (CCI) model for research, and degraded SNAP-25 by a single intrathecal administration of BoNT/A. The mechanical (MWT) and thermal withdrawal latency (TWL) were tested. The level of SNAP-25, GLT, and GAT-1 were assayed using RT-PCR and Western blotting.

RESULTSSNAP-25 was suppressed by a single intrathecal administration of 0.01U BoNT/A and the reduction of SNAP- 25 was correlated with the relief of nociceptive responses in CCI rats. MWT and TWL returned to normal from the 5th to 14th day (P < 0.05) after the administration. On the 14th day after surgery, compared to the sham group, the upregulation of SNAP-25 in CCI rats was reversed after BoNT/A treatment (P < 0.05). The decreased GLT was reversed after BoNT/A treatment but increased GAT-1 was not influenced by BoNT/A treatment.

CONCLUSIONSSNAP-25 and GLT play important roles in the development of neuropathic pain, and the mechanism may involve the imbalance of neurotransmission after peripheral nerve injury. Intrathecal administration of BoNT/A reversed the upregulation of SNAP-25 and downregulation of GLT after CCI, but had no significant effect on the expression of GAT-1.

Amino Acid Transport System X-AG ; genetics ; metabolism ; Animals ; Disease Models, Animal ; GABA Plasma Membrane Transport Proteins ; Male ; Neuralgia ; genetics ; metabolism ; Neuroglia ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Synaptic Transmission ; genetics ; physiology ; Synaptosomal-Associated Protein 25 ; genetics ; metabolism

OBJECTIVETo observe the occurrence of anastomotic bleeding following laparoscopic and open radical resection for rectal carcinoma, and to explore its contributing factors.

METHODSTwo hundred and sixty-three cases of rectal carcinoma undergone radical resection were divided into 2 groups, laparoscopic surgery (LS) group (n=86) and open surgery (OS) group (n=177). According to the different locations of anastomotic stoma and with or without preventive colostomy, the two groups were divided into AR sub-group and LAR/UAR sub-group, colostomy sub-group and non-colostomy sub-group. After analyzing the incidence of anastomotic bleeding in each sub-group, a logistic regression model was established to determine the relationships between anastomotic bleeding and three contributing factors including surgical approaches (LS or OS), location of stoma (AR or LAR/UAR) and preventive colostomy.

RESULTSAnastomotic bleeding occurred on 16 out of 263 patients with radical resection of rectal cancer (6.1%). The rates of anastomotic bleeding in LS group and OS group were 9.3% and 4.5%, in colostomy and non-colostomy were 8.1% and 5.5%, and in AR group and LAR/UAR group were 3.3% and 12.1% respectively, there were no significant differences between them (P>0.05). Comparing the two different surgical approaches (LS vs OS), the coefficient of regression, odd ratio and standard coefficient of regression for LS were 1.319, 3.741 and 0.342 respectively. In comparison of the locations of anastomosis (AR vs LAR/UAR), the three index for LAR/UAR were 2.460, 11.704, and 0.632 respectively. Comparing colostomy with non-colostomy, the three index for colostomy were -1.394, 0.248, and -0.327 respectively.

CONCLUSIONSAnastomotic bleeding after radical rectectomy is related to the choice of surgical approach, location of anastomosis and with or without preventive colostomy. Both LS and LAR/UAR are risk factors, and preventive colostomy is a protective factor. Regarding to the significance of three factors, location of anastomosis takes the first place, following by surgical method and with or without preventive colostomy.

Adult ; Aged ; Aged, 80 and over ; Anastomosis, Surgical ; adverse effects ; Colostomy ; adverse effects ; Female ; Humans ; Laparoscopy ; adverse effects ; Male ; Middle Aged ; Postoperative Hemorrhage ; etiology ; Rectal Neoplasms ; surgery

Objective To detect EIA factor expression in human rectal cancer and normal tissue and to determine whether it is correlated with invasion and metastasis of human rectal cancer. Methods Real- time RT-PCR was used to detect E1AF expression in matched rectal cancers and normal tissues from g6 in- patients.Results Among the 86 rectal cancer samples tested,55 cases E1AF mRNA overexpression was ob- served. The mRNA expression of E1AF in the sample group was remarkably different from that in the control group.In carcinomas,E1AF mRNA expression correlated significantly with histological type,depth of inva- sion,lymph node and distant metastasis and advanced Duke stage.Conclusion E1AF is correlated signifi- cantly with invasion and metastasis of human rectal cancer and may be an important factor in the invasion and metastasis.

Objective The study is to explore the extent, main-point and use of en bloc principle in re-operation of thyroid carcinoma. Methods Clinical data of 75 patients received re-operation from 2002 to 2006 because of nonstandard operation were reviewed, including 10 men, 65 women, age ranging from 21-56. lpailateral completing lobectumy, isthmectomy, ipsilateral anterior cervical muscle, and level Ⅵ dissetion were done in all the patients by en bloc principle. Results Residual tumor was found in 39 cases. There were, 10 in primary site, 8 in Ⅵ level, and 21 in beth. Therefore tumor residual rate was 52.0%. The total metastatic lymph nodes in Ⅵ level were 63. Among the eight patients with recurrent laryngeal nerves paralyses which were hurt in the first operation, 4 were found been cut completely, 4 were ligated. The ligated locations were all near the place which the nerve enter the larynx. The ligated nerves were released, and the patients voice was improved greatly. Conclusions Ipsilateral completing Iobectomy, isthmectomy, ipsilateral anterior cervical muscle and Ⅵ level dissetion is adequate for thyroid carcinoma patients who received nonstandard operation. The principle of en bloc resection can be used in the reoperation of thyroid carcinoma.

The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR, then the pPIC9K-IL-18 of fusion expression vector was constructed by inserting IL-18 fragment,and was transformed to GS115 by electroporation, multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol, SDS-PAGE was used to analyze expression product, fusion protein was purified by Sephadex G-100 column, bioactivity of IL-18 was measured by MTT assays. Experiment results show fusion protein of pIL-18 secreted by GS115,expression reaches the secretion peak of 160 mg/L at 72 h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in Pichia pastoris.
Animals ; Electrophoresis, Polyacrylamide Gel ; Electroporation ; Interleukin-18 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; analysis ; biosynthesis ; genetics ; Swine

BACKGROUNDIn patients suffering from acute pancreatitis, the pathogenesis is not completely understood, and several recent studies in vitro suggested that heat shock proteins might play an important role in cell signaling. To investigate the possible role of extracellular heat shock protein 70 (Hsp70) in pancreatitis, toll-like receptor-4 (TLR4)-deficient and wild-type mice were administered with exogenous Hsp70 during the course of cerulein-induced pancreatitis (CIP).

METHODSAcute pancreatitis was induced by 5 intraperitoneal injections of cerulein at hourly intervals, and then treated with recombinant Hsp70 through the caudal vein 4 hours after the start of cerulein injections. Subsequently serum amylase and serum cytokines levels were detected. Histologic alteration of the pancreas was evaluated. Tumor necrosis factor alpha (TNF-alpha) concentrations and myeloperoxidase (MPO) activity in both pancreas and lungs were analyzed. The nuclear factor kappa B (NF-kappaB) activation in pancreatic tissue was measured using a sensitive RelA enzyme-linked immunosorbent assay.

RESULTSTreatment with recombinant Hsp70 to wild-type mice in CIP resulted in significant aggravation of inflammation in pancreas, elevated levels of serum cytokines, up-regulation of pulmonary MPO activity and increase of lung tissues TNF-alpha concentrations. In contrast, treatment with Hsp70 to TLR4-deficient mice had little effect on serum cytokines levels, pancreatic inflammation, pulmonary MPO activity and TNF-alpha concentrations.

CONCLUSIONSThe results suggest that extracellular Hsp70 might induce systemic inflammatory response syndrome (SIRS)-like response in vivo and TLR4 might be involved in the Hsp70-mediated activation of inflammatory reaction in the progression of CIP without infection.

Acute Disease ; Animals ; Ceruletide ; toxicity ; Female ; HSP70 Heat-Shock Proteins ; physiology ; Male ; Mice ; Mice, Inbred C57BL ; Pancreatitis ; etiology ; Systemic Inflammatory Response Syndrome ; etiology ; Toll-Like Receptor 4 ; physiology

OBJECTIVETo study cardiopulmonary physiology during exercise in patients after extracardiac total cavopulmonary connection (ECTCPC).

METHODSTwenty-six patients were studied after ECTCPC by exercise testing with bicycle treadmill protocol. Heart rate (HR), blood pressure (BP), respiratory frequency (RF) and pulse oxygen saturation (SpO(2)) were measured continuously; twenty-six patients suffered from Fallot 4 underwent biventricular repair were also studied as control group.

RESULTSIn ECTCPC group, HR, BP, SpO(2) and RF all increased with exercise below 3 grade; when exceed 4 grade, BP, SpO(2) decreased and RF kept increasing. Compared with control group, HR, RF were higher (t = 2.13, P < 0.05; t = 2.31, P < 0.05), SpO(2) was lower (t = 2.46, P < 0.05) under the quiescent condition; When exceed 3 grade, HR, BP, SpO(2) decreased more significantly, but RF increased continuously. In fenestration group after ECTCPC, HR reached the top at 5 grades, but in group without fenestration it reached the top at 3 grades; In the whole process of exercising, RF kept higher and SpO(2) kept lower in fenestration group.

CONCLUSIONSThe ECTCPC patients showed obviously exercise limitation. Totally bypass of sinoatrial node in this operation may have some adverse effects on the integer regulation of HR.

Adolescent ; Adult ; Child ; Child, Preschool ; Exercise Test ; Female ; Follow-Up Studies ; Fontan Procedure ; methods ; Heart Defects, Congenital ; physiopathology ; surgery ; Humans ; Male ; Physical Endurance ; physiology ; Postoperative Period

OBJECTIVETo study the influences on mandibular development after removing the outer cortex of mandibular body in childhood minitype pig.

METHODSSix childhood minitype pigs were selected as the experimental animals. The outer cortex of mandibular body measured as 3.0 cm x 1.5 cm was removed in one side, and the other side remained intact as the control. The changes of mandibular modality and occlusion relationship as well as the histological and biomechanical changes were studied 24 weeks after operation.

RESULTSThere was no obvious difference compared with the control side in the height of the mandibular ramus and the length of the mandibular body, However, lateral deviation occlusion was found in some animals. The body thickness was thinner than that of the control side, there were no obvious biomechanical and histological differences between the two sides.

CONCLUSIONSThere was less influence on the growth of mandibular bone after removing one side of the outer cortex of the mandibular body in childhood minitype pig. But further study should be done for the cause of the lateral deviation of the mandible in part of the animals.

Animals ; Female ; Male ; Mandible ; growth & development ; surgery ; Mandibular Condyle ; surgery ; Osteotomy ; adverse effects ; Swine ; Swine, Miniature

OBJECTIVETo study the effects of inhaled nitric oxide (NO) on pulmonary vascular resistance in patients after total cavopulmonary connection (TCPC).

METHODSFifty-two patients after TCPC were evaluated, of them 24 patients were administered with inhaled nitric oxide in the early postoperative period. The cardiac index (CI) and pulmonary vascular resistance (PVR) were compared before and after inhaled NO.

RESULTSIn experimental group, after inhaled NO, partial pressure of oxygen in artery/fraction of inspired oxygen increased from 161 +/- 17 to 193 +/- 23 (t = 2.75, P < 0.01); CI from (2.86 +/- 0.24) L.min(-1).m(-2) to (3.13 +/- 0.22) L.min(-1).m(-2) (t = 2.25, P < 0.05); PVR decreased from (4.2 +/- 0.5) U/m(2) to (3.8 +/- 1.4) U/m(2) (t = 2.29, P < 0.05); central venous pressure (CVP) from (17.0 +/- 1.8) mm Hg to (15.0 +/- 1.1) mm Hg, decreased 11.7%. Compared with the control group, respirator time decreased from (86 +/- 27) h to (54 +/- 18) h (t = 2.29, P < 0.05); ICU time from (6 +/- 2) d to (4 +/- 2) d (t = 2.32, P < 0.05); But hydrothorax drainage and length of stay had no significant difference.

CONCLUSIONSThough inhaled NO, there is no significant long-term effects in patients after TCPC, but it may play an important role in the management of low cardiac output syndrome and high cava pressure caused by reactive elevated pulmonary vascular resistance in the early postoperative period of TCPC.

Administration, Inhalation ; Adolescent ; Adult ; Arteriovenous Shunt, Surgical ; methods ; Cardiac Output ; drug effects ; Child ; Child, Preschool ; Female ; Humans ; Male ; Nitric Oxide ; administration & dosage ; therapeutic use ; Postoperative Period ; Pulmonary Artery ; physiology ; surgery ; Time Factors ; Vascular Resistance ; drug effects ; Vasodilator Agents ; administration & dosage ; therapeutic use ; Venae Cavae ; surgery