Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage phiC31-derivative KC515( tsrR) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca2+ and Mg + concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10(3) /microg DNA on YMG medium supplemented with 10mmol/L MgSO4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105) were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S. hygroscopicus 17997 genome, and disruption of geldanamycin production was observed as a result of homologous recombination between the cloned insert in recombinant phage and the S. hygroscopicus 17997 genome by integration. Thiostrepton resistant transductants were selected and integration event was analyzed by Southern hybridization. The fermentation broth extracts from five resistant transductants were analyzed by TLC and HPLC. The results showed that only G16 mutant failed to produce geldanamycin. This result showed that the integration of the insert DNA fragment in recombinant phage phl6 into the chromosome of S. hygroscopicus disrupted the expression of the geldanamycin biosynthetic genes. The original cosmid pCGBK10 containing this cloned insert was predicted to encode PKS genes in the geldanamycin biosynthesis. This study laid the foundation for cloning the PKS genes involved in geldanamycin biosynthetic gene cluster from S. hygroscopicus 17997.
Alcohol Oxidoreductases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Bacteriophages ; genetics ; Benzoquinones ; metabolism ; Blotting, Southern ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Genetic Vectors ; genetics ; Lactams, Macrocyclic ; metabolism ; Multigene Family ; genetics ; physiology ; Polymerase Chain Reaction ; Streptomyces ; genetics ; metabolism

Many studies have shown that the incidence of type 1 diabetes mellitus (T1DM) is increasing with an annual of 2％ to 5％.As the same with type 2 diabetes mellitus,T1DM is also a risk factor for stroke.Although some T1DM studies have taken stroke as a component of the composite endpoint of cardiovascular events,a few studies have focused on the risk of stroke in patients with T1DM.Recent studies have shown that the risk of stroke in patients with T1DM is significantly higher than those without diabetes mellitus,especially in the population under the age of 50.In addition,the T1DM patients died of the risk of stroke are 3 to 4 times higher than the general population.This article reviews the relationship between T1DM and stroke.

Effective prevention is the best approach for reducing the burden of stroke.The reduction of low-density lipoprotein cholesterol with statins may decrease the risks of patients with the first ever stroke and ischemic stroke or recurrent stroke in patients with transient ischemic attack.In stroke prevention,the treatment of statins has become the most important advance following aspirin and antihypertensive treatment.This article reviews the roles of statins in the prevention of stroke.

Animals ; Brain-Derived Neurotrophic Factor ; metabolism ; Hypothalamus ; metabolism ; Male ; Physical Conditioning, Animal ; physiology ; Rats ; Rats, Sprague-Dawley

Adult ; Aged ; Aged, 80 and over ; Female ; Head and Neck Neoplasms ; surgery ; Humans ; Intubation, Intratracheal ; methods ; Laryngoscopes ; Male ; Middle Aged

Objective To investigate the therapeutic effect of quercetin on acute gouty arthritis and its influence in re-nal function in rats. Methods Seventy male Sprague-Dawley rats were randomly divided into normal control group, model group, colchicine group (0.5 mg/kg), allopurinol group (20 mg/kg), quercetin 100, 200 and 400 mg/kg groups (n=10 for each group). Rats were administered various drugs by oral gavage once a day for seven consecutive days throughout the experi-ment. On the fifth day, the animal model of acute arthritis was set up by giving monosodium urate crystal combined with hypo-xanthine. The inflammatory reaction was detected by measuring the circumference of right hind leg anklejoint with a tie line method at 0, 2, 6, 12, 24 and 48 h. The swelling ratio was calculated. The serum levels of uric acid (UA),β2-microglobulin (β2-MG), cystatin C (Cys-C), urea nitrogen (Urea) and creatinine (Cr) were detected by colorimetry and enzyme-linked im-munosorbent assay.Rats were sacrificed at the end of experiment, and the kidney was weighed and the renal index was calcu-lated. Results Treatment with quercetin, colchicine or allopurinol can significantly attenuate swelling rate in rats of acute gouty arthritis. The serum levels of UA were significantly higher in colchicine group and quercetin group than those of nor-mal control group and allopurinol group. The serum levels of UA was significantly lower in allopurinol group than that of nor-mal control group. After 48-h modeling, there was no significant difference in serum UA level between seven groups except allopurinol group. The levels ofβ2-MG and Cys-C were the lowest in normal control group than those of other groups. The se-rum levels of Urea and Cr and renal index were the highest in allopurinol group compared with those of other groups ( P<0.05). Conclusion Quercetin shows a significant effect of anti-inflammatory on acute gouty arthritis in rats. The model es-tablishment may lead to different degrees of renal damage. Quercetin has no protective effect against renal injury, and allopu-rinol aggravates kidney injury.

Trypanosoma is a human parasite severely affecting poor tropical areas.However,current frontline drugs for Trypanosoma treatment have severe side-effects with decreased effectiveness.Based on the fact that aminoacyl-tRNA synthetase is a bonafide drug target for several microorganisms,including bacteria and fungi,it is plausible that it may also be effective target of Trypanosoma.The Trypanosoma brucei leucyl-tRNA synthetase(tbLeuRS)was cloned,expressed and purified to develop an in vitro enzymatic assay system.The assay conditions were further optimized for the effective screening of tbLeuRS inhibitors thus establishing an anti-Trypanosoma drug screening system targeting tbLeuRS.The results indicated that this system can be employed for the effective screening of anti-Trypanosoma drugs with satisfactory specificity.In addition,this system can also be used for compound optimization,as well as IC50 testing.Using this system a series of compounds are identified that are effective Trypanosoma inhibitors without toxicity to human cells.Therefore,targeting tbLeuRS may represent a new venue for the development of anti-Trypanosoma drugs.

OBJECTIVETo investigate the clinicopathologic features, clinical progress and prognosis of the basal-like subtype of invasive lobular carcinoma (ILC) of the breast.

METHODSFour cases of ILC were analyzed by detailed histopathologic observation and immunohistochemical staining for E-cadherin, p120 catenin, ER, PR, HER2, CK5/6, EGFR, p63, p53, Ki-67 using MaxVision method. The follow-up and clinical data were analyzed.

RESULTSMorphologically, one case was mixed ILC and three cases were pleomorphic ILC. The tumor cells were negative for E-cadherin except one case with focal membrane positivity, and all showed p120 catenin cytoplasmic positivity except one case with focal membrane positivity. All cases were negative for ER, PR and HER2 (triple negative), and positive for EGFR and CK5/6. Two cases were positive for p63. The cases were partly and weakly positive for p53, and the Ki-67 positive rate was between 30% and 75%. Follow-up data showed that two cases developed chest wall metastases, and in one case, there was progression to liver and abdominal metastases.

CONCLUSIONSILC of the breast are ER, PR and HER2 "triple negative", CK5/6 and EGFR positive, indicative of basal-like characteristics. Basal-like subtype of ILC are peculiarly prone to metastasis and poor response to chemotherapy, suggesting that it is associated with poor prognosis.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Biomarkers, Tumor ; metabolism ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Cadherins ; metabolism ; Carcinoma, Lobular ; drug therapy ; metabolism ; pathology ; secondary ; surgery ; Catenins ; metabolism ; Combined Modality Therapy ; Female ; Follow-Up Studies ; Humans ; Immunohistochemistry ; Keratin-5 ; metabolism ; Keratin-6 ; metabolism ; Ki-67 Antigen ; metabolism ; Liver Neoplasms ; secondary ; Lymphatic Metastasis ; Mastectomy, Modified Radical ; Middle Aged ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism ; Thoracic Neoplasms ; secondary ; Thoracic Wall ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the effectiveness and safety of deferasirox (DFX) in the treatment of iron overload in children with β-thalassemia major.

METHODSTwenty-four β-thalassemia major children with iron overload who received regular blood transfusion were randomly enrolled. The serum feritin (SF) levels were measured in the patients after different doses of DFX treatment. The DFX treatment-related adverse events were observed. The values of cardiac MRI T2* and liver MRI T2* were compared between the patients receiving DFX treatment for 5 years and the patients treated with deferoxamine and deferiprone.

RESULTSThe patients with iron overload did not respond to DFX at the initial dose of 20-30 mg/kg•d. However, the SF level decreased significantly after the dose of DFX increased to 30-40 mg/kg•d (U=58, P<0.01). Serum liver transaminase elevation was the most common adverse effect, followed by non-progressive elevation in serum creatinine level. The mean SF level was significantly lower (1748±481 ng/mL vs 3462±1744 ng/mL; P<0.05), in contrast, the liver MRI T2* value was significantly higher (8.5±2.9 ms vs 2.7±1.9 ms; P<0.01) in patients receiving DFX treatment for 5 years than in the controls. There were no significant differences in the cardiac MRI T2* value between the two groups.

CONCLUSIONSDFX can reduce SF levels in a dose-dependent manner in children with β-thalassemia major. It can significantly lower liver iron overload but not cardiac overload. Serum liver transaminase elevation and non-progressive elevation in serum creatinine level are major adverse effects in DFX treatment.

Adolescent ; Adult ; Benzoates ; adverse effects ; therapeutic use ; Child ; Child, Preschool ; Dose-Response Relationship, Drug ; Female ; Ferritins ; blood ; Humans ; Iron Chelating Agents ; adverse effects ; therapeutic use ; Iron Overload ; drug therapy ; Male ; Transfusion Reaction ; Triazoles ; adverse effects ; therapeutic use ; beta-Thalassemia ; blood ; complications ; therapy

OBJECTIVETo discuss the risk factors of splenic arterial steal syndrome (SASS) after orthotopic liver transplantation.

METHODSTwenty-four cases who confirmed SASS after liver transplantation in Tianjin First Central Hospital between June 2005 and June 2013 were analyzed retrospectively. Another 96 cases were selected randomly from those patients of the same time with no complication of SASS patients postoperatively as control group. Clinical data of two groups including diameter of splenic artery and hepatic artery preoperatively, weight of graft, weight of recipients, cold/warm ischemia time, an hepatic period and operation time and so on were collected. Others including hepatic artery peak systolic velocity (PSV), end diastolic velocity (EDV), blood flow resistance index and portal vein average velocity (PVF) on the first day after liver transplantation, the day before diagnosis, the day when diagnosed, the 1, 3, 7 days after treatment in SASS group and on 1, 3, 7, 9, 11, 14 days after liver transplantation in control group. Statistical analysis were made between two groups.

RESULTSThe splenic artery/hepatic artery ratio preoperatively and weight of donor liver,and the GRWR in SASS group and control group were 1.26 and 1.00, 1 032 g and 1 075 g, (1.40±0.30)% and (1.82±0.21)% respectively, with significantly statistical differences (Z=-6.40, Z=-2.22, t=-6.50; all P<0.05). The warm ischemia time, the cold ischemia time, the anhepatic period and operation time in SASS group and control group were 3.5 minutes and 4.0 minutes, 10.25 hours and 10.10 hours, 43 minutes and 45 minutes, 8.7 hours and 8.7 hours, with no significantly statistical differences (all P>0.05). RI of hepatic went up gradually in the early time after transplantation while dropped obviously when spleen artery spring coils embolization was received (P<0.01) and trended to stable two weeks later.

CONCLUSIONSSplenic artery/hepatic artery ratio and GRWR are the positive and negative risk factors respectively for SASS. The gradual rising of hepatic RI in the early time after transplantation may be the warning signal SASS and spleen artery spring coils embolization is the effective strategy for SASS after liver transplantation.

Cold Ischemia ; Embolization, Therapeutic ; Hepatic Artery ; pathology ; Humans ; Liver ; surgery ; Liver Transplantation ; adverse effects ; Retrospective Studies ; Risk Factors ; Spleen ; blood supply ; Splenic Artery ; pathology ; Vascular Diseases ; epidemiology ; Warm Ischemia