With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.
Antifibrinolytic Agents ; pharmacology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fibrinolytic Agents ; metabolism ; Genetic Vectors ; genetics ; Paenibacillus ; chemistry ; enzymology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVE: To investigate the association of atrial fibrillation (AF) with hemorrhagic transformation (HT) in patients with ischemic stroke treated by intravenous thrombolysis. METHODS: Clinical data of 3272 patients treated by intravenous thrombolysis from 71 hospitals in Zhejiang Province during June 2017 and December 2018 were retrospectively reviewed. Intracranial HT was defined as intracranial hemorrhage suggested by imaging examination 24 hours after intravenous thrombolysis. Patients were dichotomized into HT group (=533) and non-HT group (=2739). The association of AF and HT was analyzed by univariate analysis and binary logistic regression. RESULTS: Compared with the non-HT group, the HT group were older, had longer onset to needle time (ONT), higher baseline National Institute of Health Stroke Scale (NIHSS) score, higher baseline glucose level, and higher AF rate (<0.05 or <0.01). Binary logistic regression analysis revealed that AF was independently associated with HT (=2.527, 95%:2.030-3.146, <0.01). CONCLUSIONS AF is independently associated with the occurrence of HT in ischemic stroke patients treated with intravenous thrombolysis.
Antifibrinolytic Agents ; adverse effects ; pharmacology ; Atrial Fibrillation ; complications ; Brain Ischemia ; complications ; drug therapy ; Humans ; Retrospective Studies ; Stroke ; complications ; drug therapy ; Thrombolytic Therapy ; adverse effects ; Treatment Outcome

The purpose of this study was to clarify the differential effect of vitamin K and vitamin D supplementation on bone mass in young rats fed a normal or low calcium diet. Ninety female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into nine groups with 10 rats in each group: baseline control, and 0.5% (normal) or 0.1% (low) calcium diet, either alone, or with vitamin K (30 mg/100g, food intake), vitamin D (25microgram/100 g, food intake), or vitamin K + vitamin D. After 10 weeks of feeding, bone histomorphometric analyses were performed on cortical bone of the tibial shaft and cancellous bone of the proximal tibia. Vitamin K supplementation increased the maturation-related cancellous bone gain and retarded the reduction in the maturation-related cortical bone gain in rats fed a low calcium diet, and increased the maturation-related cortical bone gain in rats fed a normal calcium diet. Vitamin D supplementation reduced the maturation-related cancellous bone gain, prevented the reduction in periosteal bone gain, and enhanced the enlargement of the marrow cavity, with no significant effect on the reduction in the maturation-related cortical bone gain in rats fed a low calcium diet, and increased the maturation- related cancellous and cortical bone gains with increased periosteal bone gain in rats fed a normal calcium diet. An additive effect of vitamin K and vitamin D on the maturation- related cortical bone gain was found in rats fed a normal calcium diet. This study shows the differential effects of vitamin K and vitamin D supplementation on cancellous and cortical bone mass in young rats fed a normal or low calcium diet, as well as the additive effect on cortical bone under calcium sufficient condition.
Age Factors ; Animals ; Antifibrinolytic Agents/*pharmacology ; Bone Density/*drug effects ; Bone Development/*drug effects ; Calcium, Dietary/*pharmacology ; Dietary Supplements ; Female ; Rats ; Rats, Sprague-Dawley ; Vitamin D/*pharmacology ; Vitamin K/*pharmacology