The single-chain antibody gene (ox26-scFv) to transferrin receptor (TfR) was synthesized and amplified by three-step PCR. After sequencing, the gene was cloned into prokaryotic expression vector pTIG-Trx which carried thioredoxin (Trx) gene and a C-terminal His.tag. The Ox26-scFv proteins achieved 31% yields of total bacteria proteins at 20 degrees C, after 0.02mM IPTG induction using the strain E. coli BL21 (DE3). The soluble scFv proteins in cytoplasm suspension were about 35% and the inclusion bodies were about 65%. The soluble products were purified by immobilized metal chelation affinity chromatography (Ni-NTA), a single band with molecular weight 29 kD appeared on SDS-PAGE gel. Rat GH3 cell immunocytochemistry staining showed that Ox26-scFv protein could recognize and bind to transferrin receptor. Injected SD rats with Ox26-scFv proteins by tail veins, the antibodies were detected from brain tissues specially on the brain capillaries 4 h later which indicate that Ox26-scFv proteins have a good target function to brain capillaries and can permeate the blood-brain barrier mediated by the transferrin receptors.
Animals ; Antibodies ; administration & dosage ; genetics ; metabolism ; Antibody Formation ; Base Sequence ; Blood-Brain Barrier ; drug effects ; Drug Delivery Systems ; Genetic Vectors ; Molecular Sequence Data ; Rats ; Rats, Sprague-Dawley ; Receptors, Transferrin ; immunology

KK-Ay mice with type 2 diabetes were divided into metformin plus high-fat diet,metformin plus low-fat diet,high fat diet alone and low fat diet alone groups.AMP-activated protein kinase(AMPK)activity was measured in skeletal muscle.The results showed that metformin and low-fat diet were able to increase AMPK activity.However,no additive effect on AMPK activity by metformin and low-fat diet was found.

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; pharmacokinetics ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; biosynthesis ; pharmacokinetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacokinetics

OBJECTIVETo explore the changes in spatial learning performance and long-term potentiation (LTP) which is recognized as a component of the cellular basis of learning and memory in normal and lead-exposed rats after administration of melatonin (MT) for two months.

METHODSExperiment was performed in adult male Wistar rats (12 controls, 12 exposed to melatonin treatment, 10 exposed to lead and 10 exposed to lead and melatonin treatment). The lead-exposed rats received 0.2% lead acetate solution from their birth day while the control rats drank tap water. Melatonin (3 mg/kg) or vehicle was administered to the control and lead-exposed rats from the time of their weaning by gastric gavage each day for 60 days, depending on their groups. At the age of 81-90 days, all the animals were subjected to Morris water maze test and then used for extracellular recording of LTP in the dentate gyrus (DG) area of the hippocampus in vivo.

RESULTSLow dose of melatonin given from weaning for two months impaired LTP in the DG area of hippocampus and induced learning and memory deficit in the control rats. When melatonin was administered over a prolonged period to the lead-exposed rats, it exacerbated LTP impairment, learning and memory deficit induced by lead.

CONCLUSIONMelatonin is not suitable for normal and lead-exposed children.

Animals ; Female ; Lead ; toxicity ; Learning ; drug effects ; Long-Term Potentiation ; drug effects ; Male ; Maze Learning ; drug effects ; Melatonin ; administration & dosage ; toxicity ; Rats ; Spatial Behavior ; drug effects

Objective: To examine the Quality of life among school-aged children with dyslexia in target city and to provide scientific evidence for improving the quality of life of children with dyslexia. Methods: By using cluster sampling,students from grade 3 to grade 6 from 6 primary schools in a middle-sized were selected and administered with questionnaire survey. According to the criteria of dyslexia, dyslexic children and non-dyslexic children were identified and the difference of the Quality of Life was compared. Results: Totally 3 673 children were collected, and 119 of them were identified as dyslexia(3.24%).The prevalence of dyslexia differed by gender,grades,educational level of parents(χ2=24.77,11.75,18.50,9.79,P<0.05). The Quality of Life which below the average proportion accounted for 30.3% of dyslexic children and 16.7% of normal children. Quality of life scored signiticantly different between dyslexic children and non-dyslexia children, including psychosocial functioning domain(134.54±30.88)(143.49±32.53), physical and mental health domain(2.71±0.84)(2.92±0.81) vs (2.83±0.90)(3.06±0.87), the living satisfaction domain(2.95±0.87)(3.14±0.87)(t=-6.09，-5.48，-5.44，-4.50，P<0.01),with dyslexic group significantly lower than that of non-dyslexic group. Conclusion The Quality of Life of Dyslexic children was in a poor condition.

Objective To assess efficacy and safety of oral brivudine 125 mg once daily versus 4 times daily in the treatment of herpes zoster.Methods A five-centre,randomized,double-blind,parallel- controlled study was performed on 226 patients with herpes zoster.Oral brivudine 125 mg was given once daily to 112 patients,and four times daily to 114 patients,both for 7 days.All patients were followed up for 3 weeks after the end of treatment.Results The time to the last formation of new vesicles was 3.88 days for the once daily group,and 3.79 days for the 4 times daily group,without significant differences between the two groups.There was also no significant difference between the two groups with respect to the time to total resolution of vesicles,time to first crusts,time to full crusting,time to first loss of crusts,time to full loss of crusts,time to first relief of pain,and time to complete relief of pain.Postherpetic neuralgia occurred in 34.5% of patients in the once daily group,and 30.4% of patients in the 4 times daily group.The incidence of treatment-related adverse events was 5.4% and 9.6%,in the once daily group and 4 times daily group, respectively.Conclusions Brivudine 125 mg once daily is equally effective,more convenient and safe in comparison with brivudine 125 mg 4 times daily for the treatment of herpes zoster.

OBJECTIVETo explore the distribution of lecithin-cholesterol acyltransferase gene (LCAT) 608C/T polymorphism in Chinese Han population and the relationship of the polymorphism association with the occurrence of atherosclerotic cerebral infarction.

METHODSThe lecithin:cholesterol acyltransferase gene 608C/T polymorphism is identified by polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP)and restriction fragment length polymorphism (RFLP) in 150 patients with ACI and 122 healthy controls matching age and sex.

RESULTSThe distribution of LCAT 608C/T gene polymorphism was in accordance with Hardy-Weinberg equilibrium. The CT genotype frequency (14.0%) and T allele frequency (7.0%) in ACI group were significantly higher than those in control group (P<0.05). The concentration of high density lipoprotein cholesterol (HDL-C) in 608CC subgroups were significantly higher than those in 608CT subgroups both in ACI group and in control group (P<0.05).

CONCLUSIONThe LCAT 608C/T polymorphism is possibly a predisposing factor in ACI happening of Chinese Han population. T allele frequency is possibly concerned with the metabolism of HDL-C.

Aged ; Alleles ; Cerebral Infarction ; etiology ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Intracranial Arteriosclerosis ; complications ; Male ; Middle Aged ; Phosphatidylcholine-Sterol O-Acyltransferase ; genetics ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Polymorphism, Single-Stranded Conformational ; genetics

The NS1 gene of the H5N1 subtype avian influenza virus was amplified by RT-PCR, and the am-plified product was cloned into the eukaryotic expression vector pCMV-Myc, then it was transfected into A549 cells. After 48 h, the expression of NS1 was detected by Western blot. Fluorescence and electron microscopy and flow cytometry showed that the NS1 gene of H5N1 avian influenza virus could induce apop-tosis in human pulmonary carcinoma cell line A549.
Annexin A5 ; analysis ; Apoptosis ; Cell Line, Tumor ; Cloning, Molecular ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; pathogenicity ; Lung Neoplasms ; pathology ; Viral Nonstructural Proteins ; genetics ; physiology

OBJECTIVETo investigate the effects and apoptosis of intrathecal injection of Methylprednisolone Sodium Succinate (MPss) for acute spinal cord injury (SCI) in New Zealand rabbits.

METHODSSeventy-two healthy New Zealand rabbits were used for the procedure and were randomly divided into two groups: SCI group and SHAM group, which was both divided into 6 subgroups, such as the vehicle group, the MPss intrathecal injection groups (1.5 mg/kg, 3.0 mg/kg, 6.0 mg/kg group), the MPss intravenous injection group and the combined injection group. TARLOV score was tested daily to evaluate the motor function. The rabbits were sacrificed 7 days after the surgery and the thoracic spinal cord sections and the sacral sections where MPss was injected were harvested for HE and TUNEL staining. Two-Factors Repeated Measures analysis of variance for TARLOV scores tested at various times and One-Way ANOVA analysis of variance for data between groups were used.

RESULTSeven days after surgery in SCI group, there was no statistical difference between the TARLOV scores of intrathecal injection of MPss 3.0 mg/kg group, 6.0 mg/kg group and MPss intravenous injection group (P > 0.05), which were all better than the vehicle group (F = 4.762, P < 0.05). Referring to the lymphocyte infiltration at the injury site in SCI group, there was statistical difference between MPss intrathecal injection 6.0 mg/kg group (1.33 ± 0.21) and the vehicle group (2.67 ± 0.21) (F = 5.793, P < 0.05) and no statistical difference between intrathecal injection of MPss 6.0 mg/kg group and MPss intravenous injection group (P > 0.05). As for the lymphocyte infiltration at the intrathecal injection site in SHAM group, there was statistical difference between MPss intrathecal injection 6.0 mg/kg group (2.50 ± 0.55) and the vehicle group (0.50 ± 0.55) (F = 17.333, P < 0.05). TUNEL staining in SCI group showed statistical difference between MPss intrathecal injection 6.0 mg/kg group (6.3 ± 1.5) and the vehicle group (20.3 ± 2.2) (F = 71.279, P < 0.05).

CONCLUSIONSIntrathecal injection of MPss can improve the functional recovery of lower limb and decrease apoptosis of neuron cells,which can provide same effects as the traditional intravenous injection of MPss in New Zealand rabbits.

Acute Disease ; Analysis of Variance ; Animals ; Disease Models, Animal ; Injections, Spinal ; Male ; Methylprednisolone Hemisuccinate ; administration & dosage ; therapeutic use ; Rabbits ; Recovery of Function ; Spinal Cord Injuries ; drug therapy