Optimization of the fermentation condition for human apolipoproteinA-I expression in recombinant Escherichia coli was investigated. The recombinant plasmid pBV220-ApoA-I was transformed respectively into different E.coli hosts such as JM109, BL21(DE3),DH5?, BMH7118,and TG1. The best host E.coli was DH5? in which the recombinant ApoA-I expression percentage was 21.2% corresponding to that in BL21(DE3) in flask shaker cultivation,while the ApoA-I expressed percentage in E.coli TG1 was 11%.Fed-batch cultivation was performed in FMG-5L fermentor,the optimum fermentation cultivation conditions were as following :optimum pH value was 7.0 in growth phase and 7.4 in the expression phase. The initial glucose concentration in batch phase was 3 g?L -1.The optimum C/N ratio was 2∶1.The recombinant ApoA-I reached about 40% of the total protein, and concentration of ApoA-I was 2.86 g?L -1.

The temperature effect on the recombinant protein production formation was investigated in present study. The culture temperature of growth phase is 30℃, and the culture temperature of induction phase was arranged according to three modes. Hign cell-density and high expression culture of E.coli to product recombinant human apolipoprotein A-I Milano by two temperature-shifted induction . Two temperature-shifted induction was carried out high density and high expression recombinant human ApoA-1 Milano. The recombinant protein ApoA-I Milano reached 4.8 g?L -1 with the final cell density of OD 600 150. And the two temperature-shifted induction avoided the acetic acid successfully to the influence of the high density and high expression. Two temperature-shifted induction was viable in high density culture and high expression of heterogenous protein in recombination E.coli.The sduty provides a basic work for production of recombinant ApoA-I Milano in scale.

OBJECTIVETo investigate the effets of flurothyl-induced neonatal recurrent seizures on glucocorticoid receptor (GR) expression in the rat brain.

METHODSForty-eight seven-day-old Sprague-Dawley rats were randomly divided into two groups: control and seizure. Seizures were induced by inhalant flurothyl daily for six consecutive days. Brains were sampled on postnatal days 13, 15 and 19. The expression of GR protein in the cerebral cortex was detected by Western blot and immunohistochemical method.

RESULTSThe expression of GR in the cerebral cortical plasma protein was significantly lower in the seizure group than in the control group on postnatal day 15. The expression of GR protein in the cerebral cortical nuclear protein decreased significantly in the seizure group compared with that in the control group on postnatal days 15 and 19 (p<0.05). Compared to the control group, the accumulated optical density (AOD) of GR immunoreactivity (IR) decreased significantly in the parietal cortex on postnatal day 13 (p<0.05), the AOD of GR IR decreased significantly in the parietal cortex and the temporal cortex on postnatal day 15 (p<0.05), and the AOD of GR IR decreased significantly in the parietal cortex, temporal cortex and the frontal cortex in the seizure group on postnatal day 19 (p<0.05).

CONCLUSIONSRecurrent seizures in neonatal rats result in abnormal GR expression in the cerebral cortex which might play an important role in short-term brain injury induced by early recurrent seizures.

Animals ; Blotting, Western ; Cerebral Cortex ; chemistry ; Female ; Hypothalamo-Hypophyseal System ; physiology ; Immunohistochemistry ; Male ; Pituitary-Adrenal System ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, Glucocorticoid ; analysis ; physiology ; Recurrence ; Seizures ; metabolism

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.
Apolipoprotein A-I ; biosynthesis ; genetics ; Chromatography, Ion Exchange ; methods ; Escherichia coli ; genetics ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Protein Precursors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVES: To recognize the possibility of Y fragment deletion of Amelogenin gene intuitively and simply according to the genotyping graphs. METHODS: By calculating the ratio of total peak height of genotyping graphs, the statistics of equilibrium distribution between Amelogenin and D3S1358 loci, Amelogenin X-gene and Amelogenin Y-gene, and different alleles of D3S1358 loci from 1 968 individuals was analyzed after amplified by PowerPlex^® 21 detection kit. RESULTS: Sum of peak height of Amelogenin X allele was not less than 60% that of D3S1358 loci alleles in 90.8% female samples, and sum of peak height of Amelogenin X allele was not higher than 70% that of D3S1358 loci alleles in 94.9% male samples. CONCLUSIONS The result of genotyping after amplified by PowerPlex^® 21 detection kit shows that the possibility of Y fragment deletion should be considered when only Amelogenin X-gene of Amelogenin is detected and the peak height of Amelogenin X-gene is not higher than 70% of the total peak height of D3S1358 loci.
Alleles ; Amelogenin/genetics* ; Asian People/genetics* ; DNA Fingerprinting/methods* ; Female ; Genotype ; Humans ; Male ; Mutation ; Polymerase Chain Reaction/methods* ; Population Groups

OBJECTIVE: Polycystic ovary syndrome (PCOS), a common endocrine-metabolic dysfunction in reproductive-aged women, may be involved in compromised pregnancy and offspring outcomes. This study aimed to investigate whether maternal PCOS affects fetal growth, fetal development, and placental features. METHODS: This retrospective case-control study included 60 pregnant women with PCOS (PCOS group) and 120 healthy pregnant women without PCOS (control group). Fetal magnetic resonance imaging (MRI) was performed followed by an ultrasound examination and indications for imaging, including known or suspected fetal pathology, history of fetal abnormality in previous pregnancy or in a family member, and concern for placenta accreta. Fetal MRI images were analyzed for head circumference (HC), abdomen circumference (AC), lung-to-liver signal intensity ratio (LLSIR, a prenatal marker of fetal lung maturity), lengths of liver and kidney diameters in fetuses, and placental relative signal intensity on T2-weighted single-shot fast spin echo (SSFSE) imaging (rSI RESULTS: Compared to the control group, the PCOS group showed the following characteristics: (1) smaller biparietal diameter and femur length in fetuses (P=0.026 and P=0.005, respectively), (2) smaller HC in fetuses (evident after 32 weeks; P=0.044), (3) lower LLSIR and smaller dorsoventral length of liver in fetuses (evident before 32 weeks; P=0.005 and P=0.019, respectively), and (4) smaller placental thickness (evident before 32 weeks; P=0.017). No significant differences in placental rSI CONCLUSIONS There exist alterations of fetal growth, fetal development, and placental features from women with PCOS.

We wished to establish an expert consensus on late stage of critical care (CC) management. The panel comprised 13 experts in CC medicine. Each statement was assessed based on the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) principle. Then, the Delphi method was adopted by 17 experts to reassess the following 28 statements. (1) ESCAPE has evolved from a strategy of delirium management to a strategy of late stage of CC management. (2) The new version of ESCAPE is a strategy for optimizing treatment and comprehensive care of critically ill patients (CIPs) after the rescue period, including early mobilization, early rehabilitation, nutritional support, sleep management, mental assessment, cognitive-function training, emotional support, and optimizing sedation and analgesia. (3) Disease assessment to determine the starting point of early mobilization, early rehabilitation, and early enteral nutrition. (4) Early mobilization has synergistic effects upon the recovery of organ function. (5) Early functional exercise and rehabilitation are important means to promote CIP recovery, and gives them a sense of future prospects. (6) Timely start of enteral nutrition is conducive to early mobilization and early rehabilitation. (7) The spontaneous breathing test should be started as soon as possible, and a weaning plan should be selected step-by-step. (8) The waking process of CIPs should be realized in a planned and purposeful way. (9) Establishment of a sleep-wake rhythm is the key to sleep management in post-CC management. (10) The spontaneous awakening trial, spontaneous breathing trial, and sleep management should be carried out together. (11) The depth of sedation should be adjusted dynamically in the late stage of CC period. (12) Standardized sedation assessment is the premise of rational sedation. (13) Appropriate sedative drugs should be selected according to the objectives of sedation and drug characteristics. (14) A goal-directed minimization strategy for sedation should be implemented. (15) The principle of analgesia must be mastered first. (16) Subjective assessment is preferred for analgesia assessment. (17) Opioid-based analgesic strategies should be selected step-by-step according to the characteristics of different drugs. (18) There must be rational use of non-opioid analgesics and non-drug-based analgesic measures. (19) Pay attention to evaluation of the psychological status of CIPs. (20) Cognitive function in CIPs cannot be ignored. (21) Delirium management should be based on non-drug-based measures and rational use of drugs. (22) Reset treatment can be considered for severe delirium. (23) Psychological assessment should be conducted as early as possible to screen-out high-risk groups with post-traumatic stress disorder. (24) Emotional support, flexible visiting, and environment management are important components of humanistic management in the intensive care unit (ICU). (25) Emotional support from medical teams and families should be promoted through"ICU diaries"and other forms. (26) Environmental management should be carried out by enriching environmental content, limiting environmental interference, and optimizing the environmental atmosphere. (27) Reasonable promotion of flexible visitation should be done on the basis of prevention of nosocomial infection. (28) ESCAPE is an excellent project for late stage of CC management.
Humans ; Consensus ; Critical Care/methods* ; Intensive Care Units ; Pain/drug therapy* ; Analgesics/therapeutic use* ; Delirium/therapy* ; Critical Illness