Thirty six weanling male rats fed a vitamin D-deficient diet, were divided into three groups and each of them were supplemented with 0, 5000 or 30000 IU.kg-1.w-1 of vitamin D3, respectively. In three weeks feeding, increments of body weight and tissue weight were not significantly different among the 3 groups. The zinc contents of plasma, liver and kidney of rats in the supplemented groups and the zinc content of testis in vitamin D3 5000 IU.kg-1.w-1 group were significantly higher than that of rats in the group without supplement. But there was no significant differance between the zinc levels of prostate and tibia in all groups. The results showed that the nutrition state of vitamin D could influence the zinc level of blood and tissues.

The absorption of zinc was studied with feeding method in rats. The zinc absorption of the castrated rat (44.21 ? 1.88%, x?SE) was significantly lower than that of the sham-operated rat (49.44?1.17%). The zinc absorption of the rat injected testosterone propionate (20 mg/kg, q.d.alt.) returned to the level of the sham-operated rat (49.13 ?1.88%). The results suggested that androgen could promote the absorption of zinc.

Objective To sum up the clinical characteristics and the diagnostic and therapeutic principle of severe hypertensive intracerebral hemorrhage complicated with gastroparesis.Methods The clinical data of 51 patients with severe hypertensive intracerebral hemorrhage complicated with gastroparesis were retrospectively analyzed.Results Of the patients who died,3 died of over-severe hemorrhage,and 1 died of acute respiratory distress syndrome caused by aspiration,as well as one died of respiratory failure resulting from pulmonary infection after aspiration.None died of digestive tract complication.Twenty-one patients(41.18％) needed feeding via naso-intestinal tube.Thirty patients (58.82％) were recovered within two weeks and 14 patients (27.45％) were recovered within the third week,and those who recovered beyond three weeks accounted for 3.92％ (n =2).Conclusion The gastroparesis complicating severe hypertensive intracerebral hemorrhage is considered as a functional disorder rather than mechanical obstruction.It is mainly on the basis of symptoms and signs in combination with gastroscopy or radiography that the diagnosis can be made.The conservative treatment (including nasal feeding in some patients) should be applied to the disorder.

Objective To investigate the application value of methylene blue excretion test in early reasonable nutritional support for severe traumatic brain injury.Methods One hundred and thirty-three cases of severe traumatic brain injury admitted to hospital from January 2010 to June 2012 were chosen as treatment group,while the 127 cases of similar patients admitted to hospital from January 2007 to December 2009 were chosen as control group.Patients in treatment group underwent methylene blue excretion test in 3 days,8 days,15 days after injury,and the nutritional support ways were determined according to the elimination time of methylene blue in patients' urine.The control group conventionally receive enteral nutrition support therapy firstly,after 15 days if they still cannot be tolerant of the enteral nutrition,then parenteral nutrition therapy were adopted.The weight,serum albumin and hemoglobin circumstances of the two groups were determined and the complications were recorded.Glasgow coma score (GCS) of 3 months after injury were followed up.Results There was no significant difference on the average body weigh between these two groups before treatment.The average body weight of the treatment group was significantly higher than that of control group after 3 months treatment((56.3 ± 5.5) kg vs.(52.6 ± 5.3) kg,t =5.93,P ＜ 0.01).The serum albumin and hemoglobin of 14 d,21 d after injury were significantly higher than those of the control group (serum albumin of 14 d:(32.7 ±3.4) g/L vs.(28.8 ±3.1) g/L; serum albumin of 21 d:(34.3 ±3.8) g/L vs.(30.7 ±3.3) g/L;hemoglobin of 14 d:(113.4±12.5) g/L vs.(102.2 ±11.6) g/L;hemoglobin of 21 d:(118.5 ±13.3) g/L vs.(106.7 ± 12.4) g/L.Nutritional status of treatment group was significantly better than that of the control groupall P ＜ 0.05).After three months,the effective rate of treatment group (93.23％ (124/133)) was significantly higher than that of the control group (84.25％ (107/127)),the difference was statistically significant (x2 =5.29,P ＜ 0.05).Conclusion Determining the early reasonable nutrition support ways for patients with severe traumatic brain injury according to the elimination time of methylene blue in the urine,can provide comprehensive nutrition to patients,enhance their body resistance,reduce the incidence of complications,and create an important clinical value for improving prognosis.

Objective To study the adjuvant effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) coding gene on cellular immunity induced by Japanese encephalitis (JE) virus DNA vaccine. Methods GM-CSF coding gene was amplified by nested-reverse transcriptase-polymerase chain reaction (RT-PCR) technique from BALB/c murine spleen cells. Recombinant plasmids pJME/GM-CSF and pGM-CSF were constructed by JE virus (JEV) prM-E protein with GM-CSF coding gene or GM-CSF coding gene only, respectively. The plasmids were transfected into China hamster ovary (CHO) cells by Lipofectamine 2000. The coding protein expressions and distributions were detected by immunofluorescence. The BALB/c mice were vaccinated with indicated immunogens with or without GM-CSF gene. The changes of T lymphocyte subsets in the spleen and levels of intracellular cytokines, such as interferon (IFN)-γ and interleukin (IL)-4 of splenic cells from mice immunized with different immunogens were evaluated by flow cytometry. The cytotoxicity T lymphocyte (CTL) activity was assessed by lactate dehydrogenase (LDH). The data were compared by one-factor analysis of variance and least significant difference. Results The constructed recombinant pGM-CSF and pJME/GM-CSF were confirmed by restrict enzyme digestion and DNA sequencing. The expressions of the above proteins were mainly in the cytoplasm and minor on cell membrane. The percentage of CD4+ T lymphocytes in pJME/GM-CSF vaccinated group was (33.90±0.79)%, which was significantly higher than that of in other groups (t values were 9. 818, 6. 804, 6.594, 10.061, 9.380, and 17.675, all P<0.05). The percentages of CD4+T lymphocytes in pJME +pGM-CSF (0) and pJME+pGM-CSF (-3) vaccinated groups were (29.83±0.61)% and (29.70±0.51)%, respectively, which were both higher than that in pJME+pGM-CSF (+3) vaccinated group of (27.69+0.50)% (t=3.466, t=3.255, both P<0.05). The percentages of CD8+ T cells in pJME/GM-CSF and pJME+pGM-CSF vaccinated groups were both higher than that in empty vector (pcDNA 3.1+) group and JE inactivated vaccine vaccinated group (t values were 3.811, 2.627, 10.537, and 3.811, all P<0.05). The CTL activity in pJME/GM-CSF vaccinated group was (51.48±0.10)%, which was higher than those in other groups (t values were 22.868, 13.823, 5.377, 32.287, 34.632, and 53.795, all P<0.05). The IFN-γ/IL-4 ratios in pJME/GM-CSF, pJME+pGM-CSF (0) and pJME + pGM-CSF (-3) vaccinated groups were (19.13±1.36), (12.32±0.82) and (7.05±0.43), respectively, which were higher than those in other groups (P<0.05). Conclusion GM-CSF coding gene could enhance the cellular immune response induced by Japanese encephalitis DNA vaccine.

OBJECTIVETo explore the effects of hepatitis C virus (HCV) core protein on the activity of double-stranded RNA-dependent protein kinase (PKR).

METHODSThe human hepatoma cell line BEL-7402 was transfected with the HCV core gene-containing eukaryotic expression vector pCMH6K-Core (at various concentrations), or empty vector, or no vector; a group of cells was co-transfected with the luciferase reporter plasmid pGL3-promoter. The cells were treated with interferon (IFN) a-2b to induce the expression and activation of endogenous PKR, or left untreated to serve as controls. The effect of core protein on PKR phosphorylation was detected by western blotting. Luciferase activity was detected to reflect effects of the core protein on the synthesis of cellular proteins. The t-test and F test were used for statistical analyses.

RESULTSIn the case of IFNa stimulation, PKR phosphorylation levels were significantly lower in the HCV core protein expressing cells than in the cells transfected with empty plasmid or with no vector, but the total PKR expression level was not significantly different among these three groups of cells. Cells co-transfected with luciferase plasmid and the core protein expressing vector showed significantly higher levels of luciferase expression than the cells co-transfected with the empty vector. Moreover, the luciferase activity and core protein expression levels increased in a dose-dependent manner, with the luciferase activity of the cells treated with 0.5 mug, 1.0 mug and 1.5 mug pCMH6K-Core being 1.941 ± 0.199 times, 2.868 ± 0.275 times and 3.839 ± 0.338 times higher than that of the empty vector group (all P < 0.05).

CONCLUSIONIn the human hepatoma cell line BEL-7402, the HCV core protein can inhibit the activity of endogenous PKR, thereby promoting cell protein synthesis.

Cell Line, Tumor ; Genes, Reporter ; Genetic Vectors ; Humans ; Phosphorylation ; Protein Biosynthesis ; RNA-Binding Proteins ; metabolism ; Transfection ; Viral Core Proteins ; genetics ; metabolism

Patients with severe liver diseases are prone to nosocomial infection due to serious liver dysfunction, low immune function, and reduced stress ability to infection, and such infections further aggravate patients’ conditions and directly affect their clinical outcome. This article reviews the clinical features of nosocomial infection in patients with severe liver diseases and related risk factors.

Bacillus alcalophilus DTY1, one moderate halophytic bacterium isolated from saline soil in Loess Plateau of China, was characterized with efficient production of ectoine. In this study, the gene cluster ectABC taking in charge of biosynthesizing ectoine was cloned from the genomic library of strain DTY1. Nucleotide sequencing indicated that ectA, ectB and ectC were predicted to encode peptides of 169, 428 and 132 amino acids, respectively. The deduced amino acid sequences of EctA, EctB and EctC share 59%, 81% and 81% identity to 2,4-diaminobutyric acid acetyltransferase, 2,4-diaminobutyric acid transaminase and ectoine synthase of B. halodurans C-125, respectively. A fragment containing ectABC genes was introduced into B. cereus Z, which made the transgenic Z cells increased tolerance to salt, remarkably. HPLC analysis of ectoine in the transgenic Z cells revealed that 70.1 mg/g ectoine was detected in 1.0% NaCl medium and 118.6 mg/g ectoine in 5.0% NaCl medium. Furthermore, as the concentration of salt increased, transgenic Z cells accumulated more ectoine. These results suggest that ectoine is an important facet in B. alcalophilus DTY1 to high-osmolarity surroundings, and the expression of ectABC is induced by salt strength.
Amino Acid Sequence ; Amino Acids, Diamino ; biosynthesis ; genetics ; physiology ; Bacillus ; classification ; genetics ; Bacillus cereus ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; Cloning, Molecular ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; genetics ; Molecular Sequence Data ; Osmotic Pressure ; Sodium Chloride ; metabolism ; pharmacology

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k (cat)/K (m)) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.
Aeropyrum ; chemistry ; enzymology ; Archaeal Proteins ; genetics ; metabolism ; Binding Sites ; Biocatalysis ; Caprylates ; metabolism ; Cloning, Molecular ; Dimethyl Sulfoxide ; chemistry ; Escherichia coli ; Hot Temperature ; Industrial Microbiology ; methods ; Kinetics ; Laurates ; metabolism ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; methods ; Peptide Hydrolases ; genetics ; metabolism ; Protein Binding ; Protein Conformation ; Recombinant Proteins ; genetics ; metabolism ; Solvents ; chemistry ; Substrate Specificity