To prepare anti-mouse uteroglobin binding protein (mUGBP) polyclonal antibody, two polypeptides were synthesized based on the bioinformatics analysis of mUGBP, and New Zealand white rabbits were immunized separately with each peptide coupled with keyhole limpet hemocyanin (KLH). The data indicate that a 13-amino acid polypeptide (positions 221st-233rd) was able to generate anti-peptide antibodies. The titer of the antisera detected with ELISA was 1:10(8). The antisera were then purified with immuno-affinity chromatography to obtain antibodies. Western blot analysis of mUGBP expressed as a fusion protein with a green fluorescent protein (GFP) was performed on the cell lysates of COS-1 cells with the purified antisera, suggesting that the antisera specifically recognized UGBP. By immunohistochemistry and indirect immunofluorescence analysis, we examined the expression of UGBP in the lung tissues from a patient undergoing surgical lung resection for a tumor and from normal mouse lung tissue, and found for the first time that UGBP protein was widely expressed in both mouse and human lung tissue with the most abundant expression in bronchial epithelial cells. These results suggest that the antigen epitopes of mUGBP are well predicted by using bioinformatics analysis. We have obtained anti-mUGBP polyclonal antibody, which will be useful for further investigation.
Animals ; Antibodies ; chemistry ; COS Cells ; Carrier Proteins ; chemistry ; Cercopithecus aethiops ; Computational Biology ; Enzyme-Linked Immunosorbent Assay ; Hemocyanins ; Humans ; Immune Sera ; Immunohistochemistry ; Mice ; Rabbits ; Recombinant Proteins ; chemistry ; Uteroglobin

AIMTo study the influence of VIP on the expression of SP-A and its intracellular signal transduction pathway.

METHODSThe influence of VIP on the expression of SP-A was studied by immunohistochemistry and RT-PCR. The intracellular signal transduction pathway was further investigated by using receptor antagonist, protein kinase inhibitor and antisense oligonucleotides.

RESULTS(1) VIP(10(-8) mol/L) enhanced SP-A protein expression in alveolar type II cells (ATII) and increased the content of SP-A mRNA in lung tissue. (2) VIP receptor antagonist [D-P-C1-Phe (6)-Leu (17)]-VIP (10(-6) mol/L) could suppress the VIP-induced expression of SP-A protein and SP-A mRNA. (3) c-fos antisense oligonucleotides (9 x 10(-6) mol/L) could inhibit the VIP-induced expression of SP-A protein and SP-A mRNA. (4) Protein kinase C(PKC) inhibitor H7 (10(-5) mol/L) could also depress the V1P-induced SP-A protein and SP-A mRNA.

CONCLUSIONVIP can up-regulate the expression of SP-A through its receptor. PKC and c-fos protein play important roles in the intracellular signal transduction pathway through which VIP induces the expression of SP-A.

Animals ; Epithelial Cells ; drug effects ; metabolism ; In Vitro Techniques ; Protein Kinase C ; metabolism ; Proto-Oncogene Proteins c-fos ; metabolism ; Pulmonary Alveoli ; cytology ; Pulmonary Surfactant-Associated Protein A ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; Vasoactive Intestinal Peptide ; pharmacology

OBJECTIVETo study the association between fluid overload and acute kidney injury (AKI) after congenital heart disease surgery in infants.

METHODSA retrospective analysis was performed on 88 infants aged less than 6 months who underwent a radical surgery for congenital heart disease. The treatment outcomes were compared between the infants with AKI after surgery and those without. The effect of cumulative fluid overload on treatment outcomes 2 days after surgery was analyzed. The risk factors for the development of AKI after surgery were assessed by logistic regression analysis.

RESULTSCompared with those without AKI after surgery, the patients with AKI had younger age, lower body weights, higher serum creatinine levels and higher vasoactive-inotropic score, as well as longer durations of intraoperative extracorporeal circulation and aortic occlusion (P<0.05). Compared with those without AKI after surgery, the patients with AKI had a higher transfusion volume, a higher incidence rate of low cardiac output syndrome, a longer duration of mechanical ventilation, a longer length of stay in the intensive care unit (ICU), a longer length of hospital stay, a higher application rate of extracorporeal membrane oxygenation, a higher 30-day mortality rate, and higher levels of cumulative fluid overload 2 and 3 days after surgery (P<0.05). The logistic regression analysis showed that fluid overload and low cardiac output syndrome were major risk factors for the development of AKI after surgery. The children with cumulative fluid overload >5% at 2 days after surgery had a higher incidence rate of low cardiac output syndrome, a longer duration of mechanical ventilation, a longer length of stay in the ICU, a longer length of hospital stay, and a higher mortality rate (P<0.05).

CONCLUSIONSInfants with fluid overload after surgery for congenital heart disease tend to develop AKI, and fluid overload may be associated with poor outcomes after surgery.

Acute Kidney Injury ; etiology ; Body Fluids ; metabolism ; Cardiac Output, Low ; etiology ; Female ; Heart Defects, Congenital ; surgery ; Humans ; Infant ; Infant, Newborn ; Length of Stay ; Logistic Models ; Male ; Postoperative Complications ; etiology ; Respiration, Artificial ; Retrospective Studies

OBJECTIVETo study the changes in serum cortisol levels in adolescents with type 1 diabetes (T1DM) and elevated depressive symptoms.

METHODSTwenty-eight adolescents with T1DM and 31 healthy peers were assessed for depressive symptoms using a depression self-rating scale developed by the Epidemiological Survey Center. Selected subjects were classified into four groups: T1DM with elevated depressive symptoms group (n=15), T1DM without elevated depressive symptoms group (n=13), elevated depressive symptoms without T1DM group (n=15), and normal control group (n=16). Fasting blood samples were collected in the morning, and the levels of serum cortisol were compared among the four groups. The correlations of serum levels of cortisol and glycosylated hemoglobin A1c (HbA1c) with the score of depression self-rating scale were evaluated by Pearson correlation analysis.

RESULTSThe fasting serum cortisol levels in the 28 T1DM patients were significantly higher than in the 31 healthy peers (P<0.01). The fasting cortisol levels in the T1DM with elevated depressive symptoms group were significantly higher compared with those in the elevated depressive symptoms without T1DM group and normal control group (P<0.01). In adolescents with T1DM, serum HbA1c level was positively correlated with the score of depression self-rating scale (r=0.481, P=0.010).

CONCLUSIONSThe fasting serum cortisol levels in adolescents with T1DM and elevated depressive symptoms are significantly increased, suggesting that the patients with comorbidity of T1DM and depression develop dysfunction of the corticotropin-releasing hormone-adrenocorticotropic hormone-cortisol axis. The elevated depressive symptoms may be associated with a poor control of glucose metabolism.

Adolescent ; Adrenocorticotropic Hormone ; physiology ; Child ; Corticotropin-Releasing Hormone ; physiology ; Depression ; blood ; etiology ; Diabetes Mellitus, Type 1 ; blood ; Female ; Glucose ; metabolism ; Glycated Hemoglobin A ; analysis ; Humans ; Hydrocortisone ; blood ; Male

OBJECTIVETo explore the neuro-protective effect and mechanism of qingkailing injection (QKL) against cerebral injury caused by E. coli-meningitis (CM).

METHODSThe CM model rabbits were treated by ampicillin with QKL as adjuvant. The leukocyte count and protein content in cerebral spinal fluid (CSF), the contents of water, sodium, potassium and calcium in cerebral tissues were measured before, 16 h and 26 h after Bacillus coli injection respectively. The expression of matrix metalloproteinase-9 (MMP-9) was determined at the same time.

RESULTSAdjunctive treatment with QKL can not only inhibit the increase of leukocyte cells, protein content in CSF, and water, sodium, calcium content in cerebral tissues, but also the decrease of potassium content revealed during simple antibiotic treatment. It also can decrease the expression of MMP-9 in cerebral tissues of rabbits with CM.

CONCLUSIONAs an adjunctive treatment, QKL can prevent transient inflammatory reaction and aggravation of brain injury in CM induced by simple antibiotic treatment, its mechanisms might relate with calcium antagonism and attenuation of MMP-9 expression in brain tissues.

Ampicillin ; therapeutic use ; Animals ; Anti-Bacterial Agents ; therapeutic use ; Brain ; metabolism ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Injections ; Male ; Matrix Metalloproteinase 9 ; biosynthesis ; Meningitis, Escherichia coli ; drug therapy ; Neuroprotective Agents ; therapeutic use ; Phytotherapy ; Rabbits

AIMTo investigate the influence and mechanisms of 17beta-estradiol on the CTP: phosphorylcholine cytidylyltransferase (CCT) activity from cultured lung explants without serum.

METHODSWe detected the amount of [M-14C] choline incorporation into phosphatidylcholine so as to reflect CCT activity by liquid scintillation.

RESULTS(1) 17beta-estradiol increased the CCT activity in dose-dependence and time-dependence. (2) Both the protein kinase C inhibitor H-7 and calmodulin antagonist W-7 abolished the stimulatory effect of 17beta-estradiol (3 x 10(-6) mol/L) on the CCT activity.

CONCLUSION17beta-estradiol can increase CCT activity in cultured lung explants, its mechanism is related to protein kinase C and calmodulin.

Animals ; Calmodulin ; metabolism ; Choline-Phosphate Cytidylyltransferase ; metabolism ; Culture Media, Serum-Free ; Estradiol ; pharmacology ; In Vitro Techniques ; Lung ; drug effects ; enzymology ; Male ; Protein Kinase C ; metabolism ; Rats ; Rats, Wistar

Objective： To observe the effect of Qingkailing (QKL) on brain damage induced by glutamate, in order to seek for effective drugs for antagonizing neurotoxicity of glutamate. Methods：The number and morphological metrology of neurocytes in cerebral cortex and hippocampus were detected by MIAS-300 image analyser, electron microscope and immunohistochemical methods. Results：QKL could alleviate the glutamate induced accumulation of water and sodium in brain tissue，relieve the metrological and structural damage of cerebral cells in cortex and hippocampus, reduce the percentage of c-fos positive cell in brain. Conclusion： QKL could protect brain damage induced by glutamate, which might be related to the inhibition of QKL on the enhancement of c-fos gene expression induced by glutamate.

L-glutamate (Glu) is an excitatory neurotransmitter in the mammalian central nervous system. Relatively much attention has been paid to functional expression of Glu signaling molecules in peripheral tissues very recently. The present study tested the hypothesis that the activation of group I metabotropic glutamate receptor (mGluRI) in neutrophils stimulated neutrophils adherence to endothelial cells by increasing the surface expression of certain adhesion molecules. Peripheral blood was obtained by venipuncture from healthy donors, and the neutrophils were isolated by Ficoll-Hypaque gradient centrifugation. Neutrophils floating into DMEM/F12 culture medium containing 10% fetal bovine serum were then used immediately. Immunocytochemistry and real-time quantitative RT-PCR were used to detect the expression of mGluRI (mGluR1 and mGluR5) in neutrophils. The adherence of neutrophils to cultured human normal umbilical vein endothelial cells (HUVE-12) was measured by the colorimetric method. Cell surface expression of adhesion molecule CD11a in the neutrophils was determined by flow cytometry. Immunocytochemistry and real-time quantitative RT-PCR showed that mGluR1 and mGluR5 were constitutively expressed in neutrophils. Application of mGluRI agonist S-3,5-dihydroxyphenylglycine (S-DHPG) (1x10(-8)-1x10(-6) mol/L) showed a dose-dependent stimulatory effect on the adherence of neutrophils to HUVE-12 (P<0.05 or P<0.01), with a maximum effect at 1x10(-6) mol/L (P<0.01). Incubations as short as 30 min were sufficient to induce increased adherence after the beginning of S-DHPG treatment. Following time extension (0.5-5 h), S-DHPG (1x10(-6) mol/L) increased the rate of neutrophils adhesion to HUVE-12 with a maximum effect at 0.5 h (P<0.01). However, a time-dependent effect of S-DHPG on the rate of neutrophils adhesion to HUVE-12 was not observed during the experimental period. 1x10(-6) mol/L of S-DHPG also induced an increased surface expression of adhesion molecule CD11a (P<0.01) when neutrophils were preincubated with 1x10(-6) mol/L of S-DHPG for 1 h. Furthermore, the specific mGluRI antagonist (RS)-alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG, 0.5 mmol/L) significantly abolished the stimulatory effect of S-DHPG (1x10(-6) mol/L) on the adherence of neutrophils to HUVE-12 (P<0.01). These results suggest that the activation of mGluRI in neutrophils results in increased adhesion molecule CD11a expression and thereby promotes the adherence of neutrophils to endothelial cells.
Benzoates ; pharmacology ; CD11a Antigen ; metabolism ; Cell Adhesion ; Endothelial Cells ; cytology ; Glycine ; analogs & derivatives ; pharmacology ; Human Umbilical Vein Endothelial Cells ; Humans ; Neutrophils ; cytology ; Receptor, Metabotropic Glutamate 5 ; metabolism ; Receptors, Metabotropic Glutamate ; metabolism ; Resorcinols ; pharmacology

BACKGROUNDTo investigate the frequency of circulating HBV specific T helper cell and evaluate its association with serum levels of HBV DNA before and during lamivudine treatment in patients with chronic hepatitis B.

METHODSThe frequency of circulating HBV specific T helper cells in response to HBcAg in 25 chronic HBV-infected patients was determined by Elispot assay; serum HBV DNA was quantitated by real-time PCR.

RESULTSThe frequency of HBV specific T helper cell before antiviral treatment (47.30 +/- 25.50 SFCs /1 x 10(6) PBMC) was significantly higher than that at the third month of therapy (23.10 +/- 18.45 SFCs /1 x 10(6) PBMC, P < 0.05). All 8 patients observed dynamically had decreased frequency of HBV specific T helper cell at the third month of therapy; six patients with serum HBV DNA level reduced had higher frequency of HBV specific T helper cell before treatment than 2 patients without serum HBV DNA level decrease.

CONCLUSIONHBV specific T helper cell response at the time of hepatitis flare in chronic hepatitis B patients was significantly augmented compared to that at the time of catabasis.

Adult ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; genetics ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Hepatitis B Core Antigens ; immunology ; Hepatitis B virus ; drug effects ; genetics ; immunology ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Lamivudine ; therapeutic use ; Male ; T-Lymphocytes, Helper-Inducer ; cytology ; drug effects ; immunology

We have previously shown that the binding of integrins with extracellular matrix component fibronectin (Fn) can improve the ability of bronchial epithelial cells (BECs) in resisting oxidant injury by up-regulating the activity of catalase and increasing the content of GSH. However, the molecular mechanism or its signaling pathway of this protection is still unclear. In order to examine the intracellular signaling mechanism activated by Fn-integrin binding reaction, the present study investigated the mRNA expression of catalase in primary cultured rabbit BECs using RT-PCR based on a cell-injury model made with ozone exposure. The product bands of target gene CAT were checked with Southern blot and oligonucleotide probe hybridization. The results showed that Fn (10 microg/ml) promoted the catalase mRNA transcription (P<0.01). This effect was abolished either by protein-tyrosine kinase inhibitor genistein or calmodulin inhibitor W(7) (P<0.01). These results indicate that the promotion of catalase activity induced by Fn-integrin reaction is partly due to the elevation of catalase mRNA transcription, and that its signalling are possibly relevant to tyrosine phosphorylation or calmodulin pathway.
Animals ; Bronchi ; cytology ; metabolism ; Calmodulin ; metabolism ; Catalase ; biosynthesis ; genetics ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Female ; Fibronectins ; physiology ; Integrins ; physiology ; Male ; Protein-Tyrosine Kinases ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Rabbits ; Signal Transduction ; Up-Regulation ; drug effects