Comparison of the properties of neural stem cells of the hippocampus in the tree shrew and rat in vitro.

Neural stem cells (NSCs) are characterized by the ability of self­renewal and capacity to proliferate and produce new nervous tissue. NSCs are capable of differentiating to three lineages of neural cells, including neurons, oligodendrocytes and astrocytes. Furthermore, hippocampal NSCs transplantation can improve the neurological deficits associated with expression of cytokines. Therefore, to compare the properties of NSCs of tree shrews and rats in vitro, NSCs from tree shrews (tsNSCs) and rats f(rNSCs) were isolated. Nestin was used as a marker to identify the cultured NSCs. Neuronal nuclei protein and glial fibrillary acidic protein (GFAP) were utilized to demonstrate the differentiation of NSCs towards neurons and astrocytes, respectively, in vitro. Furthermore, the expression of neurotrophin 3 (NT3), brain­derived neurotrophic factor (BDNF), glial cell­derived neurotrophic factor (GDNF) and transforming growth factor (TGF)ß1 was also investigated in tsNSCs and rNSCs. The expression of all of the aforementioned proteins was detected using immunofluorescence methods. The results demonstrated that, after 5 days of culture, the average number of neurospheres in the cultured tsNSCs was significantly lower compared with rNSCs (P=0.0031). Additionally, compared with the rNSCs, tsNSCs exhibited an enhanced differentiation ability towards neurons. Furthermore, the expression of NT3 in the tsNSCs was higher compared with rNSCs (P<0.01), while the expression of BDNF was lower (P=0.045). However, no significant differences were observed in the expression level of GDNF and TGFß1 between rNSCs and tsNSCs. Therefore, these results indicate that tsNSCs exhibit specific characteristics that are different from rNSCs, which provides novel information for the understanding of NSCs obtained from tree shrews. Overall, the results of the current study provide evidence to support the increased application of tree shrews as models for diseases of the central nervous system.

[The Improvement in the Primary Culture of Alveolar Epithelial Type of Rats].

OBJECTIVES: To improve the method of culturing and obtain purified alveolar epithelial type1 (AT1) cells of SD rats. METHODS: 1 d newborn SD rats were applied for cell culture and brains were decapitated for lung tissues obtaining after respiratory cessation. Collagenase1and DNase1 were used to digest and isolate cells. Then, cells were put into the plate coated with type1 rat tail collagen and different adherence was used to purify cells. Meanwhile, culture medium replacement was conducted after 48 h. The growth status was observed under an inverted microscope. Immunofluorescence including specific marker AQP5, SPC, BSI, Vimentin were used to identify cells. RESULTS: 2 d after incubation, the cells began to adhere to plate. At day 4 and 6, cells began to proliferate and exhibited a shape of spindle, cube and polygon. 8 d after incubation, the character of proliferation reached the highest and the cell viability was (87±8)% and purification was (87±5)%. CONCLUSION: By modifying the methods of tissues harvest, isolation and culture, we can obtain AT 1 cells with high yield, good growth state and super purity.

Comparison of pathology, blood gas and biomarkers between two rat models of acute lung injury.

An ideal animal model to explore pathogenesis and prevention of acute lung injury (ALI) is essential. The present study aims to compare the difference in pathology, blood gas values and biomarkers of two acute lung injury rat models at different time intervals. In the experiment, rats were randomly divided into three groups: lipopolysaccharide (LPS) group, oleic acid (OA) group and control group. Changes of pathology, blood gas values and blood-air barrier biomarkers were analyzed at 15 minutes, 6 hours, 12 hours and 24 hours after injection. The results showed that the two models exhibited different features. Compared with the LPS rats, OA rats exhibited significantly severe pathological changes, lower arterial oxygen partial pressure (PaO2) value and higher level of injury biomarkers. However, LPS rats boasted greater lactic acid (LAC) level and more severe acidosis than OA rats. This study suggests that LPS-induced model has greater value in researches on microcirculation dysfunction and sepsis resulting from ALI, while OA-induced model has greater repeatability in area of gas exchanging after ALI. These events may provide a new theoretical evidence for the model establishment of ALI.

[Protective effect of ulinastatin on pulmonary function after cardiopulmonary bypass].

OBJECTIVE: To investigate the effects of Ulinastatin with different doses on pulmonary protection after cardiopulmonary bypass (CPB). METHODS: Ninety patients after CPB were brought into this study and divided into low doses Ulinastatin group (L group, n=30, 5 000 U/kg), high doses Ulinastatin group (H group, n=30, 20 000 U/kg) and control group (C group, n= 30), respectively. When the patients were transferred into ICU after CPB, Ulinastatin was given intravenously to those in L and H group, while saline was given in C group. Blood samples were harvested at the time before the treatments (T0) and 12 hours (T1), 24 hours (T2) after the treatments, for the measurements of arterial pressure of oxygen (PaO2), arterial pressure of carbon monoxide (PaCO2),difference of alveoli-arterial oxygen pressure (PO(2A), oxygenation index (Ol),and tumor necrosis factor-alpha (TNF-alpha) level. Pulmonary dynamic compliance (Cd), plat pressure (Pplat) and peak pressure (Ppeak) were determined at the time of To and wean (Tw). The durations of ventilation and intubation were recorded. RESULTS: At T0, the levels of PaO2, PaCO2, PO2A-a, OI and TNF-alpha in each group showed no significantly difference (P> 0. 05). At T1 and T2, the patients in H group had higher levels of PO2, PO2A-a. and OI, lower level of TNF-alpha, shorter duration of ventilation and intubation than the patients in other two groups(P<0. 05). The parameters in L group were better than those in C group, but the differences were not stastically significant (P>0. 05). There was no significantly difference in the levels of Cd, Pplat, and Ppeak at T0 and Tw between any two groups (P>0. 05). The intubation and ventilation time in H group were shorter than that in L and C group (P<0. 05). CONCLUSION: The application of Ulinastatin could achieve pulmonary protective effect after CPB, and it seems the effect could be better with high dose (20 000 U/kg) of Ulinastatin.

[Computer-aided molecular modeling and activity estimation for ligand screening with specific phage clone as the target].

OBJECTIVE: To investigate the interaction between tumor necrosis factor alpha (TNF alpha) mimotopes and TNF alpha-binding peptides screened from random phage display peptide library with TNF alpha mimotopes displayed on phage clone as the target, the computational docking program AutoDock (with confirmation calculations using Discover) was used to predict and analyze the binding modes of LLT-18 (TNF alpha binding peptide, sequence EHMALTYPFRPP) with TNF alpha, after which LCS-7 (TNF alpha mimic phage clone, displayed positive sequence c-RRPAQSG-c) was docked to LLT-18 manually. The binding between LLT-18 and TNF alpha or LCS-7 was stabilized predominantly through electrostatic interaction and H-bond formation. The Arg residues in TNF alpha or LCS-7 were important for their interaction with LLT-18. For LLT-18, the key amino acid residues were Glu1, His2, Met3 and Tyr7. These results suggest the feasibility of screening ligand to single epitope with specific phage clone as the target, and of predicting the interaction between small peptides by computer-aided molecular modeling.

Structure-based optimization of phenylbutyrate-derived histone deacetylase inhibitors.

Previously, we developed a strategy to develop a novel class of histone deacetylase (HDAC) inhibitors by tethering short-chain fatty acids with Zn(2+)-chelating motifs, which led to N-hydroxy-4-(4-phenylbutyryl-amino)benzamide (HTPB), a hydroxamate-tethered phenylbutyrate derivative with sub-micromolar potency in inhibiting HDAC activity and cancer cell proliferation. In this study, we carried out structure-based optimization of HTPB by using the framework generated by the structure of histone deacetylase-like protein (HDLP)-trichostatin A (TSA) complexes. Docking of HTPB into the HDLP binding domain suggested that the hydrophobic microenvironment encompassed by Phe-198 and Phe-200 could be exploited for structural optimization. This premise was corroborated by the greater potency of (S)-(+)-N-hydroxy-4-(3-methyl-2-phenylbutyrylamino)-benzamide [(S)-11] (IC(50) in HDAC inhibition, 16 nM), of which the isopropyl moiety was favorable in interacting with this hydrophobic motif. (S)-11 at concentrations as low as 0.1 microM was effective in causing histone hyperacetylation and p21(WAF/CIP1) overexpression and suppressing proliferation in cancer cells.

A model of 3D-structure of H+, K+-ATPase catalytic subunit derived by homology modeling.

AIM: To build a model of 3D-structure of H(+), K(+)-ATPase catalytic subunit for theoretical study and anti-ulcer drug design. METHODS: The model was built on the basis of structural data from the Ca(2+)-ATPase. Structurally conserved regions were defined by amino acid sequence comparisons, optimum interconnecting loops were selected from the protein databank, and amino (N)- and carboxyl (C)-terminal ends were generated as random coil structures. Applying molecular mechanics method then minimized the model energy. Molecular dynamics technique was used to do further structural optimization. RESULTS: The model of 3D-structure of H(+), K(+)-ATPase was derived. The model is reasonable according to several validation criteria. There were ten transmembrane helices (TM1-TM10) in the model and inhibitor-binding site was identified on the TM5-8 riched negatively charged residues. CONCLUSION: The 3D-structure model from our study is informative to guide future molecular biology study about H(+), K(+)-ATPase and drug design based on database searching.