人胃癌细胞株裸鼠皮下移植瘤模型的建立及其生物学特性

目的初步建立不同浓度下制备人胃癌细胞株BGC-823裸鼠皮下移植瘤模型,观察其生物学特性。方法培养人胃癌细胞系BGC-823细胞,并分别以四个浓度皮下注射于裸鼠腋下每只0.2 mL。根据BGC-823细胞注射浓度将40只裸鼠随机分为四组：组一5×107个活细胞/mL（n=10）;组二1×107个活细胞/mL（n=10）;组三1×106个活细胞/mL（n=10）;组四1×105个活细胞/mL（n=10）。观察各组裸鼠摄食、活动情况、精神状态、死亡率、成瘤时间、成瘤率、肿瘤生长情况,采用免疫组化法检测肿瘤微血管密度。结果各组裸鼠摄食、精神情况正常,无死亡现象。除组四1×105个活细胞/mL浓度组成瘤率为0%外,其余各组成瘤率均为100%,瘤体出现时间在3～7 d,肿瘤血管密度MVD平均为：（123.26±31.57）个/mm2。结论初步建立了人胃癌裸鼠皮下移植瘤模型及建立此细胞系模型的最低浓度。为胃癌的进一步研究奠定了基础。     

流感病毒在Vero细胞系与MDCK细胞系增殖条件的比较

目的研究流感病毒H1N1及其他亚型在Vero细胞系和MDCK细胞系高效增殖的最适条件,比较两种细胞系对流感病毒的敏感性差异及影响敏感性差异的条件。方法在培养好的Vero细胞系与MDCK细胞系用不同的病毒感染复数(M.O.I)、胰酶浓度、病毒吸附时间、病毒维持液血清质量浓度等条件进行流感病毒在细胞上的增殖。结果在M.O.I为0.01接种流感病毒,吸附时间为1 h,胰酶质量浓度2μg/mL,血清质量浓度为8%时,流感病毒血凝素在MDCK细胞系可获得较高的滴度。结论 MDCK细胞系是适于流感病毒培养的细胞,它作为生产新型流感病毒疫苗的主要细胞基质需要进一步的研究。     

Proneural gene requirement for hair cell differentiation in the zebrafish lateral line

The lateral line system comprises an array of mechanosensory organs, the neuromasts, distributed over the body surface. Each neuromast consists of a patch of mechanosensory hair cells surrounded by support cells. We show that, in the zebrafish, two proneural genes are essential for differentiation of the hair cells, neuroD (nrd) and atonal homolog 1 (ath1). Gene knockdown experiments demonstrate that loss of function of either gene, but not of the related proneural gene neurogenin1 (ngn1), abrogate the appearance of hair cell markers. This is in contrast to other sensory systems, such as the neurons of the lateral line ganglion, where nrd is regulated by ngn1 and not by ath1. Overexpression of ath1 can induce nrd, and the phenotype produced by loss of ath1 function can be partially rescued by injection of nrd mRNA. This supports the conclusion that the activation of nrd probably requires ath1 in the hair cell lineage, whereas in sensory neurons nrd activation requires ngn1. We propose that the emergence of two atonal homologs, ath1 and ngn1, allowed the cellular segregation of mechanoreception and signal transmission that were originally performed by a single cell type as found in insects.     

High glucose and angiotensin II increase β1 integrin and integrin-linked kinase synthesis in cultured mouse podocytes

Alterations of integrin α3β1 may play a role in the development of diabetic nephropathy. We have investigated the effects of high glucose and angiotensin II on the expression of integrin α3 and β1, and whether these changes are associated with integrin-linked kinase (ILK) in cultured mouse podocytes. Integrin β1 and ILK mRNA expression and protein production were rapidly up-regulated in a dose-dependent manner by high glucose and angiotensin II stimulation. ILK mRNA levels in the mouse podocytes exposed to 30 mmol/l glucose were 1.66, 1.89, and 1.28 times higher than those in control cells at 6, 24, and 72 h exposure, respectively. ILK mRNA levels in mouse podocytes exposed to 1 nM, 10 nM, and 100 nM angiotensin II for 6 h were 1.38, 1.55, and 1.93 times higher, respectively, than those in control cells. Angiotensin-II-induced integrin β1 and ILK mRNA expression was significantly inhibited by treatment with losartan (100 μM). In addition, the up-regulation of ILK synthesis induced by these stimuli was related to β1 integrin synthesis and increased ILK kinase activity. Cell adhesion assay displayed inhibitory effects when podocytes were exposed to high concentrations of angiotensin II. Interestingly, glucose and angiotensin II stimulation induced shrinkage of the cell body and elongation of the podocyte processes, a phenotype similar to that of immature podocytes. In addition, β1 integrin showed higher levels of staining on both the cell membranes and the cell-cell contact areas. Thus, high glucose and angiotensin II may affect the regulation of the integrin-ILK system in podocytes; this system may therefore play a role in the pathogenesis of diabetic nephropathy and other renal diseases affecting podocytes. The results presented in this paper have not been published previously in whole or part, except in abstract form. This work was supported by grant R01–2002–000–00139–0 from the Basic Research Program of the Korea Science & Engineering Foundation.     

PTEN is involved in the signal transduction pathway of contact inhibition in endometrial cells

PTEN is involved in the regulation of normal cellular functions in addition to its well–known role as a tumor suppressor. In the present study, we have shown that stable transfection of the PTEN gene into PTEN–mutated endometrial carcinoma cells leads to contact inhibition accompanied by a decreased level of phosphorylated–Akt (p–Akt) expression, an increase in p27^Kip1, and a decrease in β–catenin. PTEN–induced cells with contact inhibition exhibit G0–G1 cell-cycle arrest, and the Ki–67 labeling index is reduced. These changes are canceled by transfection of a double–stranded short–interfering RNA against the PTEN gene. Normal endometrial stromal cells increase their PTEN expression when reaching confluence; this is followed by changes in the expression of Akt–related proteins in the same way as in tumor cells. These results indicate that PTEN, p–Akt, p27, and β–catenin are involved in the signal transduction of contact inhibition and suggest that PTEN may, in part, control the proliferation of endometrial carcinoma cells through the induction of contact inhibition.     

Comparison of survey methods for wintering grassland birds

ABSTRACT Although investigators have evaluated the efficacy of survey methods for assessing densities of breeding birds, few comparisons have been made of survey methods for wintering birds, especially in grasslands. In winter, social behavior and spatial distributions often differ from those in the breeding season. We evaluated the degree of correspondence between density estimates based on different survey methods. Surveys were conducted during two winters (2001–2002 and 2002–2003) on 16 grassland sites in southwestern Oklahoma. Line‐transect (using a detection function to account for birds present but not detected) and area‐search (where density was based on the total count within a given area) methods were employed. Observations on line transects were also analyzed as strip transects, where density was based on total count within a given strip width and no detection function was used. Savannah Sparrows (Passerculus sandwichensis), LeConte's Sparrows (Ammodramus leconteii), Song Sparrows (Melospiza melodia), Smith's Longspurs (Calcarius pictus), Chestnut‐collared Longspurs (C. ornatus), and Eastern Meadowlarks (Sturnella magna) were sufficiently abundant to allow comparison. Area‐search density estimates tended to be higher than line‐transect estimates for Savannah Sparrows, Song Sparrows, and Eastern Meadowlarks, suggesting that some individuals initially located close to the transect line were not detected on line transects. The area‐search and line‐transect methods gave similar density estimates for Chestnut‐collared and Smith's longspurs. Area‐search estimates of Eastern Meadowlarks were significantly higher in the second year of the study only. For this species, area‐search estimates did not differ from those of strip transects covering an equal area, so the reason for the differing meadowlark estimates is not clear. Higher density estimates using the area‐search method likely resulted from: (1) birds that might escape detection by hiding were more likely detected (flushed) during area searches because of the repeated passes through the area, and (2) birds close to the line in line transects escape detection by hiding, biasing those estimates low. We also evaluated the correspondence of density rankings for the six species as determined by the different survey methods and for the same species across survey sites. Correlations among the six species of the area‐search results with those of line transects and strip transects generally were high, increasing in 2002–2003 when densities of birds were greater. All three methods provided similar density rankings among species. Density rankings within species across sites for the four non‐longspur species generally were concordant for the three methods, suggesting that any of them will adequately reflect among‐site differences, especially when densities vary greatly across sites. Further research is needed to determine the extent to which grassland birds are missed on line transects. We suggest that workers using line transects to study these species give careful consideration and make additional efforts to satisfy the distance‐sampling assumption that all birds on or near the line are detected. If density is measured as a total count in a fixed area, we recommend that observers pass within <10 m of all points in the area.     

Expressions of polypeptide: N-acetylgalactosaminyltransferase in leukemia cell lines during 1,25-dihydroxyvitamin D3 induced differentiation

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)₂D3] on two leukemia cell lines, K562 and SHI-1, and its relation to the expression of different subtypes of polypeptide: N-acetylgalactosaminyltransferase (pp-GalNAc-T) was studied. With morphological and cell flow-cytometric method, it was found that 1,25(OH)₂D3 induced the differentiation of both leukemia cell lines toward monocytic lineage, but not affected the cell growth and apoptosis. The expressions of different subtypes of pp-GalNAc-T, the initial glycosyltransferase in O-glycan synthesis, were studied with RT-PCR before and after the treatment of different concentrations of 1,25(OH)₂D3. Among fourteen subtypes of pp-GalNAc-T (T1 ∼ T14), K562 cells obviously expressed pp-GalNAc-T2, T4, T5, T7 (T2 was the highest) and SHI-1 cells apparently expressed pp-GalNAcT1, T2, T3 and T4 (T4 was the highest) only. After K562 cells were treated 1, 25(OH)₂D3 for 72 h, pp-GalNAc-T2, T4, T5, T7 were increased in a dose dependent manner. In contrast, pp-GalNAc-T1 and T2, especially T1, were up-regulated in SHI-1 cells by 1,25(OH)₂D3, but T3 was unchanged and T4 was down-regulated. The different alterations of pp-GalNAc-Ts in these two cell lines were probably related to the different structural changes of O-glycans during 1,25(OH)₂D3 induced differentiation.     

Addition of protic nucleophiles to alkynyl methoxy carbene ligands in diiron complexes

Different protic nucleophiles (i.e.Ph₂CNH, PhSH, MeCO₂H, PhOH) can be added to the CC bond of [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C(OMe)CCTol}(Cp)₂][SO₃CF₃] (1), affording new diiron alkenyl methoxy carbene complexes.The additions of Ph₂CNH and MeCO₂H are regio and stereoselective, resulting in the formation of the 5-aza-1-metalla-1,3,5-hexatriene [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(OMe)C_βHC_γ(Tol)(NCPh₂)}(Cp)₂][SO₃CF₃] (2), and the 2-(acyloxy)alkenyl methoxy carbene complex [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(OMe)C_βHC_γ(Tol)OC(O)Me)}(Cp)₂][CF₃SO₃] (5); the E isomer of the former and the Z of the latter are formed exclusively.Conversely, the addition of PhSH is regio but not stereoselective; thus, both the E and Z isomers of [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(OMe)C_βHC_γ(Tol)(SPh)}(Cp)₂][SO₃CF₃] (3) are formed in comparable amounts.Compounds 3 and 5 are demethylated upon chromatography through Al₂O₃, resulting in the formation of the acyl complexes [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(O)C_βHC_γ(Tol)(SPh)}(Cp)₂] (4) and [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(O)C_βHC_γ(Tol)OC(O)Me}(Cp)₂] (6), respectively, both with a Z configured C_βC_γ bond.Finally, the reaction of 1 with PhOH proceeds only in the presence of an excess of Et₃N affording the 2-(alkoxy)alkenyl acyl complex [Fe₂{μ-CN(Me)(Xyl)}(μ- CO)(CO){C_α(O)C_βHC_γ(Tol)(OPh)}(Cp)₂] (7). The crystal structures of 4 · CH₂Cl₂ and 7 · 0.5CH₂Cl₂ have been determined by X-ray diffraction experiments.