The interaction of Bcl-2 and Bax regulates apoptosis in biliary epithelial cells of rats with obstructive jaundice

 A complex molecular network controls cell homeostasis by inducing apoptosis or proliferation. The balance of Bcl-2 and Bax, members of a protein family, determines whether a cell will become immortal (Bcl-2) or will undergo apoptosis (Bax). To determine the role of Bcl-2 and Bax during proliferation of biliary epithelial cells (BEC) after bile duct ligation (BDL) and their regression after biliary decompression we induced hyperplasia of BEC by BDL in male rats. Regression of hyperplastic BEC by way of apoptosis was induced by biliary decompression through a Roux-en-Y biliodigestive anastomosis. To quantify apoptosis a modified TUNEL assay was used. Expression of Bcl-2 and Bax was visualized by immunohistochemistry and quantified stereologically. BEC increased from <1% to >20% after BDL; this increase was associated with overexpression of Bcl-2 in up to 30% of hyperplastic BEC. After biliodigestive anastomosis, apoptotic BEC increased from <0.1% to a peak of 5.4% after 1 day to reach baseline again 1 week after decompression. This was associated with de novo appearance of Bax. The interaction between Bcl-2 and Bax triggers apoptosis in BEC and acts as a cell rheostat in BEC hyperplasia and its involution after biliary decompression. Received: 9 July 1998 / Accepted: 16 December 1998     

A serum-free method for culturing normal human bronchial epithelial cells at clonal density

Summary A technique for the isolation and culture of normal human bronchial epithelial (NHBE) cells is described. This procedure involves first explanting fragments of large airway tissue to initiate fibroblastic-cell-free outgrowths of epithelial cells and subsequently using a serum-free medium (LHC-9) to propagate the NHBE cells at clonal density.     

MDCK cysts: An in vitro model of epithelial cyst formation and growth

Summary MDCK cells dissociated from monolayer culture were either dispersed within medium-hydrated collagen gel or seeded atop a collagen substrate which was immediately overlaid with collagen gel. Individual cells exhibited clonal growth in three dimensions to form spherical cysts in which a simple epithelium surrounded a fluid-filled lumen. The cells of MDCK cysts were polarized with apical surface bordering the lumen. MDCK cysts increased in diameter with continued culture. Maximum cyst size was dependent on seeding density and was influenced by medium composition. MDCK cysts could be isolated from the collagen substrate by digestion with collagenase. Also, collagen gel could be dissected from the cyst wall to give unrestricted access to regions of the basolateral cell surface. This novel method of renal cell culture provides a study system to model the influence of the extracellular matrix on kidney epithelial cell structure and function. It also offers an in vitro model of general application to the study of epithelial cyst formation and growth.     

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes of the fetal mouse in vitro

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes was analysed in vitro. When fetal mouse liver fragments containing immature hepatocytes but no bile ducts were cultured organotypically, the immature hepatocytes differentiated into large hepatocytes. Some of these expressed bile duct markers such as cytokeratin and Dolichos biflorus agglutinin-binding sites, though only to a small extent, and typical intrahepatic bile duct cells failed to differentiate. Dexamethasone stimulated immature hepatocytes to differentiate into both mature hepatocyte and biliary epithelial cell lineages. Especially in the liver fragments cultured on Matrigel, dexamethasone stimulated the expression of bile duct markers (such as cytokeratin and binding sites for two types of lectin) in the immature hepatocytes. These results support the idea that immature hepatocytes can differentiate into both mature hepatocytes and biliary epithelial cells during normal development of the mouse liver, and suggest that glucocorticoids stimulate both these differentiation pathways. It also seems that basal laminar components may play a role in bile duct differentiation.     

Influenza A virus infection of primary differentiated airway epithelial cell cultures derived from Syrian golden hamsters

The ability of several different influenza A virus strains to infect and replicate in primary, differentiated airway epithelial cell cultures from Syrian golden hamsters was investigated. All virus strains tested replicated equivalently in the cultures and displayed a preference for infecting nonciliated cells. This tropism correlated with the expression of both alpha2,3- and alpha2,6-linked sialic acid on the nonciliated cells. In contrast, the ciliated cells did not have detectable alpha2,6-linked sialic acid and expressed only low amounts of alpha2,3-linked sialic acid. In contrast to clinical isolates, laboratory strains of influenza A virus infected a limited number of ciliated cells at late times post-infection. The presence of alpha2,3- and alpha2,6-linked sialic acid residues on the same cell type suggests that Syrian golden hamsters and differentiated airway epithelial cell cultures derived from hamsters may provide a system for studying the reassortment of influenza A virus strains which utilize different forms of sialic acid as a primary virus receptor.     

A co-culture system for studies of paracrine effects of stromal cells on the growth of epithelial cells

Summary We have developed a co-culture system to study paracrine effects of fetal mesenchyme cells on the growth of primary mammary epithelial cells of the mouse. The method should be adaptable for study of interaction of a variety of cell populations. The procedure is simple and inexpensive. The culture consists of four layers: a monolayer of mesenchyme cells on a plastic culture dish, a soft agarose layer overlaying the cell monolayer, epithelial cells suspended in collagen gel placed atop the agarose, and culture medium above the collagen gel. The agarose layer prevents direct contact of two different cell populations but allows soluble molecules produced by either cell population to diffuse through the system, so as to contact and interact with the other cell population.     

胰酶冷消化加刮刷法分离培养气管上皮细胞的实验研究

目的:改进气管上皮细胞的培养技术。方法:采用胰酶冷消化加刮刷法分离兔气管上皮细胞, 测定分离细胞的数量、纯度及成活率, 并与机械剥离加酶消化法、酶温消化加刮刷法进行比较。同时比较无血清与有血清培养方法对上皮细胞纯度、融合时间的影响。结果:(1)胰酶冷消化加刮刷法分离所得上皮细胞的数量[(8.217±0.443)×10⁶ cells/只]明显高于机械剥离加酶消化法[(2.198±0.533)×10⁶ cells/只], P＜0.01;成活率(91.730%±1.532%)明显高于机械剥离加酶消化法(86.29%±0.46%)和胰酶温消化加刮刷法(83.07%±1.80%), 均P＜0.05;纯度(94.08%±2.70%)接近机械剥离加酶消化法(97.02%±0.51%), P＞0.05, 但明显高于胰酶温消化加刮刷法(88.900%±1.304%), P＜0.05。(2)无血清组在培养第3d(95.0%±0.0%)、第5d(95.3%±0.3%)、第7d(95.3%±0.3%)气道上皮细胞纯度显著高于有血清组(90.1%±0.8%, 84.7%±1.6%和83.0%±0.9%), P＜0.01;且细胞生长融合时间(6-7d)明显早于有血清组(12-14d)。结论:胰酶冷消化加刮刷法是一种经济、简便、高效的兔气管上皮细胞体外分离培养方法, 无血清培养可以提高培养上皮细胞的纯度, 促进细胞融合。     

A novel method of extraction of TnC from skeletal muscle myofibrils

 Incubation of mechanically skinned barnacle myofibrillar bundles in 10 mM orthovanadate (pH 6.6) results in the loss of Ca²⁺-dependent force generation, which reduces to 0.98±0.006% (mean ±SEM, n=25) of control levels. Analysis of myofibrillar bundles by gel electrophoresis showed that tension loss is primarily due to the extraction of troponin C (TnC) (65.4±5.04% mean ±SEM, n=5). This is a novel finding, since treating cardiac fibres with orthovanadate results in the removal of both TnC and troponin I (TnI) (28). Ca²⁺ dependence was restored to the myofibrillar bundles following reconstitution with either native isoform of barnacle TnC (BTnC₁: 78.72±12.8%, n=9, BTnC₂: 82.73±20.3%, n=3). The reversible loss of Ca²⁺-dependent tension generation following the removal and replacement of TnC indicates that the regulation of contraction in the barnacle is controlled by thin-filament regulatory proteins. Received: 30 September 1998 / Accepted: 16 December 1998     

A method for making rapid changes of superfusate whilst maintaining temperature at 37°C

 We have developed a technique for making a rapid solution change, whilst at the same time maintaining the temperature of the preparation at 37°C. It is technically difficult to use rapid solution changes when experiments are performed at normal mammalian body temperature. As a solution is heated from room temperature to 37°C, gas bubbles form in the rapid-flowing solution streams, and these disturb a cell or attached recording pipettes. We describe a system that has been developed to eliminate these problems. We show how to construct the different components of the system, and we have designed an electronic circuit to control solution changes. We have performed tests to characterise the function of this system. Solution flow out of the nozzle of the device (0.88 ml min^–1, linear flow velocity 11.6 cm s^–1) caused a fall in the steady-state temperature at the experimental preparation of only 0.3°C. The device which takes between 0.5 and 1 s to completely change the superfusate of a single cell, was used routinely with five different experimental solutions. This system may be valuable in studies which require rapid solution changes to be performed at a normal mammalian body temperature. Received: 11 March 1996 / Accepted: 21 May 1996     

A method for direct patch-clamp recording from smooth muscle cells embedded in functional brain microvessels

 The aim of this project was to develop a method to enable routine application of all patch-clamp configurations to smooth muscle cells while they remain embedded in blood vessels. Small blood vessels were isolated from rabbit brain using an enzymatic and mechanical procedure. Vessels were identified under a microscope and the majority were small arterioles with a mean external diameter, in Ca²⁺-containing (1.5 mM) solution, of 29 μm and variable lengths of 100 μm or more. Arterioles excluded trypan blue, constricted in response to 60 mM K⁺ and dilated in response to levcromakalim. Patch-clamp gigaOhm seals were made regularly on smooth muscle cells embedded in arterioles. The membrane potential recorded using amphotericin-B-containing patch pipettes averaged –72 mV. Short arteriolar segments could be voltage-clamped. Injection of depolarising current or bath application of 10 mM Ba²⁺ induced constriction of the entire arteriolar segment. Cell-attached patch, inside-out patch and outside-out patch recordings were made readily and K⁺ channel unitary currents were studied. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. Notably, avoidance of single-cell isolation means that enzymatic treatment is minimised and cells can be studied within their normal environment of the blood vessel wall. Received: 26 August 1997 / Received after revision and accepted: 17 October 1997     

Moving pictures and pulsed-field gel electrophoresis show only linear mitochondrial DNA molecules from yeasts with linear-mapping and circular-mapping mitochondrial genomes

  The mobility of mitochondrial DNA (mtDNA) in pulsed-field gel electrophoresis (PFGE) and its appearance in moving pictures from fluorescence microscopy were used to investigate the mitochondrial genome structure for five Pichia and Williopsis strains of yeast. An apocytochrome b-gene hybridization probe identified only linear mtDNA molecules for each strain when total cellular DNA was fractionated by PFGE. Most of the mass of DNA isolated from mitochondria for one linear-mapping and one circular-mapping mitochondrial genome was found in linear molecules much larger than the genome size of 50 kb; some molecules were as long as 1500 kb, but only a trace amount of apparently circular mtDNA was found for the strain with the circular-mapping genome. Probes for both the apocytochrome-b and mitochondrial small rRNA subunit genes hybridized strongly to mtDNA of approximately 50–100 kb, but weakly to the larger DNA from mitochondria of these two strains. For the four linear-mapping strains, PFGE revealed two or three distinct bands of linear mtDNA, larger than the genome size, within a smear of approximately 50–100 kb, but a smear without bands was found for the circular-mapping strain. Received: 28 June 1995 / 15 January 1996     

Nonneoplastic signet-ring cell change in gastrointestinal and biliary tracts: a pitfall for overdiagnosis

Nonneoplastic signet-ring cell change (SRCC) is a rare but known phenomenon in gastrointestinal and biliary tracts and is always associated with underlying mucosal ulceration/erosion secondary to infection, ischemia, or other etiology. Because nonneoplastic SRCC closely mimics signet-ring cell adenocarcinoma (SRCA), differentiation of these 2 entities is critical because misdiagnosis of nonneoplastic SRCC as SRCA can lead to intense therapeutic interventions such as surgery and/or chemoradiation therapy. In this review, a brief overview on nonneoplastic SRCC in gastrointestinal and biliary tracts, including the spectrum of clinical presentation, important histologic features, and immunohistochemical markers that are useful in differentiating nonneoplastic SRCC from SRCA, is provided. The pathogenesis of nonneoplastic SRCC in gastrointestinal and biliary tracts is discussed.     

A comparative study of an olfactory epithelial area lacking olfactory neurons and a nearby presence of TGF-alpha-like immunoreactive olfactory neurons

Our previous study has shown that ddY mice have special patches of nasal epithelium in the posterior roof of the nasal cavity that exclusively consists of olfactory supporting cells and horizontal basal cells. Here, we extend this finding to Balb/c and DBA/2 mice, Wistar and Sprague-Dawley rats, hamsters, and guinea pigs. In the mice, rats, and hamsters studied, the patches lacked olfactory cells and their precursor, globose basal cells. In rats and hamsters, the supporting cells were arranged in a single layer, in mice as three or four layers. Horizontal basal cells were located in a single layer in these species. In the guinea pigs, the specialized roof structure was less clear and could be seen at the level of ultrastructure as an olfactory neuron-lacking area. Distinct populations of transforming growth factor (TGF)-α-like immunoreactive olfactory cells occupied an area close to the epithelial patches. In this region, the TGF-α-like immunoreactive neurons were negative for the usual olfactory markers, either OMP or protein gene product (PGP) 9.5 or β-tubulin. These cells are suggested to project to the so-called ’necklace glomeruli’ and use a different cGMP-driven, transduction pathway. Three-dimensional analysis of double- labeled (TGF-α, PGP9.5) serial sections revealed a unique relation among the epithelial patches, TGF-α-like immunoreactive neurons and olfactory epithelium. Accepted: 20 November 2000     

A reliable method for sustaining a pre-defined pre-innervation level

 Numerous experimental approaches are based on evaluating electromyographically recorded muscle activity. Some experiments require a certain level of pre-innervation in a muscle or muscle group whereas others must avoid this. Measured parameters, such as the time to onset of the muscle response to an electrical stimulus, etc., depend critically on the level of pre-innervation. The pre-innervation level is most commonly estimated from parameters such as the force generated by this muscle or the upright posture of a human subject. These methods, however, are indirect and may yield erroneous results. This paper describes an inexpensive method developed for a wide range of applications in muscle-tonus-based experiments, in which the tonus is precisely controlled. A simple electronic circuit is presented by which the level of muscle pre-innervation is directly recorded, monitored and – depending on the experimental approach – also fed back to the subject. Physiological experiments on the flexion reflex in healthy human subjects document the reliability of our electronic device. Received: 13 August 1996 / Accepted: 17 January 1997     

A microarray-based method for detecting methylated loci

 CpG island DNA methylation plays an important role in regulating gene expression in development and carcinogenesis. We developed a new microarray-based method called methylation amplification DNA chip (MAD) for detecting differences in methylation. In this method, only methylated CpG islands from the two samples that we wanted to compare were amplified and used for hybridization. The resource material for the microarray was derived from the methylated DNA library of the sample in which we wanted to detect hypermethylation. Choosing the methylated DNA library as the resource material of the microarray increased the percentage of DNA fragments derived from hypermethylated loci on the microarray. Received: March 20, 2002 / Accepted: April 23, 2002     

一种简单经济的小鼠骨髓源性肥大细胞的培养与鉴定

目的:通过一种简单经济的方法在体外获得大量高纯度的肥大细胞。方法:将小鼠骨髓细胞直接冲出加入到含有IL-3和SCF的培养基中培养,每周换一次液,四到六周后收集培养细胞,通过光镜和电镜下观察肥大细胞的表面和内膜结构特征,流式细胞术检测表面CD117和FITC-FCεRⅠα的表达情况,利用甲苯胺蓝检测其功能。结果:四周后收集培养的细胞,其大小均一,内部结构符合肥大细胞的特征,电镜下可见肥大细胞内部含有丰富的高电子密度的颗粒,其中有些可见脱颗粒的空泡。流式检测显示CD117和FITC-FCεRⅠα单阳性的表达率都在90%以上,双阳性的表达率也在80%以上。甲苯胺蓝染色发现收集的肥大细胞核被染成深蓝色,胞质染成淡紫色。通过这种方式我们获得了大量的肥大细胞,其成熟时间较短,所需培养基和相关细胞因子量较少,而且寿命较长,纯度较高。结论:在IL-3和SCF的刺激下可以诱导骨髓干细胞向肥大细胞定向分化,通过这种方式可以快速经济的获得大量高纯度的肥大细胞,用于体外更好的研究其在移植免疫领域的作用。     

A method for estabilishing primary cultures of human gastric epithelial cells

Long-term culture of human gastric epithelial cells has been difficult, and at present no normal human gastric epithelial cell lines are readily available. As part of our experiments to study pathogenesis of H. pylori, a bacterium that infects the stomach, we developed methods to culture normal human gastric epithelial cells. Primary cultures of human gastric epithelial cells can be established from gastric biopsies taken at upper G.I. endoscopy. Enzymatically isolated gastric epithelial-like cells are present in tight colonies on culture dishes within 24 hours of placing the cells in culture. Cells isolated stain positively for cytokeratin and produce neutral mucins, indicating that they are mucin secreting epithelial cells, consistent with gastric epithelial cells. Epithelial cells can be maintained up to 4 weeks in culture with evidence of DNA synthesis up through the first week of culture.