Extracellular matrix (ECM) modulates the EGF-induced migration of liver epithelial cells in serum-free,hormone-supplemented medium

Summary The influence of the extracellular matrix (ECM) glycoproteins collagen, IV laminin (LN), and fibronectin (FN) on the in vitro migration of epithelial cells was studied using the ECM migration track method (4) with preparations immunostained for LN and FN. The locomotion of rat liver epithelial cells stimulated to migrate in serum-free medium by epidermal growth factor (EGF) in the presence of the protein per cm². Neither LN nor collagen IV decreased the number of migrating cells, indicating that the inhibition is a specific effect of fibronectin. The data also indicate that the FN-mediated inhibition of migration is an additional and not alternative mechanism to the well-established contact inhibition of locomotion (1) which also occurs in liver epithelial cell cultures. The system is being used for a further analysis of the factors that influence migration of normal and neoplastic epithelial cells and the biochemical mechanisms underlying the migration reaction. Editor’s Statement This paper describes new and heretofore neglected aspects of EGF and fibronectin action on the migratory behavior of cultured cells. Gordon H. Sato     

Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes

The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5-D-4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5-D-4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5-D-4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose-rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannose oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N-acetyl-D-glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5-D-4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti-CSPG monomer and not with anti-type II collagen contain precursors of CSPG monomer not yet modified by Golgi-mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti-keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.     

Deposition of type X collagen in the cartilage extracellular matrix

In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.     

癌症新型的诊疗靶点－APOBEC

APOBEC（“载脂蛋白质B mRNA编辑催化多肽”）是一类进化保守的胞苷脱氨酶家族。在人体内,已知含有保守的DNA胞嘧啶脱氨酶结构域的基因共有11种,包括AID、APOBEC1、APOBEC2、APOBEC3基因家族APOBEC3A、APOBEC3B、APOBEC3C、APOBEC3DE、APOBEC3F、APOBEC3G、APOBEC3H（分别称为A3A、A3B、A3C、A3D、A3F、A3G和A3H）和APOBEC4。APOBEC利用其脱氨酶活性通过与RNA和/或DNA结合,催化mRNA或使DNA中的胞嘧啶核苷酸转变为尿嘧啶,或者胞嘧啶核苷酸转变为胸腺嘧啶核苷酸,进而完成各自不同的功能。目前研究发现,AID及APOBEC3（A3s）的7种脱氨酶在人类的天然免疫和适应性免疫防御过程中发挥重要的作用,且在口腔癌,肺癌（腺癌和鳞状细胞癌）,结直肠癌和乳腺癌等的诊疗过程中具有重要的潜在应用价值。AID可以通过将胞嘧啶脱氨基成尿嘧啶,来启动SHM (体细胞超突变)和CSR (类别转换重组),进而在抗体多样性方面发挥作用。它的异常表达能够使B细胞淋巴瘤等恶性肿瘤的发病频率显著增加。而A3A、A3B通过胞嘧啶到尿嘧啶转换,以及自身表达量上调而在乳腺癌和肺癌诊疗中起作用。A3G通过APOBEC3G/miR 29/MMP2为了解结直肠癌肝转移和开发治疗晚期结肠癌的有效疗法开辟了新的途径。综上所述,本文将以AID,A3A,A3B,A3G为例子,对APOBEC在癌症诊断和治疗方面的应用进行综述,以期为进一步药物研究和临床应用等提供参考。     

Cross-linked collagen surface for cell culture that is stable,uniform, and optically superior to conventional surfaces

Summary A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed. This work was supported by the Leopold Schepp Foundation, the Dysautonomia Foundation, National Institutes of Health Grants NS14768 and NS11237, and Institutional Core Grant HD06276.     

猪垂体总mRNA的提取、鉴定及其cDNA的合成

 用改进的LiCl沉淀法和寡聚(dT)-纤维素亲和层析法由猪垂体制得总mRNA。在兔网织红细胞无细胞翻译体系中进行体外翻译的结果表明,制得的总mRNA具有一定的翻译活力。翻译产物与兔抗猪生长激素抗血清发生免疫沉淀,沉淀物占总翻译产物的10％左右。SDS聚丙烯酰胺凝胶电泳的结果表明翻译产物有一条很深的带,分子量约为24,000道尔顿,与猪前生长激素的分子量相近。以制备的mRNA为模板反转录合成了双链cDNA。第一链的合成产率为10—35％,第二链的合成产率为84—115％。cDNA的平均分子长度为825bp。     

A comparison of the effects of different substrata on chondrocyte morphology and the synthesis of collagen types IX and X

Summary Embryonic chick sternal chondrocytes were cultured either within three dimensional gels of type I collagen, type II collagen or agar, or as monolayers on plastic dishes coated with air-dried films of these matrix macromolecules. It was observed that cell shape and cell growth varied markedly between the different culture conditions. Flattened monolayers of cells on plastic or films of type I or type II collagen, proliferated more rapidly and reached a higher final cell density per culture than the more rounded cells found in the cultures on agar films or within three-dimensional gels. Biosynthetic studies demonstrated that in addition to the synthesis of type II collagen, all the cultures were producing collagen types IX and X. Chondrocytes cultured on plastic or films of the different matrix macromolecules all showed a similar expression of types IX and X collagen, independent of whether they displayed a flattened or round cell morphology. In contrast, marked variations in the proportions of the minor collagens, particularly type X collagen, were observed when the cells were cultured within three-dimensional gels. The data suggest that direct interaction of the cell surface with matrix constituents displaying a particular spatial array could be an important aspect in the control of type IX and X collagen expression by chondrocytes. The financial support of the Arthritis & Rheumatism Council and the Medical Research Council is gratefully acknowledged.     

Altered cardiac tissue gene expression during acute hypoxic exposure

Summary Anoxia has been shown to induce the expression of one or more stress proteins in mammalian cells and tissues. A less severe form of oxygen depletion, hypoxic hypoxia, occurs in response to hypobaric decompression which simulates high altitude conditions. Under these conditions mouse hearts accumulate mRNAs for at least two polypeptides at substantially elevated levels. The molecular weights of these proteins, 85 kDa and 95 kDa, are similar to those reported for other mammalian stress proteins or glucose-regulated proteins. Time course experiments suggest that mRNAs for these species increase continuously for up to 16 hours of treatment, while mRNA for 71 kDa and 79 kDa polypeptides are elevated early in the treatment, but later decrease to control values. Total heart mRNA template activity is also increased by the hypobaric treatment. These results demonstrate that mouse cardiac tissue is capable of mounting a cellular stress-like response when exposed to moderately stressful conditions. It also provides a model for studying the direct effects of acute hypoxic stress on cellular gene expression, and its relationship to physiological adaptation.     

Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Isolation and characterization of a human interleukin 2 gene

An interleukin 2 (IL-2) gene was isolated from a Charon 4A human gene library. Electron microscopic examination of 15 heteroduplexes formed between the genomic DNAs and the IL-2 cDNAs demonstrated that the size of the IL-2 gene is about 5.1 +/- 0.5 kb and that there are at least two introns in this gene. Nucleotide sequence of the 5' flanking region of the IL-2 gene showed a homology with that of the corresponding region of the human immune interferon gene.