Neurons cultured from developing rat brain attach and spread preferentially to laminin

Neurons, mechanically dissociated from newborn rat brain and identified by immunostaining for neurofilaments, attached preferentially to laminin-coated coverslips without need of an underlying glial monolayer. The most extensive neurite outgrowth was seen when 20-30 micrograms/ml of laminin was used to coat the coverslips. Higher concentrations of laminin (greater than 30 micrograms/ml) supported single neurons to spread on the coverslips. Fibronectin coating of the coverslips allowed glial cells to attach more rapidly than on uncoated surfaces, but it did not support neuronal spreading or neurite outgrowth. Spreading of neurons and neurite outgrowth were completely inhibited by preincubation of laminin-coated coverslips with laminin antibodies but were unaffected by fibronectin antibodies. These results indicate that laminin is an attachment and spreading factor for central neurons in culture and suggest the presence of a laminin receptor on the neuronal cell surfaces.     

Gene products which play a role in cancer invasion and metastasis

Summary Invasion requires a number of distinct tumor cell interactions with host tissue, beginning with attachment to the matrix, followed by hydrolysis of matrix material and locomotion. Gene products which may be involved in these steps are discussed here. Laminin receptors and integrins have roles in the adhesion phase, while certain collagenases are prominent among the matrix-degrading enzymes. Autocrine motility factors, distinct from growth factors, appear to be involved in tumor cell locomotion. Finally, certain oncogenes, partricularly of theras family, are closely related with metastatic potential. A detailed understanding of the molecular biology of invasion and metastasis could ultimately lead to specific means of interfering with or even reversing these malignant processes.     

Loss of intercellular adhesion activates a transition from low- to high-grade human squamous cell carcinoma

The relationship between loss of intercellular adhesion and the biologic properties of human squamous cell carcinoma is not well understood. We investigated how abrogation of E-cadherin-mediated adhesion influenced the behavior and phenotype of squamous cell carcinoma in 3D human tissues. Cell-cell adhesion was disrupted in early-stage epithelial tumor cells (HaCaT-II-4) through expression of a dominant-negative form of E-cadherin (H-2Kd-Ecad). Three-dimensional human tissue constructs harboring either H-2Kd-Ecad-expressing or control II-4 cells (pBabe, H-2Kd-EcadDeltaC25) were cultured at an air-liquid interface for 8 days and transplanted to nude mice; tumor phenotype was analyzed 2 days and 2 and 4 weeks later. H-2Kd-Ecad-expressing tumors demonstrated a switch to a high-grade aggressive tumor phenotype characterized by poorly differentiated tumor cells that infiltrated throughout the stroma. This high-grade carcinoma revealed elevated cell proliferation in a random pattern, loss of keratin 1 and diffuse deposition of laminin 5 gamma2 chain. When II-4 cell variants were seeded into type I collagen gels as an in vitro assay for cell migration, we found that only E-cadherin-deficient cells detached, migrated as single cells and expressed N-cadherin. Function-blocking studies demonstrated that this migration was matrix metalloproteinase-dependent, as GM-6001 and TIMP-2, but not TIMP-1, could block migration. Gene expression profiles revealed that E-cadherin-deficient II-4 cells demonstrated increased expression of proteases and cell-cell and cell-matrix proteins. These findings showed that loss of E-cadherin-mediated adhesion plays a causal role in the transition from low- to high-grade squamous cell carcinomas and that the absence of E-cadherin is an important prognostic marker in the progression of this disease.     

Suspected malignant hyperthermia in a child with laminin alpha2 (merosin) deficiency in the absence of a triggering agent

Malignant hyperthermia (MH) is an inherited disorder of the skeletal muscles that can be triggered by many anesthetic agents. MH has different presentations and manifestations that makes it difficult to diagnose. Patients with laminin alpha2 deficiency have never been reported to be susceptible to MH. We present a suspected MH episode in the absence of classic triggering agents in a 7-year-old boy with laminin alpha2 (merosin) deficiency and congenital muscular dystrophy. The episode was diagnosed using the MH clinical grading scale and responded well to prompt management with dantrolene. We conclude that patients with laminin alpha2 deficiency may be susceptible to MH, and early suspicion and rapid treatment is vital in the management of MH. Anesthesiologists should be prepared to treat MH in susceptible patients even in the absence of a classical triggering agent.     

Laminin binding to a heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans

The interaction of Actinobacillus actinomycetemcomitans with the basement membrane protein, laminin, was examined in a ¹²⁵I-labeled protein-binding assay. The binding of laminin increased by lowering the pH. The ability to bind laminin was decreased in cells at the stationary phase of growth and by the presence of blood in the culture medium. Laminin binding to this bacterium was saturable, and the affinity constant was 4.6 nM. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot (ligand blot) analysis of cell-envelope and outer membrane of A. actinomycetemcomitans displayed a ¹²⁵I-laminin-reactive protein band with a molecular weight of 29 k. The laminin-binding protein was the previously described outer membrane protein A of A. actinomycetemcomitans. It was identified by its heat-modifiable property, detergent-solubility profile and reactivity with outer membrane protein A-specific polyclonal antiserum. At acidic pH, ¹²⁵I laminin bound to several cell-envelope components of A. actinomycetemcomitans , but at neutral pH, laminin bound only to the heat-modifiable protein. Despite the existence of the laminin-binding protein, cells grown in blood-containing media did not bind laminin. Several mammalian proteins interfered with laminin-bacterial interaction, including lactoferrin, which binds to the same bacterial protein that inhibited and displaced the laminin-bacterial interaction.     

Basement membrane patterns in borderline tumors of the ovary. An immunohistochemical study with antibodies to laminin and type IV collagen

Using antibodies directed against the basement membrane (BM) components laminin and type IV collagen, basement membrane patterns were studied in borderline epithelial tumors of the ovary. To determine the potential use of BM immunohistochemistry in the histopathologic diagnosis of tumors of borderline category, BM patterns were compared with those in cystadenomas and cystadenocarcinomas. In cystadenomas, regular and intact BM were found at the interface between the epithelial cells of the cysts and the adjacent stroma. Cystadenocarcinomas displayed an irregular pattern, with areas of intact BM between tumor cells and stroma, as well as areas with irregular discontinuities. Tumors of borderline malignancy shared a continuous BM pattern with cystadenomas, However, 30% of the borderline malignant tumors contained small areas with BM interruptions resembling those of invasive carcinoma. From the results of this study we conclude that BM patterns in ovarian tumors of borderline malignancy are mostly similar to those of benign epithelial tumors. Focal defects possibly indicating early invasive growth, however, occur in 30% of the cases.     

Downregulation of laminin alpha4 chain expression inhibits glioma invasion in vitro and in vivo

The laminin family is a structural constituent of the extracellular matrix that plays an essential role in promoting the motility of infiltrative tumor cells. We investigated the role of laminin alpha4 chain, a subset of laminin-8, -9 and -14, in the motile and invasive activities of human glioma cells. All malignant glioma cell lines examined expressed more mRNA for the laminin alpha4 and beta1 chains than for the beta2 chain, indicating that these cells predominantly express the laminin-8 isoform. Introducing an antisense oligonucleotide for laminin alpha4 chain (AS-Ln-alpha4) into the glioma cells resulted in downregulation of laminin alpha4 expression. AS-Ln-alpha4 also significantly suppressed glioma cell adhesion and migration. Furthermore, invasiveness was significantly reduced in cells transfected with AS-Ln-alpha4 compared to those transfected with the sense oligonucleotide (S-Ln-alpha4). Indeed, when glioma spheroids were implanted into rat brain slices, AS-Ln-alpha4-transfected cells failed to invade surrounding normal brain tissues. In addition, intracerebral injection of glioma cells transfected with AS-Ln-alpha4 into nude mice resulted in the formation of a noninvasive tumor, whereas injection of cells transfected with S-Ln-alpha4 resulted in diffuse invasion of brain tissue. These results suggest that mainly laminin-8 is essential for the invasive activity of human glioma cells; thus, a novel therapeutic strategy could target this molecule to treat patients with malignant glioma.     

Laminin Isoforms and Laminin-Producing Cells in Rat Anterior Pituitary

Laminin is a key component of the basement membrane and is involved in the structural scaffold and in cell proliferation and differentiation. Research has identified 19 laminin isoforms, which are assemblies of α, β, and γ chains (eg, the α1, β1, and γ1 chains form the laminin 111 isoform). Although laminin is known to be present in the anterior pituitary, the specific laminin isoforms have not been identified. This study used molecular biological and histochemical techniques—namely, RT-PCR, immunohistochemistry, and in situ hybridization—to identify the laminin isoforms and laminin-producing cells in rat anterior pituitary. RT-PCR showed that laminin α1, α3, and α4 genes were expressed in anterior pituitary. Immunohistochemistry revealed laminin α1 in gonadotrophs and laminin α4 in almost all vascular endothelial cells. Laminin α3 was seen in a subset of vascular endothelial cells. We then performed in situ hybridization to localize β and γ chains in these cells and found that laminin β1, β2, and γ1 were expressed in gonadotrophs and that laminin β1 and γ1 were expressed in endothelial cells. In conclusion, we identified gonadotroph-type (laminin 111 and 121) and vascular-type (laminin 411 and 311) laminin isoforms in rat anterior pituitary.     

氯氮平、氯丙嗪、利培酮对肝脏纤维化指标影响的研究

目的：了解抗精神病药物氯氮平、氯丙嗪、利培酮及其联合用药后对肝脏纤维化指标Ⅲ型前胶原（PⅢNP）、Ⅳ型胶原（Col-Ⅳ）、层黏连蛋白（LN）和透明质酸（HA）的影响。方法：选择211例服用氯氮平（93例）、氯丙嗪（47例）、利培酮（31例）或联合用药（40例）治疗超过三年的精神病患者作为对象,并选择性别、年龄相匹配的75例健康人群作为对照组,抽取外周血,用酶联免疫法检测其PⅢNP、Col-Ⅳ、LN及HA。结果：①与正常对照组比较,所有服药患者血清肝纤维化指标PⅢNP及HA存在显著差异（P〈0.01）,而Col-Ⅳ和LN则无差异;②氯氮平组、氯丙嗪组、利培酮组及联合用药组之间比较,血清肝纤维化指标均无显著差异;与对照组比较,单一用药组（氯氮平组、氯丙嗪组和利培酮组）PⅢNP及HA存在显著差异（P〈0.01）,而联合用药组则PⅢNP、HA及LN均存在差异。结论：氯氮平、氯丙嗪及利培酮均对血清肝脏纤维化指标PⅢNP和HA有影响;各药物对肝纤维化指标的影响无差异,但其联合用药对肝纤维化指标的影响更大。     

Differential localization of laminin gamma and integrin beta in primary cultures of the rat gingival epithelium

OBJECTIVES: The aim of this study was to investigate the differential immunolocalization of laminin gamma(2) and integrin beta(4) in primary cultures of the rat gingival epithelium. METHODS: The gingival epithelium was obtained from Sprague-Dawley rats and was cultured in serum-free keratinocyte growth medium (DK-SFM). Western blotting analysis, immunofluorescence, confocal laser scanning microscopy (CLSM), and immuno-gold labeling for laminin gamma(2) and integrin beta(4) were employed. CLSM images for laminin and integrin were analyzed in horizontal (x-y axis) and in vertical (x-z axis) sections. RESULTS: Both laminin gamma(2) and integrin beta(4) were detected by Western blot analysis in the gingival epithelium. Immunolocalization of laminin gamma(2) was distinct in the cytoplasm to form one or two irregular rings in gingival epithelial cells. By contrast, integrin beta(4) was localized diffusely in the cytoplasm. F-actin (indicating actin filaments) was clearly discernible at the periphery of the cytoplasm to form a cellular fringe. In x-z axis images obtained by CLSM, laminin gamma(2) was recognized as large foci in the most inner portion just above the basal plasma membrane. Integrin beta(4) existed in the area where F-actin was labeled surrounding the membrane. Immuno-electron microscopy showed that 10nm colloidal gold particles indicating laminin gamma(2) were mainly localized at the extracellular portion and in the peripheral cytoplasm, whereas integrin beta(4) was distributed in the cytoplasm close to the basal plasma membrane but not in extracellular regions. CONCLUSIONS: In primary cultures of the rat gingival epithelium, both laminin gamma(2) and integrin beta(4) may be produced by the epithelium, and irregular rings of laminin gamma(2) are formed in areas where gingival cells adhere to the extracellular matrix.