Neurite-promoting activity from fetal skeletal muscle: partial purification of a high-molecular-weight form

Neurite extension from sensory neuroblasts dissociated from chick embryo dorsal root ganglia can be stimulated by precoating the polylysine culture surface with extracts of skeletal muscle from bovine fetuses. The active factor(s) may be partially purified from cytosolic extracts of muscle by chromatography on Sepharose 6B and affinity chromatography on wheat germ agglutinin or Helix pomatia agglutinin columns. Extract concentrations of 10-50 micrograms protein per 1 ml were active in promoting neurite extension when the neurons were cultured without serum or nerve growth factor (beta NGF). However, levels of 1-10 micrograms/ml produced dramatic neurite extension when 10% (v/v) fetal or newborn calf serum or 0.5 ng/ml beta NGF was added to the medium. The biological activity was not blocked by antiserum that was raised against purified mouse laminin and that abolished the neurite-promoting activity of mouse laminin. The activity of the muscle extract was destroyed by trypsin or heparitinase, in contrast to the biological activity of purified mouse laminin, which was not abolished by heparitinase treatment. The activity could be resolved into two broad peaks on a Sepharose 2B column (apparent Mr between 2 X 10(6) and in 10 X 10(6) in native form). Treatment with dithiothreitol was necessary to dissociate the factor for electrophoresis in 4.25% polyacrylamide-SDS gels, revealing three major polypeptide bands at Mr = 160,000, 195,000 and 200,000. This preliminary characterization indicates that the neurite-promoting activity from bovine skeletal muscle tissue consists of a high-molecular-weight complex, one essential component of which is a heparan sulfate.     

Basement membrane continuity in benign, premalignant and malignant epithelial conditions of the uterine cervix

The pattern of basement membrane deposition in the uterine ectocervix was assessed in benign, premalignant and malignant conditions, using an indirect immunoperoxidase technique to detect laminin, an intrinsic basement membrane component. A semi-quantitative approach was used to assess the frequency of small breaks in the basement membrane. The cervical squamous epithelium in benign epithelial conditions has an almost completely continuous basement membrane, but the development of viral atypia or cervical intraepithelial neoplasia (CIN) is associated with the appearance of small basement membrane breaks. There is a correlation between increasingly severe CIN and increasing numbers of breaks, and there is a concurrent increase in the numbers of subepithelial inflammatory cells. The development of invasive neoplasm is associated with a sudden change to a fragmented pattern of basement membrane deposition, a finding which is of potential diagnostic use.     

Laminin supports short-term survival of rat septal neurons in low-density, serum-free cultures

Septal neurons from embryonic rats do not survive for 24 hr when dissociated and cultured at low density in a serum-free medium. Laminin at 5-40 micrograms/ml acts as a survival-promoting agent in the presence of 1 mM pyruvate. Laminin is effective in promoting survival only if it is added before or during cell plating; it does not support survival if added after cell attachment. Variations in laminin concentration do not effect the total number of cells attaching to the culture plate. The percentage of septal cells with neurites is increased in a dose-dependent manner by laminin as early as 45 min after cell plating, suggesting an effect of laminin on the rate of neurite initiation. Neurite extension at 24 hr is also dependent on laminin concentration. This study suggests that some interaction between cells and the substrate, independent of cell attachment, has a profound effect on cell physiology, increasing both cell survival and the rate of neurite extension. This study also defines the survival requirements of septal neurons in a chemically defined environment in a low-density situation where cellular interactions are at a minimum.     

VARIATION IN MEMBRANE PROPERTIES FROM THE ACTION OF LAMININ ON MEMBRANE RECEPTORS

Biophysical studies were conducted on the action of laminin through membrane receptors of cancer cells. The results showed that variations occurred in the thermodynamic properties of membrane proteins, the mobility of hydrocarbon chains of membrane lipids, and the permeshility and transportation pathways of the membrane.     

Immunolocalization of basement membrane molecules in the stroma of salivary gland pleomorphic adenoma

In order to determine the participation of basement membrane molecules in formation of its characteristic stroma, 30 cases of salivary gland pleomorphic adenoma were examined by immunohistochemical staining for type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. The stroma was histopathologically classified into four types: hyaline, fibrous, myxoid, and chondroid. Immunohistochemically, type IV collagen and laminin were more intensively localized in hyaline, fibrous and chondroid types of stroma, whereas heparan sulfate proteoglycan was more prominent in myxoid areas. The results suggest that the stroma contains these basement membrane components, which are possibly biosynthesized by epithelial tumor cells, and that histological variety of the stroma depends on proportion of local contents of each basement membrane molecule.     

Transgenic overexpression of laminin α1 chain in laminin α2 chain–deficient mice rescues the disease throughout the lifespan

Several approaches to treat laminin α2 chain–deficient congenital muscular dystrophy (MDC1A) in mouse models have been undertaken. Most have shown promising results in young animals. However, older animals have only been characterized to some extent. Herein we analyze the lifespan of laminin α2 chain–deficient mice with transgenic overexpression of laminin α1 chain. Further outcome measures included internalized myonuclei, heart fibrosis, grip strength, and serum creatine kinase activity. We show that laminin α2‐chain–deficient animals that overexpress laminin α1 chain survive to up to 1.5–2 years of age. Furthermore, they displayed improved skeletal and heart muscle morphology, near‐normal muscle strength, and normalized creatine kinase levels. Such an improvement of the dystrophic phenotype that persists to old age has not been previously demonstrated in mice. Our findings hold promise with regard to the efficient treatment of MDC1A patients in the future. Muscle Nerve, 2010     

施万细胞的层黏连蛋白表达变化及两者关系的初步研究

目的:观察层黏连蛋白(laminin,LN)在坐骨神经发育过程中的表达情况,以及LN和施万细胞(Schwann cells,SCs)的相互关系。方法:取E14、E17、P1和成年SD大鼠坐骨神经,免疫荧光组织化学染色检测LN表达情况;体外培养大鼠来源的SCs,经过外源性LN处理后,免疫荧光细胞化学染色检测LN、nidogen、type IVcollagen等细胞外基质成分的表达,酸性磷酸酶法检测SCs的黏附能力。结果:E17大鼠坐骨神经SCs有LN阳性免疫反应;经过外源性LN处理后,SCs有LN、nidogen、type IV collagen阳性免疫反应,且黏附能力增加。结论:在E17大鼠中,SCs开始分泌LN;LN具有促进SCs合成细胞外基质成分,并在其黏附过程中发挥作用。     

半乳糖凝集素1和层粘连蛋白在新疆维吾尔族子宫颈癌中作用的相关性研究

目的探讨维吾尔族宫颈癌发生发展过程中半乳糖凝集素1（Galectin-1）,层粘连蛋白（laminin,LN）的表达及意义。方法采用免疫组化S-P法检测Galectin-1,LN蛋白在慢性宫颈炎（20例）、CINⅠ（20例）、CINⅡ-Ⅲ（20例）,宫颈鳞癌（40例）中的表达。结果 Galectin-1在宫颈慢性炎症组,CINⅠ组、CINⅡ-Ⅲ组和宫颈鳞癌中的表达呈上升趋势（P〈0.05）,LN在各组中的表达呈下降趋势（P〈0.05）;并且Galectin-1与LN呈负相关（P〈0.05）。结论 Galectin-1表达上调可能是维吾尔族妇女宫颈鳞癌发生的分子事件;Galectin-1和LN在维族妇女宫颈癌发生发展中可能起协同作用。     

Detection of novel LAMA3 mutation in Herlitz junctional epidermolysis bullosa in a Jordanian family

The Herlitz junctional epidermolysis bullosa (H‐JEB) subtype usually presents as a severe lethal inherited variant of epidermolysis bullosa (EB) caused by a homozygous mutation in the genes LAMA3, LMAB3, or LAMAC3. Each gene encodes one of the three chains of heterotrimer laminin‐332 proteins (including the alpha‐3 chain, beta‐3 chain and gamma‐2‐chain) responsible for the adherence of the epidermis to the underlying dermis. The aim of this report is to add to the existing knowledge about EB by describing a novel mutation in a gene responsible for genodermatosis. A case of a Jordanian male neonate, born to healthy, first cousin consanguineous parents, who developed nonhealing blistering skin and mucous membrane lesions, crusted erosions with significant granulation tissue and dystrophic nails immediately after birth is described. The patient was diagnosed as having a novel LAMA3 mutation causing (H‐JEB) by immunofluorescence mapping and molecular analysis. Both parents and this baby's sibling were shown to be heterozygous carriers of the same mutation. Pre‐implantation diagnosis using molecular analysis for subsequent pregnancies in this family is crucial for managing any new pregnancy.     

Urinary laminin‐γ2 is a novel biomarker of non‐muscle invasive urothelial carcinoma

Lack of appropriate biomarkers has hampered early detection of urothelial cancer (UC), therefore, development of biomarkers for its diagnosis at earlier stages is of importance. Laminin‐332 (Ln‐332, formerly Ln‐5), a component of basement membranes, consists of Ln‐α3, Ln‐β3, and Ln‐γ2 polypeptides. However, monomeric Ln‐γ2 alone is frequently expressed in malignant neoplasms. If Ln‐γ2 is also expressed in UC and secreted into the urine, its detection could be useful for UC diagnosis. Here, we evaluated Ln‐γ2 levels from 60 patients with urinary diseases (including UC) by Western blotting, and detected it in approximately 53% of UC cases. Using immunohistochemistry, we confirmed Ln‐γ2 expression in UC tissues that were positive for Ln‐γ2, whereas Ln‐α3 expression was absent. We next developed a sandwich enzyme‐linked immunosorbent assay and applied it for screening 39 patients with non‐muscle invasive UC and 61 patients with benign urologic diseases. The Ln‐γ2 levels were higher in UC patients than in those with benign urologic diseases. Ln‐γ2 was detected even in patients with earlier stages of UC, such as Ta, T1, or carcinoma in situ. The sensitivity of Ln‐γ2 testing for UC was 97.4%, and the specificity was 45.9%, using a cut‐off of 0.5 μg/g∙crn. Ln‐γ2 had greater diagnostic value for detecting non‐muscle invasive UC compared to conventional urine cytology and available biomarkers for UC, and may be useful as a urine biomarker for the diagnosis and monitoring of UC.