Laminin isoform expression in breast tumors

Certain laminins of vascular basement membranes have been identified in human breast tumors and brain gliomas that share the same β1 chain. These laminins are new carcinoma angiogenic markers and might represent potential targets for antiangiogenic therapy

Single nucleotide polymorphism-based genome-wide linkage analysis in Japanese atopic dermatitis families

<p>Abstract</p> <p>Background</p> <p>Atopic dermatitis develops as a result of complex interactions between several genetic and environmental factors. To date, 4 genome-wide linkage studies of atopic dermatitis have been performed in Caucasian populations, however, similar studies have not been done in Asian populations. The aim of this study was to identify chromosome regions linked to atopic dermatitis in a Japanese population.</p> <p>Methods</p> <p>We used a high-density, single nucleotide polymorphism genotyping assay, the Illumina BeadArray Linkage Mapping Panel (version 4) comprising 5,861 single nucleotide polymorphisms, to perform a genome-wide linkage analysis of 77 Japanese families with 111 affected sib-pairs with atopic dermatitis.</p> <p>Results</p> <p>We found suggestive evidence for linkage with 15q21 (LOD = 2.01, NPL = 2.87, <it>P </it>= .0012) and weak linkage to 1q24 (LOD = 1.26, NPL = 2.44, <it>P </it>= .008).</p> <p>Conclusion</p> <p>We report the first genome-wide linkage study of atopic dermatitis in an Asian population, and novel loci on chromosomes 15q21 and 1q24 linked to atopic dermatitis. Identification of novel causative genes for atopic dermatitis will advance our understanding of the pathogenesis of atopic dermatitis.</p

Perlecan Maintains microvessel integrity in vivo and modulates their formation in vitro

Perlecan is a heparan sulfate proteoglycan assembled into the vascular basement membranes (BMs) during vasculogenesis. In the present study we have investigated vessel formation in mice, teratomas and embryoid bodies (EBs) in the absence of perlecan. We found that perlecan was dispensable for blood vessel formation and maturation until embryonic day (E) 12.5. At later stages of development 40% of mutant embryos showed dilated microvessels in brain and skin, which ruptured and led to severe bleedings. Surprisingly, teratomas derived from perlecan-null ES cells showed efficient contribution of perlecan-deficient endothelial cells to an apparently normal tumor vasculature. However, in perlecan-deficient EBs the area occupied by an endothelial network and the number of vessel branches were significantly diminished. Addition of FGF-2 but not VEGF165 rescued the in vitro deficiency of the mutant ES cells. Furthermore, in the absence of perlecan in the EB matrix lower levels of FGFs are bound, stored and available for cell surface presentation. Altogether these findings suggest that perlecan supports the maintenance of brain and skin subendothelial BMs and promotes vasculo- and angiogenesis by modulating FGF-2 function

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interac- tions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin g1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs

The cerebrovascular basement membrane: Role in the clearance of β-amyloid and cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA), the accumulation of β-amyloid (Aβ) peptides in the walls of cerebral blood vessels, is observed in the majority of Alzheimer’s disease (AD) brains and is thought to be due to a failure of the aging brain to clear Aβ. Perivascular drainage of Aβ along cerebrovascular basement membranes (CVBMs) is one of the mechanisms by which Aβ is removed from the brain. CVBMs are specialized sheets of extracellular matrix that provide structural and functional support for cerebral blood vessels. Changes in CVBM composition and structure are observed in the aged and AD brain and may contribute to the development and progression of CAA. This review summarizes the properties of the CVBM, its role in mediating clearance of interstitial fluids and solutes from the brain, and evidence supporting a role for CVBM in the etiology of CAA

Biological roles of laminins 8, 9 and 10

In order to form multicellular organisms, cells need attachment, either directly to each other through cell-cell interactions, or indirectly via a complex mixture of macro-molecules called the extracellular matrix. Basement membranes are specialized forms of this matrix. They can be seen in the electron microscope as a thin flat structure underlying epithelial or endothelial cells, or surrounding certain individual cells such as muscle, nerve and fat cells. The basement membranes provide a structural framework for individual cells, or groups of cells, delineating them from the surrounding tissues, and anchor the cells through specific interactions with cell surface receptors. These interactions are of vital importance for the development of a cell as well as for its maintenance, and in many cases, even for its survival. Basement membranes are evolutionary old inventions and can be found in very primitive organisms. Most mammalian basement membranes have a type IV collagen- network and a lamininnetwork that are thought to be interconnected via a linker molecule called nidogen. Dystroglycan and the integrin family of receptors are the most well characterized cell surface receptors for laminins. Laminins are large glycoproteins comprised of three individual polypeptide chains (alpha-beta-gamma) and to date, 5 alpha, 4 P and 3 V genetically distinct variants are known. Different chain variants combine into the laminin isoforms, and 14 have been identified in vivo so far. The different isoforms have partially overlapping expression pattems, which are complex both temporally and spatially. This study mainly focuses on the biological roles of laminin 8, (alpha4-beta1- gamma1) laminin 9, (alpha4-beta2-gamma1) and laminin 10 (alpha5-beta1-gamma1). In order to study the biological role of the laminin alpha 4 chain, its gene was targeted in mouse embryonic stein cells. The Lama 4 null mice were viable and fertile but displayed transient hemorrhages at birth, a subtle motor impairment as adult mice, as well as resistance to obesity. The hemorrhages in the newborn null animals were located in soft tissues. Mild fetal hemorrhages were also seen, but they were never as extensive as in the newborn. We therefore hypothesized that the hemorrhages were the result of microvessel damage during delivery. Electron microscopy analysis revealed defects in the basement membranes of microvessels in muscle from newborn animals. Furthermore, immunostaining of endothelial basement membranes showed that the amount of type IV collagen and nidogen were significantly reduced. It was concluded that laminin alpha 4 is important for endothelial basement membrane formation. The Lama 4 null mice also exhibited a mild motor impairment. Using neurophysiologic testing, we found muscle function to be intact and muscle histology revealed no obvious signs of dystrophy or myopathy. A detailed analysis of the neuromuscular junctions (NMJs) found these to be generally properly formed in Lama 4 null mice, but to have interesting specific defects in the apposition of the active zones to the junctional folds. In the NMJ, the laminin beta 2 and V1 chains are expressed; leading to the conclusion that laminin 9 (alpha 4-beta 2-gamma 1) is of importance for the localization of synaptic specializations in the NMJ. Resistance to diet-induced as well as age-related obesity was another finding in Lama 4 null mice. The difference in the weight of males became significant after 10 weeks on a high fat containing diet and was not due to a decrease in food intake. The adipocyte size was reduced to half of that of controls. The null animals had normal levels of serum lipids, except for cholesterol, which was not elevated to the same extent as in control animals on high-fat diet. Furthermore, mills showed increased spontaneous activity but no increase in resting metabolic rate. The hyperactivity could be part of a neurological syndrome, and is likely to contribute to the lean phenotype. The cDNA encoding the human laminin alpha 5 chain was obtained through screening of 2, phage cDNA libraries and PCR amplification of cDNA mixes. The cDNA sequence was used to make a full-length laminin alpha 5 expression vector, which was used to transfect a human embryonic kidney cell line. This cell line was also transfected with vectors expressing the laminin beta 1 and gamma 1 chains, enabling the cells to produce all the three chain components of laminin 10. The resulting cell line was shown to produce high amounts of recombinant laminin 10, which could be purified using affinity chromatography. The protein was shown to promote cell adhesion as well as cell migration. Using function-blocking antibodies, integrin alpha 3-beta 1 was identified as a main mediator of adhesion to laminin 10 in HT- 1080 cells, as well as in human saphenous vein endothelial cells

Charged colloids observed by electrophoretic and diffusion NMR

The thesis deals partly with methodology including construction of hardware and new pulse sequences in the field of electrophoretic NMR, and partly with practical use of ENMR and diffusion NMR in the investigation of charged colloidal systems. Several sources of artefacts are investigated, including gas production at the electrodes, electroosmosis and Joule heating effects that can cause convection. The electrophoretic double stimulated-echo pulse sequence is introduced to suppress these artefacts and to increase the feasible measuring range to higher electric fields and conductivities. The interaction between the non-ionic polymer poly(ethylene oxide) PEO and differently charged surfactants is investigated using the above mentioned methods. The investigated surfactants are the anionic sodium dodecyl sulphate (SDS) and potassium laurate (KC12), the cationic dodecyltrimethylammonium bromide (CTAB) and the non-ionic octyl β-D-glucoside. ENMR is also used to investigate two different mixed micelle systems, with SDS as the charged surfactant component and dodecyl malono-bis-N-methylglucamide (C12BNMG) respectively tetra(ethylene oxide) dodecyl ether (C12EO4) as the nonionic surfactant component. A method to calculate the degree of counter-ion dissociation, αdissociation, as a function of composition is demonstrated. Finally diffusion NMR is used to compare transport dynamics in gel electrolyte systems based on two differently grafted polymers; one amphiphilic system containing polymethacrylate grafted partly with polyethylene oxide and partly with fluorocarbons and the corresponding nonamphiphilic system grafted with only polyethylene oxide. Both systems contain the electrolyte lithium bis(trifluoromethylsulfonyl) imide salt dissolved in γ-butyrolactone. The results show that the system based on the amphiphilic polymer has better transport dynamics and therefore is more suited as material for batter

Charged colloids observed by electrophoretic and diffusion NMR

The thesis deals partly with methodology including construction of hardware and new pulse sequences in the field of electrophoretic NMR, and partly with practical use of ENMR and diffusion NMR in the investigation of charged colloidal systems. Several sources of artefacts are investigated, including gas production at the electrodes, electroosmosis and Joule heating effects that can cause convection. The electrophoretic double stimulated-echo pulse sequence is introduced to suppress these artefacts and to increase the feasible measuring range to higher electric fields and conductivities. The interaction between the non-ionic polymer poly(ethylene oxide) PEO and differently charged surfactants is investigated using the above mentioned methods. The investigated surfactants are the anionic sodium dodecyl sulphate (SDS) and potassium laurate (KC12), the cationic dodecyltrimethylammonium bromide (CTAB) and the non-ionic octyl β-D-glucoside. ENMR is also used to investigate two different mixed micelle systems, with SDS as the charged surfactant component and dodecyl malono-bis-N-methylglucamide (C12BNMG) respectively tetra(ethylene oxide) dodecyl ether (C12EO4) as the nonionic surfactant component. A method to calculate the degree of counter-ion dissociation, αdissociation, as a function of composition is demonstrated. Finally diffusion NMR is used to compare transport dynamics in gel electrolyte systems based on two differently grafted polymers; one amphiphilic system containing polymethacrylate grafted partly with polyethylene oxide and partly with fluorocarbons and the corresponding nonamphiphilic system grafted with only polyethylene oxide. Both systems contain the electrolyte lithium bis(trifluoromethylsulfonyl) imide salt dissolved in γ-butyrolactone. The results show that the system based on the amphiphilic polymer has better transport dynamics and therefore is more suited as material for batter