scholarly journals Biochemical alterations in collagen IV induced by in vitro glycation

1996 ◽  
Vol 319 (3) ◽  
pp. 699-704 ◽  
Author(s):  
Hans-M. RAABE ◽  
Hilke MOLSEN ◽  
Snjezana-M. MLINARIC ◽  
Yahya AÇIL ◽  
Gernot H. G. SINNECKER ◽  
...  
Keyword(s):  
Molecular Conformation ◽  
Microvascular Complications ◽  
Biochemical Properties ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Matrix Assembly ◽  
Experimental Conditions ◽  
Biochemical Alterations ◽  
Diabetic Microvascular Complications

Non-enzymic interactions of carbohydrates and proteins are a major feature of cumulative modification in basement membranes in the course of diabetic microvascular complications. To evaluate the significance of both glycation and glycoxidation reactions for subsequent alterations of biochemical properties, we examined the effects of in vitro glycation on distinct collagen IV domains under different experimental conditions. The 7 S domain and the major triple-helical domain from human placental collagen IV were incubated for various time intervals up to 14 days at 37 °C in the presence of different concentrations of either glucose or ribose under oxidative and antioxidative conditions. Carbohydrate-induced non-enzymic modification in two collagen IV domains was revealed by increased cross-linking and fluorescence. In addition, these non-enzymic modifications apparently have a major impact on molecular conformation and thermal stability of collagen IV, which in turn might influence both cell-matrix interactions and matrix assembly.

scholarly journals Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions

2018 ◽  
Vol 293 (43) ◽  
pp. 16635-16646 ◽  
Author(s):  
Christoph Howe ◽  
Felix Ho ◽  
Anja Nenninger ◽  
Patrícia Raleiras ◽  
Karin Stensjö
Keyword(s):  
Dna Binding ◽  
Iron Oxidation ◽  
Gel Filtration ◽  
Biochemical Property ◽  
Biochemical Properties ◽  
Oxidation Catalysis ◽  
Nostoc Punctiforme ◽  
Experimental Conditions ◽  
Specific Expression

DNA-binding proteins from starved cells (Dps, EC: 1.16.3.1) have a variety of different biochemical activities such as DNA-binding, iron sequestration, and H2O2 detoxification. Most bacteria commonly feature one or two Dps enzymes, whereas the cyanobacterium Nostoc punctiforme displays an unusually high number of five Dps proteins (NpDps1–5). Our previous studies have indicated physiological differences, as well as cell-specific expression, among these five proteins. Three of the five NpDps proteins, NpDps1, -2, and -3, were classified as canonical Dps proteins. To further investigate their properties and possible importance for physiological function, here we characterized and compared them in vitro. Nondenaturing PAGE, gel filtration, and dynamic light-scattering experiments disclosed that the three NpDps proteins exist as multimeric protein species in the bacterial cell. We also demonstrate Dps-mediated iron oxidation catalysis in the presence of H2O2. However, no iron oxidation with O2 as the electron acceptor was detected under our experimental conditions. In modeled structures of NpDps1, -2, and -3, protein channels were identified that could serve as the entrance for ferrous iron into the dodecameric structures. Furthermore, we could demonstrate pH-dependent DNA-binding properties for NpDps2 and -3. This study adds critical insights into the functions and stabilities of the three canonical Dps proteins from N. punctiforme and suggests that each of the Dps proteins within this bacterium has a specific biochemical property and function.

METABOLIC CHANGES IN TESTICULAR CELLS FROM RATS AFTER LONG-TERM EXPOSURE TO 37 °C IN VIVO OR IN VITRO

1980 ◽  
Vol 85 (3) ◽  
pp. 471-479 ◽  
Author(s):  
F. F. G. ROMMERTS ◽  
F. H. DE JONG ◽  
J. A. GROOTEGOED ◽  
H. J. VAN DER MOLEN
Keyword(s):  
Sertoli Cells ◽  
Leydig Cells ◽  
Biochemical Properties ◽  
Effect Of Temperature ◽  
Seminiferous Tubules ◽  
Experimental Conditions ◽  
Testicular Cells ◽  
Old Rats

Biochemical properties of isolated Leydig cells, Sertoli cells and spermatocytes from rat testes have been investigated after in-vivo or in-vitro exposure of these cells to abdominal temperature (37 °C). The rate of production of testosterone and pregnenolone by isolated Leydig cells from cryptorchid and normal testes from mature rats was not different. Production of pregnenolone by mitochondria prepared from cryptorchid testes was 6·7 times higher than production by mitochondria from normal testes. Sertoli cells prepared from immature rats and incubated in vitro at 32 or 37 °C showed, on day 1 of the culture period, an initial twofold increase in the secretion of androgen-binding protein which was absent after 6 days in culture. In contrast, incorporation of [3H]leucine into secreted proteins was stimulated twofold on day 1 as well as by day 6 of culture. Secretion of oestradiol was increased 30-fold by day 6 when compared with the level found on day 1 when cells had been cultured at 37 °C and the increased secretion of oestradiol was maintained for approximately 2 days when the temperature of incubation was decreased to 32 °C Spermatocytes isolated from seminiferous tubules incubated for 20 h at 37 °C were active in the synthesis of RNA. No degeneration of these cells was observed in testes of 25-day-old rats 5 days after experimental cryptorchidism, whereas under similar conditions massive degeneration of spermatocytes was shown in the testes of mature rats. These results suggest that the effects of temperature on the different testicular cells greatly depend on the experimental conditions used to study the effect of temperature.

scholarly journals Identification of a novel sequence element in the common promoter region of human collagen type IV genes, involved in the regulation of divergent transcription

1993 ◽  
Vol 292 (3) ◽  
pp. 687-695 ◽  
Author(s):  
G Fischer ◽  
C Schmidt ◽  
J Opitz ◽  
Z Cully ◽  
K Kühn ◽  
...  
Keyword(s):  
Promoter Region ◽  
Structural Integrity ◽  
Consensus Sequence ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Collagen Type Iv ◽  
Sequence Element ◽  
Type Iv ◽  
Binding Factor

The expression of the heterotrimeric collagen IV molecule alpha 1(IV)2 alpha 2(IV) is essential for the structural integrity and functional properties of all basement membranes. The two genes COL4A1 and COL4A2 that code for the subunits are found closely linked on chromosome 13 in a head-to-head arrangement and are transcribed in divergent directions. We have identified a novel trans-acting factor that binds in vitro to a unique homopyrimidine/homopurine stretch within the shared promoter region of the two collagen IV genes. Additional binding sites have been identified within the first introns of both genes and the consensus sequence CCCTYCCCC for efficient binding has been deduced; the factor was named therefore ‘CTC-binding factor’ or ‘CTCBF’. Mutations in the binding site of CTC-binding factor within the promoter inhibited binding in vitro and resulted in reduced transcription from both genes. The effect of mutations on the transcription of COL4A2 is more pronounced than on the transcription of COL4A1. CTC-binding factor is a nuclear factor that binds dominantly in vitro to the collagen IV promoter and is involved in regulating the expression of both collagen IV genes.

scholarly journals Endothelial Cell-Pericyte Interactions Stimulate Basement Membrane Matrix Assembly: Influence on Vascular Tube Remodeling, Maturation, and Stabilization

2011 ◽  
Vol 18 (1) ◽  
pp. 68-80 ◽  
Author(s):  
Amber N. Stratman ◽  
George E. Davis
Keyword(s):  
Growth Factor ◽  
Basement Membrane ◽  
Basement Membranes ◽  
Heparin Binding ◽  
Matrix Assembly ◽  
Matrix Deposition ◽  
Vascular Basement Membrane ◽  
Membrane Matrix

AbstractExtracellular matrix synthesis and deposition surrounding the developing vasculature are critical for vessel remodeling and maturation events. Although the basement membrane is an integral structure underlying endothelial cells (ECs), few studies, until recently, have been performed to understand its formation in this context. In this review article, we highlight new data demonstrating a corequirement for ECs and pericytes to properly deposit and assemble vascular basement membranes during morphogenic events. In EC only cultures or under conditions whereby pericyte recruitment is blocked, there is a lack of basement membrane assembly, decreased vessel stability (with increased susceptibility to pro-regressive stimuli), and increased EC tube widths (a marker of dysfunctional EC-pericyte interactions). ECs and pericytes both contribute basement membrane components and, furthermore, both cells induce the expression of particular components as well as integrins that recognize them. The EC-derived factors—platelet derived growth factor-BB and heparin binding-epidermal growth factor—are both critical for pericyte recruitment to EC tubes and concomitant vascular basement membrane formationin vitroandin vivo. Thus, heterotypic EC-pericyte interactions play a fundamental role in vascular basement membrane matrix deposition, a critical tube maturation event that is altered in key disease states such as diabetes and cancer.

Altered synthesis of laminin 1 and absence of basement membrane component deposition in (beta)1 integrin-deficient embryoid bodies

2000 ◽  
Vol 113 (2) ◽  
pp. 259-268 ◽  
Author(s):  
M. Aumailley ◽  
M. Pesch ◽  
L. Tunggal ◽  
F. Gaill ◽  
R. Fassler
Keyword(s):  
Basement Membrane ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Mouse Genetics ◽  
Embryoid Bodies ◽  
Membrane Formation ◽  
Beta 1 Integrin ◽  
Laminin 1

Basement membranes are the earliest extracellular matrices produced during embryogenesis. They result from synthesis and assembly into a defined supramolecular architecture of several components, including laminins, collagen IV, nidogen, and proteoglycans. In vitro studies have allowed us to propose an assembly model based on the polymerisation of laminin and collagen IV in two separate networks associated together by nidogen. How nucleation of polymers and insolubilisation of the different components into a basement membrane proceed in vivo is, however, unknown. A most important property of several basement membrane components is to provide signals controling the activity of adjacent cells. The transfer of information is mediated by interactions with cell surface receptors, among them integrins. Mouse genetics has demonstrated that the absence of these interactions is not compatible with development as deletion of either laminin (gamma)1 chain or integrin (beta)1 chain lead to lethality of mouse embryos at the peri-implantation stage. We have used embyoid bodies as a model system recapitulating the early steps of embryogenesis to unravel the respective roles of laminin and (beta)1 integrins in basement membrane formation. Our data show that there is formation of a basal lamina in wild-type, but not in (beta)1-integrin deficient, embryoid bodies. Surprisingly, in the absence of (beta)1 integrins, laminin 1 was not secreted in the extracellular space due to a rapid switch off of laminin (alpha)1 chain synthesis which normally drives the secretion of laminin heterotrimers. These results indicate that (beta)1 integrins are required for the initiation of basement membrane formation, presumably by applying a feed-back regulation on the expression of laminin (alpha)1 chain and other components of basement membranes.

scholarly journals Targeting pericyte–endothelial cell crosstalk by circular RNA-cPWWP2A inhibition aggravates diabetes-induced microvascular dysfunction

2019 ◽  
Vol 116 (15) ◽  
pp. 7455-7464 ◽  
Author(s):  
Chang Liu ◽  
Hui-Min Ge ◽  
Bai-Hui Liu ◽  
Rui Dong ◽  
Kun Shan ◽  
...  
Keyword(s):  
Vascular Dysfunction ◽  
Microvascular Complications ◽  
Microvascular Dysfunction ◽  
Circular Rna ◽  
In Vivo Studies ◽  
Circular Rnas ◽  
Related Stress ◽  
Diabetic Microvascular Complications

The crosstalk between vascular pericytes and endothelial cells (ECs) is critical for microvascular stabilization and remodeling; however, the crosstalk is often disrupted by diabetes, leading to severe and even lethal vascular damage. Circular RNAs are a class of endogenous RNAs that regulate several important physiological and pathological processes. Here we show that diabetes-related stress up-regulates cPWWP2A expression in pericytes but not in ECs. In vitro studies show that cPWWP2A directly regulates pericyte biology but indirectly regulates EC biology via exosomes carrying cPWWP2A. cPWWP2A acts as an endogenous miR-579 sponge to sequester and inhibit miR-579 activity, leading to increased expression of angiopoietin 1, occludin, and SIRT1. In vivo studies show that cPWWP2A overexpression or miR-579 inhibition alleviates diabetes mellitus-induced retinal vascular dysfunction. By contrast, inhibition of cPWWP2A-mediated signaling by silencing cPWWP2A or overexpressing miR-579 aggravates retinal vascular dysfunction. Collectively, this study unveils a mechanism by which pericytes and ECs communicate. Intervention of cPWWP2A or miR-579 expression may offer opportunities for treating diabetic microvascular complications.

scholarly journals Analysis of the role of Nidogen/entactin in basement membrane assembly and morphogenesis in Drosophila

2018 ◽  
Author(s):  
Jianli Dai ◽  
Beatriz Estrada ◽  
Sofie Jacobs ◽  
Besaiz J. Sánchez-Sánchez ◽  
Jia Tang ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Type Iv Collagen ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Extracellular Matrices ◽  
Tissue Specific ◽  
Type Iv

AbstractBasement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen interlinked by the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg on morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from in vitro and cultured embryonic tissues experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival, strongly questioned this proposed linking role. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the Laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen.Author SummaryBasement membranes (BMs) are thin layers of specialized extracellular matrices present in every tissue of the human body. Its main constituents are two networks of Laminin and Type IV Collagen linked by Nidogen (Ndg) and proteoglycans. They form an organized scaffold that regulates organ morphogenesis and function. Mutations affecting BM components are associated with organ dysfunction and several congenital diseases. Thus, a better comprehension of BM assembly and maintenance will not only help to learn more about organogenesis but also to a better understanding and, hopefully, treatment of these diseases. Here, we have used Drosophila to analyse the role of Ndg in BM formation in vivo. Elimination of Ndg in worms and mice does not affect survival, strongly questioning its proposed linking role, derived from in vitro experiments. Here, we show that in the fly Ndg is dispensable for BM assembly and preservation in many tissues, but absolutely required in others. Furthermore, our functional study of the different Ndg domains challenges the significance of some interactions between BM components derived from in vitro experiments, while confirming others, and reveals a new key requirement for the Rod domain in Ndg function and incorporation into BMs.

New Insights into Hyperglycemia-induced Molecular Changes in Microvascular Cells

2009 ◽  
Vol 89 (2) ◽  
pp. 116-127 ◽  
Author(s):  
S. Roy ◽  
K. Trudeau ◽  
S. Roy ◽  
Y. Behl ◽  
S. Dhar ◽  
...  
Keyword(s):  
Microvascular Complications ◽  
Vascular Complications ◽  
Diabetic Microangiopathy ◽  
Molecular Changes ◽  
Biochemical Alterations ◽  
Diabetic Vascular Complications ◽  
Basement Membrane Thickening ◽  
Microvascular Cells ◽  
Diabetic Microvascular Complications ◽  
Inflammation Cytokines

Hyperglycemia is the most prevalent characteristic of diabetes and plays a central role in mediating adverse effects on vascular cells during the progression of diabetic vascular complications. In diabetic microangiopathy, hyperglycemia induces biochemical and molecular changes in microvascular cells that ultimately progress to retinal, renal, and neural complications and extends to other complications, including advanced periodontal disease. In this review, we describe changes involving basement membrane thickening, tissue remodeling, gap junctions, inflammation, cytokines, and transcription factors, and their effects on the pathogenesis of diabetic microvascular complications. The majority of the changes described relate to retinal microangiopathy, since ultrastructural, structural, and biochemical alterations have been well-characterized in this tissue.

Evaluation of the Cytotoxicity of the First 20 MEIC Chemicals in Two Hepatoma Cell Lines with Different Xenobiotic Metabolism Capacities

1993 ◽  
Vol 21 (1) ◽  
pp. 65-72
Author(s):  
Anna Maria Bassi ◽  
Ornella Bosco ◽  
Sabrina Brenci ◽  
Daniela Adamo ◽  
Susanna Penco ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Biochemical Properties ◽  
Hep G2 ◽  
Experimental Conditions ◽  
Hepatoma Cell Lines ◽  
Wide Range

The cytotoxicity of the first 20 chemicals on the MEIC list was evaluated using two hepatoma cell lines having different xenobiotic metabolic capacities, one derived from the rat (HTC), and one of human origin (Hep G2). Two endpoints were measured to evaluate cytotoxicity: colony-forming ability (CF), and cell viability of the attached monolayer (CV), evaluated as total macromolecular content. The CF test provided the most sensitive endpoint, due to the lower number of exposed cells in comparison with the CV test. Using the CF assay, the Hep G2 cell line showed higher sensitivity than the HTC cell line to some chemicals known to be metabolised in vivo. The IC50s obtained under the different experimental conditions varied as a consequence of phenotypic differences between the cell lines, and of the nature of the endpoints. The most toxic chemicals were malathion, diazepam and amitriptyline (IC50: 0.001-0.1mM). The solvents tested produced the lowest toxic effects (IC50: > 10mM). These findings suggest that hepatoma cell lines possessing various specific enzyme activities could be usefully employed in a battery of tests designed to reproduce in vitro the wide range of biochemical properties expressed by the cells in the whole organism.

Observations of Frozen-Hydrated Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 504-505
Author(s):  
D. Chrétien ◽  
D. Job ◽  
R.H. Wade
Keyword(s):  
Fourier Transforms ◽  
Structural Complexity ◽  
Image Contrast ◽  
Phase Behaviour ◽  
Experimental Conditions ◽  
Thin Sections ◽  
Surface Lattice ◽  
Cilia And Flagella ◽  
Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

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