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At least 757 records · Page 42Linked to original sources
PubMed1997

Topoisomerase function during replication-independent chromatin assembly in yeast.

DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA. Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos. We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined biochemical and pharmacological approach. As measured by plasmid supercoiling, nucleosome deposition is severely impaired in assembly extracts from a yeast mutant with no topoisomerase I and a temperature-sensitive form of topoisomerase II (strain top1-top2). Expression of wild-type topoisomerase II in strain top1-top2 fully restored assembly-driven supercoiling, and assembly was equally efficient in extracts from strains expressing either topoisomerase I or II alone. Supercoiling in top1-top2 extract was rescued by adding back either purified topoisomerase I or II. Using the topoisomerase II poison VP-16, we show that topoisomerase II activity during chromatin assembly is the same in the presence and absence of topoisomerase I. We conclude that both topoisomerases I and II can provide the DNA relaxation activity required for efficient chromatin assembly in mitotically cycling yeast cells.

Chromatin↗
PubMed2004

Role of ADP ribosylation factor 1 in the assembly and secretion of ApoB-100-containing lipoproteins.

OBJECTIVE: We investigated the role of ADP ribosylation factor 1 (ARF1) in the assembly of very-low-density lipoproteins (VLDLs). METHODS AND RESULTS: The dominant-negative ARF1 mutant, T31N, decreased the assembly of apoB-100 VLDL 1 (Svedberg floatation units [Sf] 60 to 400) by 80%. The decrease coincided with loss of coatamer I (COPI) from the Golgi apparatus and inhibition of anterograde transport, as demonstrated by time-lapse studies of the vesicular stomatitis virus G protein. The VLDL 1 assembly was also completely inhibited at 15 degrees C. Thus, the antegrade transport is essential for the assembly of VLDL 1. Intracellular localization of N-acetylgalactosaminyl transferase 2 indicated that the Golgi apparatus was at least partly intact when the VLDL assembly was inhibited. Transient transfection with phospholipase D 1 increased the assembly of VLDL 1 and VLDL 2 (Sf 20 to 60). Overexpression of ARF1 in stably transfected McA-RH7777 cells increased the secretion of VLDL 2 but not of VLDL 1, which was dependent on the availability of oleic acid. Secretion of VLDL 1 increased with increasing amounts of oleic acid, and VLDL 2 secretion decreased simultaneously. CONCLUSIONS: Overexpression of ARF1 increased the assembly of VLDL 2 but not of VLDL 1, whose production was dependent on both anterograde transport and the availability of fatty acids.

ADP-Ribosylation Factor 1↗
PubMed2006

Depletion of zebrafish titin reduces cardiac contractility by disrupting the assembly of Z-discs and A-bands.

The genetic study of titin has been notoriously difficult because of its size and complicated alternative splicing routes. Here, we have used zebrafish as an animal model to investigate the functions of individual titin isoforms. We identified 2 titin orthologs in zebrafish, ttna and ttnb, and annotated the full-length genomic sequences for both genes. We found that ttna, but not ttnb, is required for sarcomere assembly in the heart as well as the subsequent establishment of cardiac contractility. In fact, ttna is the earliest sarcomeric mRNA that is expressed in the heart, which makes it an early molecular marker for cardiomyocyte differentiation. Surprisingly, ttna is required for later steps of sarcomere assembly, including the assembly of Z-discs and A-bands, but not for early steps such as the assembly of Z-bodies and nonstriated myosin filaments. Reduction of individual titin isoforms in vivo using morpholino-modified antisense oligonucleotides indicated that (1) both N2B exon-containing and N2A exon-containing isoforms of ttna are required for sarcomere assembly in the heart; (2) N2A exon-containing isoforms of both ttna and ttnb are required for sarcomere assembly in the somites; and (3) the N2B exon-containing isoforms of ttnb are expressed later than other titin isoforms and are probably involved in modulating their expression; however, these isoforms of ttnb are not required for sarcomere assembly. Collectively, our results reveal distinct functions of different titin isoforms and suggest that various phenotypes in "titinopathies" may be attributable to the disruption of different titin isoforms.

Alternative Splicing↗
PubMed1995

Asparagine-linked oligosaccharides facilitate human chorionic gonadotropin beta-subunit folding but not assembly of prefolded beta with alpha.

To determine the role of asparagine (N)-linked oligosaccharide chains in protein folding and assembly, the well established hCG-beta in vitro folding and assembly assays were used to analyze how the human CG (hCG) beta-subunit devoid of one or two N-linked glycans folds and assembles under different conditions. Two approaches were used: 1) site-specific mutagenesis of hCG-beta synthesized in Chinese hamster ovary cells transfected with beta-mutants lacking the asparagine glycosylation sites; and 2) enzymatic deglycosylation of hCG-beta synthesized in JAR cells with peptide N-glycosidase F or endoglycosidase H. In both cases, [35S]cysteine-labeled beta-subunits were used as substrates to measure the conversion of the hCG-beta folding intermediate p beta 1 into p beta 2 and assembly of p beta 2 with urinary alpha. Using the mutated substrates from Chinese hamster ovary cells, it was found that 60% of wild-type p beta 1 (two N-linked glycans), 60% of p beta 1 missing the Asn13-linked glycan, 40% of p beta 1 missing the Asn30-linked glycan, and 10% of p beta 1 missing two N-linked glycans were converted to the corresponding p beta 2, respectively. With the enzymatically deglycosylated substrate from JAR cells, 90% of p beta 1 (two N-linked glycans), 70% of p beta 1(1) (one N-linked glycan), and 10% of p beta 1(0) (without N-linked glycan) folded into p beta 2 under cysteamine and cystamine redox conditions with or without protein disulfide isomerase. These data demonstrate that at least one N-linked glycan is required for efficient folding of hCG-beta and that the Asn30-linked glycan is more important than Asn13-linked glycan for hCG-beta folding. It also was shown that the composition of N-linked glycans of hCG-p beta 1 did not change protein folding, since hCG-beta substrates with high mannose oligosacharides folded as efficiently as beta-substrates containing sialylated complex oligosaccharides. Moreover, assembly of the already folded, assembly-component folding intermediate, p beta 2, was not affected by removal of one or both of the N-linked glycans of the beta-subunit. These data thus show that N-linked glycans play their most important role in the folding component of the folding and assembly pathway for hCG-beta.

Animals↗
PubMed2003

Ran modulates spindle assembly by regulating a subset of TPX2 and Kid activities including Aurora A activation.

Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin beta, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 we identified nuclear targeting information, microtubule-binding and Aurora A binding activities. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevented Aurora A activation and recruitment to microtubules. In addition we identified nuclear targeting information in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly.

Active Transport, Cell Nucleus↗
PubMed2004

N-RAP scaffolds I-Z-I assembly during myofibrillogenesis in cultured chick cardiomyocytes.

N-RAP is a muscle-specific protein with an N-terminal LIM domain (LIM), C-terminal actin-binding super repeats homologous to nebulin (SR) and nebulin-related simple repeats (IB) in between the two. Based on biochemical data, immunofluorescence analysis of cultured embryonic chick cardiomyocytes and the targeting and phenotypic effects of these individual GFP-tagged regions of N-RAP, we proposed a novel model for the initiation of myofibril assembly in which N-RAP organizes alpha-actinin and actin into the premyofibril I-Z-I complexes. We tested the proposed model by expressing deletion mutants of N-RAP (i.e. constructs containing two of the three regions of N-RAP) in chick cardiomyocytes and observing the effects on alpha-actinin and actin organization into mature sarcomeres. Although individually expressing either the LIM, IB, or SR regions of N-RAP inhibited alpha-actinin assembly into Z-lines, expression of either the LIM-IB fusion or the IB-SR fusion permitted normal alpha-actinin organization. In contrast, the LIM-SR fusion (LIM-SR) inhibited alpha-actinin organization into Z-lines, indicating that the IB region is critical for Z-line assembly. While permitting normal Z-line assembly, LIM-IB and IB-SR decreased sarcomeric actin staining intensity; however, the effects of LIM-IB on actin assembly were significantly more severe, as estimated both by morphological assessment and by quantitative measurement of actin staining intensity. In addition, LIM-IB was consistently retained in mature Z-lines, while mature Z-lines without significant IB-SR incorporation were often observed. We conclude that the N-RAP super repeats are essential for organizing actin filaments during myofibril assembly in cultured embryonic chick cardiomyocytes, and that they also play an important role in removal of the N-RAP scaffold from the completed myofibrillar structure. This work strongly supports the N-RAP scaffolding model of premyofibril assembly.

Actins↗
PubMed2005

Assembly pathway of the anastral Drosophila oocyte meiosis I spindle.

Oocyte meiotic spindles of many species are anastral and lack centrosomes to nucleate microtubules. Assembly of anastral spindles occurs by a pathway that differs from that of most mitotic spindles. Here we analyze assembly of the Drosophila oocyte meiosis I spindle and the role of the Nonclaret disjunctional (Ncd) motor in spindle assembly using wild-type and mutant Ncd fused to GFP. Unexpectedly, we observe motor-associated asters at germinal vesicle breakdown that migrate towards the condensed chromosomes, where they nucleate microtubules at the chromosomes. Newly nucleated microtubules are randomly oriented, then become organized around the bivalent chromosomes. We show that the meiotic spindle forms by lateral associations of microtubule-coated chromosomes into a bipolar spindle. Lateral interactions between microtubule-associated bivalent chromosomes may be mediated by microtubule crosslinking by the Ncd motor, based on analysis of fixed oocytes. We report here that spindle assembly occurs in an ncd mutant defective for microtubule motility, but lateral interactions between microtubule-coated chromosomes are unstable, indicating that Ncd movement along microtubules is needed to stabilize interactions between chromosomes. A more severe ncd mutant that probably lacks ATPase activity prevents formation of lateral interactions between chromosomes and causes defective microtubule elongation. Anastral Drosophila oocyte meiosis I spindle assembly thus involves motor-associated asters to nucleate microtubules and Ncd motor activity to form and stabilize interactions between microtubule-associated chromosomes during the assembly process. This is the first complete account of assembly of an anastral spindle and the specific steps that require Ncd motor activity, revealing new and unexpected features of the process.

Animals↗
PubMed1995

Role of the carboxyl-terminal Fib2 domain in fibronectin matrix assembly.

A truncated form of fibronectin consisting of the N-terminal 70 kDa and C-terminal 37 kDa regions, designated r70F2, retained the ability to assemble into the extracellular matrix when expressed in cultured fibroblasts (Ichihara-Tanaka et al. (1992) FEBS Lett. 299, 155-158). To elucidate the role of the C-terminal 37 kDa region in fibronectin matrix assembly, we expressed a panel of mutant forms of r70F2 with various deletions and amino acid substitutions in mouse L cells. Although substitution of Ser for two Cys residues in the C-terminal dimerforming segment led to a marked reduction in the matrix assembly activity of r70F2, the resulting monomeric r70F2 still retained a low, but significant activity to assemble into the matrix. Neither the N-terminal 70 kDa nor the C-terminal 37 kDa regions, when expressed as monomeric forms, exhibited any residual activity, suggesting that the core domain of the 37 kDa region consisting of III15 and I10 through I12 modules, termed Fib2 domain, is actively involved in the matrix assembly of r70F2. In support of the role of Fib2 domain, the proteolytic fragment derived from the 37 kDa region inhibited the assembly of r70F2. Furthermore, en bloc deletion of the Fib2 domain or deletion of the I10 through I12 modules from r70F2 resulted in a marked decrease of the matrix assembly activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Dimples, pores, star-rings, and thin rings on growing nuclear envelopes: evidence for structural intermediates in nuclear pore complex assembly.

We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear envelopes in Xenopus egg extracts. Scattered among nuclear pore complexes were rare 'dimples' (outer membrane depressions, 5-35 nm diameter), more abundant holes (pores) with a variety of edge geometries (35-45 nm diameter; 3.3% of structures), pores containing one to eight triangular 'star-ring' subunits (2.1% of total), and more complicated structures. Neither mature complexes, nor these novel structures, formed when wheat germ agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (>500 microg/ml) directly to the assembly reaction; low concentrations (10 microg/ml) had no effect. However at intermediate concentrations (50-100 microg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of 'empty' pores, and other structures; this effect correlated with the lectin-induced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-binding nucleoporin. Another inhibitor, dibromo-BAPTA (5,5'-dibromo-1,2-bis[o-aminophenoxylethane-N,N,N',N'-tetraacetic acid), had different effects depending on its time of addition to the assembly reaction. When 1 mM dibromo-BAPTA was added at time zero, no pore-related structures formed. However, when dibromo-BAPTA was added to growing nuclei 40-45 minutes after initiating assembly, star-rings and other structures accumulated, suggesting that dibromo-BAPTA can inhibit multiple stages in pore complex assembly. We propose that assembly begins with the formation and stabilization of a hole (pore) through the nuclear envelope, and that dimples, pores, star-rings, and thin rings are structural intermediates in nuclear pore complex assembly.

Animals↗
PubMed2000

Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking.

Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2- to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Dissociation and re-assembly of the endoplasmic reticulum in live cells.

The endoplasmic reticulum (ER) of a typical interphase 3T3 fibroblast consists of a compact perinuclear arrangement of cisternae and lamellae which can be observed by immunofluorescence with anti-endoplasmin. During mitosis the reticulum dissociates into small fragments from which it appears to re-assemble in the daughter cells. When interphase 3T3 cells are exposed to calcium ionophores, but not other ionophores, there is a similar dissociation of the ER into small uniform fragments, which are dispersed throughout the cytoplasm. Electron microscopy shows that the fragments consist of small vesicular structures and that essentially all of the rough ER except the nuclear envelope is dissociated. The dissociation of the ER by calcium ionophore is a relatively specific process since other organelles and supramolecular assemblies remain unaffected. When cells with dissociated ER are returned to normal medium, there is a rapid reassembly of the fragments into the continuous reticulum. In a proportion of the cells it is possible to observe linear arrays of the fragments, which probably represent intermediates in the re-assembly process. These observations demonstrate that the ER in interphase 3T3 cells can be dissociated into, and re-assembled from, small fragments. Re-assembly of the ER from the fragments is dependent on the presence of millimolar levels of calcium in the external medium. In the presence of calcium, re-assembly is inhibited by the calcium channel blocker, verapamil. Thus calcium ions appear to play an important role in ER structure and assembly.

Animals↗
PubMed2003

Methods designed for the identification and characterization of in vitro and in vivo chromatin assembly mutants in Saccharomyces cerevisiae.

Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model system Saccharomyces cerevisiae. Modifications to an in vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s): RSP5, and a subunit of the Anaphase Promoting Complex (APC), APC5. Additional modifications are described that allow for a rapid analysis and an in vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that the in vitro and invivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks.

Journal Article↗
PubMed2006

30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15.

Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37 degrees C this DeltarpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the DeltarpsO strain at 37 degrees C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized.

Base Sequence↗
PubMed2005

Lymphocyte antigen receptor gene assembly: multiple layers of regulation.

Lymphocyte antigen receptor genes are assembled through the cutting and joining of segments of DNA in developing lymphocytes. The basic features of the biochemical steps of this assembly process, referred to as the V(D)J recombination, are similar for the assembly of all lymphocyte antigen receptor genes, yet this assembly is precisely regulated in several important contexts during lymphocyte development. It has long been appreciated that this occurs through modulation of accessibility of antigen receptor loci to the enzymatic complex that assembles antigen receptor genes. However, recent studies have suggested that some regulatory constraints may be enforced at the level of the V(D)J recombination reaction itself. This review focuses on recent advances in the understanding of the regulation of antigen receptor gene assembly, with particular attention paid to the assembly of T-cell receptor beta-chain genes during T-cell development.

Alleles↗
PubMed2001

Fibrinogen biosynthesis. Assembly, intracellular degradation, and association with lipid synthesis and secretion.

Plasma fibrinogen is synthesized primarily in hepatocytes and assembly of the three component chains (A alpha, B beta, and gamma) into its final form as a six-chain dimer (A alpha, B beta, gamma)2 occurs rapidly in the lumen of the endoplasmic reticulum (ER). Assembly takes place in a stepwise manner with single chains interacting with each other to form A alpha-gamma and B beta-gamma complexes. The two-chain complexes then acquire another chain to form half-molecules (A alpha, B beta, gamma)1, which in a final step are linked to form the six-chain (A alpha, B beta, gamma)2 complex. As with other secreted glycoproteins, N-linked glycosylation of B beta and gamma chains commences in the ER and is completed in Golgi organelles. Sulfation and phosphorylation occur at post-ER stages of the secretory process. Since some ER chaperones coisolate with nascent fibrinogen chains they have been implicated in assisting chain assembly. Studies with recombinant systems, using deletion and substitution mutants, indicate that initial chain assembly depends on hydrophobic interactions present in the C-terminal half of the coil-coil domains and that inter- and intra-disulfide bonds that stabilize fibrinogen are needed to complete chain assembly. Not all the chains that are synthesized are assembled into fibrinogen and the unassembled chains are not secreted. HepG2 cells contain surplus A alpha and gamma chains that accumulate as free gamma chains and as an A alpha-gamma complex. A alpha-gamma is degraded by lysosomes whereas the gamma chain is degraded by the proteasome-ubiquitin system. Studies with expression of single chains by COS cells confirm that gamma and B beta are hydrolyzed by proteasomes and indicate that A alpha is degraded partially both by lysosomes and proteasomes. The role of surplus chains in regulating fibrinogen assembly is not understood but overexpression of any one chain, elicited by transfection of HepG2 cells, results in the upregulation of the other two genes, increased fibrinogen synthesis and secretion, and maintenance of surplus intracellular A alpha and gamma chains. HepG2 cells, programmed in this manner to increase basal fibrinogen expression, have higher HMG-CoA reductase mRNA levels, enhanced cholesterol and cholesterol ester synthesis, and increased secretion of apolipoprotein B (apoB). Overexpression of basal levels of fibrinogen does not affect synthesis of other acute phase proteins. Enhanced secretion of apoB is due to diminished degradation of nascent apoB by proteasomes and not to increased expression. Increased secretion of apoB is associated with increased basal expression of fibrinogen and is not affected when fibrinogen expression is stimulated by interleukin-6. In HepG2 cells, a feedback mechanism exists and extracellular sterols specifically downregulate expression of the three fibrinogen genes. These studies link, at the cellular level, basal fibrinogen expression with lipid metabolism.

Animals↗
PubMed2001

Disruption of pathologic amyloid beta-protein fibril assembly on the surface of cultured human cerebrovascular smooth muscle cells.

Cerebral amyloid beta-protein (Abeta) angiopathy (CAA) is a common pathological feature of Alzheimer's disease and several related disorders. In this condition, the accumulation offibrillar Abeta deposits is associated with degeneration of smooth muscle cells within the cerebral blood vessel wall. We have been using primary cultures of human cerebrovascular smooth muscle (HCSM) cells to investigate pathogenic mechanisms of Abeta in CAA. The specific assembly of Abeta fibrils on the surface of these cell types initiates several pathologic responses including increased expression and cell surface accumulation of the Abeta precursor protein (AbetaPP) and induction of apoptotic cell death. These pathologic responses are not observed with preparations of Abeta that are assembled into fibrils in solution, further underscoring the significance of the fibril assembly process on the cell surface. Since cell surface Abeta fibril assembly is the key initiator of the cerebrovascular cellular pathology that is observed in vitro, inhibition of this process remains an attractive therapeutic target for CAA. We have tested the efficacy of a variety of compounds that have been reported to inhibit Abeta fibril assembly in solution and block the neurotoxic properties of Abeta in vitro. The vast majority of these agents were ineffective in inhibiting the cell surface fibrillar assembly of Abeta and the subsequent pathologic responses in the cultured HCSM cells. This emphasizes the likely requirement of therapeutic compounds that are effective in disrupting cell surface-driven Abeta fibril assembly in the treatment of CAA.

Alzheimer Disease↗
PubMed2002

Rho and Rho-kinase (ROCK) signaling in adherens and gap junction assembly in corneal epithelium.

PURPOSE: To examine whether Rho and its downstream target, a Rho-associated kinase (ROCK), are involved in the regulation of the assembly of cadherin-mediated cell adhesion and connexin 43 (Cx43) gap junctions in corneal epithelium. METHODS: Rho and ROCK activities in rabbit corneal epithelial cells in culture were inhibited by microinjection of a Clostridium botulinum ADP-ribosyltransferase (C3) and treatment with a ROCK specific inhibitor (Y-27632), respectively. Immunocytochemical and Western blot techniques were used to study the distribution and relative concentrations of E-cadherin and Cx43. Intercellular communication via gap junctions was measured by a dye transfer assay. RESULTS: Inhibition of Rho activity in the primary cultures of rabbit corneal epithelial cells by microinjecting them with C3 resulted in an inhibition of the assembly of E-cadherin-based cell-cell adhesion and Cx43 gap junctions. However, inhibition of the ROCK activity by treatment with Y-27632 inhibited the assembly of E-cadherin-based cell-cell adhesions but not Cx43 gap junctions. In fact, inhibition of ROCK resulted in an increase in the number of Cx43 gap junctions and in cell-cell communication. Culturing corneal epithelial cells in a low calcium medium prevented the formation of E-cadherin adherens junctions but not the Cx43 gap junctions. CONCLUSIONS: E-cadherin adherens junctions are not a prerequisite for the assembly of Cx43 gap junctions in corneal epithelial cells. Different Rho signaling pathways are involved in the regulation of the assembly of E-cadherin mediated cell-cell adhesion and Cx43 gap junctions. Although a Rho/ROCK signaling pathway influences the assembly of E-cadherin adherens junctions, its downregulation influences Cx43 gap junction assembly.

Adherens Junctions↗
PubMedDate not supplied

[Modification of fibrin assembly as a method for studying the mechanism of this process].

A study of monomeric fibrin assembly--the process at which the ordered fibres are formed and at the definite moment there arises an elastic three-dimension net (coagulation occurs) made it possible to establish the following. Fibrinogen and high-molecular fragment D formed in fibrinogen enzymatic hydrolysis inhibit the assembly and prevent from creating the regular morphological structure of the fibres. This effect is determined by the presence in fibrinogen and fragment D. of a part of those specific binding centres by means of which the fibrin molecules recognize each other and realize the assemply. The early but not late stages of the assembly are sensitive to fibrinogen and fragment D. This fact evidences for difference in the mechanisms acting at these stages of the process. A moderate increase in the ionic strength prolongs sharply the clotting time of fibrin monomer, that is connected with both retardation in the assembly and prolongation of the pathway in the transformation itself. Transition from the high ionic strength to the low one produces the opposite effect--acceleration in the assembly and shortening of the pathway. For the subsequently appearing intermediate products of the assembly there exists a threshold at which a slow stage of growth and ordering is replaced by the fast terminal reaction of fibre formation. The threshold height is not constant, it depends on the conditions under which the assembly proceeds. The hypothetic explanations of the obtained results are advanced.

Binding Sites↗