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PubMed1998

Significance of sex steroids in roles of cadherin subfamily and its related proteins in the uterine endometrium and placenta.

To know the biological functions of the adherens junction in uterine endometrium, mRNA expressions of E-cadherin and alpha- and beta-catenin. which mainly comprise the adherens junction, were determined. Furthermore, to understand various functions related to V-cadherin in the placenta. vessel permeability was determined by assessing V-cadherin mRNA expression in HUV-EC-C cells, derived from the endothelial cells in human umbilical cord. The levels of E-cadherin and alpha- and beta-catenin mRNAs in the endometria of the proliferative phase were significantly less than those of the secretory phase. Treatment with estradiol dipropionate significantly reduced their levels in the endometria of the secretory phase. These suggest that the functions of the adherens junction in endometrial epithelial cell are controlled by sex steroids. On the other hand, estradiol decreased the endothelial cell barrier properties in HUV-EC-C cells, whereas progesterone partly reversed the changes induced by estradiol. While estradiol decreased the level of V-cadherin mRNA in HUV-EC-C cells, progesterone partly reversed the level decreased by estradiol. Therefore, sex steroids play a role in placental development and function related to cadherins on the endothelial cells, probably via placental vessel permeability.

Adult↗
PubMed2004

Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2.

Prior studies have shown that PI3Ks play a necessary but incompletely defined role in platelet activation. One potential effector for PI3K is the serine/threonine kinase, Akt, whose contribution to platelet activation was explored here. Two isoforms of Akt were detected in mouse platelets, with expression of Akt2 being greater than Akt1. Deletion of the gene encoding Akt2 impaired platelet aggregation, fibrinogen binding, and granule secretion, especially in response to low concentrations of agonists that activate the G(q)-coupled receptors for thrombin and thromboxane A(2). Loss of Akt2 also impaired arterial thrombus formation and stability in vivo, despite having little effect on platelet responses to collagen and ADP. In contrast, reducing Akt1 expression had no effect except when Akt2 was also deleted. Activation of Akt by thrombin was abolished by deletion of Galpha(q) but was relatively unaffected by deletion of Galpha(i2), which abolished Akt activation by ADP. From these results we conclude that Akt2 is a necessary component of PI3K-dependent signaling downstream of G(q)-coupled receptors, promoting thrombus growth and stability in part by supporting secretion. The contribution of Akt1 is less evident except in the setting in which Akt2 is absent.

Animals↗
PubMed1997

Thyrotropin-releasing hormone gene expression by anterior pituitary cells in long-term cultures is influenced by the culture conditions and cell-to-cell interactions.

It has been suggested that TRH, synthesized by anterior pituitary (AP) cells in long-term monolayer cultures, may act as a paracrine or autocrine regulator. Because local control through messenger molecules depends on the cellular microenvironment, we were interested in studying the synthesis of TRH by AP cells in different culture systems and under various conditions. When AP cells were cultured as monolayers in medium containing 10% FCS for long periods of time (up to 3 weeks), a considerable increase in TRH content and prepro-TRHmessengerRNA (preproTRHmRNA) levels could be demonstrated by RIA and Northern blot analysis, whereas the cellular content of the TRH-like peptide pyroGlu-Glu-Pro-NH2 decreased with time in culture to undetectable levels. The release of TRH could be stimulated by depolarizing concentrations of K+ (55 mM), by the Ca++ ionophore A23187, and by GnRH, but not by CRH or GRF, indicating that TRH is stored in gonadotropes. Moreover, a combined in situ hybridization and immunocytochemical analysis demonstrated colocalization of LH in preproTRHmRNA-positive AP cells. When AP cells were cultured as reaggregates in the same (FCS-containing) medium, only a marginal increase in TRH content and preproTRHmRNA levels was observed. Irrespective of the culture systems and the culture conditions used, TRH gene expression was not observed when FCS was omitted. These results indicate that TRH gene expression more likely reflects derepression, rather than induction, of the TRH gene.

Animals↗
PubMed2005

Modifier of cell adhesion regulates N-cadherin-mediated cell-cell adhesion and neurite outgrowth.

Modifier of cell adhesion (MOCA) is a member of the dedicator of cytokinesis 180 family of proteins and is highly expressed in CNS neurons. MOCA is associated with Alzheimer's disease tangles and regulates the accumulation of amyloid precursor protein and beta-amyloid. Here, we report that MOCA modulates cell-cell adhesion and morphology. MOCA increases the accumulation of adherens junction proteins, including N-cadherin and beta-catenin, whereas reducing endogenous MOCA expression lowers cell-cell aggregation and N-cadherin expression. MOCA colocalizes with N-cadherin and actin in areas of cell-cell and cell substratum contact and is expressed in neuronal processes. MOCA accumulates during neuronal differentiation, and its expression enhances NGF-induced neurite outgrowth and morphological complexity. We conclude that MOCA regulates N-cadherin-mediated cell-cell adhesion and neurite outgrowth.

Animals↗
PubMed1993

Poorly differentiated colon carcinoma cell lines deficient in alpha-catenin expression express high levels of surface E-cadherin but lack Ca(2+)-dependent cell-cell adhesion.

Studies on several different types of carcinomas, with the notable exception of colon carcinoma, have shown that poorly differentiated tumors are frequently deficient in E-cadherin dependent cell-cell adhesion. In this study, we examined Ca(2+)-dependent cell-cell adhesion in colon carcinoma cell lines. Five poorly differentiated (Clone A, MIP 101, RKO, CCL 222, CCL 228) and four moderately-well differentiated (CX-1, CCL 235, DLD-2, CCL 187) colon carcinoma cell lines were assayed for their ability to form cell-cell aggregates and for their levels of E-cadherin expression. All of the poorly differentiated cell lines exhibited low levels of Ca(2+)-dependent cell-cell aggregation, in contrast to the moderately-well differentiated cell lines. Contrary to most previous studies, however, we observed that three of the five poorly differentiated cell lines examined expressed E-cadherin by FACS analysis and immunoprecipitation using an E-cadherin mAb. In fact, two of these cell lines expressed a 3- to 4-fold higher level of E-cadherin than that found in the moderately-well differentiated cell lines. mRNA levels for E-cadherin, as evaluated by both RT-PCR and Northern hybridization, corresponded to the levels of protein expression in each of the cell lines. Immunoprecipitation with an E-cadherin mAb, which is known to co-precipitate the catenins, demonstrated that the three poorly differentiated cell lines expressing E-cadherin did not co-precipitate alpha-catenin, although all of the moderately-well differentiated cell lines expressed both alpha- and beta-catenin. RT-PCR confirmed the absence of the alpha-catenin mRNA from two of these cell lines. Stable expression of an alpha-catenin cDNA in one of the poorly differentiated cell lines lacking alpha-catenin expression resulted in a 5-fold increase in its level of Ca(2+)-dependent cell-cell aggregation, providing evidence that alpha-catenin is directly responsible for the loss of cell-cell adhesion in some cell lines. The alpha-catenin transfectants also exhibited a marked reduction in migration on collagen I. These data indicate that loss of alpha-catenin expression, as well as E-cadherin expression, can lead to a phenotype associated with poorly differentiated colon carcinomas.

Base Sequence↗
PubMed1995

Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia.

We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti-Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a)+ father and from a healthy donor phenotyped as Max(a-), reverse-transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G-->A substitution at position 2,603, predicting a valine-->methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Mas and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA-3 allele.

Alleles↗
PubMed1999

Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.

FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first approximately 400 residues, whereas the C-terminus provides a nonspecific binding surface.

Amino Acid Sequence↗
PubMed2005

Localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway.

Tomato bushy stunt virus (TBSV), a positive-strand RNA virus, causes extensive inward vesiculations of the peroxisomal boundary membrane and formation of peroxisomal multivesicular bodies (pMVBs). Although pMVBs are known to contain protein components of the viral membrane-bound RNA replication complex, the mechanisms of protein targeting to peroxisomal membranes and participation in pMVB biogenesis are not well understood. We show that the TBSV 33-kD replication protein (p33), expressed on its own, targets initially from the cytosol to peroxisomes, causing their progressive aggregation and eventually the formation of peroxisomal ghosts. These altered peroxisomes are distinct from pMVBs; they lack internal vesicles and are surrounded by novel cytosolic vesicles that contain p33 and appear to be derived from evaginations of the peroxisomal boundary membrane. Concomitant with these changes in peroxisomes, p33 and resident peroxisomal membrane proteins are relocalized to the peroxisomal endoplasmic reticulum (pER) subdomain. This sorting of p33 is disrupted by the coexpression of a dominant-negative mutant of ADP-ribosylation factor1, implicating coatomer in vesicle formation at peroxisomes. Mutational analysis of p33 revealed that its intracellular sorting is also mediated by several targeting signals, including three peroxisomal targeting elements that function cooperatively, plus a pER targeting signal resembling an Arg-based motif responsible for vesicle-mediated retrieval of escaped ER membrane proteins from the Golgi. These results provide insight into virus-induced intracellular rearrangements and reveal a peroxisome-to-pER sorting pathway, raising new mechanistic questions regarding the biogenesis of peroxisomes in plants.

ADP-Ribosylation Factor 1↗
PubMed1987

Regulation of copper metabolism in the mottled mouse.

Menkes' kinky-hair syndrome is an X-linked recessive neurodegenerative and connective-tissue disorder, with decreased serum copper and ceruloplasmin-copper oxidase concentrations and tissue-specific increases in copper content. Clinical manifestations can be related to relative copper deficiency and reduced activity of cuproenzymes in multiple organs. An animal model is provided by mice hemizygous for mutant alleles, such as the blotchy allele, at the X-linked mottled locus. This locus may be homologous in mouse and man. The basic defect is unknown but has been thought to reside in the regulation of the function or synthesis of metallothioneins. In the blotchy mouse and in cultured skin fibroblasts derived therefrom, we showed that the mutation specifically affects the metabolism of copper and not other trace metals. Excessive accumulation and abnormal (reduced) exit kinetics were demonstrated for copper but not for the related trace metals cadmium and zinc. While metallothionein-I messenger RNA (mRNA) concentrations were elevated in blotchy fibroblasts, the elevations in metallothionein-I mRNA in response to metallothionein inducers (cadmium, copper) were similar in blotchy and control cells. Further, metallothionein-I mRNA levels were indistinguishable in mutant and control fibroblasts containing equivalent intracellular copper concentrations. Finally, metallothionein-I mRNA content was not elevated in blotchy kidneys at early developmental stages, before storage of excessive copper. The aggregate data suggest that the basic defect in the blotchy mouse--and, by analogy, in Menkes' syndrome--does not reside in defective modulation of metallothionein function and does not cause abnormal regulation of metallothionein synthesis.

Animals↗
PubMed1993

Reduction of E-cadherin levels and deletion of the alpha-catenin gene in human prostate cancer cells.

The cadherins are a family of transmembrane glycoproteins responsible for calcium-dependent cell-cell adhesion. This adhesion is mediated by a group of cytoplasmic proteins, the catenins, which act inside the cell to couple the cadherin molecule to the microfilament cytoskeleton. Dysfunction of E-cadherin-dependent cell-cell adhesion has been demonstrated to contribute to the acquisition of invasive potential of malignant adenocarcinoma cells. The potential role of alterations of catenin expression in tumor cell invasion is largely unexplored. We have previously found that E-cadherin is frequently down-regulated in clinical samples of prostate cancer (Umbas, R., Schalken, J. A., Aalders, T. W., Carter, B. S., Karthaus, H. F. M., Schaafsma, H. E., Debruyne, F. M. J., and Isaacs, W. B. Cancer Res., 52: 5104-5109, 1992). In this study, we further investigate this adhesion system in both benign and malignant human prostate cells in culture. Using antibodies to E-cadherin and its cytoplasmic accessory protein, alpha-catenin, we find that 5 of 6 human prostate cancer cell lines have reduced or absent levels of one or the other or both of these molecules when compared to normal prostatic epithelial cells. Only the LNCaP prostate cancer cell line is indistinguishable from normal prostate epithelium with respect to its E-cadherin-alpha-catenin complement. Interestingly, the PC-3 line is characterized by the presence of E-cadherin, but the complete lack of alpha-catenin found at both the RNA and protein level. This lack of alpha-catenin gene expression is explained by Southern analysis, which reveals a homozygous deletion of a large portion of the alpha-catenin gene in PC-3 cells. This loss of alpha-catenin is functionally manifested by negligible Ca(2+)-dependent aggregation of these cells in vitro, when compared to LNCaP cells. These results confirm that E-cadherin-dependent cell-cell adhesion is frequently aberrant in prostate cancer cells, and suggest that in a subset of prostate cancers, this adhesion may be inactivated by loss of alpha-catenin rather than E-cadherin itself. Furthermore, these results demonstrate that mutational inactivation of the alpha-catenin gene is one mechanism responsible for the loss of normal cell-cell adhesion in prostate cancer.

Cadherins↗
PubMed2005

Expression of the Opa1 mitochondrial protein in retinal ganglion cells: its downregulation causes aggregation of the mitochondrial network.

PURPOSE: Mutations in the mitochondrial dynamin-related GTPase OPA1 cause autosomal dominant optic atrophy (ADOA), but the pathophysiology of this disease is unknown. As a first step in functional studies, this study was conducted to evaluate the expression of Opa1 in whole retina and in isolated retinal ganglion cells (RGCs) and to test the effects of Opa1 downregulation in cultured RGCs. METHODS: Opa1 mRNA isoforms from total retina and from RGCs freshly isolated by immunopanning were determined by RT-PCR. Protein expression was examined by immunohistochemistry and Western blot with antibodies against Opa1 and cytochrome c, and the mitochondrial network was visualized with a mitochondrial marker. Short interfering (si)RNA targeting OPA1 mRNAs were transfected to cultured RGCs and mitochondrial network phenotypes were followed for 15 days, in comparison with those of cerebellar granule cells (CGCs). RESULTS: Opa1 expression did not predominate in rat postnatal RGCs as found by immunohistochemistry and Western blot analysis. The pattern of mRNA isoforms was similar in whole retina and RGCs. After a few days in culture, isolated RGCs showed fine mitochondrial punctiform structures in the soma and neurites that colocalized with cytochrome c and Opa1. Opa1 knockdown in RGCs induced mitochondrial network aggregation at a higher rate than in CGCs. CONCLUSIONS: Results suggest that the level of expression and the mRNA isoforms do not underlie the vulnerability of RGCs to OPA1 mutations. However, aggregation of the mitochondrial network induced by the downregulation of Opa1 appears more frequent in RGCs than in control CGCs.

Animals↗
PubMed2000

Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a.

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes RNA replication proteins 1a and 2a. 1a contains a C-terminal helicase-like domain and an N-terminal domain implicated in viral RNA capping, and 2a contains a central polymerase-like domain. 1a and 2a colocalize in an endoplasmic reticulum (ER)-associated replication complex that is the site of BMV-specific RNA-dependent RNA synthesis in plant and yeast cells. 1a also localizes to the ER in the absence of 2a or viral RNA replication templates. To investigate the determinants of 2a localization, we fused 2a to the green fluorescent protein (GFP), creating a functional GFP-2a fusion that supported BMV RNA replication and subgenomic mRNA transcription. In the absence of 1a, the GFP-2a fusion was found to be diffused throughout the cytoplasm and in punctate spots not associated with any cytoplasmic organelle so far tested. Formation of these spots was dependent on the C-terminal half of 2a and may represent aggregation of a fraction of 2a. When coexpressed with 1a, GFP-2a colocalized with 1a and ER-resident protein Kar2p in a partial or complete ring around the nucleus. Consistent with these results, cell fractionation showed that both the GFP-2a fusion and wild-type (wt) 2a remained soluble when expressed alone, while in cells coexpressing 1a, most of the GFP-2a fusion or wt 2a cofractionated with 1a in the rapidly sedimenting membrane fraction. Deletion analysis showed that the N-terminal 120-amino-acid segment of 2a, containing one of two 2a regions previously shown to interact with 1a, was necessary and sufficient for 1a-directed localization of GFP-2a derivatives to the ER. These results suggest that 1a, which also interacts independently with the ER and viral RNA, is a key organizer of RNA replication complex assembly.

Biological Transport↗
PubMed1986

The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy.

The nonchromatin structure or matrix of the nucleus has been studied using an improved fractionation in concert with resinless section electron microscopy. The resinless sections show the nucleus of the intact cell to be filled with a dense network or lattice composed of soluble proteins and chromatin in addition to the structural nuclear constituents. In the first fractionation step, soluble proteins are removed by extraction with Triton X-100, and the dense nuclear lattice largely disappears. Chromatin and nonchromatin nuclear fibers are now sharply imaged. Nuclear constituents are further separated into three well-defined, distinct protein fractions. Chromatin proteins are those that require intact DNA for their association with the nucleus and are released by 0.25 M ammonium sulfate after internucleosomal DNA is cut with DNAase I. The resulting structure retains most heterogeneous nuclear ribonucleoprotein (hnRNP) and is designated the RNP-containing nuclear matrix. The proteins of hnRNP are those associated with the nucleus only if RNA is intact. These are released when nuclear RNA is briefly digested with RNAase A. Ribonuclease digestion releases 97% of the hnRNA and its associated proteins. These proteins correspond to the hnRNP described by Pederson (Pederson, T., 1974, J. Mol. Biol., 83:163-184) and are distinct from the proteins that remain in the ribonucleoprotein (RNP)-depleted nuclear matrix. The RNP-depleted nuclear matrix is a core structure that retains lamins A and C, the intermediate filaments, and a unique set of nuclear matrix proteins (Fey, E. G., K. M. Wan, and S. Penman, 1984, J. Cell Biol. 98:1973-1984). This core had been previously designated the nuclear matrix-intermediate filament scaffold and its proteins are a third, distinct, and nonoverlapping subset of the nuclear nonhistone proteins. Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in matrix organization. Conventional Epon-embedded electron microscopy sections show comparatively little of the RNP-containing and RNP-depleted nuclear matrix structure. In contrast, resinless sections show matrix interior to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are covered with 20-30-nm electron dense particles which may contain the hnRNA. The large electron dense bodies, enmeshed in the interior matrix fibers, have the characteristic morphology of nucleoli. Treatment of the nuclear matrix with RNAase results in the aggregation of the interior fibers and the extensive loss of the 20-30-nm particles.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Fractionation↗
PubMed1987

Reovirus major capsid protein expressed in Escherichia coli.

A DNA copy of the open reading frame of the S4 gene of reovirus type 3 was cloned into a temperature-regulated bacterial expression vector. Induction at 42 degrees C resulted in the synthesis of a polypeptide that comigrated with virion capsid protein sigma 3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reacted with sigma 3-specific antisera. The protein was expressed in bacteria as insoluble aggregates that accumulated in polar inclusion bodies. Aggregated product also resulted when the expression system was manipulated to induce bacterial sigma 3 (b sigma 3) synthesis at temperatures below 42 degrees C. Various methods used to solubilize b sigma 3 did not yield the monomeric protein. The results indicate that sigma 3, the major surface component of reovirions, is expressed in transfected Escherichia coli as an aggregated, disulfide cross-linked protein.

Capsid↗
PubMed2005

Lack of direct role of parkin in the steady-state level and aggregation of alpha-synuclein and the clearance of pre-formed aggregates.

Mutations in parkin and alpha-synuclein (alpha-syn) are linked to heritable forms of Parkinson's disease (PD). Recently, it has been shown that parkin mitigates alpha-syn-induced neuronal cell death in animal and tissue culture models, suggesting that there is a functional relationship between these two proteins. Although the mechanism by which parkin protects cells from alpha-syn-induced cytotoxicity remains elusive, it is tempting to speculate that parkin might directly regulate the normal metabolism and aggregation of alpha-syn. In the current study, we show that neither the suppression of endogenous parkin expression nor ectopic overexpression affects the steady-state levels of endogenous alpha-syn expression, overall aggregation of this protein, or breakdown of pre-formed aggregates in human neuroblastoma cells. These results suggest that parkin is not directly involved in the metabolism of alpha-syn, its aggregation, or the clearance of pre-formed aggregates.

Analysis of Variance↗
PubMed1996

Comparison of the solution and crystal conformations of (G + C)-rich fragments of DNA.

DNA fragments crystallize in an unpredictable manner, and relationships between their crystal and solution conformations still are not known. We have studied, using circular dichroism spectroscopy, solution conformations of (G + C)-rich DNA fragments, the crystal structures of which were solved in the laboratory of one of the present authors. In aqueous trifluorethanol (TFE) solutions, all of the examined oligonucleotides adopted the same type of double helix as in the crystal. Specifically, the dodecamer d(CCCCCGCGGGGG) crystalized as A-DNA and isomerized into A-DNA at high TFE concentrations. On the other hand, the hexamer d(CCGCGG) crystallized in Z-form containing tilted base pairs, and high TFE concentrations cooperatively transformed it into the same Z-form as adopted by the RNA hexamer r(CGCGCG), although d(CCGCGG) could isomerize into Z-DNA in the NaCl + NiCl2) aqueous solution. The fragments crystallizing as B-DNA remained B-DNA, regardless of the solution conditions, unless they denatured or aggregated. Effects on the oligonucleotide conformation of 2-methyl-2,4-pentanediol and other crystallization agents were also studied. 2-Methyl-2,4-pentanediol induced the same conformational transitions as TFE but, in addition, caused an oligonucleotide condensation that was also promoted by the other crystallization agents. The present results indicate that the crystal double helices of DNA are stable in aqueous TFE rather than aqueous solution.

Base Composition↗
PubMed2005

Reactivity of apolipoprotein E4 and amyloid beta peptide: lysosomal stability and neurodegeneration.

We previously demonstrated that apolipoprotein E4 (apoE4) potentiates lysosomal leakage and apoptosis induced by amyloid beta (Abeta) peptide in cultured Neuro-2a cells and hypothesized that the low pH of lysosomes accentuates the conversion of apoE4 to a molten globule, inducing reactive intermediates capable of destabilizing cellular membranes. Here we report that neutralizing lysosomal pH with bafilomycin or NH4Cl abolished the apoE4 potentiation of Abeta-induced lysosomal leakage and apoptosis in Neuro-2a cells. Consistent with these results, apoE4 at acidic pH bound more avidly to phospholipid vesicles and disrupted them to a greater extent than at pH 7.4. Comparison of "Arctic" mutant Abeta, which forms multimers, and GM6 mutant Abeta, which remains primarily monomeric, showed that aggregation is essential for apoE4 to potentiate Abeta-induced lysosomal leakage and apoptosis. Both apoE4 and Abeta1-42 had to be internalized to exert these effects. Blocking the low density lipoprotein receptor-related protein with small interfering RNA abolished the enhanced effects of apoE4 and Abeta on lysosomes and apoptosis. In cultured Neuro-2a cells, Abeta1-42 increased lysosome formation to a greater extent in apoE3- or apoE4-transfected cells than in Neo-transfected cells, as shown by immunostaining for lysosome-associated membrane protein 1. Similarly, in transgenic mice expressing apoE and amyloid precursor protein, hippocampal neurons displayed increased numbers of lysosomes. Thus, apoE4 and Abeta1-42 may work in concert in neurons to increase lysosome formation while increasing the susceptibility of lysosomal membranes to disruption, release of lysosomal enzymes into the cytosol, and neuronal degeneration.

Amyloid beta-Peptides↗