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PubMed1991

Transcription of the myxobacterial hemagglutinin gene is mediated by a sigma 54-like promoter and a cis-acting upstream regulatory region of DNA.

Myxobacterial hemagglutinin (MBHA) is a major developmentally induced protein that accumulates during the period of cellular aggregation of the fruiting bacterium Myxococcus xanthus. In this study, DNA sequences mediating the transcriptional regulation of mbhA have been identified. Examination of nucleotide sequences upstream of the start site for mbhA transcription has indicated a region of DNA that bears strong homology to the consensus sequence for promoters recognized by the sigma 54 holoenzyme form of RNA polymerase of Escherichia coli and other eubacteria. Deletion of this sequence completely abolished mbhA transcription. Additionally, a cis-acting DNA element, affecting the efficiency of mbhA transcription, has been mapped within a region of DNA 89 to 276 nucleotides upstream of the sigma 54-like sequence. Transposon insertions, mapping within the cis element, drastically reduced mbhA transcriptional activity. These observations suggest that transcription of mbhA requires a productive interaction between a form of RNA polymerase that recognizes a sigma 54-like sequence and a transcriptional activator that binds to DNA sequences upstream of the mbhA promoter.

Base Sequence↗
PubMed2003

Contribution of the zinc finger to zinc and DNA binding by a suppressor of posttranscriptional gene silencing.

The zinc finger C(36)-X1-C(38)-X7-C(46)-X6-H(53) of the nuclearly localized C2 protein of Tomato yellow leaf curl virus China is involved in pathogenicity and suppression of posttranscriptional gene silencing (PTGS). Here, we demonstrate that the zinc finger is indispensable for the C2 protein to bind zinc and DNA. Mutation of cysteine residue C(36), C(38), or C(46) reduced the zinc and DNA binding capacity of C2 protein. When expressed from potato virus X, all three mutants, C2-C(36)R, C2-C(38)N, and C2-C(46)I, tagged with a green fluorescent protein (GFP) were still capable of transporting GFP into but aggregated abnormally in nuclei. Our data establish that zinc- and DNA-binding activity correlates with C2-mediated pathogenesis and PTGS suppression.

DNA↗
PubMedDate not supplied

The molecular weight of rRNA precursor molecules and their processing in higher plant cells.

Actively dividing callus cells of higher plants (Petroselinum crispum, Daucus carota, Acer pseudoplatanus) were used to detect the primary gene product of rDNA in vivo. Parsley and carrot cells were labelled with [32P]orthophosphate. Under non-denaturing conditions, in both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels. Its molecular weight did not exceed 2.5 x 10(6) dalton. Under denaturing conditions, 2.0--2.1 x 10(6) dalton were determined on formamide gels. This rRNA precursor was already present after a labelling period of 5--10 min. In parsley cells labelled mature rRNA (25S and 18S) arrived in the cytoplasm 45 min after onset of incubation. In Acer pseudoplatanus incubated with [3H]uridine two rapidly labelled components did emerge from polyacrylamide gels without formamide; their molecular weights were 2.3 and 3.2--3.4 x 10(6) dalton. After electrophoresis in formamide, the larger component disappeared, thus indicating that it would be an intermolecular aggregate of different RNAs. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 x 10(6) dalton.

Cells, Cultured↗
PubMed2003

RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis.

We have used RNA interference (RNAi) to examine the functional relationship between valosin-containing protein (VCP/p97/Cdc48p/TER94) ATPase and the ubiquitin-proteasome system (UPS) in Drosophila S2 and human HeLa cells. In both cell types, RNAi of VCP (and, to a lesser extent, of certain VCP-interacting proteins) caused significant accumulation of high-molecular-weight conjugates of ubiquitin, an indication of inhibited UPS function. However, decreased VCP levels did not directly inhibit proteasome activity. In HeLa cells, polyubiquitinated proteins accumulated as dispersed aggregates rather than as single aggresomes, even in the presence of proteasome inhibitors, which normally promote aggresome formation. RNAi of VCP caused extensive vacuolization of the cytoplasm, and proteasome inhibitors exaggerated this feature. RNAi of VCP had little effect on S2 cell proliferation but blocked cell-cycle progression and induced mitotic abnormalities and apoptosis in HeLa cells. These results indicate that VCP plays an important general role in mediating the function of the UPS, probably by interacting with potential proteasome substrates before they are degraded by the proteasome.

Adenosine Triphosphatases↗
PubMed2002

Identification and characterization of a soluble cadherin-7 isoform produced by alternative splicing.

We identified an alternative mRNA encoding a novel cadherin-7 isoform by reverse transcriptase-PCR of RNA from day 12 chicken embryos. The alternative mRNA contains 49 bases of insertion in the premembrane region, leading to the substitution of 14 amino acids and the introduction of a premature stop codon. Identification of a 49-bp insertion sequence in the genomic DNA corresponding to the intron of the cadherin-7 gene suggests that alternative splicing is the cause of the alternative mRNA. Transient expression of the variant form in COS-7 or 293 cells produced a soluble protein. Aggregation assays and immunoprecipitation showed that the variant protein interacts with full-length cadherin-7 in vitro and in vivo and inhibits full-length cadherin-7-mediated cell adhesion. Immunohistochemistry revealed that the variant form was strongly expressed in dermomyotomes rather than in migrating neural crest cells, in contrast to the full-length cadherin-7, suggesting differential regulation of splicing and possible roles of variant cadherin-7 in the development of dermomyotomes and other tissues.

Alternative Splicing↗
PubMed2005

Adenovirus-mediated silencing of huntingtin expression by shRNA.

Huntington's disease (HD) is an inherited autosomal dominant, neurodegenerative disease that is caused by a gain of function mutation characterized by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (htt) gene. Since hairpin small interference RNA (shRNA) technology allows inhibition of specific gene expression in vitro and in vivo, vector-mediated expression of an shRNA directed to htt mRNA could form the basis of a new treatment modality for HD. By initial plasmid transfection of 293 cells, we identified one exon 1-targeted shRNA, which efficiently inhibited expression of an htt exon 1-GFP fusion protein and the endogenous htt gene. A replication-deficient adenovirus (Ad) vector Adie-1-1 was constructed to express this shRNA from the U6 promoter. In A549 cells expressing exon 1 of htt with an expanded CAG allele, Adie- 1-1 efficiently prevented htt exon 1 expression and htt aggregate formation. In addition, in different neuronal and nonneuronal cell lines, Adie-1-1 efficiently inhibited the expression of endogenous htt. Together, this data indicates the delineation of an shRNA strategy that may become the basis for treatment of HD.

Adenoviridae↗
PubMed1997

Laminin chain expression by chick chondrocytes and mouse cartilaginous tissues in vivo and in vitro.

We have observed that laminins are expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and adult mouse knee joint by the methods of in situ hybridization, immunohistochemistry, Western blotting, and immunoprecipitation. From in situ hybridization using similar sized RNA probes for different mouse laminin chains, mRNAs for the alpha 1, alpha 2, beta 1, beta 2, and gamma 1 chains were expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and the articular cartilage cap and epiphyseal growth plate of adult mouse knee joint. Through the use of chain-specific antibodies, staining for laminins was observed in the cytoplasm of chondrocytes from chick embryo sternum, mouse limb bud, and adult mouse knee joint. Western blot analysis confirmed the presence of laminin chains in the cells and sternal tissues. Cultured chick embryonic sternal chondrocytes expressed laminin mRNAs in the proliferating stage (2-3 days of culture) but the level increased in the aggregated cells during the maturation stage (5-7 days of culture). Comparable data were also obtained after immunostaining the cells. Thus, laminins are expressed in significant amounts by chondrocytes and may have an important role in cartilage development.

Animals↗
PubMed1998

Differential modulation of cell death proteins in human brain cells by tumor necrosis factor alpha and platelet activating factor.

Programmed cell death contributes to the morbidity and mortality of several neurological disorders including stroke, Alzheimer's disease and human immunodeficiency virus (HIV)-associated dementia. Patients with HIV dementia show evidence of programmed cell death in brain. In vitro data demonstrates several neurotoxic products of macrophage infection that cause neural cell death, including tumor necrosis factor alpha (TNFalpha) and platelet activating factor (PAF). We treated human brain aggregate cultures with these cytokines and determined their effect on the mRNA and protein levels for Bcl-2, Bcl(x) and Bax alpha. TNFalpha and PAF differentially regulate the Bcl-2 family of proteins at a post-transcriptional level. Following TNFalpha treatment, Bcl-2 protein is significantly decreased, and at least one additional Bax isomer emerges. Bcl(xL) protein is slightly increased after treatment with either cytokine. We demonstrated that overexpression of Bcl-2 in brain aggregate cultures protects cells from TNFalpha-induced damage but has no effect on cell damage induced by PAF. We conclude that Bcl-2 and Bax alpha proteins play significant roles in modulating neural cell death from TNFalpha- but not from PAF-induced cell damage.

AIDS Dementia Complex↗
PubMed1979

Structural comparisons of the aggregates of tobacco mosaic virus protein.

The coat protein of tobacco mosaic virus forms numerous aggregates, including the small A-protein, the disk, and two helical forms. The structures of the disk, the helical protein forms, and the virus are compared. Most of the differences are in the conformation of the chain between residues 89 and 113, which lies in the region of protein at the center of the virus, inside the RNA. It is disordered in the disk, but has a fixed conformation in the virus and the protein helices. The differences between the virus and the two helical protein forms are largely in the conformations of arginines and carboxylic acids in this region.

Macromolecular Substances↗
PubMed1997

Human herpesvirus 8 and Kaposi's sarcoma.

In the last 2 years, the discovery that the suspected causative agent of Kaposi's sarcoma (KS) is a new gamma-herpesvirus, called human herpesvirus type 8 (HHV8) or Kaposi's sarcoma-associated herpesvirus (KSHV), has been followed by studies showing it to be a sine qua non of all clinical forms of KS and a specific marker for KS in the differential diagnosis of angioproliferative lesions. Reports that the virus is ubiquitous have been based on the polymerase chain reaction and appear to be contradicted by serological studies of blood donors and patients with acquired immunodeficiency syndrome (AIDS)-related and classical KS. Further serological surveys and the application of molecular probes in histological sections should resolve the issue. The recent descriptions of KSHV RNA molecules and several viral mimickers of human cytokines offer the chance to map the viral latent-lytic cycle and will change the direction of cytokine research in KS. These discoveries suggest that the increase of endemic classical KS noted in Nordic countries before the AIDS epidemic was due to spread of KSHV by unknown routes. The aggregate data should force a paradigm shift away from the notion that human immunodeficiency virus infection has a necessary role to play in AIDS-KS other than as a cause of progressive immunodeficiency.

Diagnosis, Differential↗
PubMed1995

Differentiation of embryonal carcinoma cells to a neural or cardiomyocyte lineage is associated with selective expression of endothelin receptors.

Endothelins (ETs) were initially characterized as potent vasoactive peptides acting through at least two distinct receptors, ETA and ETB. Subsequently, their significant growth- and hypertrophy-promoting properties in cardiac and other cells were recognized. We investigated the expression of endothelin receptors during differentiation of a pluripotential embryonal carcinoma cell line (P19) to a cardiomyocyte or a neural lineage. These cells resemble those of the inner cell mass of the blastocyst, and their differentiation is believed to closely mimic critical events in early embryogenesis. Differentiation of P19 to a cardiomyocyte lineage, by aggregation and exposure to dimethyl sulfoxide resulted in induction of ETA receptors as demonstrated by radioligand binding studies, Northern blotting, and reporter gene analysis. Moreover, the P19 differentiated to a cardiac lineage responded to ET-1 with a 3-fold increase in the secretion of atrial natriuretic peptide. In contrast, differentiation to a neural lineage, by aggregation and exposure to retinoic acid, was associated with the induction of predominantly ETB. Therefore, selective differentiation of the P19 led to the differential expression of endothelin receptors in a pattern consistent with that observed in normal myocardial and neural tissue. The induction of endothelin receptors in a model system of early embryogenesis provides strong support for the critical role of this peptide/receptor family in differentiation and development. As well, this model system is well suited for the study of the mechanisms controlling endothelin receptor expression during differentiation.

Animals↗
PubMed2000

mu-Protocadherin, a novel developmentally regulated protocadherin with mucin-like domains.

Branching morphogenesis is a central event during the development of kidneys, lungs, and other organs. We previously generated a monoclonal antibody, 3D2-E9, that inhibited branching morphogenesis and caused widespread apoptosis. We now report the purification of its antigen and cloning of its full-length cDNA. Its cDNA encodes an integral membrane protein that contains four cadherin-like ectodomains and a thrice tandemly repeated region enriched in threonine, serine, and proline, similar to those of mucins. We thus term this protein mu-protocadherin, reflecting the hybrid nature of its extracellular region. mu-Protocadherin is expressed in two forms that are developmentally regulated, with the shorter isoform lacking the mucin-like repeats. Expression of the long isoform in heterologous cells results in adhesion of the expressing cells, suggesting that it is a new cell adhesion molecule. mu-Protocadherin contains both N and O glycosylations. It is expressed at lateral and basal surfaces of epithelia during kidney and lung development and is located in coated pits. Colocalization of mu-protocadherin with beta-catenin was noted primarily at the junction of the lateral and basal membrane. The cytoplasmic domain contains four proline-rich regions, similar to SH3 binding regions. Thus, it is likely that adhesive interactions mediated by mu-protocadherin induce signaling events that regulate branching morphogenesis.

Amino Acid Sequence↗
PubMed2003

Functional analysis of PKD1 transgenic lines reveals a direct role for polycystin-1 in mediating cell-cell adhesion.

The PKD1 protein, polycystin-1, is a large transmembrane protein of uncertain function and topology. To study the putative functions of polycystin-1, conditionally immortalized kidney cells transgenic for PKD1 were generated and an interaction between transgenic polycystin-1 and endogenous polycystin-2 has been recently demonstrated in these cells. This study provides the first functional evidence that transgenic polycystin-1 directly mediates cell-cell adhesion. In non-permeabilized cells, polycystin-1 localized to the lateral cell borders with N-terminal antibodies but not with a C-terminal antibody; there was a clear difference in surface intensity between transgenic and non-transgenic cells. Compared with non-transgenic cells, transgenic cells showed a dramatic increase in resistance to the disruptive effect of a polycystin-1 antibody raised to the PKD domains of polycystin-1 (IgPKD) in both cell adhesion and cell aggregation assays. The differential effect on cell adhesion between transgenic and non-transgenic cells could be reproduced using recombinant fusion proteins encoding non-overlapping regions of the IgPKD domains. In contrast, antibodies raised to other extracellular domains of polycystin-1 had no effect on cell adhesion. Finally, the specificity of this finding was confirmed by the lack of effect of IgPKD antibody on cell adhesion in a PKD1 cystic cell line deficient in polycystin-1. These results demonstrate that one of the primary functions of polycystin-1 is to mediate cell-cell adhesion in renal epithelial cells, probably via homophilic or heterophilic interactions of the PKD domains. Disruption of cell-cell adhesion during tubular morphogenesis may be an early initiating event for cyst formation in ADPKD.

Animals↗
PubMed1993

Novel role of human chorionic gonadotropin in differentiation of human cytotrophoblasts.

Our laboratory previously demonstrated that cytotrophoblasts and syncytiotrophoblasts in human placental tissue contain hCG/LH receptors. From this finding, we postulated that one role of hCG might be to promote the differentiation of cytotrophoblasts into syncytiotrophoblasts. To test this postulate, cytotrophoblasts were isolated from human term pregnancy placentas and cultured with and without increasing concentrations of highly purified hCG. The results showed that hCG had a biphasic effect on 1) the aggregation of cells without intervening plasma membranes; 2) the expression of cadherin, a cell adhesion receptor that facilitates cellular aggregation; 3) the expression of hCG/LH receptor gene; and 4) the expression of three different hormonal markers of differentiation. The hCG effects were time and dose dependent and hormone specific. The addition of excess polyclonal hCG antibody, but not normal rabbit serum or nonspecific antirabbit immunoglobulin G, decreased basal responses as well as those to exogenous hCG. The polyclonal hCG/LH receptor antibody increased differentiation and dramatically stimulated hCG secretion in the presence or absence of exogenous hCG. (Bu)2cAMP mimicked the actions of hCG. H-89, a protein kinase-A inhibitor, decreased basal as well as exogenous hCG responses. Calphostin, a protein kinase-C inhibitor and lavendustin, a tyrosine kinase inhibitor, on the other hand, had no effect. In summary, it is novel that hCG made in human placenta can regulate the differentiation of cytotrophoblasts, which make little hCG, into syncytiotrophoblasts, which make considerable amounts of hCG.

Blotting, Northern↗
PubMed2001

IL-12 plays a pivotal role in LFA-1-mediated T cell adhesiveness by up-regulation of CCR5 expression.

The chemokine receptor CCR5 has been implicated in the recruitment of T cells to inflammatory sites. However, the regulation of CCR5 induction on T cells and its contribution to T cell adhesiveness are poorly understood. Using a Th1 clone, 2D6, that can be maintained with interleukin (IL)-12 or IL-2 alone (designated 2D6(IL-12) or 2D6(IL-2), respectively), we investigated how CCR5 is induced on T cells and whether CCR5 is responsible for up-regulating the function of adhesion molecules. 2D6(IL-12) grew, forming cell aggregates, in culture containing IL-12. This was due to lymphocyte function-associated antigen (LFA)-1-intercellular adhesion molecule (ICAM)-1 interaction, because 2D6(IL-12) expressed both LFA-1 and ICAM-1 and cell aggregation was inhibited by anti-ICAM-1 monoclonal antibody. Despite comparable levels of LFA-1 and ICAM-1 expression, 2D6(IL-2) cells did not aggregate in culture with IL-2. It is important that there was a critical difference in CCR5 expression between 2D6(IL-12) and 2D6(IL-2); the former expressed high levels of CCR5, and the latter expressed only marginal levels. Both types of cells expressed detectable albeit low levels of RANTES (regulated on activation, normal T expressed and secreted) mRNA. Unlike IL-12 or IL-2, IL-18 induced high levels of RANTES mRNA expression without modulating CCR5 expression. Therefore, combined stimulation with IL-12 and IL-18 strikingly up-regulated 2D6 cell aggregation. Notably, LFA-1-mediated aggregation of 2D6(IL-12) cells was suppressed by anti-CCR5 antibody. These results indicate that IL-12 plays a critical role in CCR5 expression on Th1 cells and consequently contributes to CCR5-mediated activation of LFA-1 molecules.

Animals↗
PubMed1994

Modulation of signaling via the B cell antigen receptor by CD21, the receptor for C3dg and EBV.

CD21 is the receptor for C3dg and EBV. Several reports have shown that these CD21 ligands, and certain anti-CD21 mAb, trigger B cell activation, particularly when combined with Ag receptor ligation. However, the characteristics, biologic functions, and importance of this CD21-signaling pathway are unknown. We have used a model we recently developed to study B cell activation induced by complex particulate Ag, such as immune complexes and viruses, to begin to examine these questions. In the current studies, we incubated purified small resting B cells with 100-nm latex beads bearing various combinations of CD21 ligands and mAbs to CD19, CD35, and the Ag receptor. CD21, CD19, and CD35 have all been implicated in modulating membrane IgM initiated signaling. Beads coated with mAb to the C3dg/EBV-binding portion of CD21, but not mAb to other portions of the CD21 molecule, triggered B cell homotypic aggregation. Beads coated with the same CD21 ligands, although inactive alone, synergized with anti-IgM mAb in greatly increasing (20- to 180-fold) mRNA levels of the c-fos nuclear proto-oncogene. Signaling via CD21 was tyrosine kinase dependent. Levels of c-myc mRNA were not altered by CD21 ligands. Anti-CD19 and anti-CD35 mAb did not augment signaling via membrane IgM as assessed by changes in c-fos mRNA levels. These findings indicate that CD21 ligands binding to the C3dg/EBV-binding site of CD21 markedly augment B cell activation initiated by Ag receptor ligation via a selective, c-fos-dependent signaling pathway.

Antibodies, Monoclonal↗
PubMed1997

CD99 (MIC2) regulates the LFA-1/ICAM-1-mediated adhesion of lymphocytes, and its gene encodes both positive and negative regulators of cellular adhesion.

Despite the fact that integrin-mediated lymphocyte adhesion is a crucial event for an appropriate immune response, little is known about the mechanisms that control the adhesion and deadhesion processes generated by the engagement of CD99 between various types of immune cells. Here we report that the CD99 gene encodes two distinct proteins with opposite functions in the LFA-1/intercellular adhesion molecule 1 (ICAM-1)-mediated cell adhesion process. The two forms of the CD99 protein are produced by alternative splicing of the CD99 gene transcript. The major form induced homotypic adhesion of the human B lymphoblastoid cell line IM-9, whereas the minor, truncated form inhibited the adhesion process. Activation of the major form of CD99 with anti-CD99 monoclonal antibodies induced rapid aggregation of IM-9 cells, which was blocked by the addition of mAbs to LFA-1 or intracellular adhesion molecule 1. Overexpression of the minor truncated form of CD99 markedly down-regulated the expression of LFA-1. The two forms of CD99 are differentially expressed in most human cells tested and are highly conserved in monkey. Taken together, these observations suggest that the two forms of CD99 function in vivo in both positive and negative regulation of LFA-1-mediated adhesion of lymphocytes during an immune response.

12E7 Antigen↗
PubMed2003

Characterization of the low pH solution structure and dynamics of the region 4 of Escherichia coli RNA polymerase sigma70 subunit.

Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and [(1)H]-(15)N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of sigma(70)(4) protein in its protonated state.

Amino Acid Sequence↗