scholarly journals In vitro demonstration of a particular affinity of glomerular basement membrane and collagen for DNA. A possible basis for a local formation of DNA-anti-DNA complexes in systemic lupus erythematosus.

1976 ◽  
Vol 144 (2) ◽  
pp. 428-443 ◽  
Author(s):  
S Izui ◽  
P H Lambert ◽  
P A Miescher
Keyword(s):  
Lupus Erythematosus ◽  
Biological Significance ◽  
Basement Membranes ◽  
Basic Site ◽  
Binding Property ◽  
Local Formation ◽  
Dna Antibodies ◽  
Low Ionic Strength

In vitro, collagen and collagen-like material in GBM, were demonstrated to have a particular high affinity for any DNA tested (mammalian, bacterial, viral, and plant). GBM fixed DNA 40-80 times more than HGG and BSA and 10-40 times more than bacterial LPS. GBM has a higher affinity for SSDNA than for DSDNA. This binding was inhibited at low pH, low ionic strength, and in the presence of anionic detergents, indicating that the highly negatively charged DNA may interact with the basic site on collagen or GBM by electrostatic forces. This interaction was competitively interfered with by DNA-binding proteins such as Clq. Complexes formed of DNA and anti-DNA antibodies did not exhibit the same binding property as free DNA. However, DNA which was already bound to GBM or to collagen could very efficiently bind anti-DNA antibodies and form immune complexes which would remain on these structures. The biological significance of the binding of DNA to GBM or to collagen should be particularly considered in relation to the pathogenesis of SLE. It is possible that DNA released from disrupted or degenerating cells would bind to surrounding collagen fibers or to basement membranes and then act as an immunoabsorbant for circulating anti-DNA antibodies. Some evidence for an in vivo binding of SSDNA to renal structures was obtained in mice treated with bacterial LPS 2 days before the injection of SSDNA.

scholarly journals Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

1996 ◽  
Vol 313 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Françoise LEVAVASSEUR ◽  
Jocelyne LIÉTARD ◽  
Kohei OGAWA ◽  
Nathalie THÉRET ◽  
Peter D. BURBELO ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Basement Membranes ◽  
Major Protein ◽  
Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

The effects of photodynamic therapy with Photodithazine on HPV 16 E6/E7 associated cervical cancer model

2011 ◽  
Vol 15 (03) ◽  
pp. 174-180 ◽  
Author(s):  
Lan Ying Wen ◽  
Su-Mi Bae ◽  
Jin Hwan Do ◽  
Kye-Shin Park ◽  
Woong Shick Ahn
Keyword(s):  
Cervical Cancer ◽  
Photodynamic Therapy ◽  
Growth Inhibition ◽  
Biological Significance ◽  
Light Irradiation ◽  
Tumor Growth Inhibition ◽  
Cancer Model ◽  
Hpv 16

Photodynamic therapy (PDT) is a promising treatment for cancer that has been recently accepted in the clinic. In this study, we examined a biological significance of PDT with a chlorin-based photosensitizer, Photodithazine, on cervical cancer model. When human papillomavirus type 16 (HPV16)- transformed mouse TC-1 cells were exposed to varied doses of Photodithazine with light irradiation (6.25 J/cm2), the significant growth inhibition of TC-1 cells was observed at 0.75 μg/mL of Photodithazine. The damaged cells by Photodithazine/PDT were categorized to be early and late apoptosis, as determined by annexin V staining. Photodithazine was primarily localized at lysosome apparatus within TC-1 cells while it was rapidly accumulated and sustained for initial 3 h in tumor tissue of TC-1 tumor bearing mice after IV injection. The tumor growth inhibition by Photodithazine/PDT with light irradiation (300 J/cm2) was examined after injection of various concentration of Photodithazine in tumor mice system. Our results show that Photodithazine/PDT might have significant advantages in the selective killing of tumor lesions in HPV 16 E6/E7 associated cervical cancer model, both in vitro and in vivo.

scholarly journals ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

2005 ◽  
Vol 79 (20) ◽  
pp. 12721-12731 ◽  
Author(s):  
Ákos Putics ◽  
Witold Filipowicz ◽  
Jonathan Hall ◽  
Alexander E. Gorbalenya ◽  
John Ziebuhr
Keyword(s):  
Active Site ◽  
Biological Significance ◽  
Virus Titer ◽  
Site Directed Mutagenesis ◽  
Replicase Gene ◽  
Active Site Residues ◽  
Nonstructural Proteins ◽  
Trna Splicing

ABSTRACT Replication of the ∼30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

PTS 50: Past, Present and Future, or Diauxie Revisited

2015 ◽  
Vol 25 (2-3) ◽  
pp. 79-93 ◽  
Author(s):  
Joseph W. Lengeler
Keyword(s):  
Catabolite Repression ◽  
Biological Significance ◽  
Cellular Growth ◽  
Gram Positive ◽  
Gram Negative ◽  
Inducer Exclusion ◽  
Gram Positive Bacteria ◽  
Cellular Control

<b><i>Past:</i></b> The title ‘PTS 50 or The PTS after 50 years' relies on the first description in 1964 of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS) by Kundig, Gosh and Roseman [Proc Natl Acad Sci USA 1964;52:1067-1074]. The system comprised proteins named Enzyme I, HPr and Enzymes II, as part of a novel PTS for carbohydrates in Gram-negative and Gram-positive bacteria, whose ‘biological significance remained unclear'. In contrast, studies which would eventually lead to the discovery of the central role of the PTS in bacterial metabolism had been published since before 1942. They are primarily linked to names like Epps and Gale, J. Monod, Cohn and Horibata, and B. Magasanik, and to phenomena like ‘glucose effects', ‘diauxie', ‘catabolite repression' and carbohydrate transport. <b><i>Present:</i></b> The pioneering work from Roseman's group initiated a flood of publications. The extraordinary progress from 1964 to this day in the qualitative and in vitro description of the genes and enzymes of the PTS, and of its multiple roles in global cellular control through ‘inducer exclusion', gene induction and ‘catabolite repression', in cellular growth, in cell differentiation and in chemotaxis, as well as the differences of its functions between Gram-positive and Gram-negative bacteria, was one theme of the meeting and will not be treated in detail here. <b><i>Future:</i></b> At the 1988 Paris meeting entitled ‘The PTS after 25 years', Saul Roseman predicted that ‘we must describe these interactions [of the PTS components] in a quantitative way [under] in vivo conditions'. I will present some results obtained by our group during recent years on the old phenomenon of diauxie by means of very fast and quantitative tests, measured in vivo, and obtained from cultures of isogenic mutant strains growing under chemostat conditions. The results begin to hint at the problems relating to future PTS research, but also to the ‘true science' of Roseman.

scholarly journals Post-embedding colloidal gold immunolocalization of laminin to the lamina rara interna, lamina densa, and lamina rara externa of renal glomerular basement membranes.

1986 ◽  
Vol 34 (7) ◽  
pp. 847-853 ◽  
Author(s):  
D R Abrahamson
Keyword(s):  
Colloidal Gold ◽  
Basement Membranes ◽  
Lamina Densa ◽  
Thin Sections ◽  
Gold Particles ◽  
Rabbit Igg ◽  
Gold Immunolocalization ◽  
Lowicryl K4m

Ultrastructural distribution of laminin within renal glomerular (GBM) and tubular basement membranes (TBM) was investigated using post-embedding immunolocalization with colloidal gold. Rat kidneys were fixed with 4% formaldehyde and embedded at 4 degrees C in Lowicryl K4M medium. Thin sections were then sequentially treated with affinity-purified rabbit anti-laminin IgG and anti-rabbit IgG conjugated to 10 nm diameter colloidal gold. Gold bound specifically to the GBM and TBM with particle densities of 690/micron2 and 731/micron2, respectively. In the GBM, the number of gold particles bound/micron2 of lamina densa greater than lamina rara externa greater than lamina rara interna. Closely similar binding patterns were found when kidneys were fixed with 0.5% glutaraldehyde plus 3% formaldehyde and embedded at 60 degrees C in L.R. White resin, but slightly less gold bound to sections overall than that seen with formaldehyde alone and Lowicryl. Taken together, these results illustrate that anti-laminin IgG, whether applied to fixed sections in vitro or introduced in vivo, bound to the lamina rara interna, lamina densa, and lamina rara externa of the GBM and throughout the TBM.

scholarly journals Characterization of a Chlamydia psittaciDNA Binding Protein (EUO) Synthesized during the Early and Middle Phases of the Developmental Cycle

1998 ◽  
Vol 66 (3) ◽  
pp. 1167-1173 ◽  
Author(s):  
Li Zhang ◽  
Annemarie L. Douglas ◽  
Thomas P. Hatch
Keyword(s):  
Promoter Region ◽  
Chlamydia Psittaci ◽  
Host Cells ◽  
Upstream Open Reading Frame ◽  
Developmental Cycle ◽  
Binding Property ◽  
Logarithmic Growth Phase ◽  
Reading Frame

ABSTRACT The EUO gene (for early upstream open reading frame) ofChlamydia psittaci was previously found to be transcribed better at 1 than at 24 h postinfection. We found that the EUO gene encodes a minor protein that is expressed within 1 h of infection of host cells with C. psittaci 6BC but that protein quantity peaks during the logarithmic growth phase of reticulate bodies (RBs), declines late in the infection (after 20 h) when RBs reorganize into elementary bodies (EBs), and is absent in infectious EBs. EUO protein lacks homology to known proteins but does contain a putative helix-turn-helix motif. We found that recombinant EUO binds to DNA in vitro with a relatively broad specificity. Using the bp −200 to +67 promoter region of the cysteine-rich envelope protein (crp) operon as a model, we show that EUO protein preferentially binds to AT-rich sequences and protects crpDNA from DNase I from approximately bp −60 to −9. We also found that native EUO protein in extracts of RBs binds to the promoter region of the crp operon, demonstrating that the DNA binding property of EUO protein is not an artifact of recombinant methods. Although EUO protein appears to bind to the crp operon with high affinity in vitro (Kd of about 15 nM), it is not known whether the protein binds the crp DNA in vivo.

scholarly journals Granulocyte aggregometry: a sensitive technique for the detection of C5a and complement activation

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 898-902 ◽  
Author(s):  
DE Hammerschmidt ◽  
TK Bowers ◽  
CJ Lammi-Keefe ◽  
HS Jacob ◽  
PR Craddock
Keyword(s):  
Lupus Erythematosus ◽  
Cytochalasin B ◽  
Normal Subjects ◽  
Standard Methods ◽  
Sensitive Technique ◽  
Fresh Serum ◽  
Induced Aggregation ◽  
Transfusion Reactions

Abstract We have previously shown that complement (C) activated plasma causes granulocyte (PMN) aggregation in vitro and that C5a is responsible. The C-induced aggregation of PMNs treated with cytochalasin-B (CB) is markedly enhanced and irreversible, and the magnitude of the response is proportional to the log (concentration of activated plasma), allowing use of this technique to detect C5a and hence C-activation. To compare the sensitivity of granulocyte aggregometry to that of more standard methods of detecting C-activation, we produced graded C- activation in vitro by treating fresh serum with varying amounts of zymosan. Aggregometry was the most sensitive index of C-activation, detecting C-activation, produced by 0.02 mg zymosan/ml of serum--1/10 that required to produce C-activation detectable by C3 immunoelectrophoresis (the next most sensitive technique). Granulocyte aggregometry may also be used to detect in vivo C-activation. We have found aggregating activity in plasmas from patients with systemic lupus erythematosus, immune vasculitis, transfusion reactions, and other conditions associated with in vivo C-activation, but not in the plasmas of normal subjects.

scholarly journals Linc00426 accelerates lung adenocarcinoma progression by regulating miR-455-5p as a molecular sponge

2020 ◽  
Vol 11 (12) ◽  
Author(s):  
Hongli Li ◽  
Qingjie Mu ◽  
Guoxin Zhang ◽  
Zhixin Shen ◽  
Yuanyuan Zhang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Molecular Mechanisms ◽  
Epithelial Mesenchymal Transition ◽  
Ectopic Expression ◽  
Tumor Development ◽  
Biological Significance ◽  
Mesenchymal Transition

AbstractIncreasing lines of evidence indicate the role of long non-coding RNAs (LncRNAs) in gene regulation and tumor development. Hence, it is important to elucidate the mechanisms of LncRNAs underlying the proliferation, metastasis, and invasion of lung adenocarcinoma (LUAD). We employed microarrays to screen LncRNAs in LUAD tissues with and without lymph node metastasis and revealed their effects on LUAD. Among them, Linc00426 was selected for further exploration in its expression, the biological significance, and the underlying molecular mechanisms. Linc00426 exhibits ectopic expression in LUAD tissues and cells. The ectopic expression has been clinically linked to tumor size, lymphatic metastasis, and tumor differentiation of patients with LUAD. The deregulation of Linc00426 contributes to a notable impairment in proliferation, invasion, metastasis, and epithelial–mesenchymal transition (EMT) in vitro and in vivo. Mechanistically, the deregulation of Linc00426 could reduce cytoskeleton rearrangement and matrix metalloproteinase expression. Meanwhile, decreasing the level of Linc00426 or increasing miR-455-5p could down-regulate the level of UBE2V1. Thus, Linc00426 may act as a competing endogenous RNA (ceRNA) to abate miR-455-5p-dependent UBE2V1 reduction. We conclude that Linc00426 accelerates LUAD progression by acting as a molecular sponge to regulate miR-455-5p, and may be a potential novel tumor marker for LUAD.

scholarly journals Quantification and characterization of the 5’exonuclease activity of the lysosomal nuclease PLD3 by a novel cell-based assay

2020 ◽  
pp. jbc.RA120.015867
Author(s):  
Cedric Cappel ◽  
Adriana Carolina Gonzalez ◽  
Markus Damme
Keyword(s):  
Lupus Erythematosus ◽  
Specific Activity ◽  
Nuclease Activity ◽  
Hydrolytic Activity ◽  
Proteolytic Processing ◽  
Exonuclease Activity ◽  
Fluorescence Quencher

Phospholipase D3 (PLD3) and phospholipase D4 (PLD4), the most recently described lysosomal nucleases, are associated with Alzheimer`s disease, spinocerebellar ataxia, and systemic lupus erythematosus. They exhibit 5’ exonuclease activity on single-stranded DNA, hydrolyzing it at the acidic pH associated with the lysosome. However, their full cellular function is inadequately understood. To examine these enzymes, we developed a robust and automatable cell-based assay based on fluorophore- and fluorescence-quencher coupled oligonucleotides for the quantitative determination of acidic 5’ exonuclease activity. We validated the assay under knockout and PLD-overexpression conditions, and then applied it to characterize PLD3 and PLD4 biochemically. Our experiments revealed PLD3 as the principal acid 5’ exonuclease in HeLa cells, where it showed a markedly higher specific activity compared to PLD4. We further used our newly developed assay to determine the substrate specificity and inhibitory profile of PLD3, and found that proteolytic processing of PLD3 is dispensable for its hydrolytic activity. We followed the expression, proteolytic processing, and intracellular distribution of genetic PLD3 variants previously associated with Alzheimer’s disease and investigated each variant's effect on the 5’ nuclease activity of PLD3, finding that some variants lead to reduced activity, but others not. The development of a PLD3/4-specific biochemical assay will be instrumental in understanding better both nucleases and their incompletely unknown roles in vitro and in vivo.

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