The effect of tumour necrosis factor-α (TNF-α) muteins on human neutrophils in vitro

Tumour necrosis factor-α (TNF-α) has been implicated as an important inflammatory mediator. In vitro, TNF-α is reported to activate human polymorphonuclear neutrophils (PMN), inducing responses such as phagocytic activity, degranulation and oxidative metabolism. Biological responses to TNF-α are initiated by its binding to specific cell surface receptors, and various studies have shown that the major TNF receptor species on PMN is the 75 kDa receptor. To verify the suggestion that the receptor binding domain includes the region close to the N-terminus of the TNF-α molecule, four TNF-α derivatives termed muteins were constructed, using a synthetic cDNA fragment substituting the N-terminal 3–7 selected hydrophilic or hydrophobic amino acids in the original TNF-α genomic DNA. Binding of muteins to PMN was assessed using monoclonal antibodies recognizing either the 55 kDa (p55) or the 75 kDa (p75) TNF receptor subtypes. Blocking by muteins of anti-p75 antibody binding to PMN was as expected from their N-terminal amino acid composition and hydrophilic properties. Hydrophilic muteins competed well with anti-TNF receptor antibodies for binding to the p75 receptor. In contrast, hydrophobic muteins were unable to block anti-p75 binding. Similarly, degranulation, chemiluminescence or enhancement of the PMN response to specific stimuli by the muteins correlated with the hydrophilic properties of the muteins. The significance of these observations in relation to the molecular structure of TNF-α is discussed

Dam methyltransferase sites located within the loop region of the oligopurine-oligopyrimidine sequences capable of forming H-DNA are undermethylated in vivo.

Several derivatives of pUC18 plasmid were constructed that contained oligopurine-oligopyrimidine (pur-pyr) motifs surrounded by Dam methylation sites. Inserts of two of the molecules (pPP1 and pPP2) were able to adopt the triple-stranded conformation in vitro and show in vivo a remarkable undermethylation of specific sites when grown in JM105 dam+ strain. Mapping experiments revealed that undermethylated GATC sequences were located exclusively within the single-stranded loop region of the sequence involved in H-DNA formation. Control molecules which either contained the pur-pyr tracts (pPPK and pKK42) or not (pUC18) and were not able to form the triple-stranded conformation were found to be normally methylated by the dam gene product in vivo. Location of GATC within the triplex forming sequence seems to be a prerequisite for achieving its in vivo undermethylation. E.coli host factors are involved in the observed phenomenon. This has been deduced from the fact that the undermethylated state of pPP1 and pPP2 does not depend on the phase of growth of host cells and is steadily maintained up to 50 hours, whereas the kinetics of Dam methylation in vitro of sites located within the triplex loop does not differ substantially from the kinetics of methylation of other sites on the vector. Full methylation can be readily achieved in vitro. Additional factor(s) that operate in vivo to control the undermethylated state are most likely proteins since the observed effect can be suppressed by chloramphenicol administration to the cell cultures

SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS– strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences

Sticky DNA: self-association properties of long GAA.TTC repeats in R.R.Y triplex structures from Friedreich's ataxia.

A novel DNA structure, sticky DNA, is described for lengths of (GAA.TTC)n found in intron 1 of the frataxin gene of Friedreich's ataxia patients. Sticky DNA is formed by the association of two purine.purine.pyrimidine (R.R.Y) triplexes in negatively supercoiled plasmids at neutral pH. An excellent correlation was found between the lengths of (GAA.TTC) (> 59 repeats): first, in FRDA patients, second, required to inhibit transcription in vivo and in vitro, and third, required to adopt the sticky conformation. Fourth, (GAAGGA.TCCTTC)65, also found in intron 1, does not form sticky DNA, inhibit transcription, or associate with the disease. Hence, R.R.Y triplexes and/or sticky DNA may be involved in the etiology of FRDA.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.info:eu-repo/semantics/publishe

The effect of tumour necrosis factor-alpha (TNF-alpha) muteins on human neutrophils in vitro.

Tumour necrosis factor-alpha (TNF-alpha) has been implicated as an important inflammatory mediator. In vitro, TNF-alpha is reported to activate human polymorphonuclear neutrophils (PMN), inducing responses such as phagocytic activity, degranulation and oxidative metabolism. Biological responses to TNF-alpha are initiated by its binding to specific cell surface receptors, and various studies have shown that the major TNF receptor species on PMN is the 75 kDa receptor. To verify the suggestion that the receptor binding domain includes the region close to the N-terminus of the TNF-alpha molecule, four TNF-alpha derivatives termed muteins were constructed, using a synthetic cDNA fragment substituting the N-terminal 3-7 selected hydrophilic or hydrophobic amino acids in the original TNF-alpha genomic DNA. Binding of muteins to PMN was assessed using monoclonal antibodies recognizing either the 55 kDa (p55) or the 75 kDa (p75) TNF receptor subtypes. Blocking by muteins of anti-p75 antibody binding to PMN was as expected from their N-terminal amino acid composition and hydrophilic properties. Hydrophilic muteins competed well with anti-TNF receptor antibodies for binding to the p75 receptor. In contrast, hydrophobic muteins were unable to block anti-p75 binding. Similarly, degranulation, chemiluminescence or enhancement of the PMN response to specific stimuli by the muteins correlated with the hydrophilic properties of the muteins. The significance of these observations in relation to the molecular structure of TNF-alpha is discussed