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Biomedical subjects

A O Summers

Publications and source records attributed to A O Summers.

At least 37 records · Page 2Linked to original sources
PubMed1995

The dental amalgam mercury controversy--inorganic mercury and the CNS; genetic linkage of mercury and antibiotic resistances in intestinal bacteria.

Mercury (Hg) vapor exposure from dental amalgam has been demonstrated to exceed the sum of all other exposure sources. Therefore the effects of inorganic Hg exposure upon cell function in the brain and in the intestinal bacteria have recently been examined. In rats we demonstrate that ADP-ribosylation of tubulin and actin brain proteins is markedly inhibited, and that ionic Hg can thus alter a neurochemical reaction involved with maintaining neuron membrane structure. In monkeys we show that Hg, specifically from amalgam, will enrich the intestinal flora with Hg-resistant bacterial species which in turn also become resistant to antibiotics.

Animals↗
PubMed1995

Mercury exposure from "silver" tooth fillings: emerging evidence questions a traditional dental paradigm.

For more than 160 years dentistry has used silver amalgam, which contains approximately 50% Hg metal, as the preferred tooth filling material. During the past decade medical research has demonstrated that this Hg is continuously released as vapor into mouth air; then it is inhaled, absorbed into body tissues, oxidized to ionic Hg, and finally covalently bound to cell proteins. Animal and human experiments demonstrate that the uptake, tissue distribution, and excretion of amalgam Hg is significant, and that dental amalgam is the major contributing source to Hg body burden in humans. Current research on the pathophysiological effects of amalgam Hg has focused upon the immune system, renal system, oral and intestinal bacteria, reproductive system, and the central nervous system. Research evidence does not support the notion of amalgam safety.

Animals↗
PubMed1993

In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. I. Metalloregulatory protein MerR mutants.

Regulation of transcriptional initiation of the Tn21 mercury resistance (mer) operon occurs at the divergent promoter region lying between the structural genes (merTPCAD) and a regulatory gene (merR). During repression, both promoters are negatively regulated by MerR bound to a dyadic operator located between the -10 and -35 hexamers of PTPCAD. Upon Hg(II) induction, MerR activates transcription only from PTPCAD and continues to repress transcription from PR. Using in vivo dimethyl sulfate and KMnO4 footprinting of the merOP region of strains carrying wild-type MerR or MerR mutants, we have dissected the steps in MerR-mediated positive and negative regulation of the divergent mer promoters. The greater sensitivity of primer extension footprinting allowed the resolution of details previously undetectable in vivo. Two MerR mutants unable to bind merOP DNA allow RNA polymerase to form an open complex preferentially at PR. The intensity of the PR open complex is considerably less than that which occurs upon MerR-Hg(II) activation of PTPCAD and considerably greater than that which occurs at PTPCAD when MerR is deleted; this is the first in vivo estimate of the relative strengths of these two promoters. Although retaining the wild-type capacity to sequester RNA polymerase in the closed complex, the four MerR mutants defective in Hg(II) binding do not distort the dyad DNA or foster open complex formation when the inducer is added. Two activation-defective MerR mutants foster closed complex formation as well as wild-type, but they do not distort the dyad DNA or foster open complex formation when the inducer is added, although they are also able to bind Hg(II). Two semiconstitutive inducible MerR mutants differ from each other in that one (which lies nearer the COOH terminus) does distort the dyad center upon Hg(II) induction, whereas the other (which lies in near the center of merR) exhibits no dyad distortion upon induction. Paradoxically, despite their relatively strong semiconstitutive expression of mer-lac transcriptional fusions, neither of these mutants has a detectable open complex in the absence of added Hg(II).

Amino Acid Sequence↗
PubMed1993

In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. II. Repressor/activator (MerR)-RNA polymerase interaction with merOP mutants.

Transcription of the Tn21 mercury resistance (mer) operon is regulated by MerR which represses and activates the mer structural genes (merTPCAD) in the absence and presence of Hg(II), respectively. The promoter for the structural genes (PTPCAD) is divergently overlapped with the promoter for the regulatory gene (PR), and a dyadic operator lies between the -10 and -35 hexamers of PTPCAD. Using in vivo dimethyl sulfate and KMnO4 footprinting of mutant mer operator-promoter (merOP) DNA to observe MerR and RNA polymerase-mediated interactions with the merOP region, we have identified three distinct domains within the palindromic mer operator. Dyad domain I consists of the outermost bases on the left arm of the operator palindrome whose alteration causes a shift, but apparently not a major loss, in occupancy by MerR, and no decrease in RNA polymerase occupancy. Mutants in dyad domain I are semiconstitutive but support additional Hg(II)-induced open complex formation at PTPCAD. Dyad domain II consists of the four highly conserved inner bases ( ... GTAC ... GTAC ... ) of the seven-base interrupted dyad, alteration of which severely modifies both MerR and RNA polymerase contacts in the promoter region. Mutants in domain II generally allow constitutive open complex formation at PR. One unusual mutant of this group retains most of the wild-type dyad's ability to repress both promoters but is unable to support activation at PTPCAD in response to Hg(II), indicating that MerR undergoes a conformational change and that the required base contacts for activation are different than those for repression. Dyad domain III is tentatively defined by a mutant in the outermost base of the right palindrome arm which is unaffected in either MerR or RNA polymerase occupancy, however, a second lesion within the PTPCAD -10 hexamer of this mutant limits effective open complex formation. Other mutations lying solely within the -10 RNA polymerase recognition hexamer of PTPCAD are similarly competent in both MerR and RNA polymerase binding, but inadequate for open complex formation. One such mutant also affects the overlapping -10 hexamer of PR and results in reduced occupancy by both MerR and RNA polymerase, likely as a result of inefficient transcriptional initiation of merR mRNA. Finally, mutations affecting the -35 hexamer of PTPCAD bind MerR but not RNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacterial Proteins↗
PubMed1993

Mercury released from dental "silver" fillings provokes an increase in mercury- and antibiotic-resistant bacteria in oral and intestinal floras of primates.

In a survey of 640 human subjects, a subgroup of 356 persons without recent exposure to antibiotics demonstrated that those with a high prevalence of Hg resistance in their intestinal floras were significantly more likely to also have resistance to two or more antibiotics. This observation led us to consider the possibility that mercury released from amalgam ("silver") dental restorations might be a selective agent for both mercury- and antibiotic-resistant bacteria in the oral and intestinal floras of primates. Resistances to mercury and to several antibiotics were examined in the oral and intestinal floras of six adult monkeys prior to the installation of amalgam fillings, during the time they were in place, and after replacement of the amalgam fillings with glass ionomer fillings (in four of the monkeys). The monkeys were fed an antibiotic-free diet, and fecal mercury concentrations were monitored. There was a statistically significant increase in the incidence of mercury-resistant bacteria during the 5 weeks following installation of the amalgam fillings and during the 5 weeks immediately following their replacement with glass ionomer fillings. These peaks in incidence of mercury-resistant bacteria correlated with peaks of Hg elimination (as high as 1 mM in the feces) immediately following amalgam placement and immediately after replacement of the amalgam fillings. Representative mercury-resistant isolates of three selected bacterial families (oral streptococci, members of the family Enterobacteriaceae, and enterococci) were also resistant to one or more antibiotics, including ampicillin, tetracycline, streptomycin, kanamycin, and chloramphenicol. While such mercury- and antibiotic-resistant isolates among the staphylococci, the enterococci, and members of the family Enterobacteriaceae have been described, this is the first report of mercury resistance in the oral streptococci. Many of the enterobacterial strains were able to transfer mercury and antibiotic resistances together to laboratory bacterial recipients, suggesting that the loci for these resistances are genetically linked. Our findings indicate that mercury released from amalgam fillings can cause an enrichment of mercury resistance plasmids in the normal bacterial floras of primates. Many of these plasmids also carry antibiotic resistance, implicating the exposure to mercury from dental amalgams in an increased incidence of multiple antibiotic resistance plasmids in the normal floras of nonmedicated subjects.

Animals↗
PubMed1993

From genotype to phenotype: computer-based modeling of gene expression with STELLA II.

STELLA II is a microcomputer program that is designed to simulate and animate the dynamics generated by biological systems in a quantitative fashion. The program employs the Macintosh user interface and includes a tool kit for assembling models, plus menus for setting parameters and selecting input or output. Quantitative relationships among components may be expressed with user-defined equations, graphical functions or data from spreadsheets. System dynamics are simulated by running the program after defining initial conditions, and results may be viewed immediately using windows for graphical or tabular plots. The present article describes how STELLA II may be used to simulate gene expression as an adjunct to experimentation or student-directed learning.

Bacteria↗
PubMed1992

Synthesis and degradation of the mRNA of the Tn21 mer operon.

The mercury resistance locus encoded by Tn21 on the monocopy IncFII plasmid R100 (merTn21) consists of a metal-responsive activator/repressor, merR, which controls initiation of a polycistronic message that includes genes for the uptake (merTPC) and reduction (merA) of Hg2+ and merD, which may also play a minor regulatory role. Comparison of the relative abundance of the 5' and 3' ends of the merTPCAD transcript revealed a strong transcriptional gradient in the operon, consistent with previous observations of lower relative abundance of the more promoter-distal gene products. In vivo mRNA degradation rates varied only slightly for the different genes: however, the rates of mRNA synthesis varied considerably from the beginning to the end of the operon. Specifically, mRNA corresponding to the promoter-proximal genes, merTPC, achieved a maximum in vivo synthesis rate between 60 and 120 seconds after induction; this rate was maintained for approximately ten minutes. In contrast, the synthesis rates of mRNA corresponding to the promoter-distal genes merA and merD, were initially fivefold lower than the rates of the promoter-proximal genes for the first five minutes after induction, and then rose gradually to approximately 50% of the merTPC synthesis rates. These data suggested that early after induction only 20% of the transcripts initiating at merT proceed beyond merC. At later times after induction approximately 50% of the transcripts proceed beyond merC. Nuclease end mapping did not reveal any discrete termination events in the merPCA region, thus, premature termination may occur at many sites.

Base Sequence↗
PubMed1992

Roles of the Tn21 merT, merP, and merC gene products in mercury resistance and mercury binding.

The mercury resistance (mer) operon of the gram-negative transposon Tn21 encodes not only a mercuric reductase and regulatory genes but also two inner membrane proteins (MerT and MerC) and a periplasmic protein (MerP). Although the merT, merP, and merC genes have been implicated in Hg(II) transport, the individual roles of these genes have not been established. We created in vitro precise deletion and frameshift mutations that eliminated each of the genes singly and in combination. Our results show that both merT and merP are required for Hg(II) binding but that merC is not. Both merT and merP are required for full expression of Hg(II) resistance, but loss of merP is less deleterious than loss of merT. Furthermore, mutations eliminating both merT and merP decrease resistance more than the single mutations do. In contrast, mutating merC had no effect on Hg(II) resistance. Both the merT and merP mutations increase the threshold Hg(II) concentration for induction of merA-lacZ transcriptional fusions and cause an increase in the maximal expression level. In contrast, the merC mutation had little effect on the threshold inducing concentration of Hg(II) but decreased the level of expression. Our results show that merT and merP alone are sufficient to specify a mercury transport system. The role of merC remains obscure.

Amino Acid Sequence↗
PubMed1992

A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity.

We constructed mercury resistance operon-luciferase (mer-lux) transcriptional fusion plasmids to evaluate in vivo gene expression rates of the mer structural gene promoter (PTPCAD) of transposon Tn21. In vivo gene expression kinetics corresponded well with those previously determined in vitro, yielding an apparent K0.5 for Hg(II)-stimulated induction by MerR of 9.3 x 10(-8) M with the same ultrasensitive threshold effect seen in vitro. We also used the mer-lux fusions to elucidate subtle variations in promoter activity brought about by altered superhelicity. Binding of inducer [Hg(II)] to the transcriptional activator MerR is known to result in DNA distortion and transcriptional activation of the mer operon; it has recently been demonstrated that this distortion is a consequence of MerR-Hg(II)-induced local DNA unwinding to facilitate RNA polymerase open complex formation at PTPCAD. Since negative supercoiling results in DNA unwinding similar to this MerR activation, we hypothesized that a global increase in plasmid supercoiling would facilitate MerR-mediated activation and compromise MerR-mediated repression, while removal of plasmid supercoils would compromise MerR's ability to induce transcription and facilitate its ability to repress transcription. Indeed, we found that increased negative supercoiling results in increased gene expression rates and decreased supercoiling results in reduced gene expression rates for the induced, repressed, and derepressed conditions of PTPCAD. Thus, luciferase transcriptional fusions can detect subtle variations in initial rates of gene expression in a real-time, nondestructive assay.

Cloning, Molecular↗
PubMed1992

Genetic analysis of the Tn21 mer operator-promoter.

The mercury resistance operon, mer, of the transposon Tn21 is transcribed from two overlapping divergent promoters: PR for the regulatory gene, merR, and PTPCAD for the structural genes, merTPCAD. Transcription of merTPCAD is repressed in the absence of Hg(II) and activated in the presence of Hg(II) by the regulatory protein, MerR. In addition, MerR represses its own expression regardless of the presence of Hg(II). MerR binds as a dimer to a single region of dyad symmetry lying between the -35 and -10 hexamers of PTPCAD. Analysis of the expression of transcriptional fusions to hydroxylamine- and oligonucleotide-generated mutants of this divergent operator-promoter region identified key bases involved in MerR-dependent repression of PTPCAD and of PR and in activation of PTPCAD. Six of the seven mutants affecting the palindromic region were altered in their ability to bind the MerR protein in vitro as measured by fragment retardation assays. These differences in in vitro MerR binding correlated well with the in vivo measurements of repression or of activation. Bases identified as functionally relevant by this genetic analysis coincide extensively with those previously identified as relevant via in vivo footprinting. Four major points emerge from this analysis: (i) transition and transversion mutations within the spacer between the -10 and -35 hexamers of PTPCAD generally have little effect on the MerR-independent (i.e., unregulated) expression of either promoter; (ii) alteration of certain bases in the MerR-binding dyad affects repression of PTPCAD differently than repression of PR; (iii) certain dyad changes can impair activation of PTPCAD more severely than repression of this promoter; and (iv) mutations in the -10 hexamer of PTPCAD which also effect PR expression define one of two potential -10 hexamers in PR as actually functional in vivo.

Bacterial Proteins↗
PubMed1990

Activator-dependent preinduction binding of sigma-70 RNA polymerase at the metal-regulated mer promoter.

Expression of the Tn21 mercury-resistance (mer) locus is controlled by the merR gene product, which represses mer structural gene (merTPCAD) transcription in the absence of mercuric ion [Hg(II)] and activates it in the presence of Hg(II). In vivo DNA methylation of the mer regulatory region (merOP) shows that, with or without the inducer Hg(II), MerR strongly protects four guanine residues in a dyadic region located between the -10 and -35 hexamers of the structural gene promoter (PTPCAD). Prior to induction by Hg(II), RNA polymerase is also bound at PTPCAD; occupancy of the uninduced promoter by RNA polymerase is dependent on MerR. Methylation and permanganate footprinting demonstrate that induction by Hg(II) results in MerR/Hg(II)-dependent promoter DNA melting in the -10 region of PTPCAD and in additional DNA structural distortions within the region of dyad symmetry. Thus, MerR fosters the binding of RNA polymerase to an inactive promoter, and upon induction, MerR/Hg(II) facilitates DNA distortions suitable for efficient formation of the active transcription complex.

Base Sequence↗
PubMed1989

Transcriptional switching by the MerR protein: activation and repression mutants implicate distinct DNA and mercury(II) binding domains.

Bacterial resistance to mercuric compounds is controlled by the MerR metalloregulatory protein. The MerR protein functions as both a transcriptional repressor and a mercuric ion dependent transcriptional activator. Chemical mutagenesis of the cloned merR structural gene has led to the identification of mutant proteins that are specifically deficient in transcriptional repression, activation, or both. Five mutant proteins have been overproduced, purified to homogeneity, and assayed for ability to dimerize, bind mer operator DNA, and bind mercuric ion. A mutation in the recognition helix of a proposed helix-turn-helix DNA binding motif (E22K) yields protein deficient in both activation and repression in vivo (a-r-) and deficient in operator binding in vitro. In contrast, mutations in three of the four MerR cysteine residues are repression competent but activation deficient (a-r+) in vivo. In vitro, the purified cysteine mutant proteins bind to the mer operator site with near wild-type affinity but are variably deficient in binding the in vivo inducer mercury(II) ion. A subset of the isolated proteins also appears compromised in their ability to form dimers at low protein concentrations. These data, taken with the results in the preceding paper (Shewchuk et al., 1989), support a model in which DNA-bound MerR dimer binds one mercuric ion and transmits this occupancy information to a protein region involved in transcriptional activation.

Amino Acid Sequence↗
PubMed1989

Translation of merD in Tn21.

All four sequenced examples of the mercury resistance (mer) operon of gram-negative bacteria have a promoter-distal reading frame, merD, whose removal has little effect on the resistance phenotype and whose translation has not previously been observed. Using merD-lacZ protein fusions, we show that merD is translated. However, Hg(II)-induced merD expression, as measured by beta-galactosidase activity and immunoblotting, is 10- to 15-fold lower than that of fusions to the gene immediately preceding it, merA.

Cloning, Molecular↗
PubMed1989

Genetic analysis of transcriptional activation and repression in the Tn21 mer operon.

Transcription of the Tn21 mercury resistance operon (mer) is controlled by the toxic metal cation Hg(II). This control is mediated by the product of the merR gene, a 144-amino-acid protein which represses transcription of the structural genes (merTPCAD) in the absence of Hg(II) and activates transcription in the presence of Hg(II). We have used a mer-lac transcriptional fusion to obtain regulatory mutants in this metal-responsive system. Some mutants were defective in Hg(II)-induced activation while retaining repression function (a- r+), others were defective in repression but not activation (a+ r-), and some had lost both functions (a- r-). Mutations in three of the four cysteine residues of merR resulted in complete loss of Hg(II)-inducible activation but retention of the repressor function, suggesting that these residues serve as ligands for Hg(II) in the activation process. Other lesions adjacent to or very near these cysteines exhibited severely reduced activation and also retained repressor function. There were two putative helix-turn-helix (HTH) domains in merR, and mutants in each had very different phenotypes. A partially dominant mutation in the more amino-terminal region of the two putative HTH regions resulted in loss of both activation and repression (a- r-), consistent with a role for this region in DNA binding. Mutations in the more centrally located HTH region resulted only in loss of Hg(II)-induced activation (a- r+). Lesions in the central and in the carboxy-terminal regions of merR exhibited both Hg(II)-independent and Hg(II)-dependent transcriptional activation, suggesting that elements important in the activation mechanism may be widely distributed in this relatively small protein. The sole cis-acting mutant obtained with this operon fusion strategy, a down-promoter mutation, lies in a highly conserved base in the -35 region of the merTPCAD promoter.

Amino Acid Sequence↗
PubMed1988

The distribution and divergence of DNA sequences related to the Tn21 and Tn501 mer operons.

The mercury resistance (mer) operons of the Gram-negative bacterial transposons, Tn21 and Tn501, are phenotypically indistinguishable and have extensive DNA identity. However, Tn21 mer has an additional coding region (merC) in the middle of the operon which is lacking in Tn501 and there is also a discrete region of the mercuric ion reductase gene (merA) which differs markedly between the two operons. DNA fragment probes were used to determine the distribution of specific mer coding regions in two distinct collections of mercury-resistant (Hgr) Gram-negative bacteria. Colony blot hybridization analysis showed that merC-positive operons occur almost exclusively in Escherichia, although merC-negative operons can also be found in this genus. The merC-negative operons were found in Citrobacter, Klebsiella, and Enterobacter and in some Pseudomonas. Most of the Pseudomonas did not hybridize detectably with either of the two operons studied, indicating that they harbor an unrelated or more distantly related class of mercury resistance locus. Southern hybridization patterns demonstrated that the merC-positive mer operon is well conserved at the DNA level, whereas the merC-negative operons are much less conserved. The presence of merC also correlated with conservation of a specific variant region of the merA gene and with an antibiotic resistance pattern similar to that of Tn21. Tn501 appears to be an atypical example of the merC-negative subgroup of Hgr loci.

Blotting, Southern↗