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1.  Escherichia coli OxyR protein represses the unmethylated bacteriophage Mu mom operon without blocking binding of the transcriptional activator C. 
Nucleic Acids Research  1996;24(20):4042-4049.
Transcription of the bacteriophage Mu mom operon requires transactivation by the phage-encoded C protein. DNase I footprinting showed that in the absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect to the transcription start site), on the top (non-transcribed) strand. This is slightly upstream from, but overlapping P1 (-49 to +16), the functional binding site for rightward transcription. Host DNA-[N6-adenine] methyltransferase (Dam) methylation of three GATCs immediately upstream of the C binding site is required to prevent binding of the E.coli OxyR protein, which represses mom transcription in dam- strains. OxyR, known to induce DNA bending, is normally in a reduced conformation in vivo, but is converted to an oxidized state under standard in vitro conditions. Using DNase I footprinting, we provide evidence supporting the proposal that the oxidized and reduced forms of OxyR interact differently with their target DNA sequences in vitro. A mutant form, OxyR-C199S, was shown to be able to repress mom expression in vivo in a dam- host. In vitro DNase I footprinting showed that OxyR-C199S protected Pmom from -104 to -46 on the top strand and produced a protection pattern characteristic of reduced wild-type OxyR. Prebinding of OxyR-C199S completely blocked RNAP binding to P2 (in the absence of C), whereas it only slightly decreased binding of C to its target site (-55 to -28, as defined by DNase I footprinting). In contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to P1. These results indicate that OxyR repression is mediated subsequent to binding by C. Mutations have been isolated that relieve the dependence on C activation and have the same transcription start site as the C-activated wild-type promoter. One such mutant, tin7, has a single base change at -14, which changes a T6 run to T3GT2. OxyR-C199S partially inhibited RNAP binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1 binding sites probably do not overlap, and in vivo expression of tin7 was reduced 5- to 10-fold in dam- cells. These results suggest that OxyR can repress tin7.
PMCID: PMC146201  PMID: 8918810
2.  Unusual Modification of Bacteriophage Mu DNA 
Journal of Virology  1979;32(2):468-475.
Bacteriophage Mu DNA was labeled after induction in the presence of [2-3H]adenine or [8-3H]adenine. Both Mu mom+·dam+ DNA and Mu mom−·dam+ DNA have similar N6-methyladenine (MeAde) contents, as well as similar frequencies of MeAde nearest neighbors. Both DNAs are sensitive to in vitro cleavage by R·DpnI but resistant to cleavage by R·DpnII. These results indicate that the mom+ protein does not alter the sequence specificity of the host dam+ methylase to produce MeAde at new sites. However, we have discovered a new modified base, denoted Ax, in Mu mom+·dam+ DNA; approximately 15% of the adenine residues are modified to Ax. Although the precise nature of the modification is not yet defined, analysis by electrophoresis and chromatography indicates that the N6-amino group is not the site of modification, and that the added moiety contains a free carboxyl group. Ax is not present in Mu mom+·dam+ or Mu mom−·dam+ phage DNA or in cellular DNA from uninduced Mu mom+·dam+ lysogens. These results suggest that expression of the dam+ and mom+ genes are required for the Ax modification and that this modification is responsible for protecting Mu DNA against certain restriction nucleases. Mu mom+·dam− DNA and Mu mom−·dam− DNA contain a very low level of MeAde (ca. 1 MeAde per 5,000 adenine residues). Since the only nearest neighbor to MeAde appears to be cytosine, we suggest that the methylated sequence is 5′... C-A*-C... 3′ and that this methylation is mediated by the EcoK modification enzyme.
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PMCID: PMC353578  PMID: 159363
3.  Role of bacteriophage Mu C protein in activation of the mom gene promoter. 
Journal of Bacteriology  1989;171(4):2019-2027.
The phage Mu C gene product is a specific activator of Mu late gene transcription, including activation of the mom operon. Fusion of the C gene to the efficient translation initiation region of the Escherichia coli atpE gene allowed significant overproduction of C protein, which was subsequently purified and assayed for DNA binding by gel retardation and nuclease footprinting techniques. C protein binds to a site immediately upstream of the -35 region both of the mom promoter and the related phage D108 mod promoter. The location of the mom promoter has been determined by primer extension. Upstream deletions extending more than 3 base pairs into the C-binding site abolished activation of the mom promoter in vivo. In vitro binding of C was not significantly affected by DNA methylation. A second, C-dependent promoter was identified just downstream of the C coding region; comparison with the mom promoter revealed common structural elements.
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PMCID: PMC209852  PMID: 2522924
4.  Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli. 
Journal of Bacteriology  1991;173(5):1789-1800.
Transcription of the pyelonephritis-associated pilus (pap) operon of Escherichia coli is subject to regulation by a phase variation control mechanism in which the pap pilin gene alternates between transcriptionally active (phase-on) and inactive (phase-off) states. Pap phase variation appears to involve differential inhibition of deoxyadenosine methylase (Dam) methylation of two pap GATC sites, GATC1028 and GATC1130, located in the regulatory region upstream of the papBA promoter. DNA from phase-on cells contains an unmethylated adenosine in the GATC1028 site, whereas DNA from phase-off cells contains an unmethylated adenosine in the GATC1130 site. papI and papB are two regulatory genes in the pap operon. Analysis of pap deletion mutants suggests that papI is required for methylation inhibition at the GATC1028 site; however, neither papI nor papB is required for inhibition of methylation at the GATC1130 site. We have identified a chromosomal locus, mbf (methylation-blocking factor), that is required for methylation protection of both the pap GATC1028 and GATC1130 sites. The mbf locus was identified after transposon mTn10 mutagenesis and mapped to 19.6 min on the E. coli chromosome. The effect of transposon mutations within mbf on pap pilin transcription was determined by using a papBAp-lac operon fusion which places lacZ under control of the papBA promoter. E. coli containing mbf::mTn10 and phase-off mbf+ E. coli cells both expressed beta-galactosidase levels about 30-fold lower than the beta-galactosidase level measured for phase-on mbf+ E. coli cells. These results indicated that mbf was necessary for pap pilin transcription and were supported by Northern (RNA) blotting and primer extension analyses. Moreover, transposon insertion within mbf greatly reduced Pap pilus expression. The mbf locus was isolated on a low-copy-number cosmid, pMBF1. Complementation analysis indicated that each of seven mbf::mTn10 mutants isolated contained a transposon insertion within the same gene or operon. The identification of the mbf locus, required for pap transcription, supports the hypothesis that pap phase variation is controlled by a mechanism involving alternation between different methylation states.
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PMCID: PMC207331  PMID: 1671857
5.  Divergent Evolution of CHD3 Proteins Resulted in MOM1 Refining Epigenetic Control in Vascular Plants 
PLoS Genetics  2008;4(8):e1000165.
Arabidopsis MOM1 is required for the heritable maintenance of transcriptional gene silencing (TGS). Unlike many other silencing factors, depletion of MOM1 evokes transcription at selected loci without major changes in DNA methylation or histone modification. These loci retain unusual, bivalent chromatin properties, intermediate to both euchromatin and heterochromatin. The structure of MOM1 previously suggested an integral nuclear membrane protein with chromatin-remodeling and actin-binding activities. Unexpected results presented here challenge these presumed MOM1 activities and demonstrate that less than 13% of MOM1 sequence is necessary and sufficient for TGS maintenance. This active sequence encompasses a novel Conserved MOM1 Motif 2 (CMM2). The high conservation suggests that CMM2 has been the subject of strong evolutionary pressure. The replacement of Arabidopsis CMM2 by a poplar motif reveals its functional conservation. Interspecies comparison suggests that MOM1 proteins emerged at the origin of vascular plants through neo-functionalization of the ubiquitous eukaryotic CHD3 chromatin remodeling factors. Interestingly, despite the divergent evolution of CHD3 and MOM1, we observed functional cooperation in epigenetic control involving unrelated protein motifs and thus probably diverse mechanisms.
Author Summary
Epigenetic regulation of transcription usually involves changes in histone modifications, as well as DNA methylation changes in plants and mammals. Previously, we found an exceptional epigenetic regulator in Arabidopsis, MOM1, acting independently of these epigenetic marks. Interestingly, MOM1 controls loci associated with bivalent chromatin marks, intermediate to active euchromatin and silent heterochromatin. Such bivalent marks are often associated with newly inserted and/or potentially active transposons, silent transgenes, and certain chromosomal loci. Notably, bivalent chromatin seems to be characteristic for embryonic stem cells, where such loci change their activity and determination of epigenetic marks during cell differentiation. Here, we provide evidence that in vascular plants, the MOM1-like proteins evolved from the ubiquitous eukaryotic chromatin remodeling factor CHD3. The domains necessary for CHD3 function degenerated in MOM1, became dispensable for its gene silencing activity, and were replaced by a novel, unrelated domain providing silencing function. Therefore, MOM1-like proteins use a different silencing mechanism compared to the ancestral CHD3s. In spite of this divergent evolution, CHD3 and MOM1 seem to retain a functional cooperation in control of transcriptionally silent loci. Our results provide an unprecedented example of an evolutionary path for epigenetic components resulting in increased complexity of an epigenetic regulatory network characteristic for multicellular eukaryotes.
doi:10.1371/journal.pgen.1000165
PMCID: PMC2507757  PMID: 18725928
6.  Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes 
PLoS Biology  2012;10(9):e1001394.
Bax promotes mitochondrial permeabilization during apoptosis via a phase-transition-like event in the membrane and oligomerization of a catalyst molecule that facilitates Bax pore formation.
Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.
Author Summary
Mitochondria are the key energy-producing structures inside cells, but are also crucial players in a common form of programmed cell death, apoptosis. A critical event in mitochondrion-driven apoptosis involves the formation of large pores in the mitochondrial outer membrane (MOM). These pores cause long-term damage to mitochondria and also allow mitochondrial proteins to escape and accelerate cell death. Previous studies have revealed that the protein Bax when activated can form pores in protein-free membranes and that it, along with Bak, is involved in the formation of mitochondrial pores, but the process remains unclear. We now show, however, that in naturally derived MOMs, Bax is assisted by another resident MOM protein, which we term the “catalyst,” and whose identity is still unknown. The mechanism involves two distinct stages. First, activated Bax activates the catalyst protein, causing multiple catalyst molecules to assemble into a larger structure (a complex). In the second stage, this catalyst complex in turn facilitates Bax-driven pore formation. Our data also reveal some unexpected details of the pore formation process; in particular, it appears that catalyst activation involves a physical change in the molecular arrangement of the membrane. Furthermore, contrary to what was previously assumed, pore formation does not require Bax molecules themselves to assemble together into larger complexes.
doi:10.1371/journal.pbio.1001394
PMCID: PMC3457932  PMID: 23049480
7.  A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development 
Genome Biology  2008;9(12):R182.
An extended ENU screen for modifiers of transgene variegation identified four new modifiers, MommeD7-D10.
Background
Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.
Results
We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.
Conclusions
These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.
doi:10.1186/gb-2008-9-12-r182
PMCID: PMC2646286  PMID: 19099580
8.  The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins. 
Molecular and Cellular Biology  1995;15(6):3382-3389.
A multisubunit complex in the mitochondrial outer membrane is responsible for targeting and membrane translocation of nuclear-encoded preproteins. This receptor complex contains two import receptors, a general insertion pore and the protein Mom22. It was unknown if Mom22 directly interacts with preproteins, and two views existed about the possible functions of Mom22: a central role in transfer of preproteins from both receptors to the general insertion pore or a more limited function dependent on the presence of the receptor Mom19. For this report, we identified and cloned Saccharomyces cerevisiae MOM22 and investigated whether it plays a direct role in targeting of preproteins. A preprotein accumulated at the mitochondrial outer membrane was cross-linked to Mom22. The cross-linking depended on the import stage of the preprotein. Overexpression of Mom22 suppressed the respiratory defect of yeast cells lacking Mom19 and increased preprotein import into mom19 delta mitochondria, demonstrating that Mom22 can function independently of Mom19. Overexpression of Mom22 even suppressed the lethal phenotype of a double deletion of the two import receptors known so far (mom19 delta mom72 delta). Deletion of the MOM22 gene was lethal for yeast cells, identifying Mom22 as one of the few mitochondrial membrane proteins essential for fermentative growth. These results suggest that Mom22 plays an essential role in the mitochondrial receptor complex. It directly interacts with preproteins in transit and can perform receptor-like activities.
PMCID: PMC230572  PMID: 7760834
9.  Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus’ molecule 1 protein 
In order to investigate its function in transcriptional gene silencing, the highly conserved motif 2 from A. thaliana Morpheus’ molecule 1 protein was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 3.2 Å.
Of the known epigenetic control regulators found in plants, the Morpheus’ molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to ∼3.2 Å resolution. They belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = 85.64, c = 292.74 Å. Structure determination is ongoing.
doi:10.1107/S1744309110021068
PMCID: PMC2917290  PMID: 20693667
Morpheus’ molecule 1; conserved MOM1 motif 2; coiled-coil domain; epigenetic; transcriptional gene silencing
10.  Formation of DNA Methylation Patterns: Nonmethylated GATC Sequences in gut and pap Operons 
Journal of Bacteriology  1998;180(22):5913-5920.
Most of the adenine residues in GATC sequences in the Escherichia coli chromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) to pap DNA was sufficient to protect the two GATC sequences in the pap regulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44.5) centered 44.5 bp upstream of the transcription start site of the gutABD operon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44.5-containing region of the gutABD operon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated that gutABD gene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of the pap GATC-I and GATC-II sites.
PMCID: PMC107665  PMID: 9811649
11.  Metal-on-Metal Total Hip Resurfacing Arthroplasty 
Executive Summary
Objective
The objective of this review was to assess the safety and effectiveness of metal on metal (MOM) hip resurfacing arthroplasty for young patients compared with that of total hip replacement (THR) in the same population.
Clinical Need
Total hip replacement has proved to be very effective for late middle-aged and elderly patients with severe degenerative diseases of the hips. As indications for THR began to include younger patients and those with a more active life style, the longevity of the implant became a concern. Evidence suggests that these patients experience relatively higher rates of early implant failure and the need for revision. The Swedish hip registry, for example, has demonstrated a survival rate in excess of 80% at 20 years for those aged over 65 years, whereas this figure was 33% by 16 years in those aged under 55 years.
Hip resurfacing arthroplasty is a bone-conserving alternative to THR that restores normal joint biomechanics and load transfer. The technique has been used around the world for more than 10 years, specifically in the United Kingdom and other European countries.
The Technology
Metal-on-metal hip resurfacing arthroplasty is an alternative procedure to conventional THR in younger patients. Hip resurfacing arthroplasty is less invasive than THR and addresses the problem of preserving femoral bone stock at the initial operation. This means that future hip revisions are possible with THR if the initial MOM arthroplasty becomes less effective with time in these younger patients. The procedure involves the removal and replacement of the surface of the femoral head with a hollow metal hemisphere, which fits into a metal acetabular cup.
Hip resurfacing arthroplasty is a technically more demanding procedure than is conventional THR. In hip resurfacing, the femoral head is retained, which makes it much more difficult to access the acetabular cup. However, hip resurfacing arthroplasty has several advantages over a conventional THR with a small (28 mm) ball. First, the large femoral head reduces the chance of dislocation, so that rates of dislocation are less than those with conventional THR. Second, the range of motion with hip resurfacing arthroplasty is higher than that achieved with conventional THR.
A variety of MOM hip resurfacing implants are used in clinical practice. Six MOM hip resurfacing implants have been issued licences in Canada.
Review Strategy
A search of electronic bibliographies (OVID Medline, Medline In-Process and Other Non-Indexed Citations, Embase, Cochrane CENTRAL and DSR, INAHTA) was undertaken to identify evidence published from Jan 1, 1997 to October 27, 2005. The search was limited to English-language articles and human studies. The literature search yielded 245 citations. Of these, 11 met inclusion criteria (9 for effectiveness, 2 for safety).
The result of the only reported randomized controlled trial on MOM hip resurfacing arthroplasty could not be included in this assessment, because it used a cemented acetabular component, whereas in the new generation of implants, a cementless acetabular component is used. After omitting this publication, only case series remained.
Summary of Findings
 
Health Outcomes
The Harris hip score and SF-12 are 2 measures commonly used to report health outcomes in MOM hip resurfacing arthroplasty studies. Other scales used are the Oxford hip score and the University of California Los Angeles hip score.
The case series showed that the mean revision rate of MOM hip resurfacing arthroplasty is 1.5% and the incidence of femoral neck fracture is 0.67%. Across all studies, 2 cases of osteonecrosis were reported. Four studies reported improvement in Harris hip scores. However, only 1 study reported a statistically significant improvement. Three studies reported improvement in SF-12 scores, of which 2 reported a significant improvement. One study reported significant improvement in UCLA hip score. Two studies reported postoperative Oxford hip scores, but no preoperative values were reported.
None of the reviewed studies reported procedure-related deaths. Four studies reported implant survival rates ranging from 94.4% to 99.7% for a follow-up period of 2.8 to 3.5 years. Three studies reported on the range of motion. One reported improvement in all motions including flexion, extension, abduction-adduction, and rotation, and another reported improvement in flexion. Yet another reported improvement in range of motion for flexion abduction-adduction and rotation arc. However, the author reported a decrease in the range of motion in the arc of flexion in patients with Brooker class III or IV heterotopic bone (all patients were men).
Safety of Metal-on-Metal Hip Resurfacing Arthroplasty
There is a concern about metal wear debris and its systemic distribution throughout the body. Detectable metal concentrations in the serum and urine of patients with metal hip implants have been described as early as the 1970s, and this issue is still controversial after 35 years.
Several studies have reported high concentration of cobalt and chromium in serum and/or urine of the patients with metal hip implants. Potential toxicological effects of the elevated metal ions have heightened concerns about safety of MOM bearings. This is of particular concern in young and active patients in whom life expectancy after implantation is long.
Since 1997, 15 studies, including 1 randomized clinical trial, have reported high levels of metal ions after THR with metal implants. Some of these studies have reported higher metal levels in patients with loose implants.
Adverse Biological Effects of Cobalt and Chromium
Because patients who receive a MOM hip arthroplasty are shown to be exposed to high concentrations of metallic ions, the Medical Advisory Secretariat searched the literature for reports of adverse biological effects of cobalt and chromium. Cobalt and chromium make up the major part of the metal articulations; therefore, they are a focus of concern.
Risk of Cancer
To date, only one study has examined the incidence of cancer after MOM and polyethylene on metal total hip arthroplasties. The results were compared to that of general population in Finland. The mean duration of follow-up for MOM arthroplasty was 15.7 years; for polyethylene arthroplasty, it was 12.5 years. The standardized incidence ratio for all cancers in the MOM group was 0.95 (95% CI, 0.79–1.13). In the polyethylene on metal group it was 0.76 (95% CI, 0.68–0.86). The combined standardized incidence ratio for lymphoma and leukemia in the patients who had MOM THR was 1.59 (95% CI, 0.82–2.77). It was 0.59 (95% CI, 0.29–1.05) for the patients who had polyethylene on metal THR. Patients with MOM THR had a significantly higher risk of leukemia. All patients who had leukemia were aged over than 60 years.
Cobalt Cardiotoxicity
 
Epidemiological Studies of Myocardiopathy of Beer Drinkers
An unusual type of myocardiopathy, characterized by pericardial effusion, elevated hemoglobin concentrations, and congestive heart failure, occurred as an epidemic affecting 48 habitual beer drinkers in Quebec City between 1965 and 1966. This epidemic was directly related the consumption of a popular beer containing cobalt sulfate. The epidemic appeared 1 month after cobalt sulfate was added to the specific brewery, and no further cases were seen a month after this specific chemical was no longer used in making this beer. A beer of the same name is made in Montreal, and the only difference at that time was that the Quebec brand of beer contained about 10 times more cobalt sulphate. Cobalt has been added to some Canadian beers since 1965 to improve the stability of the foam but it has been added in larger breweries only to draught beer. However, in small breweries, such as those in Quebec City, separate batches were not brewed for bottle and draught beer; therefore, cobalt was added to all of the beer processed in this brewery.
In March 1966, a committee was appointed under the chairmanship of the Deputy Minister of Health for Quebec that included members of the department of forensic medicine of Quebec’s Ministry of Justice, epidemiologists, members of Food and Drug Directorate of Ottawa, toxicologists, biomedical researchers, pathologists, and members of provincial police. Epidemiological studies were carried out by the Provincial Ministry of Health and the Quebec City Health Department.
The association between the development of myocardiopathy and the consumption of the particular brand of beer was proven. The mortality rate of this epidemic was 46.1% and those who survived were desperately ill, and recovered only after a struggle for their lives.
Similar cases were seen in Omaha (Nebraska). The epidemic started after a cobalt additive was used in 1 of the beers marketed in Nebraska. Sixty-four patients with the clinical diagnosis of alcoholic myocardiopathy were seen during an 18-month period (1964–1965). Thirty of these patients died. The first patient became ill within 1 month after cobalt was added to the beer, and the last patient was seen within 1 month of withdrawal of cobalt.
A similar epidemic occurred in Minneapolis, Minnesota. Between 1964 and 1967, 42 patients with acute heart failure were admitted to a hospital in Minneapolis, Minnesota. Twenty of these patients were drinking 6 to 30 bottles per day of a particular brand of beer exclusively. The other 14 patients also drank the same brand of beer, but not exclusively. The mortality rate from the acute illness was 18%, but late deaths accounted for a total mortality rate of 43%. Examination of the tissue from these patients revealed markedly abnormal changes in myofibrils (heart muscles), mitochondria, and sarcoplasmic reticulum.
In Belgium, a similar epidemic was reported in 1966, in which, cobalt was used in some Belgian beers. There was a difference in mortality between the Canadian or American epidemic and this series. Only 1 of 24 patients died, 1.5 years after the diagnosis. In March 1965, at an international meeting in Brussels, a new heart disease in chronic beer drinkers was described. This disease consists of massive pericardial effusion, low cardiac output, raised venous pressure, and polycythemia in some cases. This syndrome was thought to be different from the 2 other forms of alcoholic heart disease (beriberi and a form characterized by myocardial fibrosis).
The mystery of the above epidemics as stated by investigators is that the amount of cobalt added to the beer was below the therapeutic doses used for anemia. For example, 24 pints of Quebec brand of beer in Quebec would contain 8 mg of cobalt chloride, whereas an intake of 50 to 100 mg of cobalt as an antianemic agent has been well tolerated. Thus, greater cobalt intake alone does not explain the occurrence of myocardiopathy. It seems that there are individual differences in cobalt toxicity. Other features, like subclinical alcoholic heart disease, deficient diet, and electrolyte imbalance could have been precipitating factors that made these patients susceptible to cobalt’s toxic effects.
In the Omaha epidemic, 60% of the patients had weight loss, anorexia, and occasional vomiting and diarrhea 2 to 6 months before the onset of cardiac symptoms. In the Quebec epidemic, patients lost their appetite 3 to 6 months before the diagnosis of myocardiopathy and developed nausea in the weeks before hospital admission. In the Belgium epidemic, anorexia was one of the most predominant symptoms at the time of diagnosis, and the quality and quantity of food intake was poor. Alcohol has been shown to increase the uptake of intracoronary injected cobalt by 47%. When cobalt enters the cells, calcium exits; this shifts the cobalt to calcium ratio. The increased uptake of cobalt in alcoholic patients may explain the high incidence of cardiomyopathies in beer drinkers’ epidemics.
As all of the above suggest, it may be that prior chronic exposure to alcohol and/or a nutritionally deficient diet may have a marked synergistic effect with the cardiotoxicity of cobalt.
Conclusions
MOM hip resurfacing arthroplasty has been shown to be an effective arthroplasty procedure as tested in younger patients.
However, evidence for effectiveness is based only on 7 case series with short duration of follow-up (2.8–3.5 years). There are no RCTs or other well-controlled studies that compare MOM hip resurfacing with THR.
Revision rates reported in the MOM studies using implants currently licensed in Canada (hybrid systems, uncemented acetabular, and cemented femoral) range from 0.3% to 3.6% for a mean follow-up ranging from 2.8 to 3.5 years.
Fracture of femoral neck is not very common; it occurs in 0.4% to 2.2% of cases (as observed in a short follow-up period).
All the studies that measured health outcomes have reported improvement in Harris Hip and SF-12 scores; 1 study reported significant reduction in pain and improvement in function, and 2 studies reported significant improvement in SF-12 scores. One study reported significant improvement in UCLA Hip scores.
Concerns remain on the potential adverse effects of metal ions. Longer-term follow-up data will help to resolve the inconsistency of findings on adverse effects, including toxicity and carcinogenicity.
Ontario-Based Economic Analysis
The device cost for MOM ranges from $4,300 to $6,000 (Cdn). Traditional hip replacement devices cost about $2,000 (Cdn). Using Ontario Case Costing Initiative data, the total estimated costs for hip resurfacing surgery including physician fees, device fees, follow-up consultation, and postsurgery rehabilitation is about $15,000 (Cdn).
Cost of Total Hip Replacement Surgery in Ontario
MOM hip arthroplasty is generally recommended for patients aged under 55 years because its bone-conserving advantage enables patients to “buy time” and hence helps THRs to last over the lifetime of the patient. In 2004/2005, 15.9% of patients who received THRs were aged 55 years and younger. It is estimated that there are from 600 to 1,000 annual MOM hip arthroplasty surgeries in Canada with an estimated 100 to 150 surgeries in Ontario. Given the increased public awareness of this device, it is forecasted that demand for MOM hip arthroplasty will steadily increase with a conservative estimate of demand rising to 1,400 cases by 2010 (Figure 10). The net budget impact over a 5-year period could be $500,000 to $4.7 million, mainly because of the increasing cost of the device.
Projected Number of Metal-on-Metal Hip Arthroplasty Surgeries in Ontario: to 2010
PMCID: PMC3379532  PMID: 23074495
12.  Functionally distinct RNA polymerase binding sites in the phage Mu mom promoter region. 
Nucleic Acids Research  1992;20(11):2777-2784.
Transcription of the phage Mu com/mom operon is trans-activated by another phage gene product, C, a site-specific DNA binding protein. To gain insight into the mechanism by which C activates transcription, we carried out footprinting analyses of Escherichia coli RNA polymerase (= RNAP) binding to various com-lacZ fusion plasmids. KMnO4-sensitive sites (diagnostic of the melted regions in open-complexes) and DNase I-sensitive sites were located by primer-extension analysis. The results are summarized as follows: (i) in vivo, in the absence of C, RNAP bound in the wild-type (wt) promoter region at a site designated P2; in vitro DNase I-footprinting showed that P2 extends from -74 to -24 with respect to transcription initiation. This overlaps a known strong C-binding site (at -35 to -54). RNAP bound at P2 appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (ii) In contrast, when C was present in vivo, RNAP bound in the wt promoter region at a different site, designated P1, located downstream and partially overlapping P2. RNAP bound at P1 also appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (iii) Two C-independent mutants, which initiate transcription at the same position as the wt, were also analyzed. In vivo, in the absence of C, RNAP bound mutant tin7 (contains a T to G substitution at -14) predominantly at P1; in vitro DNase I-footprinting showed that P1 extends from -56 to +21. With mutant tin6 (a 63 base-pair deletion removing P2, as well as part of P1 and the C-binding site from -35 to -54), RNAP bound to P1 independent of C. We conclude that P1 is the 'functional' RNAP binding site for mom-transcription initiation, and that C activates transcription by promoting binding at P1, while blocking binding at P2.
Images
PMCID: PMC336922  PMID: 1535436
13.  Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence. 
Journal of Bacteriology  1989;171(4):2003-2018.
Late transcription of bacteriophage Mu, which results in the expression of phage morphogenetic functions, is dependent on Mu C protein. Earlier experiments indicated that Mu late RNAs originate from four promoters, including the previously characterized mom promoter. S1 nuclease protection experiments were used to map RNA 5' ends in the three new regions. Transcripts were initiated at these points only in the presence of C and were synthesized in a rightward direction on the Mu genome. Amber mutant marker rescue analysis of plasmid clones and limited DNA sequencing demonstrated that these new promoters are located between C and lys, upstream of I, and upstream of P within the N gene. A comparison of the promoter sequences upstream from the four RNA 5' ends yielded two conserved sequences: the first (tA . . cT, where capital and lowercase letters indicate 100 and 75% base conservation, respectively), at approximately -10, shares some similarity with the consensus Escherichia coli sigma 70 -10 region, while the second (ccATAAc CcCPuG/Cac, where Pu indicates a purine), in the -35 region, bears no resemblance to the E. coli -35 consensus. We propose that these conserved Mu late promoter consensus sequences are important for C-dependent promoter activity. Plasmids containing transcription fusions of these late promoters to lacZ exhibited C-dependent beta-galactosidase synthesis in vivo, and C was the only Mu product needed for this transactivation. As expected, the late promoter-lacZ fusions were activated only at late times after induction of a Mu prophage. The C-dependent activation of lacZ fusions containing only a few bases of the 5' end of Mu late RNA and the presence of altered promoter sequences imply that C acts at the level of transcription initiation.
Images
PMCID: PMC209851  PMID: 2522923
14.  Silencing of toxic gene expression by Fis 
Nucleic Acids Research  2012;40(10):4358-4367.
Bacteria and bacteriophages have evolved DNA modification as a strategy to protect their genomes. Mom protein of bacteriophage Mu modifies the phage DNA, rendering it refractile to numerous restriction enzymes and in turn enabling the phage to successfully invade a variety of hosts. A strong fortification, a combined activity of the phage and host factors, prevents untimely expression of mom and associated toxic effects. Here, we identify the bacterial chromatin architectural protein Fis as an additional player in this crowded regulatory cascade. Both in vivo and in vitro studies described here indicate that Fis acts as a transcriptional repressor of mom promoter. Further, our data shows that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. We propose that a combined repressive effect of Fis and previously characterized negative regulatory factors could be responsible to keep the gene silenced most of the time. We thus present a new facet of Fis function in Mu biology. In addition to bringing about overall downregulation of Mu genome, it also ensures silencing of the advantageous but potentially lethal mom gene.
doi:10.1093/nar/gks037
PMCID: PMC3378877  PMID: 22287621
15.  Impact of Community-Based Maternal Health Workers on Coverage of Essential Maternal Health Interventions among Internally Displaced Communities in Eastern Burma: The MOM Project 
PLoS Medicine  2010;7(8):e1000317.
Mullany and colleagues report outcomes from a project involving delivery of community-based maternal health services in eastern Burma, and report substantial increases in coverage of care.
Background
Access to essential maternal and reproductive health care is poor throughout Burma, but is particularly lacking among internally displaced communities in the eastern border regions. In such settings, innovative strategies for accessing vulnerable populations and delivering basic public health interventions are urgently needed.
Methods
Four ethnic health organizations from the Shan, Mon, Karen, and Karenni regions collaborated on a pilot project between 2005 and 2008 to examine the feasibility of an innovative three-tiered network of community-based providers for delivery of maternal health interventions in the complex emergency setting of eastern Burma. Two-stage cluster-sampling surveys among ever-married women of reproductive age (15–45 y) conducted before and after program implementation enabled evaluation of changes in coverage of essential antenatal care interventions, attendance at birth by those trained to manage complications, postnatal care, and family planning services.
Results
Among 2,889 and 2,442 women of reproductive age in 2006 and 2008, respectively, population characteristics (age, marital status, ethnic distribution, literacy) were similar. Compared to baseline, women whose most recent pregnancy occurred during the implementation period were substantially more likely to receive antenatal care (71.8% versus 39.3%, prevalence rate ratio [PRR] = 1.83 [95% confidence interval (CI) 1.64–2.04]) and specific interventions such as urine testing (42.4% versus 15.7%, PRR = 2.69 [95% CI 2.69–3.54]), malaria screening (55.9% versus 21.9%, PRR = 2.88 [95% CI 2.15–3.85]), and deworming (58.2% versus 4.1%, PRR = 14.18 [95% CI 10.76–18.71]. Postnatal care visits within 7 d doubled. Use of modern methods to avoid pregnancy increased from 23.9% to 45.0% (PRR = 1.88 [95% CI 1.63–2.17]), and unmet need for contraception was reduced from 61.7% to 40.5%, a relative reduction of 35% (95% CI 28%–40%). Attendance at birth by those trained to deliver elements of emergency obstetric care increased almost 10-fold, from 5.1% to 48.7% (PRR = 9.55 [95% CI 7.21–12.64]).
Conclusions
Coverage of maternal health interventions and higher-level care at birth was substantially higher during the project period. The MOM Project's focus on task-shifting, capacity building, and empowerment at the community level might serve as a model approach for similarly constrained settings.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Every minute, somewhere in the world, a woman dies of complications related to pregnancy and childbirth. Access to essential maternal and reproductive health care (including family planning) is particularly bad in war-torn countries. In Burma, for example, where there have been decades of conflict between the military junta and ethnic minority resistance groups, the maternal mortality rate (the number of deaths among women from pregnancy-related causes per 100,000 live births) is around 380, whereas in neighboring Thailand it is only 44. Maternal health is even worse in the Shan, Mon, Karen, and Karenni regions of eastern Burma where ethnic conflicts and enforced village relocations have internally displaced more than half a million people. Here, the maternal mortality rate is around 1,200. In an effort to improve access to maternal health services in these regions, community-based organizations in Burma, the Johns Hopkins Center for Public Health and Human Rights, and the Global Health Access Program undertook an innovative pilot project—the Mobile Obstetric Medics (MOM) project—between 2005 and 2008. Local health workers from 12 communities in eastern Burma received training in antenatal care, obstetrics (the care of women during childbirth), postnatal care, and family planning at the Mae Tao Clinic in Mae Sot, Thailand. These “maternal health workers” then returned to Burma where they trained local health workers and traditional birth attendants to provide maternal health care to their communities.
Why Was This Study Done?
Before the MOM project started, nearly 3,000 women living in the study communities were surveyed to evaluate the coverage of essential antenatal care interventions such as urine testing for infections during pregnancy, screening for malaria, and deworming; Urinary tract infections, malaria, and hookworm infections all increase the risk of poor maternal and neonatal outcomes. The preproject survey also evaluated how many births were attended by people able to deal with complications, and the provision of postnatal care and family planning services. In this study, the researchers undertake a similar postproject survey to evaluate the impact of MOM on the coverage of essential maternal health interventions among internally displaced communities in eastern Burma.
What Did the Researchers Do and Find?
Between October 2008 and December 2008, trained survey workers asked nearly 2,500 ever-married women of reproductive age from the project's study communities about their access to antenatal and postnatal care, skilled birth attendants, and family planning. The results of the postproject survey were then compared with those of the “baseline,” preproject survey. The general characteristics (age, marital status, ethnicity, and literacy) of the women included in the two surveys were very similar. However, 71.8% of the women whose most recent pregnancy occurred during the implementation period of the MOM project had received antenatal care compared to only 39.3% of women surveyed at baseline. Similarly, among the women questioned during the postproject survey, 42.4% had had their urine tested and 55.9% had been screened for malaria during pregnancy compared to only 15.7% and 21.9%, respectively, of the women questioned in the preproject survey. Deworming had increased from 4.1% to 58.2% during the project, postnatal care visits within 7 days had doubled, and attendance at birth by people trained to deal with obstetric emergencies had increased 10-fold from 5.1% to 48.7%. Finally, the use of modern contraception methods (slow-release contraceptives, oral contraceptives, and condoms) had increased from 23.9% to 45.0%.
What Do These Findings Mean?
These findings reveal a substantial improvement in access to maternal and reproductive health care in the study communities during the MOM project. However, because the study compared two independent groups of women before and after implementation of the MOM project rather than concurrently comparing groups of women who did and did not receive the services provided by the MOM project, this study does not prove that the MOM approach was the cause of the changes in the coverage of essential maternal health care. Nevertheless, these findings suggest that the type of approach used in the MOM project—the expansion of interventions (including components of emergency obstetric care) delivered outside healthcare facilities by community-based providers—might be an effective way to deliver maternal and reproductive health services in other parts of Burma and in other places where there are ongoing conflicts.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000317.
More information about the MOM project is available in previous publications by the researchers in PLoS Medicine, in Reproductive Health Matters, and in Social Science and Medicine
Additional resources are also available on the MOM Project
The Reproductive Health Response in Conflict Consortium provides information on how conflicts affect reproductive health
The World Health Organization provides information on all aspects of health in Burma (in several languages)
The Mae Tao clinic also provides general information about Burma and its health services
The Burma Campaign UK and Human Rights Watch both provide detailed information about human rights violations, including those that affect maternal health in Burma
The United Nations Population Fund provides information about safe motherhood and maternal and reproductive health during conflicts and among refugees (in several languages)
doi:10.1371/journal.pmed.1000317
PMCID: PMC2914639  PMID: 20689805
16.  Escherichia coli OxyR modulation of bacteriophage Mu mom expression in dam+ cells can be attributed to its ability to bind hemimethylated Pmom promoter DNA. 
Nucleic Acids Research  1997;25(21):4385-4388.
Transcription of the bacteriophage Mu mom operon is strongly repressed by the host OxyR protein in dam - but not dam + cells. In this work we show that the extent of mom modification is sensitive to the relative levels of the Dam and OxyR proteins and OxyR appears to modulate the level of mom expression even in dam + cells. In vitro studies demonstrated that OxyR is capable of binding hemimethylated P mom , although its affinity is reduced slightly compared with unmethylated DNA. Thus, OxyR modulation of mom expression in dam + cells can be attributed to its ability to bind hemimethylated P mom DNA, the product of DNA replication.
PMCID: PMC147061  PMID: 9336472
17.  In vitro transcriptional activation of the phage Mu mom promoter by C protein. 
Journal of Bacteriology  1994;176(10):2885-2891.
The phage Mu gene C encodes a 16.5-kDa site-specific DNA-binding protein that functions as a trans-activator of the four phage "late" operons, including mom. We have overexpressed and purified C and used it for DNase I footprinting and transcription analyses in vitro. The footprinting results are summarized as follows. (i) As shown previously (V. Balke, V. Nagaraja, T. Gindlesperger, and S. Hattman, Nucleic Acids Res. 12:2777-2784, 1992) in vivo, Escherichia coli RNA polymerase (RNAP) bound the wild-type (wt) mom promoter at a site slightly upstream from the functionally active site bound on the C-independent tin7 mutant promoter. (ii) In the presence of C, however, RNAP bound the wt promoter at the same site as tin7. (iii) C and RNAP were both bound by the mom promoter at overlapping sites, indicating that they were probably on different faces of the DNA helix. The minicircle system of Choy and Adhya (H. E. Choy and S. Adhya, Proc. Natl. Acad. Sci. USA 90:472-476, 1993) was used to compare transcription in vitro from the wt and tin7 promoters. This analysis showed the following. (i) Few full-length transcripts were observed from the wt promoter in the absence of C, but addition of increasing amounts of C greatly stimulated transcription. (ii) RNA was transcribed from the tin7 promoter in the absence of C, but addition of C had a small stimulatory effect. (iii) Transcription from linearized minicircles or restriction fragment templates was greatly reduced (although still stimulated by C) with both the wt and tin7 promoters. These results show that C alone is capable of activating rightward transcription in vitro by promoting RNAP binding at a functionally active site. Additionally, DNA topology plays an important role in transcriptional activation in vitro.
Images
PMCID: PMC205443  PMID: 8188589
18.  An Epigenetic Switch Involving Overlapping Fur and DNA Methylation Optimizes Expression of a Type VI Secretion Gene Cluster 
PLoS Genetics  2011;7(7):e1002205.
Type VI secretion systems (T6SS) are macromolecular machines of the cell envelope of Gram-negative bacteria responsible for bacterial killing and/or virulence towards different host cells. Here, we characterized the regulatory mechanism underlying expression of the enteroagregative Escherichia coli sci1 T6SS gene cluster. We identified Fur as the main regulator of the sci1 cluster. A detailed analysis of the promoter region showed the presence of three GATC motifs, which are target of the DNA adenine methylase Dam. Using a combination of reporter fusion, gel shift, and in vivo and in vitro Dam methylation assays, we dissected the regulatory role of Fur and Dam-dependent methylation. We showed that the sci1 gene cluster expression is under the control of an epigenetic switch depending on methylation: fur binding prevents methylation of a GATC motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. A model is proposed in which the sci1 promoter is regulated by iron availability, adenine methylation, and DNA replication.
Author Summary
DNA methylation plays an important role in the regulation of genes involved in assembly of cell surface adhesins or appendages. Methylation at a GATC motif by the Dam methylase influences binding of transcriptional regulators, leading to variation in the gene expression pattern. In several cases, this may lead to different cell subpopulations allowing a rapid adaptation to varying environments. In this work, we uncover the regulatory mechanism controlling expression of the sci1 Type VI secretion gene cluster in entero-aggregative Escherichia coli, which encodes a structure required for inter-bacterial interaction. We showed that this gene cluster is repressed by Fur in iron-replete conditions and that Fur binding on the promoter prevents methylation of a GATC motif. In iron-limited conditions, Fur is relieved from the promoter allowing expression of the gene cluster and methylation of the GATC motif. Methylation prevents de novo Fur binding allowing constitutive expression. Our findings support a model in which the expression of the Type VI secretion gene cluster is regulated by a non-stochastic epigenetic switch: switch from the OFF to ON phases depends on iron availability whereas the ON to OFF switch depends on DNA replication and competition between Dam-dependent methylation and Fur binding.
doi:10.1371/journal.pgen.1002205
PMCID: PMC3145626  PMID: 21829382
19.  Dam- and OxyR-Dependent Phase Variation of agn43: Essential Elements and Evidence for a New Role of DNA Methylation 
Journal of Bacteriology  2002;184(12):3338-3347.
Phase variation of the outer membrane protein Ag43 in E. coli requires deoxyadenosine methylase (Dam) and OxyR. Previously, it was shown that OxyR is required for repression of the Ag43-encoding gene, agn43, and that Dam-dependent methylation of three GATC target sequences in the regulatory region abrogates OxyR binding. Here we report further characterization of agn43 transcription and its regulation. Transcription was initiated from a σ70-dependent promoter at the G residue of the upstream GATC sequence. Template DNA and RNA polymerase were sufficient to obtain transcription in vitro, but DNA methylation enhanced the level of transcription. Analyses of transcription in vivo of agn′-lacZ with mutated Dam target sequences support this conclusion. Since methylation also abrogates OxyR binding, this indicates that methylation plays a dual role in facilitating agn43 transcription. In vitro transcription from an unmethylated template was repressed by OxyR(C199S), which resembles the reduced form of OxyR. Consistent with this and the role of Dam in OxyR binding, OxyR(C199S) protected from DNase I digestion the agn43 regulatory region from −16 to +42, which includes the three GATC sequences. Deletion analyses of the regulatory region showed that a 101-nucleotide region of the agn43 regulatory region containing the promoter and this OxyR binding region was sufficient for Dam- and OxyR-dependent phase variation
doi:10.1128/JB.184.12.3338-3347.2002
PMCID: PMC135096  PMID: 12029051
20.  Bacteriophage Mu-induced modification of DNA is dependent upon a host function. 
Journal of Bacteriology  1978;136(1):423-428.
The DNA of bacteriophage Mu, extracted from induced lysates, is partially resistant to digestion by the endonuclease BalI. This modification of DNA is controlled by the Mu modification function (mom), which acts in conjunction with the dam (DNA-adenine methylation) function of Escherichia coli. Since the BalI recognition site is apparently different from the dam recognition site, these results imply that either the specificity of the dam function is changed by the mom function or the mom function requires the dam function for its activity.
Images
PMCID: PMC218675  PMID: 361700
21.  Metal-on-Metal Hip Resurfacing Arthroplasty 
Background
Metal-on-metal (MOM) hip resurfacing arthroplasty (HRA) is in clinical use as an appropriate alternative to total hip arthroplasty in young patients. In this technique, a metal cap is placed on the femoral head to cover the damaged surface of the bone and a metal cup is placed in the acetabulum.
Objectives
The primary objective of this analysis was to compare the revision rates of MOM HRA using different implants with the benchmark set by the National Institute of Clinical Excellence (NICE). The secondary objective of this analysis was to review the literature regarding adverse biological effects associated with implant material.
Review Methods
A literature search was performed on February 13, 2012, to identify studies published from January 1, 2009, to February 13, 2012.
Results
The revision rates for MOM HRA using 6 different implants were reviewed. The revision rates for MOM HRA with 3 implants met the NICE criteria, i.e., a revision rate of 10% or less at 10 years. Two implants had short-term follow-ups and MOM HRA with one of the implants failed to meet the NICE criteria.
Adverse tissue reactions resulting in failure of the implants have been reported by several studies. With a better understanding of the factors that influence the wear rate of the implants, adverse tissue reactions and subsequent implant failure can be minimized. Many authors have suggested that patient selection and surgical technique affect the wear rate and the risk of tissue reactions.
The biological effects of high metal ion levels in the blood and urine of patients with MOM HRA implants are not known. Studies have shown an increase in chromosomal aberrations in patients with MOM articulations, but the clinical implications and long-term consequences of this increase are still unknown. Epidemiological studies have shown that patients with MOM HRA implants did not have an overall increase in mortality or risk of cancer. There is insufficient clinical data to confirm the teratogenicity of MOM implants in humans.
Conclusions
Metal-on-metal HRA can be beneficial for appropriately selected patients, provided the surgeon has the surgical skills required for performing this procedure.
Plain Language Summary
There are many young patients with hip diseases who need to have hip replacement surgery. Although a traditional hip replacement is an acceptable procedure for these patients, some surgeons prefer using a newer technique in young patients called hip resurfacing. In this technique, instead of removing the head of the femoral bone, a metal cap is placed on the femoral head to cover the damaged surface of the bone and a metal cup is placed in the hip socket, similar to the cups used in traditional hip replacement.
The analysis of the revision rates (i.e., how soon and in how many patients the surgery needs to be redone) and safety of resurfacing implants showed that generally these implants can last 10 years or more for the majority of young people. Good outcomes can be expected when skilled surgeons perform the surgery in properly selected patients.
However, since these implants are made of metal (cobalt and chromium alloy), there is concern about excess metal debris production due to friction between the 2 metal components leading to high levels of metal ions in the blood and urine of patients. The production of metal debris may result in inflammation in the joint or development of a benign soft tissue mass leading to implant failure. However, it has been shown that this risk can be reduced by proper positioning of the implant and the careful selection of patients for this procedure.
Little is known about the long-term biological effects of high levels of metal ions in the blood and urine of patients who have received metal implants. There is concern about potential increases in the risk of cancer and the risk of fetal abnormalities, but these effects have not been established yet. However, since cobalt and chromium can pass the placental barrier, implants that are not metal-on-metal are recommended for women at childbearing ages if they need a hip replacement.
PMCID: PMC3440005  PMID: 23074429
22.  Epigenetic Gene Regulation in the Bacterial World 
Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions.
doi:10.1128/MMBR.00016-06
PMCID: PMC1594586  PMID: 16959970
23.  Localization and DNA sequence analysis of the C gene of bacteriophage Mu, the positive regulator of Mu late transcription. 
Nucleic Acids Research  1986;14(12):4881-4897.
The C gene of bacteriophage Mu, required for transcription of the phage late genes, was localized by construction and analysis of a series of deleted derivatives of pKN50, a plasmid containing a 9.4 kb Mu DNA fragment which complements Mu C amber mutant phages for growth. One such deleted derivative, pWM10, containing only 0.5 kb of Mu DNA, complements C amber phages and transactivates the mom gene, one of the Mu late genes dependent on C for activation. The DNA sequence of the 0.5 kb fragment predicts a single long open reading frame coding for a 140 amino acid protein. Sequence analysis of DNA containing a C amber mutation located the base change to the second codon of this reading frame. Generation of a frameshift mutation by filling in a BglII site spanning codon 114 of this reading frame resulted in the loss of C complementation and transactivation activity. These results indicate that this open reading frame encodes the Mu C gene product. Comparison of the predicted amino acid sequence of the C protein with those of other transcriptional regulatory proteins revealed some similarity to a region highly conserved among bacterial sigma factors.
Images
PMCID: PMC311498  PMID: 3014438
24.  Targeting of a Tail-anchored Protein to Endoplasmic Reticulum and Mitochondrial Outer Membrane by Independent but Competing Pathways 
Molecular Biology of the Cell  2001;12(8):2482-2496.
Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b5, a TA protein existing in two, MOM or ER localized, versions. Substitution of the seven C-terminal residues of the ER isoform or of green fluorescent protein reporter constructs with one or two arginines resulted in MOM-targeted proteins, whereas a single C-terminal threonine caused promiscuous localization. To investigate whether targeting to MOM occurs from the cytosol or after transit through the ER, we tagged a MOM-directed construct with a C-terminal N-glycosylation sequence. Although in vitro this construct was efficiently glycosylated by microsomes, the protein expressed in vivo localized almost exclusively to MOM, and was nearly completely unglycosylated. The small fraction of glycosylated protein was in the ER and was not a precursor to the unglycosylated form. Thus, targeting occurs directly from the cytosol. Moreover, ER and MOM compete for the same polypeptide, explaining the dual localization of some TA proteins.
PMCID: PMC58608  PMID: 11514630
25.  Access To Essential Maternal Health Interventions and Human Rights Violations among Vulnerable Communities in Eastern Burma 
PLoS Medicine  2008;5(12):e242.
Background
Health indicators are poor and human rights violations are widespread in eastern Burma. Reproductive and maternal health indicators have not been measured in this setting but are necessary as part of an evaluation of a multi-ethnic pilot project exploring strategies to increase access to essential maternal health interventions. The goal of this study is to estimate coverage of maternal health services prior to this project and associations between exposure to human rights violations and access to such services.
Methods and Findings
Selected communities in the Shan, Mon, Karen, and Karenni regions of eastern Burma that were accessible to community-based organizations operating from Thailand were surveyed to estimate coverage of reproductive, maternal, and family planning services, and to assess exposure to household-level human rights violations within the pilot-project target population. Two-stage cluster sampling surveys among ever-married women of reproductive age (15–45 y) documented access to essential antenatal care interventions, skilled attendance at birth, postnatal care, and family planning services. Mid-upper arm circumference, hemoglobin by color scale, and Plasmodium falciparum parasitemia by rapid diagnostic dipstick were measured. Exposure to human rights violations in the prior 12 mo was recorded. Between September 2006 and January 2007, 2,914 surveys were conducted. Eighty-eight percent of women reported a home delivery for their last pregnancy (within previous 5 y). Skilled attendance at birth (5.1%), any (39.3%) or ≥ 4 (16.7%) antenatal visits, use of an insecticide-treated bed net (21.6%), and receipt of iron supplements (11.8%) were low. At the time of the survey, more than 60% of women had hemoglobin level estimates ≤ 11.0 g/dl and 7.2% were Pf positive. Unmet need for contraceptives exceeded 60%. Violations of rights were widely reported: 32.1% of Karenni households reported forced labor and 10% of Karen households had been forced to move. Among Karen households, odds of anemia were 1.51 (95% confidence interval [CI] 0.95–2.40) times higher among women reporting forced displacement, and 7.47 (95% CI 2.21–25.3) higher among those exposed to food security violations. The odds of receiving no antenatal care services were 5.94 (95% CI 2.23–15.8) times higher among those forcibly displaced.
Conclusions
Coverage of basic maternal health interventions is woefully inadequate in these selected populations and substantially lower than even the national estimates for Burma, among the lowest in the region. Considerable political, financial, and human resources are necessary to improve access to maternal health care in these communities.
Luke Mullany and colleagues examine access to essential maternal health interventions and human rights violations within vulnerable communities in eastern Burma.
Editors' Summary
Background.
After decades of military rule, Burma has one of the world's worst health-care systems and high levels of ill health. For example, maternal mortality (deaths among women from pregnancy-related causes) is around 360 per 100,000 live births in Burma, whereas in neighboring Thailand it is only 44 per 100,000 live births. Maternal health is even worse in the Shan, Karenni, Karen and Mon states in eastern Burma where ethnic conflicts and enforced village relocations have internally displaced more than half a million people. Here, maternal mortality is thought to be about 1000 per 100, 000 live births. In an effort to improve access to life-saving maternal health interventions in these states, Burmese community-based health organizations, the Johns Hopkins Center for Public Health and Human Rights and the Global Health Access Program in the USA, and the Mae Tao Clinic (a health-worker training center in Thailand) recently set up the Mobile Obstetric Maternal Health Workers (MOM) Project. In this pilot project, local health workers from 12 communities in eastern Burma received training in antenatal care, emergency obstetrics (the care of women during childbirth), blood transfusion, and family planning at the Mae Tao Clinic. Back in Burma, these maternal health workers trained additional local health workers and traditional birth attendants. All these individuals now provide maternal health care to their communities.
Why Was This Study Done?
The effectiveness of the MOM project can only be evaluated if accurate baseline information on women's access to maternal health-care services is available. This information is also needed to ensure the wise use of scarce health-care resources. However, very little is known about reproductive and maternal health in eastern Burma. In this study, the researchers analyze the information on women's access to reproductive and maternal health-care services that was collected during the initial field implementation stage of the MOM project. In addition, they analyze whether exposure to enforced village relocations and other human rights violations affect access to maternal health-care services.
What Did the Researchers Do and Find?
Trained survey workers asked nearly 3000 ever-married women of reproductive age in the selected communities about their access to antenatal and postnatal care, skilled birth attendants, and family planning. They measured each woman's mid-upper arm circumference (an indicator of nutritional status) and tested them for anemia (iron deficiency) and infection with malaria parasites (a common cause of anemia in tropical countries). Finally, they asked the women about any recent violations of their human rights such as forced labour or relocation. Nearly 90% of the women reported a home delivery for their last baby. A skilled attendant was present at only one in 20 births and only one in three women had any antenatal care. One third of the women received postnatal care and only a third said they had access to effective contraceptives. Few women had received iron supplements or had used insecticide-treated bednets to avoid malaria-carrying mosquitos. Consequently, more than half the women were anemic and 7.2% were infected with malaria parasites. Many women also showed signs of poor nutrition. Finally, human rights violations were widely reported by the women. In Karen, the region containing most of the study communities, forced relocation tripled the risk of women developing anemia and greatly decreased their chances of receiving any antenatal care.
What Do These Findings Mean?
These findings show that access to maternal health-care interventions is extremely limited and that poor nutrition, anemia, and malaria, all of which increase the risk of pregnancy complications, are widespread in the communities in the MOM project. Because these communities had some basic health services and access to training in Thailand before the project started, these results probably underestimate the lack of access to maternal health-care services in eastern Burma. Nevertheless, it is clear that considerable political, financial, and human resources will be needed to improve maternal health in this region. Finally, the findings also reveal a link between human rights violations and reduced access to maternal health-care services. Thus, the scale of human rights violations will need to be considered when evaluating programs designed to improve maternal health in Burma and in other places where there is ongoing conflict.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050242.
This research article is further discussed in a PLoS Medicine Perspective by Macaya Douoguih
The World Health Organization provides information on all aspects of health in Burma (in several languages)
The Mae Tao Clinic also provides general information about Burma and its health services
More information about the MOM project is available in a previous publication by the researchers
The Burma Campaign UK and Human Rights Watch both provide detailed information about human rights violations in Burma
The United Nations Population Fund provides information about safe motherhood and ongoing efforts to save mothers' lives around the world
doi:10.1371/journal.pmed.0050242
PMCID: PMC2605890  PMID: 19108601

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