Combined hormonal contraception and risk of venous thromboembolism within the first year following pregnancy. Danish nationwide historical cohort 1995-2009.

Estimating the risk of venous thromboembolism (VTE) associated with combined hormonal contraceptives following early terminated pregnancies or birth, a Danish nationwide retrospective cohort observing a one-year follow-up was defined using three unique registries. All Danish women with confirmed pregnancies aged 15-49 during the period of 1995-2009 were included. The main outcomes were relative and absolute risks of first time venous thromboembolism in users as well as non-users of combined hormonal contraceptives. In 985,569 person-years, 598 venous thromboembolisms were recorded. After early terminated pregnancies and births, respectively, 113 and 485 events occurred in 212,552 and 773,017 person-years. After early terminated pregnancies, the crude VTE incidence ratios were similar, and the numbers needed to harm were equal between groups that did or did not use combined hormonal contraceptives throughout the follow-up year. After childbirth, individuals that used combined hormonal contraceptives were more likely than non-users to experience VTE depicted by crude incidence ratios; however, the difference was only significant after 14 weeks. This implied that the numbers needed to harm were lower for those that used compared to those that did not use combined oral contraceptives in the initial 14 weeks postpartum. In conclusion, the use of combined hormonal contraceptives after early terminated pregnancies was not detrimental, but during the puerperal period, they should be used with caution.

Fusarin C acts like an estrogenic agonist and stimulates breast cancer cells in vitro.

Fusarin C is a mycotoxin produced by several Fusarium species and has been associated with esophageal cancer due to its carcinogenic effects. Here, we report that fusarin C stimulates growth of the breast cancer cell line MCF-7. This suggests that fusarin C can act as an estrogenic agonist and should be classified as a mycoestrogen. MCF-7 cells were stimulated in the range between 0.1 and 20µM and inhibited when the concentration exceeded 50µM. The toxicity of fusarin C is comparable to other mycoestrogens such as zearalenone, but the chemical structure of fusarin C is very different from other known estrogen agonists. Furthermore, the toxicity of fusarin C was tested in five additional human cell lines Caco 2, U266, PC3, MDA-MB-231 and MCF-10a which were all inhibited when the concentration of fusarin C exceeded 10µM. To the best of our knowledge this is the first report on the mycoestrogenic properties of fusarin C.

Changes in rRNA levels during stress invalidates results from mRNA blotting: fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels.

Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.

Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs.

The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for sigma(70) promoters were identified, suggesting that these pmo genes are recognized by sigma(70) and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmo genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the gamma-Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies of pmoC in both M. trichosporium OB3b and Methylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.

Negative in vitro selection identifies the rRNA recognition motif for ErmE methyltransferase.

Erm methyltransferases modify bacterial 23S ribosomal RNA at adenosine 2058 (A2058, Escherichia coli numbering) conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics. The motif that is recognized by Erm methyltransferases is contained within helix 73 of 23S rRNA and the adjacent single-stranded region around A2058. An RNA transcript of 72 nt that displays this motif functions as an efficient substrate for the ErmE methyltransferase. Pools of degenerate RNAs were formed by doping 34-nt positions that extend over and beyond the putative Erm recognition motif within the 72-mer RNA. The RNAs were passed through a series of rounds of methylation with ErmE. After each round, RNAs were selected that had partially or completely lost their ability to be methylated. After several rounds of methylation/selection, 187 subclones were analyzed. Forty-three of the subclones contained substitutions at single sites, and these are confined to 12 nucleotide positions. These nucleotides, corresponding to A2051-A2060, C2611, and A2614 in 23S rRNA, presumably comprise the RNA recognition motif for ErmE methyltransferase. The structure formed by these nucleotides is highly conserved throughout bacterial rRNAs, and is proposed to constitute the motif that is recognized by all the Erm methyltransferases.

ErmE methyltransferase recognition elements in RNA substrates.

Dimethylation by Erm methyltransferases at the N-6 position of adenine 2058 (A2058, Escherichia coli numbering) in domain V of bacterial 23 S rRNA confers resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics. The ErmE methyltransferase from Saccharopolyspora erythraea methylates a 625 nucleotide transcript of domain V as efficiently as it methylates intact 23 S rRNA. By progressively truncating domain V, the motif required for specific recognition by the enzyme has been localized to a helix and single-stranded region adjacent to A2058. The smallest RNA transcript that shows methyl-accepting activity is a 27-nucleotide stem-loop, corresponding to the 23 S rRNA sequences 2048 to 2063 and 2610 to 2620 (helix 73), with A2058 situated within the hairpin loop. Methylation of A2058 in the truncated RNAs is optimal in the absence of magnesium, and the efficiency of methylation is halved by the presence of 2 to 3 mM magnesium. Magnesium serves to stabilize a conformation in the truncated RNA that prevents efficient methylation. This contrasts to the intact domain V RNA, where 2 mM magnesium ions support a conformation at A2058 that is most readily recognized by ErmE. Methylation of domain V RNA is generally far less susceptible to ionic conditions than the truncated RNAs. The effects of monovalent cations on the methylation of truncated transcripts suggest that RNA structures outside helix 73 support the ErmE interaction. However, interaction with these structures is not essential for specific ErmE recognition of A2058.

Copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium.

The methanotrophic bacteria Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b convert methane to methanol using the enzyme, methane monooxygenase (MMO). These bacteria are able to express two distinct MMOs: a cytoplasmic or soluble form (sMMO) and a membrane-bound or particulate form (pMMO). Differential expression of sMMO and pMMO is regulated by the amount of copper ions available to the cells; sMMO is expressed at low copper-biomass ratios, whereas pMMO is expressed at high copper-biomass ratios. In both methanotrophs, transcription of the sMMO gene cluster is negatively regulated by copper ions. Data suggest that transcription of the M. trichosporium OB3b sMMO gene cluster is directed from a sigma54-like and a sigma70-like promoter. The pMMO (pmo) genes of M. capsulatus (Bath) are transcribed into a polycistronic mRNA of 3.3 kb. The synthesis of this mRNA was activated by copper ions. Activation of pmo transcription by copper ions was concomitant with repression of sMMO gene transcription in both methanotrophs. This suggests that a common regulatory pathway may be involved in the transcriptional switch between sMMO and pMMO gene expression.

Mechanism of post-segregational killing by hok-homologue pnd of plasmid R483: two translational control elements in the pnd mRNA.

The pnd system of plasmid R483 mediates plasmid stabilization by killing of plasmid-free cells. The pnd mRNA is very stable and can be translated into PndA protein, a cell toxin which kills the cells from within by damaging the cell membrane. Translation of the pnd mRNA is inhibited by the PndB antisense, a small labile RNA of 63 nt. The rapid decay of the PndB antidote leads to onset of PndA synthesis in plasmid-free segregants or after addition of rifampicin. Surprisingly however, the full-length pnd mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was found to be active. We have therefore suggested previously, that the 3'-end of the full-length pnd mRNA encodes a fold-back inhibitory sequence (fbi), which prevents its translation. Here we present an analysis of the metabolism of the pnd mRNAs. A mutational analysis shows that single point mutations in the fbi motif results in more rapid truncation. The fbi mutations could not be complemented by second-site mutations in either of the pndA or pndC Shine-Dalgarno (SD) elements. Surprisingly, mutations in the pndC SD element also lead to a more rapid truncation. The effect of these latter mutations was, however, complemented by mutations in a proposed anti-SD element upstream of the pndC SD. Mutations in the anti-SD element were lethal. These results show, that the pnd mRNA contains two negative control elements, one located in its very 3'-end (fbi), and one located just upstream of the pndC SD region (the anti-SD element). These observations add to the complexity of the induction scheme previously proposed to explain activation of pndA expression in plasmid-free cells: In addition to its negative effect of translation, the fbi structure also maintains a reduced processing rate in the 3'-end of the mRNA. This permits the accumulation of a reservoir of pnd mRNA, which can be activated by 3'-end processing in plasmid-free cells. The anti-SD may prevent translation of the pnd mRNA during transcription, thus preventing detrimental synthesis of toxin.

Mechanism of post-segregational killing: translation of Hok, SrnB and Pnd mRNAs of plasmids R1, F and R483 is activated by 3'-end processing.

The gene systems hok/sok of R1, srnB of F and pnd of R483 mediate plasmid maintenance by killing of plasmid-free segregants. Translation of the very stable mRNAs encoding the killer proteins is regulated by small unstable antisense RNAs. The differential decay rates of the inhibitory antisense RNAs and the mRNAs encoding the killer proteins is the basis for the onset of killer mRNA translation in newborn plasmid-free segregants and the killing of these cells. We have suggested previously that this requires that the killer mRNAs occur in two forms. A translationally inactive form was proposed to be converted into a 3'-truncated, translationally active mRNA. In the presence of the antisense RNA, translation from this killer mRNA should be inhibited. In this communication we present in vivo and in vitro evidence that support this model. The requirement for 3'-processing for killer gene expression is demonstrated. By using in vitro techniques it is shown that full-length Hok mRNA is translationally inactive, whereas a 3'-end truncated version of the Hok mRNA is translationally active. In vitro secondary structure probing suggests that the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of the mok gene and thereby inhibits translation of the mRNA. By inference we conclude that the Pnd and SrnB mRNAs are regulated by a similar mechanism.

DNA amplification on cerebrospinal fluid for diagnosis of cerebral toxoplasmosis among HIV-positive patients with signs or symptoms of neurological disease.

Reactivation of Toxoplasma gondii can lead to a life-threatening intracerebral infection in immunocompromised HIV-positive patients. Due to the current diagnostic limitations for establishing an exact diagnosis of cerebral toxoplasmosis, a nested PCR system was developed for direct detection of T. gondii in cerebrospinal fluid. A storage temperature for samples of -20 degrees C and sample preparation using Proteinase K appeared to be critical for obtaining a high sensitivity of PCR. A total of 56 samples from 38 HIV-positive patients and 12 HIV-negative patients with symptoms or signs of neurological disease were evaluated by PCR. 5 of the 38 HIV-positive patients were diagnosed as having cerebral toxoplasmosis and PCR was positive in samples from all 5 patients. In the remaining 33, PCR was positive in one case and negative in 32. An exact etiological diagnosis other than cerebral toxoplasmosis was established in 5 patients. PCR performed on cerebrospinal fluid samples seems to be a fast, sensitive, specific and valuable tool for establishing the diagnosis of cerebral toxoplasmosis among HIV-positive patients at the time of presentation of symptoms or signs of neurological disease.

The rifampicin-inducible genes srnB from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by killing of plasmid-free segregants.

The gene systems srnB of plasmid F and pnd of plasmid R483 were discovered because of their induction by rifampicin. Induction caused membrane damage, RNase I influx, degradation of stable RNA and, consequently, cell killing. We show here that the srnB and pnd systems mediate efficient stabilization of a mini-R1 test-plasmid. We also show that the killer genes srnB' and pndA are regulated by antisense RNAs, and that the srnC- and pndB-encoded antisense RNAs, denoted SrnC- and PndB-RNAs, are unstable molecules of approximately 60 nucleotides. The srnB and pndA mRNAs were found to be very stable. The differential decay rates of the inhibitory antisense RNAs and the killer-gene-encoding mRNAs explain the induction of these gene systems by rifampicin. Furthermore, the observed plasmid-stabilization phenotype associated with the srnB and pnd systems is a consequence of this differential RNA decay: the newborn plasmid-free cells inherit the stable mRNAs, which, after decay of the unstable antisense RNAs, are translated into killer proteins, thus leading to selective killing of the plasmid-free segregants. Thus our observations lead us to conclude that the F srnB and R483 pnd systems are phenotypically indistinguishable from the R1 hok/sok system, despite a 50% dissimilarity at the level of DNA sequence.

[Cerebral symptoms in hyponatremia. Treatment and course]. / Cerebrale symptomer ved hyponatriaemi. Behandling og forløb.

Pronounced hyponatraemia results in serious neurological symptoms and the untreated condition is associated with a great mortality. An account is presented of nine hyponatraemic patients (plasma sodium 101-119 mmol/l) without cardiac, renal or hepatic disease who were admitted to a department for intensive care. In all of the patients, consciousness was compromised. Three patients had generalized convulsions and six patients had respiratory involvement which required respirator treatment in two patients. In one patient, the hyponatraemia had developed acutely. The remaining cases had developed chronically. One patient was confused for one month after admission and another patient developed classical central pontine myelinolysis after a latent period of seven days and which required admission for rehabilitation for several months. The remaining patients, including the acute patient, had recovered completely after a few days. The rate of correction for plasma sodium was calculated for the patients. Animal experimental and clinical accounts concerning the pathological physiology and treatment of hyponatraemia are reviewed. It is important to differentiate between acutely and chronically developed hyponatraemia as treatments differ considerably. Severe chronic hyponatraemia should be treated with a rate of correction of plasma sodium of 0.5 mmol/l/hour, and the normal range for plasma sodium should be aimed at after a couple of days. The risk involved in more rapid normalization is development of central pontine myelinolysis with severe neurological sequelae. Hypertonic sodium chloride must only be employed in cases of acutely developed hyponatraemia with convulsions. Acute hyponatraemia may otherwise be corrected with a rate of correction of plasma sodium of 1 mmol/l/hour or more.

The hok killer gene family in gram-negative bacteria.

The seven members of the hok killer gene family in Gram-negative bacteria are described here. The members of this gene family have been sequenced and include hok/sok from plasmid R1, flm and srnB from plasmid F, pnd from plasmids R483 and R16, and gef and relF, which are located on the Escherichia coli chromosome. The killer proteins encoded by these loci are highly toxic polypeptides of 50 to 52 amino acids. The proteins kill the cells from the inside by interfering with a vital function in the cell membrane. On the basis of their relatedness, the killer proteins and their corresponding loci are divided into four subfamilies. The members of one subfamily, hok/sok and flm, mediate plasmid maintenance by killing plasmid-free cells. The pnd and srnB subfamilies were discovered through their abilities to cause membrane damage and degradation of stable RNA. gef and relF, which constitute the chromosomal subfamily, were found because of their sequence similarity at the DNA and protein levels with other members of the hok gene family. However, no function has been described for the proteins belonging to this subfamily. Although the four subfamilies are distantly related in terms of DNA and protein sequence similarity, the overall genetic organization of the different loci has been well conserved during evolution. The expression of all of the members of the hok gene family is regulated post-transcriptionally. Thus, the expression of the hok and flm genes is regulated by small antisense RNAs that inhibit the translation of the stable hok and flm mRNAs. On the basis of structural and functional similarities, we suggest that each of the related plasmid-encoded killer genes is regulated by antisense RNAs. The conservation of this widespread gene family in Gram-negative bacteria suggests that the genes are important to the genomes that carry them.