Severe infectious mononucleosis in immunocompetent adults.

OBJECTIVES: To determine the risk factors for severe infectious mononucleosis (IM) occurrence in immunocompetent adults. METHODS: We performed a multicenter, retrospective case series including immunocompetent adults presenting with confirmed IM between 2001 and 2011. Severe presentations were compared with uncomplicated presentations using Stata® 9 software. The significance level was set at 5%. RESULTS: In univariate analysis, age over 30 years (n=13 or 41.9% vs. n=5 or 12.8%; P=0.006), prior use of non-steroidal anti-inflammatory drugs (NSAIDs) (n=7 or 87.5% vs. n=1 or 12.5%; P=0.009), and smoking (n=13 or 68.4% vs. n=6 or 31.6%; P=0.013) were associated with severe IM onset. In multivariate analysis, only age over 30 years (OR=3.55; P=0.05) and prior use of NSAIDs (OR=15; P=0.05) remained associated with severe IM onset, without reaching significance level (P=0.05). CONCLUSION: Our study confirmed that age over 30 years is a risk factor for severe IM onset. Prior use of NSAIDs also seems to be correlated with severe presentations. This new data needs to be confirmed in a prospective study.

The major protein fraction of mouse milk revisited using proven proteomic tools.

The PRM/Alf inbred mice exhibit a huge intestinal lengthening. Since milk contains bioactive factors implied in numerous biological processes, one hypothesis is that PRM/Alf milk contains intestinotrophic factors contributing to this remarkable phenotype. A comparison between the milk from PRM/Alf and C57BL/6J (as a control) strains could be helpful in the identification of such factors, including proteins. However, a complete description of the mouse milk major protein fraction is still missing. Hence we adapted a reliable technique to separate and identify the major mouse milk proteins. This approach was achieved through the protein study of milk from C57BL/6J and PWK/Pas strains representative of two Mus musculus subspecies, M. m. domesticus and M. m. musculus respectively. C57BL/6J milk samples were first skimmed and fractionated by reverse phase-HPLC (RP-HPLC). The protein content of each chromatographic peak was analysed by SDS-PAGE and identified by mass spectrometry. This methodological approach allowed characterization of nine major mouse milk proteins: alpha(s1), beta, gamma, epsilon and kappa-caseins, Whey Acidic Protein, lactoferrin, Serum Albumin, Fatty Acid Binding Protein, as well as an alpha(s1)-casein isoform. Then, RP-HPLC patterns of C57BL/6J milk proteins were compared with those obtained starting from the milk of PWK/Pas females. This comparison revealed a protein polymorphism for the alpha(s1)-casein.

Respiratory diseases in French cork workers.

A cross-sectional study on suberosis was conducted in the Champagne-Ardenne County, France, to determine the prevalence of respiratory symptoms, the level of pulmonary function, and the presence of precipitins against Penicillium frequentans. Thirteen of the 33 workers exposed to cork dust had respiratory symptoms excluding hypersensitivity pneumonitis. The respiratory symptoms were not correlated with tobacco habits or duration of exposure. The levels of pulmonary function were not significantly impaired. No precipitin arc against Penicillium frequentans was found in the sera of exposed workers. The varied symptomatology of suberosis may point to several different diseases, each with its own determining factor. In the present study, exposure to weak humidity and low level of cork dust were related to asthma and chronic bronchitis only, excluding hypersensitivity pneumonitis.

Incomplete RNA polymerase II phosphorylation in Xenopus laevis early embryos.

Phosphorylation of RNA polymerase II largest subunit on its C-terminal domain (CTD) heptapeptide repeats has been shown to play a key role in the regulation of mRNA synthesis and processing. In many higher metazoans, early embryos do not synthesise mRNAs during the first cell cycles following fertilisation. Transcription resumes and becomes an absolute requirement for development after several cell cycles characteristic of each species. Therefore, CTD phosphorylation has been investigated during early development of the African clawed-frog Xenopus laevis. Fertilisation is shown to trigger an abrupt dephosphorylation of the CTD. Phosphorylation of the CTD resumes concurrently with the mid-blastula transition (MBT). Both are advanced with polyspermy and increased temperatures; they do not occur when replication is impaired with aphidicolin. In Xenopus laevis somatic cells, a set of monoclonal antibodies defined distinct phosphoepitopes on the CTD. Two of them were absent before the MBT indicating that the CTD lacks the phosphorylation at the serine-2 position of the heptapeptide. The possible contribution of RNA polymerase II phosphorylation to the developmental-regulation of maternal mRNA processing in embryos is discussed.

Nuclear translocation and carboxyl-terminal domain phosphorylation of RNA polymerase II delineate the two phases of zygotic gene activation in mammalian embryos.

In mammalian embryos, zygotic gene transcription initiates after a limited number of cell divisions through a two-step process termed the zygotic gene activation (ZGA). Here we report that RNA polymerase II undergoes major changes in mouse and rabbit preimplantation embryos during the ZGA. In transcriptionally inactive unfertilized oocytes, the RNA polymerase II largest subunit is predominantly hyperphosphorylated on its carboxy-terminal domain (CTD). The CTD is markedly dephosphorylated several hours after fertilization, before the onset of a period characterized by a weak transcriptional activity. The largest subunit of RNA polymerase II then lacks immunological and drug-sensitivity characteristics related to its phosphorylation by the TFIIH-associated kinase and gradually translocates into the nuclei independently of DNA replication and mitosis. A phosphorylation pattern of the largest subunit, close to that observed in somatic cells, is established in both mouse and rabbit embryos at the stage when transcription becomes a requirement for further development (respectively at the 2- and 8/16-cell stage). As these events occurred in the presence of actinomycin D, the nuclear translocation of RNA polymerase II and the phosphorylation of the CTD might be major determinants of ZGA.

Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation.

Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34(cdc2)off kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells.

Heat-shock induced protein modifications and modulation of enzyme activities.

Upon heat stress, the cell physiology is profoundly altered. The extent of the alterations depends on the severity of the stress and may lead to cell death. The heat shock response is an array of metabolic changes characterized by the impairment of major cellular functions and by an adaptative reprogramming of the cell metabolism. The enhanced synthesis of the HSPs is a spectacular manifestation of this reprogramming. Numerous post translational modifications of proteins occur in response to heat stress and can be related to altered cellular functions. Some proteins are heat-denatured and temporarily inactivated. Heat-denaturation is reversible, chaperones may contribute to the repair. The extent of heat-denaturation depends on the cell metabolism: (a) it is attenuated in thermotolerant cells or in cells overexpressing the appropriate chaperones (b) it is enhanced in energy-deprived cells. Covalent modifications may also rapidly alter protein function. Changes in protein glycosylation, methylation, acetylation, farnesylation, ubiquitination have been found to occur during stress. But protein phosphorylation is the most studied modification. Several protein kinase cascades are activated, among which the various mitogen activated protein kinase (MAP kinase) cascades which are also triggered by a wide range of stimuli. As a possible consequence, stress modifies the phosphorylation status and the activity of components from the transcriptional and translational apparatuses. The same kinases also target key enzymes of the cellular metabolism. Protein denaturation results in constitutive hsp titration, this titration is a signal to trigger the heat-shock gene transcription and to activate some of the protein kinase cascades.

Phosphorylation state of the RNA polymerase II C-terminal domain (CTD) in heat-shocked cells. Possible involvement of the stress-activated mitogen-activated protein (MAP) kinases.

RNA polymerase (RNAP) II is a multisubunit enzyme composed of several different subunits. Phosphorylation of the C-terminal domain (CTD) of the largest subunit is tightly regulated. In quiescent or in exponentially growing cells, both the unphosphorylated (IIa) and the multiphosphorylated (IIo) subunits of RNAP II are found in equivalent amounts as the result of the equilibrated antagonist action of protein kinases and phosphatases. In Drosophila and mammalian cells, heat shock markedly modifies the phosphorylation of the RNAP II CTD. Mild heat shocks result in dephosphorylation of the RNAP II CTD. This dephosphorylation is blocked in the presence of actinomycin D, as the CTD dephosphorylation observed in the presence of protein kinase inhibitors. Thus, heat shock might inactivate CTD kinases which are operative at normal growth temperatures, as some protein kinase inhibitors do. In contrast, severe heat shocks are found to increase the amount of phosphorylated subunit independently of the transcriptional activity of the cells. Mild and severe heat shocks activate protein kinases, which then phosphorylate, in vitro and in vivo, the CTD fused to beta-galactosidase. Most of the heat-shock-activated CTD kinases present in cytosolic lysates co-purify with the activated mitogen-activated protein (MAP) kinases, p42mapk and p44mapk. The weak CTD kinase activation occurring upon mild heat shock might be insufficient to compensate for the heat inactivation of the already existing CTD kinases. However, under severe stress, the MAP kinases are strongly heat activated and might prevail over the phosphatases. A survey of different cells and different heat-shock conditions shows that the RNAP II CTD hyperphosphorylation rates follow the extent of MAP kinase activation. These observations lead to the proposal that the RNAP II CTD might be an in vivo target for the activated p42mapk and p44mapk MAP kinases.

Inhibitors of transcription such as 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole and isoquinoline sulfonamide derivatives (H-8 and H-7) promote dephosphorylation of the carboxyl-terminal domain of RNA polymerase II largest subunit.

The RNA polymerase IIO and IIA differ by the extent of phosphorylation in the carboxyl-terminal domain (CTD) of the largest subunit. It has been proposed that the IIA form of RNA polymerase II interacts with the promoter to form a stable preinitiation complex whereas the IIO form would be generated upon entry into initiation of transcription. Phosphorylation of the CTD might be required to release the interaction between the polymerase and the promoter binding factors. In this paper, we show that in the presence of actinomycin D, the phosphorylated IIO form accumulates. In contrast, the dephosphorylated IIA form accumulates while the amount of phosphorylated IIo form decreases in cells treated with CTD-kinase inhibitors such as the nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or the isoquinoline sulfonamide derivatives H-7* or H-8. These changes are fast and suggest a very rapid phosphate turnover on the CTD. Transcription is inhibited in intact cells by drug concentrations that are effective in altering CTD phosphorylation, although no causal relationship is established yet. These effects do not concern other cellular functions such as protein synthesis. Thus isoquinoline sulfonamide derivatives might be helpful to further dissect the role of CTD phosphorylation in transcription.

Phosphorylation of the RNA polymerase II largest subunit during heat shock and inhibition of transcription in HeLa cells.

The phosphorylation of the C-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II has been investigated in HeLa cells exposed to heat shock. In control cells, the phosphorylated subunit, IIo, and the dephosphorylated subunit, IIa, were found in similar amounts. During heat shock, however, the phosphorylated subunit, IIo, accumulated, whereas the amount of IIa subunit decreased. Since phosphorylation of the CTD had been suggested to play a role in the initiation of transcription and since heat shock was known to perturb gene expression at the level of transcription, the phosphorylation state of RNA polymerase II was examined in cells that had been treated with various inhibitors of transcription. Under normal growth temperature, actinomycin D (over 0.1 microgram/ml) and okadaic acid, a phosphatase inhibitor, were found to inhibit polymerase dephosphorylation. Whereas 5,6-dichlorobenzimidazole riboside (DRB), N-(2-[Methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), and actinomycin D (over 5 micrograms/ml) were found to inhibit polymerase phosphorylation. Actinomycin D concentrations, which inhibited the dephosphorylation process, were lower than those required to inhibit the phosphorylation process. In contrast, during heat shock or exposure to sodium arsenite, a chemical inducer of the heat-shock response, the phosphorylated subunit, IIo, accumulated even in the presence of inhibitors of transcription such as DRB, H-8, and actinomycin D. These experiments demonstrated the existence of a heat-shock-induced CTD-phosphorylation process that might contribute to the regulation of transcription during stress.