Refinement by linkage analysis in two large families of the candidate region of the third locus (SCA3) for autosomal dominant cerebellar ataxia type I

The autosomal dominant cerebellar ataxias (ADCA) are clinically and genetically heterogeneous. To date, several loci (SCAI-V) have been identified for ADCA type I. We have studied two large families from the northern part of The Netherlands with ADCA type I with a broad intra-familial variation of symptoms. In both families significant linkage is shown of the disease to the markers of the SCA3 locus on chromosome 14. Through recombinations, the candidate region for SCA3 could be refined to a 13-cM range between D14S256 and D14S81. No recombinations were detected with the markers D14S291 and D14S280, which suggests that the SCA3 gene lies close to these loci. This finding will benefit the individuals at risk in these two families who are seeking predictive testing or prenatal diagnosis.     

Dendritic cells require multidrug resistance protein 1 (ABCC1) transporter activity for differentiation

Dendritic cells (DC) express the ATP-binding cassette (ABC) transporters P-glycoprotein (ABCB1) and multidrug resistance protein 1 (MRP1; ABCC1). Functionally, both these transporters have been described to be required for efficient DC and T cell migration. In this study, we report that MRP1 activity is also crucial for differentiation of DC. Inhibition of MRP1, but not P-glycoprotein, transporter activity with specific antagonists during in vitro DC differentiation interfered with early DC development. Impaired interstitial and Langerhans DC differentiation was characterized by 1) morphological changes, reflected by dropped side scatter levels in flow cytometric analysis and 2) phenotypic changes illustrated by maintained expression of the monocytic marker CD14, lower expression levels of CD40, CD86, HLA-DR, and a significant decrease in the amount of cells expressing CD1a, CD1c, and Langerin. Defective DC differentiation also resulted in their reduced ability to stimulate allogeneic T cells. We identified the endogenous CD1 ligands sulfatide and monosialoganglioside GM1 as MRP1 substrates, but exogenous addition of these substrates could not restore the defects caused by blocking MRP1 activity during DC differentiation. Although leukotriene C(4) was reported to restore migration of murine Mrp1-deficient DC, the effects of MRP1 inhibition on DC differentiation appeared to be independent of the leukotriene pathway. Though MRP1 transporter activity is important for DC differentiation, the relevant MRP1 substrate, which is required for DC differentiation, remains to be identified. Altogether, MRP1 seems to fulfill an important physiological role in DC development and DC functions.     

Selection of tetracycline inducible self-cleaving ribozymes as synthetic devices for gene regulation in yeast

Synthetic regulatory devices are key components for the development of complex biological systems and the reprogramming of cellular functions and networks. Here we describe the selection of tetracycline inducible hammerhead ribozymes. A tetracycline aptamer was fused to the full-length hammerhead ribozyme via a variable linker region. 11 rounds of in vitro selection were applied to isolate linker sequences that mediate tetracycline dependent hammerhead cleavage. We identified allosteric ribozymes that cleave in the presence of 1 μM tetracycline as fast as the full-length ribozyme whereas cleavage is inhibited up to 333-fold in the absence of tetracycline. Reporter gene assays indicate that the allosteric ribozymes can be employed to control gene expression in yeast.     

Thermodynamic characterization of an engineered tetracycline-binding riboswitch

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.     

Discomfort: not pain but still unpleasant feelings from the gut

Most of the factors initiating food or fluid intake have already been studied, but much less is known about those terminating ingestion. We have hypothesised that discomfort originating from the gastrointestinal system may be one of those factors. Gut distension cause pain if the intestinal volume changes but merely discomfort if only the tension of the gut wall increases. It seems that mild unpleasantness (i.e. discomfort) arising from the gut as a result of moderate (quasi-isometric) distension, among and in concordance with other factors, may significantly reduce intake and hence contribute to physiological satiety. The arising discomfort can be detected by measuring the amount and rate of the ingestion, by recording and analysing ingestive behavior by taste-aversivity and taste-reactivity tests, etc. Conclusions of all experiments point to the same direction: tension increase in the gut wall causes discomfort and results in decrease of intake, i.e. satiety.     

Stress survival islet 1 (SSI-1) survey in Listeria monocytogenes reveals an insert common to listeria innocua in sequence type 121 L. monocytogenes strains

Listeria monocytogenes strains (n = 117) were screened for the presence of stress survival islet 1 (SSI-1). SSI-1(+) strains (32.5%) belonged mainly to serotypes 1/2c, 3b, and 3c. All sequence type 121 (ST-121) strains included (n = 7) possessed homologues to Listeria innocua genes lin0464 and lin0465 instead of SSI-1.     

Booster vaccination against tetanus and diphtheria: insufficient protection against diphtheria in young and elderly adults

We have recently demonstrated that single shot vaccinations against tetanus and diphtheria do not lead to long-lasting immunity against diphtheria in elderly persons despite administration at 5 year intervals. In the present study we have immunized a group of young adults against tetanus and diphtheria to compare the pre- and 28 days post-vaccination immune responses in the young group with results of the same vaccination performed in an elderly group of a previous study. We also studied protection in both groups 5 years after vaccination. We compared antibody titers at all three time points and also analyzed the T cell responses in both age groups 5 years after vaccination.Before vaccination 9 % of the elderly persons were not protected against tetanus, and 48 % did not have protection against diphtheria. In the young group all participants were protected against tetanus, but 52 % were also unprotected against diphtheria before vaccination. 28 days after vaccination 100 % of all participants had protective antibody concentrations against tetanus and only a small percentage in each age group (<10 %) was unprotected against diphtheria. 5 years later, 100 % of both cohorts were still protected against tetanus, but 24 % of the young and 54 % of the elderly group were unprotected against diphtheria. Antibody concentrations against diphtheria measured by ELISA correlated well with their neutralizing capacity. T cell responses to tetanus and diphtheria did not differ between young and old persons. We conclude that booster vaccinations against tetanus and diphtheria according to present recommendations provide long-lasting protection only against tetanus, but not against diphtheria, independently of age. In elderly persons, the level of protection is even lower, probably due to intrinsic age-related changes within the immune system and/or insufficient vaccination earlier in life.