A cross-sectional single-centre study on Pompe disease in 42 German patients: Molecular analysis of the GAA gene, manifestation and genotype-phenotype correlations

ABSTRACT: BACKGROUND: Pompe disease (Glycogen storage disease type II, GSD II, acid alpha-glucosidase deficiency, acid maltase deficiency, OMIM # 232300) is an autosomal-recessive lysosomal storage disorder due to a deficiency of acid alpha-glucosidase (GAA, acid maltase, EC 3.2.1.20, Swiss-Prot P10253). Clinical manifestations are dominated by progressive weakness of skeletal muscle throughout the clinical spectrum. In addition, the classic infantile form is characterised by hypertrophic cardiomyopathy. Methods: In a cross-sectional single-centre study we clinically assessed 3 patients with classic infantile Pompe disease and 39 patients with non-classic presentations, measured their acid alpha-glucosidase activities and analysed their GAA genes. Results: Classic infantile patients had nearly absent residual enzyme activities and a typical clinical course with hypertrophic cardiomyopathy until the beginning of therapy. The disease manifestations in non-classic patients were heterogeneous. There was a broad variability in the decline of locomotive and respiratory function. The age of onset ranged from birth to late adulthood and correlated with enzyme activities. Molecular analysis revealed as many as 33 different mutations, 14 of which are novel. All classic infantile patients had two severe mutations. The most common mutation in the non-classic group was c.-32-13T>G. It was associated with a milder course in this subgroup. Conclusion: Disease manifestation strongly correlates with the nature of the GAA mutations, while the variable progression in non-classic Pompe disease is likely to be explained by yet unknown modifying factors. This study provides the first comprehensive dataset on the clinical course and the mutational spectrum of Pompe disease in Germany.     

Infection of myeloid dendritic cells with Listeria monocytogenes leads to the suppression of T cell function by multiple inhibitory mechanisms

Myeloid dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. In this study we provide evidence that myeloid DC respond to infection with Listeria monocytogenes with simultaneous induction of multiple stimulatory and inhibitory molecules. However, the overall impact of infected DC during T cell encounter results in suppression of T cell activation, indicating that inhibitory pathways functionally predominate. Inhibitory activity of infected DC is effected mainly by IL-10 and cyclooxygenase 2-mediated mechanisms, with soluble CD25 acting as an IL-2 scavenger as well as by the products of tryptophan catabolism. These inhibitory pathways are strictly TNF-dependent. In addition to direct infection, DC bearing this regulatory phenotype can be induced in vitro by a combination of signals including TNF, TLR2, and prostaglandin receptor ligation and by supernatants derived from the infected cells. Both infection-associated DC and other in vitro-induced regulatory DC are characterized by increased resistance to infection and enhanced bactericidal activity. Furthermore, myeloid DC expressing multiple regulatory molecules are identified in vivo in granuloma during listeriosis and tuberculosis. Based on the in vivo findings and the study of in vitro models, we propose that in granulomatous infections regulatory DC may possess dual function evolved to protect the host from disseminating infection via inhibition of granuloma destruction by T cells and control of pathogen spreading.     

Targeted mutagenesis of Burkholderia thailandensis and Burkholderia pseudomallei through natural transformation of PCR fragments

Burkholderia pseudomallei is the causative agent of melioidosis, an overwhelming, rapidly fatal septic infection, and B. thailandensis is a closely related, less virulent species. Both organisms are naturally competent for DNA transformation, and this report describes a procedure exploiting this property for the rapid generation of marked deletion mutations by using PCR products. The method was employed to create 61 mutant strains. Several selectable elements were employed, including elements carrying loxP and FRT recombinase recognition sites to facilitate resistance marker excision. Chromosomal mutations could also be transferred readily between strains by transformation. The availability of simple procedures for creating defined chromosomal mutations and moving them between strains should facilitate genetic analysis of virulence and other traits of these two Burkholderia species.     

Response probability and response time: a straight line, the Tagging/Retagging interpretation of short term memory, an operational definition of meaningfulness and short term memory time decay and search time

The functional relationship between correct response probability and response time is investigated in data sets from Rubin, Hinton and Wenzel, J Exp Psychol Learn Mem Cogn 25:1161–1176, 1999 and Anderson, J Exp Psychol [Hum Learn] 7:326–343, 1981. The two measures are linearly related through stimulus presentation lags from 0 to 594 s in the former experiment and for repeated learning of words in the latter. The Tagging/Retagging interpretation of short term memory is introduced to explain this linear relationship. At stimulus presentation the words are tagged. This tagging level drops slowly with time. When a probe word is reintroduced the tagging level has to increase for the word to be properly identified leading to a delay in response time. The tagging time is related to the meaningfulness of the words used—the more meaningful the word the longer the tagging time. After stimulus presentation the tagging level drops in a logarithmic fashion to 50% after 10 s and to 20% after 240 s. The incorrect recall and recognition times saturate in the Rubin et al. data set (they are not linear for large time lags), suggesting a limited time to search the short term memory structure: the search time for recall of unusual words is 1.7 s. For recognition of nonsense words the corresponding time is about 0.4 s, similar to the 0.243 s found in Cavanagh (1972).     

Determination of zinc in vegetal tissue microsamples by platinum-wire loop in flame atomization atomic absorption spectrometry

The zinc content of 3 μL of vegetal samples (tree leaves, lichens and grape sap) atomized from a Pt-wire in the methane–air flame has been determined by atomic absorption spectrometry. The effect of gas flow rates and the atomization height in the flame on the absorption of zinc was evaluated at 213.9 nm. The best results were obtained at a height of 5 mm and gas flow rates of 200 L/h air and 26 L/h methane, respectively. The effect of Na, K, Ca, Mg, SO₄²⁻, and PO₄³⁻ on the absorption of zinc was studied too. The detection limit of 0.40 ± 0.21 ng was obtained at a significance level of 0.05, using the two-step Neyman–Pearson criterion. The zinc content of the samples has been determined with continuous nebulization and by atomization from the Pt-wire, using both the standard calibration curve and the standard addition method. The results of the two procedures agree within the determination errors.     

Cellulose conversion in dry grind ethanol plants

The expansion of the dry grind ethanol industry provides a unique opportunity to introduce cellulose conversion technology to existing grain to ethanol plants, while enhancing ethanol yields by up to 14%, and decreasing the volume while increasing protein content of distiller's grains. The technologies required are cellulose pretreatment, enzyme hydrolysis, fermentation, and drying. Laboratory data combined with compositional analysis and process simulations are used to present a comparative analysis of a dry grind process to a process with pretreatment and hydrolysis of cellulose in distiller's grains. The additional processing steps are projected to give a 32% increase in net present value if process modifications are made to a 100 million gallon/year plant.     

Mutational Analysis of the Class IIa Bacteriocin Curvacin A and Its Orientation in Target Cell Membranes

To analyze the orientation in target cell membranes of the pediocin-like bacteriocin (antimicrobial peptide) curvacin A, 55 variants were generated by site-directed mutagenesis and their potencies against four different target cells determined. The result suggest that the somewhat hydrophilic short central helix (residues 19 to 24), along with the N-terminal β-sheet-like structure (residues 1 to 16), inserts in the interface region of the target cell membrane, with Ala22 close to the hydrophobic core of the membrane. The following hinge region, with Gly28 as an important residue, may then form a turn wherein Gly28 becomes positioned near the border between the interface and the hydrophobic regions, thus permitting the longer and more-hydrophobic C-terminal helix (residues 29 to 41) to insert into the hydrophobic core of the membrane. This helix contains three glycine residues (G33, G37, and G40) that form a putative helix-helix-interacting GxxxGxxG motif. The replacement of any of these glycines with a larger residue was very detrimental, suggesting their possible involvement in helix-helix interactions with a membrane-embedded receptor protein.     

Die Bedeutung der Züchtung des Lieschgrases (Timothee) auf Rostresistenz

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