Transport, enzymatic activity, and stability of mutant sulfamidase (SGSH) identified in patients with mucopolysaccharidosis type III A

Mucopolysaccharidosis type IIIA (MPSIIIA) is an autosomal recessive lysosomal storage disease caused by mutations in the N-sulfoglucosamine sulfohydrolase gene (SGSH; encoding sulfamidase, also sulphamidase) leading to the lysosomal accumulation and urinary excretion of heparan sulfate. Considerable variation in the onset and severity of the clinical phenotype is observed. We report here on expression studies of four novel mutations: c.318C>A (p.Ser106Arg), c.488T>C (p.Leu163Pro), c.571G>A (p.Gly191Arg), and c.1207_1209delTAC (p.Tyr403del), and five previously known mutations: c.220C>T (p.Arg74Cys), c.697C>T (p.Arg233X), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) identified in MPSIIIA patients. Transient expression of mutant sulfamidases in BHK or CHO cells revealed that all the mutants were enzymatically inactive with the exception of c.318C>A (p.Ser106Arg), which showed 3.3% activity of the expressed wild-type enzyme. Western blot analysis demonstrated that the amounts of expressed mutant sulfamidases were significantly reduced compared with cells expressing wild type. No polypeptides were immunodetectable in extracts of cells transfected with the cDNA carrying the c.697C>T (p.Arg233X) nonsense mutation. In vitro translation and pulse-chase experiments showed that rapid degradation rather than a decrease in synthesis is responsible for the low, steady-state level of the mutant proteins in cells. The amounts of secreted mutant precursor forms, the cellular stability, the proteolytic processing, and data from double-label immunofluorescence microscopy suggest that the degradation of the majority of newly synthesized c.220C>T (p.Arg74Cys), c.571G>A (p.Gly191Arg), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) mutant proteins probably occurs in the ER, whereas c.488T>C (p.Leu163Pro) mutant protein showed instability in the lysosomes.     

Engulfment of mast cell secretory granules on skin inflammation boosts dendritic cell migration and priming efficiency

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Identification of a new cluster of T-cell receptor delta recombining elements

Within the human T-cell receptor delta (TCRD) gene we have identified a new cluster of seven delta recombining elements (deltaRec2.1-2.7), located 2.6-5.2 kilobases downstream of the Vdelta2 gene segment. The deltaRec2 elements are isolated recombining signal sequences (RSS), which were shown to rearrange with the Ddelta3 and Jdelta1 segments of the TCRD gene as well as with the psiJalpha of the TCRA gene. Rearrangements involving the deltaRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100-fold lower than that of classical deltaRec rearrangements. The total frequency of deltaRec2 rearrangements was lower in PB T lymphocytes, as compared with thymocytes, suggesting that they are deleted during T-cell development. The decrease of the frequency of the deltaRec2-Ddelta3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the deltaRec2-Jdelta1 rearrangements contained the Ddelta3 segment in the junctional region, we assume that they are derived from the deltaRec2-Ddelta3 rearrangements. In contrast, the majority of deltaRec2-psiJalpha rearrangements did not contain the Ddelta3 segment, indicating that they are single step rearrangements. The deltaRec2-Jdelta1 and deltaRec2-psiJalpha rearrangements seem to be T-lineage specific, but the deltaRec2-Ddelta3 rearrangements were also found at very low frequencies in B lymphocytes and natural killer cells. Our results suggest that deltaRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Valpha-Jalpha rearrangements. We hypothesize, that in a similar manner to the classical deltaRec rearrangements, the deltaRec2 rearrangements might also contribute to T-cell differentiation towards the TCR-alphabeta lineage.     

Electrical stimulation of rhesus monkey nucleus reticularis gigantocellularis

Saccade kinematics are altered by ongoing head movements. The hypothesis that a head movement command signal, proportional to head velocity, transiently reduces the gain of the saccadic burst generator (Freedman 2001, Biol Cybern 84:453-462) can account for this observation. Using electrical stimulation of the rhesus monkey nucleus reticularis gigantocellularis (NRG) to alter the head contribution to ongoing gaze shifts, two critical predictions of this gaze control hypothesis were tested. First, this hypothesis predicts that activation of the head command pathway will cause a transient reduction in the gain of the saccadic burst generator. This should alter saccade kinematics by initially reducing velocity without altering saccade amplitude. Second, because this hypothesis does not assume that gaze amplitude is controlled via feedback, the added head contribution (produced by NRG stimulation on the side ipsilateral to the direction of an ongoing gaze shift) should lead to hypermetric gaze shifts. At every stimulation site tested, saccade kinematics were systematically altered in a way that was consistent with transient reduction of the gain of the saccadic burst generator. In addition, gaze shifts produced during NRG stimulation were hypermetric compared with control movements. For example, when targets were briefly flashed 30 degrees from an initial fixation location, gaze shifts during NRG stimulation were on average 140% larger than control movements. These data are consistent with the predictions of the tested hypothesis, and may be problematic for gaze control models that rely on feedback control of gaze amplitude, as well as for models that do not posit an interaction between head commands and the saccade burst generator.     

Analysis of the behavior of eryC mutants of Brucella suis attenuated in macrophages

The facultatively intracellular pathogen Brucella, characterized by its capacity to replicate in professional and non professional phagocytes, also causes abortion in ruminants. This property has been linked to the presence of erythritol in the placenta, as brucellae preferentially utilize erythritol. The ery operon encodes enzymes involved in erythritol metabolism, and a link with virulence has since been discussed. Allelic exchange mutants in eryC of Brucella suis were erythritol sensitive in vitro with a MIC of 1 to 5 mM of erythritol. Their multiplication in macrophage-like cells was 50- to 90-fold reduced, but complementation of the mutant restored wild-type levels of intracellular multiplication and the capacity to use erythritol as a sole carbon source. In vivo, the eryC mutant colonized the spleens of infected BALB/c mice to a significantly lower extent than the wild type and the complemented strain. Interestingly, eryC mutants that were in addition spontaneously erythritol tolerant nevertheless exhibited wild-type-like intramacrophagic and intramurine replication. We concluded from our results that erythritol was not an essential carbon source for the pathogen in the macrophage host cell but that the inactivation of the eryC gene significantly reduced the intramacrophagic and intramurine fitness of B. suis.     

Age-related accumulation of congophilic fibrillar inclusions in endocrine cells

Summary Intracellular fibrillar congophilic inclusions are well known as neurofibrillary tangles in neurons and as Biondi bodies in choroid plexus epithelial cells. Recently similar amyloid-like inclusions in adrenal cortical cells were described (Eriksson and Westermark 1990). This study on 150 adrenal glands confirms these observations. In our material the age-related accumulation of congophilic inclusions starts earlier (in the sixth decade) and reaches a higher incidence (42.7%). We found similar intracellular inclusions in other endocrine organs, for example in the anterior lobe of the pituitary, in the cells of parathyroid glands and in Sertoli cells. The age-related incidence of these fibrillar inclusions in the pituitary was 68%; the co-incidence with interstitial amyloid deposits was 49.5%. Thus the intracellular accumulation of congophilic fibrils in old age is a widespread phenomenon and occurs not only in neurons but also in endocrine cells (adrenal, pituitary and parathyroid glands) and in active secretory cells (choroid plexus and Sertoli cells).     

Modulation of Whole-Cell Currents in Plasmodium Falciparum-Infected Human Red Blood Cells by Holding Potential and Serum

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum -infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole-cell configuration of the patch-clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole-cell recordings in malaria-infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4-fold and inward currents by approximately 2-fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.     

Metabolic functions of duplicate genes in Saccharomyces cerevisiae

The roles of duplicate genes and their contribution to the phenomenon of enzyme dispensability are a central issue in molecular and genome evolution. A comprehensive classification of the mechanisms that may have led to their preservation, however, is currently lacking. In a systems biology approach, we classify here back-up, regulatory, and gene dosage functions for the 105 duplicate gene families of Saccharomyces cerevisiae metabolism. The key tool was the reconciled genome-scale metabolic model iLL672, which was based on the older iFF708. Computational predictions of all metabolic gene knockouts were validated with the experimentally determined phenotypes of the entire singleton yeast library of 4658 mutants under five environmental conditions. iLL672 correctly identified 96%-98% and 73%-80% of the viable and lethal singleton phenotypes, respectively. Functional roles for each duplicate family were identified by integrating the iLL672-predicted in silico duplicate knockout phenotypes, genome-scale carbon-flux distributions, singleton mutant phenotypes, and network topology analysis. The results provide no evidence for a particular dominant function that maintains duplicate genes in the genome. In particular, the back-up function is not favored by evolutionary selection because duplicates do not occur more frequently in essential reactions than singleton genes. Instead of a prevailing role, multigene-encoded enzymes cover different functions. Thus, at least for metabolism, persistence of the paralog fraction in the genome can be better explained with an array of different, often overlapping functional roles.     

Oral DNA vaccination with a plasmid encoding soluble ICAM-1 modulates cytokine expression profiles in nonobese diabetic mice

Adhesion molecules are important for leukocyte extravasation and for the delivery of costimulatory signals in T cell activation. We therefore interfered in the immune process leading to islet inflammation in diabetes prone NOD mice by oral vaccination with plasmid DNA encoding soluble ICAM-1. Female NOD mice were treated orally with ICAM-1, TGF-beta, or control plasmid DNA and received a single injection of cyclophosphamide for synchronization and acceleration of the disease process in the pancreas. Quantitative RT-PCR analysis of pancreatic mRNA showed that cyclophosphamide induced the expression of Th1 cytokines (IFN-gamma and IL-12p40) in vehicle- or control plasmid-treated mice. Treatment with ICAM-1 and TGF-beta DNA resulted in increased levels of IL-10 mRNA in the pancreas, indicating an anti-inflammatory regulatory immune response. Histological analysis of pancreatic islets showed that the DNA treatment did not alter islet infiltration in response to cyclophosphamide. Hence vaccination with the ICAM-1 plasmid had not suppressed leukocyte migration but rather modulated lymphocyte activity, similarly as seen for the TGF-beta-encoding plasmid. Neither of the three plasmids caused recognizable changes in cytokine expression in the small intestine, Peyer's patches, or mesenteric lymph nodes. We conclude that oral vaccination with DNA encoding immunoregulatory molecules such as ICAM-1 and TGF-beta represents an approach for modulating the ongoing inflammatory process in the pancreas of diabetes prone NOD mice.