A numerical taxonomic study of Propionibacterium strains from dairy sources

T.J. BRITZ AND K-H.J. RIEDEL. 1991. Phenotypic results from 81 tests conducted on 73 propionibacteria, including five type strains, 22 reference strains, unidentified propionibacteria and strains isolated from dairy sources, were analysed by numerical taxonomy. Characters giving uniform results were excluded. With the simple matching coefficient and the single linkage cluster analysis, 61 cultures were recovered in five major clusters. Final linkage of all the Propionibacterium cultures examined was at the 77% S-level. The results clearly showed that it was possible to distinguish between the 'classical' and 'cutaneous' Propionibacterium spp., corresponding with the type habitat of each group. The major 'classical' clusters were equated with the P. freudenreichii, P. thoenii, P. jensenii and P. acidipropionici species, while the only major 'cutaneous' cluster was equated with the P. acnes species. The major clusters were identified by relating them to specific type strains and by comparing phenotypic characteristics. The differentiating characteristics of each cluster were determined. The largest cluster, representing 37% of the strains, was equated with P. jensenii but contained cultures that produced an atypical brown/red pigment. These strains, although positively identified as P. jensenii , could also be identified as the 'old' P. rubrum ' species. Thus if pigmentation is used as differential characteristic two distinct groups of propionibacteria could be identified within the P. jensenii species.     

Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization--RING-FISH

Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 10(4)-10(5) copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (10(1)-10(3) copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method--a halo-like, ring-shaped concentration of fluorescence in the cell periphery--we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH.     

The Helmholtz Network for Bioinformatics: an integrative web portal for bioinformatics resources

SUMMARY: The Helmholtz Network for Bioinformatics (HNB) is a joint venture of eleven German bioinformatics research groups that offers convenient access to numerous bioinformatics resources through a single web portal. The 'Guided Solution Finder' which is available through the HNB portal helps users to locate the appropriate resources to answer their queries by employing a detailed, tree-like questionnaire. Furthermore, automated complex tool cascades ('tasks'), involving resources located on different servers, have been implemented, allowing users to perform comprehensive data analyses without the requirement of further manual intervention for data transfer and re-formatting. Currently, automated cascades for the analysis of regulatory DNA segments as well as for the prediction of protein functional properties are provided. AVAILABILITY: The HNB portal is available at http://www.hnbioinfo.de     

Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a major signaling molecule implicated in the regulation of various ion transporters and channels. Here we show that PIP(2) and intracellular MgATP control the activity of the KCNQ1/KCNE1 potassium channel complex. In excised patch-clamp recordings, the KCNQ1/KCNE1 current decreased spontaneously with time. This rundown was markedly slowed by cytosolic application of PIP(2) and fully prevented by application of PIP(2) plus MgATP. PIP(2)-dependent rundown was accompanied by acceleration in the current deactivation kinetics, whereas the MgATP-dependent rundown was not. Cytosolic application of PIP(2) slowed deactivation kinetics and also shifted the voltage dependency of the channel activation toward negative potentials. Complex changes in the current characteristics induced by membrane PIP(2) was fully restituted by a model originally elaborated for ATP-regulated two transmembrane-domain potassium channels. The model is consistent with stabilization by PIP(2) of KCNQ1/KCNE1 channels in the open state. Our data suggest a striking functional homology between a six transmembrane-domain voltage-gated channel and a two transmembrane-domain ATP-gated channel.     

Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson''s disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson''s disease progression, particularly in the context of bioenergetic dysfunction.Parkinson''s disease (PD), the second most common neurodegenerative disorder, is characterized by impairment of the motor system and associated non-motor clinical manifestations.¹ Age² and exposure to environmental toxins³ constitute the most important non-genetic risk factors in the development of sporadic disease. Neuronal loss is progressive, primarily (but not exclusively) dopaminergic, and accompanied by the accumulation of intracellular proteinaceous inclusions known as Lewy bodies and Lewy neurites.⁴ α-Synuclein (aSyn) is the main protein constituent of these inclusions⁵ and numerous findings attribute to it a central role in the pathogenesis of PD.^{6, 7, 8, 9} Both missense mutations (p.A30P, p.E46K, p.H50Q,¹⁰ p.G51D,¹¹ p.A53T, p.A53E¹²) and increased copy number (duplication¹³ or triplication¹⁴) of the SNCA gene encoding aSyn (PARK1/4 locus) cause early onset autosomal dominant PD. In addition, multiple genome-wide association studies have established that variations at the SNCA locus contribute significantly to the etiology of sporadic disease.^{15, 16, 17}The induced pluripotent stem cell (iPSC) technology offers a unique and valuable tool for defining the early mechanisms underlying PD and the development of early diagnostics and new therapeutics.^{18, 19, 20} Cell lines have been generated from fibroblasts obtained from patients with a variety of neurodegenerative diseases and neurons differentiated therefrom reproduce specific features of those diseases in vitro.²⁰ Comparisons between patient-derived and appropriately selected healthy control lines are feasible, but unfortunately phenotypic differences unrelated to the disease mechanisms arise due to the high clonal variability inherent in the generation of iPSCs and differences in the genetic background of the iPSC lines.^{21, 22, 23} Lines manipulated by single gene mutation have demonstrated the power of iPS technology for disease modeling^{18, 19, 20} with possible therapeutic potential.^{24, 25}We have examined in this study the effects of increased aSyn expression on the differentiation capacity and phenotypic signatures of two iPS clones derived from a patient with a triplication of the SNCA gene, and compared them with (i) lines generated by lentiviral infection of the patient cells by an shRNA construct targeting aSyn, and (ii) two control iPSC lines one from an unaffected age-matched sibling²⁶ and the other from an unrelated healthy individual.²⁷ All lines were differentiated by defined protocols into neurons that exhibited cardinal neuronal markers. These paradigms were used to assess differentiation capacity, cell survival, neurite outgrowth and electrophysiological properties. The results establish aSyn-dosage as an important modulator of developmental fitness of neuronal progenitor cells and support our previous findings from studies of PD patient fibroblasts²⁸ and neural-committed induced pluripotent stem cells (NiPSCs) (including the knockdown lines featured in this report)²⁹ exposed to toxins: (i) quantifiable reduction in viability under starvation and stress and (ii) decreased mitochondrial function and upregulated catabolism.     

Na+ -D-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb d-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts.     

MSC-DC interactions: MSC inhibit maturation and migration of BM-derived DC

BACKGROUND: Mesenchymal stromal cells (MSC) comprise one of the BM stromal cells that are known to support hematopoiesis. It has also been suggested recently that MSC display immunosuppressive capacities through inhibiting the differentiation of monocyte-derived DC. DC travel to the lymph nodes (LN) to present Ag to T cells, and CCL21 is the chemokine that plays an important role in DC migration into the T-cell area of LN. We addressed the effect of MSC on this chemotactic activity of DC, one of the typical characteristics upon maturation. METHODS: BM cells were isolated and then cultured for generation of myeloid DC in the presence of GM-CSF and/or lipopolysaccharide with or without MSC. MSC were identified by flow cytometry of the immunologic markers and by performing colony-forming unit fibroblast assay. Migration of DC was observed with a newly developed time-lapse video microscopic technique. RESULTS: MSC co-culture inhibited the initial differentiation of DC, as well as their maturation. The matured DC actively migrated directionally in response to CCL21, a powerful DC-attracting chemokine, whereas the MSC co-cultured DC did not. DISCUSSION: Collectively, the findings of these experiments raise the possibility that MSC suppress the migratory function of DC and so they may serve immunoregulatory activities through the modulation of the Ag-presenting function of DC.