Reversibility of thiamine deficiency-induced partial necrosis and mitochondrial uncoupling by addition of thiamine to neuroblastoma cell suspensions

Culture of neuroblastoma cells in the presence of low thiamine concentration (6 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired. When 10 µM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h. Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration. Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional. (Mol Cell Biochem 174: 121–124, 1997)     

Temporal Patterns in Sheep Fetal Heart Rate Variability Correlate to Systemic Cytokine Inflammatory Response: A Methodological Exploration of Monitoring Potential Using Complex Signals Bioinformatics

Fetal inflammation is associated with increased risk for postnatal organ injuries. No means of early detection exist. We hypothesized that systemic fetal inflammation leads to distinct alterations of fetal heart rate variability (fHRV). We tested this hypothesis deploying a novel series of approaches from complex signals bioinformatics. In chronically instrumented near-term fetal sheep, we induced an inflammatory response with lipopolysaccharide (LPS) injected intravenously (n = 10) observing it over 54 hours; seven additional fetuses served as controls. Fifty-one fHRV measures were determined continuously every 5 minutes using Continuous Individualized Multi-organ Variability Analysis (CIMVA). CIMVA creates an fHRV measures matrix across five signal-analytical domains, thus describing complementary properties of fHRV. We implemented, validated and tested methodology to obtain a subset of CIMVA fHRV measures that matched best the temporal profile of the inflammatory cytokine IL-6. In the LPS group, IL-6 peaked at 3 hours. For the LPS, but not control group, a sharp increase in standardized difference in variability with respect to baseline levels was observed between 3 h and 6 h abating to baseline levels, thus tracking closely the IL-6 inflammatory profile. We derived fHRV inflammatory index (FII) consisting of 15 fHRV measures reflecting the fetal inflammatory response with prediction accuracy of 90%. Hierarchical clustering validated the selection of 14 out of 15 fHRV measures comprising FII. We developed methodology to identify a distinctive subset of fHRV measures that tracks inflammation over time. The broader potential of this bioinformatics approach is discussed to detect physiological responses encoded in HRV measures.     

Microinjection of glycosaminoglycan-degrading enzymes in the chicken blastoderm

The relationship between the presence of glycosaminoglycans (GAGs) and the morphology of the middle layer or mesoblast was examined by performing transmission electron microscopy of chicken blastoderms microinjected with GAG-degrading enzymes. The controls included microinjections with saline or trypsin, as well as solid-phase assays for proteolytic activity in commercially available GAG-degrading preparations. The results indicate that, in normal as well as in saline-injected blastoderms, middle-layer cells are rounded or cuboidal in shape, and are linked to each other by small intercellular junctions in the primitive-streak region. As they migrate laterally along the basal lamina, they appear as typical mesenchymal cells, being separated by large intercellular spaces and covered by cell processes. The removal of hyaluronate (by the microinjection of hyaluronidases) led to compaction of the middle-layer cells in the area lateral to the primitive streak. These cells lost their mesenchymal aspect and retracted their processes, and intercellular junctions were observed. The presence of proteolytic activity in the enzyme preparations did not interfere with the results. On the basis of the results obtained using this microinjection technique, we were able to confirm at the ultrastructural level that hyaluronate, due to its space-creating properties, promotes the detachment of ingressed primitive-streak cells and preserves the mesenchymal aspect of the middle layer during the lateral migration of single cells along the basal lamina. Whether the presence of hyaluronate is necessary to allow positioning of the mesoblast could not be inferred using our experimental procedure. We present evidence that this molecule, as well as having physicochemical properties, is also involved in the modulation of tissue interactions during gastrulation.     

Isolation of an IS1 flanked kanamycin resistance transposon from R1drd19

Summary We have isolated and identified an IS1-flanked transposon from the plasmid R1drd19. This transposon specifies resistance to kanamycin and is 10.4 kb long. It exhibits a frequency of transposition two orders of magnitude lower than that of the smaller, IS1-flanked transposon Tn9. We have named it Tn2350.     

Development of early erythroid precursors (BFUe) from mouse bone marrow in a serum-free culture media. Effect of hemin

In culture medium containing albumin, iron saturated transferrin, phospholipids, cholesterol and erythropoietin, BFUe growth requires foetal Calf serum. Without serum the BFUe development is advantageously restored by the addition of hemin associated with spleen conditioned medium. In erythropoietin supplemented serum-free medium the growth of primitive erythroid precursor cells is closely dependent on growth factor supply found in spleen conditioned medium. Using this medium allows us to clearly distinguish between the respective effects of erythropoietin and of BPA on erythroid progenitor cells.     

Identification of Two Critically Deleted Regions within Chromosome Segment 7q35-q36 in EVI1 Deregulated Myeloid Leukemia Cell Lines

Chromosomal rearrangements involving the EVI1 proto-oncogene are a recurrent finding in myeloid leukemias and are indicative of a poor prognosis. Rearrangements of the EVI1 locus are often associated with monosomy 7 or cytogenetic detectable deletions of part of 7q. As EVI1 overexpression alone is not sufficient to induce leukemia, loss of a 7q tumour suppressor gene might be a required cooperating event. To test this hypothesis, we performed high-resolution array comparative genomic hybridization analysis of twelve EVI1 overexpressing patients and three EVI1 deregulated cell lines to search for 7q submicroscopic deletions. This analysis lead to the delineation of two critical regions, one of 0.39 Mb on 7q35 containing the CNTNAP2 gene and one of 1.33 Mb on chromosome bands 7q35–q36 comprising nine genes in EVI1 deregulated cell lines. These findings open the way to further studies aimed at identifying the culprit EVI1 implicated tumour suppressor genes on 7q.     

NITZSCHIA ALICAE SP. NOV. AND N. PURIFORMIS SP. NOV., NEW DIATOMS FROM EUROPEAN RIVERS AND COMPARISON WITH THE TYPE MATERIAL OF N. SUBLINEARIS AND N. PURA1

Nitzschia sublinearis Hustedt and N. pura Hustedt are common oligosaprobic freshwater diatom species that frequently occur in diatom inventories, thus being important in water quality studies. Both are considered as species with overlapping diagnostic criteria in several floras, which is typical of the whole genus Nitzschia. The type material of Hustedt of N. sublinearis and N. pura was examined using LM and EM in order to document the range of variation within the type populations and to compare it with populations occurring in different European rivers. Detailed observations allowed recognition of two new freshwater diatom species: N. alicae sp. nov., occurring in mesotrophic up to eutrophic conditions, and N. puriformis sp. nov., mostly occurring in oligotrophic habitats, both in rivers and streams at middle and high altitudes. The most reliable taxonomic features that separate both new species from the most similar taxa are the density of fibulae and striae, valve shape, and valve width as well as the shape of areolae. Morphological examination of different populations indicates that N. puriformis is relatively common in European rivers and has been overlooked to date and confounded with N. pura by several researchers. By contrast, N. alicae has, to date, been collected only in Slovakia and Northern Italy, but with a high frequency of occurrence and sometimes in high abundance at sites.