Mixed culture of killer and sensitive Saccharomyces cerevisiae strains in batch and continuous fermentations

This paper presents a kinetic study of the dynamics of the population of two Saccharomyces cerevisiae strains (designated K1 and 522D) in mixed culture. These two strains are commonly used in wine making. The K1 strain (killer yeast) secretes a glycoprotein (killer toxin) which causes the death of the 522D strain (sensitive yeast). Initially, the mixed cultures were realized in batch fermentations. Initial concentrations of killer yeast were 5 and 10% of the total population. The influence of the killer strain on the sensitive cultures was measured in comparison with a reference fermentation. The reference fermentation was inoculated only with the sensitive strain. Results show that an initial concentration of 10% of killer strain affects the microbial population balance and the rate of ethanol production. However the fermentation was only slightly disturbed when the proportion of killer to sensitive yeast at the beginning of mixed culture was 5%. To achieve total displacement by the killer yeast at low concentrations, the mixed cultures were carried out in a continuous system. The results obtained in continuous fermentations with the same strains have shown that a level of contamination as low as 0.8% of killer strain was sufficient to completely displace the original sensitive population after 150 h incubation.     

Reovirus intermediate subviral particles constitute a strategy to infect intestinal epithelial cells by exploiting TGF‐β dependent pro‐survival signaling

Intestinal epithelial cells (IECs) constitute the primary barrier that separates us from the outside environment. These cells, lining the surface of the intestinal tract, represent a major challenge that enteric pathogens have to face. How IECs respond to viral infection and whether enteric viruses have developed strategies to subvert IECs innate immune response remains poorly characterized. Using mammalian reovirus (MRV) as a model enteric virus, we found that the intermediate subviral particles (ISVPs), which are formed in the gut during the natural course of infection by proteolytic digestion of the reovirus virion, trigger reduced innate antiviral immune response in IECs. On the contrary, infection of IECs by virions induces a strong antiviral immune response that leads to cellular death. Additionally, we determined that virions can be sensed by both TLR and RLR pathways while ISVPs are sensed by RLR pathways only. Interestingly, we found that ISVP infected cells secrete TGF‐β acting as a pro‐survival factor that protects IECs against virion induced cellular death. We propose that ISVPs represent a reovirus strategy to initiate primary infection of the gut by subverting IECs innate immune system and by counteracting cellular‐death pathways.     

GDF-15 Is Elevated in Children with Mitochondrial Diseases and Is Induced by Mitochondrial Dysfunction

BackgroundWe previously described increased levels of growth and differentiation factor 15 (GDF-15) in skeletal muscle and serum of patients with mitochondrial diseases. Here we evaluated GDF-15 as a biomarker for mitochondrial diseases affecting children and compared it to fibroblast-growth factor 21 (FGF-21). To investigate the mechanism of GDF-15 induction in these pathologies we measured its expression and secretion in response to mitochondrial dysfunction.MethodsWe analysed 59 serum samples from 48 children with mitochondrial disease, 19 samples from children with other neuromuscular diseases and 33 samples from aged-matched healthy children. GDF-15 and FGF-21 circulating levels were determined by ELISA.ResultsOur results showed that in children with mitochondrial diseases GDF-15 levels were on average increased by 11-fold (mean 4046pg/ml, 1492 SEM) relative to healthy (350, 21) and myopathic (350, 32) controls. The area under the curve for the receiver-operating-characteristic curve for GDF-15 was 0.82 indicating that it has a good discriminatory power. The overall sensitivity and specificity of GDF-15 for a cut-off value of 550pg/mL was 67.8% (54.4%-79.4%) and 92.3% (81.5%-97.9%), respectively. We found that elevated levels of GDF-15 and or FGF-21 correctly identified a larger proportion of patients than elevated levels of GDF-15 or FGF-21 alone. GDF-15, as well as FGF-21, mRNA expression and protein secretion, were significantly induced after treatment of myotubes with oligomycin and that levels of expression of both factors significantly correlated.ConclusionsOur data indicate that GDF-15 is a valuable serum quantitative biomarker for the diagnosis of mitochondrial diseases in children and that measurement of both GDF-15 and FGF-21 improves the disease detection ability of either factor separately. Finally, we demonstrate for the first time that GDF-15 is produced by skeletal muscle cells in response to mitochondrial dysfunction and that its levels correlate in vitro with FGF-21 levels.     

Hemolytic,cytotoxic and complement inactivating properties of extracts of different species of Aspergillus

Some of the biological properties of saline extracts of the mycelia of several species of the Aspergillus genus, namely, A. fumigatus, A. flavus, A. niger, A. nidulans, A. parasiticus and A. glaucus, were studied. Only the extract prepared from A. fumigatus was found to be hemolytic for sheep red blood cells. In contrast, all the extracts with the only exception of that of A. glaucus, had cytotoxic effects on Vero cells. Both, the hemolytic and cytotoxic constituents of the extracts were removable by adsorption with activated carbon. Heating of the extracts at 100°C for 30 minutes also resulted in detoxification. In vivo studies, performed only with detoxified extracts of A. fumigatus, showed these were capable of depleting complement levels in guinea pigs. Complement inactivation was also found to occur in vitro and was caused by all the extracts tested. Also triggered by the extracts was the conversion of serum C3 but not of purified C3, indicating that other serum factors are essential in the process. Despite the similarity in this respect with cobra venom factor, differences in activity after heating-negative in cobra venom factor-indicate that the complement inactivating substance/s present in the Aspergillus extracts differ from those of the snake product.     

Occurrence of 5''-Deoxy-5''-Methylthioadenosine Phosphorylase in the Mammalian CNS: Distribution and Kinetic Studies on the Rat Brain Enzyme

5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase catalyzes the cleavage of MTA, a secondary product of polyamine biosynthesis, to 5-methylthioribose-1-phosphate and adenine. The occurrence and the general properties of the enzyme were studied in mammalian brain with the following results. (1) Cerebral tissues contained levels of MTA phosphorylase that were comparable to those occurring in other mammalian tissues. (2) Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in pig brain. Moreover, the enzyme seemed to be generally more concentrated in the cerebellar fractions. (3) Rat brain MTA phosphorylase was highly localized in the cellular soluble fraction. In the first days of rat life, its specific activity in the whole brain was observed to decline significantly from a value of 17.6 units/mg at 1-5 days of age to 13.7 units/mg at 6-10 days of age, remaining then fairly constant up to maturity. (4) Kinetic studies performed with the soluble enzyme extracted from rat brain showed: a pH optimum of 7.4; a Km value for MTA of about 10 microM; an inhibitory effect of the MTA analog 5'-deoxy-5'-isobutylthioadenosine; and a remarkable resistance of the enzyme to heat treatment.     

Developmental Regulation of β-Thymosins in the Rat Central Nervous System

HPLC analysis of guanidinium hydrochloride extracts of neonatal and adult rat brain revealed a polypeptide that is present in high concentration in the immature nervous system, but whose levels decline dramatically in the adult. This polypeptide has been isolated and its complete amino acid sequence determined by gas-phase Edman degradation following specific chemical and enzymatic cleavages. The molecule is identified as thymosin beta 10, a member of a multigene family that encodes a structurally conserved series of small acidic polypeptides of uncertain function. Thymosin beta 10 is present in the developing nervous system as early as embryonic day 9. Levels subsequently increase to peak values between embryonic day 15 and postpartum day 3, before falling to adult values (about a 20-fold reduction) by postpartum day 14. The elevated levels of thymosin beta 10 in fetal and neonatal brain correlate with high levels of thymosin beta 10 mRNA, whereas the low values of the polypeptide in the adult and juvenile are mirrored by an approximate 15-fold reduction in specific mRNA. In comparison, the levels of thymosin beta 4 polypeptide, a homologue of thymosin beta 10, only decline by about 20% during the same developmental period. However, the mRNA encoding thymosin beta 4 is elevated in fetal brain, and its levels decrease approximately four-fold to a stable value around the time of birth. The reason for this discrepancy between thymosin beta 4 protein and mRNA levels is unknown. Thymosin beta 10 can also be detected by HPLC in fetal liver, where levels are approximately 5% of those in brain. In liver, thymosin beta 10 also declines following birth. It is concluded that beta-thymosin expression (as measured by steady-state mRNA and polypeptide levels) is both up- and down-regulated during different phases of maturation of the mammalian nervous system.     

Monoclonal antibody-defined surface markers of effector cells involved in human monocyte cytotoxicity

Human adherent peripheral blood mononuclear cells were cytotoxic in vitro against the murine TU5 line in a 48-hr [3H]thymidine-release assay. Monocyte-enriched adherent cell preparations contain a small and variable (usually less than 5%) contamination with large granular lymphocytes as assessed by morphology and staining with monoclonal antibody markers B73.1 and HNK1. To assess whether killing was in fact mediated by monocytes, mononuclear cells or monocyte-enriched preparations were separated using monoclonal antibodies directed against mononuclear phagocytes (Mo2, UCHM1, B44.1) or natural killer (NK) cells (B73.1 and HNK1), and a fluorescence-activated cell sorter. Cells positive for monocyte markers were highly cytotoxic against TU5, whereas negative cells were not. B73.1+ or HNK1+ cells had little or no activity. Cytotoxicity of cells positive for monocyte markers (Mo2, UCHM1, B44.1) was augmented by in vitro exposure to lymphokines or less frequently to interferon (IFN). However, cells negative for these monocytes markers were also stimulated to kill TU5 by lymphokine or IFN to an extent similar or greater than that of positive ones. IFN or lymphokines induced killing of TU5 by monocyte-depleted, B73.1-positive, lymphoid cells. These observations demonstrate that human monocytes do kill tumor cells, either in the absence of deliberate stimulation or after exposure to agents such as lymphokines. However, the possible contribution to "monocyte" cytotoxicity of minor NK cell contaminants must be taken into account particularly when agents such as IFN and lymphokines are applied, even when a relatively NK-cell-resistant target such as TU5 is used.     

Lack of TdT and immunoglobulin and T-cell receptor gene rearrangements in Hodgkin's disease

To study the pathogenesis of Hodgkin's disease (HD), which today remains obscure, we have undertaken a combined experimental approach: determination of TdT and molecular analysis of rearrangements of immunoglobulin heavy chain (IgH), T-cell receptor (TCR) beta chain and the T-cell rearranging gamma (TRG) genes. TdT determination indicate would the presence of immature cells that are not detected in the normal lymphnode; molecular analysis of the rearrangements of these genes would reveal the presence of even a small monoclonal population of both T and B lineages in the lymphnodes. We believe that the combination of these two types of analysis can indicate whether an expanding lymphoid clone is responsible for this disease. TdT determination was negative in all 41 cases tested. Gene rearrangements were studied in 10 cases for IgH and TCR beta genes and in 5 cases for the TRG gene. No abnormal band beside the germ-line ones was detected in any of our cases, ruling out the presence of a minor neoplastic population. We can explain these results in at least three ways: first, the neoplastic population could represent less than 1% of the total, thus escaping detection by current techniques; second, the neoplastic population is not lymphoid in nature or is composed of mature cells that do not rearrange Ig and TCR genes and therefore belongs to a true non-B, non-T lineage; third, the pathogenesis of HD is completely different from that of non-Hodgkin's lymphomas (NHL) and does not involve the clonal expansion of a cell frozen at a particular maturative stage as is thought to happen in most NHL.