M13 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp.

The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.     

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement.

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.     

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix

Naringenin (NGEN), a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth. It has been also shown to access the brain and there is an increasing interest in its therapeutic applications. The up-regulated expression of Cx43 leads to the suppression of tumorigenicity with promoted apoptotic events. In this study, we investigated the in vivo effect of NGEN in fostering apoptosis in cerebrally implanted C6 glioma cells rat model. We analysed the expression of Cx43, caspase-3, caspase-9, Cyt C, Bcl-2 and Bax in vivo by immunoblot analysis and the ultra structure of brain cells by transmission electron microscopy. Supplementation of NGEN to experimental animals modulated Bcl-2/Bax ratio and up-regulation of caspase-3 and 9. NGEN was also found to up-regulate the expression of Cx43. These findings provide evidence that NGEN’s apoptotic effect, modulation of Bcl-2/Bax ratio leads to release of Cyt C from mitochondria, thereby activation of caspase-3 and caspase-9 is mediated by enhanced expression of Cx43. These observations were well supported by the transmission electron microscopic results which showed the characteristic apoptotic features. In conclusion, our findings demonstrate that NGEN promotes apoptosis in rat C6 glioma model.     

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.     

Effect of carbon disulphide on the cardiovascular system

Biochemical, histological and electron-microscopic examinations of the heart and the aorta of albino rats exposed to carbon disulphide in concentrations of 10, 50, 100 and 200 mg . m-3 were carried out. Changes in the metabolic and energetic processes in the myocardium and in the quantitative and qualitative characteristics of the connective tissue of the aortal vessel wall were observed. The established disorders follow the dose-effect dependence. The authors studied the effect of carbon disulphide in concentrations of 10 and 50 mg . m-3 on the electrophoretic spectrum of serum proteins, the quantitative and qualitative characteristics of fibrous structures in the myocardium and the aorta of albino rats fed on atherogenic diet (cholesterol, cholic acid and vitamin D2). The combination of carbon disulphide with the atherogenic factor leads to intensification of changes in the cardiovascular system and in serum proteins, observed after independent exposure to either of the factors. The combination of atherogenic diet and 10 mg . m-3 carbon disulphide induced symptoms of intoxication, decreased survival of the animals, and the developing sclerotic process was found to be more severe and to progress more rapidly than in animals subject to atherogenic diet alone. The obtained results testify to cardiovascular effect of carbon disulphide. The observed atherogenic effect of carbon disulphide in low concentrations is most probably connected with its direct effect on the myocardium and the vessels.     

Elevational gradients during the Late-Glacial/Holocene vegetational transition in southern Bulgaria

The Late Glacial and early-Holocene vegetational history of a newly dated pollen and macrofossil diagram from Besbog, a cirque lake at 2250 m just above the forest limit in the Pirin Mountains of southwestern Bulgaria, is compared with a newly dated pollen diagram for the mire Shiroka Polyana at 1400 m in the conifer forest of the nearby Rhodope Mountains in order to investigate the chronology of major changes in the vegetation at different elevations. In the Lake Besbog record the non-arboreal pollen assemblage of the Late Glacial changed abruptly to that of Betula, Quercus and other deciduous types. The date for this change is about 11.6 ka cal b.p. The Quercus assemblage may be composed of pollen blown from intermediate elevations, to which deciduous forest had expanded because of higher summer temperatures related to high summer insolation. At Shiroka Polyana (1400 m) in the modern conifer belt, a similar change did not occur until about 8.8 ka cal b.p. The persistence of the dry steppe or steppe forest in the early Holocene at this lower site can also be attributed to high summer insolation. Thus as atmospheric temperature increased at the end of the Late Glacial, deciduous forests expanded first at intermediate elevations in the Pirin Mountains and only later in the Rhodope Mountains at lower elevations as summer insolation decreased.     

A mutation at the Ala122 position of acetohydroxyacid synthase (AHAS) located on chromosome 6D of wheat: improved resistance to imidazolinone and a faster assay for marker assisted selection

A new mutation at the acetohydroxyacid synthase (AHAS) locus on chromosome 6D of wheat was analyzed in detail because it conferred an improved resistance to the imidazolinone group of herbicides. Sequence analysis showed that the mutation was at the Ala122 position (A122T), a position in AHAS which has not to date been identified in imidazolinone resistant wheat lines even though the position has been identified in other plants and is associated with resistance. An allele-specific assay for the mutation (in the wheat line Brookton-8) was developed and used in a genetic analysis. Two mapping populations were analysed and the doubled haploid progeny from the cross Brookton-8 × Clearfield STL proved to be most informative. The AHAS^Ala122 mutation (A122T) was allelic to the AHAS^Ser653 mutation (S653N) in Clearfield STL (Imi1, on chromosome 6D) and hence was assigned to the chromosome 6D locus. The analysis of the doubled haploid lines in the mapping population demonstrated the greater resistance conferred by the A122T mutation because lines from the same cross and carrying either the A122T or S653N mutations could be directly compared. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role of antioxidant enzymes in survival of conidiospores of Aspergillus niger 26 under conditions of temperature stress

AIMS: A better understanding of the role of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) in the protection of Aspergillus niger spores against thermal stress. METHODS AND RESULTS: Conidiospores from A. niger 26 were subjected to wide range of temperatures (30, 50, 60 and 80 degrees C). The stress response was investigated by the determination of spore germination and mycelial growth of survivors under submerged cultivation. Exposure to any temperature above the optimal value induced an increase in SOD and CAT activities. PAGE demonstrated enhanced level of Cu/ZnSOD under stress conditions. We compared the influence of heat shock and superoxide-generating agent paraquat on growth and antioxidant enzyme defence and found different response to the both type of stresses. CONCLUSIONS: Heat stress elicits the enhanced synthesis of enzymes whose functions are to scavenge reactive oxygen species. These results suggested an association between thermal and oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence is provided for the possibility that oxidative stress plays a major role in the effect of heat in low eucaryotes such as A. niger. This knowledge may be of importance in controlling both fermentation and pathogenicity.     

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as “genomic storm” in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous “danger” signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.     

Testing biological activity of model Maillard reaction products: studies on gastric smooth muscle tissues

Water-soluble Maillard reaction products obtained from five different model systems were investigated for their effects upon the mechanical activity of rat gastric smooth muscle. Most of the total Maillard reaction products applied at concentration of 1.5 mg/ml evoked contractions; among them the product obtained from arginine and glucose (Arg-Glc) produced the most powerful contractions. The product obtained from glycine and ascorbic acid (Gly-AsA) was the only one that brought about relaxation response. The high molecular weight fractions (>3,500 Da) isolated from the reaction systems Arg-Glc and Gly-AsA demonstrated effects similar in type and amplitude to those evoked by non-fractioned reaction products. The results obtained suggest that moieties of molecules acting upon the muscle tonus originate mainly from lysine and arginine residues; that these structures are available in both low and high molecular pools in similar concentrations, and most likely these fragments act upon membrane-located cellular structures involved in calcium transport.