Ecophysiological factors affecting growth,sporulation and survival of the biocontrol agent Penicillium oxalicum

The effect of temperature, pH, water potential and sources of nitrogen and carbon on the biocontrol agent Penicillium oxalicum were studied in vitro. The fungus is xerotolerant, mesophillic and has a wide pH tolerance. The parameters evaluated (germination, germ tube length, growth rate and sporulation) showed different sensitivities to the environmental factors. Peptone and free amino acids gave the highest growth rates and high levels of sporulation. Xylose, mannose and fructose gave the highest growth rates and mannose induced strong sporulation. The effect of nutrients (mannose + arginine) and water potential was also studied in vivo. The xerotolerant character of the fungus was confirmed. From this study we consider Penicillium oxalicum ecologically competent to perform effectively as a biocontrol agent in the soil environment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

Summary The 10-kb chromosomal fragment of Streptococus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome.A spontaneous deletion beginning 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (10^-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed.Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanil-amide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.     

The anti-fibrotic effect of liver growth factor is associated with decreased intrahepatic levels of matrix metalloproteinases 2 and 9 and transforming growth factor beta 1 in bile duct-ligated rats

Liver growth factor (LGF), a mitogen for liver cells, behaves as an anti-fibrotic agent even in extrahepatic sites, but its mechanistic basis is unknown. We aimed to determine the intrahepatic expression pattern of key modulators of liver fibrosis in bile duct-ligated rats (BDL) after injection of LGF. BDL rats received either LGF (4.5 microg/ratXdose, two doses/week, at time 0 or 2 or 5w after operation, depending on the group (BDL+LGF groups, n=20) or saline (BDL+S groups, n=20). Groups were compared in terms of fibrosis (histomorphometry), liver function (aminopyrine breath test), matrix metalloproteinases MMP-2 and MMP-9, transforming growth factor beta 1 (TGF-beta1) and liver endoglin content (Western blotting), and serum tissue inhibitor of metalloproteinases 1 (TIMP-1) levels (ELISA). In BDL+LGF rats, the fibrotic index was significantly lower at 5w, p=0.006, and at 8w, p=0.04, than in BDL+S rats. Liver function values in BDL+LGF rats were higher than those obtained in BDL+S rats (80% at 5w and 79% at 8w, versus 38% and 29%, p<0.01, taking healthy controls as 100%). Notably, in BDL+LGF rats the intrahepatic expression levels of both MMPs were lower at 2w (MMP-2, p=0.03; MMP-9, p=0.05) and 5w (MMP-2, p=0.05, MMP-9, p=0.04). In addition, the hepatic TGF-beta1 level in BDL+LGF rats was lower at 2w (36%, p=0.008), 5w (50%) and 8wk (37%), whereas intrahepatic endoglin expression remained constant in all BDL rats studied. LGF ameliorates liver fibrosis and improves liver function in BDL rats. The LGF-induced anti-fibrotic effect is associated with a decreased hepatic level of MMP-2, MMP-9 and TGF-beta1 in fibrotic rats.     

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

The Response to Stimulation in Neurons and Astrocytes

Neurochemical Research - The brain uses mainly glucose as fuel with an index of glucose to oxygen utilization close to 6, the maximal index if all glucose was completely oxidized. However, this...     

The Diamine Oxidase Gene Is Associated with Hypersensitivity Response to Non-Steroidal Anti-Inflammatory Drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) are the drugs most frequently involved in hypersensitivity drug reactions. Histamine is released in the allergic response to NSAIDs and is responsible for some of the clinical symptoms. The aim of this study is to analyze clinical association of functional polymorphisms in the genes coding for enzymes involved in histamine homeostasis with hypersensitivity response to NSAIDs. We studied a cohort of 442 unrelated Caucasian patients with hypersensitivity to NSAIDs. Patients who experienced three or more episodes with two or more different NSAIDs were included. If this requirement was not met diagnosis was established by challenge. A total of 414 healthy unrelated controls ethnically matched with patients and from the same geographic area were recruited. Analyses of the SNPs rs17740607, rs2073440, rs1801105, rs2052129, rs10156191, rs1049742 and rs1049793 in the HDC, HNMT and DAO genes were carried out by means of TaqMan assays. The detrimental DAO 16 Met allele (rs10156191), which causes decreased metabolic capacity, is overrepresented among patients with crossed-hypersensitivity to NSAIDs with an OR  = 1.7 (95% CI  = 1.3–2.1; Pc  = 0.0003) with a gene-dose effect (P = 0.0001). The association was replicated in two populations from different geographic areas (Pc  = 0.008 and Pc  = 0.004, respectively).

Conclusions and implications

The DAO polymorphism rs10156191 which causes impaired metabolism of circulating histamine is associated with the clinical response in crossed-hypersensitivity to NSAIDs and could be used as a biomarker of response.     

Characterization of <Emphasis Type="Italic">Penicillium</Emphasis> Species by Ribosomal DNA Sequencing and BOX,ERIC and REP-PCR Analysis

The genus Penicillium is one of the largest and widely distributed fungal genera described to date. As a result, its taxonomic classification and species discrimination within this genus has become complicated. In this study, 52 isolates that belonged to the Penicillum genus and other related genera were characterized using two DNA-based methods: (i) analysis of the nucleotide sequences of internal transcribed spacers in ribosomal DNA and (ii) analysis of DNA fingerprints that were generated by polymerase chain reactions with specific primers for enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) sequences, and BOX elements. Using both methods, Penicillium species were discriminated from other fungal genera. Furthermore, Penicillium species that include strains which are used as biocontrol agents, such as P. glabrum, P. purpurogenum, and P. oxalicum, could be distinguished from other Penicillium species using these techniques. Based on our findings, we propose that a polyphasic approach that includes analysis of the nucleotide sequences of ribosomal DNA and detecting the presence of highly conserved, repeated nucleotide sequences can be used to determine the genetic relationships between different Penicillium species. Furthermore, we propose that our results can be used as a start point to develop a strategy to monitor the environmental presence of particular strains of Penicillium species when they are used as biocontrol agents.     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^−/− mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^−/− mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.