The Sigma-1 Receptor Is a Novel Target for Improving Cold Preservation in Rodent Kidney Transplants.

Kidney transplantation is the preferred treatment for patients with end-stage kidney disease. Maintaining organ viability between donation and transplantation, as well as minimizing ischemic injury, are critically important for long-term graft function and survival. Moreover, the increasing shortage of transplantable organs is a considerable problem; thus, optimizing the condition of grafts is a pivotal task. Here, rodent models of kidney transplantation and cold storage were used to demonstrate that supplementation of a preservation solution with Sigma-1 receptor (S1R) agonist fluvoxamine (FLU) reduces cold and warm ischemic injury. Post-transplant kidney function was improved, histological injury was mitigated, and mRNA expression of two tubular injury markers-kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin-was robustly reduced. In addition, renal inflammation was diminished, as shown by reduced leukocyte infiltration and pro-inflammatory cytokine expression. In the cold ischemia model, FLU ameliorated structural injury profoundly after 2 h as well as 24 h. The reduced number of TUNEL-positive and Caspase 3-positive cells suggests the anti-apoptotic effect of FLU. None of these beneficial effects of FLU were observed in S1R-/- mice. Of note, organ damage in FLU-treated kidneys after 24 h of cold storage was similar to just 2 h without FLU. These results indicate that S1R agonists can prolong storage time and have great potential in improving organ preservation and in alleviating the problem of organ shortages.

The role of Sigma-1 receptor in sex-specific heat shock response in an experimental rat model of renal ischaemia/reperfusion injury.

We previously showed that female rats are more protected against renal ischaemia/reperfusion (I/R) injury than males, which is partly attributed to their more pronounced heat shock response. We recently described that Sigma-1 receptor (S1R) activation improves postischaemic survival and renal function. 17ß-estradiol activates S1R, thus here we investigated the role of sex-specific S1R activation and heat shock response in severe renal I/R injury. Proximal tubular cells were treated with 17ß-estradiol, which caused direct S1R activation and subsequent induction of heat shock response. Uninephrectomized female, male and ovariectomized female (Ovx) Wistar rats were subjected to 50-min renal ischaemia followed by 2 (T2) and 24 (T24) hours of reperfusion. At T24 renal functional, impairment was less severe and structural damage was less prominent in females versus males or Ovx. Postischaemic increase in S1R, pAkt, HSF-1, HSP72 levels were detected as early as at T2, while pHSP27 was elevated later at T24. Abundance of heat shock proteins was higher in healthy female rats and remained higher at T2 and T24 (female versus male or Ovx; resp.). We propose a S1R-dependent mechanism, which contributes to the relative renoprotection of females after I/R injury by enhancing the heat shock response.

σ1-Receptor Agonism Protects against Renal Ischemia-Reperfusion Injury.

Mechanisms of renal ischemia-reperfusion injury remain unresolved, and effective therapies are lacking. We previously showed that dehydroepiandrosterone protects against renal ischemia-reperfusion injury in male rats. Here, we investigated the potential role of σ1-receptor activation in mediating this protection. In rats, pretreatment with either dehydroepiandrosterone or fluvoxamine, a high-affinity σ1-receptor agonist, improved survival, renal function and structure, and the inflammatory response after sublethal renal ischemia-reperfusion injury. In human proximal tubular epithelial cells, stimulation by fluvoxamine or oxidative stress caused the σ1-receptor to translocate from the endoplasmic reticulum to the cytosol and nucleus. Fluvoxamine stimulation in these cells also activated nitric oxide production that was blocked by σ1-receptor knockdown or Akt inhibition. Similarly, in the postischemic rat kidney, σ1-receptor activation by fluvoxamine triggered the Akt-nitric oxide synthase signaling pathway, resulting in time- and isoform-specific endothelial and neuronal nitric oxide synthase activation and nitric oxide production. Concurrently, intravital two-photon imaging revealed prompt peritubular vasodilation after fluvoxamine treatment, which was blocked by the σ1-receptor antagonist or various nitric oxide synthase blockers. In conclusion, in this rat model of ischemia-reperfusion injury, σ1-receptor agonists improved postischemic survival and renal function via activation of Akt-mediated nitric oxide signaling in the kidney. Thus, σ1-receptor activation might provide a therapeutic option for renoprotective therapy.

The homeobox BcHOX8 gene in Botrytis cinerea regulates vegetative growth and morphology.

Filamentous growth and the capacity at producing conidia are two critical aspects of most fungal life cycles, including that of many plant or animal pathogens. Here, we report on the identification of a homeobox transcription factor encoding gene that plays a role in these two particular aspects of the development of the phytopathogenic fungus Botrytis cinerea. Deletion of the BcHOX8 gene in both the B. cinerea B05-10 and T4 strains causes similar phenotypes, among which a curved, arabesque-like, hyphal growth on hydrophobic surfaces; the mutants were hence named Arabesque. Expression of the BcHOX8 gene is higher in conidia and infection cushions than in developing appressorium or mycelium. In the Arabesque mutants, colony growth rate is reduced and abnormal infection cushions are produced. Asexual reproduction is also affected with abnormal conidiophore being formed, strongly reduced conidia production and dramatic changes in conidial morphology. Finally, the mutation affects the fungus ability to efficiently colonize different host plants. Analysis of the B. cinerea genome shows that BcHOX8 is one member of a nine putative homeobox genes family. Available gene expression data suggest that these genes are functional and sequence comparisons indicate that two of them would be specific to B. cinerea and its close relative Sclerotinia sclerotiorum.

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma.

BACKGROUND: Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma. RESULTS: Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei. CONCLUSIONS: The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizoctonia solani, the causal agent of rice sheath blight.

In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.

Infectious keratitis caused by Aspergillus tubingensis.

PURPOSE: To report 2 cases of keratomycosis caused by Aspergillus tubingensis. METHODS: The therapeutic courses were recorded for 2 male patients, 52 and 78 years old, with fungal keratitis caused by black Aspergillus strains. Morphological examination of the isolates was carried out on malt extract agar plates. A segment of the beta-tubulin gene was used for molecular identification. Antifungal susceptibilities were determined by the E test method for molds and the broth microdilution technique National Committee for Clinical Laboratory Standards M38-A. RESULTS: A 52-year-old man presented with complaints of pain and redness in the right eye. The patient was successfully treated with natamycin and econazole eyedrops, itraconazole eye ointment, and oral ketoconazole. A 78-year-old man presented with total corneal necrosis in the right eye. A therapeutic keratoplasty was performed, and topical natamycin and econazole were applied. At the postoperative visit after 3 weeks, almost the full corneal graft was clear with formed anterior chamber. Black Aspergillus strains were isolated from the corneal scrapings of both cases and initially identified as Aspergillus niger based on culture characteristics. Sequence analysis of a segment of the beta-tubulin gene revealed that the isolates are representatives of A. tubingensis. CONCLUSIONS: Aspergillus tubingensis is closely related with A. niger, the differentiation of these 2 species is difficult by classical morphological criteria. To our knowledge, the presented cases of fungal keratitis are the first reports on ocular infection caused by A. tubingensis.

Alternative reproductive strategies of Hypocrea orientalis and genetically close but clonal Trichoderma longibrachiatum, both capable of causing invasive mycoses of humans.

The common soil fungus Trichoderma (teleomorph Hypocrea, Ascomycota) shows increasing medical importance as an opportunistic human pathogen, particularly in immunocompromised and immunosuppressed patients. Regardless of the disease type and the therapy used, the prognosis for Trichoderma infection is usually poor. Trichoderma longibrachiatum has been identified as the causal agent in the majority of reported Trichoderma mycoses. As T. longibrachiatum is very common in environmental samples from all over the world, the relationship between its clinical and wild strains remains unclear. Here we performed a multilocus (ITS1 and 2, tef1, cal1 and chit18-5) phylogenetic analysis of all available clinical isolates (15) and 36 wild-type strains of the fungus including several cultures of its putative teleomorph Hypocrea orientalis. The concordance of gene genealogies recognized T. longibrachiatum and H. orientalis to be different phylogenetic species, which are reproductively isolated from each other. The majority of clinical strains (12) were attributed to T. longibrachiatum but three isolates belonged to H. orientalis, which broadens the phylogenetic span of human opportunists in the genus. Despite their genetic isolation, T. longibrachiatum and H. orientalis were shown to be cosmopolitan sympatric species with no bias towards certain geographical locations. The analysis of haplotype association, incongruence of tree topologies and the split decomposition method supported the conclusion that H. orientalis is sexually recombining whereas strict clonality prevails in T. longibrachiatum. This is a rare case of occurrence of sexual reproduction in opportunistic pathogenic fungi. The discovery of the different reproduction strategies in these two closely related species is medically relevant because it is likely that they would also differ in virulence and/or drug resistance. Genetic identity of environmental and clinical isolates of T. longibrachiatum and H. orientalis suggests the danger of nosocomial infections by Hypocrea/Trichoderma and highlights the need for ecological studies of spore dispersal as source of invasive human mycoses.

Purification and preliminary characterization of a cold-adapted extracellular proteinase from Trichoderma atroviride.

Eleven cold-tolerant Trichoderma isolates were screened for the production of proteolytic activities at 10 degrees C. Based on the activity profiles determined with paranitroanilide substrates at 5 degrees C, strain T221 identified as Trichoderma atroviride was selected for further investigations. The culture broth of the strain grown at 10 degrees C in casein-containing culture medium was concentrated by lyophilization and subjected to gel filtration, which was followed by chromatofocusing of the fraction showing the highest activity on N-benzoyl-Phe-Val-Arg-paranitroanilide. The purified enzyme had a molecular weight of 24 kDa, an isoelectric point of 7.3 and a pH optimum of 6.2. The temperature optimum of 25 degrees C and the low thermal stability suggested that it is a true cold-adapted enzyme. Substrate specificity data indicate that the enzyme is a proteinase with a preference for Arg or Lys at the P1 position. The effect of proteinase inhibitors suggests that the enzyme has a binding pocket similar to the one present in trypsin.

[Neonatal lupus erythematosus: case report and review of the literature]. / Neonatalis lupus erythematosus: irodalmi összefoglalás egy esetbemutatás kapcsán.

Neonatal lupus erythematosus (NLE) is a disease of the first few months of infancy. It is caused by anti-SSA and anti-SSB antibodies, which are products of maternal autoimmune disorders (SLE, Sjögren, rheumatoid arthritis) and can be passively transported across the placenta. The prevalence of NLE is low. The major clinical findings are cutaneous (typical annular erythematous plaques), cardiac, hepatic and hematologic alterations. Its most severe consequence is third-degree heart block, which is irreversible, requires pacemaker-implantation and responsible for the 20-30% mortality rate. Symptoms usually resolve spontaneously at age of 6-9 months in association with disappearance of maternal antibodies from the infant's serum. In our case the typical cutaneous manifestations covered virtually the whole body, were present at birth, however, no conduction defects developed. The fact that the mother's sickness was not known at birth made it difficult to establish the diagnosis. The significant thrombocytopaenia, progressive skin-changes and the elevated liver function tests necessitated systemic steroid treatment.

Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion.

The sensitivity of two cold-tolerant Trichoderma strains belonging to the species T. harzianum and T. atroviride was determined to a series of pesticides widely used in agriculture. From the 16 pesticides tested, seven fungicides: copper sulfate, carbendazim, mancozeb, tebuconazole, imazalil, captan and thiram inhibited colony growth of the test strains significantly with minimal inhibitory concentrations of 300, 0.4, 50, 100, 100, 100 and 50 microg/ml, respectively. Mutants resistant to carbendazim and tebuconazole were produced from both wild type strains by means of UV-mutagenesis. The cross-resistance capabilities and in vitro antagonistic properties of the mutants were determined. Carbendazim-resistant mutants showed total cross-resistance to benomyl and thiabendazole at a concentration of 20 microg/ml. Intraspecific protoplast fusion was carried out between carbendazim- and tebuconazole-resistant mutants of both parental strains, and putative haploid recombinants with stable resistance to both pesticides were produced in the case of T. atroviride. These pesticide-polyresistant progenies are potential candidates for application in an integrated pest management system.

Intraspecific mitochondrial DNA polymorphism within the emerging filamentous fungal pathogen Trichoderma longibrachiatum.

The genetic diversity of the emerging fungal pathogen Trichoderma longibrachiatum was examined at the level of mitochondrial DNA. The 17 investigated strains, comprising nine clinical and eight non-clinical isolates, exhibited seven and ten different mitochondrial DNA profiles by using the restriction enzymes BsuRI and Hin6I, respectively. The sizes of mitochondrial DNAs varied from 34.9 to 39.5 kb. The discriminatory power of the method was higher than that of internal transcribed spacer sequence analysis and therefore should be more suitable for identification and epidemiological investigations. However, clinical and non-clinical isolates did not form separate clusters on the resulting dendrogram and thus there was no indication of a correlation between genetic structure and pathogenicity of the isolates.

Peptaibols and related peptaibiotics of Trichoderma. A review.

Peptaibols and the related peptaibiotics are linear, amphipathic polypeptides. More than 300 of these secondary metabolites have been described to date. These compounds are composed of 5-20 amino acids and are generally produced in microheterogeneous mixtures. Peptaibols and peptaibiotics with unusual amino acid content are the result of non-ribosomal biosynthesis. Large multifunctional enzymes known as peptide synthetases assemble these molecules by the multiple carrier thiotemplate mechanism from a remarkable range of precursors, which can be N-methylated, acylated or reduced. Peptaibols and peptaibiotics show interesting physico-chemical and biological properties including the formation of pores in bilayer lipid membranes, as well as antibacterial, antifungal, occasionally antiviral activities, and may elicit plant resistance. The three-dimensional structure of peptaibols and peptaibiotics is characterized predominantly by one type of the helical motifs alpha-helix, 3(10)-helix and beta-bend ribbon spiral. The aim of this review is to summarize the data available about the biosynthesis, biological activity and conformational properties of peptaibols and peptaibiotics described from Trichoderma species.

Extracellular proteases of Trichoderma species. A review.

Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

The interaction between resistance modifiers such as pyrido[3,2-g]quinoline, aza-oxafluorene and pregnane derivatives with DNA, plasmid DNA and tRNA.

Various resistance mechanisms such as complex formation with DNA, tRNA and MDR1 p-glycoprotein were modified in bacteria and cancer cells in presence of pregnane, pyridoquinoline, and aza-oxafluorene derivatives. Interaction between the compounds, plasmid DNA and tRNA was shown and compared to the interaction with calf thymus DNA. Complex formation with MDR1 p-glycoprotein and drug accumulation increased in cancer cells. Both plasmid DNA and p-gp complex formation were related to the chemical structures of the resistance modifiers.

Comparative study of potential virulence factors in human pathogenic and saprophytic Trichoderma longibrachiatum strains.

Potential virulence factors of 9 saprophytic and 12 clinical Trichoderma longibrachiatum strains were examined in the present study, in order to compare their capacity to cause infection in humans. All of the strains were able to grow at temperatures up to 40 degrees C and at pH values ranging from 2.0 to 9.0. Carbon and nitrogen source utilization experiments revealed that all of the strains were able to utilize a series of basic amino acids both as sole carbon and nitrogen sources. The MIC values of the tested antifungal drugs were found to be 0.016-8 microg/ml for amphotericin B, 64-256 microg/ml for fluconazole, 0.5-32 microg/ml for itraconazole and 0.008-1 microg/ml for ketoconazole in the case of the examined isolates. Metabolites of the strains inhibited the growth of different bacteria, furthermore, compounds produced by three clinical isolates reduced the motility of boar spermatozoa, indicating their toxicity to mammalian cells as well. On the whole, there were no significant differences in the examined features between strains derived from clinical or soil samples. The question, however, whether all environmental Trichoderma longibrachiatum strains have the capacity to cause infections or not, remains still unanswered.

Etest for assessing the susceptibility of filamentous fungi.

The purpose of this study was to evaluate the Etest as an in vitro antifungal susceptibility test method for different moulds originating from human samples and from the environment. A total of 50 isolates (1 Acremonium, 18 Aspergillus, 2 Cladosporium, 1 Epicoccum, 15 Penicillium, 2 Scopulariopsis and 11 Trichoderma strains) were tested by the Etest. Forty-six of the tested moulds (92%) were resistant to fluconazole with minimal inhibitory concentrations (MICs) > or = 256 microg ml(-1). There were strains resistant to ketoconazole among Aspergillus niger, A. ochraceus and Cladosporium spp. with MICs > 32 microg ml(-1). For fluconazole, no differences were observed using two different inocula, while for itraconazole, ketoconazole and amphotericin B, a 1 or less step 2-fold dilution difference in MIC was seen for the most of 10 selected strains. The MICs of fluconazole and amphotericin B obtained for Trichoderma strains by the Etest and the agar dilution method were also compared. MICs for fluconazole were in agreement, while MICs for amphotericin B were higher with 1 or 2 steps of 2-fold dilutions for most of Trichoderma strains in the case of the agar dilution method.

Production of extracellular proteases by human pathogenic Trichoderma longibrachiatum strains.

Species belonging to the filamentous fungal genus Trichoderma are well known as potential candidates for the biological control of plant pathogenic fungi and as cellulase producers of biotechnological importance. Several data were published in the last decade also about the clinical importance of this genus, indicating that Trichoderma strains may be potential opportunistic pathogens in immunocompromised patients. However, there is a lack of information about the potential virulence factors of clinical Trichoderma strains. This study was designed to examine the extracellular proteolytic enzymes of six clinical T. longibrachiatum isolates. Supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. The production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities cleaving N-Benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide, N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, and N-Succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, respectively, was common among the strains examined. Separation of trypsin- and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. The pH-dependence of these two protease systems was also studied. Based on the results, the different isoenzymes seem to have different optimal pH values. Extracellular proteolytic enzymes may be involved in the pathogenecity of Trichoderma strains as facultative human pathogens.

Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags.

We report on the development of a new PCR technique for the isolation of genomic fragments that flank known DNA sequences. This technique, single oligonucleotide nested (SON)-PCR, relies on only two amplification reactions with two or three nested sequence-specific primers. It allows the isolation of DNA regions located on either side of a known DNA sequence, with high specificity. DNA products of 2 kb in size can be generated that all contain one copy of the same primer at both ends. Sequence analysis of these products indicates that the binding of the primers to non-specific DNA sites mainly depends on their overall complementarity to the target sequence. Moreover, analysis shows that short extensions of the primers can occur during the first amplification reaction and that a 2-bp overlap between subsequent primers can target their annealing to their predecessor's sequence. Ninety percent of the DNA products larger than 0.5 kb correspond to fragments of interest and we obtained successful results with various templates and primer sets. SON-PCR therefore seems a very efficient and widely applicable method for the rapid identification of large unknown DNA regions. Based on available expressed sequence tags, this technique was applied to isolate the palH and pacC genes of the phytopathogenic fungus Botrytis cinerea, with their 5' or 3' flanking regions.