Addition Of Cycloheximide Research Articles

Effect of the antagonistic yeast Hannaella sinensis on the degradation of Patulin

Patulin (PAT) is a toxic fungal metabolite that is widely distributed in food and poses a serious health risk to humans and animals. Hannaella sinensis has been found to be effective in controlling post-harvest diseases of apples. However, it is not known whether H. sinensis can degrade PAT. The aim of this study was to investigate the degradation efficacy of PAT by H. sinensis and the possible mechanisms involved. The experimental results showed that H. sinensis degraded 100 % of PAT in NYDB medium within 30 h. The living cells of H. sinensis did not absorb PAT, and the cell wall and extracellular metabolites of H. sinensis were not involved in the degradation of PAT. The addition of cycloheximide to the culture medium inhibited the degradation of PAT by H. sinensis. Our results indicated that H. sinensis could degrade PAT and that the intracellular enzymes of H. sinensis played a crucial role in the degradation process. This result provides a new solution for the degradation of PAT.

STUDY OF XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS ISOLATES ORIGINATING FROM SOIL AND BRASSICA SPP. SEEDS

Xanthomonas campestris pv. campestris (Xcc) is a harmful pathogen of the Brassicaceae agricultural plants in Serbian agroecological conditions. The bacterium survives in soil, plant residues, seeds, and wild and weedy plants. This study aimed to characterize Xcc isolates that persist in field soil and roots where they represent a risk to causing disease of Brassicaceae plants. Collection of soil samples was carried out in the period 2015 - 2017 in four locations regions Vojvodina with cabbage cultivation in monoculture or crop rotations. The cabbage seed samples were collected in the same period in Futog and Despotovo locations (agricultural farms) and local farmer markets. All isolations were done on SX and YDC agar plates with the addition of cycloheximide at 250 mg/l. Based on the pathogenicity tests and morphological and biochemical characteristics, 28 identified Xcc isolates were selected, 12 from soil and 16 from seeds. Molecular confirmation of the Xcc isolates was done using multiplex PCR (m-PCR) with Xcc-specific primer sets DLH 120/125 and Zup 2309/2310. In all selected isolates and the reference Xcc strain NCPPB1144, the expected 370 and 619 bp PCR fragments were amplified. Characterizing isolates by rep-PCR fingerprinting using BOX A1R and (GTG)5 primers resulted in informative patterns. Cluster analysis grouped the strain Xcc NCPPB 1144 with all Xcc isolates, while other tested Xanthomonads were clustered separately. Based on the similarity level of 90%, we obtained three out of 12 representative isolates from soil and four out of 16 representative seed isolates. The characterization of Xcc isolates from soil and seeds will enable their comparison with those pathogenic isolated from leaves of Brassicaceae plants.

Analysis of Cancer‐associated Mutations in the Novel PKC Theta

The diacylglycerol‐regulated novel protein kinase C theta is highly druggable, but how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Cancer‐associated mutations in Ca2+‐regulated (conventional) PKC isozymes are generally loss‐of‐function, reframing PKC as having a tumor suppressive property, but whether this is the case for PKC Theta (θ) has not been addressed. Here we examine several cancer‐associated mutations in PKCθ, focusing on those at domain interfaces that are likely to impair autoinhibition, to begin to explore whether this isozyme of the PKC family functions to promote or suppress oncogenic signaling. PKCθ is selectively expressed in hematopoietic cells, namely platelets and T lymphocytes, where it regulates inflammatory signaling. Unsurprisingly, deregulated PKCθ has been implicated in many diseases including cancer where dozens of mutations have already been identified. One such residue was R145 in the autoinhibitory pseudosubstrate segment of PKCθ, whose mutation to His or Cys has been reported for two different cancers. This Arg interacts with Asp465 and Asp508 in the substrate binding cavity to maintain effective autoinhibition in the absence of the allosteric activator, diacylglycerol. We determined the effect of R145H and R145C mutation on the cellular activity and stability of PKCθ. Specifically, we used a genetically‐encoded FRET‐based reporter, C kinase activity reporter 2 (CKAR2) to measure the basal and agonist‐evoked activity of PKCθ. Whereas wild‐type PKCθ was effectively autoinhibited and required addition of agonist (here the phorbol ester, PDBu) for activation, both the R145H and R145C mutants were almost maximally activated under basal conditions. However, addition of cycloheximide to inhibit protein synthesis revealed that the half‐time of turn‐over of the mutants was 3‐ and 8‐fold faster than that of wild‐type enzyme, respectively. Taken together, these data suggest that the R145H and R145C mutants fail to autoinhibit resulting in a PKC with constitutive activity that is unstable and subject to the quality‐control degradation we have previously reported for Ca2+‐regulated PKC isozymes. Thus, these pseudosubstrate mutations in PKCθ are paradoxically loss‐of‐function because the constitutively active kinases are sensitive to degradation.

Biodegradation of mycotoxin patulin by the yeast Meyerozyma guilliermondii

Patulin is a well-known mycotoxin widely found in rotten fruits, produced by a series of toxigenic fungi, particularly Penicillium expansum. Patulin is hazardous to human and animal health. Meyerozyma guilliermondii is an antagonistic yeast that has been found to have biocontrol properties against blue mold disease in pears. But, it is not clear whether M. guilliermondii can degrade patulin. The present study aimed to investigate M. guilliermondii to degrade patulin and the possible mechanisms involved. The results showed that neither uptake of patulin by cells nor adsorption by cell wall components were involved in patulin elimination by M. guilliermondii. Extracellular metabolites also do not degrade patulin. The addition of cycloheximide to the M. guilliermondii cells decreased the capacity of patulin degradation. Enzymes induced by patulin had a more substantial degradation effect on patulin, which indicated that the patulin-degradation ability of M. guilliermondii was inducible. Our results confirmed that M. guilliermondii could degrade patulin. The intracellular enzymes of M. guilliermondii, particularly a short-chain dehydrogenase (SDR), carried out the degradation process. The degradation ability of M.guilliermondii towards patulin would be a novel approach for the elimination of patulin contamination.

Effect of Rhodotorula mucilaginosa on patulin degradation and toxicity of degradation products

ABSTRACT Rhodotorula mucilaginosa is an antagonistic yeast for which our research team has recently reported interesting biocontrol activities against blue mould decay of apples and a strong ability to decrease the patulin concentration in vivo. However, the possible mechanisms of patulin degradation by R. mucilaginosa and the toxicity of patulin degradation products remain unclear. In this study, the effect of R. mucilaginosa on patulin degradation and toxicity of degradation products were investigated, the results showed that viable cells of R. mucilaginosa are essential to patulin degradation. Also, R. mucilaginosa eliminated patulin without adsorbing it through its cell wall. The extracellular metabolites of R. mucilaginosa stimulated by patulin showed little degradation activity for patulin. Cycloheximide addition into the medium significantly decreased the patulin degradation capacity of R. mucilaginosa cells. The main patulin degradation product by R. mucilaginosa was ascladiol, which was proved non-toxic to human hepatoma (HepG2) cells at 0.625–10 g/mL. Furthermore, toxicological analysis using a confocal laser scanning microscope revealed that the degradation product induced cellular apoptosis to a lesser extent than patulin itself. This result offers an innovative method to detoxify patulin and limit the risks of patulin in fruits and vegetables using R. mucilaginosa.

Antifungal Efficacy and Safety of Cycloheximide as a Supplement in Optisol-GS.

PurposeThe incidence of fungal infection after corneal transplant has increased significantly in recent years, especially Candida spp. This study aimed to evaluate the efficacy and safety of the addition of cycloheximide in Optisol-GS media in decreasing the growth of Candida spp. strains.MethodsThis in vitro laboratory efficacy study measured fungal colony growth in 24 vials of Optisol-GS that were divided into 6 groups of 4 vials each, as follows: (1) MIC/2 cycloheximide, (2) MIC cycloheximide, (3) MICx5 cycloheximide, (4) MICx10 cycloheximide, from MIC values obtained for each strain, (5) unsupplemented optisol-GS as a positive control (added inoculum), and (6) unsupplemented optisol-GS as a negative control (no inoculum). In each group was added Candida albicans, C. glabrata and C. parapsilosis, except in the negative control. The evaluated variables were fungal colony growth from the Optisol-GS vials, corneal endothelial cell density and endothelial cell viability at different concentrations of cycloheximide.ResultsIn the efficacy study, all strains showed a reduction in fungal cell growth from the second day at all evaluated concentrations of optisol-GS supplemented with cycloheximide, even at subinhibitory concentrations (MIC/2). For C. glabrata, the colony count was reduced to 99%. No evidence of corneal endothelial toxicity was found at any concentration, in the safety study, compared with the paired control.ConclusionThe addition of cycloheximide to optisol-GS decreased the fungal growth, demonstrating fungicide action against C. glabrata and fungistatic action against C. albicans and C.parapsilosis. This drug did not demonstrate toxicity to the corneal endothelium at different concentrations.

Analysis of Protein Stability by Synthesis Shutoff.

In this protocol, we describe the analysis of protein stability over time, using synthesis shutoff. As an example, we express HA-tagged yeast mitofusin Fzo1 in Saccharomyces cerevisiae and inhibit translation via cycloheximide (CHX). Proteasomal inhibition with MG132 is performed, as an optional step, before the addition of CHX. Proteins are extracted via trichloroacetic acid (TCA) precipitation and subsequently separated via SDS-PAGE. Immunoblotting and antibody-decoration are performed to detect Fzo1 using HA-specific antibodies. We have adapted the method of blocking protein translation with cycloheximide to analyze the stability of high molecular weight proteins, including post-translational modifications and their impact on protein turnover.

Multicentre validation of a EUCAST method for the antifungal susceptibility testing of microconidia-forming dermatophytes.

Terbinafine resistance is increasingly reported in Trichophyton, rendering susceptibility testing particularly important in non-responding cases. We performed a multicentre evaluation of six EUCAST-based methods. Ten laboratories susceptibility tested terbinafine, itraconazole, voriconazole and amorolfine against a blinded panel of 38 terbinafine WT and target gene mutant isolates. E.Def 9.3.1 modifications included: medium with/without addition of chloramphenicol and cycloheximide (CC), incubation at 25°C to 28°C for 5-7 days and three MIC endpoints [visually and spectrophotometrically (90%/50% inhibition)], generating 7829 MICs. Quality control (QC) strains were Aspergillus flavus ATCC 204304 and CNM-CM1813. Eyeball, ECOFFinder (where ECOFF stands for epidemiological cut-off) and derivatization WT upper limits (WT-ULs), very major errors (VMEs; mutants with MICs ≤WT-ULs) and major errors (MEs; WT isolates with MICs >WT-ULs) were determined. MICs fell within the QC ranges for ATCC 204304/CNM-CM1813 for 100%/96% (voriconazole) and 84%/84% (itraconazole), respectively. Terbinafine MICs fell within 0.25-1 mg/L for 96%/92%, suggesting high reproducibility. Across the six methods, the number of terbinafine MEs varied from 2 to 4 (2.6%-5.2%) for Trichophyton rubrum and from 0 to 2 (0%-2.0%) for Trichophyton interdigitale. Modes for WT and mutant populations were at least seven 2-fold dilutions apart in all cases. Excluding one I121M/V237I T. rubrum mutant and two mixed WT/mutant T. interdigitale specimens, the numbers of VMEs were as follows: T. rubrum: CC visual, 1/67 (1.5%); CC spectrophotometric 90% inhibition, 3/59 (5.1%); and CC spectrophotometric 50% inhibition, 1/67 (1.5%); and T. interdigitale: none. Voriconazole and amorolfine MICs were quite uniform, but trailing growth complicated determination of itraconazole visual and spectrophotometric 90% inhibition MIC. Although none of the laboratories was experienced in dermatophyte testing, error rates were low. We recommend the CC spectrophotometric 50% inhibition method and provide QC ranges and WT-ULs for WT/non-WT classification.

Successively expressed cuticular protein genes at the prepupal stage in wing discs of Bombyx mori

BackgroundWe classified cuticular protein genes expressed at prepupal stage in wing discs of Bombyx mori into six groups according to their developmental expression and ecdysone responsiveness. Their expression pattern is suggested to be regulated by ecdysone-responsive transcription factors, whose transcripts showed resemblance with those of cuticular protein gene expression.ResultGroup1 and Group2 CP genes showed peak expression at stage W2. Group3 CP genes showed high expression at stage W3E and W3L and were upregulated by 20E addition, showing a peak 12 h after 20E pulse treatment. Group4 CP gene transcripts started expression from stage V5 and peaked at stage W3L. Some genes showed significant increase 4 or 6 h after 20E addition and were induced 6 h and showed a peak 18 h after the 20E pulse treatment. Group5 CP gene transcripts peaked at the same stage W3L. Some Group5 genes showed significant increase 6 h after the 20E addition, while others were not induced by the 20E addition. These different sub-groups showed different expression profiles in the feeding stage. Transcripts of this group genes were induced 12 h and showed a peak 18 h after the 20E pulse treatment. Group6 CP genes peaked at the stage P0, were not induced by the 20E addition, and showed a peak 24 h after the 20E pulse treatment. Group3, 4, 5, and 6 CP genes are suggested to be regulated by BHR4, BR-C, E74A, and βFTZ-F1, respectively. ERTFs showed different responsiveness to 20E concentration. BR-C was most and E74A was least insensitive. The addition of cycloheximide inhibited BR-C, E74A, and βFTZ-F1 expression depending on the length of treatment after ecdysone pulse treatment, which suggests that BHR4 induced BR-C, E74A, and βFTZ-F1.ConclusionExpression patterns of CPs were determined by the ecdysone-responsiveness and the related ERTFs expressed in the prepupal stage in B. mori wing discs.

Fate of generic and Shiga toxin-producing Escherichia coli (STEC) in Central Florida surface waters and evaluation of EPA Worst Case water as standard medium

Agricultural water is considered as one of the main contamination source for produce prior to harvest. The purpose of study was to evaluate the fate of Shiga toxin-producing Escherichia coli (STEC), and generic E. coli in Central Florida agricultural surface water at different temperatures and the potential use of EPA Worst Case water as a standardized media. Cocktails of STEC (O145, O104, O111, O103, O157), and generic E. coli K-12 were inoculated into agricultural surface water samples (non-sterile and sterilized) and EPA Worst Case water, and enumerated for up to 168 days. E. coli was held at 15 and 25 ± 1 °C. Tested microorganisms decreased most rapidly in non-sterile surface water. At day 168, E. coli populations decreased to ≤2.5 log CFU/100 ml in non-sterile surface water and were 4.8 ≤ and ≤ 8.5 log CFU/100 ml in sterile surface water and EPA Worst Case water. Populations were significantly (P ≤ .05) higher in sterile surface water and EPA Worst Case water at all sampling points starting from Day 28. Rate of declines in non-sterile surface waters were between 32.8 and 50 days at both tested temperatures and microorganisms. Addition of cycloheximide to non-sterile surface waters resulted in no significant effect on behavior of E. coli populations. Monitoring generic E. coli (represented by K-12) population changes is a reasonable indicator of STEC survival in agricultural water. EPA Worst Case water is a suitable standard control for surface water microcosms.

Water Filtered Infrared A and Visible Light (wIRA/VIS) Irradiation Reduces Chlamydia trachomatis Infectivity Independent of Targeted Cytokine Inhibition.

Chlamydia trachomatis is the major cause of infectious blindness and represents the most common bacterial sexually transmitted infection worldwide. Considering the potential side effects of antibiotic therapy and increasing threat of antibiotic resistance, alternative therapeutic strategies are needed. Previous studies showed that water filtered infrared A alone (wIRA) or in combination with visible light (wIRA/VIS) reduced C. trachomatis infectivity. Furthermore, wIRA/VIS irradiation led to secretion of pro-inflammatory cytokines similar to that observed upon C. trachomatis infection. We confirmed the results of previous studies, namely that cytokine secretion (IL-6, IL-8, and RANTES/CCL5) upon wIRA/VIS treatment, and the subsequent reduction of chlamydial infectivity, are independent of the addition of cycloheximide, a host protein synthesis inhibitor. Reproducible cytokine release upon irradiation indicated that cytokines might be involved in the anti-chlamydial mechanism of wIRA/VIS. This hypothesis was tested by inhibiting IL-6, IL-8, and RANTES secretion in C. trachomatis or mock-infected cells by gene silencing or pharmaceutical inhibition. Celastrol, a substance derived from Trypterygium wilfordii, used in traditional Chinese medicine and known for anti-cancer and anti-inflammatory effects, was used for IL-6 and IL-8 inhibition, while Maraviroc, a competitive CCR5 antagonist and anti-HIV drug, served as a RANTES/CCL5 inhibitor. HeLa cell cytotoxicity and impact on chlamydial morphology, size and inclusion number was evaluated upon increasing inhibitor concentration, and concentrations of 0.1 and 1 μM Celastrol and 10 and 20 μM Maraviroc were subsequently selected for irradiation experiments. Celastrol at any concentration reduced chlamydial infectivity, an effect only observed for 20 μM Maraviroc. Triple dose irradiation (24, 36, 40 hpi) significantly reduced chlamydial infectivity regardless of IL-6, IL-8, or RANTES/CCL5 gene silencing, Celastrol or Maraviroc treatment. Neither gene silencing nor pharmaceutical cytokine inhibition provoked the chlamydial stress response. The anti-chlamydial effect of wIRA/VIS is independent of cytokine inhibition under all conditions evaluated. Thus, factors other than host cell cytokines must be involved in the working mechanism of wIRA/VIS. This study gives a first insight into the working mechanism of wIRA/VIS in relation to an integral component of the host immune system and supports the potential of wIRA/VIS as a promising new tool for treatment in trachoma.

Four TRPM4 Cation Channel Mutations Found in Cardiac Conduction Diseases Lead to Altered Protein Stability.

Transient receptor potential melastatin member 4 (TRPM4), a non-selective cation channel, mediates cell membrane depolarization in immune response, insulin secretion, neurological disorders, and cancer. Pathological variants in TRPM4 gene have been linked to several cardiac phenotypes such as complete heart block (CHB), ventricular tachycardia, and Brugada syndrome (BrS). Despite recent findings regarding the functional implications of TRPM4 in cardiac diseases, the molecular and cellular mechanisms leading to altered conduction are poorly understood. In the present study, we identify and characterize four novel TRPM4 variants found in patients with CHB or ventricular fibrillation. Three of them, p.A101T, p.S1044C and a double variant p.A101T/P1204L, led to a decreased expression and function of the channel. On the contrary, the variant p.Q854R showed an increase in TRPM4 current. Recent evidence indicates that altered degradation rate of mutant proteins represents a pathogenic mechanism underlying genetic diseases. In consequence, protein turnover of WT-TRPM4 and TRPM4 variants overexpressed in HEK293 cells was analyzed using cycloheximide, an inhibitor of protein biosynthesis. Upon addition of cycloheximide, WT-TRPM4 decayed with a half-life of ~20 h, while loss-of-expression variants showed a ~30% increase in degradation rate, with a half-life close to 12 h. Together, the gain-of-expression variant showed a higher stability and a doubled half-life compared to WT-TRPM4. In conclusion, decreased or increased protein expression of several TRPM4 variants linked to cardiac conduction disorders or ventricular arrhythmias were found to be caused by altered TRPM4 half-life compared to the WT form.

Endoplasmic Reticulum Network Formation with Xenopus Egg Extracts.

The endoplasmic reticulum (ER) consists of morphologically distinct domains, including a polygonal network of tubules that is connected by three-way junctions. This network is found in all eukaryotic cells. Extracts from Xenopus laevis eggs contain stockpiles of components that allow the assembly of an ER network in vitro. Here we provide protocols for assembly of ER networks in extracts that are arrested at different stages of the cell cycle. Unfertilized Xenopus laevis eggs contain a cytostatic factor (CSF) that keeps them in the metaphase stage of the cell cycle. Disruption of the eggs by low-speed centrifugation releases calcium and the eggs cycle into interphase. This state can then be maintained by the addition of cycloheximide, which prevents the synthesis of cyclin B. CSF extracts can be also prepared in the presence of a calcium chelator, thus keeping the extract in metaphase. In this protocol, we outline procedures for the assembly of an ER network using either interphase- or metaphase-arrested Xenopus egg extracts. The network assembled is strikingly similar to the network observed in tissue culture cells. The extract allows easy biochemical manipulation, permitting the effects of purified proteins or small molecules, or the depletion of cytosolic components to be tested.

Relative Contribution of nirK- and nirS- Bacterial Denitrifiers as Well as Fungal Denitrifiers to Nitrous Oxide Production from Dairy Manure Compost.

The relative contribution of fungi, bacteria, and nirS and nirK denirifiers to nitrous oxide (N2O) emission with unknown isotopic signature from dairy manure compost was examined by selective inhibition techniques. Chloramphenicol (CHP), cycloheximide (CYH), and diethyl dithiocarbamate (DDTC) were used to suppress the activity of bacteria, fungi, and nirK-possessing denitrifiers, respectively. Produced N2O were surveyed to isotopocule analysis, and its 15N site preference (SP) and δ18O values were compared. Bacteria, fungi, nirS, and nirK gene abundances were compared by qPCR. The results showed that N2O production was strongly inhibited by CHP addition in surface pile samples (82.2%) as well as in nitrite-amended core samples (98.4%), while CYH addition did not inhibit the N2O production. N2O with unknown isotopic signature (SP = 15.3-16.2‰), accompanied by δ18O (19.0-26.8‰) values which were close to bacterial denitrification, was also suppressed by CHP and DDTC addition (95.3%) indicating that nirK denitrifiers were responsible for this N2O production despite being less abundant than nirS denitrifiers. Altogether, our results suggest that bacteria are important for N2O production with different SP values both from compost surface and pile core. However, further work is required to decipher whether N2O with unknown isotopic signature is mostly due to nirK denitrifiers that are taxonomically different from the SP-characterized strains and therefore have different SP values rather than also being interwoven with the contribution of the NO-detoxifying pathway and/or of co-denitrification.

Human neutrophils are targets to paracoccin, a lectin expressed by Paracoccidioides brasiliensis.

Paracoccin (PCN), a lectin expressed by Paracoccidioides brasiliensis (Pb), is known to exert activities on the fungal biology, as well as different immune cells of myeloid origin. The aim of this study was to investigate the direct interaction of the recombinant form of the lectin (rPCN) with neutrophils, a neglected area. Freshly isolated human neutrophils from healthy donors were used. Neutrophils were incubated with rPCN in vitro. After the treatment, the production of reactive oxygen species (ROS), DNA release, IL-8, TNF, IFN-γ, IL-10, IL-12p40, TGF-β and IL-1β production, fungicidal ability, apoptosis and de novo protein synthesis was determined. rPCN was found to induce ROS production as well as DNA release. Using the ROS inhibitor, diphenyleneiodium, both ROS production and DNA release were significantly inhibited. In addition, rPCN was found to induce IL-8 and IL1-β production, inhibit apoptosis and induce de novo protein synthesis. Addition of cycloheximide, a protein synthesis inhibitor, drastically reversed the antiapoptotic effect of rPCN. Finally, the ability to kill Pb yeasts by human neutrophils was significantly increased after rPCN stimulation. rPCN can alter the biology of human neutrophils increasing their fungicidal ability. Moreover, the ability of rPCN to increase DNA release and to induce suppression of neutrophil apoptosis occurs by a ROS- and de novo protein synthesis-dependent mechanism, respectively.

Epidemiologic Study of Opportunistic Fungi Contaminating Soil of Schools, Mosques and Hotels in Diwaniya City - Iraq

Soil mycoflora in several schools, mosques and hotels in Diwaniya city were examined using dilution plate technique and three different culture media with and without supplementation of cycloheximide antifungal. A total number of fungal isolates observed in all experiments were 2589 that represent 57 species related to 48 genus, in addition to the white and dark sterile mycelium. Cycloheximide substantially affected the frequency of isolated fungi. Where Penicillium chrysogenum was the most frequent species (44.73%) on culture media free from cycloheximide. Acremonium kiliense was the most with more capability than other examined fungi in tested blood which took 3 days only while this ability of other species was varied from 4 to 12 days . frequent one (26.85%) on media supplemented with this antifungal. In contrast the frequency and occurrence of P. chrysogenum in both soils of school (41.26%) and (95.95%) respectively and of mosques (43.63%) and(100%) respectively was higher than other species, also this species was the most occurring (90%) in hotels soil. Memoneilla subimplex was the most frequent species (18.87%) in this soil. Number of isolates were varied according to the addition of cycloheximide and the composition of culture media: highest number of fungal isolates (337) were recorded when potato dextrose agar used, in contrast the greatest numbers of fungal species (21) were identified when sabouraude dextrose agar was used. In culture media free of cycloheximide the number of fungal isolates and fungal species were substantially increased to 720 and 37 respectively. The results of this experiment revealed a negative correlation between soil fungi and pH ( as r= 0.307 with fungal isolates and 0.185 with the number of fungal species ) while this correlation was positive with organic content of soil salinity (r= 0.042 and 0.130 with fungal isolates and fungal species respectively ) moreover the results showed negative correlation between fungal isolates and soil salinity (r=0.202) while the number of species of fungal society was positively correlated ( r= 0.109). The hemolytic activity of 30 species in vitro using blood agar culture:18 species showed variable capabilities in hemolysis. Aspergillus fumigatus was

Loss of ΔNp63α promotes mitotic exit in epithelial cells.

Volume 585, Issue 17, Pages 2720–2726, First published online: 3 August 2011 https://doi.org/10.1016/j.febslet.2011.07.030 In this paper by Hau et al. [1], there are errors in Fig. 4B concerning the western blots of HA (Cdc20), and HA (Cdh1). The western blots of HA (Cdc20) and HA (Cdh1) were erroneously used as the western blots for Actin and ΔNp63α expression respectively. The corrected figure 4B is shown below. These mistakes do not alter the conclusion that APC/CCdh1 targets ΔNp63α for degradation during mitosis. Fig. 4B. 293 cells were cotransfected with ΔNp63α and either with HA-cdc20 (upper panel) or HA-cdh1 (lower panel) expressing vectors. The cells were treated with 10 μg·mL−1 cycloheximide (CHX) and harvested according to the time specified. For the rescue experiment, 10 μm MG132 was added 1 h before addition of cycloheximide. Cell extract was prepared and subjected to immunoblot with anti-HA (12CA5) and anti-p63 (4A4) antibodies.

MicroRNA-142-3p and let-7g Negatively Regulates Augmented IL-6 Production in Neonatal Polymorphonuclear Leukocytes.

Neonatal PMN are qualitatively impaired in functions, yet they frequently reveal augmented inflammatory reactions during sepsis. Here, we hypothesized that PMN from newborns produce more IL-6 than those from adults under LPS stimulation, in which transcriptional or posttranscriptional regulation is involved in the altered expression. We found that neonatal PMN produced significantly higher IL-6 mRNA and protein than adult PMN. The higher IL-6 expression was not related to transcriptional but posttranscriptional regulation as the IL-6 expression was affected by the addition of cycloheximide but not actinomycin. To examine whether miRNA was involved in the IL-6 regulation of neonatal PMN, we surveyed differential displays of miRNAs that could potentially regulate IL-6 expression before and after LPS stimulation. Four miRNAs: hsa-miR-26a, hsa-miR-26b, hsa-miR-142-3p and hsa-let 7g decreased or increased after LPS treatment for 4 h. Further validation by qRT-PCR identified miR-26b, miR-142-3p and let-7g significantly changed in neonatal PMN after LPS stimulation. The functional verification by transfection of miR-142-3p and let-7g precursors into neonatal PMN significantly repressed the IL-6 mRNA and protein expression, suggesting that miR-142-3p and let-7g negatively regulate IL-6 expression in neonatal PMN. Modulation of miRNA expression may be used to regulate IL-6 production in newborns with altered inflammatory reactions.

Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins.

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.

The Possible Mechanisms Involved in Degradation of Patulin by Pichia caribbica.

In this work, we examined the mechanisms involved in the degradation of patulin by Pichia caribbica. Our results indicate that cell-free filtrate of P. caribbica reduced patutlin content. The heat-killed cells could not degrade patulin. However, the live cells significantly reduced the concentration of the patulin. In furtherance to this, it was observed that patulin was not detected in the broken yeast cells and cell wall. The addition of cycloheximide to the P. caribbica cells decreased the capacity of degradation of patulin. Proteomics analyses revealed that patulin treatment resulted in an upregulated protein which was involved in metabolism and stress response processes. Our results suggested that the mechanism of degradation of patulin by P. caribbica was not absorption; the presence of patulin can induce P. caribbica to produce associated intracellular and extracellular enzymes, both of which have the ability to degrade patulin. The result provides a new possible method that used the enzymes produced by yeast to detoxify patulin in food and feed.