Effects of sugars and polyols on basidiocarp formation in a phosphoglucose isomerase mutant of Coprinus cinereus

1984 ◽  
Vol 30 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Hiroshi Nyunoya ◽  
Tsuneo Takemaru ◽  
Tatsuo Ishikawa
Keyword(s):  
Culture Medium ◽  
Complete Recovery ◽  
Biochemical Properties ◽  
Phosphoglucose Isomerase ◽  
Coprinus Cinereus ◽  
Partial Recovery ◽  
Wild Type ◽  
In The Wild ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Basidiocarp Formation

Several biochemical properties of a mutant deficient in phosphoglucose isomerase (pgi) of Coprinus cinereus were examined in connection with its ability to produce basidiocarps. Mycelium of the pgi mutant accumulated glucose-6-phosphate and showed higher glucose-6-phosphate dehydrogenase activity than wild type. A conventional fruiting medium did not support basidiocarp formation by the homozygous dikaryon pgi/pgi, but the addition of a reduced amount of glucose plus a compensatory amount of fructose to the culture medium resulted in partial recovery of fruiting ability. This modification of the culture medium decreased the intracellular glucose-6-phosphate to almost the same level as in the wild type. The addition of polyols such as mannitol resulted in complete recovery of fruiting ability by the dikaryon pgi/pgi without affecting the level of glucose-6-phosphate. Mycelium of the mutant showed an elevated activity of NAD-linked polyol dehydrogenase and an elevated intracellular NAD level, irrespective of whether the mycelium was grown in the presence or absence of polyol.

A role for rhamnolipid in biofilm dispersion

Biofilms ◽  
2004 ◽  
Vol 1 (2) ◽  
pp. 91-99 ◽  
Author(s):  
S. R. Schooling ◽  
U. K. Charaf ◽  
D. G. Allison ◽  
P. Gilbert
Keyword(s):  
Surface Tension ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Culture Medium ◽  
High Cell Density ◽  
Culture Media ◽  
Homoserine Lactone ◽  
Wild Type ◽  
In The Wild ◽  
Biofilm Dispersion

Biofilms are often considered as localized zones of high cell density. Quorum sensing provides a means for control of population processes and has been implicated in the regulation of biofilm activities. We present a role for quorum sensing in programmed detachment and dispersal processes. Biofilms of Pseudomonas aeruginosa PAO1 and its isogenic homoserine lactone (HSL) mutant P. aeruginosa PAO-JP2 were grown in batch culture on glass substrata; differences were found in the rate and extent of formation of biofilm. Climax communities were observed for PAO1 at 24 h. These were later accompanied by foaming, a drop in the surface tension of culture media and dispersal of the biofilm, after which no subsequent biofilm accretion occurred. PAO-JP2 cultures reformed biofilm post-detachment and did not foam. Prevention of biofilm reformation in the wild type was related to some component excreted into the culture medium. Rhamnolipid, a biosurfactant regulated by quorum sensing, was detected in PAO1 cultures. When rhamnolipid was added to freshly inoculated substrata, biofilm formation was inhibited. At 20 h, PAO1 biofilms were transferred to medium with added rhamnolipid: biofilm was relatively unaffected. Biofilm events were also studied in medium supplemented with N-butyryl-L-homoserine lactone, which is involved in the regulation of rhamnolipid synthesis. Both strains exhibited similar trends of rapid biofilm formation and dramatic changes in the rate and extent of biofilm accretion. In both cases, there was premature foaming, lowered surface tension and elevated rhamnolipid levels. A role for HSLs in maintenance of biofilm and events leading to dispersion of cells is proposed. This role would encompass dispersion but not necessarily detachment of cells from biofilm and supports a new function for rhamnolipid in pathogenesis, whereby rhamnolipid would promote the dissemination of cells from a nidus of infection.

scholarly journals ISOLATION AND STUDY OF MUTANTS LACKING A DEREPRESSIBLE PHOSPHATASE IN CHLAMYDOMONAS REINHARDI

Genetics ◽  
1975 ◽  
Vol 80 (2) ◽  
pp. 239-250
Author(s):  
R F Matagne ◽  
R Loppes
Keyword(s):  
Alkaline Phosphatase ◽  
Inorganic Phosphate ◽  
Double Mutant ◽  
Culture Medium ◽  
Acid Phosphatases ◽  
Wild Type ◽  
Optimal Activity ◽  
Wide Range ◽  
In The Wild ◽  
Definition Of

ABSTRACT In the green alga Chlamydomonas reinhardi, removal of inorganic phosphate from the culture medium results in the increase of phosphatase activity (derepression) in the wild-type (WT) strain as well as in a double mutant (P2Pa) lacking the two main constitutive acid phosphatases. Following treatment of WT and P2Pa with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), mutants were recovered which display very low phosphatase activities when grown in the absence of phosphate; as shown by electrophoresis, they lack one non-migrating phosphatase (PD mutants). This enzyme is active over a wide range of pH with an optimum at pH 7.5. The comparison of electropherograms from WT and mutants grown on media with or without phosphate allowed us to provide a tentative definition of the pool of derepressible phosphatases in Chlamydomonas : in addition to the neutral phosphatase lacking in PD mutants, Chlamydomonas produces two electrophoretic forms of alkaline phosphatase showing an optimal activity at pH 9.5.

scholarly journals Microglia-dependent presynaptic disruption in an organotypic hippocampal slice culture model of neuroinflammation

2018 ◽  
Author(s):  
Olivia Sheppard ◽  
Michael P Coleman ◽  
Claire S Durrant
Keyword(s):  
Culture Medium ◽  
Molecular Mechanisms ◽  
Hippocampal Slice ◽  
Synaptic Proteins ◽  
Slice Culture ◽  
Partial Recovery ◽  
Wild Type ◽  
Aseptic Inflammation ◽  
Operative Trauma ◽  
Hippocampal Slice Culture

Background: Systemic inflammation, such as occurs during sepsis, bone fracture, infections or post-operative trauma, has been linked to synapse loss and cognitive decline in human patients and animal models. Organotypic hippocampal slice cultures (OHSCs) represent an underused tool in neuroinflammation; retaining much of the neuronal architecture, synaptic connections and diversity of cell types present in the hippocampus in vivo whilst providing convenient access to manipulate and sample the culture medium and observe cellular reactions as in other in vitro methods. Here we report the development of an OHSC model of synaptic disruption after aseptic inflammation and investigate the underlying mechanism. Methods: OHSCs were generated from P6-P9 C57BL/6, the APP transgenic TgCRND8 model, or wild-type littermate mice according to the interface method. Aseptic inflammation was induced via addition of lipopolysaccharide (LPS) and cultures were analysed for changes in synaptic proteins via western blot. qPCR and ELISA analysis of the slice tissue and culture medium respectively determined changes in gene expression and protein secretion. Microglia were selectively depleted using the toxin clodronate and the effect of IL1β was assessed using a specific neutralising monoclonal antibody. Results: Addition of LPS caused a loss of the presynaptic protein synaptophysin via a mechanism dependent on microglia and involving IL1β. Washout of LPS via medium exchange allows for partial recovery of synaptic protein levels after 2 weeks. TgCRND8 OHSCs do not show alterations in IL1β expression at a timepoint where they exhibit spontaneous synaptophysin loss, and LPS does not alter levels of APP or Aβ in wild-type OHSCs. This indicates that although synaptophysin loss is seen in both systems, there is likely to be distinct underlying pathogenic mechanisms between the neuroinflammatory and amyloid models. Conclusions: We report the development of an OHSC model of LPS-induced synaptophysin loss and demonstrate a key role for microglia and involvement of IL1β. We propose that distinct molecular mechanisms lead to synaptophysin protein loss in LPS- exposed versus TgCRND8 OHSCs and provide a new experimental paradigm for assessing chronic changes in synaptic proteins, and synaptic plasticity, following acute inflammatory insults.

scholarly journals Engineering of Primary Carbon Metabolism for Improved Antibiotic Production in Streptomyces lividans

2002 ◽  
Vol 68 (10) ◽  
pp. 4731-4739 ◽  
Author(s):  
Michael J. Butler ◽  
Per Bruheim ◽  
Srdjan Jovetic ◽  
Flavia Marinelli ◽  
Pieter W. Postma ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Lividans ◽  
Pentose Phosphate ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild ◽  
Commercially Important ◽  
Biochemical Product ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Wild Type Control

ABSTRACT Deletions were made in Streptomyces lividans in either of two genes (zwf1 and zwf2) encoding isozymes of glucose-6-phosphate dehydrogenase, the first enzyme in the oxidative pentose phosphate pathway (PPP). Each mutation reduced the level of Zwf activity to approximately one-half that observed in the wild-type strain. When the mutants were transformed with multicopy plasmids carrying the pathway-specific transcriptional activator genes for either the actinorhodin (ACT) or undecylprodigiosin (RED) biosynthetic pathway, they produced higher levels of antibiotic than the corresponding wild-type control strains. The presumed lower flux of carbon through the PPP in each of the Δzwf mutants may allow more efficient glucose utilization via glycolysis, resulting in higher levels of antibiotic production. This appears to occur without lowering the concentration of NADPH (the major biochemical product of the oxidative PPP activity) to a level that would limit antibiotic biosynthesis. Consistent with this hypothesis, deletion of the gene (devB) encoding the enzyme that catalyzes the next step in the oxidative PPP (6-phosphogluconolactonase) also resulted in increased antibiotic production. However, deletion of both zwf genes from the devB mutant resulted in reduced levels of ACT and RED production, suggesting that some of the NADPH made by the PPP is utilized, directly or indirectly, for antibiotic biosynthesis. Although applied here to the model antibiotics ACT and RED, such mutations may prove to be useful for improving the yield of commercially important secondary metabolites.

Collismycin A biosynthesis in Streptomyces sp. CS40 is regulated by iron levels through two pathway-specific regulators

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 467-478 ◽  
Author(s):  
Natalia M. Vior ◽  
Carlos Olano ◽  
Ignacio García ◽  
Carmen Méndez ◽  
José A. Salas
Keyword(s):  
Gene Cluster ◽  
Culture Medium ◽  
Wild Type Strain ◽  
Transcriptional Regulator ◽  
Moderate Increase ◽  
Wild Type ◽  
Environmental Stimulus ◽  
Fourfold Increase ◽  
In The Wild ◽  
Regulatory Model

Two putative pathway-specific regulators have been identified in the collismycin A gene cluster: ClmR1, belonging to the TetR-family, and the LuxR-family transcriptional regulator ClmR2. Inactivation of clmR1 led to a moderate increase of collismycin A yields along with an early onset of its production, suggesting an inhibitory role for the product of this gene. Inactivation of clmR2 abolished collismycin A biosynthesis, whereas overexpression of ClmR2 led to a fourfold increase in production yields, indicating that ClmR2 is an activator of collismycin A biosynthesis. Expression analyses of the collismycin gene cluster in the wild-type strain and in ΔclmR1 and ΔclmR2 mutants confirmed the role proposed for both regulatory genes, revealing that ClmR2 positively controls the expression of most of the genes in the cluster and ClmR1 negatively regulates both its own expression and that of clmR2. Additionally, production assays and further transcription analyses confirmed the existence of a higher regulatory level modulating collismycin A biosynthesis in response to iron concentrations in the culture medium. Thus, high iron levels inhibit collismycin A biosynthesis through the repression of clmR2 transcription. These results have allowed us to propose a regulatory model that integrates the effect of iron as the main environmental stimulus controlling collismycin A biosynthesis.

Freeze-substituted fungal cells of Nectria haematococca: A comparison of the macroconidial walls of the adhesion-competent wild type with an adhesion-reduced mutant

1990 ◽  
Vol 48 (3) ◽  
pp. 688-689
Author(s):  
Thecan Caesar-Ton That ◽  
Lynn Epstein
Keyword(s):  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Wild Type ◽  
Nectria Haematococca ◽  
Fruit Extract ◽  
Dialysis Tubing ◽  
Tube Emergence ◽  
Contrast Microscopy ◽  
In The Wild ◽  
Freeze Damage

Nectria haematococca mating population I (anamorph, Fusarium solani) macroconidia attach to its host (squash) and non-host surfaces prior to germ tube emergence. The macroconidia become adhesive after a brief period of protein synthesis. Recently, Hickman et al. (1989) isolated N. haematococca adhesion-reduced mutants. Using freeze substitution, we compared the development of the macroconidial wall in the wild type in comparison to one of the mutants, LEI.Macroconidia were harvested at 1C, washed by centrifugation, resuspended in a dilute zucchini fruit extract and incubated from 0 - 5 h. During the incubation period, wild type macroconidia attached to uncoated dialysis tubing. Mutant macroconidia did not attach and were collected on poly-L-lysine coated dialysis tubing just prior to freezing. Conidia on the tubing were frozen in liquid propane at 191 - 193C, substituted in acetone with 2% OsO4 and 0.05% uranyl acetate, washed with acetone, and flat-embedded in Epon-Araldite. Using phase contrast microscopy at 1000X, cells without freeze damage were selected, remounted, sectioned and post-stained sequentially with 1% Ba(MnO4)2 2% uranyl acetate and Reynold’s lead citrate. At least 30 cells/treatment were examined.

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals Mass Spectrometric Profiling of Extraocular Muscle and Proteomic Adaptations in the mdx-4cv Model of Duchenne Muscular Dystrophy

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 595
Author(s):  
Stephen Gargan ◽  
Paul Dowling ◽  
Margit Zweyer ◽  
Jens Reimann ◽  
Michael Henry ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Biochemical Properties ◽  
Mass Spectrometric ◽  
Cellular Stress Response ◽  
Diaphragm Muscle ◽  
Spectrometric Analysis ◽  
Mitochondrial Enzymes ◽  
Wild Type ◽  
Potential Marker

Extraocular muscles (EOMs) represent a specialized type of contractile tissue with unique cellular, physiological, and biochemical properties. In Duchenne muscular dystrophy, EOMs stay functionally unaffected in the course of disease progression. Therefore, it was of interest to determine their proteomic profile in dystrophinopathy. The proteomic survey of wild type mice and the dystrophic mdx-4cv model revealed a broad spectrum of sarcomere-associated proteoforms, including components of the thick filament, thin filament, M-band and Z-disk, as well as a variety of muscle-specific markers. Interestingly, the mass spectrometric analysis revealed unusual expression levels of contractile proteins, especially isoforms of myosin heavy chain. As compared to diaphragm muscle, both proteomics and immunoblotting established isoform MyHC14 as a new potential marker in wild type EOMs, in addition to the previously identified isoforms MyHC13 and MyHC15. Comparative proteomics was employed to establish alterations in the protein expression profile between normal EOMs and dystrophin-lacking EOMs. The analysis of mdx-4cv EOMs identified elevated levels of glycolytic enzymes and molecular chaperones, as well as decreases in mitochondrial enzymes. These findings suggest a process of adaptation in dystrophin-deficient EOMs via a bioenergetic shift to more glycolytic metabolism, as well as an efficient cellular stress response in EOMs in dystrophinopathy.

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