Use of insertional mutagenesis to tag putative parasitic fitness genes in the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi

2008 ◽  
Vol 54 (9) ◽  
pp. 797-802 ◽  
Author(s):  
Karine V. Plourde ◽  
Volker Jacobi ◽  
Louis Bernier
Keyword(s):  
Insertional Mutagenesis ◽  
Dutch Elm Disease ◽  
Parasitic Fitness ◽  
Flanking Sequences ◽  
Fungal Protoplasts ◽  
Nuclear Loci ◽  
Mutant Phenotypes ◽  
Kinetics Of ◽  
Single Insertion

We used insertional mutagenesis to produce genetically tagged mutants of the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi. We first optimized transformation of O. novo-ulmi protoplasts by the restriction enzyme mediated integration method. A concentration of 80 U of HindIII with 108 fungal protoplasts and 5 μg of plasmid DNA was the most efficient for generating a high number of O. novo-ulmi mutants carrying a single insertion in their genome. Mycelium- and yeast-like growth kinetics of 24 O. novo-ulmi mutants were evaluated in vitro. Flanking sequences were successfully recovered in 8% of the transformants analyzed. Some mutant phenotypes appeared to result from gene disruption events, whereas others likely involved modifications of noncoding regions. Several nuclear loci that control vegetative growth and could potentially impact parasitic fitness were successfully tagged.

Ty element-induced temperature-sensitive mutations of Saccharomyces cerevisiae.

Genetics ◽  
1992 ◽  
Vol 131 (4) ◽  
pp. 821-832 ◽  
Author(s):  
K Kawakami ◽  
B K Shafer ◽  
D J Garfinkel ◽  
J N Strathern ◽  
Y Nakamura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
High Temperatures ◽  
Insertional Mutagenesis ◽  
Trna Gene ◽  
Protein A ◽  
Temperature Sensitive ◽  
Mutant Phenotypes

Abstract Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Acquired Haemophilia: Functional Study of Antibodies to Factor VIII

1981 ◽  
Vol 45 (03) ◽  
pp. 285-289 ◽  
Author(s):  
J P Allain ◽  
A Gaillandre ◽  
D Frommel
Keyword(s):  
Factor Viii ◽  
Functional Study ◽  
Factor Viii Inhibitor ◽  
Acquired Haemophilia ◽  
Viii Inhibitor ◽  
Kinetics Of ◽  
Antibody Function ◽  
Native Plasma

SummaryFactor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR.Ag levels were found in all patients. VIII:WF levels were 50% of those of VTIIRrAg, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers.Antibody function has been characterized by kinetics of VIII :C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed:1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (> 1).2) Antibodies from other patients, usually with lower titres, inactivated VIII :C according to complex order kinetics, were not saturable, and had a less steep affinity slope (< 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

EFFECT OF EXOGENOUS METHIONINE ON DIGESTIBILITY IN VITRO AND ENZYMOLYSIS KINETICS OF CYPRINUS CARPIO

2013 ◽  
Vol 36 (6) ◽  
pp. 1200-1204
Author(s):  
Ling ZHANG ◽  
Wei-Dong ZHANG ◽  
Xiao-Ling ZHANG ◽  
Kai LIU ◽  
Qing-Li LI ◽  
...  
Keyword(s):  
Cyprinus Carpio ◽  
Kinetics Of

Vancomycin Adsorption During in vitro Model of Hemoperfusion with HA380 Cartridge

Nephron ◽  
2021 ◽  
pp. 1-7
Author(s):  
Ilaria Godi ◽  
Anna Lorenzin ◽  
Silvia De Rosa ◽  
Gianlorenzo Golino ◽  
Maira Knust ◽  
...  
Keyword(s):  
Complete Removal ◽  
Potential Interaction ◽  
Blood Purification ◽  
Therapeutic Monitoring ◽  
High Concentration ◽  
Langmuir Isotherm Model ◽  
Vitro Model ◽  
Kinetics Of ◽  
Balanced Solution

<b><i>Introduction:</i></b> A critical point for using blood purification during sepsis may be the potential interaction with antimicrobial therapy, the mainstay of sepsis treatment. The aim of our study was to investigate the vancomycin removal during hemoperfusion (HP) using HA380 cartridge. <b><i>Methods:</i></b> This is an experimental study, in which 500 mL of solution was circulated in a closed-circuit (blood flow of 250 mL/min) simulating HP ran using HA380. Vancomycin was added to reach a through concentration or a very high concentration to evaluate the removal ratio (RR) during 120 min of HP. Comparison between blood-crystalloid solution and balanced solution was performed by using Kruskal-Wallis test. The kinetics of vancomycin removal and the adsorption isotherm were evaluated. <b><i>Results:</i></b> We found a complete removal of vancomycin at baseline through concentration of 23.0 ± 7.4 mg/L. Using extremely high concentration (baseline 777.0 ± 62.2 mg/L), RR was 90.1 ± 0.6% at 5 min and 99.2 ± 0.6% at 120 min. No difference in terms of RR was found between blood-crystalloid mixture and balanced solution. The kinetics of the vancomycin reduction followed an exponential decay. Repeated boluses (total amount of 2,000 mg) resulted in cumulative adsorption of 1,919.4 mg with RR of 96.6 ± 1.4%, regardless of the amount injected (100 vs. 500 mg). Vancomycin adsorption onto HA380 followed the Langmuir isotherm model. <b><i>Conclusions:</i></b> A considerable amount of vancomycin was rapidly removed during in vitro HP with HA380. Clinical studies are needed to determine whether this may lead to underdosing. Drug therapeutic monitoring is highly recommended when using HA380 for blood purification in patients receiving vancomycin.

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