Sequential cold-sensitive mutations in Aspergillus fumigatus. III. Mechanism of cold sensitivity

1981 ◽  
Vol 27 (3) ◽  
pp. 304-310 ◽  
Author(s):  
Anthony J. Arseneau ◽  
Kenneth F. Gregory
Keyword(s):  
Aspergillus Fumigatus ◽  
Rna Synthesis ◽  
Feedback Inhibition ◽  
Ribosomal Subunit ◽  
Cold Sensitivity ◽  
Nonpermissive Temperature ◽  
Cold Sensitive ◽  
Ribosomal Rna Processing

The mechanism of cold sensitivity of Aspergillus fumigatus ON5, a 37 °C-sensitive mutant derived from A. fumigatus I-21 (ATCC 32722) by five sequential mutations, was investigated. The rate of in vivo protein synthesis by ON5 was not affected for 2 h following a shift from 45 to 34 °C, but the rate of in vivo RNA synthesis dropped almost immediately. The RNA polymerases of ON5 possessed wild-type activity in vitro at a nonpermissive temperature (34 °C) indicating that the reduction in the rate of in vivo RNA synthesis did not result from cold sensitivity in transcription, but was possibly a result of rapid feedback inhibition of transcription. Mutant ON5 was not able to produce ribosomes at a nonpermissive temperature as evidenced by the fact that no 3H-labelled amino acids were incorporated into the monosome, large ribosomal subunit, or small ribosomal subunit at 34 °C. Ribosomal subunit assembly or ribosomal RNA processing appears, therefore, to be the cold-sensitive cellular function in ON5.

Sequential cold-sensitive mutations in Aspergillus fumigatus

1978 ◽  
Vol 24 (2) ◽  
pp. 84-88 ◽  
Author(s):  
Ole Nielsen ◽  
Kenneth F. Gregory
Keyword(s):  
Aspergillus Fumigatus ◽  
Type A ◽  
Maximum Growth ◽  
Cold Sensitivity ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Cold Sensitive ◽  
Wild Type Parent ◽  
Thermotolerant Fungus

Mutants of the thermotolerant fungus Aspergillus fumigatus I-21 (ATCC 32722) unable to grow at 37 °C were sought. Cold-sensitive mutants were enriched from progeny spores of γ-irradiated conidia by two or more incubations at various nonpermissive temperatures alternating with filtrations through cheesecloth. The approximate minimum, optimum, and maximum growth temperatures of the parent were 12, 40, and 50 °C, respectively. Mutants unable to grow at 37 °C were not successfully isolated directly from the wild type. A mutant unable to grow at 25 °C was isolated and mutations further increasing the cold sensitivity by increments of 3–5 °C were found to occur. Mutants completely unable to grow at 37 °C were obtained by five sequential mutations. All mutants grew as fast as the wild-type parent at 45 °C and higher. Each mutant produced revenants able to grow not only at the nonpermissive temperature used for its isolation but also at lower temperatures.

scholarly journals Mobility restriction in vivo of the heavy ribosomal subunit in a secretory cell.

1976 ◽  
Vol 70 (3) ◽  
pp. 573-580 ◽  
Author(s):  
U Lönn ◽  
J E Edström
Keyword(s):  
Endoplasmic Reticulum ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Ribosomal Subunit ◽  
Chironomus Tentans ◽  
Salivary Gland Cells ◽  
Unique Parameter ◽  
Labeled Rna

Analysis in insect (Chironomus tentans) salivary gland cells of labeled RNA as a function of time after precursor injection and its distance to the nuclear membrane, cytoplasmic zone analysis, could previously be used to demonstrate the presence of short-lasting gradients in newly labeled ribosomal RNA. Since the gradients were sensitive to puromycin, they are likely to be a result of diffusion restriction due to an engagement of the subunits into polysomes. In the present paper the possibility was explored of recording gradients that were caused by labeled subunits in puromycin-resistant associations, which, in all probability, involve the endoplasmic reticulum. It was found that labeled 28 S and 5 S RNA but not 18 S RNA were present in radioactivity gradients lasting for at least 2 days but less than 6 days. The gradients also remained during inhibition of RNA synthesis by actinomycin, and they were completely resistant to puromycin whether given in vivo or in vitro. The semipermanent gradients formed fhere offer a unique parameter for the in vivo study of conditions for formation and maintenance of heavy subunits in puromycin-resistant bonds. An explanation for these and previous results is that the light subunit, although restricted in movement by engagement to polysomes, is nevertheless free to exchange and spread between rounds of translation, whereas at least part of the heavy subunit population is bound to the endoplasmic reticulum and less free to spread. These results offer a good in vivo correlate to previous results on in vitro exchangeability of subunits.

scholarly journals Pleiotropic Defects Caused by Loss of the Proteasome-Interacting Factors Rad23 and Rpn10 of Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 69-79
Author(s):  
David Lambertson ◽  
Li Chen ◽  
Kiran Madura
Keyword(s):  
Slow Growth ◽  
26S Proteasome ◽  
Cold Sensitivity ◽  
Genetic Studies ◽  
Single Mutant ◽  
M Phase ◽  
Cold Sensitive ◽  
Increased Sensitivity

Abstract Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Δ rpn10Δ displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Δ rpn10Δ displays similar sensitivity to DNA damage as a rad23Δ single mutant, deletion of RAD23 in rpn10Δ significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Δ single mutant. A mutant Rad23 that is unable to bind the proteasome (ΔUbLrad23) does not suppress the canavanine or cold-sensitive defects of rad23Δ rpn10Δ, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Δ rpn10Δ suggest that proteasome function is altered.

scholarly journals Spb1p Is a Yeast Nucleolar Protein Associated with Nop1p and Nop58p That Is Able To BindS-Adenosyl-l-Methionine In Vitro

2000 ◽  
Vol 20 (4) ◽  
pp. 1370-1381 ◽  
Author(s):  
Lionel Pintard ◽  
Dieter Kressler ◽  
Bruno Lapeyre
Keyword(s):  
Ribosomal Subunit ◽  
Restrictive Temperature ◽  
Protein Sequence Analysis ◽  
Nucleolar Proteins ◽  
Cold Sensitive ◽  
Binding Protein Gene ◽  
Sensitive Allele

ABSTRACT We present here the characterization of SPB1, an essential yeast gene that is required for ribosome synthesis. A cold-sensitive allele for that gene (referred to here asspb1-1) had been previously isolated as a suppressor of a mutation affecting the poly(A)-binding protein gene (PAB1) and a thermosensitive allele (referred to here asspb1-2) was isolated in a search for essential genes required for gene silencing in Saccharomyces cerevisiae. The two mutants are able to suppress the deletion of PAB1, and they both present a strong reduction in their 60S ribosomal subunit content. In an spb1-2 strain grown at the restrictive temperature, processing of the 27S pre-rRNA into mature 25S rRNA and 5.8S is completely abolished and production of mature 18S is reduced, while the abnormal 23S species is accumulated. Spb1p is a 96.5-kDa protein that is localized to the nucleolus. Coimmunoprecipitation experiments show that Spb1p is associated in vivo with the nucleolar proteins Nop1p and Nop5/58p. Protein sequence analysis reveals that Spb1p possesses a putative S-adenosyl-l-methionine (AdoMet)-binding domain, which is common to the AdoMet-dependent methyltransferases. We show here that Spb1p is able to bind [3H]AdoMet in vitro, suggesting that it is a novel methylase, whose possible substrates will be discussed.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

scholarly journals Super Cooling Point Phenotypes and Cold Resistance in Hyles euphorbiae Hawk Moths from Different Climate Zones

Diversity ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 207
Author(s):  
Hana Daneck ◽  
Matthias Benjamin Barth ◽  
Martin Geck ◽  
Anna K. Hundsdoerfer
Keyword(s):  
Environmental Conditions ◽  
Cold Hardiness ◽  
Cold Treatment ◽  
Underlying Mechanism ◽  
Frost Tolerance ◽  
Cold Sensitivity ◽  
Cold Hardy ◽  
Hyles Euphorbiae

The spurge hawkmoth Hyles euphorbiae L. (Sphingidae) comprises a remarkable species complex with still not fully resolved taxonomy. Its extensive natural distribution range covers diverse climatic zones. This predestinates particular populations to cope with different local seasonally unfavorable environmental conditions. The ability of the pupae to overcome outer frosty conditions is well known. However, the differences between two main ecotypes (‘euphorbiae’ and ‘tithymali’) in terms of the inherent degree of frost tolerance, its corresponding survival strategy, and underlying mechanism have not been studied in detail so far. The main aim of our study was to test the phenotypic exhibition of pupae (as the relevant life cycle stadia to outlast unfavorable conditions) in response to combined effects of exogenous stimuli, such as daylight length and cooling regime. Namely, we tested the turnout of subitan (with fast development, unadapted to unfavorable conditions) or diapause (paused development, adapted to unfavorable external influences and increased resistance) pupae under different conditions, as well as their mortality, and we measured the super cooling point (SCP) of whole pupae (in vivo) and pupal hemolymph (in vitro) as phenotypic indicators of cold acclimation. Our results show higher cold sensitivity in ‘tithymali’ populations, exhibiting rather opportunistic and short-termed cold hardiness, while ‘euphorbiae’ produces a phenotype of seasonal cold-hardy diapause pupae under a combined effect of short daylight length and continuous cold treatment. Further differences include the variability in duration and mortality of diapause pupae. This suggests different pre-adaptations to seasonal environmental conditions in each ecotype and may indicate a state of incipient speciation within the H. euphorbiae complex.

Comparison of the hemodynamic effects of nitric oxide and endothelium-dependent vasodilators in intact lungs

1990 ◽  
Vol 68 (2) ◽  
pp. 735-747 ◽  
Author(s):  
S. L. Archer ◽  
K. Rist ◽  
D. P. Nelson ◽  
E. G. DeMaster ◽  
N. Cowan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Methylene Blue ◽  
Pulmonary Hypertension ◽  
Feedback Inhibition ◽  
Pressor Response ◽  
Pulmonary Vasculature ◽  
Hemodynamic Effects ◽  
Isolated Lung

The effects of endothelium-dependent vasodilation on pulmonary vascular hemodynamics were evaluated in a variety of in vivo and in vitro models to determine 1) the comparability of the hemodynamic effects of acetylcholine (ACh), bradykinin (BK), nitric oxide (NO), and 8-bromo-guanosine 3′,5′-cyclic monophosphate (cGMP), 2) whether methylene blue is a useful inhibitor of endothelium-dependent relaxing factor (EDRF) activity in vivo, and 3) the effect of monocrotaline-induced pulmonary hypertension on the responsiveness of the pulmonary vasculature to ACh. In isolated rat lungs, which were preconstricted with hypoxia, ACh, BK, NO, and 8-bromo-cGMP caused pulmonary vasodilation, which was not inhibited by maximum tolerable doses of methylene blue. Methylene blue did not inhibit EDRF activity in any model, despite causing increased pulmonary vascular tone and responsiveness to various constrictor agents. There were significant differences in the hemodynamic characteristics of ACh, BK, and NO. In the isolated lung, BK and NO caused transient decreases of hypoxic vasoconstriction, whereas ACh caused more prolonged vasodilation. Pretreatment of these lungs with NO did not significantly inhibit ACh-induced vasodilation but caused BK to produce vasoconstriction. Tachyphylaxis, which was agonist specific, developed with repeated administration of ACh or BK but not NO. Tachyphylaxis probably resulted from inhibition of the endothelium-dependent vasodilation pathway proximal to NO synthesis, because it could be overcome by exogenous NO. Pretreatment with 8-bromo-cGMP decreased hypoxic pulmonary vasoconstriction and, even when the hypoxic pressor response had largely recovered, subsequent doses of ACh and NO failed to cause vasodilation, although BK produced vasoconstriction. These findings are compatible with the existence of feedback inhibition of the endothelium-dependent relaxation by elevation of cGMP levels. Responsiveness to ACh was retained in lungs with severe monocrotaline-induced pulmonary hypertension. Many of these findings would not have been predicted based on in vitro studies and illustrate the importance for expanding studies of EDRF to in vivo and ex vivo models.

Azole-resistant and -susceptible Aspergillus fumigatus isolates show comparable fitness and azole treatment outcome in immunocompetent mice

2017 ◽  
Vol 56 (6) ◽  
pp. 703-710
Author(s):  
Michaela Lackner ◽  
Günter Rambach ◽  
Emina Jukic ◽  
Bettina Sartori ◽  
Josef Fritz ◽  
...  
Keyword(s):  
Body Weight ◽  
Aspergillus Fumigatus ◽  
Severe Disease ◽  
Weight Ratio ◽  
Clinical Parameters ◽  
Fitness Advantage ◽  
Significant Difference ◽  
Immunocompetent Mice

Abstract No data are available on the in vivo impact of infections with in vitro azole-resistant Aspergillus fumigatus in immunocompetent hosts. Here, the aim was to investigate fungal fitness and treatment response in immunocompetent mice infected with A. fumigatus (parental strain [ps]) and isogenic mutants carrying either the mutation M220K or G54W (cyp51A). The efficacy of itraconazole (ITC) and posaconazole (PSC) was investigated in mice, intravenously challenged either with a single or a combination of ps and mutants (6 × 105 conidia/mouse). Organ fungal burden and clinical parameters were measured. In coinfection models, no fitness advantage was observed for the ps strain when compared to the mutants (M220K and G54W) independent of the presence or absence of azole-treatment. For G54W, M220K, and the ps, no statistically significant difference in ITC and PSC treatment was observed in respect to fungal kidney burden. However, clinical parameters suggest that in particular the azole-resistant strain carrying the mutation G54W caused a more severe disease than the ps strain. Mice infected with G54W showed a significant decline in body weight and lymphocyte counts, while spleen/body weight ratio and granulocyte counts were increased. In immunocompetent mice, in vitro azole-resistance did not translate into therapeutic failure by either ITC or PSC; the immune system appears to play the key role in clearing the infection.

scholarly journals Feedback Inhibition of the epithelial Na+ channel (ENaC): in vitro vs. in vivo

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Ankit Bharat Patel ◽  
Gustavo Frindt ◽  
Su Deng ◽  
Lawrence G. Palmer
Keyword(s):  
Feedback Inhibition ◽  
Na Channel ◽  
Epithelial Na Channel
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