scholarly journals Structure-Function Study of MalF Protein by Random Mutagenesis

1999 ◽  
Vol 181 (7) ◽  
pp. 2267-2272 ◽  
Author(s):  
María Isabel Tapia ◽  
Michaël Mourez ◽  
Maurice Hofnung ◽  
Elie Dassa
Keyword(s):  
Steady State ◽  
Subcellular Localization ◽  
Cytoplasmic Membrane ◽  
Random Mutagenesis ◽  
State Level ◽  
Mutant Proteins ◽  
Transmembrane Segments ◽  
Functional Regions ◽  
Large N ◽  
Steady State Levels

ABSTRACT MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of malFmutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism.

scholarly journals Cyclin-Dependent Kinase Activity Is Required for Efficient Expression and Posttranslational Modification of Human Cytomegalovirus Proteins and for Production of Extracellular Particles

2006 ◽  
Vol 80 (12) ◽  
pp. 5886-5896 ◽  
Author(s):  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Steady State ◽  
Human Cytomegalovirus ◽  
Virus Titer ◽  
State Level ◽  
Early Gene ◽  
Tegument Protein ◽  
Cyclin Dependent Kinase ◽  
Viral Dna ◽  
Infected Cells ◽  
Steady State Levels

ABSTRACT We have previously shown that the addition of the cyclin-dependent kinase (cdk) inhibitor Roscovitine at the beginning of infection of cells with human cytomegalovirus (HCMV) significantly disrupts immediate-early gene expression and the progression of the infection. In the present study, we have examined the effects of cdk inhibition on late viral events by delaying addition of Roscovitine until 24 h postinfection. Although viral DNA replication was inhibited two- to threefold by treatment of infected cells with Roscovitine, the drop did not correspond to the 1- to 2-log-unit decrease in virus titer. Quantification of viral DNA in the supernatant from cells revealed that there was a significant reduction in the production or release of extracellular particles. We observed a lag in the expression of several viral proteins but there was a significant decrease in the steady-state levels of IE2-86. Likewise, the steady-state level of the essential tegument protein UL32 (pp150) was reduced. The levels of pp150 and IE2-86 mRNA were not greatly affected by treatment with Roscovitine and thus did not correlate with the reduced levels of protein. In contrast, the expression of the tegument protein ppUL69 was higher in drug-treated samples, and the protein accumulated in a hyperphosphorylated form. ppUL69 localized to intranuclear aggregates that did not overlap with viral replication centers in cells treated with Roscovitine. Taken together, these data indicate that cdk activity is required at multiple steps during HCMV infection, including the expression, modification, and localization of virus-encoded proteins.

scholarly journals Proteasomal Degradation of the Papillomavirus E2 Protein Is Inhibited by Overexpression of Bromodomain-Containing Protein 4

2009 ◽  
Vol 83 (9) ◽  
pp. 4127-4139 ◽  
Author(s):  
David Gagnon ◽  
Simon Joubert ◽  
Hélène Sénéchal ◽  
Amélie Fradet-Turcotte ◽  
Sabrina Torre ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Steady State ◽  
Luciferase Assay ◽  
Viral Gene ◽  
Transactivation Domain ◽  
Interaction Domain ◽  
E2 Protein ◽  
Mutant Proteins ◽  
Steady State Levels

ABSTRACT The E2 protein of human papillomavirus (HPV) binds to specific sites in the viral genome to regulate its transcription, replication, and maintenance in infected cells. Like most regulatory proteins, E2 is rapidly turned over. A high-throughput assay was developed to quantify the expression and stability of E2 in vivo, based on its fusion to Renilla luciferase (RLuc). The steady-state levels of Rluc-E2 were quantified by measuring the amounts of associated luciferase activity, and its degradation was measured by monitoring the decrease in enzymatic activity occurring after a block of translation with cycloheximide. Using this assay, the E2 proteins from a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) type were found to have short half-lives of 60 min in C33A cervical carcinoma cells and to be ubiquitinated and degraded by the proteasome. Analysis of mutant proteins showed that the instability of E2 is independent of its DNA-binding and transcriptional activities but is encoded within its transactivation domain, the region that binds to the cellular chromatin factor bromodomain-containing protein 4 (Brd4) to regulate viral gene transcription. Overexpression of Brd4, or of its C-terminal E2-interaction domain, was found to increase the steady-state levels and stability of wild-type E2 but not of E2 mutants defective for binding Brd4. These results indicate that the stability of E2 is increased upon complex formation with Brd4 and highlight the value of the luciferase assay for the study of E2 degradation.

Steady-State Serum Concentrations of Dothiepin and Northiaden after Two Dosage Regimens of Dothiepin Hydrochloride (Prothiaden)

1973 ◽  
Vol 1 (2) ◽  
pp. 391-397
Author(s):  
B R S Nakra ◽  
R C Glass ◽  
J A Rees
Keyword(s):  
Steady State ◽  
Parent Drug ◽  
State Level ◽  
Serum Levels ◽  
Single Daily Dose ◽  
Serum Concentrations ◽  
Dosage Regimens ◽  
Daily Dose ◽  
Steady State Serum ◽  
Steady State Levels

Five healthy volunteers took part in a crossover study which examined the serum concentrations of dothiepin and northiaden after a 25 mg three times a day and a 75 mg once a day dosage regimen of Prothiaden. The inter-individual variation of serum levels was large after either schedule which is to be expected with this group of drugs. The minimum steady-state level of dothiepin tended to be lower after the single daily dose, but the differences were small and not statistically significant. The approximate maximum steady-state levels of dothiepin showed large intra- and inter-subject variation and no obvious trend. The values of the desmethylated metabolite, northiaden, tended to follow the dothiepin concentrations but were lower than the parent drug. Average steady-state levels tended, with one exception, to be very similar after both regimens with no evidence of any trend when comparing the two regimens. The study showed that the two regimens yielded similar steady-state serum concentrations both of drug and metabolite but inter-individual differences were large.

scholarly journals An orthogonal c-Cbl recognition mode targets LynA for rapid degradation and builds specificity into the LynA checkpoint

2019 ◽  
Author(s):  
Ben F. Brian ◽  
Myra G. Nunez ◽  
Kathryn L. Schwertfeger ◽  
Tanya S. Freedman
Keyword(s):  
Steady State ◽  
Ubiquitin Ligase ◽  
Biochemical Analysis ◽  
Cell Activation ◽  
Critical Factor ◽  
Myeloid Cell ◽  
State Level ◽  
Cell Specificity ◽  
Steady State Levels ◽  
Insert Region

AbstractThe activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is critical factor regulating myeloid-cell activation. In a previous paper (Freedman et al., 2015) we showed in macrophages that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation, functioning as a rheostat regulating ITAM signaling. We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic and biochemical analysis, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via tyrosine 32 in its unique insert region. This orthogonal mode of c-Cbl recognition depresses the steady-state level of macrophage LynA. Mast cells, however, express little c-Cbl and have correspondingly high steady-state levels of LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression therefore builds cell specificity into the LynA checkpoint.

scholarly journals Role of STE genes in the mating factor signaling pathway mediated by GPA1 in Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (9) ◽  
pp. 3777-3783 ◽  
Author(s):  
N Nakayama ◽  
Y Kaziro ◽  
K Arai ◽  
K Matsumoto
Keyword(s):  
Steady State ◽  
Signaling Pathway ◽  
State Level ◽  
G1 Arrest ◽  
Gene Products ◽  
Mating Efficiency ◽  
Protein Functions ◽  
Steady State Levels ◽  
Mating Factor

The ste mutants (ste2, ste4, ste5, ste7, ste11, and ste12) are insensitive to mating factors and are, therefore, sterile. Roles of the STE gene products in the GPA1-mediated mating factor signaling pathway were studied by using ste gpa1 double mutants. Mating efficiency of a ste2 mutant defective in the alpha-factor receptor increased 1,000-fold in a gpa1 background, while G1 arrest and aberrant morphology (shmoo) caused by gpa1 were not suppressed by ste2. Furthermore, the steady-state level of the FUS1 transcript, which normally increases in response to mating factors, was also elevated when the GPA1 function was impaired. These results suggest that the GPA1 protein functions downstream of the STE2 receptor. Conversely, the sterility of ste4, ste5, ste7, ste11, and ste12 mutants was not suppressed by gpa1, but the lethal phenotype of gpa1 was suppressed by these ste mutations. Northern (RNA) blotting analysis revealed that the ste7, ste11, and ste12 mutations caused reductions of 50 to 70% in the steady-state levels of the GPA1 transcript, while ste4 had a slight effect and ste5 had no effect. This implies that the suppression by ste7, ste11, and ste12 could be due to reduced syntheses of additional components, including an effector, and that suppression by ste4 and ste5 may result from direct effects on the signaling pathway. The STE4, STE5, STE7, STE11, and STE12 products, therefore, appear to specify components of the signal transduction machinery, directly or indirectly, which function together with or downstream of GPA1.

scholarly journals NEMO score in nailfold videocapillaroscopy is a good tool to assess both steady state levels and overtime changes of disease activity in patients with systemic sclerosis: a comparison with the proposed composite indices for this disease status entity

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Francesca Pignataro ◽  
Wanda Maglione ◽  
Antonina Minniti ◽  
Domenico Sambataro ◽  
Gianluca Sambataro ◽  
...  
Keyword(s):  
Steady State ◽  
Systemic Sclerosis ◽  
Disease Activity ◽  
State Level ◽  
Disease Status ◽  
Cumulative Number ◽  
Nailfold Videocapillaroscopy ◽  
Roc Curve Analysis ◽  
Good Tool ◽  
Steady State Levels

Abstract Background In previous studies, we demonstrated that the NEMO score, i.e. the cumulative number of microhaemorrhages (MHEs) and microthromboses (MTs), observed in nailfold videocapillaroscopy was a good indicator of the steady state level of disease activity (DA) in patients with systemic sclerosis (SSc) when the European Scleroderma Study Group (EScSG) index was considered the gold standard. Aim of the study To verify whether the NEMO score could be (i) a valid tool to assess DA, even when the modified European Scleroderma Trials and Research (EUSTAR) index was considered to be the comparator, and (ii) a sensitive method to capture the DA overtime changes. Patients and methods The NEMO score and the EScSG and EUSTAR indices were contemporarily assessed at baseline (T0) and after a follow-up of 4–56 months (T1) in 98 patients with SSc. The differences (Δ) between the T1 and T0 values of the NEMO score and the EScSG and EUSTAR indices were calculated and compared to each other. Results NEMO score values were very closely correlated with the corresponding values of the EScSG and EUSTAR indices both at T0 and T1 observations (p < 0.0001 in all cases with the exception of the correlation with EScSG values at T1 (p < 0.03)). The values of the two composite DA indices were also strictly related to each other in both T0 and T1 observations (p < 0.0001). Receiver operating characteristic (ROC) curve analysis showed the NEMO score had a good sensitivity and specificity in classifying patients with a predefined level of DA (scores ≥ 3.0 and ≥ 2.5 for the EScSG and EUSTAR indices, respectively, p < 0.0001 in both cases). Δ values of the NEMO score were significantly correlated with the corresponding values of both the EScSG and EUSTAR indices. Weighted Cohen’s k level of agreement between Δ values of the NEMO score and those of the EScSG and EUSTAR indices was moderate (0.55 and 0.59, respectively). Conclusions NEMO score proves to be a feasible, non-invasive, and valid tool to assess steady state levels and changes over time of DA in patients with SSc. Thus, it can represent an alternative or complementary method to measure this disease status entity in this disorder.

Induction of gene expression during involution of the lactating mammary gland of the rat

1994 ◽  
Vol 12 (1) ◽  
pp. 47-60 ◽  
Author(s):  
R S Guenette ◽  
H B Corbeil ◽  
J Léger ◽  
K Wong ◽  
V Mézl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Steady State ◽  
Epithelial Cells ◽  
Tissue Transglutaminase ◽  
State Level ◽  
Ultrastructural Changes ◽  
Steady State Level ◽  
Hsp 27 ◽  
Steady State Levels

ABSTRACT After weaning, the mammary gland ceases lactation and involutes. The wet weight of the gland decreases by 70% within 4 days of weaning. This involves significant tissue remodelling as the ducts regress and return to the resting state. The presence of apoptotic bodies in the luminal epithelial compartment 2 to 3 days after weaning provides clear evidence that a substantial proportion of the regression is attributable to the induction of active cell death (ACD) of the epithelial cells. These changes in the architecture of the gland were found to be mirrored by changes in gene expression. The steady-state level of β-casein mRNA decreased rapidly after weaning from the high levels seen during lactation to undetectable levels by 8 days after weaning. The steady-state levels of expression of a number of genes associated with ACD, including TRPM-2, tissue transglutaminase (TGase) and poly(ADP-ribose) polymerase (PARP), increased transiently during this time-frame. The steady-state level of TRPM-2 mRNA increased 2 days after weaning, reaching a peak on day 4, and decreasing to undetectable levels by day 8 after weaning. The steady-state levels of two other mRNAs, TGase and PARP, showed very similar kinetics. In contrast, the mRNA for Hsp 27, which has been shown to be induced during prostate regression, was not significantly induced in the regressing mammary gland. In-situ hybridization demonstrated that the TRPM-2, TGase and PARP genes were expressed predominantly in the luminal epithelial cells of the ducts. These cells expressed β-casein mRNA during lactation, and underwent ACD after weaning. While the ultrastructural changes in the mammary gland after weaning, and the induction of TRPM-2, TGase and PARP mRNAs, are reminiscent of apoptosis in the prostate, several features of the process are different. Most notably, the disruption of the secretory processes and the lack of increased expression of Hsp 27 in the regressing mammary gland suggest that there may be a number of important events in ACD that are not common to all cells.

scholarly journals Melatonin Mitigates the Infection of Colletotrichum gloeosporioides via Modulation of the Chitinase Gene and Antioxidant Activity in Capsicum annuum L.

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 7
Author(s):  
Muhammad Ali ◽  
Anthony Tumbeh Lamin-Samu ◽  
Izhar Muhammad ◽  
Mohamed Farghal ◽  
Abdul Mateen Khattak ◽  
...  
Keyword(s):  
Antioxidant Enzymes ◽  
Steady State ◽  
Colletotrichum Gloeosporioides ◽  
State Level ◽  
Pathogen Resistance ◽  
Chitinase Gene ◽  
Pathogenesis Related ◽  
Steady State Levels ◽  
Plant Pathogen Interactions ◽  
Pepper Plants

Anthracnose, caused by Colletotrichum gloeosporioides, is one of the most damaging pepper (Capsicum annum L.) disease. Melatonin induces transcription of defense-related genes that enhance resistance to pathogens and mediate physiological activities in plants. To study whether the melatonin-mediated pathogen resistance is associated with chitinase gene (CaChiIII2), pepper plants and Arabidopsis seeds were treated with melatonin, then CaChiIII2 activation, hydrogen peroxide (H2O2) levels, and antioxidant enzymes activity during plant–pathogen interactions were investigated. Melatonin pretreatment uncoupled the knockdown of CaChiIII2 and transiently activated its expression level in both control and CaChiIII2-silenced pepper plants and enhanced plant resistance. Suppression of CaChiIII2 in pepper plants showed a significant decreased in the induction of defense-related genes and resistance to pathogens compared with control plants. Moreover, melatonin efficiently enabled plants to maintain intracellular H2O2 concentrations at steady-state levels and enhanced the activities of antioxidant enzymes, which possibly improved disease resistance. The activation of the chitinase gene CaChiIII2 in transgenic Arabidopsis lines was elevated under C. gloeosporioides infection and exhibited resistance through decreasing H2O2 biosynthesis and maintaining H2O2 at a steady-state level. Whereas melatonin primed CaChiIII2-overexpressed (OE) and wild-type (WT) Arabidopsis seedlings displayed a remarkable increase in root-length compared to the unprimed WT plants. Using an array of CaChiIII2 knockdown and OE, we found that melatonin efficiently induced CaChiIII2 and other pathogenesis-related genes expressions, responsible for the innate immunity response of pepper against anthracnose disease.

scholarly journals Down-regulation of actin genes precedes microfilament network disruption and actin cleavage during p53-mediated apoptosis

1997 ◽  
Vol 110 (4) ◽  
pp. 489-495 ◽  
Author(s):  
I. Guenal ◽  
Y. Risler ◽  
B. Mignotte
Keyword(s):  
Steady State ◽  
Interleukin 1 ◽  
Simian Virus 40 ◽  
State Level ◽  
Simian Virus ◽  
T Antigen ◽  
Mrna Levels ◽  
Down Regulation ◽  
Actin Genes ◽  
Steady State Levels

Inactivation of Simian Virus 40 large T antigen, in cells immortalized with conditional mutants, leads to activation of p53 and apoptosis. We used the mRNA differential display method to identify genes differentially expressed during this process. We found that steady-state levels of mRNA for cytoplasmic actins decreased early during apoptosis. We also showed that, although the steady-state level of the corresponding proteins is not profoundly affected, they are substrates for an interleukin 1-beta converting enzyme (ICE)-like protease activated during the process. However, only a very small fraction of actin is proteolysed during the early stages of apoptosis. The microfilament network is affected and non polymerized actin accumulates in apoptotic bodies after the decrease of mRNA levels, but before a significant amount of actin is cleaved. This suggests that down-regulation of actin genes may be involved in microfilament rearrangements during p53-mediated apoptosis.

scholarly journals Non-canonical localization of RubisCO under high light conditions in the toxic cyanobacteriumMicrocystis aeruginosaPCC7806

2019 ◽  
Author(s):  
Tino Barchewitz ◽  
Arthur Guljamow ◽  
Sven Meissner ◽  
Stefan Timm ◽  
Manja Henneberg ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Cytoplasmic Membrane ◽  
High Cell Density ◽  
High Light ◽  
Membrane Association ◽  
High Cell ◽  
Light Conditions ◽  
Wild Type ◽  
Electron Microscopic ◽  
Steady State Levels

AbstractThe frequent production of the hepatotoxin microcystin and its impact on the life-style of bloom-forming cyanobacteria are poorly understood. Here we report that microcystin interferes with the assembly and the subcellular localization of RubisCO, inMicrocystis aeruginosaPCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes inM. aeruginosaand relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of microcystin to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2fixation product 3-phosphoglycerate are significantly higher in the microcystin-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction ofM. aeruginosawith its heterotrophic microbial community.

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