scholarly journals Modulation of the fate of cytoplasmic mRNA by AU-rich elements: key sequence features controlling mRNA deadenylation and decay.

1997 ◽  
Vol 17 (8) ◽  
pp. 4611-4621 ◽  
Author(s):  
N Xu ◽  
C Y Chen ◽  
A B Shyu
Keyword(s):  
Mammalian Cells ◽  
Class Ii ◽  
Class I ◽  
Untranslated Regions ◽  
Sequence Motif ◽  
Class Iii ◽  
Decay Kinetics ◽  
Close Proximity

Regulation of cytoplasmic deadenylation has a direct impact on the fate of mRNA and, consequently, its expression in the cytoplasm. AU-rich elements (AREs) found in the 3' untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. AREs direct accelerated deadenylation as the first step in mRNA turnover. Recently we have proposed that AREs can be divided into three different classes. mRNAs bearing either the class I AUUUA-containing ARE or the class III non-AUUUA ARE display synchronous poly(A) shortening, whereas class II ARE-containing mRNAs are deadenylated asynchronously, with the formation of poly(A)- intermediates. In this study, we have systematically characterized the deadenylation kinetics displayed by various AREs and their mutant derivatives. We find that a cluster of five or six copies of AUUUA motifs in close proximity forming various degrees of reiteration is the key feature that dictates the choice between processive versus distributive deadenylation. An AU-rich region 20 to 30 nucleotides long immediately 5' to this cluster of AUUUA motifs can greatly enhance the destabilizing ability of the AUUUA cluster and is, therefore, an integral part of the class I and class II AREs. These two features are the defining characteristics of class II AREs. Our results are consistent with the interpretation that the pentanucleotide AUUUA, rather than the nonamer UUAUUUA(U/A)(U/A), is both an essential and the minimal sequence motif of AREs. Our study provides the groundwork for future characterization of ARE-binding proteins identified by in vitro gel shift assays in order to stringently define their potential role in the ARE-mediated decay pathway. Moreover, transformation of deadenylation kinetics from one type to the other by mutations of AREs implies the existence of cross talk between the ARE and 3' poly(A) tail, which dictates the decay kinetics.

295 ASSESSMENT OF CYTOTOXICITY AND INTERFERENCE OF ATELEIA GLAZIOVIANA IN BOVINE HERPESVIRUS TYPE 1 (BoHV-1) INTERACTION WITH IN VITRO-MATURED BOVINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 304
Author(s):  
D. L. Pavão ◽  
M. M. Piccolomini ◽  
A. C. Góes ◽  
R. Harakava ◽  
M. Haraguchi ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Class Ii ◽  
Class I ◽  
Class Iii ◽  
Herpesvirus Type ◽  
Action Analysis ◽  
Bovine Herpesvirus Type 1 ◽  
Bovine Oocytes ◽  
Class Iv

In vitro embryo production (IVP), as well as having a biotechnical importance, is a valuable tool for studies of gamete and/or embryo interaction with pathogens and xenobiotics. In consequence, it has become an excellent model not only for investigations about sanitary aspects, but also for aspects related to toxic processes. The aim of this study was to evaluate the effect of cytotoxic aqueous extract of Ateleia glazioviana and its interference on the interaction of bovine herpesvirus type 1 (BoHV-1) with bovine oocytes during the In vitro maturation (IVM) period. The statistical analysis of the experiments was made according to Student’s t-test (P < 0.05). The parameters used for this experiment were based on the morphological, physiological, and clastogenic action analysis of the bovine oocytes. The oocytes were collected from ovaries from slaughterhouse and divided into control group (G1, n = 214), a group infected with BoHV-1 (Los Angeles sample 105.5 TCID50 mL-1(G2, n = 210), a group exposed to the extract of A. glazioviana, 0.24 g mL-1; G3, n = 228), and a group simultaneously exposed to the virus and to the extract (G4, n = 210). For IVM, the oocytes were kept in TCM-199 supplemented with hormones and incubated at 38°C, 5%CO2, and 95% humidity for 24 h. The oocytes in G1 showed high expansion of the cumulus cells and ooplasm uniform in appearance; oocytes in G2 showed uniform but moderate expansion of cumulus cells and retraction of ooplasm; the G3 group showed low and irregular expansion with degeneration of cumulus cells and retraction of ooplasm with a granular aspect; and oocytes in G4 showed degeneration of cumulus cells, retracted and granular ooplasm. We observed maturation rates of 81.3% in G1, 31.0% in G2, 5.7% in G3, and 1.4% in G4. As for the clastogenic action analysis, an additional group of oocytes, named in natura (n = 210), was evaluated and presented 41.9% of comets class 0 (zero), 34.8% class I, 12.4% class II, 7.1% class III, and 3.8% class IV G1 (n = 211) presented 6.1% of comets class 0, 47.8% class I, 31.3% class II, 11.0% class III, and 3.8% class IV Oocytes belonging to G3 (217) presented 0.5% of comets class 0, 19.8% class I, 28.1% class II, 34.1% class III, and 17.5% class IV G2 (n = 229) presented 4.4% of comets class 0, 61.2% class I, 26.6% class II, 4.8% class III, and 3.0% class IV Oocytes in G4 (n = 206) presented 3.9% of comets class 0, 26.2% class I, and similar amounts of comets level II (23.8%), III (22.8%), and IV (23.3%). The statistical analysis presented a significant difference in the final results. Such results show the cytotoxic effect of A. glazioviana in bovine oocytes. The simultaneous exposure to the virus and the extract aggravated the effect of the virus, suggesting an increase of the pathogen within the gametic cell. Vitrocel/Embriolife.

scholarly journals Quantitative Cytochemical Observations on the Control of Respiration in Polymorphonuclear Neutrophil Leucocytes of Amphiuma Tridactylum

1972 ◽  
Vol 10 (3) ◽  
pp. 719-747
Author(s):  
G. B. DAVID ◽  
JAMIE M. McMULLEN
Keyword(s):  
Reductase Activity ◽  
Class Ii ◽  
Enzymic Activity ◽  
Class I ◽  
Polymorphonuclear Neutrophil ◽  
Amoeboid Movement ◽  
Class Iii ◽  
High Concentration ◽  
In Cells

Quantitative effects of altering oxidative phosphorylation and respiration on the activity of the enzyme menadione reductase (NAD[P]H2:2-methyl-1,4-naphthoquinone oxidoreductase, E.C. 1 6.5.2), in stabilized polymorphonuclear neutrophil leucocytes of Amphiuma tridactylum, were studied by amplitude-contrast microscopy and microspectrophotometry. In a general way, the rate of enzymic activity was proportional to ADP concentration and inversely proportional to the concentration of ATP. Terminal respiratory blocking by azide produced selective subtotal inhibition. Uncoupling of phosphorylation by dinitrophenol produced complex results. Neutrophils of A. tridactylum, irrespective of their stage of maturation in the circulating blood, could be subdivided into 3 metabolic classes: Class I cells, of low enzymic activity (predominantly mitochondrial), greatly activated by ADP, somewhat activated by ATP, and only slightly inhibited by dinitrophenol; Class II cells, twice as active as Class I (in which the endoplasmic reticulum and idiozome were as active as the mitochondria), further activated by ADP used alone or with dinitrophenol, and unaffected by ATP or dinitrophenol; Class III, hyperactive cells (enzymic localization identical with that in Class II), inhibited by ATP and dinitrophenol, and not activated by ADP. Some of the mitochondria of Class III neutrophils retained nearly a third of their reductase activity when the reaction mixture contained 10-1 azide. There is reason to believe that Class I neutrophils may form a reserve population of vegetative cells; in vitro, they can be transformed into Class II cells when a high concentration of ADP is added. Class II and Class III cells are potentially capable of amoeboid movement and phagocytosis. The metabolic mobilization of neutrophils could be interpreted as being controlled by 2 different feedback mechanisms: activation by ADP in cells of Classes I and II, and inhibition by ATP in cells of Class III.

The next-generation pan-RAF inhibitor, KIN-2787, is active in class II and class III BRAF mutant models.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3116-3116
Author(s):  
Aleksandra Franovic ◽  
Nichol Miller ◽  
Paul Severson ◽  
Toufike Kanouni ◽  
Noelito Timple ◽  
...  
Keyword(s):  
Class Ii ◽  
Class I ◽  
Braf Inhibitors ◽  
Class Iii ◽  
Braf Gene ◽  
Braf Mutations ◽  
Braf Mutant ◽  
Gene Alterations

3116 Background: Oncogenic BRAF gene alterations, leading to aberrantly activated BRAF monomers (Class I mutations) or dimers (Class II and Class III mutations), are observed in approximately 6% of all human cancers. First-generation BRAF inhibitors targeting Class I BRAF mutants, including dabrafenib, encorafenib, and vemurafenib, provide significant clinical benefit to patients with BRAF V600 mutation-driven melanoma and select solid tumors as monotherapies or in combination with other targeted therapies. The currently approved BRAF inhibitors have not, however, proven to be effective in patients with Class II or III BRAF alterations which account for a large proportion (34%) of BRAF mutations. KIN-2787 is an orally available, potent and selective small molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers, in addition to Class I mutants. Methods: The efficacy and tolerability of the pan-RAF inhibitor, KIN-2787, was evaluated in vitro and in vivo in Class I, II, and III BRAF mutation-driven human cancer models. Results: In biochemical assays, KIN-2787 showed low nanomolar to picomolar potency against RAF1, BRAF, and ARAF (IC50 0.06-3.46 nM) with minimal activity towards non-RAF kinases. In cell-based assays, KIN-2787 inhibited RAF activity, as measured by inhibition of downstream ERK phosphorylation (pERK), across multiple BRAF mutant cancer cell lines. Class II and III BRAF mutant cell lines were the most responsive when treated with KIN-2787 (IC50 < 50 nM); 19- and 7-fold more sensitive compared to cells harboring wild-type BRAF, respectively. Dose-dependent inhibition of A-375 (Class I), BxPC-3 (Class II), and WM3629 (Class III) BRAF mutant human xenograft tumor growth was attained with daily KIN-2787 treatment and was well-tolerated. A trend towards greater tumor responses was observed with twice daily (BID) compared to once daily (QD) dosing of KIN-2787; however, the two dosing regimens led to similar tumor growth inhibition (TGI) and regressions (mean TGI up to 101-118%; p ≤0.0001) at equivalent total daily doses. Furthermore, KIN-2787 led to a significant in vivo pharmacodynamic response using either regimen, however, prolonged target coverage, as measured by pERK, was achieved with BID dosing. The impact of KIN-2787 treatment on additional biomarkers, including transcriptional changes and MAPK pathway modulation in cell-based models and patient-derived samples, will be presented at the meeting. Conclusions: KIN-2787 is a next-generation pan-RAF inhibitor with pronounced in vitro and in vivo activity against human cancers driven by Class II and III BRAF mutations. A phase 1 dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 monotherapy in patients with advanced or metastatic solid tumors harboring BRAF gene alterations, including Class II and III mutations, is expected to initiate in 2021.

scholarly journals Comparative study of Two In Vitro Methods for Assessing Drug Absorption: Sartorius SM 16750 Apparatus Versus Everted Gut Sac

2011 ◽  
Vol 14 (1) ◽  
pp. 117 ◽  
Author(s):  
Mohamed Ali Lassoued ◽  
Fathia Khemiss ◽  
Souad Sfar
Keyword(s):  
Drug Absorption ◽  
Intestinal Barrier ◽  
Class Ii ◽  
In Vitro Models ◽  
Class I ◽  
Oral Drug ◽  
Class Iii ◽  
Apparent Permeability ◽  
Everted Gut Sac

– Purpose. Oral drug administration remains the most common and most convenient way used in clinical therapy. The availability of a simple, rapid, economic and reproducible in vitro method to assess drug absorption is a very helpful tool. The purpose of this study is to compare the performance of Sartorius SM 16750 Absorption Simulator apparatus to Everted Gut Sac (EGS) technique in terms of predicting drug absorption. Methods. Permeation studies across these two in vitro models were performed with six drugs selected across the Biopharmaceutics Classification System (BCS) categories: Tramadol (class I of BCS), doxcycycline (class I of BCS), diclofenac (class II of BCS), clopidogrel (class II of BCS), metformin (class III of BCS) and chlorothiazide (class IV of BCS). Results. Apparent permeability coefficient (Papp) and diffusion profiles obtained with EGS and Sartorius SM 16750 apparatus were similar for diclofenac and metformine. But there were differences in results, for the other molecules. Conclusion. It could be concluded that the Sartorius SM 16750 apparatus, easier to carry out, could be an alternative to EGS when drug passage across intestinal barrier occurs by passive diffusion and when no efflux system is implicated in limiting the transepithelial passage.

scholarly journals GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors

2002 ◽  
Vol 365 (2) ◽  
pp. 369-378 ◽  
Author(s):  
Hiroyuki TAKATSU ◽  
Kaori YOSHINO ◽  
Kyoko TODA ◽  
Kazuhisa NAKAYAMA
Keyword(s):  
Membrane Trafficking ◽  
Class Ii ◽  
Small Gtpases ◽  
Class I ◽  
Class Iii ◽  
Limited Region ◽  
Adp Ribosylation ◽  
Early Endosomes

ADP-ribosylation factors (ARFs) are a family of small GTPases that are involved in various aspects of membrane trafficking events. These include ARF1—ARF6, which are divided into three classes on the basis of similarity in the primary structure: Class I, ARF1—ARF3; Class II, ARF4 and ARF5; and Class III, ARF6. Previous studies identified a novel family of potential ARF effectors, termed GGA1—GGA3, which interact specifically with GTP-bound ARF1 and ARF3 and are localized to the trans-Golgi network (TGN) or its related compartment(s) (GGA is an abbreviation for Golgi-localizing, γ-adaptin ear homology domain, ARF-binding protein). In the present study we have shown that ARF proteins belonging to the three classes, ARF1, ARF5 and ARF6, can interact with all GGA proteins in a yeast two-hybrid assay, in vitro and in vivo. Segmentation of GGA proteins and isolation of GGA mutants defective in ARF binding have revealed that a limited region within the GGA homology domain, which is conserved in the GGA family, is essential for ARF binding. Expression in cells of GTPase-restricted mutants of ARF1 and ARF5 blocks dissociation of GGA proteins from membranes induced by brefeldin A. However, neither of the ARF mutants recruits GGA mutants defective in ARF binding. On the basis of these observations, we conclude that at least ARF1 (Class I) and ARF5 (Class II) in their GTP-bound state cause recruitment of GGA proteins on to TGN membranes. In contrast, on the basis of similar experiments, ARF6 (Class III) may be involved in recruitment of GGA proteins to other compartments, possibly early endosomes.

scholarly journals Environmental Cadmium Exposure and Dental Indices in Orthodontic Patients

Healthcare ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 413
Author(s):  
Hui-Ling Chen ◽  
Jason Chen-Chieh Fang ◽  
Chia-Jung Chang ◽  
Ti-Feng Wu ◽  
I-Kuan Wang ◽  
...  
Keyword(s):  
Cadmium Concentration ◽  
Class Ii ◽  
Cadmium Exposure ◽  
Class I ◽  
P Value ◽  
Tooth Decay ◽  
Class Iii ◽  
Orthodontic Patients ◽  
Urine Cadmium ◽  
The Mean

Background. Previous studies have shown that environmental cadmium exposure could disrupt salivary gland function and is associated with dental caries and reduced bone density. Therefore, this cross-sectional study attempted to determine whether tooth decay with tooth loss following cadmium exposure is associated with some dental or skeletal traits such as malocclusions, sagittal skeletal pattern, and tooth decay. Methods. Between August 2019 and June 2020, 60 orthodontic patients with no history of previous orthodontics, functional appliances, or surgical treatment were examined. The patients were stratified into two groups according to their urine cadmium concentrations: high (>1.06 µg/g creatinine, n = 28) or low (<1.06 µg/g creatinine, n = 32). Results. The patients were 25.07 ± 4.33 years old, and most were female (female/male: 51/9 or 85%). The skeletal relationship was mainly Class I (48.3%), followed by Class II (35.0%) and Class III (16.7%). Class I molar relationships were found in 46.7% of these patients, Class II molar relationships were found in 15%, and Class III molar relationships were found in 38.3%. The mean decayed, missing, and filled surface (DMFS) score was 8.05 ± 5.54, including 2.03 ± 3.11 for the decayed index, 0.58 ± 1.17 for the missing index, and 5.52 ± 3.92 for the filled index. The mean index of complexity outcome and need (ICON) score was 53.35 ± 9.01. The facial patterns of these patients were within the average low margin (26.65 ± 5.53 for Frankfort–mandibular plane angle (FMA)). There were no significant differences in the above-mentioned dental indices between patients with high urine cadmium concentrations and those with low urine cadmium concentrations. Patients were further stratified into low (<27, n = 34), average (27–34, n = 23), and high (>34, n = 3) FMA groups. There were no statistically significant differences in the urine cadmium concentration among the three groups. Nevertheless, a marginally significant p-value of 0.05 for urine cadmium concentration was noted between patients with low FMA and patients with high FMA. Conclusion. This analysis found no association between environmental cadmium exposure and dental indices in our orthodontic patients.

scholarly journals Characterization of a Cytotoxic Factor in Culture Filtrates of Serratia marcescens

2002 ◽  
Vol 70 (3) ◽  
pp. 1121-1128 ◽  
Author(s):  
Kent B. Marty ◽  
Christopher L. Williams ◽  
Linda J. Guynn ◽  
Michael J. Benedik ◽  
Steven R. Blanke
Keyword(s):  
Cytotoxic Activity ◽  
Serratia Marcescens ◽  
Mammalian Cells ◽  
Divalent Cations ◽  
Recombinant Expression ◽  
Cytotoxic Factor ◽  
Culture Filtrates ◽  
Type Strains

ABSTRACT Serratia marcescens culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of S. marcescens culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that S. marcescens mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the S. marcescens 56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic Escherichia coli strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in S. marcescens culture filtrates.

scholarly journals Upstream basal promoter element important for exclusive RNA polymerase III transcription of the EBER 2 gene.

1993 ◽  
Vol 13 (5) ◽  
pp. 2655-2665 ◽  
Author(s):  
J G Howe ◽  
M D Shu
Keyword(s):  
Rna Polymerase ◽  
Consensus Sequence ◽  
Rna Polymerase Iii ◽  
Class Ii ◽  
Transcription Unit ◽  
Tata Box ◽  
Promoter Element ◽  
Class Iii ◽  
Polymerase Iii

The Epstein-Barr virus-encoded small RNA (EBER) genes are transcribed by RNA polymerase III, but their transcription unit appears to contain both class II and class III promoter elements. One of these promoter element, a TATA-like box which we call the EBER TATA box, or ETAB, is located in a position typical for a class II TATA box but contains G/C residues in the normal T/A motif and a conserved thymidine doublet. Experiments using chloramphenicol acetyltransferase constructs and mutations in the TATA box of the adenovirus major late promoter showed that the ETAB promoter element does not substitute for a class II TATA box. However, when the ETAB promoter element sequence was changed to a class II TATA box consensus sequence, the EBER 2 gene was transcribed in vitro by both RNA polymerases II and III. From these results, we conclude that the ETAB promoter element is important for the exclusive transcription of the EBER 2 gene by RNA polymerase III.

scholarly journals A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

1976 ◽  
Vol 159 (2) ◽  
pp. 245-257 ◽  
Author(s):  
R Norris ◽  
K Brocklehurst
Keyword(s):  
Convenient Method ◽  
Specific Activity ◽  
Class Ii ◽  
High Reactivity ◽  
Class I ◽  
Thiol Groups ◽  
Class Iii ◽  
Active Centre ◽  
Class Ib ◽  
Covalent Chromatography

1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 ± 1.47 kat/kg, Km = 3.32 ± 0.05 mM; kcat. = 2.15 × 104 ± 0.05 × 104s-1 at pH7.0 and 38°C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin.11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and ε280 = 2.84 × 105 litre·mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six ‘essential’ thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25°C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 × 104M-1·s-1 and (b) pKa = 8.1, k = 8.05 × 102M-1·s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 × 102M-1·s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases as the pH is raised according to a pKa value ≥ approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the ‘essential’ thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.

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