scholarly journals Molecular mechanisms of the irreversible thermal denaturation of guinea-pig liver transglutaminase

1990 ◽  
Vol 266 (2) ◽  
pp. 487-490 ◽  
Author(s):  
S Nury ◽  
J C Meunier
Keyword(s):  
Thermal Denaturation ◽  
Molecular Mechanisms ◽  
Molecular Events ◽  
Sds Page ◽  
Irreversible Denaturation ◽  
Enzyme Molecules ◽  
Kinetics Of ◽  
Guinea Pig Liver ◽  
Step Mechanism ◽  
Rule Out

When transglutaminase is heated at temperatures above 40 degrees C, it loses its activity according to a two-step mechanism [Nury, Meunier & Mouranche (1989) Eur. J. Biochem. 180, 161-166]: N→X(TD)→D However, the nature of the molecular events responsible for the irreversible denaturation is still unknown. Investigation of the effects of dithiothreitol and 5,5′-dithiobis-2-nitrobenzoate on the kinetics of inactivation, titrations of ammonia released by deamidation and of thiol groups on the native and denatured enzymes and SDS/PAGE rule out the involvement of covalent processes during the denaturation of transglutaminase at 55 degrees C and pH 7. Of the two possible kinds of non-covalent events, i.e. unfolding of the polypeptide chain and aggregation of enzyme molecules, we show that both occur, though only the former process is responsible for the denaturation. The latter process, aggregation, follows the unfolding of the molecules.

Isotopic Effect on the Kinetic of Thermal Denaturation of Ceruloplasmin

1985 ◽  
Vol 40 (7-8) ◽  
pp. 551-554 ◽  
Author(s):  
L. Sportelli ◽  
A. Desideri ◽  
A. Campaniello
Keyword(s):  
Thermal Denaturation ◽  
Charge Transfer Band ◽  
Type I ◽  
Protein Solution ◽  
First Order ◽  
Irreversible Denaturation ◽  
Optical Density Change ◽  
Kinetics Of ◽  
Protein Active Site ◽  
Denaturation Process

Abstract The kinetics of thermal denaturation of ceruloplasmin in water and in water with different percentage of D20 in the temperature range 25 - 85 °C, following the optical density change of the 610 nm charge transfer band of the protein has been investigated. A temperature Tt ≃ 65 °C above which an irreversible denaturation process of the protein active site takes place has been found. The kinetics of the denaturation process of the protein are fitted by two first order reactions, which have been assigned to a different thermal denaturation behaviour of the two Cu2+ type I sites existing in the protein. Addition of D2O to the protein solution affects the two kinetics in a different way, i.e. the rate of one of them is increased whilst that of the other is decreased. The different effect of D2O on the kinetics of disruption of the two copper sites is discussed in terms of different location and degree of hydrophobicity of the two Cu2+ type I sites.

scholarly journals High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Betty Ha ◽  
Kevin P. Larsen ◽  
Jingji Zhang ◽  
Ziao Fu ◽  
Elizabeth Montabana ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Viral Entry ◽  
Reverse Transcription ◽  
Molecular Mechanisms ◽  
Dissociation Kinetics ◽  
Structural Mechanism ◽  
Medium Resolution ◽  
Kinetics Of ◽  
Hiv 1

AbstractReverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC–nevirapine, and RTIC–efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA–tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

scholarly journals Prospective Evaluation of Serum β-Glucan Testing in Patients With Probable or Proven Fungal Diseases

2016 ◽  
Vol 3 (3) ◽  
Author(s):  
Cécile Angebault ◽  
Fanny Lanternier ◽  
Frédéric Dalle ◽  
Cécile Schrimpf ◽  
Anne-Laure Roupie ◽  
...  
Keyword(s):  
Clinical Utility ◽  
Clinical Suspicion ◽  
Fungal Diseases ◽  
Prospective Evaluation ◽  
Invasive Fungal Diseases ◽  
Undetectable Serum ◽  
Antifungal Use ◽  
Kinetics Of ◽  
Rule Out

Abstract Background.  Early diagnosis and treatment are crucial in invasive fungal diseases (IFD). Serum (1-3)-β-d-glucan (BG) is believed to be an early IFD marker, but its diagnostic performance has been ambiguous, with insufficient data regarding sensitivity at the time of IFD diagnosis (TOD) and according to outcome. Whether its clinical utility is equivalent for all types of IFD remains unknown. Methods.  We included 143 patients with proven or probable IFD (49 invasive candidiasis, 45 invasive aspergillosis [IA], and 49 rare IFD) and analyzed serum BG (Fungitell) at TOD and during treatment. Results.  (1-3)-β-d-glucan was undetectable at TOD in 36% and 48% of patients with candidemia and IA, respectively; there was no correlation between negative BG results at TOD and patients' characteristics, localization of infection, or prior antifungal use. Nevertheless, patients with candidemia due to Candida albicans were more likely to test positive for BG at TOD (odds ratio = 25.4, P = .01) than patients infected with other Candida species. In 70% of the patients with a follow-up, BG negativation occurred in >1 month for candidemia and >3 months for IA. A slower BG decrease in patients with candidemia was associated with deep-seated localizations (P = .04). Thirty-nine percent of patients with rare IFD had undetectable BG at TOD; nonetheless, all patients with chronic subcutaneous IFD tested positive at TOD. Conclusions.  Undetectable serum BG does not rule out an early IFD, when the clinical suspicion is high. After IFD diagnostic, kinetics of serum BG are difficult to relate to clinical outcome.

Abstract 126: High Efficiency Reprogramming of Fibroblasts Into Cardiomyocytes Requires Suppression of Pro-fibrotic Signaling

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Kunhua Song ◽  
Yuanbiao Zhao ◽  
Pilar Londono ◽  
Emily Sharpe ◽  
Joshua R Clair ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
High Efficiency ◽  
Transforming Growth Factor ◽  
Cardiac Fibroblasts ◽  
Rho Kinase ◽  
Transforming Growth Factor Β ◽  
Direct Reprogramming ◽  
Cardiac Gene Expression ◽  
Cardiac Gene ◽  
Kinetics Of

The mammalian heart is composed of ~30% cardiomyocytes which have limited capacity to regenerate and ~70% non-cardiomyocytes including endothelial cells and cardiac fibroblasts. Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic transcription factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. Despite extensive efforts, the efficiency of direct reprogramming of embryonic or adult fibroblasts into cardiomyocytes has yet to exceed 20%, or 0.1% respectively, leading many in the field to question the clinical translatability of this method. Here, we demonstrate that pro-fibrotic signaling events governed by transforming growth factor-β (TGF-β) and Rho kinase (ROCK) are concomitantly activated in GHMT-expressing fibroblasts, leading to potent suppression of cardiac reprogramming ( Figure 1 ). Remarkably, pharmacological inhibition of TGF-β, or ROCK leads to conversion of ≥ 60% of fibroblasts into highly functional cardiomyocytes, displaying global cardiac gene expression, spontaneous contractility, action potentials and calcium transients. Furthermore, inhibition of TGF-β, or ROCK dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than two weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts, and should enhance efforts to generate cardiomyocytes for clinical applications.

A calorimetric approach to the kinetics of the irreversible thermal denaturation of proteins

1992 ◽  
Vol 199 ◽  
pp. 147-157 ◽  
Author(s):  
María L. Galisteo ◽  
Francisco Conejero-Lara ◽  
Josefa Núñez ◽  
Jose M. Sánchez-Ruiz ◽  
Pedro L. Mateo
Keyword(s):  
Thermal Denaturation ◽  
Kinetics Of ◽  
Denaturation Of Proteins

scholarly journals Role of maltase in the utilization of sucrose by Candida albicans

1993 ◽  
Vol 291 (3) ◽  
pp. 765-771 ◽  
Author(s):  
P R Williamson ◽  
M A Huber ◽  
J E Bennett
Keyword(s):  
Candida Albicans ◽  
Intracellular Localization ◽  
Cross Reactivity ◽  
Neutral Sugar ◽  
Sequence Specificity ◽  
Western Blots ◽  
Sds Page ◽  
Molecular Masses ◽  
Kinetics Of

Two isoenzymes of maltase (EC 3.2.1.20) were purified to homogeneity from Candida albicans. Isoenzymes I and II were found to have apparent molecular masses of 63 and 66 kDa on SDS/PAGE with isoelectric points of 5.0 and 4.6 respectively. Both isoenzymes resembled each other in similar N-terminal sequence, specificity for the alpha(1-−>4) glycosidic linkage and immune cross-reactivity on Western blots using a maltase II antigen-purified rabbit antibody. Maltase was induced by growth on sucrose whereas beta-fructofuranosidase activity could not be detected under similar conditions. Maltase I and II were shown to be unglycosylated enzymes by neutral sugar assay, and more than 90% of alpha-glucosidase activity was recoverable from spheroplasts. These data, in combination with other results from this laboratory [Geber, Williamson, Rex, Sweeney and Bennett (1992) J. Bacteriol. 174, 6992-6996] showing lack of a plausible leader sequence in genomic or mRNA transcripts, suggest an intracellular localization of the enzyme. To establish further the mechanism of sucrose assimilation by maltase, the existence of a sucrose-inducible H+/sucrose syn-transporter was demonstrated by (1) the kinetics of sucrose-induced [14C]sucrose uptake, (2) recovery of intact [14C]sucrose from ground cells by t.l.c. and (3) transport of 0.83 mol of H+/mol of [14C]sucrose. In total, the above is consistent with a mechanism whereby sucrose is transported into C. albicans to be hydrolysed by an intracellular maltase.

scholarly journals Epigenetic Effects Induced by Methamphetamine and Methamphetamine-Dependent Oxidative Stress

2018 ◽  
Vol 2018 ◽  
pp. 1-28 ◽  
Author(s):  
Fiona Limanaqi ◽  
Stefano Gambardella ◽  
Francesca Biagioni ◽  
Carla L. Busceti ◽  
Francesco Fornai
Keyword(s):  
Molecular Mechanisms ◽  
Neuropsychiatric Disorders ◽  
Behavioral Alterations ◽  
Neuronal Phenotype ◽  
Cellular Events ◽  
Epigenetic Effects ◽  
Molecular Events ◽  
Epigenetic Events ◽  
Genetic Modifications ◽  
Altered Gene

Methamphetamine is a widely abused drug, which possesses neurotoxic activity and powerful addictive effects. Understanding methamphetamine toxicity is key beyond the field of drug abuse since it allows getting an insight into the molecular mechanisms which operate in a variety of neuropsychiatric disorders. In fact, key alterations produced by methamphetamine involve dopamine neurotransmission in a way, which is reminiscent of spontaneous neurodegeneration and psychiatric schizophrenia. Thus, understanding the molecular mechanisms operated by methamphetamine represents a wide window to understand both the addicted brain and a variety of neuropsychiatric disorders. This overlapping, which is already present when looking at the molecular and cellular events promoted immediately after methamphetamine intake, becomes impressive when plastic changes induced in the brain of methamphetamine-addicted patients are considered. Thus, the present manuscript is an attempt to encompass all the molecular events starting at the presynaptic dopamine terminals to reach the nucleus of postsynaptic neurons to explain how specific neurotransmitters and signaling cascades produce persistent genetic modifications, which shift neuronal phenotype and induce behavioral alterations. A special emphasis is posed on disclosing those early and delayed molecular events, which translate an altered neurotransmitter function into epigenetic events, which are derived from the translation of postsynaptic noncanonical signaling into altered gene regulation. All epigenetic effects are considered in light of their persistent changes induced in the postsynaptic neurons including sensitization and desensitization, priming, and shift of neuronal phenotype.

scholarly journals Molecular Events Involved in Influenza A Virus-Induced Cell Death

2022 ◽  
Vol 12 ◽  
Author(s):  
Rui Gui ◽  
Quanjiao Chen
Keyword(s):  
Cell Death ◽  
Influenza A Virus ◽  
Influenza A ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Tissue Destruction ◽  
Inflammatory Reactions ◽  
Molecular Events ◽  
Host Death

Viral infection usually leads to cell death. Moderate cell death is a protective innate immune response. By contrast, excessive, uncontrolled cell death causes tissue destruction, cytokine storm, or even host death. Thus, the struggle between the host and virus determines whether the host survives. Influenza A virus (IAV) infection in humans can lead to unbridled hyper-inflammatory reactions and cause serious illnesses and even death. A full understanding of the molecular mechanisms and regulatory networks through which IAVs induce cell death could facilitate the development of more effective antiviral treatments. In this review, we discuss current progress in research on cell death induced by IAV infection and evaluate the role of cell death in IAV replication and disease prognosis.

scholarly journals The transglutaminase in vascular cells and tissues could provide an alternate pathway for fibrin stabilization

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 702-709
Author(s):  
CS Greenberg ◽  
KE Achyuthan ◽  
MJ Borowitz ◽  
MA Shuman
Keyword(s):  
Guinea Pig ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Vascular Cells ◽  
Pig Liver ◽  
Alternate Pathway ◽  
Sds Page ◽  
Immunohistochemical Techniques ◽  
Plasmin Inhibitor ◽  
Guinea Pig Liver

A thrombin-independent transglutaminase (TG) has been identified in vascular cells and tissues from human, rabbit, rat, porcine, and bovine sources. The vascular TG had several properties that were similar but not identical to guinea pig liver TG. Both enzymes had similar chromatographic and electrophoretic properties, preferentially cross- linked the alpha-chains of fibrinogen, and reacted with polyclonal and monoclonal anti-guinea-pig liver TG antibodies. However, the TG from adult bovine aortic endothelial (ABAE) cells exhibited a novel Ca2+/Mg2+ dependence for enzymatic activity that was distinct from that of purified guinea pig liver TG. The mol wt of the vascular TG (79 +/- 3 kd) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was slightly lower than the purified guinea pig liver TG (85 +/- 9 kd). The TG antigen was detected by immunohistochemical techniques in association with the endothelial and smooth muscle cells of arteries, veins, venules, and capillaries. The TG antigen also codistributed with the fibronectin antigen along the hepatic sinusoids. The ABAE cell TG cross-linked alpha 2-plasmin inhibitor to fibrinogen and caused the modified fibrinogen to be 40- fold more resistant to plasminolysis. A thrombin-independent TG in vascular cells of blood vessels could provide an alternate pathway to inhibit fibrinolysis and promote fibrin stabilization.

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