Lysosomal sulphate transport is dependent upon sulphydryl groups

Hsu-Fang CHOU; Merry PASSAGE; J. Adam JONAS

doi:10.1042/bj3300713

Lysosomal sulphate transport is dependent upon sulphydryl groups

Biochemical Journal ◽

10.1042/bj3300713 ◽

1998 ◽

Vol 330 (2) ◽

pp. 713-717 ◽

Cited By ~ 6

Author(s):

Hsu-Fang CHOU ◽

Merry PASSAGE ◽

J. Adam JONAS

Keyword(s):

Alkylating Agent ◽

Fold Increase ◽

Substrate Affinity ◽

Transport Activity ◽

Inhibitory Effects ◽

Control Value ◽

Sulphate Transport ◽

Sulphydryl Groups ◽

Affinity Resin

Using thiol blocking agents, we examined the role of sulphydryl groups for function of the lysosomal sulphate transport system. Monothiol binding reagents, p-hydroxymercuribenzoic acid (p-HMB) and p-chloromercuribenzene sulphonic acid (p-CMBS), dithiol binding reagents such as CuCl2, the alkylating agent, N-ethylmaleimide (NEM), and NADH all inhibited lysosomal sulphate transport. The inhibitory effects of NEM and Cu2+ were not additive, suggesting that they both act upon the same critical sulphydryl group(s). Unlike the case for NEM, the inhibitory effects of Cu2+ were reversed by the reducing agent, dithiothreitol. Exposure to NEM resulted in a seven-fold increase in Km to 867 μM versus a control value of 126 μM and a modest decrease in Vmax to 99 pmol per unit β-hexosaminidase per 30 s versus a control value of 129 pmol per unit β-hexosaminidase per 30 s. Similar although somewhat less dramatic results were obtained using Cu2+ with an increase of Km to 448 μM and a Vmax of 77 pmol per unit β-hexosaminidase per 30 s. The sulphate transport activity of detergent solubilized lysosomal membranes could be bound to a p-chloromercuribenzoic acid (p-CMB)-Sepharose sulphydryl affinity resin and eluted with mercaptoethanol. Sulphydryl groups thus appear to play a role in sulphate transport through effects on substrate affinity. Sulphydryl-binding appears to be a strategy that may be useful for purification of the transporter.

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Influence of C-ANF receptor and neutral endopeptidase on pharmacokinetics of ANF in rats

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1991.260.1.r208 ◽

1991 ◽

Vol 260 (1) ◽

pp. R208-R216 ◽

Cited By ~ 1

Author(s):

P. J. Chiu ◽

G. Tetzloff ◽

M. T. Romano ◽

C. J. Foster ◽

E. J. Sybertz

Keyword(s):

High Performance ◽

Fold Increase ◽

Neutral Endopeptidase ◽

Volume Of Distribution ◽

Fourfold Increase ◽

Control Value ◽

Enzymatic Pathways ◽

Hydrolysis Of

The role of C-atrial natriuretic factor (ANF) receptors and neutral endopeptidase (NEP) in the pharmacokinetics and hydrolysis of 125I-labeled ANF was evaluated in rats by using C-ANF and SCH 39370 to block the nonenzymatic and enzymatic pathways, respectively. After a bolus injection of 125I-ANF, the resulting area under the plasma concentration curve (AUC) with C-ANF treatment was seven times the control value with regard to trichloroacetic acid-precipitable (TCA-ppt) radioactivity (intact ANF). SCH 39370 tended to increase AUC, but the changes were not significant. Nevertheless, SCH 39370 suppressed the appearance of TCA-soluble radioactivity (hydrolytic products), indicating that in vivo inhibition of ANF degradation had occurred. SCH 39370 plus C-ANF produced a 15-fold increase in AUC for TCA-ppt radioactivity and a reduction in plasma TCA-soluble radioactivity. High-performance liquid chromatography (HPLC) analysis confirmed that combination treatment increased intact ANF and reduced hydrolytic products in the plasma. SCH 39370 reduced clearance (C) without altering volume of distribution in steady state (Vss) and half-life (t1/2). C-ANF decreased both C and Vss leading to a fourfold increase in t1/2, which was further prolonged by SCH 39370 (7.5 times control). Bilateral nephrectomy caused a proportionally similar decrease in Vss and C without changing t1/2, suggesting significant extrarenal metabolism of ANF. SCH 39370 systemically inhibits ANF hydrolysis; the resulting increase in ANF, however, is masked by the great capacity of ANF clearance receptors but can be revealed with excess C-ANF, suggesting that the plasma ANF concentrations are determined by the interplay of the C-ANF receptor and NEP systems.

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The role of OXA-1 β-lactamase Asp66 in the stabilization of the active-site carbamate group and in substrate turnover

Biochemical Journal ◽

10.1042/bj20070573 ◽

2008 ◽

Vol 410 (3) ◽

pp. 455-462 ◽

Cited By ~ 15

Author(s):

David A. Leonard ◽

Andrea M. Hujer ◽

Brian A. Smith ◽

Kyle D. Schneider ◽

Christopher R. Bethel ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Fold Increase ◽

Luria Bertani ◽

Saturation Mutagenesis ◽

Substrate Affinity ◽

Luria Bertani Broth ◽

Class D ◽

Carbamate Group

The OXA-1 β-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser67. In class A β-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp66 in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp66 mutants expressed in Escherichia coli DH10B ranged from ≤8 μg/ml for cysteine, proline and the basic amino acids to ≥256 μg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys70, that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria–Bertani-broth agar resulted in a ≥16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp66 forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO2 bound to Lys70 and thereby profoundly affects substrate turnover.

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Plasminogen Activation In Vivo upon Intravenous Infusion of DDAVP

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648390 ◽

1992 ◽

Vol 67 (01) ◽

pp. 111-116 ◽

Cited By ~ 64

Author(s):

Marcel Levi ◽

Jan Paul de Boer ◽

Dorina Roem ◽

Jan Wouter ten Cate ◽

C Erik Hack

Keyword(s):

Monoclonal Antibodies ◽

Plasminogen Activator ◽

Plasma Levels ◽

Fold Increase ◽

Fibrinolytic System ◽

Plasminogen Activation ◽

Plasminogen Activator Activity ◽

Insight Into

SummaryInfusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established.A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor α2 antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated α2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system.Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex.We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

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The SOS Error-Prone DNA Polymerase V Mutasome and β-Sliding Clamp Acting in Concert on Undamaged DNA and during Translesion Synthesis

Cells ◽

10.3390/cells10051083 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1083

Author(s):

Adhirath Sikand ◽

Malgorzata Jaszczur ◽

Linda B. Bloom ◽

Roger Woodgate ◽

Michael M. Cox ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Pathogenic Bacteria ◽

Fold Increase ◽

Translesion Dna Synthesis ◽

Sliding Clamp ◽

Pol V ◽

Dna Polymerase V ◽

Acting In Concert

In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin.

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Health research capacity in Nepal: Analysis of the trend and the role of local researchers

Tropical Doctor ◽

10.1177/0049475520982770 ◽

2021 ◽

pp. 004947552098277

Author(s):

Madhu Kharel ◽

Alpha Pokharel ◽

Krishna P Sapkota ◽

Prasant V Shahi ◽

Pratisha Shakya ◽

...

Keyword(s):

Health Research ◽

Fold Increase ◽

Research Capacity ◽

Rapid Review ◽

Evidence Based ◽

Middle Income ◽

Middle Income Countries ◽

Health Related ◽

Evidence Based Decision Making

Evidence-based decision-making is less common in low- and middle-income countries where the research capacity remains low. Nepal, a lower-middle-income country in Asia, is not an exception. We conducted a rapid review to identify the trend of health research in Nepal and found more than seven-fold increase in the number of published health-related articles between 2000 and 2018. The proportion of articles with Nepalese researchers as the first authors has also risen over the years, though they are still only in two-thirds of the articles in 2018.

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An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression

Cell Death and Disease ◽

10.1038/s41419-021-03934-y ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Thao Thi Thanh Nguyen ◽

Masato Shingyoji ◽

Michiko Hanazono ◽

Boya Zhong ◽

Takao Morinaga ◽

...

Keyword(s):

Wild Type ◽

Inhibitory Effects ◽

Cytotoxic Effects ◽

P53 Expression ◽

Nuclear Factor I ◽

Infected Cells ◽

Wild Type P53 ◽

Mdm2 Inhibitor ◽

P16 Expression

AbstractA majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

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Seed Gamma Irradiation of Arabidopsis thaliana ABA-Mutant Lines Alters Germination and Does Not Inhibit the Photosynthetic Efficiency of Juvenile Plants

Dose-Response ◽

10.1177/1559325820979249 ◽

2020 ◽

Vol 18 (4) ◽

pp. 155932582097924

Author(s):

Darya Babina ◽

Marina Podobed ◽

Ekaterina Bondarenko ◽

Elizaveta Kazakova ◽

Sofia Bitarishvili ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Growth Response ◽

Dose Range ◽

Fine Tuning ◽

Plant Tolerance ◽

Inhibitory Effects ◽

Γ Irradiation ◽

Photosynthetic Responses ◽

Mutant Lines

Plant growth response to γ-irradiation includes stimulating or inhibitory effects depending on plant species, dose applied, stage of ontogeny and other factors. Previous studies showed that responses to irradiation could depend on ABA accumulation and signaling. To elucidate the role of ABA in growth and photosynthetic responses to irradiation, lines Col-8, abi3-8 and aba3 -1 of Arabidopsis thaliana were used. Seeds were γ-irradiated using 60Co in the dose range 50-150 Gy. It was revealed that the dose of 150 Gy affected germination parameters of aba3 -1 and Col-8 lines, while abi3-8 line was the most resistant to the studied doses and even showed faster germination at early hours after γ-irradiation at 50 Gy. These results suggest that susceptibility to ABA is probably more important for growth response to γ-irradiation than ABA synthesis. The photosynthetic functioning of 16-day-old plants mainly was not disturbed by γ-irradiation of seeds, and no indication of photosystem II photoinhibition was noticed, revealing the robustness of the photosynthetic system of A. thaliana. Glutathione peroxidase activity and ABA concentrations in plant tissues were not affected in the studied dose range. These results contribute to the understanding of germination and photosynthesis fine-tuning and of mechanisms of plant tolerance to ionizing radiation.

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Amphetamine-Decreased Progesterone and Estradiol Release in Rat Granulosa Cells: The Regulatory Role of cAMP- and Ca2+-Mediated Signaling Pathways

Biomedicines ◽

10.3390/biomedicines9050493 ◽

2021 ◽

Vol 9 (5) ◽

pp. 493

Author(s):

Chung-Yu Chen ◽

Chien-Rung Chen ◽

Chiao-Nan Chen ◽

Paulus S. Wang ◽

Toby Mündel ◽

...

Keyword(s):

Granulosa Cells ◽

Prostaglandin F2α ◽

Inhibitory Effect ◽

Inhibitory Effects ◽

Steroidogenic Enzyme ◽

Estradiol Secretion ◽

Basal Release ◽

Ca2 Channels

The purpose of this study is to evaluate the amphetamine effects on progesterone and estradiol production in rat granulosa cells and the underlying cellular regulatory mechanisms. Freshly dispersed rat granulosa cells were cultured with various test drugs in the presence of amphetamine, and the estradiol/progesterone production and the cytosolic cAMP level were measured. Additionally, the cytosolic-free Ca2+ concentrations ([Ca2+]i) were measured to examine the role of Ca2+ influx in the presence of amphetamine. Amphetamine in vitro inhibited both basal and porcine follicle-stimulating hormone-stimulated estradiol/progesterone release, and amphetamine significantly decreased steroidogenic enzyme activities. Adding 8-Bromo-cAMP did not recover the inhibitory effects of amphetamine on progesterone and estradiol release. H89 significantly decreased progesterone and estradiol basal release but failed to enhance a further amphetamine inhibitory effect. Amphetamine was capable of further suppressing the release of estradiol release under the presence of nifedipine. Pretreatment with the amphetamine for 2 h decreased the basal [Ca2+]i and prostaglandin F2α-stimulated increase of [Ca2+]i. Amphetamine inhibits progesterone and estradiol secretion in rat granulosa cells through a mechanism involving decreased PKA-downstream steroidogenic enzyme activity and L-type Ca2+ channels. Our current findings show that it is necessary to study the possibility of amphetamine perturbing reproduction in females.

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Arsenic Removal by Advanced Electrocoagulation Processes: The Role of Oxidants Generated and Kinetic Modeling

Catalysts ◽

10.3390/catal10080928 ◽

2020 ◽

Vol 10 (8) ◽

pp. 928

Author(s):

Micah Flor V. Montefalcon ◽

Meliton R. Chiong ◽

Augustus C. Resurreccion ◽

Sergi Garcia-Segura ◽

Joey D. Ocon

Keyword(s):

Kinetic Modeling ◽

Arsenic Removal ◽

Fold Increase ◽

Electrochemical Treatment ◽

Promising Alternative ◽

Iron Electrodes ◽

Removal Capacity ◽

Naturally Occurring ◽

Treatment Technologies

Arsenic (As) is a naturally occurring element in the environment that poses significant risks to human health. Several treatment technologies have been successfully used in the treatment of As-contaminated waters. However, limited literature has explored advanced electrocoagulation (EC) processes for As removal. The present study evaluates the As removal performance of electrocoagulation, electrochemical peroxidation (ECP), and photo-assisted electrochemical peroxidation (PECP) technologies at circumneutral pH using electroactive iron electrodes. The influence of As speciation and the role of oxidants in As removal were investigated. We have identified the ECP process to be a promising alternative for the conventional EC with around 4-fold increase in arsenic removal capacity at a competitive cost of 0.0060 $/m3. Results also indicated that the rate of As(III) oxidation at the outset of electrochemical treatment dictates the extent of As removal. Both ECP and PECP processes reached greater than 96% As(III) conversion at 1 C/L and achieved 86% and 96% As removal at 5 C/L, respectively. Finally, the mechanism of As(III) oxidation was evaluated, and results showed that Fe(IV) is the intermediate oxidant generated in advanced EC processes, and the contribution of •OH brought by UV irradiation is insignificant.

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