scholarly journals Kinetics of Enzymatic Mercury Methylation at Nanomolar Concentrations Catalyzed by HgcAB

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
Swapneeta S. Date ◽  
Jerry M. Parks ◽  
Katherine W. Rush ◽  
Judy D. Wall ◽  
Stephen W. Ragsdale ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Anaerobic Bacteria ◽  
Significant Risk ◽  
Health Concern ◽  
Global Public Health ◽  
Microbial Conversion ◽  
Experimental Conditions ◽  
Cell Lysates ◽  
Kinetics Of

ABSTRACTMethylmercury (MeHg) is a potent bioaccumulative neurotoxin that is produced by certain anaerobic bacteria and archaea. Mercury (Hg) methylation has been linked to the gene pairhgcAB, which encodes a membrane-associated corrinoid protein and a ferredoxin. Although microbial Hg methylation has been characterizedin vivo, the cellular biochemistry and the specific roles of the gene products HgcA and HgcB in Hg methylation are not well understood. Here, we report the kinetics of Hg methylation in cell lysates ofDesulfovibrio desulfuricansND132 at nanomolar Hg concentrations. The enzymatic Hg methylation mediated by HgcAB is highly oxygen sensitive, irreversible, and follows Michaelis-Menten kinetics, with an apparentKmof 3.2 nM andVmaxof 19.7 fmol · min−1· mg−1total protein for the substrate Hg(II). Although the abundance of HgcAB in the cell lysates is extremely low, Hg(II) was quantitatively converted to MeHg at subnanomolar substrate concentrations. Interestingly, increasing thiol/Hg(II) ratios did not impact Hg methylation rates, which suggests that HgcAB-mediated Hg methylation effectively competes with cellular thiols for Hg(II), consistent with the low apparentKm. Supplementation of 5-methyltetrahydrofolate or pyruvate did not enhance MeHg production, while both ATP and a nonhydrolyzable ATP analog decreased Hg methylation rates in cell lysates under the experimental conditions. These studies provide insights into the biomolecular processes associated with Hg methylation in anaerobic bacteria.IMPORTANCEThe concentration of Hg in the biosphere has increased dramatically over the last century as a result of industrial activities. The microbial conversion of inorganic Hg to MeHg is a global public health concern due to bioaccumulation and biomagnification of MeHg in food webs. Exposure to neurotoxic MeHg through the consumption of fish represents a significant risk to human health and can result in neuropathies and developmental disorders. Anaerobic microbial communities in sediments and periphyton biofilms have been identified as sources of MeHg in aquatic systems, but the associated biomolecular mechanisms are not fully understood. In the present study, we investigate the biochemical mechanisms and kinetics of MeHg formation by HgcAB in sulfate-reducing bacteria. These findings advance our understanding of microbial MeHg production and may help inform strategies to limit the formation of MeHg in the environment.

scholarly journals Kinetics of enzymatic mercury methylation at nanomolar concentrations catalyzed by HgcAB

2019 ◽  
Author(s):  
Swapneeta S. Date ◽  
Jerry M. Parks ◽  
Katherine W. Rush ◽  
Judy D. Wall ◽  
Stephen W. Ragsdale ◽  
...  
Keyword(s):  
Anaerobic Bacteria ◽  
Desulfovibrio Desulfuricans ◽  
Experimental Conditions ◽  
Cell Lysates ◽  
Oxygen Sensitive ◽  
Kinetics Of ◽  
Substrate Concentrations ◽  
Insight Into

ABSTRACTMethylmercury (MeHg) is a potent neurotoxin that bioaccumulates in fish. MeHg is generated by anaerobic bacteria and archaea possessing the gene pair hgcAB. Although bacterial mercury (Hg) methylation has been characterized in vivo, the specific role of HgcAB in catalyzing Hg methylation is not well understood. Here we report the kinetics of HgcAB-mediated Hg methylation in cell lysates of Desulfovibrio desulfuricans ND132 at nanomolar Hg concentrations. The enzymatic Hg methylation mediated by HgcAB is highly oxygen-sensitive, irreversible, and follows Michaelis-Menten kinetics with an apparent KM of 3.2 nM and Vmax of 19.7 fmol·min-1·mg-1 total protein for the substrate Hg(II). Although the abundance of HgcAB in the cell lysates is extremely low, Hg(II) was quantitatively converted to MeHg at subnanomolar substrate concentrations. Supplementation with ATP, methyltetrahydrofolate, or pyruvate did not enhance MeHg production under the experimental conditions. Insight into the kinetics of Hg methylation catalyzed by HgcAB advances our understanding of the complex global Hg cycle.

Different mechanisms in the attachment of cells to native and denatured collagen

1979 ◽  
Vol 38 (1) ◽  
pp. 267-281
Author(s):  
S.L. Schor ◽  
J. Court
Keyword(s):  
Cell Attachment ◽  
Experimental Conditions ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Independent Mechanism ◽  
Cell Matrix Interactions ◽  
Serum Dependent ◽  
Kinetics Of ◽  
Dependent Mechanism

The attachment of cells to collagen has been reported previously to require the presence of serum and the particular serum protein involved in this process, variously known as CIG, CAP or fibronectin, has been isolated. This conclusion that cell attachment to collagen requires serum (or more precisely, fibronectin) is based on experiments measuring the kinetics of cell attachment to films of collagen. We have measured the kinetics of attachment of HeLa and attachment to films of collagen-containing substrata under a variety of experimental conditions and present evidence that the serum-dependent mechanism of cell attachment described by others is actually only the case for films of denatured collagen, while cell attachment to native collagen fibres occurs by a different, serum-independent, mechanism. The possible relevance of these findings to cell-matrix interactions in vivo is discussed.

scholarly journals Lived experiences of caregivers of children with autism spectrum disorder in Kenya

2019 ◽  
Vol 8 ◽  
Author(s):  
Lizahn G. Cloete ◽  
Evans O. Obaigwa
Keyword(s):  
Autism Spectrum Disorder ◽  
Developmental Disorders ◽  
Phenomenological Study ◽  
Autism Spectrum ◽  
Spectrum Disorder ◽  
Health Concern ◽  
Global Public Health ◽  
African Countries ◽  
Children With Asd ◽  
Occupational Therapy Interventions

Background: Autism spectrum disorder (ASD) is a global public health concern. In African countries such as Kenya, there is a greater need for establishing support services for developmental disorders such as ASD. The emotional, social and economic burden of ASD on caregivers is unknown because of a number of challenges. Citizens of Kenya have a unique view of disability and inclusion.Objectives: To explore the perspectives of caregivers who are responsible for caring for both family and children living with ASD and to highlight the needs of children with ASD as well as the needs of their caregivers.Method: A qualitative, descriptive phenomenological study utilising focus group discussions (FGDs) was conducted. Verbatim transcription was used. QSR N ’Vivo 10 was used to organise and analyse the data. Content analysis was used to identify important ideas and concepts.Results: One theme, namely ‘the burden of caring for children with ASD’, was identified. Children with ASD and their caregivers experience isolation and stigmatisation.Conclusion: Occupational therapists in Kenya should collaborate with the relevant national and global stakeholders for the promotion of the inclusion of children with ASD and their families. Responsive and context-appropriate occupational therapy interventions may begin to address service barriers.

Kinetics of Homologous Urea-soluble 131I-Fibrin and 125I-Fibrinogen in Rabbits

1975 ◽  
Author(s):  
G. Müller-Berghaus ◽  
I. Mahn ◽  
G. Kövecker
Keyword(s):  
Exponential Decay ◽  
Distribution Volume ◽  
Fibrinolytic System ◽  
Experimental Conditions ◽  
Soluble Fibrin ◽  
Kinetics Of ◽  
Deutsche Forschungsgemeinschaft

In order to study the in vivo behaviour of soluble fibrin, purified rabbit 13I-fibrinogen was clotted in the presence of EDTA (0.005 M) and aprotinin (100 units/ml), the generated clot dissolved in buffered urea (3.0 M, pH 7.4), and the formed ureasoluble 131I-fibrin injected into rabbits. The behaviour of homologous 125I-fibrinogen was simultaneously studied in the same animals. Results: The distribution volume of 131I-fibrin (44 ml/kg) as well as that of 125I-fibrinogen (45 ml/kf) was nearly identical indicating that the injected soluble fibrin was homogenously distributed in the circulation immediately after injection. The elimination of soluble fibrin from the circulating blood did not represent a monophasic exponential decay. 84% (range: 77–94%) of the injected soluble fibrin disappeared from the circulation during the first 24 hours after injection, whereas only 54% (range: 41–72%) of the injected fibrinogen disappeared during that interval. Under these experimental conditions, the T/2 of fibrinogen was 46 hours. The clearance of urea-soluble fibrin did not influence the kinetics of 13I-fibrinogen. The disappearance of soluble fibrin could not be influenced by treating the animals with aprotinin for fibrinolysis inhibition. The experiments demonstrate that soluble fibrin can be traced in the circulation and cleared from the blood by a mechanism independent of the fibrinolytic system.(Supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany.)

Extracellular ATP regulates transcervical permeability by modulating two distinct paracellular pathways

1997 ◽  
Vol 272 (5) ◽  
pp. C1602-C1610 ◽  
Author(s):  
G. I. Gorodeski ◽  
J. Goldfarb
Keyword(s):  
Phase I ◽  
Phase Ii ◽  
Extracellular Atp ◽  
Acetoxymethyl Ester ◽  
Experimental Conditions ◽  
Mucus Production ◽  
Atp Regulation ◽  
Junctional Resistance ◽  
Kinetics Of

Extracellular ATP stimulates a biphasic change in transepithelial electrical resistance (RTE) across cultures of human cervical epithelial cells: an acute decrease (phase I), followed by a delayed increase in resistance (phase II). The objective of this study was to determine the contributions of changes in the lateral intercellular space resistance (RLIS) and the tight junctional resistance (RTJ) to the changes in RTE. Phase I and phase II effects were uncoupled by treatment with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester, which blocks the ATP-induced increases in cytosolic Ca2+ and abolishes phase I. BAPTA-loaded cells differed from control cells in that 1) phase I began when ATP was added, in contrast to a delay of 1.5-3.5 min in phase II, 2) phase I decreases in RLIS followed a simple exponential pattern, in contrast to the complex kinetics of phase II, and 3) the magnitude of phase II varied between 20 and 100% for increases of RTJ in day 2-6 cultures; the phase I decrease of 50% in RLIS was unrelated to different experimental conditions. These results indicate that phase I and phase II are induced simultaneously and independently by ATP, and they contribute to the total changes in RTE. We conclude that ATP regulation of RLIS and RTJ may be important mechanisms of modulating cervical mucus production in vivo.

scholarly journals Contraction kinetics of intact and skinned frog muscle fibers and degree of activation. Effects of intracellular Ca2+ on unloaded shortening.

1985 ◽  
Vol 86 (4) ◽  
pp. 479-500 ◽  
Author(s):  
J Gulati ◽  
A Babu
Keyword(s):  
Experimental Conditions ◽  
Bathing Medium ◽  
Internal Load ◽  
Skinned Fiber ◽  
Fiber Properties ◽  
Partial Activation ◽  
A Minor ◽  
Kinetics Of ◽  
Speed Of Shortening

This study addresses a long-standing controversy on the effects of the degree of activation on cross-bridge kinetics in vivo, by utilizing isolated intact and skinned fiber preparations. Steady force levels ranging from 0.1 to 0.76 P0 were achieved at 0 degrees C with temperature-step stimulation of intact fibers by varying the amount of caffeine in the bathing medium. The speed of unloaded shortening (by slack test) was found to be practically constant, which suggests that intracellular Ca2+ in the intact preparation has relatively little effect on isotonic shortening. Along with the results on tetanically stimulated fibers (force, P0), we observed a minor but significant trend for the speed to decline with lowered force levels. This trend is explained by the presence of a constant internal load equaling approximately 1% P0. The effect of Ca2+ on the shortening behavior of skinned fibers was examined at 0 and 10 degrees C. At 0 degrees C, there was practically no effect of Ca2+ on the shortening response in slack tests. At 10 degrees C, there was also no Ca2+ effect during the first activation cycle, but in subsequent cycles the speed of shortening was reduced during partial activation, which indicates that there were permanent changes in the fiber properties under these experimental conditions. The latter result could be explained if the internal load had increased to approximately 5% P0 in the modified skinned fiber (compared with 1% P0 in intact fiber). These findings show that isotonic contraction of frog fibers is intrinsically unaffected by the variations in intracellular Ca2+ that modulated the force over a nearly complete range. The results provide support for the idea that Ca2+ influences the force development in vivo by on-off switching mechanisms.

Absorption of Galactose by the Rat Small Intestine in Vivo: Proximal—Distal Kinetic Gradients and a New Method to Express Absorption Per Enterocyte

1976 ◽  
Vol 50 (6) ◽  
pp. 499-509 ◽  
Author(s):  
R. M. Batt ◽  
T. J. Peters
Keyword(s):  
Small Intestine ◽  
Absorptive Capacity ◽  
Proximal Jejunum ◽  
Rat Small Intestine ◽  
Experimental Conditions ◽  
Carrier Mediated Transport ◽  
Saturation Kinetics ◽  
Kinetics Of ◽  
Diffusive Component

1. The absorption in vivo of d-galactose by the rat small intestine has been examined in proximal jejunum and distal ileum by use of a recirculation—perfusion technique. 2. Multiple sequential perfusions over 4 h produced no subsequent functional or morphological damage in the perfused segments. 3. Absorption of galactose from 8 and 64 mmol/l solutions was found to be independent of flow rate over the range 1·0–6·5 ml/min. 4. Galactose absorption in both the jejunum and the ileum exhibited saturation kinetics of the Michaelis—Menten type, and phlorrhizin sensitivity. Sorbose was only absorbed minimally. These observations demonstrate that galactose is absorbed by carrier-mediated transport and that there is no significant passive diffusive component in vivo. 5. Under the stated experimental conditions, the maximum absorptive capacity was 4·5 times greater in the jejunum than in the ileum. The Michaelis constant for galactose was higher in the jejunum than in the ileum. 6. Enterocytes were isolated from perfused segments and quantified by DNA assay with a correction for yield. In this manner, the absorptive capacity per enterocyte was calculated. 7. The maximum absorptive capacity per enterocyte was 3·5 times greater in the jejunum than in the ileum.

Functional Connectivity: Application to Developmental Disorders

2013 ◽  
pp. 995-1009
Author(s):  
Luke Bloy ◽  
Ragini Verma ◽  
Timothy P.L. Roberts
Keyword(s):  
Functional Connectivity ◽  
Developmental Disorders ◽  
Brain Activity ◽  
Structural Connectivity ◽  
Autism Spectrum ◽  
Great Success ◽  
Experimental Conditions ◽  
Neuronal Ensembles ◽  
The Brain

Noninvasive imaging and electrophysiological methods have been developed to facilitate the in vivo investigation of brain function and dysfunction. Such methods have been employed, with great success, to the functional mapping of the brain, as well as the characterization of the temporal activity of these regions during a variety of tasks and experimental conditions. These methods are however inadequate to fully capture our current understanding of brain activity, as the complex interplay of structurally and functionally connected networks of neuronal ensembles. Here we offer an overview of the methodological advancements that have been made to better facilitate the investigation of connectivity in the brain and its relationship to development and pathology. We have focused primarily on in vivo modalities that have been most widely adopted, namely fMRI and electro/magneto encephalography for the investigation of functional connectivity and diffusion MRI for structural connectivity. Finally, the application of these methodologies to the study of neurodevelopmental disorders, such as the autism spectrum disorders, schizophrenia and attention deficit hyperactivity disorder, is presented.

scholarly journals Addressing Latent Tuberculosis: New Advances in Mimicking the Disease, Discovering Key Targets, and Designing Hit Compounds

2020 ◽  
Vol 21 (22) ◽  
pp. 8854
Author(s):  
André Campaniço ◽  
Shrika G. Harjivan ◽  
Digby F. Warner ◽  
Rui Moreira ◽  
Francisca Lopes
Keyword(s):  
Drug Discovery ◽  
Latent Tuberculosis ◽  
Experimental Methods ◽  
Health Concern ◽  
Global Public Health ◽  
Public Health Concern ◽  
Physiological Factor ◽  
Current Therapies

Despite being discovered and isolated more than one hundred years ago, tuberculosis (TB) remains a global public health concern arch. Our inability to eradicate this bacillus is strongly related with the growing resistance, low compliance to current drugs, and the capacity of the bacteria to coexist in a state of asymptomatic latency. This last state can be sustained for years or even decades, waiting for a breach in the immune system to become active again. Furthermore, most current therapies are not efficacious against this state, failing to completely clear the infection. Over the years, a series of experimental methods have been developed to mimic the latent state, currently used in drug discovery, both in vitro and in vivo. Most of these methods focus in one specific latency inducing factor, with only a few taking into consideration the complexity of the granuloma and the genomic and proteomic consequences of each physiological factor. A series of targets specifically involved in latency have been studied over the years with promising scaffolds being discovered and explored. Taking in account that solving the latency problem is one of the keys to eradicate the disease, herein we compile current therapies and diagnosis techniques, methods to mimic latency and new targets and compounds in the pipeline of drug discovery.

scholarly journals Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential

2021 ◽  
Vol 118 (25) ◽  
pp. e2103984118
Author(s):  
Ziqi Zhou ◽  
Kenrie P. Y. Hui ◽  
Ray T. Y. So ◽  
Huibin Lv ◽  
Ranawaka A. P. M. Perera ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Arabian Peninsula ◽  
Ex Vivo ◽  
Prototype Strain ◽  
Health Concern ◽  
Epithelial Cell Line ◽  
Global Public Health ◽  
Human Bronchus

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV–infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

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