scholarly journals Removal Efficiency of Cr6+by IndigenousPichiasp. Isolated from Textile Factory Effluent

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Pablo M. Fernández ◽  
María M. Martorell ◽  
Julia I. Fariña ◽  
Lucia I. C. Figueroa
Keyword(s):  
Culture Medium ◽  
Incubation Temperature ◽  
Reductase Activity ◽  
Chromate Reduction ◽  
Intact Cells ◽  
Chromate Reductase ◽  
Optimum Ph ◽  
Associated Proteins ◽  
Textile Factory

Resistance of the indigenous strainsP. jadiniiM9 andP. anomalaM10, to high Cr6+concentrations and their ability to reduce chromium in culture medium was studied. The isolates were able to tolerate chromium concentrations up to 104 μg mL−1. Growth and reduction of Cr6+were dependent on incubation temperature, agitation, Cr6+concentration, and pH. Thus, in both studied strains the chromium removal was increased at 30°C with agitation. The optimum pH was different, with values of pH 3.0 and pH 7.0 in the case ofP. anomalaM10 and pH 7.0 usingP. jadiniiM9. Chromate reduction occurred both in intact cells (grown in culture medium) as well as in cell-free extracts. Chromate reductase activity could be related to cytosolic or membrane-associated proteins. The presence of a chromate reductase activity points out a possible role of an enzyme in Cr6+reduction.

scholarly journals Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1834
Author(s):  
Tomoko Okada ◽  
Toshihiko Ogura
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Related Gene ◽  
Autophagosome Formation ◽  
Intact Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Difference ◽  
Related Proteins ◽  
Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

Vectorial transport of proteins by membrane-bound ribosomes of nongrowing and growing Jerusalem artichoke tuber cells

1978 ◽  
Vol 56 (3) ◽  
pp. 167-173
Author(s):  
Joachim Sparkuhl ◽  
George Setterfield
Keyword(s):  
Reductase Activity ◽  
Jerusalem Artichoke ◽  
Jerusalem Artichoke Tuber ◽  
Protease Digestion ◽  
Associated Proteins ◽  
Membrane Bound ◽  
Nadh Cytochrome ◽  
Vectorial Transport

Both nongrowing (water-incubated) and growing (hormonally stimulated) Jerusalem artichoke tuber cells contain membrane-bound (mb) ribosomes. Using a rapid flotation procedure, a membrane fraction was prepared from both types of cells. This fraction was enriched in mb ribosomes, contained NADH cytochrome c reductase activity, had RNA:phospholipid and RNA:protein ratios similar to those reported for rough microsomes from animal tissues, and supported synthesis of preinitiated proteins in vitro. Using puromycin and detergent release, vectorial transport of labelled polypeptides was measured in the in vitro system. Of proteins made by mb ribosomes from nongrowing cells, only 12% remained associated with microsome membranes following chain termination. The comparable figure for proteins from mb ribosomes of growing tissue was 42%. The membrane-associated proteins were preferentially protected from protease digestion. Some possible reasons are suggested for the correlation between cell growth and the association of newly synthesized proteins with microsomes. The role of proteins synthesized by mb ribosomes but not vectorially transported, in both growing and nongrowing cells, is unknown.

scholarly journals Purification to Homogeneity and Characterization of a Novel Pseudomonas putida Chromate Reductase

2000 ◽  
Vol 66 (5) ◽  
pp. 1788-1795 ◽  
Author(s):  
C. H. Park ◽  
M. Keyhan ◽  
B. Wielinga ◽  
S. Fendorf ◽  
A. Matin
Keyword(s):  
Enzyme Activity ◽  
Pseudomonas Putida ◽  
Reductase Activity ◽  
Gel Filtration ◽  
Enzyme Reaction ◽  
Exchange Chromatography ◽  
Chromate Reduction ◽  
Biochemical Engineering ◽  
Edge Structure ◽  
Chromate Reductase

ABSTRACT Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80°C and 5, respectively; and theKm was 374 μM, with aV max of 1.72 μmol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50°C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.

scholarly journals Microbial abilities to detoxify chromate by reduction

2001 ◽  
Author(s):  
◽  
Neroshini Maistry
Keyword(s):  
Mass Balance ◽  
Reductase Activity ◽  
Batch Reactor ◽  
Reduction Rate ◽  
Water Soluble ◽  
Chromate Reduction ◽  
Cytoplasmic Fraction ◽  
Chromate Reductase ◽  
Pseudomonas Maltophilia ◽  
Reducing Bacteria

Hexavalent chromium [Cr(VI)] or chromate, is a toxic, water-soluble contaminant present in many soils and industrial eflluents. As a result of contaminated discharges from industrial applications, and inappropriate wastedisposal practices, significant amounts of chromate have found their way into the environment. This poses a health risk to man as well as animals and plants due to the carcinogenicity, mutagenicity, and teratogenicity of chromate. In man, acute, high level exposures to Cr(VI) can result in ulceration of the skin, eyes, and mucous membranes. Exposure of plants to Cr(VI) can result in reduced biomass production, and in extreme cases, death. Upon reduction ofCr(VI) to trivalent chromium [Cr(III], the toxic effects are significantly decreased because of a decrease in the solubility and bioavailability of Cr(III). Traditionally, Cr(VI) has been recovered from aqueous systems using processes exploiting the differential solubility properties described above. The use of chromate reducing bacteria represents a potential mechanism for the development of an efficacious, cost effective alternative to traditional chemical/physical processes for Cr(VI) recovery from the environment. Therefore, the aim of this research was to isolate and identify chromate reducing bacteria from soil, and characterise the chromate reductase enzyme in order to determine the potential of bacteria to detoxify chromate by reduction. Bacteria from soils and wastewater were examined for chromate reducing potential and identified on the basis of biochemical tests and API 20E. Organisms were isolated by the spread plate technique. Species of Pseudomonas maltophilia, Bacillus subtilis, Acinetobacter calcoaceticus, and Cellumonas cellasea were capable of catalyzing the reduction ofCr(VI) to Cr(IlI) in batch experiments. Reduction capability as high as 99% by the isolates was detected from an initial Cr(VI) concentration of 150 mg.L' in batch cultures. Chromate reduction was determined by means of the diphenylcarbazide method and total chromium was measured by atomic absorption spectroscopy. Pseudomonas maltophilia was observed to be the most suited organism for the efficient detoxification ofCr(VI) due to its wide temperature and pH requirements, low substrate utilization, and tolerance to heavy metal ions of'Cu', Cd2+,Zn2+,and Ni2+which commonly appear in industrial eflluents along with Cr(VI). Reduction rate in a batch reactor for this organism was calculated to be 1.75 mg.g+h'. Comparison of the rates of chromate reduction by Cr(VI) grown cells and cells grown without chromate indicated that the chromate reductase activity is constitutive. Reductase activity was detected by means of the lysozyme-EDTA method in aerobically grown cells, with highest specific activity in the cytoplasmic fraction of the cell. The Cr(VI)-reductase was found to be NAD(p)H-dependent and yielded an activity of 3.24 ml.I.mg' of protein in the cytoplasmic fraction. Once optimization of the parameters in the batch reactor was achieved, cells of Ps. maltophilia was immobilized into polyacrylamide gel and packed in a column. Mass balance studies indicated that ca 147 mg.L' chromate passing through the column undergoes reduction with an initial Cr(VI) concentration of 150 mg.L' resulting in a Cr(VI) reduction efficiency of98%. An amount of 0.11 mg.L' remained in the cells, 0.11 mg.L' in the cell wash water, and 1.65 mg.L' was unaccounted for in the mass balance. Chromate reduction rate in the continuous-upflow reactor system was calculated to be 5.34 mg.g'l.h', which was 3-fold higher than that calculated for the batch reactor. Chromium-contaminated industrial eflluent obtained from Sheffield, Natal, and Saayman Danks Electroplaters was pumped into the continuous-upflow reactor containing immobilized cells of Ps. maltophilia to determine the industrial applicability of the reactor to treat chromate-containing effluents. Complete Cr(VI) reduction

scholarly journals NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Audrey G. O’Neill ◽  
Brett A. Beaupre ◽  
Yuanzhang Zheng ◽  
Dali Liu ◽  
Graham R. Moran
Keyword(s):  
Dissociation Constant ◽  
Catalytic Reduction ◽  
Reductase Activity ◽  
Sequence Similarity ◽  
Flavin Mononucleotide ◽  
Chromate Reduction ◽  
Content Type ◽  
Product Release ◽  
Chromate Reductase ◽  
Redox Active

ABSTRACT Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s−1. Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s−1. The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes. IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.

Follicle-stimulating hormone-dependent estrogen secretion by rat Sertoli cells in vitro: Modulation by calcium

1991 ◽  
Vol 125 (3) ◽  
pp. 280-285 ◽  
Author(s):  
J. Alan Talbot ◽  
Ann Lambert ◽  
Robert Mitchell ◽  
Marek Grabinski ◽  
David C. Anderson ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Culture Medium ◽  
Follicle Stimulating Hormone ◽  
Dibutyryl Camp ◽  
Immature Rat ◽  
Estradiol Secretion ◽  
Old Rats ◽  
Stimulating Hormone

Abstract We have investigated the role of Ca2+ in the control of FSH-induced estradiol secretion by Sertoli cells isolated from 8-10 days old rats. Exogenous Ca2+ (4-8 mmol/1) inhibited FSH-stimulated E2 secretion such that, with 8 mmol/l Ca2+ and FSH (8 IU/l) E2 secretion decreased from 2091±322 to 1480±84 pmol/l (p<0.002), whilst chelation of Ca2+ in the culture medium with EGTA (3 mmol/l) increased E2 secretion from 360±45 to 1242±133 pmol/l) in the absence of FSH. Further, EGTA (3 mmol/l) markedly potentiated FSH (8 IU/l), forskolin (1 μmol/l) and dibutyryl cAMP (1 mmol/l)-stimulated E2 secretion. Addition of the Ca2+ ionophores, ionomycin (2-5 μmol/l) and A23187 (2 μmol/l), inhibited FSH (8 IU/l)-stimulated E2 secretion by >80%. The effect of ionomycin was totally reversible, whereas that of A23187 was irreversible. Ionomycin (5 μmol/l) had no effect on EGTA-induced E2 secretion in the absence of FSH, but reduced EGTA-provoked E2 secretion by 59% in the presence of FSH (8 IU/l). Similarly, forskolin- and dibutyryl cAMP-provoked E2 production was inhibited 46-50% by ionomycin (5 μmol/l). We conclude that FSH-induced E2 secretion from immature rat Sertoli cells is modulated by intra- and extracellular Ca2+.

scholarly journals A novel putative microtubule-associated protein is involved in arbuscule development during arbuscular mycorrhiza formation

2021 ◽  
Author(s):  
Tania Ho-Plágaro ◽  
Raúl Huertas ◽  
María I Tamayo-Navarrete ◽  
Elison Blancaflor ◽  
Nuria Gavara ◽  
...  
Keyword(s):  
Plant Root ◽  
Arbuscular Mycorrhizal ◽  
Host Cells ◽  
Root Cells ◽  
Filament Dynamics ◽  
Fungal Structure ◽  
Genes Encoding ◽  
Associated Proteins ◽  
Mycorrhizal Development

Abstract The formation of arbuscular mycorrhizal (AM) symbiosis requires plant root host cells to undergo major structural and functional reprogramming in order to house the highly branched AM fungal structure for the reciprocal exchange of nutrients. These morphological modifications are associated with cytoskeleton remodelling. However, molecular bases and the role of microtubules (MTs) and actin filament dynamics during AM formation are largely unknown. In this study, the tomato tsb gene, belonging to a Solanaceae group of genes encoding MT-associated proteins for pollen development, was found to be highly expressed in root cells containing arbuscules. At earlier stages of mycorrhizal development, tsb overexpression enhanced the formation of highly developed and transcriptionally active arbuscules, while tsb silencing hampers the formation of mature arbuscules and represses arbuscule functionality. However, at later stages of mycorrhizal colonization, tsb OE roots accumulate fully developed transcriptionally inactive arbuscules, suggesting that the collapse and turnover of arbuscules might be impaired by TSB accumulation. Imaging analysis of the MT cytoskeleton in cortex root cells overexpressing tsb revealed that TSB is involved in MT-bundling. Taken together, our results provide unprecedented insights into the role of novel MT-associated protein in MT rearrangements throughout the different stages of the arbuscule life cycle.

scholarly journals Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

2021 ◽  
Author(s):  
Jonathon A Ditlev
Keyword(s):  
Phase Separation ◽  
Cell Biology ◽  
Cellular Environment ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Available Technology ◽  
And Function ◽  
Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

The neuro-cardiac junction defines an extracellular microdomain required for neurotrophic signaling

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M Mongillo ◽  
M Franzoso ◽  
V Prando ◽  
L Dokshokova ◽  
A Di Bona ◽  
...  
Keyword(s):  
Culture Medium ◽  
Heart Diseases ◽  
Sympathetic Neurons ◽  
Confocal Imaging ◽  
Public Institution ◽  
Mdx Mice ◽  
Mouse Heart ◽  
Funding Source ◽  
Neurotrophic Signaling

Abstract Background Sympathetic neurons (SNs) innervate the myocardium with a defined topology that allows physiological modulation of cardiac activity. Neurotrophins released by cardiac cells control SN viability and myocardial distribution, which are impaired in heart diseases with reduced (e.g. heart failure) or heterogenous sympathetic stimulation (e.g. arrhythmias). We previously demonstrated that SNs interact directly with cardiomyocytes (CMs) at neuro-cardiac junctions (NCJ), and such structured contact sites allow neurons to efficiently activate β-adrenoceptors on the myocyte membrane. Aims We here asked whether NCJs are functional for retrograde (myocyte to neuron) neurotrophic signaling. Methods and results Electron microscopy and immunofluorescence on mouse heart slices and SN/CM co-cultures showed that the NGF receptor, TrkA, is preferentially found in correspondence of the NCJ. Consistently, neurons taking structured contact with CMs showed fast TrkA activation and its retrograde transport to the soma, which was monitored using live confocal imaging in cells expressing TrkA-RFP. In accord with NGF dependent effects, CM-contacted SN showed larger synaptic varicosities and did not require NGF supplementation in the culture medium. In support that NGF locally released at NCJs sustains SN viability, the neurotrophin concentration in the culture medium was 1.61 pg/mL, and did not suffice to maintain neuronal viability, which was also perturbed (66% decrease of neuronal density) by silencing NGF expression in CMs. These results support that the NCJ is essential for intercellular neurotrophin signaling. Consistently, by applying competitive inhibition of TrkA with increasing doses of K252a, we estimated NGF concentration at the contact site to be about 1000-fold higher than that released by CM in the culture medium. To seek for the structural determinants of the NCJ, we focused on dystrophin, based on the finding that the protein accumulates on the CM membrane portion contacted by SNs, as observed in mouse heart slices, and co-cultured CMs. In support of a role of CM-expressed dystrophin in neurotrophic signaling, hearts from dystrophin-KO (mdx) mice showed 74.36% decrease of innervation, with no significant changes of NGF expression. In line with the purported role of NCJs, in co-cultures between wild type SNs and mdx CMs, TrkA activation (TrkA movements toward SN soma (%): WTCM-WTSN=18±4; MDXCM-WTSN= 12±3; p&lt;0,05) and neuronal survival were reduced. Conclusions Taken together, our results suggest that NGF-dependent signaling to SNs requires a direct and specialized interaction with myocytes, and that loss of dystrophin at the CM membrane impairs retrograde signaling to the neurons leading to cardiac sympathetic dys-innervation. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): University of Padova

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