Regulatory properties of yeast nitrate reductase in situ

1976 ◽  
Vol 22 (1) ◽  
pp. 35-42 ◽  
Author(s):  
V. Prabhakara Choudary ◽  
G. Ramananda Rao
Keyword(s):  
Amino Acids ◽  
Nitrate Reductase ◽  
Nitrogen Starvation ◽  
Yeast Cells ◽  
Vitro Assay ◽  
Wide Range ◽  
In Situ Assay

A simple and rapid procedure to make yeast cells permeable by agitating with toluene–ethanol, (TE) 1:4, v/v was developed. The permeated cells retained their ability to catalyze certain enzyme reactions. Temperature and duration of agitation during TE treatment played an important role in retention of the catalytic potential of permeated cells. The in situ assay using permeated cell preparations was more sensitive even in the absence of added cofactors than the in vitro assay in detecting assimilatory nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) (NAR) activity in Candida utilis.Using in situ assay technique, different mechanisms regulating the biosynthesis of NAR in C. utilis were investigated. Nitrogen starvation did not lead to derepression of NAR. NO3− ions were absolutely essential for induction and maintenance of high levels of NAR activity. Cells grown on ammonium nitrate possessed relatively lower levels of NAR. Kinetics of NAR induction were followed as a function of time and inducer concentration. The influence of various cations on the induction of NAR by nitrate was investigated. A wide range of D-amino acids induced NAR synthesis. Of 22 L-amino acids tested only phenylalanine induced significant levels of NAR. Various intermediates of the pathway of nitrate reduction influenced the rate of NAR induction. There was a rapid disappearance of in vivo activity of the enzyme of induced yeast cells on nitrogen starvation, and the rate of loss was accelerated by the presence of NH4+.

Minimum intracellular PO2 for maximum cytochrome turnover in red muscle in situ

1987 ◽  
Vol 252 (5) ◽  
pp. H906-H915 ◽  
Author(s):  
T. E. Gayeski ◽  
R. J. Connett ◽  
C. R. Honig
Keyword(s):  
Energy Demand ◽  
Concentration Ratio ◽  
Probability Distributions ◽  
Michaelis Constant ◽  
Cytochrome Aa3 ◽  
Wide Range ◽  
Red Muscle

Probability distributions of myoglobin (Mb) saturation and intracellular PO2 were determined with subcellular spatial resolution in dog gracilis muscles during steady-state twitch contraction at 5-100% of maximal rate of O2 consumption (VO2). Calculations (Clark, A., and P. A.A. Clark. Biophys. J. 48: 931-938, 1985) and measurements (Gayeski, T. E. J., and C. R. Honig. Adv. Exp. Med. Biol. 200: 487-494, 1986) indicate that the PO2 in equilibrium with Mb is virtually identical to the PO2 at cytochrome aa3. Median intracellular PO2 and PO2 in the lower tails of probability distributions were poorly correlated with VO2. The variability of cell PO2 was greatly diminished when median PO2 was less than the PO2 for half saturation of MB, since Mb acts as a PO2 buffer. The lower tails of PO2 distributions contained almost no anoxic loci even when median PO2 was less than 1 Torr. VO2 was well correlated with the concentration ratio of phosphocreatine to free creatine (PCr/Crf) over a wide range of PO2. PO2 greater than or equal to 0.5 Torr supported maximal VO2 and energy demand. We conclude that 1) the mechanism of action of cytochrome aa3 is the same in red muscle in vivo as in mitochondria in vitro, and 2) an upper bound on the apparent Michaelis constant for maximal VO2 of red muscle is approximately 0.06 Torr.

In vitro techniques to replace in vivo methods for estimating amino acid supply

1998 ◽  
Vol 22 ◽  
pp. 131-144 ◽  
Author(s):  
T. Hvelplund ◽  
M. R. Weisbjerg
Keyword(s):  
Protein Synthesis ◽  
Alternative Methods ◽  
Microbial Protein ◽  
Microbial Protein Synthesis ◽  
Wide Range ◽  
Intestinal Digestibility ◽  
Protein Degradability

Abstract Expressing the protein value of a food involves measurements of several of its characteristics. Many in vivo studies have shown, that the protein degradability in the rumen varies substantially both between and within foods and therefore estimation of protein degradability in the rumen is an important task in protein evaluation. The most common method used has been the in situ (in sacco, nylon bag) method but many in vitro methods have been introduced and are based on use of either buffer solubility, chemical methods, rumen fluid or enzymes. None of these in vitro methods has proven to be of general use. In further development of in vitro methods as well as the in situ method a major problem is lack of a set of samples with a ‘true’ in vivo degradability which can be used for calibration of alternative methods. Microbial protein synthesis in the rumen has to be related to food characteristics which can be analysed easily. In vitro methods which can predict organic matter digestibility in foods are available and can be used to predict microbial protein synthesis in the rumen. Intestinal digestibility of undegraded dietary protein varies substantially both between and within foods and easy methods to estimate intestinal digestibility are therefore essential. The mobile bag method is easy to use and seems to give reliable results on most foods but requires access to duodenal cannulated animals which prevents this method from being routine. Alternative in vitro methods have been developed but further research is required for validation of these methods on a wide range of foods before they can be accepted for general use.

scholarly journals Three-dimensional ultrastructure of the septin filament network inSaccharomyces cerevisiae

2012 ◽  
Vol 23 (3) ◽  
pp. 423-432 ◽  
Author(s):  
Aurélie Bertin ◽  
Michael A. McMurray ◽  
Jason Pierson ◽  
Luong Thai ◽  
Kent L. McDonald ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Yeast Cells ◽  
Lipid Monolayers ◽  
Morphological Description ◽  
Section Electron ◽  
Subunit Organization

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P2-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo–electron tomography. We found networks of filaments both perpendicular and parallel to the mother–bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.

The Development of an In Situ Assay for bFGF Release

1993 ◽  
Vol 331 ◽  
Author(s):  
S. K. Hobbs ◽  
L. M. Periolat ◽  
L. G. Cima ◽  
M. Nugent ◽  
M. Leunig ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Dorsal Skinfold Chamber ◽  
Control Growth ◽  
Vessel Growth ◽  
And Control ◽  
Tissue Reactivity ◽  
In Situ Assay

AbstractThere is a need for an in situ assay to quantify tissue reactivity to sustained release of bFGF to better understand and control growth factor-induced angiogenesis. To this end we have adapted the alginate/heparin-sepharose release system for use in the mouse dorsal skinfold chamber. A mathematical model was used to predict the time dependence of bFGF release as a function of bFGF loading, heparin concentration, and device geometry. The model predictions agreed well with previously reported in vitro data. In vivo studies to correlate blood vessel growth as a function of release rate are in progress.

scholarly journals TRA1, a Novel mRNA Highly Expressed in Leukemogenic Mouse Monocytic Sublines But Not in Nonleukemogenic Sublines

Blood ◽  
1997 ◽  
Vol 89 (8) ◽  
pp. 2975-2985 ◽  
Author(s):  
Takashi Kasukabe ◽  
Junko Okabe-Kado ◽  
Yoshio Honma
Keyword(s):  
Amino Acids ◽  
Cdna Library ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
S2 Cells ◽  
Normal Tissues ◽  
Wide Range ◽  
P Cells

Abstract Mouse monocytic Mm-A, Mm-P, Mm-S1, and Mm-S2 cells are sublines of mouse monocytic and immortalized Mm-1 cells derived from spontaneously differentiated, mouse myeloblastic M1 cells. Although these subline cells retain their monocytic characteristics in vitro, Mm-A and Mm-P cells are highly leukemogenic to syngeneic SL mice and athymic nude mice, whereas Mm-S1 and Mm-S2 cells are not or are only slightly leukemogenic. To better understand the molecular mechanisms of these levels of leukemogenicity, we investigated putative leukemogenesis-associated genes or oncogenes involved in the maintenance of growth, especially in vivo, by means of differential mRNA display. We isolated a fragment clone (15T01) from Mm-P cells. The mRNA probed with 15T01 was expressed at high levels in leukemogenic Mm-P and Mm-A cells but not in nonleukemogenic Mm-S1 and Mm-S2 cells. The gene corresponding to 15T01, named TRA1, was isolated from an Mm-P cDNA library. The longest open reading frame of the TRA1 clone predicts a peptide containing 204 amino acids with a calculated molecular weight of 23,049 D. The predicted TRA1 protein is cysteine-rich and contains multiple cysteine doublets. A putative normal counterpart gene, named NOR1, was also isolated from a normal mouse kidney cDNA library and sequenced. NOR1 cDNA predicts a peptide containing 234 amino acids. The sequence of 201 amino acids from the C-terminal NOR1 was completely identical to that of TRA1, whereas the remaining N-terminal amino acids (33 amino acids) were longer than that (3 amino acids) of TRA1 and the N-terminus of NOR1 protein contained proline-rich sequence. A similarity search against current nucleotide and protein sequence databases indicated that the NOR1/TRA1 gene(s) is conserved in a wide range of eukaryotes, because apparently homologous genes were identified in Caenorhabditis elegans and Saccharomyces cerevisiae genomes. Northern blotting using TRA1-specific and NOR1-specific probes indicated that TRA1 mRNA is exclusively expressed in leukemogenic but not in nonleukemogenic Mm sublines and normal tissues and also indicated that NOR1 mRNA is expressed in normal tissues, especially in kidney, lung, liver, and bone marrow cells but not in any Mm sublines. After leukemogenic Mm-P cells were induced to differentiate into normal macrophages by sodium butyrate, the normal counterpart, NOR1, was expressed, whereas the TRA1 level decreased. Furthermore, transfection of TRA1 converted nonleukemogenic Mm-S1 cells into leukemogenic cells. These results indicate that the TRA1 gene is associated at least in part with the leukemogenesis of monocytic Mm sublines.

In Vitro and In Vivo Approaches for Screening the Potential of Anti Cancer Agents: A Review

2022 ◽  
Vol 19 ◽  
Author(s):  
Rakhi Mishra ◽  
Prem Shankar Mishra ◽  
Shruti Varshney ◽  
Rupa Mazumder ◽  
Avijit Mazumder
Keyword(s):  
In Vivo Studies ◽  
Chemical Toxicity ◽  
Vitro Assay ◽  
Wide Range ◽  
Anti Cancer ◽  
Assay Methods ◽  
New Anticancer Drugs ◽  
Hallmark Feature

Background: Anticancer drug development is a tedious process, requiring several in vitro, in vivo, and clinical studies. To avoid chemical toxicity in animals during an experiment, it is necessary to envisage toxic doses of screened drugs in vivo at different concentrations. Several in vitro and in vivo studies have been reported to discover the management of cancer. Materials and Methods: This study has focused on bringing together a wide range of in vivo and in vitro assay methods, developed to evaluate each hallmark feature of cancer. Result: This review provides elaborated information about target-based and cell-based screening of new anticancer drugs in the molecular targeting period. This would help to incite an alteration from the preclinical screening of pragmatic compound-orientated to target-orientated drug selection. Conclusion: Selection methodologies for finding anticancer activity have importance for tumor-specific agents. In this study, advanced rationalization of the cell-based assay is explored along with broad applications of the cell-based methodologies considering other opportunities also.

scholarly journals Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

1996 ◽  
Vol 16 (2) ◽  
pp. 475-480 ◽  
Author(s):  
X Mao ◽  
B Schwer ◽  
S Shuman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Yeast Cells ◽  
Terminal Deletion ◽  
Methyltransferase Activity

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.

The High Potential of Ozone Gas to Inactivate Echinococcus granulosus Protoscoleces During Hydatid Cyst Surgery

2020 ◽  
Vol 20 (5) ◽  
pp. 708-712
Author(s):  
Hossein Mahmoudvand ◽  
Majid Fasihi Harandi ◽  
Massumeh Niazi ◽  
Abdolreza Rouientan ◽  
Fazel Mohammadi-Moghadam ◽  
...  
Keyword(s):  
Hydatid Cyst ◽  
Ex Vivo ◽  
Human Subjects ◽  
Oral Bacteria ◽  
Vitro Assay ◽  
Low Concentrations ◽  
Wide Range ◽  
Resistant Strains

Background: In medicine, ozone therapy is effectively used in a broad spectrum of diseases. Reviews have shown that ozone gas demonstrates potent antimicrobial effects against a wide range of pathogenic microorganisms, such as oral bacteria, fungi, viruses, and parasite even in resistant strains. The present investigation was designed to assess the protoscolicidal effects of ozone gas on hydatid cysts protoscoleces in vitro and in vivo. Methods: Hydatid cyst protoscoleces were acquired from sheep livers that were slaughtered at Kerman slaughterhouse, Iran. The viability of protoscoleces was assessed by the eosin exclusion examination after exposure with ozone gas for 1 to 14 min in vitro and ex vivo. Results: In this study, in vitro assay showed that ozone gas at the concentration of 20 mg/L killed 85 and 100% of hydatid cyst protoscoleces after 4 and 6 min of treatment, respectively. However, in the ex vivo analysis, a longer time was needed to confirm a potent protoscolicidal activity such that ozone gas after an exposure time of 12 min, 100% of the protoscoleces were killed within the hydatid cyst. Conclusion: : In conclusion, the findings of the present study showed that ozone gas at low concentrations (20 mg/L) and short times (4-6 min) might be used as a novel protoscolicidal drug for use in hydatid cyst surgery. However, more clinical surveys are required to discover the precise biological activity of ozone gas in animal and human subjects.

A muscle-powered energy delivery system and means for chronic in vivo testing

1999 ◽  
Vol 86 (6) ◽  
pp. 2106-2114 ◽  
Author(s):  
Dennis R. Trumble ◽  
James A. Magovern
Keyword(s):  
In Vitro Tests ◽  
Access Port ◽  
Wide Range ◽  
Piston Displacement ◽  
In Vivo Testing ◽  
Implant Testing

Electrically stimulated skeletal muscle represents a potentially unlimited source of energy for the actuation of motor prostheses. Devices to harvest and deliver contractile power have proven mechanically feasible, but long-term efficacy has not been demonstrated. This report describes recent refinements in muscle energy converter (MEC) design and details the development of an implantable afterload chamber (IAC) designed to facilitate implant testing. The IAC comprises a fluid-filled bladder housed within a titanium cylinder that connects directly to the MEC. A vascular access port allows percutaneous measurement and adjustment of air pressure within the housing and provides a means both to monitor MEC function and to control hydraulic loading conditions. Data from in vitro tests show that IAC pressure mirrors changes in MEC-piston displacement over a wide range of actuation speeds and stroke lengths. Stroke lengths and actuation forces calculated from IAC pressure readings were typically found to be within 5% of measured values. This testing scheme may yield important information in regard to the ability to harness energy from in situ muscle over prolonged periods.

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