scholarly journals Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis

1984 ◽  
Vol 217 (2) ◽  
pp. 435-440 ◽  
Author(s):  
P C Rumsby ◽  
P C Campbell ◽  
L A Niswander ◽  
J N Davidson
Keyword(s):  
Pyrimidine Biosynthesis ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Multifunctional Protein ◽  
Aggregation Site ◽  
Aspartate Carbamoyltransferase ◽  
Low Concentrations ◽  
Proteinase A

When the multifunctional protein that catalyses the first three steps of pyrimidine biosynthesis in hamster cells is treated with staphylococcal V8 proteinase, a single cleavage takes place. The activities of carbamoyl-phosphate synthetase (EC 6.3.5.5), aspartate carbamoyltransferase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3) and the allosteric inhibition by UTP are unaffected. One fragment, of Mr 182000, has the first and third enzyme activities, whereas the other fragment, of Mr 42000, has aspartate carbamoyltransferase activity and an aggregation site. A similar small fragment is observed in protein digested with low concentrations of trypsin. A similar large fragment is seen after digestion with trypsin and as the predominating form of this protein in certain mutants defective in pyrimidine biosynthesis. These results indicate that a region located adjacent to the aspartate carbamoyltransferase domain is hypersensitive to proteinase action in vitro and may also be sensitive to proteolysis in vivo.

scholarly journals Citrulline synthesis in rat tissues and liver content of carbamoyl phosphate and ornithine

1974 ◽  
Vol 138 (2) ◽  
pp. 225-232 ◽  
Author(s):  
Luisa Raijman
Keyword(s):  
Rat Liver ◽  
Soluble Fraction ◽  
Maximal Activity ◽  
Rat Tissues ◽  
Ornithine Carbamoyltransferase ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Citrulline Synthesis

Rat liver ornithine carbamoyltransferase appears to be located exclusively in the mitochondria; the activity that is found in the soluble fraction is indistinguishable from mitochondrial ornithine carbamoyltransferase by simple kinetic criteria, and seems to result from breakage of mitochondria during homogenization. Of several rat tissues studied, only the liver and the mucosa of small intestine contain significant amounts of ornithine carbamoyltransferase; the activity in intestinal mucosa is less than one thousandth of that in liver. Qualitatively, this distribution coincides with that of carbamoyl phosphate synthetase I and its cofactor, acetylglutamate. The rat liver contents of carbamoyl phosphate and ornithine were 0.1 and 0.15μmol/g wet wt. of tissue respectively. On the basis of these values, it is proposed that in vivo the ornithine carbamoyltransferase activity of liver may be much lower than its maximal activity in vitro might suggest.

scholarly journals A Carbamoyl Phosphate Synthetase II (CPSII) Deletion Mutant of Toxoplasma gondii Induces Partial Protective Immunity in Mice

2021 ◽  
Vol 11 ◽  
Author(s):  
Xunhui Zhuo ◽  
Kaige Du ◽  
Haojie Ding ◽  
Di Lou ◽  
Bin Zheng ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
De Novo ◽  
Parasite Growth ◽  
Pyrimidine Biosynthesis ◽  
Mrna Levels ◽  
Biosynthesis Pathway ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate

Toxoplasma gondii is an obligate intracellular protozoan parasite. T. gondii primarily infection in pregnant women may result in fetal abortion, and infection in immunosuppressed population may result in toxoplasmosis. Carbamoyl phosphate synthetase II (CPSII) is a key enzyme in the de novo pyrimidine-biosynthesis pathway, and has a crucial role in parasite replication. We generated a mutant with complete deletion of CPSII via clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 in type-1 RH strain of T. gondii. We tested the intracellular proliferation of this mutant and found that it showed significantly reduced replication in vitro, though CPSII deletion did not completely stop the parasite growth. The immune responses induced by the infection of RHΔCPSII tachyzoites in mice were evaluated. During infection in mice, the RHΔCPSII mutant displayed notable defects in replication and virulence, and significantly enhanced the survival of mice compared with survival of RH-infected mice. We tracked parasite propagation from ascitic fluid in mice infected with the RHΔCPSII mutant, and few tachyzoites were observed at early infection. We also observed that the RHΔCPSII mutant induced greater accumulation of neutrophils. The mutant induced a higher level of T-helper type-1 cytokines [interferon (IFN)-γ, interleukin (IL)-12]. The mRNA levels of signal transducer and activator of transcription cellular transcription factor 1 and IFN regulatory factor 8 were significantly higher in the RHΔCPSII mutant-infected group. Together, these data suggest that CPSII is crucial for parasite growth, and that strains lack the de novo pyrimidine biosynthesis pathway and salvage pathway may become a promising live attenuated vaccine to prevent infection with T. gondii.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

scholarly journals In Bacillus subtilis, the Sirtuin Protein Deacetylase, Encoded by the srtN Gene (Formerly yhdZ), and Functions Encoded by the acuABC Genes Control the Activity of Acetyl Coenzyme A Synthetase

2009 ◽  
Vol 191 (6) ◽  
pp. 1749-1755 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
New Information ◽  
Low Concentrations ◽  
Dependent Protein ◽  
Protein Deacetylase

ABSTRACT This report provides in vivo evidence for the posttranslational control of the acetyl coenzyme A (Ac-CoA) synthetase (AcsA) enzyme of Bacillus subtilis by the acuA and acuC gene products. In addition, both in vivo and in vitro data presented support the conclusion that the yhdZ gene of B. subtilis encodes a NAD+-dependent protein deacetylase homologous to the yeast Sir2 protein (also known as sirtuin). On the basis of this new information, a change in gene nomenclature, from yhdZ to srtN (for sirtuin), is proposed to reflect the activity associated with the YdhZ protein. In vivo control of B. subtilis AcsA function required the combined activities of AcuC and SrtN. Inactivation of acuC or srtN resulted in slower growth and cell yield under low-acetate conditions than those of the wild-type strain, and the acuC srtN strain grew under low-acetate conditions as poorly as the acsA strain. Our interpretation of the latter result was that both deacetylases (AcuC and SrtN) are needed to maintain AcsA as active (i.e., deacetylated) so the cell can grow with low concentrations of acetate. Growth of an acuA acuC srtN strain on acetate was improved over that of the acuA + acuC srtN strain, indicating that the AcuA acetyltransferase enzyme modifies (i.e., inactivates) AcsA in vivo, a result consistent with previously reported in vitro evidence that AcsA is a substrate of AcuA.

In vitro and in vivo effects of leflunomide, Brequinar, and cyclosporine on pyrimidine biosynthesis

1997 ◽  
Vol 29 (1-2) ◽  
pp. 1292-1293 ◽  
Author(s):  
H.T. Silva ◽  
R.A. Shorthouse ◽  
M. Löffler ◽  
R.E. Morris ◽  
W. Cao
Keyword(s):  
Pyrimidine Biosynthesis ◽  
In Vivo Effects

Aerobic resistance of Trichomonas vaginalis to metronidazole induced in vitro

Parasitology ◽  
1993 ◽  
Vol 106 (1) ◽  
pp. 31-37 ◽  
Author(s):  
J. Tachezy ◽  
J. Kulda ◽  
E. Tomková
Keyword(s):  
Parent Strain ◽  
Trichomonas Vaginalis ◽  
Drug Resistant ◽  
Ferredoxin Oxidoreductase ◽  
Low Concentrations ◽  
Percent Concentration ◽  
Resistant Strains ◽  
Pyruvate Ferredoxin Oxidoreductase

SUMMARYAerobic resistance of Trichomonas vaginalis to metronidazole was induced in vitro by anaerobic cultivation of drug-susceptible trichomonads with low concentrations of the drug (2–3 μg/ml) for 50 days. Minimal lethal concentrations (MLC) for metronidazole of the resistant derivatives were high in aerobic susceptibility assays (MLC = 216–261.5 μg/ml) but low in anaerobic assays (MLC = 4.2–6.3 μg/ml), surpassing MLC values of their parent strain approximately 50-fold and 3-fold under aerobiosis and anaerobiosis, respectively. Sensitivity to metronidazole under anaerobic conditions and activity of the hydrogenosomal enzyme pyruvate: ferredoxin oxidoreductase indicated that the resistance was of the aerobic type. Dependence of the resistance manifestation on O2 was further confirmed by susceptibility assays in vitro performed in defined gas mixtures of different oxygen content (1–20%). Five percent concentration of O2 proved to be the threshold required for resistance demonstration and the MLC values further increased with increasing O2 concentrations. The in vitro-induced resistance was also demonstrated in vivo by subcutaneous mouse assay. The dose of metronidazole needed to cure 50% of infected mice (DC50) was 223 mg/kg × 3 for resistant derivative MR-3a but 6.6 mg/kg × 3 only for its drug-susceptible parent strain. The metronidazole – resistant strains developed in this study correspond by their properties to drug-resistant T. vaginalis strains isolated from patients refractory to treatment, and promise to be a useful tool in the study of 5-nitroimidazole aerobic resistance.

scholarly journals The Whisking Rhythm Generator: A Novel Mammalian Network for the Generation of Movement

2007 ◽  
Vol 97 (3) ◽  
pp. 2148-2158 ◽  
Author(s):  
Nathan P. Cramer ◽  
Ying Li ◽  
Asaf Keller
Keyword(s):  
Firing Rate ◽  
Serotonin Receptor ◽  
Central Pattern Generators ◽  
Rhythm Generator ◽  
Low Concentrations ◽  
Rhythmic Firing ◽  
Pattern Generators ◽  
Cortical Inputs

Using the rat vibrissa system, we provide evidence for a novel mechanism for the generation of movement. Like other central pattern generators (CPGs) that underlie many movements, the rhythm generator for whisking can operate without cortical inputs or sensory feedback. However, unlike conventional mammalian CPGs, vibrissa motoneurons (vMNs) actively participate in the rhythmogenesis by converting tonic serotonergic inputs into the patterned motor output responsible for movement of the vibrissae. We find that, in vitro, a serotonin receptor agonist, α-Me-5HT, facilitates a persistent inward current (PIC) and evokes rhythmic firing in vMNs. Within each motoneuron, increasing the concentration of α-Me-5HT significantly increases the both the magnitude of the PIC and the motoneuron's firing rate. Riluzole, which selectively suppresses the Na+ component of PICs at low concentrations, causes a reduction in both of these phenomena. The magnitude of this reduction is directly correlated with the concentration of riluzole. The joint effects of riluzole on PIC magnitude and firing rate in vMNs suggest that the two are causally related. In vivo we find that the tonic activity of putative serotonergic premotoneurons is positively correlated with the frequency of whisking evoked by cortical stimulation. Taken together, these results support the hypothesized novel mammalian mechanism for movement generation in the vibrissa motor system where vMNs actively participate in the rhythmogenesis in response to tonic drive from serotonergic premotoneurons.

scholarly journals Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 930-936 ◽  
Author(s):  
I Max-Audit ◽  
U Testa ◽  
D Kechemir ◽  
M Titeux ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Cell Lines ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Low Concentrations ◽  
Erythroleukemia Cell ◽  
Erythroleukemic Cell ◽  
High Level

To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

scholarly journals Direct modulation of basal and angiotensin II-stimulated aldosterone secretion by hydrogen ions

2000 ◽  
Vol 166 (1) ◽  
pp. 183-194 ◽  
Author(s):  
RE Kramer ◽  
TV Robinson ◽  
EG Schneider ◽  
TG Smith
Keyword(s):  
Angiotensin Ii ◽  
Free Calcium ◽  
Acid Base Balance ◽  
Aldosterone Secretion ◽  
Plasma Aldosterone Concentration ◽  
Low Concentrations ◽  
Effects Of Ph ◽  
Glomerulosa Cells

Disturbances in acid-base balance in vivo are associated with changes in plasma aldosterone concentration, and in vitro changes in extracellular pH (pH(o)) influence the secretion of aldosterone by adrenocortical tissue or glomerulosa cells. There is considerable disparity, however, as to the direction of the effect. Furthermore, the mechanisms by which pH(o) independently affects aldosterone secretion or interacts with other secretagogues are not defined. Thus, bovine glomerulosa cells maintained in primary monolayer culture were used to examine the direct effects of pH(o) on cytosolic free calcium concentration ([Ca(2+)](i))( )and aldosterone secretion under basal and angiotensin II (AngII)-stimulated conditions. pH(o) was varied from 7.0 to 7.8 (corresponding inversely to changes in extracellular H(+) concentration from 16 nM to 100 nM). Whereas an elevation of pH(o) from 7.4 to 7.8 had no consistent effect, reductions of pH(o) from 7.4 to 7.2 or 7.0 caused proportionate increases in aldosterone secretion that were accompanied by increases in transmembrane Ca(2+) fluxes and [Ca(2+)](i). These effects were abolished by removal of extracellular Ca(2+). A decrease in pH(o) from 7.4 to 7.0 also enhanced AngII-stimulated aldosterone secretion. This effect was more pronounced at low concentrations of AngII and was manifested as an increase in the magnitude of the secretory response with no effect on potency. In contrast to its effect on AngII-stimulated aldosterone secretion, a reduction of pH(o) from 7.4 to 7.0 inhibited the Ca(2+) signal elicited by low concentrations (</=1x10(-10) M) of AngII, but did not affect the increase in [Ca(2+)](i) caused by a maximal concentration (1x10(-8) M) of AngII. These data suggest that pH(o) (i.e. H(+)) has multiple effects on aldosterone secretion. It independently increases aldosterone secretion through a mechanism involving Ca(2+) influx and an increase in [Ca(2+)](i). Also, it modulates the action of AngII by both decreasing the magnitude of the AngII-stimulated Ca(2+) signal and increasing the sensitivity of a more distal site to intracellular Ca(2+). The latter action appears to be a more important determinant in the effects of pH(o) on AngII-stimulated aldosterone secretion.

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