Thymidine-requiring mutants of Dictyostelium discoideum

1984 ◽  
Vol 4 (12) ◽  
pp. 2784-2791
Author(s):  
G Podgorski ◽  
R A Deering
Keyword(s):  
Dictyostelium Discoideum ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Normal Growth ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Cell Extracts ◽  
Dna Replication And Repair

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.

scholarly journals Thymidine-requiring mutants of Dictyostelium discoideum.

1984 ◽  
Vol 4 (12) ◽  
pp. 2784-2791 ◽  
Author(s):  
G Podgorski ◽  
R A Deering
Keyword(s):  
Dictyostelium Discoideum ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Normal Growth ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Cell Extracts ◽  
Dna Replication And Repair

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.

XV.—The metabolism of DNA in the liver during precocious induction of metamorphosis in Rana catesbeiana

1969 ◽  
Vol 70 (4) ◽  
pp. 295-310 ◽  
Author(s):  
Ailsa M. Campbell ◽  
Martita H. Corrance ◽  
J. N. Davidson ◽  
H. M. Keir
Keyword(s):  
Dna Polymerase ◽  
Nuclear Dna ◽  
Rana Catesbeiana ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Polymerase Activity ◽  
Nucleic Acid Metabolism ◽  
Cell Extracts

SynopsisStudies have been made on the incorporation of [8H]-deoxythymidine into the DNA of the livers of Rana catesbeiana tadpoles. When metamorphosis was induced with tri-iodothyronine, the specific activity of the nuclear DNA rose 5 days after administration of the hormone. In contrast, the specific activity of the DNA from the mitochondrial fraction rose grave–2 days after hormone administration.In order to determine whether the in vivo change was due to alterations in the pool sizes of the DNA precursors, in vitro studies on DNA polymerase were carried out. It was found that under conditions where the enzyme activity was not limited by availability of template or substrates, there was a rise in the DNA polymerase activity in crude cell extracts from the tadpole liver. Fractionation of the cell components showed that little of this increment in activity appeared to be located in the nucleus, but that a large percentage alteration in activity occurred in the mitochondrial and cell sap fractions.A possible interpretation of these results is that an increase in the mitochondrial DNA polymerase is one of the early effects of thyroid hormone. This possibility is discussed in relation to the other known effects of thyroid hormones in tadpoles, with particular reference to nucleic acid metabolism and also to mitochondrial hyperplasia.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

Gastric cancer is the world's second-largest death cause. Peptides can be used to deliver radiation or other fatal chemicals to tumors

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gastric Cancer ◽  
High Efficiency ◽  
Specific Activity ◽  
High Specific Activity ◽  
Systemic Circulation ◽  
Efficient Delivery ◽  
Liver And Kidney ◽  
Targeting Ability ◽  
Therapeutic Research

Gastric cancer is the world's second-largest death cause. Developing suitable medical therapies can help individuals live longer. So far, GC treatment has depended on several pharmaceutical techniques. Chemotherapy and surgery are GC patients' most frequent treatment choices. The most major hurdles to effective GC therapy are chemotherapeutic resistance and non-selective targeting. Recent GC-targeted therapeutic research has focused on building more selective and effective anti-GC pharmacological approaches. Because molecular focused therapy can greatly exacerbate the current inefficacy of normal GC therapy procedures, peptide base synthesis can be used as a carrier to deliver radiation or other fatal chemicals to tumor locations with precise protein overexpression. Different types of peptides with special binding affinity to GC overexpressed receptors have been identified for targeted therapy and imaging. Although some of these peptides have excellent GC targeting ability, they also need great GC penetration capacity and no systemic in vivo toxicity before they can be employed in clinical studies. One of these peptides' most notable limitations is their short plasma half-life, limiting their efficient delivery to tumor locations. Sluggish binding pharmacokinetics, along with in vivo instability, can produce targeted treatment failure. Using an appropriate modification strategy to boost blood circulation time may be advantageous.The key to producing successful, innovative anti-cancer targeting drugs with specific targeting capabilities is to mark the peptide with distinct diagnostic and therapeutic radioisotopes. Although a peptide's radiolabeling or enzymatic degradation may not affect its targeting capabilities, the radiation dose delivery impact on it is obvious. Selecting an appropriate type of radionuclide to achieve high-specific activity, using a simple and high-efficiency radiolabeling process, and selecting an adequate spacer and chelator to manage peptide biodistribution are all important considerations when designing a peptide-based radiopharmaceutical. High internalization and significant systemic circulation washout are other essential tumor targeting needs. Many of the peptides described in this work lack these critical features. The radiolabeled peptide should also remain intact and have a short blood washout period, allowing targeted imaging and therapy. SPECT and PET are the most extensively used technologies in nuclear medicine. Although PET has a greater resolution, SPECT technology gives a comparable sensitivity at a lesser cost. Combining fast binding pharmacokinetics with suitable stability in vivo can result in efficient tumor contrast. Non-target liver and kidney accumulation is required when employing radiolabeled peptides to target GC. When a radiolabeled peptide accumulates more in the liver and intestine than in the GC tumor, the image quality degrades. However, using the proper chelator and spacer can assist decrease non-target accumulation in the kidneys. Finally, considering all these conditions and being positive, it is conceivable to produce a unique peptide with avid binding to GC cells.

In vivo quantification of pulmonary beta-adrenoceptor density in humans with (S)-[11C]CGP-12177 and PET

1993 ◽  
Vol 75 (2) ◽  
pp. 559-565 ◽  
Author(s):  
J. Ueki ◽  
C. G. Rhodes ◽  
J. M. Hughes ◽  
R. De Silva ◽  
D. C. Lefroy ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Specific Activity ◽  
Regional Distribution ◽  
High Specific Activity ◽  
Normal Subjects ◽  
Normal Male ◽  
Tissue Density ◽  
Beta Adrenoceptor ◽  
Cgp 12177

The in vivo regional distribution of pulmonary beta-adrenoceptors was imaged and quantified in humans with the hydrophilic beta-adrenoceptor antagonist (S)-CGP-12177 labeled with carbon-11 [(S)-[11C]CGP-12177] and positron emission tomography (PET). Six normal male volunteers and eight patients with hypertrophic cardiomyopathy were studied. PET scanning consisted of transmission (tissue density), C15O (blood volume), and (S)-[11C]CGP-12177 (beta-adrenoceptor) emission scans. High-specific-activity (S)-[11C]-CGP-12177 (7.1 +/- 2.0 micrograms, 6.5 +/- 2.1 GBq/mumol) was given intravenously followed by a low-specific-activity (S)-[11C]CGP-12177 injection (34.0 +/- 4.8 micrograms, 2.3 +/- 0.8 GBq/mumol). Binding capacity (Bmax) was calculated in each region of interest as picomoles per gram by normalizing it to the local extravascular tissue density. In normal subjects, average Bmax for all regions of interest was 14.8 +/- 1.6 (SD) pmol/g, which is similar to previously reported in vitro values. In both groups there were no differences in beta-adrenoceptor density between peripheral and central regions nor between right and left lungs. In patients with hypertrophic cardiomyopathy, extravascular tissue density was 24% higher than in normal subjects; Bmax per milliliter thoracic volume was correspondingly higher but was not different from that in normal subjects when expressed per gram tissue (15.8 +/- 2.6 pmol/g). These data suggest that in vivo beta-adrenoceptor density may be quantifiable in humans with the use of PET. This should offer a means to study physiological regulation through repeat measurements.

scholarly journals In vitro and in vivo hematopoietic effect of mutant human granulocyte colony-stimulating factor [see comments]

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1788-1793 ◽  
Author(s):  
M Okabe ◽  
M Asano ◽  
T Kuga ◽  
Y Komatsu ◽  
M Yamasaki ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Intravenous Injection ◽  
Specific Activity ◽  
High Specific Activity ◽  
Daily Injection ◽  
Total Leukocyte Count ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor

About 100 derivatives of human recombinant granulocyte colony- stimulating factor (rhG-CSF) were created by various gene-mutagenic techniques, and KW-2228, in which amino acids were replaced at five positions of N-terminal region of intact rhG-CSF, was picked up and evaluated for its biologic and physicochemical properties in comparison with intact rhG-CSF. KW-2228 showed two to four times higher specific activity than that of intact rhG-CSF in mouse and/or human bone marrow progenitor cells by colony-forming unit assay in soft agar, and by cell- proliferation assay in liquid culture. KW-2228 showed a potency to increase peripheral neutrophil counts when it was administered to normal C3H/He mice by single intravenous injection. Increase of total leukocyte count and neutrophils was observed, with peak level at 8 to 12 hours at low doses (0.5 to 1.0 micrograms/mouse), and the highest level was maintained for 24 to 30 hours at high doses (5 to 10 micrograms/mouse). The granulopoietic effect of KW-2228 was examined by several doses of single course (once daily for 10 days) or multiple courses (twice daily injection for 5 days followed by cessation for 9 days on one cycle, 3 cycles in total) of treatment. KW-2228 showed higher activity than that of rhG-CSF, especially at sub-optimal doses of multiple courses of treatment. Furthermore, KW-2228 was found to be more stable physicochemically and biologically than intact rhG-CSF, especially under thermal conditions at 56 degrees C and in the human plasma at 37 degrees C, suggesting a protease resistancy. Pharmacokinetic study showed that plasma concentration of KW-2228 assayed for its bioactivity maintained a higher level than that of intact rhG-CSF for 60 minutes after intravenous injection of this protein to normal mice. Those results suggest that KW-2228 might show a superior in vivo hematopoietic effect to intact rhG-CSF due to its high specific activity to progenitor cells, and also due to its improved physicochemical, biologic, and pharmacokinetic stability in host animals.

scholarly journals A New Technic for the Production of an in Vivo Labeled Fibrinogen

Blood ◽  
1967 ◽  
Vol 29 (4) ◽  
pp. 517-525 ◽  
Author(s):  
HENRY GANS ◽  
JAMES MC LEOD ◽  
JAMES T. LOWMAN
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
Entire Period ◽  
Turnover Rates ◽  
Prolonged Infusion ◽  
Slow Infusion

Abstract The fact that in vitro labeled proteins, as a rule, exhibit faster turnover rates than in vivo labeled materials led us to explore means of obtaining in vivo labeled fibrinogen of high specific activity. It was found that defibrination of the rat provides a stimulus for the liver to regenerate fibrinogen at an accelerated rate. Administration of seleno75 methionine shortly after thrombin-induced defibrination of the animal resulted in the incorporation of large quantities of the label. The rate of incorporation was further increased if the amino acid was administered as a slow infusion during the entire period of fibrinogen regeneration. In addition, prior nephrectomy of the animal would appear to result in a slight increase in specific activity of the fibrinogen preparation obtained. The results of these studies indicate that defibrination, nephrectomy, and the prolonged infusion of the labeled amino acid selenomethionine provided us with a technic for obtaining a biosynthetically labeled, γ-emitting, fibrinogen preparation of high specific activity.

Copper transport in rats involving a new plasma protein

1985 ◽  
Vol 249 (1) ◽  
pp. E77-E88 ◽  
Author(s):  
K. C. Weiss ◽  
M. C. Linder
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
High Specific Activity ◽  
Apparent Molecular Weight ◽  
Female Rats ◽  
Whole Body ◽  
Peripheral Tissues ◽  
Chelex 100

The time course of distribution of high-specific activity 67CuCl2 to tissues and plasma components was followed in adult, female rats. Immediately after intubation or injection, tracer 67Cu associated with two components of the blood plasma separable on columns of Sephadex G-150: albumin and another (larger) component, which was not ceruloplasmin. The latter, tentatively named transcuprein, had an apparent molecular weight of 270,000 and a high affinity for Cu2+, as judged by processing through Chelex-100, dilution, and exchange with albumin copper, in vitro and in vivo. It was capable of donating copper to tumor cells in serum-free medium. Analysis of "cold" plasma by furnace atomic absorption confirmed the presence of 10-15% of plasma copper in this peak. Plots of percent dose and 67Cu specific activity against time showed that copper followed a very specific pathway after binding to albumin and transcuprein, entering mainly the liver, then reappearing in the plasma on ceruloplasmin, and then achieving peak distribution in peripheral tissues (muscles, brain, etc.). 67Cu disappeared from liver and kidney with an apparent half-life of 4.5 days, the same exponential rate found for whole body turnover. Apparent turnover of ceruloplasmin copper was more rapid. Even after 7-12 days, tracer copper in plasma was still found exclusively with ceruloplasmin. The results indicate that copper follows a carefully prescribed path, on entering the blood and binding to a new transport protein.

Sign in / Sign up

Export Citation Format

Share Document