DNA Synthesis in Isolated Yeast Mitochondria

1974 ◽  
Vol 52 (11) ◽  
pp. 941-949 ◽  
Author(s):  
L. Zeman ◽  
C. V. Lusena
Keyword(s):  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Base Composition ◽  
Nuclear Dna ◽  
Sedimentation Velocity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Denatured State ◽  
Saccharomyces Cerevisiae Mitochondria

Isolated yeast (Saccharomyces cerevisiae) mitochondria incorporate radioactive precursors into mitochondrial DNA. This in vitro labelled DNA was characterized by isopycnic and sedimentation velocity centrifugation both in the native and denatured state. The profiles of isopycnic CsCl gradients obtained by centrifugation in a fixed-angle rotor are skewed toward high density. The skew is neither due to the presence of in vitro labelled nuclear DNA nor due to random breaks in mitochondrial DNA which would reveal, then, its heterogeneity in base composition. The in vitro labelled DNA is reproducibly recovered as a class of molecules sedimenting at about 5–8 S, indicating a molecular weight of 1 × 105 – 4 × 105 daltons, while the smallest in vivo labelled fragments sediment at about 13–14 S, corresponding to 1.6 × 106 – 2.0 × 106 daltons. After denaturation, the in vitro labelled DNA molecules sediment at about 2–5 S, corresponding to a single-strand molecular weight of 1 × 104 – 7 × 104 daltons, which is about one hundred times less than the observed size of the denatured in vivo labelled molecules.

scholarly journals Mitochondrial DNA Instability and Peri-Implantation Lethality Associated with Targeted Disruption of Nuclear Respiratory Factor 1 in Mice

2001 ◽  
Vol 21 (2) ◽  
pp. 644-654 ◽  
Author(s):  
Lei Huo ◽  
Richard C. Scarpulla
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Dna ◽  
Staining Intensity ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1 ◽  
Mitochondrial Dna Instability ◽  
Neomycin Cassette ◽  
Nuclear Dna Fragmentation

ABSTRACT In vitro studies have implicated nuclear respiratory factor 1 (NRF-1) in the transcriptional expression of nuclear genes required for mitochondrial respiratory function, as well as for other fundamental cellular activities. We investigated here the in vivo function of NRF-1 in mammals by disrupting the gene in mice. A portion of the NRF-1 gene that encodes the nuclear localization signal and the DNA-binding and dimerization domains was replaced through homologous recombination by a β-galactosidase–neomycin cassette. In the mutant allele, β-galactosidase expression is under the control of the NRF-1 promoter. Embryos homozygous for NRF-1 disruption die between embryonic days 3.5 and 6.5. β-Galactosidase staining was observed in growing oocytes and in 2.5- and 3.5-day-old embryos, demonstrating that the NRF-1 gene is expressed during oogenesis and during early stages of embryogenesis. Moreover, the embryonic expression of NRF-1 did not result from maternal carryover. While most isolated wild-type and NRF-1+/− blastocysts can develop further in vitro, the NRF-1−/− blastocysts lack this ability despite their normal morphology. Interestingly, a fraction of the blastocysts from heterozygous matings had reduced staining intensity with rhodamine 123 and NRF-1−/− blastocysts had markedly reduced levels of mitochondrial DNA (mtDNA). The depletion of mtDNA did not coincide with nuclear DNA fragmentation, indicating that mtDNA loss was not associated with increased apoptosis. These results are consistent with a specific requirement for NRF-1 in the maintenance of mtDNA and respiratory chain function during early embryogenesis.

Effects of Unfractionated and Low Molecular Weight Heparins on Platelet Thromboxane Biosynthesis “In Vivo”

1994 ◽  
Vol 72 (06) ◽  
pp. 942-946 ◽  
Author(s):  
Raffaele Landolfi ◽  
Erica De Candia ◽  
Bianca Rocca ◽  
Giovanni Ciabattoni ◽  
Armando Antinori ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Low Molecular Weight Heparin ◽  
Primary Source ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
Heparin Administration ◽  
To Receive

SummarySeveral “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.

Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

1964 ◽  
Vol 12 (01) ◽  
pp. 232-261 ◽  
Author(s):  
S Sasaki ◽  
T Takemoto ◽  
S Oka
Keyword(s):  
Molecular Weight ◽  
Dextran Sulfate ◽  
Anticoagulant Activity ◽  
Molecular Structures ◽  
Severe Reaction ◽  
Clinical Use ◽  
Molecular Weights ◽  
Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

Neutralization of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin by Protamine Sulfate in Rats

1986 ◽  
Vol 56 (03) ◽  
pp. 318-322 ◽  
Author(s):  
V Diness ◽  
P B Østergaard
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Thrombus Formation ◽  
Experimental Models ◽  
Protamine Sulfate ◽  
Low Molecular Weight ◽  
Antithrombotic Effect ◽  
Higher Doses

SummaryThe neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized.In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.

Studies on Metabolism of Urokinase and Mechanism of Thrombolysis by Urokinase

1979 ◽  
Vol 42 (03) ◽  
pp. 885-894 ◽  
Author(s):  
Tatsuo Ueno ◽  
Norio Kobayashi ◽  
Tadashi Maekawa
Keyword(s):  
Molecular Weight ◽  
Plasma Clearance ◽  
Radioactive Material ◽  
Optimal Dosage ◽  
Plasma Clot ◽  
Optimal Dosage Regimen ◽  
Arterial Thrombus ◽  
Contact Experiment

SummaryPharmacokinetics of intravenously injected 125I-labeled urokinase (125I-UK) of a molecular weight of 33,000 daltons in normal rabbits and patients with various diseases were investigated. The plasma clearance of 125I-UK in rabbits was described by a biexponential curve within six hours with a half-life of 8 minutes, 2.3 hours, respectively. The radioactivity in the liver and kidneys 15 minutes after iv injection with 125I-UK was 9.6% and 14.0% of the radioactivity injected, respectively. Approximately 80% of the total radioactive material injected was excreted in the urine in 18 hours. No increase in activator activity in the urine was observed after a large amount of UK injection. Activity uptake of 125I-UK by experimentally induced arterial thrombus was little. Lysis of the stasis thrombus was produced by injecting 7.5 × 104 IU of UK in only one out of 8 rabbits. In vitro contact experiment revealed that transfer of 125I-UK to plasma clot is slow (24 hours for 10% of 125I-UK by plasma clot). In 4 patients plasma clearance of 125I-UK was essentially similar to that in rabbits. From the results obtained optimal dosage regimen of UK administration for complete thrombolysis in vivo was discussed.

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1356-1363 ◽  
Author(s):  
Barbara P. Schick ◽  
David Maslow ◽  
Adrianna Moshinski ◽  
James D. San Antonio
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Tandem Repeats ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

scholarly journals Novel Role for Albumin in Innate Immunity: Serum Albumin Inhibits the Growth of Blastomyces dermatitidis Yeast Form In Vitro

2003 ◽  
Vol 71 (11) ◽  
pp. 6648-6652 ◽  
Author(s):  
Steven Giles ◽  
Charles Czuprynski
Keyword(s):  
Molecular Weight ◽  
Inhibitory Activity ◽  
Mammalian Species ◽  
Blastomyces Dermatitidis ◽  
Low Molecular Weight ◽  
Rat Serum ◽  
Yeast Form ◽  
Domain Iii

ABSTRACT In this study we found that serum inhibitory activity against Blastomyces dermatitidis was principally mediated by albumin. This was confirmed in experiments using albumin from several mammalian species. Analbuminemic rat serum did not inhibit B. dermatitidis growth in vivo; however, the addition of albumin restored inhibitory activity. Inhibitory activity does not require albumin domain III and appears to involve binding of a low-molecular-weight yeast-derived growth factor.

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