scholarly journals Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography.

1979 ◽  
Vol 82 (2) ◽  
pp. 485-493 ◽  
Author(s):  
R Hand ◽  
J R Gautschi
Keyword(s):  
Replication Fork ◽  
Radioactive Tracer ◽  
Cell System ◽  
Unit Size ◽  
Brij 58 ◽  
Thymidine Triphosphate ◽  
Average Size ◽  
Made In

We have used fiber autoradiography to examine the DNA product made in vitro in a lysed cell system. CHO cells were treated with 0.01% Brij-58 and the lysates were incubated at 30 degrees C in a complete reaction mixture for in vitro DNA synthesis with [3H]thymidine triphosphate ([3H]TTP) as the radioactive tracer. Fiber autoradiograms prepared from the DNA showed that it was synthesized on tandemly arranged replication units that were of average size of 20 micrometers, very similar to the size of units found in vivo. The rate of replication fork movement was 25--50% of the in vivo rate. More than 80% of forks stopped functioning by 15 min, and 95% stopped by 60 min. This suggests that synthesis is halted by premature terminations. Evidence for new initiations was provided by replication units with labeled origins in DNA synthesized in an in vitro reaction in which radioactivity was omitted for the first 10 min of incubation. This, plus the observations that the distance between initiation points (replication unit size) is not increased and that premature termination accounts largely for the cessation of synthesis, suggest that significant initiation takes place in this in vitro replication system.

scholarly journals Features of hemostatic activity of 2-[3-methyl-1-h-propyl-7-(1,1-dioxothiethanyl-3) xantinyl-8-thio] acetic acid cyclohexilammonium salt

2013 ◽  
Vol 94 (4) ◽  
pp. 549-552
Author(s):  
F Kh Kamilov ◽  
G A Timirkhanova ◽  
A I Samorodova ◽  
F A Khaliullin ◽  
R A Gubaeva
Keyword(s):  
Acetic Acid ◽  
Male Rats ◽  
Control Group ◽  
Hemostatic Activity ◽  
Platelet Aggregates ◽  
Aggregation Parameters ◽  
Average Size ◽  
Made In

Aim. To study the systemic hemostatic activity of firstly synthesized 2-[3-methyl-1-h-propyl-7-(1,1-dioxotiethanyl-3) xantinyl-8-thio] acetic acid cyclohexilammonium salt in vitro and in vivo. Methods. Experiments in vitro using the blood samples form healthy male donors, and in vivo on male rats by intraperitoneal injection of the equimolar concentrations of researched substances. The effect of the firstly synthesized xantine and etamsylate derivative on the platelet functional activity in vitro and in vivo was studied using a platelet aggregation laser analyzer «Biola 230 LA» (Russia). 20 mkg/ml of adenosinediphosphate and 5 mg/ml of collagen (produced by «Technologia-Standart» company, Russia) were used as an aggregation inducers. Aggregation was analyzed using the AGGR software. General aggregation parameters, maximal aggregation value, maximal aggregation speed, average size of platelet aggregates were analyzed. Experimental assessment of specific systemic hemostatic activity in vivo was made in accordance with the parenchymal hemorrhage model on mature male rats. Coagulation time and blood loss volume were registered. Results. 2-[3-Methyl-1-h-propyl-7-(1,1-dioxotiethanyl-3) xantinyl-8-thio] acetic acid cyclohexilammonium salt showed a pro-aggregative effect both in vitro and in vivo. 2-[3-Methyl-1-h-propyl-7-(1,1-dioxotiethanyl-3) xantinyl-8-thio] acetic acid cyclohexilammonium salt pro-aggregative effect which was observed both in vitro and in vivo increased the systemic hemostatic activity, exceeding the results of the control group and etamsylate group. Conclusion. The findings reveal potentially high systemic hemostatic activity of 2-[3-methyl-1-h-propyl-7-(1,1-dioxotiethanyl-3) xantinyl-8-thio] acetic acid cyclohexilammonium salt and confirm the need for further studies of this compound and its equivalents in order to create highly effective selective hemostasis correctors on their basis.

scholarly journals Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

1986 ◽  
Vol 6 (7) ◽  
pp. 2663-2673 ◽  
Author(s):  
M C Strobel ◽  
J Abelson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trna Gene ◽  
Nuclear Extract ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Specific Sequence ◽  
Made In

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

scholarly journals An In Vitro–In Vivo Simulation Approach for the Prediction of Bioequivalence

Materials ◽  
2021 ◽  
Vol 14 (3) ◽  
pp. 555
Author(s):  
Marilena Vlachou ◽  
Vangelis Karalis
Keyword(s):  
Mathematical Description ◽  
Development Process ◽  
Bioequivalence Study ◽  
In Vitro Dissolution ◽  
Monte Carlo Techniques ◽  
In Vivo Kinetics ◽  
Probability Of Success ◽  
Made In

The aim of this study was to develop a new in vitro–in vivo simulation (IVIVS) approach in order to predict the outcome of a bioequivalence study. The predictability of the IVIVS procedure was evaluated through its application in the development process of a new generic product of amlodipine/irbesartan/hydrochlorothiazide. The developed IVIVS methodology is composed of three parts: (a) mathematical description of in vitro dissolution profiles, (b) mathematical description of in vivo kinetics, and (c) development of joint in vitro–in vivo simulations. The entire programming was done in MATLAB® and all created scripts were validated through other software. The IVIVS approach can be implemented for any number of subjects, clinical design, variability and can be repeated for thousands of times using Monte Carlo techniques. The probability of success of each scenario is recorded and finally, an overall assessment is made in order to select the most suitable batch. Alternatively, if the IVIVS shows reduced probability of BE success, the R&D department is advised to reformulate the product. In this study, the IVIVS approach predicted successfully the BE outcome of the three drugs. During the development of generics, the IVIVS approach can save time and expenses.

Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast

1985 ◽  
Vol 5 (5) ◽  
pp. 1093-1099
Author(s):  
R J Schmidt ◽  
N W Gillham ◽  
J E Boynton
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Apparent Molecular Weight ◽  
Protein L ◽  
Mature Form ◽  
Chloroplast Protein Synthesis ◽  
Made In

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.

scholarly journals A Selective TSH Receptor Antagonist Inhibits Stimulation of Thyroid Function in Female Mice

Endocrinology ◽  
2014 ◽  
Vol 155 (1) ◽  
pp. 310-314 ◽  
Author(s):  
Susanne Neumann ◽  
Eshel A. Nir ◽  
Elena Eliseeva ◽  
Wenwei Huang ◽  
Juan Marugan ◽  
...  
Keyword(s):  
Small Molecule ◽  
Sodium Iodide ◽  
Cell System ◽  
Tsh Receptor ◽  
Free T4 ◽  
Serum Free ◽  
Stimulation Of ◽  
Lowered Serum

Because the TSH receptor (TSHR) plays an important role in the pathogenesis of thyroid disease, a TSHR antagonist could be a novel treatment. We attempted to develop a small molecule, drug-like antagonist of TSHR signaling that is selective and active in vivo. We synthesized NCGC00242364 (ANTAG3) by chemical modification of a previously reported TSHR antagonist. We tested its potency, efficacy, and selectivity in a model cell system in vitro by measuring its activity to inhibit stimulation of cAMP production stimulated by TSH, LH, or FSH. We tested the in vivo activity of ANTAG3 by measuring its effects to lower serum free T4 and thyroid gene expression in female BALB/c mice continuously treated with ANTAG3 for 3 days and given low doses of TRH continuously or stimulated by a single administration of a monoclonal thyroid-stimulating antibody M22. ANTAG3 was selective for TSHR inhibition; half-maximal inhibitory doses were 2.1 μM for TSHR and greater than 30 μM for LH and FSH receptors. In mice treated with TRH, ANTAG3 lowered serum free T4 by 44% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 75% and 83%, respectively. In mice given M22, ANTAG3 lowered serum free T4 by 38% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 73% and 40%, respectively. In conclusion, we developed a selective TSHR antagonist that is effective in vivo in mice. This is the first report of a small-molecule TSHR antagonist active in vivo and may lead to a drug to treat Graves' disease.

Hemodynamics in the abdominal aorta: a comparison of in vitro and in vivo measurements

1994 ◽  
Vol 76 (4) ◽  
pp. 1520-1527 ◽  
Author(s):  
J. E. Moore ◽  
S. E. Maier ◽  
D. N. Ku ◽  
P. Boesiger
Keyword(s):  
Abdominal Aorta ◽  
Velocity Profiles ◽  
Flow Reversal ◽  
Infrarenal Aorta ◽  
Complex Flow ◽  
Flow Measurements ◽  
In Vivo Measurements ◽  
Made In

In vivo measurements of blood velocity profiles are difficult to obtain and interpret, since the parameters that govern the normally highly complex flow situation may not be fully quantified or understood at the time of measurement. In vitro flow models have been used often to better understand vascular hemodynamics. The assumptions made in the design of these models limit the applicability of the results. In this study, in vitro flow measurements made in a carefully designed model of the abdominal aorta were compared with in vivo measurements obtained with magnetic resonance imaging. In the suprarenal aorta, the velocity profiles were mostly forward and axisymmetric in both the in vitro and in vivo cases. In the infrarenal aorta, there was extensive flow reversal noted near the posterior wall in both cases. In the aortic bifurcation, two peaks of flow reversal were noted near the lateral posterior walls, and M-shaped velocity profiles were observed in late diastole. The in vitro and in vivo measurements exhibited good qualitative agreement. The in vitro model was accurate in modeling the in vivo hemodynamics of the abdominal aorta. The complex phenomena observed in vivo were explained on the basis of knowledge gained from the in vitro study.

scholarly journals The research significance of concomitant use of CAR-CD138-NK and CAR-CD19-NK to target multiple myelomas

2018 ◽  
Vol 16 ◽  
pp. 205873921878896 ◽  
Author(s):  
Songbo Zhao ◽  
Zhichao Han ◽  
Cheng Ji ◽  
Gangli An ◽  
Huimin Meng ◽  
...  
Keyword(s):  
Nk Cells ◽  
Lymphoblastic Leukemia ◽  
Small Subset ◽  
Novel Drugs ◽  
Prevalent Disease ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Multiple Myelomas ◽  
Made In

Multiple myeloma (MM) is a type of cancer characterized by abnormal proliferation of clonal cells; it is the very dangerous and highly prevalent disease. Although significant progress has been made in clinical research, especially with novel drugs such as bortezomib, lenalidomide, and carfilzomib, most of the patients with MM still suffer from often fetal relapses due to drug resistance. In this study, we aimed to develop immune cells that could specifically target and destroy MM cells. Chimeric antigen receptor–modified NK-92 (CAR-NK92) cells have been very effective against B-cell acute lymphoblastic leukemia (B-ALL); as MM shows high expression of CD138, we constructed CD138-directed CAR-NK-92MI cells (CAR-CD138). It 2is reported that there is a small subset of CD138–/CD19+ MM cells showing, to some extent, stem cell qualities. We therefore generated the CD19-directed CAR-NK-92MI cells (CAR-CD19) as well. These two CAR-NK cells showed strong in vitro biological activity in specifically killing target tumor cells. Thus, the concomitant use of these CAR-NK cells may achieve excellent results in vivo.

Recent findings on oogenesis of Drosophila melanogaster

Development ◽  
1977 ◽  
Vol 38 (1) ◽  
pp. 125-138
Author(s):  
F. Giorgi ◽  
J. Jacob
Keyword(s):  
Drosophila Melanogaster ◽  
Fat Body ◽  
Radioactive Tracer ◽  
Superficial Layer ◽  
Ruthenium Red ◽  
Time Interval ◽  
Intracellular Location ◽  
Short Time

Vitellogenic ovaries from Drosophila melanogaster flies have been exposed, either in in vivo or in vitro conditions, to various extracellular tracers in an attempt to determine the possible route of entry of the yolk precursors. Ruthenium red and lanthanum nitrate have been shown to gain access to the oocyte surface by initially passing through the intercellular spaces of the follicle layer. Both these tracers, however, never attain an intracellular location within any of the cells forming the ovarian chamber. Colloidal Thorotrast when injected into adult females has never been detected within any of the ovarian chambers examined, irrespective of their stage. Vitellogenic oocytes exposed to peroxidase in in vivo conditions exhibit the oolemma and all the structural elements present in the cortical ooplasm well labelled within a very short time after the injection. Moreover, with gradually increasing exposure times to peroxidase, the labelled yolk platelets increase progressively in number. At each time interval after the injection, the label over the yolk platelets remains restricted to the superficial layer and never gets into the associated body. The pattern of tritiated lysine incorporation into vitellogenic oocytes has been studied over a period of 20 h. A few hours after injection of the radioactive tracer, the silver grains located over the ooplasm appear distributed at random. A predominant labelling of the yolk platelets as compared to the rest of the ooplasm, becomes evident only with a 6 h delay since the time of injection. When analysed by electrophoresis and isolectrofocusing, the vitellogenic ovary is seen to exhibit a number of protein bands which are common to those of other tissues as, for instance, haemolymph and fat body. The evidence obtained in the present study is discussed in relation to the hypothesis of an extraovarian origin of the yolk precursors and their sequestration into forming yolk platelets.

Kinematic models of the buccal mass of Aplysia californica.

1998 ◽  
Vol 201 (10) ◽  
pp. 1563-1583 ◽  
Author(s):  
R F Drushel ◽  
D M Neustadter ◽  
I Hurwitz ◽  
P E Crago ◽  
H J Chiel
Keyword(s):  
Neural Control ◽  
Kinematic Model ◽  
Aplysia Californica ◽  
Relative Location ◽  
Marine Mollusc ◽  
Buccal Mass ◽  
Kinematic Models ◽  
Made In

The feeding behavior of the marine mollusc Aplysia californica is an intensively studied model system for understanding the neural control of behavior. Feeding movements are generated by contractions of the muscles of the buccal mass. These muscles are internal and cannot be visualized during behavior. In order to infer the movements of the muscles of the buccal mass, two kinematic models were constructed. The first kinematic model assumed that the complex consisting of the pincer-like radula and the underlying odontophore was spherical in shape. In this model, the radula/odontophore was moved anteriorly or posteriorly and the more superficial buccal muscles (I1/I3 and I2) were fitted around it. Although the overall buccal mass shapes predicted by this model were similar to those observed in vivo during protraction, the shapes predicted during retraction were very different. We therefore constructed a second kinematic model in which the shape of the radula/odontophore was based on the shapes assumed by those structures in vitro when they were passively forced into protraction, rest or retraction positions. As each of these shapes was rotated, the second kinematic model generated overall shapes of the buccal mass that were similar to those observed in vivo during swallowing and tearing, and made predictions about the antero-posterior length of the buccal mass and the relative location of the lateral groove. These predictions were consistent with observations made in vivo and in vitro. The kinematic patterns of intrinsic buccal muscles I1 and I2 in vivo were estimated using the second model. Both models make testable predictions with regard to the functions and neural control of intrinsic buccal muscles I2 and I3.

Prolonged Anti-Inflammatory Activity of Topical Melatonin by Niosomal Encapsulation

2014 ◽  
Vol 902 ◽  
pp. 70-75 ◽  
Author(s):  
Aroonsri Priprem ◽  
Vassana Netweera ◽  
Pramote Mahakunakorn ◽  
Nutjaree Pratheepawanit Johns ◽  
Jeffrey Roy Johns
Keyword(s):  
Skin Permeation ◽  
Entrapment Efficiency ◽  
Rapid Decline ◽  
Tail Flick ◽  
Tail Flick Test ◽  
Topical Gel ◽  
Anti Inflammatory ◽  
Average Size

Melatonin, encapsulated and non-encapsulated, in a topical gel, was comparatively investigated for its in vitro permeation and in vivo anti-inflammatory properties. An average size of the melatonin-encapsulated niosomes of 197 nm with a zeta potential of-78.8 mV and an entrapment efficiency of 92.7% was incorporated into a gel base. In vitro skin permeation of the same gel base incorporated with non-encapsulated melatonin or melatonin niosomes at 5% was comparatively evaluated through porcine skin using Franz diffusion cells and analyzed by spectroflurometry at λex 278 and λem 348 nm. From the same gel base, the permeation rate of non-encapsulated melatonin was about 2.5 times greater than that of melatonin-encapsulated niosomes. In comparison to piroxicam gel and hydrocortisone cream used as the positive controls, topical applications of melatonin and melatonin niosome gels tested in croton oil-induced ear edema in mice suggested that its anti-inflammatory activities were prolonged by the niosomal encapsulation. Similarly, analgesic effect of melatonin was prolonged by niosomal encapsulation using tail flick test in mice. Therefore, its immediate permeation through the skin was retarded by niosomal encapsulation which could also prolong its rapid decline in exerting anti-inflammatory and analgesic activities in vivo.

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