Direct exposure of mouse embryonic limb-buds to 5-bromodeoxyuridine in vitro and its effect on chondrogenesis: increasing resistance to the analog at successive stages of development

Development ◽  
1976 ◽  
Vol 36 (3) ◽  
pp. 639-652
Author(s):  
Narsingh D. Agnish ◽  
Devendra M. Kochhar
Keyword(s):  
Developmental Stage ◽  
Prolonged Exposure ◽  
Inhibitory Effect ◽  
Limb Buds ◽  
Embryonic Mouse ◽  
Continuous Growth ◽  
Somite Stage ◽  
Stage Of Development ◽  
Short Period

The inhibitory effect of 5-bromodeoxyuridine (BudR) — an analog of thymidine — on embryonic mouse limb-buds was studied in vitro employing an organ-culture system. The effect was found to be dose-related and also depended on the developmental stage of the donor embryos. Limbs at an early stage of development (early llth-day embryos, somite stage 26–29) were extremely sensitive to the analog. Treatment with low levels (2–4µml) and for a relatively short period of time in culture (2–3 days) completely and irreversibly suppressed chondrogenesis in these explants. Limbs from older embryos (somite stage 40 and up) were found to be much less sensitive to the inhibitory effect of the drug; a prolonged exposure to a much higher dose (100–150 µml) resulted in an incomplete suppression of chondrogenesis. Only a 20% inhibition was observed in the cultures of limbs from mid-13th-day mouse embryos. After continuous growth in vitro, the limbs became progressively resistant to the analog and towards the end of the culture period had become refractory to the drug. The time of complete insensitivity appeared earlier in the cultures of the limbs taken from older embryos than in the explants of younger limbs. These studies show that as limbs continue to differentiate in vivo or in vitro, they become increasingly resistant to the inhibitory effect of BudR in at least as far as the effect on the process of chondrogenesis is concerned. It is suggested that the relative sensitivity or insensitivity to the inhibitory effect of BudR may prove to be a useful parameter in evaluating the developmental stage of an organ.

scholarly journals Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hayato Mizuta ◽  
Koutaroh Okada ◽  
Mitsugu Araki ◽  
Jun Adachi ◽  
Ai Takemoto ◽  
...  
Keyword(s):  
Gene Rearrangement ◽  
Kinase Inhibitors ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Resistance Mutations ◽  
Lung Cancer Patients ◽  
Short Period ◽  
Tki Resistance

AbstractALK gene rearrangement was observed in 3%–5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI–resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.

Regulation of Trypanosoma cruzi infectivity by alpha- and beta-adrenergic agonists: desensitization produced by prolonged treatments or increasing agonist concentrations

Parasitology ◽  
1990 ◽  
Vol 100 (3) ◽  
pp. 429-434 ◽  
Author(s):  
A. Ayala ◽  
F. Kierszenbaum
Keyword(s):  
Trypanosoma Cruzi ◽  
Prolonged Exposure ◽  
Inhibitory Effect ◽  
Tissue Level ◽  
Adrenergic Agonists ◽  
Adrenergic Agonist ◽  
Beta Adrenergic ◽  
Beta Adrenergic Agonists ◽  
Alpha Adrenergic Agonist

SUMMARYWe previously reported that blood forms of Trypanosoma cruzi express alpha- and beta-adrenergic receptors and that binding of specific agonists to these receptors modifies the infective capacity of the parasite in vitro. The present study has revealed that the inhibitory effect of the beta-adrenergic agonist L-isoproterenol and the stimulatory effect of the alpha-adrenergic agonist L-phenylephrine are not produced when the parasite is subjected to prolonged exposure to otherwise effective doses of these agonists or when supraoptimal doses of these agonists are used. We refer to these phenomena as ‘desensitization’ because of their analogy with vertebrate cells becoming desensitized by prolonged exposure to, or relatively high concentrations of, adrenergic agonists. At a constant agonist concentration, T. cruzi desensitization was time-dependent and, when the time of parasite treatment with the agonists was not changed, the higher concentrations of the agonist tested were the most effective in producing desensitization. The reduced infectivity resulting from treatment with optimal doses of L-isoproterenol was accompanied by elevated levels of cyclic adenosine mono- phosphate (cAMP) which were not detectable when L-isoproterenol concentrations producing desensitization were used. This finding implicated cAMP as a likely second signal in the inhibitory mechanisms of this agonist. No significant change in cAMP was detectable in parasites treated with L-phenylephrine, leaving open the question about how optimal doses of this alpha-adrenergic agonist enhance T. cruzi infectivity. Parasite responsiveness to alpha- and beta-adrenergic agonists as well as the desensitization effects define a system which regulates infectivity and could be modified at the host tissue level by naturally occurring agonists.

Glucocorticoid modulation of growth hormone secretion in vitro. Evidence for a biphasic effect on GH-releasing hormone mediated release

1987 ◽  
Vol 114 (4) ◽  
pp. 465-469 ◽  
Author(s):  
Gian Paolo Ceda ◽  
Robert G. Davis ◽  
Andrew R. Hoffman
Keyword(s):  
Growth Hormone ◽  
Prolonged Exposure ◽  
Hormone Secretion ◽  
Growth Hormone Secretion ◽  
Inhibitory Effect ◽  
Pituitary Cells ◽  
Gh Secretion ◽  
Glucocorticoid Action

Abstract. Glucocorticoids have been shown to have both stimulatory and suppressive effects on GH secretion in vitro and in vivo. In order to study the kinetics of glucocorticoid action on the somatotrope, cultured rat pituitary cells were exposed to dexamethasone for varying periods of time. During short-term incubations (≤ 4 h), dexamethasone inhibited GHRH and forskolin-elicited GH secretion, but during longer incubation periods, the glucocorticoid enhanced both basal and GHRH-stimulated GH release. The inhibitory effect of brief dexamethasone exposure was also seen in cells which previously had been exposed to dexamethasone. In addition, growth hormone secretion from cultured rat and human somatotropinoma cells was inhibited by a brief exposure to dexamethasone. Thus, the nature of glucocorticoid action on the isolated cultured somatotrope is biphasic, with brief exposure inhibiting, and more prolonged exposure stimulating GH secretion.

scholarly journals Leptin has concentration and stage-dependent effects on embryonic development in vitro

Reproduction ◽  
2006 ◽  
Vol 132 (2) ◽  
pp. 247-256 ◽  
Author(s):  
Muren Herrid ◽  
Van Ly Nguyen ◽  
Geoff Hinch ◽  
James R McFarlane
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Culture Medium ◽  
Leptin Receptor ◽  
Inhibitory Effect ◽  
Differential Sensitivity ◽  
Cell Stage ◽  
Paracrine Signalling ◽  
Stage Of Development

There is accumulating evidence that leptin may be directly involved in pre-implantation embryonic development, however, it is unclear whether there is a concentration and stage-dependent regulatory pattern. In this study, the addition of 10 ng/ml human recombinant leptin to the culture medium significantly increased the percentage of two-cell mouse embryos that developed into blastocysts and hatched blastocysts, whereas in the presence of 100 ng/ml leptin, the development rate was significantly inhibited. The total cell numbers in the hatched blastocysts were significantly higher in the presence of 10 ng/ml leptin compared with controls and higher concentrations. The differential sensitivity to leptin was found to vary among embryos at different stages of development. Supplementation of leptin (10 ng/ml) to culture medium at two- to eight-cell stages resulted in a consistent stimulatory effect on embryo development. Most interestingly, the inhibitory effect of high leptin concentration (100 ng/ml) on embryo development was diminished when it was added to the culture medium at the eight-cell stage of development. The concentration-dependent regulation pattern was confirmed using sheep embryos, under similar conditions although sheep embryos appeared to be more sensitive in responding to leptin. Having established the effect of exogenous leptin on embryo development, the expression pattern of leptin and its receptors were also investigated. Leptin mRNA was not detected in mouse two-, four-, eight-cell and blastocyst stage embryos, whereas three isoforms of leptin receptor (Ob-Ra, Ob-Rb and Ob-Re) were identified in these cells, indicating that leptin is likely to modulate embryo development via a paracrine signalling system.

scholarly journals Young but not defenceless: antifungal activity during embryonic development of a social insect

2020 ◽  
Vol 7 (8) ◽  
pp. 191418
Author(s):  
Erin L. Cole ◽  
Haley Bayne ◽  
Rebeca B. Rosengaus
Keyword(s):  
Antifungal Activity ◽  
Developmental Stage ◽  
Immature Stage ◽  
Selective Pressures ◽  
Fungistatic Activity ◽  
Stage Of Development ◽  
Zootermopsis Angusticollis ◽  
Endogenous Proteins ◽  
Dampwood Termite

Termites live in environments heavily colonized by diverse microorganisms, including pathogens. Eggs laid within the nest are likely to experience similar pathogenic pressures as those experienced by older nest-mates. Consequently, eggs may be under selective pressures to be immune-competent. Through in vitro experiments using developing embryos of the dampwood termite, Zootermopsis angusticollis , we tested the ontogeny, location and strength of their antifungal activity against the fungus, Metarhizium brunneum . Exterior washes of the chorion (extra-chorionic) and components within the chorion (intra-chorionic) were incubated with fungal conidia, which were then scored for viability. The fungistatic activity was location and developmental stage dependent. Extra-chorionic washes had relatively weak antifungal activity. Intra-chorionic homogenates were highly antifungal, exhibiting increased potency through development. The positive correlation between intra-chorionic fungistasis and developmental stage is probably due to the expression of endogenous proteins during embryogenesis. Boiling of both the extra-chorionic washes and the intra-chorionic contents rescued conidia viability, indicating the antifungal agent(s) is (are) heat-sensitive and probably proteinaceous. This study is the first to address embryonic antifungal activity in a hemimetabolous, eusocial taxon. Our results support the hypothesis that microbes have been significant agents of selection in termites, fostering the evolution of antifungal properties even in the most immature stage of development.

Transport of the cationic fluorochrome rhodamine 123 in an insect's Malpighian tubule: indications of a reabsorptive function of the secondary cell type

1992 ◽  
Vol 101 (2) ◽  
pp. 349-361
Author(s):  
W. Meulemans ◽  
A. De Loof
Keyword(s):  
Lucifer Yellow ◽  
Malpighian Tubule ◽  
Inhibitory Effect ◽  
Malpighian Tubules ◽  
Rhodamine 123 ◽  
Primary Cells ◽  
Short Period ◽  
Selective Accumulation

The pathway of rhodamine 123 was examined after injection into Sarcophaga flies and after in vitro labeling of the Malpighian tubules. After in vitro labeling the primary cells only retained this potential-sensitive dye for a short period while all secondary cells accumulated the dye from the tubule lumen. In vivo the secondary cells also accumulated rhodamine 123 from the lumen, but the primary cells in the distal parts of all four tubules retained the dye for prolonged periods. This was most pronounced in the distal part of the anterior Malpighian tubules, where rhodamine 123 was eventually precipitated on the luminal concretions. Rhodamine 123 initially accumulated in the secondary cell mitochondria and eventually in intensely fluorescing vesicles, probably lysosomes. No evidence for endocytotic processes from the lumen was found using Lucifer Yellow CH, fluorescent dextrans and fluorescent albumin. Prior incubation with the ionophores valinomycin, nigericin, CCCP (all 1 micrograms/ml), dinitrophenol (1 mM) and NaN3 (10(−2) M) inhibited the selective accumulation of rhodamine 123 to a large extent while monensin (1–5 micrograms/ml) showed little inhibitory effect. Furthermore, only cationic and no anionic or neutral dyes were accumulated by the secondary cells. In the fleshfly Calliphora and the fruitfly Drosophila, the dye rhodamine 123 also selectively accumulated in the secondary cells, as well in vitro as in vivo.

A kinetic study of the effects of δ-aminolevulinic acid upon the synthesis of embryonic and fetal hemoglobins in the blood islands of the developing chick blastodisc

1970 ◽  
Vol 48 (4) ◽  
pp. 400-406 ◽  
Author(s):  
S. D. Wainwright ◽  
Lillian K. Wainwright
Keyword(s):  
Aminolevulinic Acid ◽  
Fetal Hemoglobin ◽  
Primitive Streak ◽  
Stages Of Development ◽  
In Ovo ◽  
Rapid Synthesis ◽  
Somite Stage ◽  
Stage Of Development ◽  
Solid Media

Chick blastodiscs contained and synthesized a functional (embryonic) hemoglobin as early as the stage of the definitive primitive streak. The rate of hemoglobin synthesis in ovo rose dramatically at about the stage of the 3-somite embryo.Embryos explanted onto solid media at the 3-somite stage of development continued to synthesize hemoglobin in vitro for at least 12 h. The rate of synthesis rose markedly at the 6-somite stage of development, coincident with the onset of rapid synthesis of fetal hemoglobin. A supplement of exogenous aminolevulinic acid markedly stimulated the synthesis of hemoglobin before the 6-somite stage of development, but had little or no effect thereafter. These responses were obtained on both minimal and rich media. Levels of hemoglobin formed were characteristic of the final stages of development attained, regardless of initial stage of development, medium used, or time of incubation required to attain the final level of development.A new regulatory process resulting in elimination of a requirement for continuous presence of egg homogenate for maximal rates of hemoglobin was revealed. This took place between the 6- and 8-somite stages of development at the time of onset of rapid synthesis of fetal hemoglobin.

scholarly journals Hst-1 (FGF-4) antisense oligonucleotides block murine limb development.

1995 ◽  
Vol 130 (4) ◽  
pp. 997-1003 ◽  
Author(s):  
T Ochiya ◽  
H Sakamoto ◽  
M Tsukamoto ◽  
T Sugimura ◽  
M Terada
Keyword(s):  
Organ Culture ◽  
Limb Development ◽  
Culture System ◽  
Inhibitory Effect ◽  
Defined Medium ◽  
Limb Bud ◽  
Embryonic Mouse ◽  
Organ Culture System ◽  
Mouse Limb

The initiation of limb development depends on the site specific proliferation of the mesenchyme by the signals from the apical ectodermal ridge (AER) in embryonic mouse. We have previously reported that the local expression of Hst-1/Fgf-4 transcripts in AER of the mouse limb bud is developmentally regulated, expressed at 11 and 12 days post coitus (p.c.) embryo. In an effort to further understand the role of Hst-1/FGF-4 in mouse limb development, an antisense oligodeoxynucleotides (ODNs) study was performed. We first established a novel organ culture system to study mouse limb development in vitro. This system allows mouse limb bud at 9.5-10-d p.c. embryo, when placed on a sheet of extracellular matrix in a defined medium, to differentiate into a limb at 12.5-d p.c. embryo within 4.5 d. Using this organ culture system, we have shown that exposure of 9.5-10-d p.c. embryonal limb bud explants to antisense ODNs of Hst-1/FGF-4 blocks limb development. In contrast, sense and scrambled ODNs have no inhibitory effect on limb outgrowth, suggesting that Hst-1/FGF-4 may work as a potent inducing factor for mouse limb development.

Development of four-cell hamster embryos to the blastocyst stage in vitro and its regulation by components of the culture milieu

1990 ◽  
Vol 2 (1) ◽  
pp. 1 ◽  
Author(s):  
H Monis ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Co2 Concentration ◽  
Inhibitory Effect ◽  
Defined Medium ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Stage Of Development

Constituents of the culture milieu known to influence development of hamster 2-cell and 8-cell embryos were examined for effects on the 4-cell stage. Embryos were collected at the mid 4-cell stage (approx. 45-46 h after egg activation) from superovulated females and cultured for 24 h in a chemically defined medium (TLP-PVA). As with the 2-cell stage, inorganic phosphate (Pi) strongly inhibited development of 4-cell embryos, although some (14%) were able to reach the 8-cell stage or further in the presence of Pi. However, unlike 2-cell embryos, no significant inhibitory effect of glucose on development of 4-cell embryos was found. In the absence of glucose and Pi, development of 4-cell embryos was sensitive to amino acids in the medium: the mean cell number was increased using 21 amino acids compared with 4 amino acids, similarly to the 2-cell stage; however, late blastocyst development (blastocele formation) from 4-cell embryos was reduced using 21 compared with 4 amino acids, as with 8-cell embryos. Similarly to the 2-cell and 8-cell stages, raising the CO2 concentration from 5% to 10% in the gas atmosphere for culture increased the percentage of total blastocysts developing from the 4-cell stage, but did not affect the proportions of late-stage blastocysts. These data show that 4-cell-stage hamster embryos are somewhat similar to 2-cell embryos with respect to the regulation of development by constituents of the culture milieu, but, to some extent, the 4-cell embryo is a transitional stage of development.

scholarly journals 252A COMPARATIVE EXPRESSION ANALYSIS OF GENES IN PREIMPLANTATION DEVELOPMENTAL STAGES OF BOVINE EMBRYOS PRODUCED IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 246 ◽  
Author(s):  
D. Tesfaye ◽  
K. Wimmers ◽  
M. Gilles ◽  
S. Ponsuksili ◽  
K. Schellander
Keyword(s):  
Developmental Stage ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
The Novel ◽  
Cell Stage ◽  
Novel Transcript ◽  
Stage Of Development ◽  
Significant Difference

A comparative analysis of mRNA expression patterns between embryos produced under different in vitro and in vivo culture systems allows the isolation of genes associated with embryo quality and investigation of the effect of culture environment on the embryonic gene expression. In this study, expression analysis of four known (PSCD2, TCF7L2, NADH-subunit and PAIP1) genes and one novel transcript, derived from differential display PCR, was performed in in vitro (Ponsuksili et al., 2002, Theriogenology 57, 1611–1624) or in vivo- (Moesslacher et al., 2001 Reprod. Dom. Anim. 32, 37) produced bovine 2-, 4-, 8-, 16-cell, morula and blastocyst stage embryos using real time PCR technology. Poly(A) RNA was isolated from four separate individual embryos from each developmental stage and embryo group (in vitro or in vivo) using Dynabeads mRNA kit (Dynal, Oslo, Norway). After reverse transcription, quantitative PCR was performed with sequence specific primers in an ABI PRISM® 7000 Sequence Detection System instrument (Applied Biosystems, Foster City, CA, USA) using SYBR® Green as a double-strand DNA-specific fluorescent dye. Standard curves were generated for target and endogenous genes using serial dilutions of plasmid DNA. Final quantification was done using the relative standard curve method, and results were reported as relative expression or n-fold difference to the calibrator cDNA (i.e., the blastocyst stage) after normalization with the endogenous control (Histone2a). Data were analyzed using SAS version 8.0 (SAS Institute Inc., NC, USA) software package. Analysis of variance was performed with the main effects being the developmental stage and embryo source (in vitro or in vivo) and their interactions followed by multiple pairwise comparisons using Tukey’s test. No significant difference was observed in the relative abundance of the PSCD2 gene between the two embryo groups. However, its expression was higher (20-fold) (P<0.05) at the 8-cell stage than the other developmental stages among in vitro embryos. Higher expression (P<0.05) of NADH-subunit mRNA was detected in vivo than in vitro at the 2-cell stage of development. The TCF7L2 mRNA was expressed in the in vitro embryos but not in the in vivo ones. PAIP1 mRNA was higher (P<0.05) in in vitro (1500-fold) than in the in vivo embryos (500-fold) at the 2-cell developmental stage compared to the calibrator. The novel transcript was also detected at higher level (P<0.05) in the in vitro than in the in vivo embryos at the 2-cell stage of development. However, the PAIP1 and the novel transcript showed no significant difference in their expression between the two embryo groups beyond the 2-cell developmental stage. Both PAIP1 and the novel transcript were detected only up to 8-cell stage in both embryo groups, suggesting their maternal origin. In conclusion, the variations in the expression of studied genes between in vitro and in vivo may reflect the effect of the two culture systems on the transcriptional activity of early embryos.

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