Polarization and density of Na-pumps in the inner ear of the chick embryo during early stages of development

Development ◽  
1987 ◽  
Vol 100 (2) ◽  
pp. 271-278
Author(s):  
F. Giraldez ◽  
J.J. Represa ◽  
L. Borondo ◽  
E. Barbosa
Keyword(s):  
Binding Sites ◽  
Fluid Transport ◽  
Developmental Stages ◽  
Otic Vesicle ◽  
Transient Stage ◽  
Highly Sensitive ◽  
Proliferative Growth ◽  
K Concentration ◽  
Otic Vesicles ◽  
Vectorial Transport

The otic vesicle consists of a pseudostratified epithelium with some features of transporting epithelia. The present work questions whether Na-pumps are polarized in this epithelium and what is the relation between the location or density of pumps and development. This was done by measuring the binding of [3H]ouabain to isolated otic vesicles in developmental stages 16 to 22. The results show the presence of specific ouabain-binding sites located in the inward-facing membrane of the otic vesicle epithelium. Binding was saturable at increasing concentrations of ouabain and was highly sensitive to the external K+ concentration with half-maximal inhibition below 0.5 mM, indicating that the binding sites and Na-pump sites are identical. A transient stage-dependent increase in the density of Na-pumps during the period that precedes growth was observed. Evidence is given against this being directly related to an increased net fluid transport rate despite the fact that pump sites were always polarized throughout these stages. The conclusions are that (1) the otic vesicle epithelium is a polarized structure with the possibility of vectorial transport of solutes and water and (2) the increased number of pumps may operate as a regulatory mechanism during normal proliferative growth.

Bombesin and other growth factors activate cell proliferation in chick embryo otic vesicles in culture

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 87-96 ◽  
Author(s):  
J.J. Represa ◽  
C. Miner ◽  
E. Barbosa ◽  
F. Giraldez
Keyword(s):  
Cell Proliferation ◽  
Growth Factors ◽  
Chick Embryo ◽  
Calf Serum ◽  
Cell Number ◽  
Otic Vesicle ◽  
Thymidine Uptake ◽  
Normal Pattern ◽  
Proliferative Growth ◽  
Otic Vesicles

The ability of the mitogenic peptide bombesin and other growth factors to trigger and support early development of the inner ear was studied on chick embryo otocysts in culture. The normal pattern of development was preserved in cultured otic vesicles in the presence of 20% fetal calf serum in the medium. Differentiation proceeded from stage 18 to 22 during the first 24 h and further to stage 24 in 48 h. Estimates of cell number and mitotic rates revealed a distinct period of proliferative growth which was maximum at the 24 h period of incubation. This was coincident with a high rate of DNA synthesis as measured by the acid-precipitable incorporation of [3H]thymidine. Development could be arrested by deprivation of serum during 24h. It could then be reactivated by readmission of serum to proceed with the normal pattern of morphological differentiation and cell proliferation. Bombesin (100 nM) was able to reactivate development in growth-arrested vesicles. Its effect was dose-dependent, saturable and potentiated by insulin (5 micrograms ml-1) which was ineffective if used alone. When associated with insulin, bombesin carried differentiation to stage 21 and stimulated mitotic activity above the level of serum as judged from estimates of cell number and [3H]thymidine uptake. EGF and PDGF were also effective in reinitiating development although their potency was smaller than bombesin. The reactivation by serum or bombesin was blocked by amiloride. The results show that (1) the otic vesicle can provide a useful model for studying the mechanisms that control proliferative growth and differentiation during normal development and (2) bombesin and other growth factors are able to activate growth in embryonic developing tissues.

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

scholarly journals Genomic and cDNA selection-amplification identifies transcriptome-wide binding sites for the Drosophila protein sex-lethal

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0250592
Author(s):  
Hiren Banerjee ◽  
Ravinder Singh
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Developmental Stages ◽  
Rna Binding Proteins ◽  
Systematic Analysis ◽  
Downstream Targets ◽  
Drosophila Protein ◽  
Sex Lethal ◽  
Female Specific

Background Downstream targets for a large number of RNA-binding proteins remain to be identified. The Drosophila master sex-switch protein Sex-lethal (SXL) is an RNA-binding protein that controls splicing, polyadenylation, or translation of certain mRNAs to mediate female-specific sexual differentiation. Whereas some targets of SXL are known, previous studies indicate that additional targets of SXL have escaped genetic screens. Methodology/Principal findings Here, we have used an alternative molecular approach of GEnomic Selective Enrichment of Ligands by Exponential enrichment (GESELEX) using both the genomic DNA and cDNA pools from several Drosophila developmental stages to identify new potential targets of SXL. Our systematic analysis provides a comprehensive view of the Drosophila transcriptome for potential SXL-binding sites. Conclusion/Significance We have successfully identified new SXL-binding sites in the Drosophila transcriptome. We discuss the significance of our analysis and that the newly identified binding sites and sequences could serve as a useful resource for the research community. This approach should also be applicable to other RNA-binding proteins for which downstream targets are unknown.

Retinoic acid modulation of the early development of the inner ear is associated with the control of c-fos expression

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1081-1090 ◽  
Author(s):  
J. Represa ◽  
A. Sanchez ◽  
C. Miner ◽  
J. Lewis ◽  
F. Giraldez
Keyword(s):  
Cell Proliferation ◽  
Retinoic Acid ◽  
Inner Ear ◽  
Early Development ◽  
Otic Vesicle ◽  
Low Concentrations ◽  
Full Effect ◽  
20 Nm ◽  
Otic Vesicles

The effects of retinoic acid (RA) on the early development of the inner ear were studied in vitro using isolated chick embryo vesicles. Low concentrations of RA (1–50 nM) inhibited vesicular growth in stage 18 otic vesicles that were made quiescent and then reactivated by either serum or bombesin. Growth inhibition was concentration-dependent and was paralleled by a reduction in the rate of DNA synthesis as measured by [3H]thymidine incorporation. Half-inhibition occurred between 1 and 10 nM RA, and the full effect at 20 nM. Retinoic acid, in the presence of serum, induced the precocious differentiation of (1) secretory epithelium, the tegmentum vasculosum and endolymphatic sac and (2) early sensory and supporting epithelia. These structures were positioned in their corresponding normal presumptive areas. The overall direction of growth was reversed by RA and the ratio of the internal to the external vesicular surface area increased with RA concentration. The expression of the nuclear proto-oncogene c-fos in the developing otic vesicle was transient and stage-dependent. High levels of c-fos mRNA were positively correlated with cell proliferation. Incubation of growth-arrested otic vesicles with bombesin plus insulin at concentrations that induced cell proliferation produced a strong induction of c-fos. This mitogen-induced expression was suppressed by 25 nM RA. The results suggest (1) a role for retinoic acid in controlling the early development of the inner ear and (2) that this control is effected through the regulation of the proto-oncogene c-fos.

Evaluating Headline Fungicide on Alfalfa Production and Sensitivity of Pathogens to Pyraclostrobin

2013 ◽  
Vol 14 (1) ◽  
pp. 10 ◽  
Author(s):  
Deborah A. Samac ◽  
Bill Halfman ◽  
Bryan Jensen ◽  
Fritz Brietenbach ◽  
Lisa Behnken ◽  
...  
Keyword(s):  
Return On Investment ◽  
Crude Protein ◽  
Field Experiments ◽  
Developmental Stages ◽  
Forage Yield ◽  
Fungicide Application ◽  
Highly Sensitive ◽  
Disease Pressure ◽  
Foliar Pathogens

Headline fungicide was recently registered for management of foliar diseases on alfalfa. The effect of disease control on yield, forage quality, and potential return on investment for fungicide application was determined for field experiments conducted at five locations in 2012, three in Wisconsin and two in Minnesota. Headline reduced defoliation in 12 out of 14 harvests and increased forage yield and return on investment in 5 out 12 harvests compared to the untreated control. Headline plus the insecticide Warrior II reduced defoliation in 10 out of 14 harvests and increased yield and return on investment in four harvests compared to Warrior II alone. Two common foliar pathogens were highly sensitive to pyraclostrobin, the active ingredient in Headline. For Phoma medicaginis, the in vitro EC50 was 2.3 ng pyraclostrobin/ml and for Stemphylium globuliferum the EC50 was 52 ng pyraclostrobin/ml. The results indicate that fungicide application can increase yields under higher disease pressure and increase crude protein when the crop is harvested at later developmental stages. Accepted for publication 25 June 2013. Published 17 September 2013.

scholarly journals Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system

Reproduction ◽  
2005 ◽  
Vol 130 (5) ◽  
pp. 669-679 ◽  
Author(s):  
A S Lopes ◽  
L H Larsen ◽  
N Ramsing ◽  
P Løvendahl ◽  
M Räty ◽  
...  
Keyword(s):  
Respiration Rate ◽  
Embryo Quality ◽  
Developmental Stages ◽  
Embryo Morphology ◽  
The Novel ◽  
Non Invasive ◽  
Respiration Rates ◽  
Morphological Evaluation ◽  
Highly Sensitive

Oxygen consumption is a useful parameter for evaluating embryo quality, since it provides a valuable indication of overall metabolic activity. Over the years, several approaches have been used to measure the respiration rates of individual embryos, but a convincing method has not yet been reported. In this study, we introduce and have validated a novel high resolution microsensor technology to determine the respiration rates of individual embryos at different developmental stages. We have employed this technology to investigate the correlation between respiration rate and embryo morphology, diameter and sex. Following morphological evaluation, individual respiration rates of day 3 (n = 18) and day 7 (n = 60) bovine in vitro-produced embryos were determined. Of the measured embryos, 64 were lysed for sex diagnosis by PCR. Average respiration rates of day 7 embryos (1.30 ± 0.064 nl/h) were 3.4-fold higher than day 3 embryos (0.38 ± 0.011 nl/h). On day 7, the average respiration rate of quality 1 blastocysts was significantly higher than the respiration rates of the lower qualities. For both day 3 and day 7 embryos, respiration rates were directly influenced by embryo diameter but did not differ between sexes. These results have demonstrated that the novel microsensor technology can be used to accurately and rapidly (8 min) measure the respiration rates of individual embryos at different developmental stages. Respiration rates were only in partial agreement with embryo morphology, suggesting a slight discrepancy between these two methods in assessing embryo quality. It is likely that a combined assessment of embryo respiration and morphology would improve embryo classification and subsequent selection.

scholarly journals Study on sex-organ development. Oestrogen-receptor translocation in the developing chick Müllerian duct

1976 ◽  
Vol 154 (1) ◽  
pp. 1-9 ◽  
Author(s):  
C S Teng ◽  
C T Teng
Keyword(s):  
Binding Sites ◽  
Developmental Stages ◽  
Dissociation Constants ◽  
Duct Cell ◽  
Mullerian Duct ◽  
Embryonic Chick ◽  
Müllerian Duct ◽  
Initial Content

After oestradiol administration in vivo, 87-95% of the initial concentration of oestradiol receptor in the cytoplasm of the embryonic-chick Müllerian-duct cell was translocated into the nucleus. The process of translocation depends on the amount of oestardiol administered in vivo. At 6 h after oestradiol administration in vivo, about 30% replenishment of the initial content of the cytosol receptor was observed in the cytoplasm. The Müllerian-duct nuclei, after exposure to non-radioactive oestradiol, exhibit saturable exchange with [3H]oestradiol in vitro. The exchange of oestradiol is temperature- and time-dependent. The optimal temperature and time for exchange are 37-41 degrees C and 2h respectively. The [3H]oestradiol-receptor complex extracted from the exchanged nuclei is present in 5-6S form, and its isoelectric point is 6.8. The number of nuclear oestradiol-binding sites of the developing Müllerian duct are 1.66, 2.22, 2.63, and 2.50 pmol/mg of DNA respectively for embryos of 10, 12, 15 and 18 days. The dissociation constants of the nuclear oestradiol receptor of the four observed developmental stages range from 3.0 to 3.1 nM.

scholarly journals A novel detection technique of polyamide binding sites by photo-induced electron transfer in BrU substituted DNA

2015 ◽  
Vol 51 (77) ◽  
pp. 14485-14488 ◽  
Author(s):  
Abhijit Saha ◽  
Fumitaka Hashiya ◽  
Seiichiro Kizaki ◽  
Sefan Asamitsu ◽  
Kaori Hashiya ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Binding Sites ◽  
Detection Technique ◽  
Sensitive Detection ◽  
Highly Sensitive ◽  
Photo Induced Electron Transfer ◽  
Highly Sensitive Detection ◽  
Sensitive Detection Technique

We demonstrate a highly sensitive detection technique of polyamide binding sites using the photochemistry of BrU labeled DNA.

scholarly journals Size control of the inner ear via hydraulic feedback

2018 ◽  
Author(s):  
Kishore R. Mosaliganti ◽  
Ian A. Swinburne ◽  
Chon U Chan ◽  
Nikolaus D. Obholzer ◽  
Amelia A. Green ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
Inner Ear ◽  
Fluid Transport ◽  
Quantitative Imaging ◽  
Size Variation ◽  
Size Control ◽  
Otic Vesicle ◽  
Physiological Measurement ◽  
Cellular Processes ◽  
Pressure Driven

SUMMARYAnimals make organs of precise size, shape, and symmetry despite noise in underlying molecular and cellular processes. How developing organs manage this noise is largely unknown. Here, we combine quantitative imaging, physical theory, and physiological measurement of hydrostatic pressure and fluid transport in zebrafish to study size control of the developing inner ear. We find that fluid accumulation creates hydrostatic pressure in the lumen leading to stress in the epithelium and expansion of the otic vesicle. Pressure, in turn, inhibits fluid transport into the lumen. This negative feedback loop between pressure and transport allows the otic vesicle to change growth rate to control natural or experimentally-induced size variation. Spatiotemporal patterning of contractility modulates pressure-driven strain for regional tissue thinning. Our work connects moleculardriven mechanisms, such as osmotic pressure driven strain and actomyosin tension, to the regulation of tissue morphogenesis via hydraulic feedback to ensure robust control of organ size.

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