scholarly journals The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus

2019 ◽  
Author(s):  
Caitlin Armstrong ◽  
Lynne Cassimeris ◽  
Claire Da Silva Santos ◽  
Yagmur Micoogullari ◽  
Bettina Wagner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Equus Caballus ◽  
Pcr Analysis ◽  
Common Disease ◽  
Basal Cells ◽  
Rt Pcr ◽  
Keratin Gene ◽  
Equine Hoof ◽  
Keratin Proteins

AbstractThe equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three monoclonal antibodies generated against N- and C-terminal K124 peptides detected a band of the appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-K124 monoclonal antibodies.

Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants

Development ◽  
1999 ◽  
Vol 126 (20) ◽  
pp. 4405-4419 ◽  
Author(s):  
R. Ruiz-Medrano ◽  
B. Xoconostle-Cazares ◽  
W.J. Lucas
Keyword(s):  
Higher Plants ◽  
Phloem Transport ◽  
The Body ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Long Distance ◽  
Rna Molecules ◽  
Long Distance Transport ◽  
Meristem Development

Direct support for the concept that RNA molecules circulate throughout the plant, via the phloem, is provided through the characterisation of mRNA from phloem sap of mature pumpkin (Cucurbita maxima) leaves and stems. One of these mRNAs, CmNACP, is a member of the NAC domain gene family, some of whose members have been shown to be involved in apical meristem development. In situ RT-PCR analysis revealed the presence of CmNACP RNA in the companion cell-sieve element complex of leaf, stem and root phloem. Longitudinal and transverse sections showed continuity of transcript distribution between meristems and sieve elements of the protophloem, suggesting CmNACP mRNA transport over long distances and accumulation in vegetative, root and floral meristems. In situ hybridization studies conducted on CmNACP confirmed the results obtained using in situ RT-PCR. Phloem transport of CmNACP mRNA was proved directly by heterograft studies between pumpkin and cucumber plants, in which CmNACP transcripts were shown to accumulate in cucumber scion phloem and apical tissues. Similar experiments were conducted with 7 additional phloem-related transcripts. Collectively, these studies established the existence of a system for the delivery of specific mRNA transcripts from the body of the plant to the shoot apex. These findings provide insight into the presence of a novel mechanism likely used by higher plants to integrate developmental and physiological processes on a whole-plant basis.

scholarly journals The identification and characterization of a testis-specific cDNA during spermatogenesis

2006 ◽  
Vol 11 (1) ◽  
Author(s):  
Ying Chen ◽  
Jiarui Hu ◽  
Ping Song ◽  
Wuming Gong
Keyword(s):  
Experimental Validation ◽  
Rna Binding ◽  
Pcr Analysis ◽  
Splicing Factors ◽  
Rt Pcr ◽  
Rich Domain ◽  
Pachytene Spermatocytes ◽  
Identification And Characterization

AbstractUsing bioinformatics and experimental validation, we obtained a cDNA (named srsf) which was exclusively expressed in the mouse testes. RT-PCR analysis showed that srsf mRNA was not expressed in the gonad during the sex determination period or during embryogenesis. In developing mouse tests, srsf expression was first detected on post-natal day 10, reached its highest level on day 23, and then reduced to and remained at a moderate level throughout adulthood. In situ hybridization analysis demonstrated that srsf mRNA was expressed in pachytene spermatocytes and round spermatids in the testes. The predicted protein contains one RNA-binding domain (RBD) and a serine-arginine rich domain (RS), which are characterized by some splicing factors of SR family members. These findings indicate that srsf may play a role during spermatogenesis.

Molecular identification of Ca2+-activated K+ channels in parotid acinar cells

2003 ◽  
Vol 284 (2) ◽  
pp. C535-C546 ◽  
Author(s):  
Keith Nehrke ◽  
Claire C. Quinn ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Acinar Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Parotid Acinar Cells ◽  
Large Conductance Channel ◽  
Intracellular Ca

We used molecular biological and patch-clamp techniques to identify the Ca2+-activated K+ channel genes in mouse parotid acinar cells. Two types of K+ channels were activated by intracellular Ca2+ with single-channel conductance values of 22 and 140 pS (in 135 mM external K+), consistent with the intermediate and maxi-K classes of Ca2+-activated K+ channels, typified by the mIK1 ( Kcnn4) and mSlo ( Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo β-subunits ( Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each β-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from β1 knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the β4-subunit.

scholarly journals Up-regulation of Per1 expression by estradiol and progesterone in the rat uterus

2007 ◽  
Vol 194 (3) ◽  
pp. 511-519 ◽  
Author(s):  
Pei-Jian He ◽  
Masami Hirata ◽  
Nobuhiko Yamauchi ◽  
Masa-aki Hattori
Keyword(s):  
Stromal Cells ◽  
Clock Genes ◽  
Staining Intensity ◽  
Reproductive Organ ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Rat Uterus ◽  
Ovarian Steroids ◽  
Direct Regulation

It has been established that estrogen can alter circadian rhythms in behavior and endocrine physiology in rodents. The uterus is a reproductive organ that is critically dependent on regulation by ovarian steroids. Here, we examined the expression of Per1 in different compartments of the uterus, and explored whether the ovarian steroids could regulate Per1 expression employing ovariectomized rat uterus. RT-PCR analysis showed that Per1 was cyclically expressed in the uterus. As revealed by in situ hybridization, the staining intensity of Per1 mRNA was stronger at ZT 8 than at ZT 0 in the uterine luminal epithelium (LE), stroma (S), and myometrium (M) compartments, but was not changed in the glandular epithelium (GE). Both in situ hybridization and immunofluorescence analyses revealed that estradiol (E2) administration induced high expression of Per1 in the LE, GE, and M, and less expression in the S compartment. Progesterone (P4) treatment resulted in an obvious enhancement of Per1 expression in the LE, GE, and S, but unchanged in the M compartment. Furthermore, the E2- and P4-activated Per1 expression was significantly repressed by their respective antagonists, ICI182 780 and RU486. These findings were further supported by RT-PCR analysis of Per1 expression in cultured uterine stromal cells. Collectively, the present data indicate that E2 and P4 might be involved in modification of circadian rhythm via direct regulation of the expression of clock genes.

scholarly journals Occurrence of Two Distinct Urotensin II-Related Peptides in Zebrafish Provides New Insight into the Evolutionary History of the Urotensin II Gene Family

Endocrinology ◽  
2011 ◽  
Vol 152 (6) ◽  
pp. 2330-2341 ◽  
Author(s):  
Caroline Parmentier ◽  
Emilie Hameury ◽  
Christophe Dubessy ◽  
Feng B. Quan ◽  
Damien Habert ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Close Contact ◽  
Ventral Side ◽  
Pcr Analysis ◽  
Urotensin Ii ◽  
Rt Pcr ◽  
History Of ◽  
The Brain ◽  
Insight Into

The urotensin II (UII) family is currently known to consist of two paralogous peptides, namely UII and UII-related peptide (URP). In contrast to UII, which has been identified in all vertebrate classes so far, URP has only been characterized in tetrapods. We report here the occurrence of two distinct URP genes in teleosts, which we have named URP1 and URP2. Synteny analysis revealed that teleost URP1 and URP2 genes and tetrapod URP genes represent three distinct paralog genes that, together with the UII gene, probably arose from the two rounds of tetraploidization, which took place early in vertebrate evolution. The absence of URP in fish indicates that the corresponding gene has been lost in the teleost lineage, whereas it is likely that both the URP1 and URP2 genes have been lost in the tetrapod lineage. Quantitative RT-PCR analysis revealed that the URP2 gene is mainly expressed in the spinal cord and the brain in adult zebrafish. In situ hybridization experiments showed that in zebrafish embryos, URP2 mRNA-containing cells are located in the floor plate of the neural tube. In adult, URP2-expressing cells occur in close contact with the ventral side of the ependymal canal along the whole spinal cord, whereas in the brain, they are located below the fourth ventricle. These URP-expressing cells may correspond to cerebrospinal fluid-contacting neurons. In conclusion, our study reveals the occurrence of four distinct UII paralogous systems in vertebrates that may exert distinct functions, both in tetrapods and teleosts.

Gene expression of Na+/H+ exchanger in zebrafish H+-ATPase-rich cells during acclimation to low-Na+ and acidic environments

2007 ◽  
Vol 293 (6) ◽  
pp. C1814-C1823 ◽  
Author(s):  
Jia-Jiun Yan ◽  
Ming-Yi Chou ◽  
Toyoji Kaneko ◽  
Pung-Pung Hwang
Keyword(s):  
Amino Acid Sequences ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Acid Base ◽  
Phylogenetic Tree Analysis ◽  
Major Player ◽  
C Subunit ◽  
Proximal Tubular ◽  
Acidic Environments

In mammalian nephrons, most of the Na+ and HCO3− is reabsorbed by proximal tubular cells in which the Na+/H+ exchanger 3 (NHE3) is the major player. The roles of NHEs in Na+ uptake/acid-base regulation in freshwater (FW) fish gills are still being debated. In the present study, functional genomic approaches were used to clone and sequence the full-length cDNAs of the nhe family from zebrafish ( Danio rerio). A phylogenetic tree analysis of the deduced amino acid sequences showed that zNHE1–8 are homologous to their mammalian counterparts. By RT-PCR analysis and double/triple in situ hybridization/immunocytochemistry, only zebrafish NHE3b was expressed in zebrafish gills and was colocalized with V-H+-ATPase but not with Na+-K+-ATPase, indicating that H+-ATPase-rich (HR) cells specifically express NHE3b. A subsequent quantitative RT-PCR analysis demonstrated that acclimation to low-Na+ FW caused upregulation and downregulation of the expressions of znhe3b and zatp6v0c (H+-ATPase C-subunit), respectively, in gill HR cells, whereas acclimation to acidic FW showed reversed effects on the expressions of these two genes. In conclusion, both NHE3b and H+-ATPase are probably involved in Na+ uptake/acid-base regulation in zebrafish gills, like mammalian kidneys, but the partitioning of these two transporters may be differentially regulated depending on the environmental situation in which fish are acclimatized.

scholarly journals Generation of Thyrotropin-Releasing Hormone Receptor 1-Deficient Mice as an Animal Model of Central Hypothyroidism

2004 ◽  
Vol 18 (6) ◽  
pp. 1450-1460 ◽  
Author(s):  
Roland Rabeler ◽  
Jens Mittag ◽  
Lars Geffers ◽  
Ulrich Rüther ◽  
Michael Leitges ◽  
...  
Keyword(s):  
Animal Model ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Pcr Analysis ◽  
Target Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Central Hypothyroidism ◽  
R1 Gene

Abstract To provide an animal model of central hypothyroidism, mice deficient in the TRH-receptor 1 (TRH-R1) gene were generated by homologous recombination. The pituitaries of TRH-R1−/− mice are devoid of any TRH-binding capacity, demonstrating that TRH-R1 is the only receptor localized on TRH target cells of the pituitary. With the exception of some retardation in growth rate, TRH-R1−/− mice appear normal, but compared with control animals they exhibit a considerable decrease in serum T3, T4, and prolactin (PRL) levels but not in serum TSH levels. In situ hybridization histochemistry and real-time RT-PCR analysis revealed that in adult TRH-R1−/− animals TSHβ-mRNA expression is not impaired whereas PRL mRNA and GH mRNA levels are considerably reduced compared with control mice. The numbers of thyrotropes, somatotropes, and lactotropes, however, are not affected by the deletion of the TRH-R1 gene. The mutant mice are fertile, and the dams nourish their pups well, indicating that TRH is not a decisive factor for suckling-induced PRL release. In situ hybridization and quantitative RT-PCR analysis, furthermore, revealed that, as in control animals, pituitary PRL-mRNA expression in TRH-R1−/− is considerably increased during lactation, albeit strongly reduced as compared with lactating control animals.

Intragraft mRNA Expression of Cytokines and Growth Factors in Human Kidney Allograft Biopsies by In Situ RT-PCR Analysis

2005 ◽  
Vol 37 (2) ◽  
pp. 767-769 ◽  
Author(s):  
D. Kaminska ◽  
B. Tyran ◽  
O. Mazanowska ◽  
W. Letachowicz ◽  
A. Kochman ◽  
...  
Keyword(s):  
Growth Factors ◽  
Mrna Expression ◽  
Human Kidney ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Kidney Allograft

Microarray analysis of normal cervix, carcinoma in situ, and invasive cervical cancer: identification of candidate genes in pathogenesis of invasion in cervical cancer

2008 ◽  
Vol 18 (5) ◽  
pp. 1051-1059 ◽  
Author(s):  
J. Y. Song ◽  
J. K. Lee ◽  
N. W. Lee ◽  
H. H. Jung ◽  
S. H. Kim ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Candidate Genes ◽  
Expression Profiles ◽  
Invasive Cervical Cancer ◽  
Pcr Analysis ◽  
Cervix Carcinoma ◽  
Rt Pcr ◽  
Cancer Group ◽  
Normal Cervix

The objective of this study was to identify genes that are related to pathogenesis of carcinoma in situ (CIS) to invasive cervical cancer with the use of oligonucleotide microarray and reverse transcription-polymerase chain reaction (RT-PCR). Each two cases of normal cervix, CIS, and invasive cervical cancer were investigated with DNA microarray technology. Differential gene expression profiles among them were analyzed. Expression levels of selected genes from the microarray results were confirmed by RT-PCR. The expressions of 15,286 genes were compared and 458 genes were upregulated or downregulated by twofold or more compared with each other group. Among 458 genes, 22 genes were upregulated and 40 genes were downregulated by twofold or more in invasive cervical cancer group compared with CIS group. RT-PCR analysis confirmed upregulation of 18 genes and downregulation of 5 genes in invasive cervical cancer group. RBP1, TFRC, SPP1, SAA1, ARHGAP8, and NDRG1, which were upregulated, and GATA3, PLAGL1, APOD, DUSP1, and CYR61, which were downregulated, were considered as candidate genes associated with invasion of cervical cancer.

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