Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development

ABSTRACT The eyes absent 1 protein (Eya1) plays an essential role in the development of various organs in both invertebrates and vertebrates. Mutations in the human EYA1 gene are linked to BOR (branchio-oto-renal) syndrome, characterized by kidney defects, hearing loss, and branchial arch anomalies. For a better understanding of Eya1's function, we have set out to identify new Eya1-interacting proteins. Here we report the identification of the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as novel interaction partners of Eya1. We confirmed the interactions by glutathione S-transferase (GST) pulldown analysis and coimmunoprecipitation. A first mechanistic insight is provided by the demonstration that Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Using reverse transcriptase PCR (RT-PCR) and in situ hybridization, we show that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. By morpholino-mediated knockdown, we demonstrate that the Sipl1 and Rbck1 orthologs are involved in different aspects of zebrafish development. In particular, knockdown of one Sipl1 ortholog as well as one Rbck1 ortholog led to a BOR syndrome-like phenotype, with characteristic defects in ear and branchial arch formation.

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Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162545 ◽

1996 ◽

Vol 54 ◽

pp. 16-17

Author(s):

J. R. Hully ◽

K. R. Luehrsen ◽

K. Aoyagi ◽

C. Shoemaker ◽

R. Abramson

Keyword(s):

Nucleic Acids ◽

Viral Infections ◽

Anatomical Location ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Cellular Source ◽

Gene Transcripts ◽

Initial Description ◽

In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

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UP‐REGULATION OF TRANSFORMING GROWTH FACTOR BETA‐1 (TGF β 1 ) IN FOCAL AND SEGMENTAL GLOMERULOSCLEROSIS (FSGS). A STUDY OF HUMAN RENAL BIOPSY TISSUE USING COMPETITIVE RT‐PCR, AND IN‐SITU HYBRIDISATION

Nephrology ◽

10.1046/j.1440-1797.2000.0abs6.x ◽

2000 ◽

Vol 5 (3) ◽

pp. A68-A68

Author(s):

Langham R ◽

Egan M ◽

Dowling J ◽

Gilbert R ◽

Thomson N.

Keyword(s):

Growth Factor ◽

Renal Biopsy ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

In Situ Hybridisation ◽

Rt Pcr ◽

Focal And Segmental Glomerulosclerosis ◽

Biopsy Tissue ◽

Growth Factor Beta

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RNA expression as a prognostic tool in idiopathic nephrotic syndrome

Orvosi Hetilap ◽

10.1556/oh.2007.27978 ◽

2007 ◽

Vol 148 (23) ◽

pp. 1067-1075

Author(s):

Krisztina Fischer ◽

Orsolya Galamb ◽

Béla Molnár ◽

Zsolt Tulassay ◽

András Szabó

Keyword(s):

Nephrotic Syndrome ◽

Northern Blot ◽

Idiopathic Nephrotic Syndrome ◽

Minimal Change ◽

Ribonuclease Protection Assay ◽

Rna Expression ◽

Rt Pcr ◽

Protection Assay ◽

Ribonuclease Protection

A gyermekkori nephrosis 90%-a idiopathiás nephrosis szindróma. Az idetartozó három kórkép, a minimal change betegség, a mesangialis proliferatio és a focalis sclerosis hasonló klinikai képpel jelentkező, eltérő prognózisú és terápiás válaszú betegség. Dolgozatunk célja az idiopathiás nephrosis szindrómába tartozó kórképek kialakulásával, progressziójával összefüggő genetikai ismeretek, génexpressziós változások áttekintése és funkcionális csoportosítása. A génexpressziós változások meghatározásának eszközeként, dolgozatunk röviden összefoglalja a northern blot, a ribonuclease protection assay, az in situ RNS-hibridizáció, a kvantitatív RT-PCR és a microarray módszerek lényegét. Az eddig elvégzett vizsgálatok a DNS-szintézis és repair gének, növekedési faktorok, extracelluláris mátrix, extracelluláris ligandreceptorok, extracelluláris jelátvitel zavarai mellett kiemelik a metabolikus és transzporter gének, illetve az immunszabályozó gének molekuláris eltéréseit, amelyek összefüggésben vannak az idiopathiás nephrosis szindróma eddig megismert molekuláris hátterével. A chiptechnológia fejlődésével és elterjedésével ezek a markerek és a hagyományos vizsgálati módszerek párhuzamos alkalmazása rutindiagnosztikai szempontból is fontossá válhat.

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Phosphorylation of GAPVD1 Is Regulated by the PER Complex and Linked to GAPVD1 Degradation

International Journal of Molecular Sciences ◽

10.3390/ijms22073787 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3787

Author(s):

Hussam Ibrahim ◽

Philipp Reus ◽

Anna Katharina Mundorf ◽

Anna-Lena Grothoff ◽

Valerie Rudenko ◽

...

Keyword(s):

Circadian Clock ◽

Transcriptional Repression ◽

Small Gtpase ◽

Main Role ◽

Interacting Proteins ◽

Casein Kinase 1 ◽

Bona Fide ◽

Kinetics Of

Repressor protein period (PER) complexes play a central role in the molecular oscillator mechanism of the mammalian circadian clock. While the main role of nuclear PER complexes is transcriptional repression, much less is known about the functions of cytoplasmic PER complexes. We found with a biochemical screen for PER2-interacting proteins that the small GTPase regulator GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), which has been identified previously as a component of cytoplasmic PER complexes in mice, is also a bona fide component of human PER complexes. We show that in situ GAPVD1 is closely associated with casein kinase 1 delta (CSNK1D), a kinase that regulates PER2 levels through a phosphoswitch mechanism, and that CSNK1D regulates the phosphorylation of GAPVD1. Moreover, phosphorylation determines the kinetics of GAPVD1 degradation and is controlled by PER2 and a C-terminal autoinhibitory domain in CSNK1D, indicating that the regulation of GAPVD1 phosphorylation is a novel function of cytoplasmic PER complexes and might be part of the oscillator mechanism or an output function of the circadian clock.

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Overexpression of P-glycoprotein, MRP2, and CYP3A4 impairs intestinal absorption of octreotide in rats with portal hypertension

BMC Gastroenterology ◽

10.1186/s12876-020-01532-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Xiaoyu Sun ◽

Shunxiong Tang ◽

Binbin Hou ◽

Zhijun Duan ◽

Zhen Liu ◽

...

Keyword(s):

Liver Cirrhosis ◽

Portal Hypertension ◽

Western Blot ◽

In Vitro Experiments ◽

Rt Pcr ◽

P Glycoprotein ◽

Intestinal Microsomes ◽

First Pass

Abstract Background Portal hypertension (PH) is the main cause of complications and death in liver cirrhosis. The effect of oral administration of octreotide (OCT), a drug that reduces PH by the constriction of mesenteric arteries, is limited by a remarkable intestinal first-pass elimination. Methods The bile duct ligation (BDL) was used in rats to induce liver cirrhosis with PH to examine the kinetics and molecular factors such as P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2) and cytochrome P450 3A4 (CYP3A4) influencing the intestinal OCT absorption via in situ and in vitro experiments on jejunal segments, transportation experiments on Caco-2 cells and experiments using intestinal microsomes and recombinant human CYP3A4. Moreover, RT-PCR, western blot, and immunohistochemistry were performed. Results Both in situ and in vitro experiments in jejunal segments showed that intestinal OCT absorption in both control and PH rats was largely controlled by P-gp and, to a lesser extent, by MRP2. OCT transport mediated by P-gp and MRP2 was demonstrated on Caco-2 cells. The results of RT-PCR, western blot, and immunohistochemistry suggested that impaired OCT absorption in PH was in part due to the jejunal upregulation of these two transporters. The use of intestinal microsomes and recombinant human CYP3A4 revealed that CYP3A4 metabolized OCT, and its upregulation in PH likely contributed to impaired drug absorption. Conclusions Inhibition of P-gp, MRP2, and CYP3A4 might represent a valid option for decreasing intestinal first-pass effects on orally administered OCT, thereby increasing its bioavailability to alleviate PH in patients with cirrhosis.

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