ABSTRACT
Tyrosine kinase activity of the asymmetric EGFR homodimer is negatively regulated via ERK-mediated phosphorylation of Thr-669 in the juxtamembrane domain. In the present study, we investigated in human breast cancer cells whether a similar mechanism plays a role in the feedback regulation of the ErbB2/ErbB3 heterodimer, the most potent ErbB receptor dimer. Constitutive tyrosine phosphorylation of ErbB2 and ErbB3 was significantly decreased in phorbol ester- and growth factor-treated BT-474 and MDA-MB-453 cells. In contrast to the decreased tyrosine phosphorylation, Phos-tag Western blot analysis revealed that TPA induced phosphorylation of ErbB2 in an ERK-dependent manner. The target threonine residue corresponding to EGFR Thr-669 and the surrounding residues are highly conserved in ErbB2, but not in ErbB3. Therefore, we demonstrated ERK-mediated phosphorylation of ErbB2 at Thr-677 by generating phospho-specific monoclonal antibodies. Moreover, treatment with trametinib and SCH772984, inhibitors of the MEK-ERK pathway, and substitution of Thr-677 to alanine impaired the feedback inhibition of ErbB2 and ErbB3. These results demonstrated that ERK-mediated phosphorylation of the conserved threonine is a common mechanism for the negative feedback control of active ErbB receptor dimers.
Subject(s)
Breast Neoplasms/enzymology , Extracellular Signal-Regulated MAP Kinases/metabolism , Receptor, ErbB-2/metabolism , Threonine/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/genetics , Female , HEK293 Cells , Humans , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Phosphorylation/genetics , Protein Domains , Receptor, ErbB-2/genetics , Receptor, ErbB-3/genetics , Receptor, ErbB-3/metabolism , Threonine/geneticsABSTRACT
In an attempt to develop a suitable freezing method for Poodle dog sperm, an experiment was conducted to investigate semen collection methods of digital stimulation and an artificial vagina (AV), using Tris and trehalose-egg yolk extender, on the characteristics and cryopreservation of sperm. Two dogs (dogs A and B) were subjected to semen collection by digital stimulation and AV. The volume, sperm concentration, sperm motility index (SMI) and acrosome status of ejaculates were determined immediately after collection. The remainder was frozen as pellets in Tris and trehalose-egg yolk extender. Sperm motility index was evaluated after thawing and during a thermal resistance test, and acrosome integrity was also assessed. No significant differences regarding sperm concentrations, SMI and acrosome integrity were observed between semen collected by AV and digital stimulation. However, when dog sperm were collected by an AV and frozen in trehalose-egg yolk extender, the motility index of frozen-thawed sperm was significantly improved compared to sperm frozen in Tris-egg yolk extender which were collected by digital stimulation. In conclusion, semen collected by an AV and frozen in trehalose-egg yolk extender was effective in enhancing the freezability of Poodle dog sperm.
Subject(s)
Cryopreservation/veterinary , Cryoprotective Agents/pharmacology , Dogs , Specimen Handling/veterinary , Spermatozoa/drug effects , Trehalose/pharmacology , Acrylates/pharmacology , Animals , Citric Acid/pharmacology , Freezing , Glucose/pharmacology , Male , Methylamines/pharmacologyABSTRACT
A number of North American and European studies have observed a higher proportion of winter births in schizophrenia patients. Thus, seasonal fluctuation of unknown environmental factors may affect brain development in ways that alter susceptibility to schizophrenia. Specification of these factors may help elucidate the etiopathological mechanism of the disease, about which little is certain. A small number of studies have investigated this issue in Asian populations, and the findings are not as consistent as those of Western populations. No remarkable excess of winter births has been observed in Japanese or Korean studies, while some studies have reported a significant decrease of summer births. We further investigated the issue in Japanese patients with schizophrenia (n=3927). No significant excess of winter births was observed, but a decrease in the summer births was found in male subjects. This is largely consistent with previous Japanese studies; however, when the subjects were confined to those born in a colder and higher latitude area of Japan (n=1338), a consistent trend for both a winter increase, and a summer decrease, was found. However, the results did not consistently reach statistical significance, possibly due to the lack of statistical power. Environmental factors that correlate with latitude might play a role in the development of the seasonality of births in schizophrenia. Further studies in a larger sample size are required to test these possibilities.
