DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate beta-catenin stability.
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The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37% identity with the C-terminal region of Axin, and this related region is named the DIX domain. The functions of the DIX domains of Dvl-1 and Axin were investigated. By yeast two-hybrid screening, the DIX domain of Dvl-1 was found to interact with Dvl-3, a second mammalian Dishevelled relative. The DIX domains of Dvl-1 and Dvl-3 directly bound one another. Furthermore, Dvl-1 formed a homo-oligomer. Axin also formed a homo-oligomer, and its DIX domain was necessary. The N-terminal region of Dvl-1, including its DIX domain, bound to Axin directly. Dvl-1 inhibited Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 was required for this inhibitory activity. Expression of Dvl-1 in L cells induced the nuclear accumulation of beta-catenin, and deletion of the DIX domain abolished this activity. Although expression of Axin in SW480 cells caused the degradation of beta-catenin and reduced the cell growth rate, expression of an Axin mutant that lacks the DIX domain did not affect the level of beta-catenin or the growth rate. These results indicate that the DIX domains of Dvl-1 and Axin are important for protein-protein interactions and that they are necessary for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin. (+info)
Glutamatergic projection to RVLM mediates suppression of reflex bradycardia by parabrachial nucleus.
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We investigated the role of glutamatergic projection from the parabrachial nucleus (PBN) complex to the rostral ventrolateral medulla (RVLM) in the PBN-induced suppression of reflex bradycardia in adult Sprague-Dawley rats that were maintained under pentobarbital anesthesia. Under stimulus conditions that did not appreciably alter the baseline systemic arterial pressure and heart rate, electrical (10-s train of 0.5-ms pulses, at 10-20 microA and 10-20 Hz) or chemical (L-glutamate, 1 nmol) stimulation of the ventrolateral regions and Koelliker-Fuse (KF) subnucleus of the PBN complex significantly suppressed the reflex bradycardia in response to transient hypertension evoked by phenylephrine (5 micrograms/kg iv). The PBN-induced suppression of reflex bradycardia was appreciably reversed by bilateral microinjection into the RVLM of the N-methyl-D-aspartate (NMDA)-receptor antagonist MK-801 (500 pmol) or the non-NMDA-receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (50 pmol). Anatomically, most of the retrogradely labeled neurons in the ventrolateral regions and KF subnucleus of the ipsilateral PBN complex after microinjection of fast blue into the RVLM were also immunoreactive to anti-glutamate antiserum. These results suggest that a direct glutamatergic projection to the RVLM from topographically distinct regions of the PBN complex may participate in the suppression of reflex bradycardia via activation of both NMDA and non-NMDA receptors at the RVLM. (+info)
Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats.
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We studied the effects of microinjection of leptin into the ventromedial hypothalamus (VMH) and lateral hypothalamus (LH) on glucose uptake in peripheral tissues in unanesthetized rats. The rate of glucose uptake was assessed in vivo by 2-[3H]deoxyglucose incorporation. Single injection of leptin into VMH increased glucose uptake in brown adipose tissue (BAT), heart, skeletal muscles, and spleen but not in white adipose tissue or skin. On the other hand, microinjection of leptin into LH had little effect on glucose uptake in those tissues. The plasma concentrations of glucose and insulin were unaltered by intrahypothalamic injection of leptin into either VMH or LH. Among skeletal muscles, the increase in glucose uptake induced by intrahypothalamic injection of leptin was greater in the soleus than in the extensor digitorum longus. Likewise, the increased glucose uptake in the gastrocnemius in response to leptin was more prominent in the red part than in the white part of the tissue. When surgical sympathetic denervation of the interscapular BAT was performed, the enhanced glucose uptake by BAT in response to intrahypothalamic leptin was completely suppressed. These findings suggest that intrahypothalamic injection of leptin preferentially increases glucose uptake by some peripheral tissues through activation of the VMH-sympathetic (or its neighboring medial hypothalamus-sympathetic) nervous system, thereby contributing to the maintenance of energy balance. (+info)
Incidence and perinatal outcome of multiple pregnancies after intracytoplasmic sperm injection compared to standard in vitro fertilization.
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PURPOSE: Our purpose was to assess the incidence of multiple pregnancies and their obstetric outcome after intracytoplasmic sperm injection. METHODS: The study group comprised women who delivered twins or triplets after intracytoplasmic sperm injection and standard in vitro fertilization. The incidence and main perinatal outcome of 140 multiple pregnancies resulting from intracytoplasmic sperm injection or standard in vitro fertilization treatment were analyzed. RESULTS: A total of 60 multiple pregnancies was obtained after intracytoplasmic sperm injection (3.4 +/- 1.1 embryos/cycle) and 80 after standard in vitro fertilization (3.3 +/- 2.0 embryos/cycle). The incidence of multiple pregnancy, i.e., 22.6 compared to 20.7%, respectively, was calculated. The obstetric outcome of 47 multiple pregnancies after intracytoplasmic sperm injection was 39 twin deliveries at between 27 and 37 weeks of gestation (mean, 36 +/- 3.3) and 8 successful triplet deliveries between 26 and 36 weeks of gestation (mean 32.6 +/- 2.4). The outcome after regular in vitro fertilization was similar. No major malformations were observed. CONCLUSIONS: The results of this study showed that the incidence of multiple pregnancies after intracytoplasmic sperm injection was similar to that after standard, conventional in vitro fertilization. The perinatal outcome did not differ between both groups. (+info)
A retrospective follow-up study on intracytoplasmic sperm injection.
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PURPOSE: Genetic aspects of male subfertility and the novelty of intracytoplasmic sperm injection (ICSI) as a new technique can influence the development of zygotes and children born after ICSI. Therefore, we evaluated the outcome of ICSI compared to in vitro fertilization (IVF). METHODS: Data from medical records of 233 total pregnancies and the follow-up of 132 children born after IVF and 120 after ICSI were retrospectively analyzed. RESULTS: No differences were found between ICSI and IVF for early embryonic development and obstetric outcome. In both groups the rate of women undergoing prenatal chromosomal diagnosis was low, 30.0%. The congenital malformation rate was 3.0% after IVF and 1.7% after ICSI, which was not significantly different. Follow-up on development of children born after IVF and ICSI also showed no significant differences. CONCLUSIONS: Our results indicate that at this moment ICSI is a safe procedure. However, a consistent prospective follow-up is still mandatory to exclude possible risks. (+info)
Detection of azoospermic factor genes in Chinese men with azoospermia or severe oligozoospermia.
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PURPOSE: We investigated the prevalence of deletions in the azoospermic factor (AZF) region of chromosome Yq11 in Chinese men with infertility due to idiopathic azoospermia or severe oligozoospermia. The DAZ gene cluster was also examined for mutations. METHODS: Sixty-eight men with azoospermia or severe oligozoospermia taking part in an intracytoplasmic sperm injection program were recruited. Four loci specific for AZFa, AZFb, and AZFc were amplified from genomic DNA via polymerase chain reaction to determine whether deletions were present in the AZF region. Direct DNA sequencing of amplified products was also performed to look for mutations or polymorphism from exon 2 to exon 6 of the DAZ gene cluster. RESULTS: Six (9%) of the 68 patients had AZF deletions. None had mutations in exons 2 to 6 of DAZ. CONCLUSIONS: The prevalence of AZF deletions in our study was similar to those in Western reports, as was the lack of DAZ mutations. (+info)
Paradoxical stimulation of a DEG/ENaC channel by amiloride.
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Extracellular amiloride inhibits all known DEG/ENaC ion channels, including BNC1, a proton-activated human neuronal cation channel. Earlier studies showed that protons cause a conformational change that activates BNC1 and exposes residue 430 to the extracellular solution. Here we demonstrate that, in addition to blocking BNC1, amiloride also exposes residue 430. This result suggested that, like protons, amiloride might be capable of activating the channel. To test this hypothesis, we introduced a mutation in the BNC1 pore that reduces amiloride block, and found that amiloride stimulated these channels. Amiloride inhibition was voltage-dependent, suggesting block within the pore, whereas stimulation was not, suggesting binding to an extracellular site. These data show that amiloride can have two distinct effects on BNC1, and they suggest two different interaction sites. The results suggest that extracellular amiloride binding may have a stimulatory effect similar to that of protons in BNC1 or extracellular ligands in other DEG/ENaC channels. (+info)
Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals.
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Various proteins with different biological activities have been observed to be translocated from the nucleus to the cytoplasm in an energy- and signal-dependent manner in eukaryotic cells. This nuclear export is directed by nuclear export signals (NESs), typically characterized by hydrophobic, primarily leucine, amino acid residues. Moreover, it has been shown that CRM1/exportin 1 is an export receptor for leucine-rich NESs. However, additional NES-interacting proteins have been described. In particular, eukaryotic initiation factor 5A (eIF-5A) has been shown to be a critical cellular cofactor for the nuclear export of the HIV type 1 (HIV-1) Rev trans-activator protein. In this study we compared the nuclear export activity of NESs of different origin. Microinjection of export substrates into the nucleus of somatic cells in combination with specific inhibitors indicated that specific nuclear export pathways exist for different NES-containing proteins. In particular, inhibition of eIF-5A blocked the nuclear export of NESs derived from the HIV-1 Rev and human T cell leukemia virus type I Rex trans-activators, whereas nucleocytoplasmic translocation of the protein kinase inhibitor-NES was unaffected. In contrast, however, inhibition of CRM1/exportin 1 blocked the nuclear export of all NES-containing proteins investigated. Our data confirm that CRM1/exportin 1 is a general export receptor for leucine-rich NESs and suggest that eIF-5A acts either upstream of CRM1/exportin 1 or forms a complex with the NES and CRM1/exportin 1 in the nucleocytoplasmic translocation of the HIV-1 Rev and human T cell leukemia virus type I Rex RNA export factors. (+info)