GATA2 functions in adrenal chromaffin cells

Catecholamine synthesized in the sympathoadrenal system, including sympathetic neurons and adrenal chromaffin cells, is vital for cardiovascular homeostasis. It has been reported that GATA2, a zinc finger transcription factor, is expressed in murine sympathoadrenal progenitor cells. However, a physiological role for GATA2 in adrenal chromaffin cells has not been established. In this study, we demonstrate that GATA2 is specifically expressed in adrenal chromaffin cells. We examined the consequences of Gata2 loss- of- function mutations, exploiting a Gata2 conditional knockout allele crossed to neural crest- specific Wnt1- Cre transgenic mice (Gata2 NC- CKO). The vast majority of Gata2 NC- CKO embryos died by embryonic day 14.5 (e14.5) and exhibited a decrease in catecholamine- producing adrenal chromaffin cells, implying that a potential catecholamine defect might lead to the observed embryonic lethality. When intercrossed pregnant dams were fed with synthetic adrenaline analogs, the lethality of the Gata2 NC- CKO embryos was partially rescued, indicating that placental transfer of the adrenaline analogs complements the lethal catecholamine deficiency in the Gata2 NC- CKO embryos. These results demonstrate that GATA2 participates in the development of neuroendocrine adrenaline biosynthesis, which is essential for fetal survival.GATA2 is specifically expressed in adrenal chromaffin cells in which GATA2 plays a role for catecholamine biosynthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162786/2/gtc12795_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162786/1/gtc12795.pd

Heatstroke risk informing system using wearable perspiration ratemeter on users undergoing physical exercise

Abstract We constructed an informing system to users for the heatstroke risk using a wearable perspiration ratemeter and the users’ thirst responses. The sweating ratemeter was constructed with a capacitive humidity sensor in the ventilated capsule. The timing point for informing heatstroke risk was decided to change from positive to negative on the second derivative of sweating curve. In addition, a wearable self-identification and -information system of thirst response was constructed with a smartphone. To evaluate the validity of wearable apparatus, we aimed to conduct human experiments of 16 healthy subjects with the step up and down physical exercises. The blood and urine samples of the subjects were collected before and after the 30-min physical exercise. The concentrations of TP, Alb, and RBC increased slightly with the exercise. In contrast, the concentrations of vasopressin in all subjects remarkably increased with the exercise. In almost subjects, they identified their thirst response until several min after the informing for heatstroke risk. In conclusion, the wearable ratemeter and self-information system of thirst response were suitable for informing system of heatstroke risk. The validity of timing point for informing heatstroke risk was confirmed with changes in the thirst response and concentrations of vasopressin in blood

Comprehensive Analysis of Prokaryotes in Environmental Water Using DNA Microarray Analysis and Whole Genome Amplification

The microflora in environmental water consists of a high density and diversity of bacterial species that form the foundation of the water ecosystem. Because the majority of these species cannot be cultured in vitro, a different approach is needed to identify prokaryotes in environmental water. A novel DNA microarray was developed as a simplified detection protocol. Multiple DNA probes were designed against each of the 97,927 sequences in the DNA Data Bank of Japan and mounted on a glass chip in duplicate. Evaluation of the microarray was performed using the DNA extracted from one liter of environmental water samples collected from seven sites in Japan. The extracted DNA was uniformly amplified using whole genome amplification (WGA), labeled with Cy3-conjugated 16S rRNA specific primers and hybridized to the microarray. The microarray successfully identified soil bacteria and environment-specific bacteria clusters. The DNA microarray described herein can be a useful tool in evaluating the diversity of prokaryotes and assessing environmental changes such as global warming

SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

<div><p>Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.</p></div

Inhibition of SDF-1α-induced platelet aggregation and Akt phosphorylation by raft disruption with methyl-β-cyclodextrin.

<p>(A) 500 ng/ml SDF-1α-induced platelet aggregation with or without pretreatment with 2% methyl-β-cyclodextrin (MβCD) for 15 min (2 donors, n = 4: a duplicated measurement from 2 donors on the same day). Bar represents 1 min. Western blotting (B) and measurement of Akt phosphorylation at Thr308 (C) and Ser473 (D) in resting platelets (lane 1), 500 ng/ml SDF-1α-treated platelets (lane 2), and 500 ng/ml SDF-1α-treated platelets with pretreatment with 2% MβCD for 15 min (lane 3). Phosphorylation was quantified by densitometry. Data are presented as the mean +/- SD of triplicates. **Statistically significant difference (<i>P</i>< .01).</p

SDF-1α-induced Akt phosphorylation at Thr308 and Ser473 and Akt-dependent platelet aggregation.

<p>(A) Washed human platelets were treated with 500 ng/ml SDF-1α for 10 min at 37^°C. Resting platelets (left lane) and SDF-1α-treated platelets (right lane) were lysed in the Laemmli sample buffer containing phosphatase inhibitors and separated by SDS-PAGE. Phosphorylations of Akt at Thr308 (upper panel), Akt at Ser473 (middle panel), and Akt protein (lower panel) were detected by western blotting using specific Akt antibodies. (B) Dose- and time-dependent phosphorylation of Akt in platelets on SDF-1α treatment. Washed human platelets were treated for 10 min at the indicated concentrations (a-c) or at 500 ng/ml SDF-1α for the indicated times (d-f). Cells were lysed in the Laemmli sample buffer containing phosphatase inhibitors and separated by SDS-PAGE. Phosphorylations of Akt at Thr308 and Ser473 were detected by western blotting (a,d). Phosphorylation of Akt at Thr308 (b,e) and Ser473 (c,f) was quantified by densitometry. Data are presented as the mean +/- SD of triplicates. Statistically significant differences (<i>P</i>< .05 shown by *, <i>P</i>< .01 shown by **). (C) Effect of CXCR4 antagonist and PI3 kinase inhibitor on SDF-1α-induced Akt phosphorylation. Western blotting (a) and measurement of Akt phosphorylation at Thr308 (b) and Ser473 (c) in resting platelets (lane 1), 500 ng/ml SDF-1α-treated platelets (lane 2), and 500 ng/ml SDF-1α-treated platelets with pretreatment with 5 μg/ml AMD3100 (lane 3), or 30 μM LY294002 (lane 4) for 10 min. Data are presented as the mean +/- SD of triplicates. **Statistically significant differences (<i>P</i>< .01). (D) Inhibition of SDF-1α-induced platelet aggregation by Akt inhibitor. SDF-1α(200 ng/ml)-induced platelet aggregation without or with pretreatment with 0.3, 1, or 3 μM MK-2206, an Akt inhibitor, for 10 min (2 donors, n = 3: a single measurement from one donor and a duplicate measurement from another donor on the same day). Bar represents 2 min.</p