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Changes in the expression of hyperpolarization-activated cyclic nucleotide (HCN)-gated channels and I(f) currents during the differentiation of embryonic stem cells into cardiac cells remain unknown. We examined changes of HCN genes in expression and function during the differentiation of Nkx2.5-positive cardiac precursor cells derived from mouse ES cells using cell sorting, RTPCR, immunofluorescence and whole cell patch-clamp techniques. Cs(+)-induced inhibition of automaticity and transcription of HCN genes increased during differentiation. Expressions of Nkx2.5, a marker of cardiac progenitor cell, and Flk1, a marker of hemangioblast, were mutually exclusive. Messenger RNA and proteins encoded by HCN1 and 4 genes were predominantly observed in Nkx2.5-positive cells on day 15, although Flk1-positive cells did not express genes of the HCN family on that day. Cs(+)-induced prolongation of the cycle of spontaneous action potentials and I(f) currents were predominantly observed on day 15. These results suggested that a fraction of Nkx2.5-positive cardiac precursor cells was committed to pacemaking cells expressing I(f) channels predominantly encoded by HCN 1 and 4 genes.

The mouse ES cell line hcgp7/#21, which carries a human chromosome 21 (hChr.21), was used as an in vitro model to examine the effects of hChr.21 on cardiomyocyte differentiation. Cardiomyocytes derived from hcgp7/#21 showed a significant delay in the onset of spontaneous beating. The number of Nkx2.5/GFP(+) cardiac progenitor cells was comparable to that in control ES cells and they also expressed comparable mRNA levels for mesodermal markers, cardiac specific transcription factors, contractile proteins, and L-type Ca 2+ channels. However, cells from hcgp7/#21 expressed significantly reduced levels of mRNA for Cav3.1 and Cav3.2, which was consistent with the decreased number of cells expressing T-type Ca 2+ channels and decreased T-type Ca 2+ channel currents. These findings suggest that the presence of human chromosome 21 suppresses expression of T-type Ca 2+ channels in cardiomyocytes during differentiation, which may be responsible for delayed onset of spontaneous beating.

Proteasomes are the multicatalytic protein complexes with huge molecular weight. It is well known that the ubiquitin proteasome system plays an important role in regulated proteolysis. The 26S proteasome is composed of two 19S regulating components and a 20S proteasome. The 20S proteasome forms barrel shape and is composed of four rings, α- and β-rings; each ring contains highly homological seven α-subunits (1~7) and seven β-subunits (1~7), N-terminal tails of α-ring subunit form a gate to facilitate substrate entry. Each of 1, 2 and 5 β–subunit has different enzyme activity; 1 is caspase-, 2 tryptic- and 5 chymotryptic- like activity, respectively. We found a new protease inhibitor compound-A, which interacts weakly against proteasome. In order to elucidate the binding mode of the compound-A to proteasome, structure determination of the 20S proteosme complexed with compound-A was carried out. Isolated and concentrated 20S proteasome was co-crystallized with compound-A. Sitting drop vapor diffusion method was applied using 0.1M MES-NaOH (pH 6.5), 35% MPD, 50 mM magnesium acetate as a reservoir solution and 10 mg/ml proteasome and 20 mM compound-A as a sample solution. Crystals are isomorphous with the previous report [1]. Diffraction images were recorded at 100 K by using ADSC Quantum 210r CCD detector at NW12A of Photon Factory, Tsukuba, Japan. Initial phases were determined by molecular replacement method using the structure of the yeast 20S proteasome [1RYP] as a starting model and the structure model with a ligand was refined by using Refmac5 in CCP4 program package with an R value of 16.5% at 2.85 Å. The electron densities have been observed at the active site as mentioned above in the β-ring. Binding site of compound-A is closed to Tyr170 and Thr1 of the β5 subunit. The compound-A binds weakly to their residues by hydrogen bondings. It is quite different from the binding mode of the known potent proteasome inhibitor bortezomib [2].

Congenital Adrenal Hyperplasia (CAH) is an adrenal disorders due to impaired activity of one of the enzymes required for cortisol and aldosterone biosynthesis. One of the subtypes of CAH is the salt-wasting (SW) form which there is a renal salt loss due to aldosterone deficiency. Plasma Renin Activity (PRA) is the main index used to evaluate the mineralocorticoid control in CAH. PRA testing is almost very rare done for the CAH patients due to high cost, sophisticated laboratory is not available in all region and no compulsary health insurance in Indonesia. The objective of this research was to describe PRA level in patient with Congenital Adrenal Hyperplasia. This study is a part of CAH cohort study in Center for Biomedical Research (CEBIOR), Semarang, Indonesia. Eighteen patients diagnosed as CAH were drawned blood samples for hormonal test, including 17 OHP, Plasma Renin Activity, Cortisol, and Electrolytes. Clinical history and physical examination was performed to each patients. All 18 patients (17 female and 1 male) have high 17-OHP level and normal electrolites levels, but only 14 patients have PRA data. Four patients did not show their PRA level due to fail in too much blood collection. Out of 14 patient, four patients which had history of SW have very high PRA level, while six patients have PRA level more than normal without history of salt wasting. The mean of PRA level in patients with history of SW (47.72 (SD 18.63)) are higher than the patient without history of SW (13.97 (SD 13.3)). This study suggest that PRA level might be useful for evaluating mineralocorticoid level in CAH. It is proposed to the government to subsidize and provide PRA and other hormonal testing for CAH in many regions with affordable cost.