1H and 13C NMR assignments for the glycans in glycoproteins by using 2H/13C-labeled glucose as a metabolic precursor

In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-¹³C₆,²H₇]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with ¹³C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-¹³C₆,²H₇]glucose.     

Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.     

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.     

Serum melatonin levels and insomnia in patients with Machado-Joseph Disease

We previously reported a patient with Machado-Joseph Disease (MJD) who had severe insomnia and a low serum melatonin (MLT) level, and whose insomnia was alleviated by oral MLT replacement therapy. The aims of this study were to examine whether patients with MJD are likely to have insomnia, and whether there is a relationship between the degree of insomnia and the serum MLT level among patients with MJD. This study included 8 patients with MJD. A 58-year-old-patient with cervical spondylosis was also included in this study to check the condition of the test room for sleeping. All patients filled out the Japanese version of Pittsburgh Sleep Quality Index (PSQI-J) questionnaire. We obtained blood samples at 12:00 and 24:00 hours to measure the MLT level. We checked the sleep condition of the patient once an hour and recorded the grade in sleep-logs: the grades of sleep condition were asleep, sleepy, or awake. Statistical analyses were performed to search for correlations between the PSQI score and the serum MLT level or actual sleep time using Spearman’s rank correlation coefficient. Seven of the 8 MJD patients had a total PSQI score of above 5.5 (cut-off level). The daytime MLT level (at 12:00 hours) was below 2.8 pg/mL in all 8 patients, whereas the mean night-time MLT level (at 24:00 hours) of the MJD patients (23.6 ± 17.5 pg/mL) was lower than that of the control patient (43.0 pg/mL) and also lower than the reported cut-off level among healthy people aged 30–50 years (55.5 pg/mL). There was a negative correlation between the total PSQI score and the serum MLT level among the MJD patients (P < 0.05). Our results show that a low serum MLT level may contribute to insomnia in patients with MJD.

     

5a-Carba-β-D-glucopyranose derivatives as novel sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors for the treatment of type 2 diabetes

5a-Carba-β-D-glucopyranose derivatives were synthesized and identified as novel SGLT2-selective inhibitors. These inhibitors exhibited potent SGLT2 inhibition with high selectivity over SGLT1. Among the tested compounds, 6f indicated the most potent hSGLT2 inhibition and the highest selectivity over hSGLT1. Moreover, the pharmacokinetics data also showed that 6h, which had the same aglycon structure as sergliflozin-active (3-active), had a threefold longer half-life time (T(1/2)) than sergliflozin (3) with a high distribution volume in db/db mice. Subsequently, 6h lowered blood glucose levels as much as 3 and showed longer hypoglycemic action than 3 in db/db mice.     

Morphology of Gambierdiscus scabrosus sp. nov. (Gonyaulacales): a new epiphytic toxic dinoflagellate from coastal areas of Japan

A new epiphytic dinoflagellate is described, G ambierdiscus scabrosus sp. nov., from tidal pools and rocky shores along the coastal areas of Japan. Cells are 63.2 ± 5.7 μm in depth, 58.2 ± 5.7 μm in width, and 37.3 ± 3.5 μm in length. The plate formula of G . scabrosus is Po, 4′, 0a, 6′′, 6c, ?s, 5′′′, 0p, and 2′′′′. Morphologically, G . scabrosus resembles G . belizeanus as follows: anterioposteriorly compressed cell shape, narrow 2′′′′ plate, and areolated surface. Despite this similarity, the cells of G . scabrosus can be distinguishable by the presence of the asymmetric shaped 3′′ plate and the rectangular shaped 2′ plate.