Development of an optimized 5-stage protocol for the in vitro preparation of insulin-secreting cells from mouse ES cells

In order to produce insulin-secreting cells with a high value of glucose-stimulated insulin secretion (GSIS) from mouse embryonic stem cells, we have developed an optimized 5-stage protocol by referring to culture conditions so far reported elsewhere. This protocol is characterized by 4 points: (1) use of an activin-free medium in the first stage, (2) use of gelatin/fibronectin coated culture dishes in 1–4 stages throughout, (3) removal of undifferentiated cells by cell sorter at the end of 4th stage, and (4) sedimental culture in the 5th stage. GSIS value of the produced cells reached 2.4, that was at a higher rank of those so far reported. The produced cells were transplanted in diabetes model mice but no remedy effect was observed. Then transplantation was conducted in pre-diabetes model mice, in which GSIS was impaired without affecting insulin producing function. The transplantation of 5 × 10⁶ cells resulted in a marked improvement of glucose tolerance within 20 days. This effect decreased but was still observed at 120 days post-transplantation. This demonstrates the feasibility of the novel optimized protocol.     

Multifunctional polymeric micelles with folate-mediated cancer cell targeting and pH-triggered drug releasing properties for active intracellular drug delivery

A new type of multifunctional polymeric micelle drug carrier for active intracellular drug delivery was prepared and characterized in this study. The micelle is a nano-supramolecular assembly with a spherical core-shell structure, and its surface and core were modified with piloting molecules for cancer cells and pH-sensitive drug binding linkers for controlled drug release, respectively. In order to prepare such micelles, self-assembling amphiphilic block copolymers, folate-poly(ethylene glycol)-poly(aspartate hydrazone adriamycin) [Fol-PEG-P(Asp-Hyd-ADR)], were specially designed and synthesized by installing a molecular promoter to enhance intracellular transport, folate (Fol), at the end of the shell-forming PEG chain and conjugating the anticancer drug, adriamycin (ADR), to the side chain of the core-forming PAsp segment through an acid-sensitive hydrazone bond. Because folate-binding proteins (FBP) are selectively overexpressed on the cancer cell membranes, the folate-bound micelles (FMA) can be guided to the cancer cells in the body, and after the micelles enter the cells, hydrazone bonds are cleaved by the intracellular acidic environment (pH 5-6) so that the drug release profile of the micelles is controlled pH-dependently. In this regard, FBP-binding selectivity of the prepared FMA was evaluated by surface plasmon resonance (SPR) measurements. The tetrazolium dye method (MTT assay) using human pharyngeal cancer cells (KB cell) revealed that FMA significantly improved cell growth inhibitory activity in spite of a short exposure time due to the selective and strong interaction between folate molecules and their receptors. Subsequent flow cytometric analysis showed that cellular uptake of FMA significantly increased. Consequently, these findings would provide one of the most effective approaches for cancer treatment using intracellular environment-targeting supramolecular drug carriers.     

Metabotropic Glutamate Receptor in C6BU-1 Glioma Cell Has NMDA Receptor-Ion Channel Complex-Like Properties and Interacts with Serotonin2 Receptor-Stimulated Signal Transduction

Abstract: We found in cultured glioma (C6BU-1) cells that excitatory amino acids (EAAs) such as glutamate, N-methyl-d -aspartate (NMDA), aspartate, and metabotropic glutamate receptor agonist trans-(±)-1-amino-1,3-cyclopentanedicarboxylate caused an increase in the inositol 1,4,5-trisphosphate formation and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Mg²⁺ and Ca²⁺. Pertussis toxin treatment abolished this glutamate-induced [Ca²⁺]_i increase. Various antagonists against NMDA receptor-ion channel complex, such as Mg²⁺, d -2-amino-5-phosphonovalerate (d -APV), HA-966, and MK-801, also inhibited the increase in [Ca²⁺]_i induced by glutamate. These results indicate that these metabotropic EAA receptors coupled to pertussis toxin-susceptible GTP-binding protein and phospholipase C system in C6BU-1 glioma cells have the pharmacological properties of NMDA receptor-ion channel complexes. We also found that in the presence of Mg²⁺ these metabotropic receptors resemble the NMDA receptor-ion channel complex interacted with 5-hydroxytryptamine₂ (5-HT₂) receptor signaling. EAAs inhibited 5-HT₂ receptor-mediated intracellular Ca²⁺ mobilization and inositol 1,4,5-trisphosphate formation in a concentration-dependent manner. The inhibitory effect of glutamate was reversed by various NMDA receptor antagonists (d -APV, MK-801, phencyclidine, and HA-966), but l -APV failed to block the inhibitory effect of glutamate. The same result was observed in the absence of extracellular Ca²⁺. In addition, this inhibitory effect on 5-HT₂ receptor-mediated signal transduction was abolished by treatment of C6BU-1 cells with pertussis toxin, whereas 5-HT₂ receptor-mediated [Ca²⁺]_i increase was not abolished by pertussis toxin treatment. We can, therefore, conclude that the inhibitory effect of glutamate is not a result of the influx of Ca²⁺ through the ion channel and that it operates via metabotropic glutamate receptors, having NMDA receptor-ion channel complex-like properties and being coupled with pertussis toxin-sensitive GTP-binding protein and phospholipase C.     

Possible Mechanism of Dantrolene Stabilization of Cultured Neuroblastoma Cell Plasma Membranes

Abstract: Some reports have suggested that dantrolene interacts directly with the membrane bilayer. We investigated effects of dantrolene on changes in membrane properties induced by compound 48/80 (C48/80), a membrane stimulator. The addition of C48/80 for 1 min elicited a rapid, dose-dependent Ca²⁺ influx, which was reduced to 14% by the absence of external Ca²⁺. Dantrolene inhibited the C48/80-induced increase in Ca²⁺ permeability of plasma membranes in a concentration-dependent manner (0.33–10 µ M , IC₅₀ value was 5 µ M ). We next examined C48/80-induced changes in structural and dynamic membrane properties by electron spin resonance (ESR). The ratio h ₀/ h ₋₁ was determined to evaluate membrane fluidity. C48/80 increased the membrane fluidity in a concentration-dependent manner (0.1–0.56 mg/ml). Dantrolene (10 µ M ) itself did not change the membrane fluidity, but it significantly reduced the C48/80-induced increase in membrane fluidity (0.56 mg/ml). Moreover, the C48/80-induced increase in fluidity was dependent on extracellular Ca²⁺. We conclude that dantrolene protects neuroblastoma cell plasma membrane from C48/80-induced membrane perturbation, which causes Ca²⁺ influx and an increase in membrane fluidity. These findings strongly suggest that dantrolene directly stabilizes the neuronal plasma membrane.     

Identification of short side chain bile acids in urine of patients with cerebrotendinous xanthomatosis

Urine from patients with cerebrotendinous xanthomatosis (CTX) was found to contain a number of minor bile acids along with three major bile acids, 7-epicholic acid, norcholic acid, and cholic acid. The following minor bile acids were identified by combined gas-liquid chromatography-mass spectrometry: 7-ketobisnordeoxycholic acid; 12-ketobisnorchenodeoxycholic acid; 7-ketonordeoxycholic acid; 12-ketochenodeoxycholic acid; 7-ketodeoxycholic acid; 12-ketochendeoxycholic acid; bisnorcholic acid; allonorcholic acid; allocholic acid; 1 beta-hydroxybisnorcholic acid; 1 beta-hydroxynorcholic acid; 1 beta-hydroxycholic acid; 2 beta-hydroxybisnorcholic acid; 2 beta-hydroxy-norcholic acid; 2 beta-hydroxycholic acid. The presence of C22 and C23 bile acids in urine of the CTX patients suggests that bile alcohols having a hydroxyl group at C22 or C23 in the side chain may be further degraded to these bile acids.     

In Vivo Determination of 5-Hydroxytryptamine Receptor-Stimulated Phosphoinositide Turnover in Rat Brain

Phosphoinositide turnover stimulated by 5-hydroxytryptamine (5-HT) receptors in the intact rat brain was studied using an in vivo method. Phosphoinositides in the rat brain were prelabeled with [3H]inositol injected into the lateral cerebral ventricles. The rats were killed by microwave irradiation after 48 h and the contents in the frontal cortex of 3H-inositol phosphates, [3H]inositol-1-monophosphate [( 3H]IP1), [3H]inositol-1,4-bisphosphate [( 3H]IP2), and a mixture of [3H]inositol-1,4,5-trisphosphate and [3H]inositol-1,3,4-trisphosphate [( 3H]IP3) were assayed by HPLC. Lithium treatment (10 mEq/kg, i.p., 2 h before) increased the content of [3H]IP1 and [3H]IP2. 5-Methoxy-N,N-dimethyltryptamine (5-MeODMT) and quipazine, 5-HT agonists, significantly increased the amount of 3H-inositol phosphates under lithium pretreatment. The response to 5-MeODMT was inhibited by ritanserin, a 5-HT2 antagonist, but not by (-)-propranolol, a 5-HT1 antagonist. These results suggest that phosphoinositide turnover in the rat frontal cortex in vivo is stimulated by 5-HT2 receptor activation. It is considered that this method will be useful for measurement of 5-HT2 receptor-stimulated phosphoinositide turnover in vivo to examine the in vivo effects of various psychotropic drugs such as antidepressants.     

Serum Procalcitonin Level and SOFA Score at Discharge from the Intensive Care Unit Predict Post-Intensive Care Unit Mortality: A Prospective Study

Purpose

The final decision for discharge from the intensive care unit (ICU) is uncertain because it is made according to various patient parameters; however, it should be made on an objective evaluation. Previous reports have been inconsistent and unreliable in predicting post-ICU mortality. To identify predictive factors associated with post-ICU mortality, we analyzed physiological and laboratory data at ICU discharge.

Methods

Patients admitted to our ICU between September 2012 and August 2013 and staying for critical care>2 days were included. Sequential Organ Failure Assessment (SOFA) score; systemic inflammatory response syndrome score; white blood cell count; and serum C reactive protein, procalcitonin (PCT), interleukin-6 (IL-6), lactate, albumin, and hemoglobin levels were recorded. The primary end point was 90-day mortality after ICU discharge. Two hundred eighteen patients were enrolled (195 survivors, 23 non-survivors).

Results

Non-survivors presented a higher SOFA score and serum PCT, and IL-6 levels, as well as lower serum albumin and hemoglobin levels. Serum PCT, albumin, and SOFA score were associated with 90-day mortality in multiple logistic regression analysis. Hosmer-Lemeshow test showed chi-square value of 6.96, and P value of 0.54. The area under the curve (95% confidence interval) was 0.830 (0.771–0.890) for PCT, 0.688 (0.566–0.810) for albumin, 0.861 (0.796–0.927) for SOFA score, and increased to 0.913 (0.858–0.969) when these were combined. Serum PCT level at 0.57 ng/mL, serum albumin at 2.5 g/dL and SOFA score at 5.5 predict 90-day mortality, and high PCT, low albumin and high SOFA groups had significantly higher mortality. Serum PCT and SOFA score were significantly associated with survival days after ICU discharge in Cox regression analysis.

Conclusions

Serum PCT level and SOFA score at ICU discharge predict post-ICU mortality and survival days after ICU discharge. The combination of these two and albumin level might enable accurate prediction.     

Association of TNFAIP3 interacting protein 1, TNIP1 with systemic lupus erythematosus in a Japanese population: a case-control association study

Introduction

TNFAIP3 interacting protein 1, TNIP1 (ABIN-1) is involved in inhibition of nuclear factor-κB (NF-κB) activation by interacting with TNF alpha-induced protein 3, A20 (TNFAIP3), an established susceptibility gene to systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Recent genome-wide association studies revealed association of TNIP1 with SLE in the Caucasian and Chinese populations. In this study, we investigated whether the association of TNIP1 with SLE was replicated in a Japanese population. In addition, association of TNIP1 with RA was also examined.

Methods

A case-control association study was conducted on the TNIP1 single nucleotide polymorphism (SNP) rs7708392 in 364 Japanese SLE patients, 553 RA patients and 513 healthy controls.

Results

Association of TNIP1 rs7708392C was replicated in Japanese SLE (allele frequency in SLE: 76.5%, control: 69.9%, P = 0.0022, odds ratio [OR] 1.40, 95% confidence interval [CI] 1.13-1.74). Notably, the risk allele frequency in the healthy controls was considerably greater in Japanese (69.9%) than in Caucasians (24.3%). A tendency of stronger association was observed in the SLE patients with renal disorder (P = 0.00065, OR 1.60 [95%CI 1.22-2.10]) than in all SLE patients (P = 0.0022, OR 1.40 [95%CI 1.13-1.74]). Significant association with RA was not observed, regardless of the carriage of human leukocyte antigen DR β1 (HLA-DRB1) shared epitope. Significant gene-gene interaction between TNIP1 and TNFAIP3 was detected neither in SLE nor RA.

Conclusions

Association of TNIP1 with SLE was confirmed in a Japanese population. TNIP1 is a shared SLE susceptibility gene in the Caucasian and Asian populations, but the genetic contribution appeared to be greater in the Japanese and Chinese populations because of the higher risk allele frequency. Taken together with the association of TNFAIP3, these observations underscore the crucial role of NF-κB regulation in the pathogenesis of SLE.     

Arginine-mediated dissociation of single cells and cell sheets from a polystyrene culture dish

ABSTRACT

Here, we report a novel non-enzymatic cell dissociation method, based on our finding that adherent cells dissociate rapidly from the polystyrene culture dish when incubated in an l- or d-arginine-containing solution. We also demonstrate the successful detachment of confluent NIH/3T3 cell monolayers from the culture dish as a cell sheet by the addition of an arginine solution.