Phenotypic characterization of a new Grin1 mutant mouse generated by ENU mutagenesis

In the RIKEN large‐scale N‐ethyl‐N‐nitrosourea (ENU) mutagenesis project we screened mice with a dominant mutation that exhibited abnormal behavior in the open‐field test, passive avoidance test and home‐cage activity test. We tested 2045 progeny of C57BL/6J males treated with ENU and untreated DBA/2J females in the open‐field test and isolated behavioral mutant M100174, which exhibited a significant increase in spontaneous locomotor activity. We identified a missense mutation in the Grin1 gene, which encodes NMDA receptor subunit 1, and designated the mutant gene Grin1^Rgsc174. This mutation results in an arginine to cysteine substitution in the C0 domain of the protein. Detailed analyses revealed that Grin1^Rgsc174 heterozygote exhibited increased novelty‐seeking behavior and slight social isolation in comparison with the wild type. In contrast to other Grin1 mutant mice, this mutant exhibited no evidence of heightened anxiety. These results indicate that this is a unique behavioral Grin1 gene mutant mouse that differs from the known Grin1 mutant mice. The results of immunohistochemical and biochemical analyses suggested that impaired interaction between the glutamatergic pathway and dopaminergic pathway may underlie the behavioral phenotypes of the Grin1^Rgsc174 mutant.     

Measurement of density and affinity for dopamine D2 receptors by a single positron emission tomography scan with multiple injections of [11C]raclopride

Positron emission tomography (PET) with [¹¹C]raclopride has been used to investigate the density (B_max) and affinity (K_d) of dopamine D₂ receptors related to several neurological and psychiatric disorders. However, in assessing the B_max and K_d, multiple PET scans are necessary under variable specific activities of administered [¹¹C]raclopride, resulting in a long study period and unexpected physiological variations. In this paper, we have developed a method of multiple-injection graphical analysis (MI-GA) that provides the B_max and K_d values from a single PET scan with three sequential injections of [¹¹C]raclopride, and we validated the proposed method by performing numerous simulations and PET studies on monkeys. In the simulations, the three-injection protocol was designed according to prior knowledge of the receptor kinetics, and the errors of B_max and K_d estimated by MI-GA were analyzed. Simulations showed that our method could support the calculation of B_max and K_d, despite a slight overestimation compared with the true magnitudes. In monkey studies, we could calculate the B_max and K_d of diseased or normal striatum in a 150 mins scan with the three-injection protocol of [¹¹C]raclopride. Estimated B_max and K_d values of D₂ receptors in normal or partially dopamine-depleted striatum were comparable to the previously reported values.     

Glutamate‐induced calcium increase mediates magnesium release from mitochondria in rat hippocampal neurons

Excess administration of glutamate is known to induce Ca²⁺ overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg²⁺ in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg²⁺ in the excitotoxicity, we simultaneously measured intracellular Ca²⁺ and Mg²⁺, using fluorescent dyes, Fura red, a fluorescent Ca²⁺ probe, and KMG‐104, a highly selective fluorescent Mg²⁺ probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg²⁺ concentration ([Mg²⁺]_i). Extracellular Mg²⁺ was not required for this glutamate‐induced increase in [Mg²⁺]_i, and no increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) or [Mg²⁺]_i was observed in neurons in nominally Ca²⁺‐free medium. Application of 5 μM carbonyl cyanide p‐(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca²⁺]_i and [Mg²⁺]_i. Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg²⁺]_i but did induce an additive increase in [Ca²⁺]_i. Moreover, the glutamate‐induced increase in [Mg²⁺]_i was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg²⁺ efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, prevented this [Mg²⁺]_i increase. These results indicate that glutamate‐induced increases in [Mg²⁺]_i result from the Mg²⁺ release from mitochondria and that Ca²⁺ accumulation in the mitochondria is required for this Mg²⁺ release. © 2010 Wiley‐Liss, Inc.     

Impact of age at Kasai operation on short- and long-term outcomes of type III biliary atresia at a single institution

Purpose

We reevaluated the impact of age at Kasai operation on the short- and long-term outcomes of type III biliary atresia (BA).

Patients and Methods

From 1953 to 2009, 242 patients with type III BA underwent Kasai operation at ages ranging between 12 and 421 days (average, 79.7 days). The relationship between age at Kasai operation and jaundice disappearance rates (JDRs), and 10-, 20-, and 30-year native liver survival rates (NLSRs) were assessed retrospectively (JDR [%] = the number of patients in whom jaundice disappeared/the number of patients in each group × 100).

Results

Age at Kasai operation had a significant impact on the JDRs (P < .001). However, there was no statistical relationship between long-term NLSR of the patients in whom jaundice disappeared after Kasai operation and operative age. From the results of the cumulative NLSRs estimated by Kaplan-Meier method, each survival rate was quite dependent on the age at operation until 30 years after Kasai operation, but the difference became much smaller in the later period provided age at operation was 4 months or younger.

Conclusion

The operative age as a prognostic factor might be less significant in the long-term outcome than in the short-term outcome.     

Population pharmacokinetics of a propofol bolus administered in patients with major burns

Objective

In operating theaters and burn units, propofol is commonly used for sedation and anesthesia in patients with major burns. This study determined the population pharmacokinetics of propofol in burns and identified clinically significant covariates.

Method

Seventeen adults, age 42 ± 10 (mean ± SD) years, with 41 ± 19% total body surface area burns, were enrolled at 16 ± 14 days after-burn. Non-burn adults (n = 19) served as controls. After an intravenous bolus of 2 mg/kg propofol, the plasma concentration was determined at designated times for up to 4.5 h. Concentration–time profiles were analyzed using nonlinear mixed-effect modeling.

Results

A three-compartment model gave the best fit. The volume of distribution of the central compartment (V₁) was considerably greater in the burned than non-burned group (48.4 L vs. 27.6 L, respectively). The clearances of the central (CL₁) and slow peripheral (CL₃) compartments were higher in burn patients (4.2 L/min vs. 1.7 L/min and 3.6 L/min vs. 1.1 L/min, respectively). Adding the covariates BURN to V₁, CL₁, and CL₃ and WT (weight) to CL₁ significantly improved the model performance.

Conclusion

The pharmacokinetic characteristics of a propofol bolus administered in patients with major burns were enhanced clearance and expanded volume of distribution. BURN and WT were the important covariates. For sedation or anesthesia induction, a higher than recommended dose of propofol may be required to maintain therapeutic plasma drug concentrations in patients with severe burns. Vigilance regarding the burned individual and careful titration of hypnotics to the desired effect cannot be overemphasized.     

The surgical procedure and clinical results of subtotal stomach preserving pancreaticoduodenectomy (SSPPD) in comparison with pylorus preserving pancreaticoduodenectomy (PPPD)

BACKGROUND: Subtotal stomach preserving pancreaticoduodenectomy (SSPPD) is compared retrospectively with pylorus preserving pancreaticoduodenectomy (PPPD). METHODS: During 2002-2005, 21 patients (13 female, 8 male) underwent SSPPD. The mean age was 64.3 (range 33-80). PPPD was performed for 12 patients after 1999. Days of hospital stay, operation time, operative blood loss, postoperative morbidity and mortality, days of nasogastric intubation, days until liquid diet, delayed gastric emptying, postoperative change of serum Albumin value, were compared between SSPPD and PPPD. Clinical characteristics (age, gender, benign, or malignant condition, presence of preoperative jaundice, preoperative value of serum Albumin) were analyzed in both procedures. RESULTS: In comparison of clinical characteristics, all factors were similar between PPPD and SSPPD. There were also quite similar results in days of hospital stay, operation time, operative blood loss, postoperative morbidity and mortality. Days of nasogastric intubation, days until liquid diet in PPPD were significantly longer than those in SSPPD and the incidence of delayed gastric emptying in PPPD was significantly higher than that in SSPPD. Finally, PPPD and SSPPD postoperative change of serum Albumin value were statistically similar. CONCLUSIONS: We consider SSPPD as one of the most favorable procedures in patients who undergo pancreaticoduodenectomy.     

Development and characterization of IL-21-producing CD4+ T cells

It has recently been shown that interleukin (IL)-21 is produced by Th17 cells, functions as an autocrine growth factor for Th17 cells, and plays critical roles in autoimmune diseases. In this study, we investigated the differentiation and characteristics of IL-21-producing CD4(+) T cells by intracellular staining. Unexpectedly, we found that under Th17-polarizing conditions, the majority of IL-21-producing CD4(+) T cells did not produce IL-17A and -17F. We also found that IL-6 and -21 potently induced the development of IL-21-producing CD4(+) T cells without the induction of IL-4, IFN-gamma, IL-17A, or IL-17F production. On the other hand, TGF-beta inhibited IL-6- and IL-21-induced development of IL-21-producing CD4(+) T cells. IL-2 enhanced the development of IL-21-producing CD4(+) T cells under Th17-polarizing conditions. Finally, IL-21-producing CD4(+) T cells exhibited a stable phenotype of IL-21 production in the presence of IL-6, but retained the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions. These results suggest that IL-21-producing CD4(+) T cells exhibit distinct characteristics from Th17 cells and develop preferentially in an IL-6-rich environment devoid of TGF-beta, and that IL-21 functions as an autocrine growth factor for IL-21-producing CD4(+) T cells.     

Ca(2+) influx through P2X receptors induces actin cytoskeleton reorganization by the formation of cofilin rods in neurites

In physiological and pathological events, extracellular ATP plays an important role by controlling several types of purinergic receptors and changing cytoskeleton dynamics. To know the process of ATP-dependent cytoskeleton remodeling, we focused on cofilin, a key regulator of actin cytoskeleton, and investigated the dynamics of cofilin in PC12 cells through fluorescent protein-labeled cofilin and actin, Ca(2+) imaging, and fluorescence resonance energy transfer (FRET) techniques. As a result, ATP induced intracellular Ca(2+) increase, following cofilin rods' formation. ATP-induced cofilin rods' formation was not observed in cells expressing unphosphorylatable variant of cofilin. A P2X receptor agonist, but not P2Y, induced the formation of cofilin rods, whereas calmodulin and calcineurin inhibitors suppressed it. These results indicate that Ca(2+) influx through P2X receptors induces the formation of cofilin rods via calcineurin-dependent dephosphorylation of cofilin. This pathway might be one candidate to explain the effects of ATP on neuronal development and injury.