Amino acid taste receptor regulates insulin secretion in pancreatic β-cell line MIN6 cells

Amino acids such as L-glutamate and L-arginine are potent stimuli for insulin secretion from pancreatic β-cells. However, the precise molecular mechanisms of amino acid-induced insulin secretion have only partly understood. In this study, we show here that mouse pancreatic β-cell line MIN6 cells expressed amino acid taste receptor (heterodimer of type 1 taste G protein-coupled receptor member 1 and member 3; Tas1R1 and Tas1R3, respectively). We found that administration of L-glutamate or L-arginine to MIN6 cells caused the increase in free intracellular concentrations of both inositol-1,4,5-triphosphate (IP(3)) and Ca(2+) , and umami substance inocinate enhanced the effects of l-glutamate. Effects of amino acids on intracellular IP(3) and Ca(2+) concentration were diminished by application of lactisole, a Tas1R3 receptor antagonist. Furthermore, we investigated the effect of amino acids on the insulin release from MIN6 cells by both ELISA and total internal reflection fluorescence microscopy. Application of L-glutamate or L-arginine significantly increased the release of insulin, whereas inhibited by the application of lactisole. Based on these findings, we propose that heterodimer of Tas1R1 and Tas1R3 is the fundamental receptor for the sensing amino acids and regulates the amino acid-induced insulin secretion in pancreatic β-cells.     

Involvement of aquaporin-5 in differentiation of human gastric cancer cells

Litttle is known about the function of aquaporin (AQP) water channels in human gastric cancer. In the upper or middle part of human stomach, we found that expression level of AQP5 protein in intestinal type of adenocarcinoma was significantly higher than that in accompanying normal mucosa. AQP5 was localized in the apical membrane of the cancer cells. On the other hand, both AQP3 and AQP4 were not up-regulated in the adenocarcinoma. To elucidate the role of AQP5 in cancer cells, AQP5 was exogenously expressed in a cell line of poorly differentiated human gastric adenocarcinoma (MKN45). The AQP5 expression significantly increased the proportion of differentiated cells with a spindle shape, the activity of alkaline phosphatase, a marker for the intestinal epithelial cell type of cancer cells, and the expression level of laminin, an epithelial cell marker. Treatment of the MKN45 cells stably expressing AQP5 with HgCl₂, an inhibitor of aquaporins, significantly decreased the proportion of differentiated cells and the activity of alkaline phosphatase. Our results suggest that up-regulation of AQP5 may be involved in differentiation of human gastric cancer cells. T. Watanabe and T. Fujii have equally contributed to this work.     

Mutations in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene cause autosomal recessive nonsyndromic hearing loss

In two large Turkish consanguineous families, a locus for autosomal recessive nonsyndromic hearing loss (ARNSHL) was mapped to chromosome 6p21.3 by genome-wide linkage analysis in an interval overlapping with the loci DFNB53 (COL11A2), DFNB66, and DFNB67. Fine mapping excluded DFNB53 and subsequently homozygous mutations were identified in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene, also named tetraspan membrane protein of hair cell stereocilia (TMHS) gene, which was recently shown to be mutated in the "hurry scurry" mouse and in two DFNB67-linked families from Pakistan. In one family, we found a homozygous one-base pair deletion, c.649delG (p.Glu216ArgfsX26) and in the other family we identified a homozygous transition c.494C>T (p.Thr165Met). Further screening of index patients from 96 Turkish ARNSHL families and 90 Dutch ARNSHL patients identified one additional Turkish family carrying the c.649delG mutation. Haplotype analysis revealed that the c.649delG mutation was located on a common haplotype in both families. Mutation screening of the LHFPL5 homologs LHFPL3 and LHFPL4 did not reveal any disease causing mutation. Our findings indicate that LHFPL5 is essential for normal function of the human cochlea.     

Calcification by MC3T3-E1 cells on RGD peptide immobilized on titanium through electrodeposited PEG

The effect of a cell-adhesive peptide containing Arg-Gly-Asp (RGD) immobilized through poly(ethylene glycol) (PEG) on titanium (Ti) on calcification by MC3T3-E1 cells was investigated to develop a new surface modification technique using biofunctional molecules. RGD was immobilized on Ti through PEG, both terminals of which were terminated with –NH₂ and –COOH to combine with the Ti surface and RGD. PEG was immobilized on Ti with electrodeposition, and RGD, with immersion. For comparison, glycine was employed because it is the simplest molecule containing both –NH₂ and –COOH at its terminals. MC3T3-E1 cells were cultured and differentiation-induced on each specimen, and the cell calcification properties were investigated. As a result, there was no significant difference in the morphology and extension of MC3T3-E1 cells cultured on each specimen, while the number of cells cultured on RGD/PEG/Ti was the largest. After differentiation-induction, there was no significant difference in the ALP activity among all specimens. On the other hand, the level of cell calcification on RGD/PEG/Ti was the highest. Therefore, the hard tissue compatibility of Ti is improved by immobilizing RGD through functional molecules which have a long molecular chain.     

New variant groups identified from HGV isolates

Summary.  We have determined the primary sequence of the 5^′ noncoding region (5^′ NCR) and putative helicase regions (NS-3) of hepatitis G virus (HGV) and GB virus C (GBV-C) that were isolated in Japan from suspected cases of nonA-nonB and/or nonA-nonB-nonC viral hepatitis by using RT-PCR, and we compared the newly isolated sequences with three established isolates. The addition of a "G" residue was found at the 5^′ terminus of all 8 Japanese isolates. These isolates were more clearly distinguished from the prototype viruses by comparison with the 5^′ NCR sequence than by comparison with the NS-3 region. Our results suggested that at least three distinct genomic variants of HGV exist. Genotyping of HGV by using RT-PCR based on the sequence of the 5^′ NCR seems highly feasible. Accepted November 18, 1996 Received September 11, 1996     

Recruitment and rate coding organisation for soleus motor units across entire range of voluntary isometric plantar flexions

Unlike upper limb muscles, it remains undocumented as to how motor units in the soleus muscle are organised in terms of recruitment range and discharge rates with respect to their recruitment and de-recruitment thresholds. The possible influence of neuromodulation, such as persistent inward currents (PICs) on lower limb motor unit recruitment and discharge rates has also yet to be reported. To address these issues, electromyographic (EMG) activities from the soleus muscle were recorded using selective branched-wire intramuscular electrodes during ramp-and-hold contractions with intensities up to maximal voluntary contraction (MVC). The multiple single motor unit activities were then derived using a decomposition technique. The onset–offset hysteresis of motor unit discharge, i.e. a difference between recruitment and de-recruitment thresholds, as well as PIC magnitude calculated by a paired motor unit analysis were used to examine the neuromodulatory effects on discharge behaviours, such as minimum firing rate, peak firing rate and degree of increase in firing rate. Forty-two clearly identified motor units from five subjects revealed that soleus motor units are recruited progressively from rest to contraction strengths close to 95% of MVC, with low-threshold motor units discharging action potentials slower at their recruitment and with a lower peak rate than later recruited high-threshold units. This observation is in contrast to the 'onion skin phenomenon' often reported for the upper limb muscles. Based on positive correlations of the peak discharge rates, initial rates and recruitment order of the units with the magnitude of the onset–offset hysteresis and not PIC contribution, we conclude that discharge behaviours among motor units appear to be related to a variation in an intrinsic property other than PICs.     

Appearance of the LAT protein at an early stage of B-cell development and its possible role

The linker protein LAT is expressed mainly in T and natural killer (NK) cells. LAT-deficient mice have an arrest of intrathymic T-cell development at the CD4+ CD8+ stage and lack mature T cells in the periphery. However, no gross abnormality in development and function of the B and NK cells has been described. Here we report that LAT is expressed in mouse progenitor B (pro-B) and precursor B (pre-B) cells, but not in immature or mature B cells. LAT in pre-B cells becomes tyrosine phosphorylated upon cross-linking of the pre-B-cell receptor (pre-BCR) by anti- micro antibody. Incubation of 1xN/2b (mouse pre-B-cell line) cells or bone marrow cells from microMT/ microMT mice, which lack B cells after the small pre-B-cell stage, with anti-Ig beta antibody resulted in the downregulation of LAT expression. Transgenic mice which expressed LAT protein in B-lineage cells showed an increased proportion of pro- and large pre-B cells in the bone marrow and a remarkable reduction in the numbers of mature B cells in peripheral lymphoid tissues. Collectively, the present results indicate that LAT is expressed in the cells at the early stages of B-lineage development, but is absent in immature and mature B cells. LAT may play a crucial role in the negative regulation of B-cell development at the transition from pre-B to mature B-cell stages, and signal(s) via the pre-BCR may extinguish LAT expression, thus allowing pre-B-cell differentiation towards the mature B-cell stage.     

Characterization of the receptors for peptide-YY and avian pancreatic polypeptide in chicken and pig brains

We have previously identified the peptide-YY (PYY) receptor on porcine brain membranes as a 50-kDa protein after chemical cross-linking. PYY receptors are discretely distributed in the brain of various mammals, to which neuropeptide-Y (NPY), but not pancreatic polypeptide (PP), bind with great specificity. The present study was carried out in order 1) to identify and characterize the PYY receptor in the avian brain, 2) to compare it with the APP receptor that had been demonstrated in the cerebellum, and 3) to examine [125I]APP-binding activity in the porcine brain. [125I]PYY was bound to chicken brain membranes via high affinity (Kd = 2.19 x 10(-10) M) and low affinity (Kd = 1.93 x 10(-7) M) components. The binding sites were highly specific for PYY and APP as well as for NPY and PPP, coupled to a guanine nucleotide regulatory protein, and distributed in various brain areas, including the cerebellum. The C-terminal fragments of PYY, PYY-(17-36) and PYY-(24-36), exhibited low potency in inhibiting binding, but behaved like full agonists. Porcine brain membranes, on the other hand, possessed two orders of the APP-binding sites, a high affinity component (Kd = 4.24 x 10(-9) M) and a low affinity component (Kd = 3.08 x 10(-7) M). APP binding showed a high specificity for APP, but not for PPP, NPY, or PYY. The binding activity was highest in the pituitary gland, followed by the hippocampus, amygdala, cerebral cortex, hypothalamus, and cerebellum. Guanosine 5'-O-thiotriphosphate, a nonhydrolyzable GTP analog, did not inhibit the binding of [125I]APP to porcine or chicken brain membranes, which ran counter to the results of PYY receptors in both species. Cross-linking studies have demonstrated that receptor-bound [125I]APP is cross-linked to a protein of 67 kDa without disulfide-linked subunits in both porcine and chicken brain membranes. In the latter species, [125I]PYY and [125I]NPY were also cross-linked to the same 67-kDa proteins, which were different from the receptor proteins (50 kDa) in mammalian species. These results indicate that chicken brain has receptors specific for PYY and NPY, as was found in mammalian brains, and that PYY, NPY, and PP act in the brain through interaction at multiple receptor sites, which are similar to and shared by other members of the PP family. Furthermore, the finding that APP-binding sites in porcine brain are more specific than those in avian brain suggests that an endogenous peptide similar to APP may exist in porcine brain.     

Decreased resting energy expenditure in patients with Duchenne muscular dystrophy

Background

Skeletal muscle metabolism is a major determinant of resting energy expenditure (REE). Although the severe muscle loss that characterizes Duchenne muscular dystrophy (DMD) may alter REE, this has not been extensively investigated.

Methods

We studied REE in 77 patients with DMD ranging in age from 10 to 37 years using a portable indirect calorimeter, together with several clinical parameters (age, height, body weight (BW), body mass index (BMI), vital capacity (VC), creatine kinase, creatinine, albumin, cholinesterase, prealbumin), and assessed their influence on REE. In addition, in 12 patients maintaining a stable body weight, the ratio of energy intake to REE was calculated and defined as an alternative index for the physical activity level (aPAL).

Results

REE (kcal/day, mean ± SD) in DMD patients was 1123 (10–11 years), 1186 ± 188 (12–14 years), 1146 ± 214 (15–17 years), 1006 ± 136 (18–29 years) and 1023 ± 97 (?30 years), each of these values being significantly lower than the corresponding control (p < 0.0001). VC (p < 0.001) was the parameter most strongly associated with REE, followed by BMI (p < 0.01) and BW (p < 0.05). The calculated aPAL values were 1.61 (10–11 years), 1.19 (12–14 years), 1.16 (15–17 years), and 1.57 (18–29 years).

Conclusion

The REE in DMD patients was significantly lower than the normal value in every age group, and strongly associated with VC. Both the low REE and PAL values during the early teens, resulting in a low energy requirement, might be related to the obesity that frequently occurs in this age group. In contrast, the high PAL value in the late stage of the disease, possibly due to the presence of respiratory failure, may lead to a high energy requirement, and thus become one of the risk factors for development of malnutrition.     

Predictive coding and pitch processing in the auditory cortex

In this work, we show that electrophysiological responses during pitch perception are best explained by distributed activity in a hierarchy of cortical sources and, crucially, that the effective connectivity between these sources is modulated with pitch strength. Local field potentials were recorded in two subjects from primary auditory cortex and adjacent auditory cortical areas along the axis of Heschl's gyrus (HG) while they listened to stimuli of varying pitch strength. Dynamic causal modeling was used to compare system architectures that might explain the recorded activity. The data show that representation of pitch requires an interaction between nonprimary and primary auditory cortex along HG that is consistent with the principle of predictive coding.