Responses to Apical and Basolateral Application of Glutamate in Mouse Fungiform Taste Cells with Action Potentials

In taste bud cells, glutamate may elicit two types of responses, as an umami tastant and as a neurotransmitter. Glutamate applied to apical membrane of taste cells would elicit taste responses whereas glutamate applied to basolateral membrane may act as a neurotransmitter. Using restricted stimulation to apical or basolateral membrane of taste cells, we examined responses of taste cells to glutamate stimulation, separately. Apical application of monosodium glutamate (MSG, 0.3 M) increased firing frequency in some of mouse fungiform taste cells that evoked action potentials. These cells were tested with other basic taste compounds, NaCl (salty), saccharin (sweet), HCl (sour), and quinine (bitter). MSG-sensitive taste cells could be classified into sweet-best (S-type), MSG-best (M-type), and NaCl or other electrolytes-best (N- or E/H-type) cells. Furthermore, S- and M-type could be classified into two sub-types according to the synergistic effect between MSG and inosine-5′-monophosphate (S1, M1 with synergism; S2, M2 without synergism). Basolateral application of glutamate (100 μM) had almost no effect on the mean spontaneous firing rates in taste cells. However, about 10% of taste cells tested showed transient increases in spontaneous firing rates (>mean + 2 standard deviation) after basolateral application of glutamate. These results suggest the existence of multiple types of umami-sensitive taste cells and the existence of glutamate receptor(s) on the basolateral membrane of a subset of taste cells.     

Characterization of cysteine protease induced by oxidative stress in cells of Chlamydomonas sp. strain W80

Unlike known Chlamydomonas species, Chlamydomonas sp. strain W80, which was isolated from seawater, shows tolerance to salt and cadmium. In this study, we purified and characterized cysteine protease from Chlamydomonas sp. strain W80 cells and also investigated their response to oxidative stress. The protease was purified 2760-fold with a yield of 2.6% by five steps of successive chromatography. This protease had a pH optimum of 8.0 and was specific only for tert -butoxycarbonyl (Boc)-Leu-Arg-Arg-4-methylcoumaryl-7-amide (MCA) (Boc-LRR-MCA) and Boc-Val-Leu-Lys-MCA as substrates among eight fluorogenic peptides tested. The K _m value was estimated to be 44.4 μ M for Boc-LRR-MCA. The molecular weight of the protease was determined to be approximately 102 kDa by Superdex 200 gel filtration and 60 kDa by SDS–PAGE, suggesting that this enzyme is a dimer. This enzyme was inhibited by the cysteine protease inhibitors leupeptin and N-ethylmaleimide but neither inhibited by phenylmethylsulfonyl fluoride or ethylenediaminetetraacetic acid nor activated by metal cations. These findings indicate that this enzyme is likely a cysteine protease. When strain W80 was grown under oxidative stress in the presence of methyl viologen and cadmium chloride, cysteine protease activity was about 30–90% higher than normal, whereas no changes were observed in carbon enrichment or senescence. It is likely that this protease is upregulated in response to oxidative stress and plays a role in the maintenance of cell metabolism under oxidative stress conditions.     

Within-farm variability in age structure of breeding-female pigs and reproductive performance on commercial swine breeding farms

This study investigated relationships between herd age structure and herd productivity in breeding herds; it also investigated a pattern in parity proportions of females over 2 years and its relationship with herd productivity in commercial swine herds. This study was based on data from 148 commercial farms in North America stored in the swine database program at the University of Minnesota. The primary selection criterion was fluctuations in breeding-female pig (female) inventories over a 2-year interval. Productivity measurements and parity proportions of females were extracted from the database. A 24-month time-plot in proportions of Parity 0 and Parities 3-5 females (mid-parity) was charted for each farm. Using these charts, a change in proportions of Parity 0 and mid-parity for each farm was categorized into patterns: FLUCTUATE (Parity 0 and mid-parity proportion lines crossed) or STABLE (the two proportion lines never crossed). Higher proportions of mid-parity sows were correlated with greater pigs weaned per female per year (PWFY; P < 0.01). Farms with a FLUCTUATE (73% of the 148 farms) pattern had lower PWFY than those with a STABLE pattern (P < 0.01). The STABLE farms had higher proportions of mid-parity sows, higher parity at culling, higher frequency of gilt deliveries per year, and lower replacement rate than the FLUCTUATE farms (P < 0.01). In conclusion, maintaining stable subpopulations with mid-parity and Parity 0 are recommended to optimize herd productivity.     

Effects of Y132H and F145L substitutions on the activity, azole resistance and spectral properties of Candida albicans sterol 14-demethylase P450 (CYP51): a live example showing the selection of altered P450 through interaction with environmental compounds

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.     

Cinnamon extract inhibits the postprandial overproduction of apolipoprotein B48-containing lipoproteins in fructose-fed animals

We have reported previously that a cinnamon extract (CE), high in type A polyphenols, prevents fructose feeding-induced decreases in insulin sensitivity and suggested that improvements of insulin sensitivity by CE were attributable, in part, to enhanced insulin signaling. In this study, we examined the effects of CE on postprandial apolipoprotein (apo) B-48 increase in fructose-fed rats, and the secretion of apoB48 in freshly isolated intestinal enterocytes of fructose-fed hamsters. In an olive oil loading study, a water-soluble CE (Cinnulin PF, 50 mg/kg body weight, orally) decreased serum triglyceride (TG) levels and the over production of total- and TG-rich lipoprotein-apoB48. In ex vivo ³⁵S labeling study, significant decreases were also observed in apoB48 secretion into the media in enterocytes isolated from fructose-fed hamsters. We also investigated the molecular mechanisms of the effects of CE on the expression of genes of the insulin signaling pathway [insulin receptor (IR), IR substrate (IRS)1, IRS2 and Akt1], and lipoprotein metabolism [microsomal TG transfer protein (MTP), sterol regulatory element-binding protein (SREBP1c) in isolated primary enterocytes of fructose-fed hamsters, using quantitative real-time polymerase chain reaction. The CE reversed the expression of the impaired IR, IRS1, IRS2 and Akt1 mRNA levels and inhibited the overexpression of MTP and SREBP1c mRNA levels of enterocytes. Taken together, our data suggest that the postprandial hypertriglycerides and the overproduction of apoB48 can be acutely inhibited by a CE by a mechanism involving improvements of insulin sensitivity of intestinal enterocytes and regulation of MTP and SREBP1c levels. We present both in vivo and ex vivo evidence that a CE improves the postprandial overproduction of intestinal apoB48-containing lipoproteins by ameliorating intestinal insulin resistance and may be beneficial in the control of lipid metabolism.     

Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate.

The LAL test is inhibited or enhanced by many substances. To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This method is composed of two steps. The first step is the adsorption of endotoxins. Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit. The second step is the reaction of adsorbed endotoxins with the LAL reagent. The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay. The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95%. The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.     

Synthesis of Chiral NADH Model Compounds Containing a Sulfur Atom and Their Asymmetric Reduction

Oryzalexins A, B and C were isolated as a group of novel phytoalexins from rice (Oryza sativa) blast leaves infected with Pyricularia oryzae. The basis of the structures of Oryzalexins A, B and C was laid by spectroscopic methods and their inhibitory activities against spore germination and germ tube growth of P. oryzae were assayed.

Oryzalexins A, B and C, isolated from rice leaves infected with P. oryzae as a group of novel phytoalexins, were confirmed to be (+)-sandaracopimaradiene derivatives by chemical and spectroscopic studies, i.e. A: 3-oxy-7-oxo- (I); B: 3-oxo-7-oxy- (II) and C: 3,7-dioxo-(+)-sandaracopimaradiene (III).