An artificial sweetener stimulates the sweet taste in insect: dual effects of glycyrrhizin in Phormia regina.

Glycyrrhizin, found in the root of licorice (Glycyrrhizia glabra), has been used extensively as a non-sugar sweetener for humans and also as a medicine. As far as we know, the present work is the first report describing that a non-sugar sweetener for humans induces a sweet taste in insects. In behavioural experiments, we found that glycyrrhizin induced the feeding response, including full proboscis extension in the blowfly, Phormia regina. Glycyrrhizin also induced impulses of the sugar receptor cell in the labellar chemosensillum, which is highly specialized for the tastes of sugars and nucleotides. The optimum concentration of glycyrrhizin was 3.0 mM, which is much lower than that of sucrose. It has been established that multiple receptor sites, the pyranose receptor site (P site) and the furanose receptor site (F site), are present in the sugar receptor cell of the blowfly and the fleshfly. The inhibitors specific to the P site, starch and PCMB (p-chloromercuribenzoate), partially inhibited glycyrrhizin-induced responses but not levan (an inhibitor to the F site), indicating that the P site on the sugar receptor cell is involved in the glycyrrhizin action but not the F site. When 30 s stimulation with 3.0 mM glycyrrhizin was repeated with an interval of 3--10 min, the impulse frequency to the second stimulus was higher than that to the first one and doubled within 6 min. The first stimulus lasting longer than 10 s potentiated the impulse generation and reduced the adaptation rate during the second stimulus. These results suggest that, in addition to the action via the P site, an additional mechanism, possibly in the signal transduction cascade of the sugar receptor cell, may be involved in the action of glycyrrhizin.

Auxin is a positive regulator for ethylene-mediated response in the growth of Arabidopsis roots.

The requirement of auxin for the ethylene-mediated growth response in the root of Arabidopsis thaliana seedlings was investigated using two ethylene-resistant mutants, aux1-7 and eir1-1, whose roots have been shown to have a defect in the auxin influx and efflux carriers, respectively. A 50% inhibition of growth (I(50)) was achieved with 0.84 microl liter(-1) ethylene in wild-type roots, but 71.3 microl liter( -1) ethylene was required to induce I(50) in eir1-1 roots. In aux1-7 roots, I(50) was not obtained even at 1,000 microl liter(-1) ethylene. By contrast, in the presence of 10 nM 1-naphthaleneacetic acid (NAA), the concentrations of ethylene required to induce I(50) in eir1-1 and aux1-7 roots were greatly reduced nearly to the level required in wild-type roots. Since the action of NAA to restore the ethylene response in aux1-7 roots was not replaced by IAA, an increase in the intracellular level of auxin is likely to be the cause for the restoration of ethylene response. NAA at 10 nM did not inhibit root growth when applied solely, but it was the optimum concentration to recover the ethylene response in the mutant roots. These results suggest that auxin is a positive regulator for ethylene-induced inhibition in root elongation.

Chromosaponin I specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots.

We have found that chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea (Pisum sativum L. cv Alaska), specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots. Application of 60 microM CSI disrupts the vertically oriented elongation of wild-type roots grown on agar plates but orients the elongation of agravitropic mutant aux1-7 roots toward the gravity. The CSI-induced restoration of gravitropic response in aux1-7 roots was not observed in other agravitropic mutants, axr2 and eir1-1. Because the aux1-7 mutant is reduced in sensitivity to auxin and ethylene, we examined the effects of CSI on another auxin-resistant mutant, axr1-3, and ethylene-insensitive mutant ein2-1. In aux1-7 roots, CSI stimulated the uptake of [(3)H]indole-3-acetic acid (IAA) and induced gravitropic bending. In contrast, in wild-type, axr1-3, and ein2-1 roots, CSI slowed down the rates of gravitropic bending and inhibited IAA uptake. In the null allele of aux1, aux1-22, the agravitropic nature of the roots and IAA uptake were not affected by CSI. This close correlation between auxin uptake and gravitropic bending suggests that CSI may regulate gravitropic response by inhibiting or stimulating the uptake of endogenous auxin in root cells. CSI exhibits selective influence toward IAA versus 1-naphthaleneacetic acid as to auxin-induced inhibition in root growth and auxin uptake. The selective action of CSI toward IAA along with the complete insensitivity of the null mutant aux1-22 toward CSI strongly suggest that CSI specifically interacts with AUX1 protein.

Effects of age and blood sugar levels on the proboscis extension of the blow fly Phormia regina.

In some insects the proboscis is extended to imbibe a sugar solution if the concentration of sugar applied to the chemosensilla exceeds the behavioural threshold value. Recently, I found a reversal of the threshold values of this "proboscis extension reflex" (PER) in the blow fly (Phormia regina M.) for glucose and fructose. It depended on maturation and physiological conditions, both of which are explicable in terms of changing concentration of haemolymph trehalose. The direct injection of trehalose into the fly haemocoele brought about a dramatic shift of the threshold values of PER measured on tarsi or labellar sensilla, suggesting a strong dependence of PER on the blood sugar level. Using the tip-recording method, the dose-response (impulse frequency) curves for glucose and fructose were obtained on individual largest labellar chemosensilla. The curves for glucose and fructose crossed at one point because the former had a steeper gradient and higher maximum response than the latter. Injection experiments with trehalose were also carried out to test for changes in gustatory response. The shifting of the behavioural dose-response curves for glucose and fructose two hours after injection of 1 M trehalose (2 µl) into the haemocoele of the fly was associated with significant reduction in responsiveness of labellar chemosensilla to glucose, but less so to fructose. No change in responsiveness was found following injection of mannose. A hypothesis to explain the reversal relation of the PER thresholds, based on a shift in the firing rate in gustatory sensilla and possibly also interneurons, is discussed.

Chromosaponin I stimulates the sugar taste receptor cells of the blowfly, Phormia regina.

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, stimulates the growth of roots in a variety of plants. In the present work, we introduce CSI as a sugar taste substance for the blowfly, Phormia regina. The blowfly has taste chemosensilla on the labellum. The sensory receptor cells in the chemosensillum are highly specialized for the tastes of sugar, salt and water, respectively. Application of CSI induced the feeding response of blowflies including full proboscis extension. CSI also induced impulses of the sugar taste receptor cell in the LL-type sensillum. The optimum concentration of CSI in these responses was 0.1 mM which is much lower than that of sucrose. Based on the comparison of dose-response relationships, CSI is 100 times more effective than sucrose in stimulating the sugar taste receptor cells. CSI-induced impulses appeared after a significant latency compared with sucrose. As far as we know, this is the first report describing that a natural saponin induces sugar responses in insects. CSI is a unique saponin because of its bifunctional property in plants and insects.

Involvement of ethylene and gibberellin signalings in chromosaponin I-induced cell division and cell elongation in the roots of Arabidopsis seedlings.

Chromosaponin I (CSI), a triterpenoid saponin isolated from pea, stimulates the growth of roots in Arabidopsis thaliana seedlings on wetted filter paper in the light for 14 d. The growth rates of roots in Columbia (Col) and Landsberg erecta (Ler) wild-types were 0.92 and 0.26 mm d(-1), respectively, and they were accelerated to 3.46 (Col) and 2.20 (Ler) mm d(-1) by treating with 300 microM CSI. The length of mature epidermal cells was increased by 1.8-fold (Col) and 2.81-fold (Ler) compared with control and the number of epidermal cells was increased by a factor of 1.65 (Col) and 2.12 (Ler). Treatment with 2-aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, also increased cell length but not cell number. The effects of CSI on root growth were not detected in the ethylene-insensitive mutant ein2-1. CSI did not inhibit ethylene production but stimulated the growth of roots in ctr1-1, the constitutive triple response mutant for ethylene, indicating that CSI inhibits ethylene signaling, especially downstream of CTR1. In the GA-insensitive mutant gai and the mutant spy-3, in which the basal level of GA signaling is activated, CSI did not increase cell number, although both CSI and AVG stimulated cell elongation in these mutants. These results suggest that the inhibition of ethylene signaling is the cause of CSI-induced cell elongation. A possible involvement of both GA and ethylene signalings is discussed for the CSI-induced cell division.

Beneficial effect of salmon roe phosphatidylcholine in chronic liver disease.

Phosphatidylcholine (PC), especially dilinoleoyl-PC, has been reported to be effective in preventing hepatic fibrosis in chronically alcohol-fed baboons. Continuous hepatic inflammation predisposes the structure of the liver to fibrosis. Since n-3 polyunsaturated fatty acids (PUFA) have been shown to exhibit an anti-inflammatory effect, we tested the hypothesis that n-3 PUFA PC as a dietary supplement has a beneficial effect on chronic liver disease susceptible to fibrosis. Salmon roe phospholipids, 90% of which are PC, were extracted and encapsulated. Almost a third of the PC fatty acids were docosahexaenoic acid (22:6 n3) and 10% were eicosapentanoic acid (20:5 n3). About 1600 mg/day of the phospholipids was administered for six months to six chronic liver disease patients, four with hepatitis B infection (three with cirrhosis, one with chronic hepatitis), one with hepatitis C virus cirrhosis and one with alcoholic cirrhosis. There was no change in the results of blood chemistry studies related to liver function, except in globulin, which decreased from 3.80 g/dl to 3.67 g/dl (p < 0.05). Among the lipid parameters, HDL-cholesterol, apolipoprotein A-I and apolipoprotein E increased significantly. Although this was a small trial, n-3 PUFA PC may be beneficial in the treatment of chronic liver diseases.

Results of screening for cancer in Japanese in the prime of life--an analysis of nationwide MHTS and human dry dock statistics--Preventive Medicine Committee of the Japan Hospital Association.

The nationwide survey of institutions with MHTS and human dry dock capabilities was analyzed and the following results have been obtained. 1) The relative rates of cancer detection by sex and organ were the stomach > colon > rectum > lung > kidney > esophagus for men and the stomach > breast > uterus > colon > thyroid > lung for women. 2) Gastric cancer takes first place in the ranking of rates of cancer detection in the population of both sexes, followed by colon cancer. The difference in rate of detection between these cancers has been narrowed from year to year. In 1997, the ratio of gastric to colon cancers was 10:7. 3) Early cancers account for 74% of gastric cancer patients and 75% of colon cancer patients. 4) Since gastric and colon cancers are detected early, the proportions of persons with gastrointestinal symptoms are as low as 28% for gastric cancer patients and 26% for colon cancer patients. 5) The relative rates of cancer detection by the degree obesity are normal > obese > lean person. The rates of gastric and colon cancers are 2- and 3-fold higher for obese persons than for lean persons, respectively. Gastric and colon cancers are detected with higher frequency in well-nourished persons. The present review of the national MHTS and human dry dock statistics has confirmed the efficacy of MHTS and human dry dock, especially in the examination for gastrointestinal cancers.

The relationship between lipoprotein(a) and low density lipoprotein receptors during the treatment of hyperthyroidism.

To determine whether the low density lipoprotein (LDL) receptor pathway is involved in the catabolism of plasma lipoprotein (a) [Lp(a)], serum lipids, Lp(a), and LDL receptor activity were measured in seven patients with hyperthyroidism before and after methimazole treatment given hyperthyroidism is associated with enhanced LDL receptor activity. LDL receptor activity in patients was estimated by the equation using the serum concentrations of apolipoprotein (apo) B and C-II. When euthyroidism was achieved after treatment, not only did serum total cholesterol, high density lipoprotein-cholesterol, apo B, and LDL-cholesterol (LDL-Ch) levels rise, but Lp(a) significantly increased and calculated LDL receptor activity significantly decreased. The changes in LDL receptor activity were significantly correlated with the changes in LDL-Ch as expected, but not with changes in Lp(a). These results suggest that the serum concentration of Lp(a) is lowered in hyperthyroidism, probably by a mechanism other than the enhanced activity of the LDL receptor, and that the LDL receptor pathway is involved in the catabolism of Lp(a) to a limited extent.

Two types of sugar-binding protein in the labellum of the fly. Putative taste receptor molecules for sweetness.

Flies have taste cells specifically sensitive to sweetness. It has been suggested that the cells possess two types of receptor sites covering the receptive field of sweetness. By affinity electrophoresis with the site-specific inhibitory polysaccharides, two types of sugar-binding protein were isolated from the labellar extract of the blowfly. These proteins showed consistent sugar-binding specificities and affinities with the two types of receptor sites for sweetness, respectively. The dissociation constant of the protein-sugar complex varies 100-400 mM and the molecular weight of one type of the protein is 27,000, while that of the other is 31,000 or 32,000. Both proteins were water insoluble and were also detected in the isolated chemosensilla. Thus they are probably located on the taste receptor membrane, and the proteins are likely to act as the taste receptor molecules for sweetness in the fly.

Adaptation-promoting effect of IP3, Ca2+, and phorbol ester on the sugar taste receptor cell of the blowfly, Phormia regina.

The fly has a receptor cell highly specialized for the taste of sugars. We introduced inositol 1,4,5-trisphosphate (IP3), Ca2+, or a phorbol ester, 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA), into the cell and investigated their effects on the response to sucrose. The sugar receptor cell generates impulses during constant stimulation with sucrose, but the impulse frequency gradually declines as the cell adapts to the stimulus. Thus, this gradual reduction of the impulse frequency is a direct manifestation of adaptation of the cell. These reagents accelerated the gradual reduction of the impulse frequency, although the initial impulse frequency was little affected. In contrast to these reagents, glycoletherdiamine-tetraacetate (EGTA) retarded the gradual reduction of the impulse frequency. Moreover, when IP3 and DPBA were applied together, the gradual reduction of the impulse frequency was more accelerated than when either IP3 or DPBA was applied. When IP3 and EGTA were applied together, however, the accelerating effect of IP3 tended to be canceled. Based on these results, we hypothesized that an intracellular cascade involving inositol phospholipid hydrolysis, intracellular Ca2+ mobilization, and protein kinase C-mediated phosphorylation promotes adaptation of the sugar receptor cell.

Hereditary complete deficiency of lactate dehydrogenase H-subunit.

We report the second known case of a patient, a 45-year-old Japanese woman, with hereditary complete deficiency of lactate dehydrogenase (LDH; EC 1.1.1.27) H-subunit. Total LDH activity in her serum was abnormally low (35 U/L, normal reference interval 195-360). LDH activity in her erythrocytes was also low, but all the other glycolytic enzyme activities in her erythrocytes were within normal limits. Electrophoresis of her serum, erythrocytes, lymphocytes, thrombocytes, and saliva showed only one band, the LDH M4 isoenzyme. LDH activities in her saliva and lymphocytes exceeded the reference interval. Her erythrocytes contained fructose 1,6-diphosphate 26 mumol/L (normal range 4-13), dihydroxyacetone phosphate 75 mumol/L (normal range 8-22), glyceraldehyde 3-phosphate 57 mumol/L (normal range 4-14), and pyruvate 45 mumol/L (normal range 31-63). The family study of three generations showed that this deficiency was inherited in an autosomal recessive mode.

Phosphatidylinositol stimulates phosphorylation of protein components I and II in rod outer segments of frog photoreceptors.

We studied the effect of phosphoinositides on the phosphorylation of endogenous proteins in the soluble fraction of the frog photoreceptor rod outer segments (ROS). Phosphatidylinositol (PI) stimulated the phosphorylation of two low molecular weight proteins, components I and II (12 and 11 kDa) which are known to be the preferential substrates of the cyclic GMP (cGMP)-dependent protein kinase in the ROS. Polyphosphoinositides (PPI) specifically inhibited the PI-dependent phosphorylation of these two components. On the other hand, PPI stimulated the phosphorylation of 38, 48 and 52 kDa proteins in the absence of PI. These data suggest that PI and PPI may function in the ROS by regulating the phosphorylation of some enzymes or regulator proteins in the transduction mechanism in the ROS.