The clock gene cryptochrome of Bactrocera cucurbitae (Diptera: Tephritidae) in strains with different mating times.

Differences in mating time between populations can give rise to premating reproductive isolation. Tephritid fruit flies exhibit large variation in mating time among intra- or inter-specific populations. We previously cloned the clock gene period from two strains of melon fly, Bactrocera cucurbitae; in one the individuals mate early during the day, whereas in the other the individuals mate later. These strains were originally established by divergent artificial selection for developmental time, 'short' and 'long', with early and late mating times, respectively. The deduced amino acid sequences of PERIOD proteins for these two strains were reported to be identical. Here we cloned another clock gene cryptochrome (cry) from the two strains, and found two stable amino acid substitutions in the strains. In addition, the allele frequency at the two polymorphic sites of cry gene correlated with the circadian locomotor period (tau) across strains, whereas the expression pattern of cry mRNA in the heads of flies taken from the short strain significantly differed from that from the long strain. These findings suggest that variation in the cry gene is related to differences in the circadian behaviour in the two strains, thus implying that the cry gene may have an important role in reproductive isolation.

Relationship between the acid-inhibitory effects of two proton pump inhibitors and CYP2C19 genotype in Japanese subjects: a randomized two-way crossover study.

This two-way crossover study investigated possible differences between the proton pump inhibitors, omeprazole and rabeprazole, in their effect on gastric acid secretion in Japanese subjects with differing cytochrome P450, family 2, subfamily C, polypeptide 19 (CYP2C19) genotypes. A total of 23 Helicobacter pylori-negative healthy volunteers received omeprazole 20 mg/day and rabeprazole 10 mg/day. Each drug treatment was given for a continuous 7-day period allocated in random order, with an interval of at least 1 week between drug treatment periods to allow for wash-out. Intragastric pH was measured on days 1 and 7. Overall median intragastric pH levels at 7 and 8 h after the first administration were significantly higher with omeprazole. There was no significant difference in intragastric pH in homozygous extensive metabolizers, whereas intragastric pH was significantly higher with omeprazole in combined data from heterozygous extensive metabolizers and poor metabolizers at 6, 7 and 8 h after the first drug administration. There were no significant differences in intragastric pH between omeprazole and rabeprazole irrespective of genotype on day 7 of administration. In conclusion, on day 1 the time to onset of the antisecretory action of 20 mg/day omeprazole was more rapid than that of 10 mg/day rabeprazole in Japanese individuals who have a higher incidence of the CYP2C19 poor metabolizer genotype, however by day 7 no difference in antisecretory effect was found, regardless of genotype.

Molecular identification of a taste receptor gene for trehalose in Drosophila.

The molecular nature of sweet taste receptors has not been fully explored. Employing a differential screening strategy, we identified a taste receptor gene, Tre1, that controls the taste sensitivity to trehalose in Drosophila melanogaster. The Tre1 gene encodes a novel protein with similarity to G protein-coupled seven-transmembrane receptors. Disruption of the Tre1 gene lowered the taste sensitivity to trehalose, whereas sensitivities to other sugars were unaltered. Overexpression of the Tre1 gene restored the taste sensitivity to trehalose in the Tre1 deletion mutant. The Tre1 gene is expressed in taste sensory cells. These results provide direct evidence that Tre1 encodes a putative taste receptor for trehalose in Drosophila.

Droplet countercurrent chromatography.

A new form of countercurrent chromatography, named droplet counter-current chromatography, has been developed. This all-liquid separation technique is based on the partitioning of solute between a steady stream of droplets of moving phase and a column of surrounding stationary liquid phase. Milligram quantities of dinitrophenyl (DNP) amino acids were separated with an efficiency comparable to that of gas chromatography.

Inverse silica opal photonic crystals for optical sensing applications.

This work reports fabrication of inverse silica opal photonic crystal structures from direct polystyrene micro sphere opals using low-temperature sol-gel infiltration of silica, and examines performance of these photonic crystals as environmental refractive index sensors. Sensitivity of the spectral position and optical attenuation of photonic stop gaps is found to allow detection of the index changes by the amount of ~10(-3). The high value of sensitivity, which is comparable with those of other optical sensing techniques, along with simplicity of the optical detection setup required for sensing, and the low-temperature, energy-efficient fabrication process make inverse silica opals attractive systems for optical sensing applications.

timrit Lengthens circadian period in a temperature-dependent manner through suppression of PERIOD protein cycling and nuclear localization.

A fundamental feature of circadian clocks is temperature compensation of period. The free-running period of ritsu (timrit) (a novel allele of timeless [tim]) mutants is drastically lengthened in a temperature-dependent manner. PER and TIM protein levels become lower in timrit mutants as temperature becomes higher. This mutation reduces per mRNA but not tim mRNA abundance. PER constitutively driven by the rhodopsin1 promoter is lowered in rit mutants, indicating that timrit mainly affects the per feedback loop at a posttranscriptional level. An excess of per+ gene dosage can ameliorate all rit phenotypes, including the weak nuclear localization of PER, suggesting that timrit affects circadian rhythms by reducing PER abundance and its subsequent transportation into nuclei as temperature increases.

Targeted expression of ced-3 and Ice induces programmed cell death in Drosophila.

CED-3 is a cysteine protease required for programmed cell death in the nematode, Caenorhabditis elegans, and shares a sequence similarity with mammalian ICE (interleukin-1beta converting enzyme) family proteases. Both CED-3 and ICE family proteases can induce programmed cell death in mammalian cells. Structural and functional similarities between CED-3 and ICE family proteases indicate that the mechanism of cell death is evolutionarily conserved, suggesting the presence of a similar mechanism involving CED-3/ICE-like proteases in Drosophila. Here we determined whether CED-3 or ICE functions to induce programmed cell death in Drosophila. We have generated transformant lines in which ced-3 or Ice is ectopically expressed using the GAL4-UAS system. Expression of CED-3 and ICE can elicit cell death in Drosophila and the cell death was blocked by coexpressing the p35 gene which encodes a viral inhibitor of CED-3/ICE proteases. Results support the idea that the mechanism of programmed cell death controlled by CED-3/ICE is conserved among widely divergent animal species including Drosophila, and the system described provides a tool to dissect cell death mechanism downstream of CED-3/ICE proteases.

Stereospecificity of multiple receptor sites in a labellar sugar receptor of the fleshfly for amino acids and small peptides.

N-Formylation and N-methylation of the alpha-amino group of L-phenylalanine result in extremely decreased responses of the labellar sugar receptor of the fleshfly, whereas the same structural alteration of L-valine hardly affects the response. Methyl esterification of the alpha-carboxyl group of phenylalanine, on the other hand, maintains the response to some extent, but similar treatment of valine completely diminishes the response. The aromatic structure in phenylalanine is not essential for stimulation. These results suggest a substantial difference in the stereospecificities and functional group specificities of the furnase (F) and aliphatic carboxylate (T) sites in the sugar receptor. The effect of small peptides on the sugar receptor was examined systematically. Their effectiveness depends mainly on the place of the constituent amino acids rather than on their composition, indicating the decisive role that certain aliphatic amino acids in the C-terminal position play in stimulation. Remarkable regularities in the stimulating effectiveness of small peptides exactly correspond to the stereospecificity of each receptor site. We propose two hypothetical models of the F and T sites, which involve three and two subsites, respectively, that are capable of hydrogen bond formation. The F and T sites also have a hydrophobic subsite that discriminates the R groups of the stimulants and a few spatial barriers.

Dependency on light and vitamin A derivatives of the biogenesis of 3-hydroxyretinal and visual pigment in the compound eyes of Drosophila melanogaster.

When the fruitfly, Drosophila melanogaster, was reared on media deficient in carotenoids and retinoids, the level of 3-hydroxyretinal (the chromophore of fly rhodopsin) in the retina decreased to less than 1% compared with normal flies. The level of 3-hydroxyretinal increased markedly in flies that were given a diet supplemented with retinoids or carotenoids. The retinas of flies fed on all-trans retinoids and maintained in the dark predominantly contained the all-trans form of 3-hydroxyretinal, and showed no increase in the level of either the 11-cis isomer or the visual pigment. Subsequent illumination of the flies converted substantial amounts of all-trans 3-hydroxyretinal to its 11-cis isomer. The action spectrum of the conversion by illumination showed the optimum wavelength to be approximately 420 nm, which is significantly greater than the absorption maximum of free, all-trans 3-hydroxyretinal. Flies that were fed on carotenoids showed a rapid increase of the levels of 11-cis 3-hydroxyretinal and of visual pigment in the absence of light.

Taste sensitivity to trehalose and its alteration by gene dosage in Drosophila melanogaster.

The taste sensitivity to the disaccharide trehalose of Drosophila melanogaster is under the genetic control by the Tre gene on the X chromosome. The gene is genetically dimorphic for high and low sensitivity and is likely to be functioning in the primary step of chemoreception. We have determined the cytological localization of the Tre gene to be between 5A10 and 5B1-3 by analyzing the sensitivity to trehalose in flies which are segmentally aneuploid bearing either deficiencies or duplicated fragments of T(X;Y) translocations. We also constructed flies which are aneuploidy and thus carry different dosage of Tre and/or Tre(+) alleles in order to examine the gene dosage effect on trehalose sensitivity and to deduce the nature of the gene's action. Trehalose sensitivity decreased in females carrying half the normal dosage of a given Tre allele, but a proportional increase in sensitivity was not observed in flies bearing a duplication of the Tre alleles. The changes in sensitivity in various aneuploid flies suggest that there is an upper limit to the number of molecules that can be incorporated into the receptor membrane. Genetic evidence strongly suggests that Tre is the structural gene for the trehalose receptor. We present a model to account for the mechanism of genetical control on the sensitivity to trehalose.

Glucose absorption and utilization by rat embryos.

There is little doubt that glucose plays a significant nutritional role in early somite embryos. The high glucose utilization of anaerobic glycolysis drops as the activity of the Kreb's cycle and terminal electron transport increase. Concurrently, maturation of mitochondrial cristae and dependence on oxygen supply are taking place. The neuroepithelium of the early somite rat embryo responds in vitro during culture by microvilliar lengthening when exposed to glucose levels of 50 mg/dl or more. At lower glucose concentrations both in whole embryo culture and inside the closed neural tube the microvilli are shorter. Lengthening of the microvilli at room temperature is produced only by d-glucose and 2-deoxyglucose, two hexoses that are absorbed and phosphorylated. Cytochalasin D which disrupts actin polymerization causes ballooning of the microvilli. A role of this microvillar elongation in degenerative changes seen in uncontrolled diabetes and on function of the immune system is proposed. The amniotic cavity is one major portal of entry for glucose during the early somite embryo stage. The 7-fold increase in volume of the amniotic cavity after day 10 allows the rat embryo to convert its axis from dorsal to ventral flexion.

Isolation of acein-2, a novel angiotensin-I-converting enzyme inhibitory peptide derived from a tryptic hydrolysate of human plasma.

We previously described a novel angiotensin-I-converting enzyme (ACE) inhibitory peptide, designated Acein-1, that was isolated from a tryptic hydrolysate of human plasma. We now report a second such inhibitory peptide, Acein-2 obtained from the same hydrolysate. The peptide was purified by gel filtration and cation exchange chromatography followed by reversed-phase gradient and isocratic high performance liquid chromatography. Acein-2 was found to be a tripeptide, Leu-Ile-Tyr, which is thought to correspond to f(518-520) of human alpha2-macroglobulin. The synthetic tripeptide showed a potent dose-dependent inhibition of ACE, with an IC(50) value of 0.82 micromol/l. Lineweaver-Burk plots suggested that Acein-2 as well as the previously described Acein-1 are non-competitive inhibitors.

Acein-1, a novel angiotensin-I-converting enzyme inhibitory peptide isolated from tryptic hydrolysate of human plasma.

A novel angiotensin-I-converting enzyme (ACE) inhibitory peptide, designated acein-1, was isolated from the tryptic hydrolysate of human plasma. Gel filtration and cation exchange chromatography were performed to purify this peptide, followed by reversed-phase gradient and isocratic high-performance liquid chromatography. Acein-1 was found to be a heptapeptide, Tyr-Leu-Tyr-Glu-Ile-Ala-Arg, corresponding to f(138-144) of human serum albumin. The synthetic heptapeptide, hexapeptide (Tyr-Leu-Tyr-Glu-Ile-Ala, des-7R acein-1) and octapeptide (Tyr-Leu-Tyr-Glu-Ile-Ala-Arg-Arg, acein-1R) showed dose-dependent inhibitions of ACE, and their IC50 values were 16 micromol/l, 500 micromol/l and 86 micromol/l, respectively. Acein-1 might be a non-competitive inhibitor, while acein-1R may be an uncompetitive inhibitor, as shown by Lineweaver-Burk plots.

The action of ranatensin, a new polypeptide from amphibian skin, on the blood pressure of experimental animals.

1. The blood pressure response to ranatensin, an undecapeptide from the skin of the frog, Rana pipiens, has been studied in various experimental animals.2. Ranatensin raised blood pressure in the dog and rabbit. The response was not altered by atropine, phentolamine, propranolol or hexamethonium, suggesting a direct peripheral vasoconstrictor action. In both animals ranatensin was about one-tenth as potent as angiotensin. Tachyphylaxis to ranatensin did occur, but there was no cross-tachyphylaxis with angiotensin, bradykinin, or noradrenaline.3. The peptide lowered blood pressure in the monkey, being as potent as eledoisin. The response was not altered by atropine, phentolamine, propranolol, tripelennamine, tetraethylammonium, bretylium, or methysergide. This again suggests a direct peripheral action on vascular smooth muscle. There was no tachyphylaxis to the depressor action, nor was there cross-tachyphylaxis with angiotensin, eledoisin, bradykinin, or noradrenaline.4. Ranatensin did not alter the blood pressure in cats and had a variable action in the guinea-pig with a rapid onset of tachyphylaxis.5. Ranatensin has a variable effect on the blood pressure in the rat that is related to the basal level of blood pressure. When the blood pressure is high, the response to the peptide is hypotension. Ranatensin raises blood pressure in the rat when the basal blood pressure is low. The pressor response to ranatensin appears to be due, in part, to the release of noradrenaline from peripheral sympathetic nerve endings.6. The composite action of ranatensin on blood pressure of various experimental animals is unlike that of any other peptide. Its hypertensive action in the dog or rabbit, together with a potent hypotensive action in the monkey, readily distinguishes it from all other vasoactive peptides.

Effects of ranatensin, a polypeptide from frog skin, on isolated smooth muscle.

1. The actions of bretylium tosylate on neuromuscular transmission in the rat phrenic nerve diaphragm preparation have been investigated by electro-2. On the guinea-pig ileum, threshold doses elicit repeated maximal spike contractions which are blocked by atropine. In the presence of atropine, higher concentrations of ranatensin elicit small contraction spikes superimposed on a relatively weak sustained contraction. These latter two actions are not blocked by increasing the concentration of atropine.3. Other smooth muscle preparations respond as follows: rat uterus, rhythmic contractions; rat duodenum, relaxation; rabbit aortic strip, contraction. Atropine has no effect on the above responses. The rat aortic strip does not respond to ranatensin. Ranatensin is four times as active as bradykinin on the rat uterus.4. Ranatensin can be readily distinguished from other known peptides such as angiotensin, bradykinin and the eledoisin-like peptides, by bioassay.

Purification and partial characterization of three forms of alpha-glucosidase from the fruit fly Drosophila melanogaster.

Three forms of alpha-glucosidase, I, II, and III, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2.1, 5.3, and 6.7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of alpha-glucosidases I, II, and III were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that alpha-glucosidases II and III were each composed of a single polypeptide chain, whereas alpha-glucosidase I was composed of two identical subunits. Both alpha-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-alpha-D-glucoside (PNPG), but alpha-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of alpha-glucosidases I, II, and III were pH 6.0, 5.0, and 6.0 and the Km values were 13.1, 8.9, and 10 mM, respectively. For PNPG the pH optima of alpha-glucosidases II and III were pH 5.5 and 6.5 and the Km values were 0.77 and 0.21 mM, respectively.

Effects of prenatal aflatoxin B1 exposure on behaviors of rat offspring.

The effects of prenatal aflatoxin B1 (AFB) exposure on eight behavioral parameters in Jcl:Wistar rat offspring were assessed. Pregnant rats were injected subcutaneously with 0.3 mg/kg/day of AFB dissolved in dimethylsulfoxide on days 11-14 (Group A) or 15-18 (Group B) of gestation. Controls received the vehicle similarly on days 11-18 of gestation. Before weaning, the offspring were examined using the cliff avoidance response (5 days of age), the negative geotaxis reflex (7 days), and swimming development (6, 8, and 10 days). After weaning, animals were examined using the rotarod test (5 weeks of age), the open field test (6 weeks), a conditioned avoidance learning test (14 weeks), an underwater T-maze test (15 weeks), and a reproduction test (16 weeks). The preweaning offspring in the AFB-A group showed significantly lower success rates than controls in cliff avoidance responses. In swimming development, the offspring in the AFB-A group had significantly lower scores than controls for swimming direction. In the rotarod test, the AFB-A group remained on the rod for a significantly shorter time than the controls at 15 rpm on both the first and second trial days. The avoidance performance of the rats in AFB-A and AFB -B groups was significantly poorer than that of controls. These results indicate that prenatal exposure to AFB produced a delay of early response development, impaired locomotor coordination, and impaired learning ability in the offspring of rats exposed to AFB during middle pregnancy, and the early gestational exposure appears to produce more effects than latter exposure.

Effects of prenatal rubratoxin-B exposure on behaviors of mouse offspring.

The effects of prenatal rubratoxin-B (RB) exposure on 8 behavioral parameters in JCL:ICR mice were assessed. Pregnant mice were injected intraperitoneally with 0.1 or 0.2 mg/kg/day of RB dissolved in propylene glycol water solution on days 7-9 (Group A) or 10-12 (Group B) of gestation. Controls received the vehicle similarly on days 7-12 of gestation. Before weaning, the offspring of both sexes were examined to test their the surface righting reflex (5 days of age), cliff avoidance response (6 days), negative geotaxis response (7 days), and swimming development (8, 10, and 12 days). After weaning, male animals were examined using the rotarod test (6 weeks of age), the open-field test (7 weeks), the shuttle-box-avoidance-learning test (9 weeks), and the water E-maze test (10 weeks). The preweanling offspring in the 0.2 mg/kg-B group showed significantly lower success rates and longer response times than controls in the cliff-avoidance response. In swimming development, the offspring in the 0.2 mg/kg B group had significantly lower scores than controls for swimming angle at 10 and 12 days of age. The avoidance learning of the mice in all RB-exposed A and B groups was significantly poorer than that of controls. These results indicate that prenatal exposure to RB produced a delay of early response development and impaired learning ability in the offspring of mice exposed to RB during middle pregnancy.

Mechanized assay of serum cholinesterase by specific colorimetric detection of released acid.

An automated assay method has been developed for the measurement of serum cholinesterase activity. The samples were prepared by an automated liquid handling unit and incubated for 9.7 min at 30 degrees C, followed by automatic injection into a colorimetric flow injection determination system for acetic acid liberated from acetylcholine by cholinesterase catalytic activity. The coloration reaction employed is based upon the formation of 2-nitrophenylhydrazide utilizing a water-soluble carbodiimide and has a high selectivity for carboxylic acids. The coefficients of variation of the proposed method were 2.4% for within-run analysis (n = 14) and 2.6% for between-run analysis (n = 6). Sera of 55 hospitalized patients were analyzed and the values obtained correlated well with an automated differential pH method (gamma = 0.989).

Myelomeningocele before birth.

The authors report a study of 92 human embryos and four fetuses with myeloschisis. The characteristics of embryonic myeloschisis compared with spina bifida cystica in infants are: 1) the lesion is often more diffuse, involving the whole spinal cord (12 embryos); 2) the cervical cord is frequently affected (23 of the remaining 80 embryos); 3) holoprosencephaly is frequently associated (18 embryos); 4) meningocele is not found; and 5) hydrocephalus and Arnold-Chiari malformation are not yet developed. Hydrocephalus and Arnold-Chiari malformation are found in myeloschistic fetuses. Almost all embryos with diffuse and cervical myeloschisis or with holoprosencephaly are extruded before birth by spontaneous abortion. Absence of meningocele in the embryonic period implies that its appearance is deferred to the fetal period. The development of hydrocephalus and Arnold-Chiari malformation also seems to be delayed until the fetal period. Our observation implies that myelomeningocele is induced by non-closure of the neural tube, not by rupture once it was closed. "Neural overgrowth" and disturbed "recanalization process" are discussed in relation to the pathogenesis of myelomeningocele.