The extracellular matrix in the mature brain: its reactions to endo-alpha-N-acetylgalactosaminidase and collagenase.

The hippocampal subiculum in the adult rat brain contains many neurons with nerve cell surface glycoproteins which are linked by collagenous ligands to perineuronal proteoglycans. The nerve cell surface glycoproteins or their terminal N-acetylgalactosamines are digested by endo-alpha-N-acetylgalactosaminidase. The terminal N-acetylgalactosamines linked by the collagenous ligands are not digested by endo-alpha-N-acetylgalactosasminidase. The collagenous ligands associated with the terminal N-acetylgalactosamines were digested by collagenase. The newly exposed terminal N-acetylgalactosamines by this collagenase incubation were digested by endo-alpha-N-acetyl-galactosaminidase. These findings on the rat agree with those obtained in our previous studies of the adult mouse brain samples. Furthermore they emphasize our hypothetical model that the perineuronal proteoglycans are related--via collagen ligands--with the nerve cell surface glycoproteins.

Application of the gene search system to screen for longevity genes in Drosophila.

We have developed a strategy using Drosophila as a model system to identify genes that are crucial for extension of longevity. A collection of transgenic lines with a P-element based gene search (GS) vector containing UAS (Upstream Activating Sequence) was screened for longevity in combination with an hsp70 promoter-driven GAL4 transgene. Misexpression of the vector-flanking sequence was induced throughout the adult stage to assess its effects on the aging process rather than development. We showed that the longevity was greatly affected by GS inserts, and it was positively correlated with paraquat resistance. Of 646 GS inserts, we selected 23 inserts with relatively longer longevity for further molecular analysis. All of the misexpressed sequences matched either known genes or ESTs (Expressed Sequence Tags). Among 13 genes whose functions are already known or suggested, six were related to stress resistance or redox balance (DmGST2, hsp26, nla, and Drosophila homologs of mammalian TRX, GILT and POSH), suggesting the importance of stress resistance for the extension of longevity. This is the first demonstration that a systematic gain-of-function screen could efficiently detect longevity genes.

Neural-specific overexpression of drosophila plenty of SH3s (DPOSH) extends the longevity of adult flies.

Extended longevity mutants are extremely useful to understand the molecular mechanism of longevity determination. Here we report identification and characterization of the Drosophila Plenty of SH3s (DPOSH) gene, a candidate that might be associated with the extended longevity phenotype. DPOSH encodes a protein containing a RING finger domain and four SH3 domains. We showed that neural-specific overexpression of DPOSH could extend the mean longevity of adult flies by 14% at 25 degrees C without affecting viability or morphology. In contrast, forced expression of DPOSH in developing imaginal discs produced various phenotypes including lethality and morphological defects such as loss of crossvein, notched wing, and disordered hair polarity. Puckered, a target gene of JNK/SAPK pathway, was activated by overexpression of DPOSH and the forced expression phenotypes were suppressed by introducing a mutation of Drosophila JNK (bsk) or JNKK (hep), suggesting that the JNK/SAPK signaling pathway is one of the critical elements in the determination of longevity.

Male-killing Wolbachia in Drosophila: a temperature-sensitive trait with a threshold bacterial density.

Inherited microorganisms that disturb the reproduction of their host have been characterized from a number of host taxa. To understand the general principles underlying the genetic and mechanistic basis of interactions, study of different agents in model host species is required. To this end, the nature and genetics of the maternally inherited sex-ratio trait of Drosophila bifasciata were investigated. Successful curing of affected lines with antibiotics demonstrated this trait was associated with the presence of a bacterium, and molecular systematic analysis demonstrated an association between the presence of the trait and infection with an A group Wolbachia. The penetrance and heritability of the trait did not vary with maternal age. Exposure to elevated temperatures did reduce trait penetrance but did not affect heritability. Examination of the effect of temperature on bacterial density in eggs revealed a decrease in bacterial density following exposure of the parent to elevated temperature, consistent with the hypothesis that male killing in D. bifasciata requires a threshold density of Wolbachia within eggs. The male offspring produced following exposure to elevated temperatures were infected with Wolbachia on emergence as adults. Crossing studies demonstrated a weak cytoplasmic incompatibility phenotype exhibited by Wolbachia in these males. The results are discussed with respect to the incidence of male killing within the clade Wolbachia, the general nature of Wolbachia-host interactions, and the prospects for using this association to investigate the mechanism of male killing.

Enhanced visualization of weak colloidal iron signals with Bodian's protein silver for demonstration of perineuronal nets of proteoglycans in the central nervous system.

The present study aimed for a clear visualization of faintly deposited colloidal iron in tissue sections for light microscopy. Paraffin blocks containing paraformaldehyde-fixed brain tissue from healthy adult mice were cut into sections 10-15 microm thick. After deparaffinization, the sections were stained with fine cationic iron colloid at a pH value of 1.0-1.5, and treated with a mixture of potassium ferrocyanide and hydrochloride for Prussian blue reaction. Some sections were further treated with Bodian's protein silver after the Prussian blue reaction. This sensitized development of Prussian blue reaction with Bodian's protein silver more clearly visualized the faintly deposited cationic colloidal irons than the demonstration by Prussian blue reaction alone, and allowed an enhanced visualization of the perineuronal nets of sulfated proteoglycans in the brain. Thus, such fine perineuronal sulfated proteoglycans as those in the CA3 field of the hippocampus, which are weakly stained with cationic iron colloid and usually overlooked by a demonstration with only a Prussian blue reaction, could be clearly visualized with striking contrast by the sensitized development with Bodian's protein silver after the Prussian blue reaction. Preliminary hyaluronidase digestion erased Bodian's protein silver development of perineuronal sulfated proteoglycans. Though some axonal fibers were also additionally stained with Bodian's protein silver itself, this sensitized development is useful to enhance such weak colloidal iron signals as are hardly detectable by only Prussian blue reaction.

Syndrome of microcephaly, Dandy-Walker malformation, and Wilms tumor caused by mosaic variegated aneuploidy with premature centromere division (PCD): report of a new case and review of the literature.

We report a male infant with multiple congenital anomalies and mosaic variegated aneuploidy; a rare cytogenetic abnormality characterized by mosaicism for several different aneuploidies involving many different chromosomes. He had prenatal-onset growth retardation, microcephaly, dysmorphic face, seizures, hypotonia, feeding difficulty, and developmental delay. In addition, he developed bilateral Wilms tumors. Neuroradiological examination revealed Dandy-Walker malformation and hypoplasia of the cerebral hemisphere and pons. Cytogenetic analysis revealed various multiple numerical aneuploidies in blood lymphocytes, fibroblasts, and bone marrow cells, together with premature centromere division (PCD). Peripheral blood chromosome analysis from his parents also showed PCD, but no aneuploid cells. The clinical phenotype and multiple aneuploidies of the patient may be a consequence of the homozygous PCD trait inherited from his parents. Comparison with previously reported cases of multiple aneuploidy suggests that mosaic variegated aneuploidy with PCD may be a clinically recognizable syndrome with major phenotypes being mental retardation, microcephaly, structural brain anomalies (including Dandy-Walker malformation), and possible cancer predisposition.

Successful transcutaneous arterial embolization of a giant hemangioma associated with high-output cardiac failure and Kasabach-Merritt syndrome in a neonate: a case report.

We describe the case of a patient with a neonatal giant cutaneous hemangioma with high-output cardiac failure and Kasabach-Merritt syndrome and successfully treated with transcutaneous arterial embolization aimed at controlling severe congestive heart failure and consumption coagulopathy. A patient was admitted to the neonatal care unit on the first day of age because of a large hemangioma on his right lateral chest wall and respiratory distress, associated with cardiac failure resulting from arteriovenous shunting. On the second day of age the platelet count decreased to 5.7 x 10(4)/microliter and fibrinogen level was 85 mg/dl. The values of prothrombin time and activated partial thromboplastin time were prolonged. Intravenous predonisone therapy was started immediately, but bleeding tendency was getting worse and the evidence of congestive heart failure persisted. On the third day the patient then underwent embolization of feeding arteries with microcoils. The cardiac failure and thrombocytopenic coagulopathy had improved significantly without complications. We conclude that transcutaneous arterial embolization is an effective and safe treatment in this neonate and should be considered for the treatment of control high-output cardiac failure and coagulopathy in infants with hemangioma and Kasabach-Merritt syndrome.

Ovulation stimulating substances in Drosophila biarmipes males: their origin, genetic variation in the response of females, and molecular characterization.

Ovulation responses of Drosophila biarmipes females to an injection of methanolic extract from conspecific males vary with the strains of females. This strain difference seems to be controlled by a small number of autosomal genes, with low responsiveness being recessive. Strangely, all D. biarmipes strains show a high level of ovulation after mating. We pursued the reason for this discrepancy and found that D. biarmipes males produce two different substances with ovulation-inducing activity. One of them is derived from the accessory glands and effective in females of all strains. Another originates in the ejaculatory duct and is inactive in some strains. In an active HPLC fraction of the ejaculatory duct extract, we found a peptide consisting of 32 amino acids. Its C-terminal region has a striking similarity to the sex-peptide of D. melanogaster, but the N-terminal region was entirely different. Evolutionary implications of these findings are discussed.

Ovulation and the suppression of mating in Drosophila melanogaster females: behavioral basis.

Virgin females of Drosophila melanogaster that are ectopically expressing the sex-peptide gene show a high level of ovulation and are unreceptive to males. However, if they are genetically deprived of eggs, receptivity is considerably restored (Fuyama, 1995). These females, whether they have eggs or not, extrude their ovipositors toward courting males as frequently as do fertilized females. However, this rejection behavior was ineffective in suppressing male courtship. Of females with eggs, about half of them could suppress male courtship. Females lacking eggs could not suppress male courtship and continued to elicit vigorous courtship. This difference seems to account for the increased mating frequency in sterilized females. Courtship behavior by mutant males defective in olfaction or learning suggested that females are capable of repelling males by emitting a volatile pheromone(s) with an inhibitory effect on male courtship.

Long-term follow up of abdominal aortic aneurysm complicating Kawasaki disease: comparison of the effectiveness of different imaging methods.

A 2 month old boy with Kawasaki disease developed the rare complication of abdominal aortic aneurysm (AAA). He was followed up over 7 years by ultrasonography (2D-ECHO) with and without Doppler flow evaluation, angiography and computed tomography. Calcification was noted 33 months after the onset of the disease but the aneurysm did not decrease in size. 2D-ECHO was adequate for evaluating the size of an aneurysm, but did not show thrombus formation or calcification. Doppler flow studies did not show abnormally high blood flow velocities either at the inflow or outlet of aneurysms, which are indicative of stenosis. On the other hand an abdominal aortogram provided information regarding the luminal shape and abnormalities in flow pattern suggestive of thrombus formation. Fluoroscopy cannot demonstrate calcification in the early stages. Computed tomography (CT) was the imaging method of choice for the evaluation of obstructive or calcific changes; an organizing thrombus was clearly demonstrated and early detection of calcification was possible.

Genetic evidence that ovulation reduces sexual receptivity in Drosophila melanogaster females.

Drosophila females start ovulation shortly after mating, and at the same time they become unreceptive to courting males. Both physiological changes are induced by the "sex-peptide" derived from the male accessory glands. It is conceivable, therefore, that the first effect of the peptide is to induce ovulation, and some signal derived from ovulated eggs makes females unreceptive. To test this hypothesis, I examined the mating receptivity of virgin D. melanogaster females homozygous for lozenge mutants that showed a high-frequency spontaneous ovulation. These females were reluctant to mate. However, when mature eggs were genetically deprived using nonallelic female sterile mutants, their receptivity increased significantly, although mating speed was still slower than that of normal virgin females. Essentially the same was found with the females that were ectopically expressing the sex-peptide gene. The results indicate that ovulation induced by the sex-peptide has an effect of reducing the sexual receptivity of mated females.

Drosophila suzukii contains a peptide homologous to the Drosophila melanogaster sex-peptide and functional in both species.

A peptide homologous to the Drosophila melanogaster sex-peptide (SP) was isolated from Drosophila suzukii accessory glands and its amino acid sequence determined. The D. suzukii peptide contains 41 amino acids and has a calculated molecular weight of 5100 Da. Comparison of the sequences reveals strong homologies in the N-terminal and C-terminal parts of the peptides. In the D. suzukii sex-peptide, however, five additional amino acids are inserted after amino acid 7. Based on the sequence of the peptide, a cDNA coding for the D. suzukii peptide was isolated by PCR. Sequence analysis of the cDNA confirmed the SP amino acid sequence determined by peptide sequencing. Furthermore, based on the cDNA sequence, we isolated the D. suzukii sex-peptide gene by inverse PCR. The D. suzukii sex-peptide gene contains an intron and codes for a 60 amino acid precursor. The D. melanogaster and the D. suzuki sex-peptides elicit rejection behaviour in the presence of males and an increased egg laying in virgin females of both species.

Genetics of food preference in Drosophila sechellia. I. Responses to food attractants.

To reveal the genetic mechanism of host selection in a monophagous fruit fly Drosophila sechellia, olfactory responses and oviposition preferences of this species were compared with those of closely related polyphagous species, D. simulans and D. melanogaster. Adult flies of D. sechellia were strongly attracted to the ripe fruit of Morinda citrifolia which is known to be the sole breeding site of this species. They were also attracted to the odor of n-caproic acid which is contained in the ripe fruit of M. citrifolia and is presumably responsible for the characteristic odor of the fruit. In contrast, D. simulans and D. melanogaster showed a strong repulsion to n-caproic acid. In parallel with the olfactory responses, D. sechellia females laid eggs preferentially on a medium containing n-caproic acid, to which the other two species showed an aversion. Genetic analyses using the hybrid progeny between D. sechellia and D. simulans suggested that the species differences in these behaviors are controlled by gene(s) located on the second chromosome.

Genetics of Parthenogenesis in DROSOPHILA MELANOGASTER. II. Characterization of a Gynogenetically Reproducing Strain.

A strain of Drosophila melanogaster, named gyn-F9, can reproduce by gynogenesis. On mating with a male sterile mutant, ms( 3)K81, gyn-F9 females produced impaternate progeny at a rate of about 15 flies per female, which was almost 2000 times as frequent as that of the control. When the females were mated with normally fertile males, the number of offspring varied extremely from parent to parent, with average fertility being much lower than that of normal females. Nearly one-third of these bisexual progeny were either triploid females or intersexes. Among the rest of the progeny, some were diploid impaternates having developed without syngamy. The gynogenetic property of gyn-F9 is primarily governed by a few genes, most likely two recessive genes, one each located on the second and third chromosomes. The impaternates were found to restore their diploidy by the fusion of two nonsister nuclei out of the four egg pronuclei which result from the second meiotic division (central fusion). Although nondisjunction occurs frequently in the meiosis of gyn-F9, this is unlikely to bring about an appreciable number of diploid gametes developing into impaternates. Possible mechanisms of the evolutionary origin of parthenogenesis are discussed in relation to these findings.

Genetics of Parthenogenesis in DROSOPHILA MELANOGASTER. I. the Modes of Diploidization in the Gynogenesis Induced by a Male-Sterile Mutant, ms(3)K81.

Sperm that are produced by males homozygous for ms(3)K81 , a male sterile mutant of Drosophila melanogaster, are defective in syngamy but are capable of activating eggs to develop gynogenetically. The activated eggs usually produce haploid embryos, but a small fraction (10( -4)-10(-5)) of them give rise to diploid impaternate adults. To know the cytological mechanisms by which these impaternates restore diploidy, the genotypes of impaternate progeny obtained from females doubly heterozygous for visible markers were examined. The results show that, as generally found among parthenogenetic Drosophila, diploidy is restored after completing meiosis either by pronuclear fusion or by gamete duplication (doubling of a haploid cleavage nucleus). The fusion of two nonsister nuclei following meiosis II (central fusion) was indicated to be a predominant mode of diploidization in this species. Two meiotic mutants, mei-9 and mei-S332, which are known to greatly increase meiotic nondisjunction, did not cause an increased incidence of impaternates. This seems to exclude the possibility that some impaternates might have been derived from diploid egg nuclei produced through nondisjunction.

Behavior genetics of choice of oviposition sites in Drosophila melanogaster. I. Genetic variability and analysis of behavior.

Females of Drosophila melanogaster were given a choice of oviposition site either on the surface of the medium or on the surface of paper positioned vertically on the medium. A significant difference was seen in the proportion of eggs deposited on the paper among wild strains of different geographic origins. Bidirectional selection for oviposition on these two sites was effective. These selected lines were examined under various conditions to determine the factors involved in this site selection for oviposition. The lines that chose medium laid eggs only on substrates into which eggs could be inserted. The lines preferring paper showed no strict requirement for burying their eggs. Tarsal sensillae were involved in site discrimination.

Behavior genetics of olfactory responses in Drosophila. II. An odorant-specific variant in a natural population of Drosophila melanogaster.

Homozygous second chromosome lines were extracted from a natural population of Drosophila melanogaster and tested for their olfactory responses to ethyl acetate. The chromosome lines were highly heterogeneous for this behavior, and the variability was also specific to other esters and ketones such as ethyl propionate, 2-butanone, 2-pentanone, and 3-pentanone. The responses to these odorants negatively correlated with the response to another odorant, lactic acid. A large part of this odorant-specific variation seemed to be controlled by genes located on the right arm of the second chromosome.

Behavior genetics of olfactory responses in Drosophila. I. Olfactometry and strain differences in Drosophila melanogaster.

A simple olfactometer was constructed for measuring the responses of adult Drosophila to odoriferous substances, and an index (attractability index, AI) is proposed to evaluate the response. Five strains of Drosophila melanogaster of different geographic origins were tested for their responses to five chemical attractants: ethyl alcohol, acetic acid, lactic acid, ethyl acetate, and n-butyraldehyde. Statistical analysis has shown that the differences of AI among strains are highly significant for all the odorants except acetic acid and that male flies are, in general, less sensitive than females. The number of flies which were inactive in the olfactometer also varied with strain, sex, and odorant, suggesting the involvement of some other behaviors, and this raises some difficulties in interpreting the heterogeneities of AI. Nevertheless, these results suggest that a considerable amount of genetic variation for olfactory response exists in natural population of D. melanogaster.