Tissue-specific promoter usage and diverse splicing variants of found in neurons, an ancestral Hu/ELAV-like RNA-binding protein gene of insects, in the direct-developing insect Gryllus bimaculatus.

Hu/ELAV-like RNA-binding proteins (RBPs) are involved in the post-transcriptional regulation of RNA metabolism including splicing, transport, translational control and turnover. The Hu/ELAV-like RBP genes are predominantly expressed in neurons, and are therefore used as common neuronal markers in many animals. Although the expression patterns and functions of the Hu/ELAV-like RBP genes have been extensively studied in the model insect Drosophila melanogaster, little is known in basal direct-developing insects. In the present study, we performed an identification and expression analysis of the found in neurons (fne) gene, an ancestral insect Hu/ELAV-like RBP gene, in the cricket Gryllus bimaculatus. Contrary to expectation that the Gryllus fne transcript would be predominantly expressed in the nervous system, expression analysis revealed that the Gryllus fne gene is expressed broadly. In addition, we discovered that alternative promoter usage directs tissue-specific and embryonic stage-dependent regulation of fne expression, and that alternative splicing contributes to the generation of diverse sets of fne transcripts. Our data provide novel insights into the evolutionary diversification of the Hu/ELAV-like RBP gene family in insects.

Characteristics and osteogenic differentiation of stem/progenitor cells in the human dental follicle analyzed by gene expression profiling.

The dental follicle is an ectomesenchymal tissue that surrounds developing tooth germ and that contains osteoblastic-lineage-committed stem/progenitor cells. We examined the osteogenic potential of human dental follicle cells (hDFC) by microarray analysis. We first compared the characteristics of hDFC with those of human bone marrow mesenchymal stem cells (hMSC). Like hMSC, hDFC expressed stem cell markers such as STRO-1 and Notch-1 and differentiated not only into the osteoblastic lineage, but also into the adipogenic lineage. We analyzed the gene expression profiles of hDFC and hMSC that were not differentiated toward the osteogenic lineage. The expression of cell markers and growth factor receptors by hDFC and hMSC was similar, whereas the expression pattern of homeobox genes differed between hDFC and hMSC. Next, we investigated gene expression in hDFC during osteogenic differentiation. Gene expression profiles were analyzed in hDFC cultured in osteogenic induction medium (OIM) or in growth medium (GM) for 3 and 10 days. Many genes whose expression was regulated under these conditions were functionally categorized as "transcription" genes. Osteogenic markers were up-regulated in hDFC during osteogenic differentiation, whereas neurogenic markers were down-regulated. The genes whose expression was regulated in hDFC during osteogenic differentiation were further analyzed by ingenuity pathway analysis and real-time polymerase chain reaction. Bone morphogenetic protein and transforming growth factor-ß signaling pathways were activated in hDFC cultured in OIM for 3 days. This study indicates that the dental follicle contains stem cells and/or osteoblastic progenitor cells and is a potential cellular resource for bone regeneration therapy.

Identification and expression analyses of a novel serotonin receptor gene, 5-HT2ß, in the field cricket, Gryllus bimaculatus.

Biogenic amine serotonin (5-HT) modulates various aspects of behaviors such as aggressive behavior and circadian behavior in the cricket. In our previous report, in order to elucidate the molecular basis of the cricket 5-HT system, we identified three genes involved in 5-HT biosynthesis, as well as four 5-HT receptor genes (5-HT1A, 5-HT1B, 5-HT2α, and 5-HT7) expressed in the brain of the field cricket Gryllus bimaculatus DeGeer [7]. In the present study, we identified Gryllus 5-HT2ß gene, an additional 5-HT receptor gene expressed in the cricket brain, and examined its tissue-specific distribution and embryonic stage-dependent expression. Gryllus 5-HT2ß gene was ubiquitously expressed in the all examined adult tissues, and was expressed during early embryonic development, as well as during later stages. This study suggests functional differences between two 5-HT2 receptors in the cricket.

Octopaminergic system in the brain controls aggressive motivation in the ant, Formica japonica.

The ant, Formica japonica, is polyphagous and workers hunt other insects as foods. In this study, interspecific aggression was examined in the workers and queens. Behavior experiments demonstrated that interspecific aggressiveness was significantly higher in workers than queens. Workers showed predatory aggressive behavior towards crickets, on the other hand, queens elicited threat behavior but they didn't attack crickets. In order to investigate neuronal mechanisms underlying regulation of aggressive motivation, the role of biogenic amine in the brain in evoking aggressive behavior was examined by measuring biogenic amine using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). No significant difference in the octopamine (OA) level was found between workers and queens, but the level of N-acetyloctopamine (NacOA) in the brain of queens was significantly higher than that of workers. This study suggests that OAergic system in the brain must involve in controlling aggressive motivation in the ants.

Aggressive behavior of the white-eye mutant crickets, Gryllus bimaculatus.

Aggressive behavior of white-eye mutant crickets was investigated and compared with that of wild-type crickets. In the dark, wild-type pairs performed long-lasting fights with significantly higher aggressive levels compared to those in the light. In contrast, fights between two white-eye mutants were not significantly different with those between two wild-type crickets both in duration and the aggressive levels. Ethograms of aggressive behavior showed that the mutants could show typical sequentially escalating fight with the same behavioral categories as the wild-type crickets. These results indicate that the white-eye mutants are able to express normal aggressive behavior.

Mathematical analysis of the honeybee waggle dance.

A honeybee informs her nestmates of the location of a flower by doing a waggle dance. The waggle dance encodes both the direction of and distance to the flower from the hive. To reveal how the waggle dance benefits the colony, we created a Markov model of bee foraging behavior and performed simulation experiments by incorporating the biological parameters that we obtained from our own observations of real bees as well as from the literature. When two feeders were each placed 400 m away from the hive in different directions, a virtual colony in which honeybees danced and correctly transferred information (a normal, real bee colony) made significantly greater numbers of successful visits to the feeders compared to a colony with inaccurate information transfer. Howerer, when five feeders were each located 400 m from the hive, the inaccurate information transfer colony performed better than the normal colony. These results suggest that dancing's ability to communicate accurate information depends on the number of feeders. Furthermore, because non-dancing colonies always made significantly fewer visits than those two colonies, we concluded that dancing behavior is beneficial for hives' ability to visit food sources.

Identification and expression analysis of the genes involved in serotonin biosynthesis and transduction in the field cricket Gryllus bimaculatus.

Serotonin (5-HT) modulates various aspects of behaviours such as aggressive behaviour and circadian behaviour in the cricket. To elucidate the molecular basis of the cricket 5-HT system, we identified 5-HT-related genes in the field cricket Gryllus bimaculatus DeGeer. Complementary DNA of tryptophan hydroxylase and phenylalanine-tryptophan hydroxylase, which convert tryptophan into 5-hydroxy-L-tryptophan (5-HTP), and that of aromatic L-amino acid decarboxylase, which converts 5-HTP into 5-HT, were isolated from a cricket brain cDNA library. In addition, four 5-HT receptor genes (5-HT(1A) , 5-HT(1B) , 5-HT(2α) , and 5-HT(7) ) were identified. Expression analysis of the tryptophan hydroxylase gene TRH and phenylalanine-tryptophan hydroxylase gene TPH, which are selectively involved in neuronal and peripheral 5-HT synthesis in Drosophila, suggested that two 5-HT synthesis pathways co-exist in the cricket neuronal tissues. The four 5-HT receptor genes were expressed in various tissues at differential expression levels, suggesting that the 5-HT system is widely distributed in the cricket.

Nitric oxide-cyclic guanosine monophosphate signaling in the local circuit of the cricket abdominal nervous system.

The distribution of potential nitric oxide (NO) donor neurons and NO-responsive target neurons was revealed in the terminal abdominal ganglion (TAG) of the cricket. The expression of nitric oxide synthase (NOS) in the nervous system was examined by Western blotting using universal nitric oxide synthase (uNOS) antibody that gave about a 130 kDa protein band. Immunohistochemistry using the uNOS antibody detected neurons whose cell bodies are located at the lateral region of the TAG. These neurons expanded their neuronal branches into the dorsal-median region or the dorsal-lateral region of the TAG. NADPH-diaphorase histochemistry was performed to confirm the distribution of NOS-containing neurons. The distributions of cell bodies and stained neuronal branches were similar to those revealed by uNOS immunohistochemistry. NO-induced cGMP immunohistochemistry was performed to reveal NO-responsive target neurons. Most of the cell bodies stained by immunohistochemistry appeared at the dorsal side of the TAG. At the dorsal-median region, some unpaired neuronal cell bodies were strongly stained. Some efferent neurons whose axon innervate into each nerve root were strongly stained. The generation of NO in the TAG was detected by NO electrode. We found that NO is generally produced to maintain a basal concentration of 70 nM. Hemoglobin scavenged released NO from the ganglion. The concentration of NO was partly recovered when hemoglobin was replaced by normal saline. Application of 10 microM L-arginine that is a substrate of NOS increased NO release by approximately 10 nM. Furthermore, an excitatory neurotransmitter acetylcholine (ACh) also increased NO generation by approximately 40-50 nM in concentration in addition to the basal level of 70 nM. Optical imaging with fluorescent NO-indicator demonstrated that ACh-induced enhancement of NO release was transiently observed in the outer-edge region of TAG, where cell bodies of NOS-immunoreactive neurons were located. These results suggest that ACh accelerates NO production via neuronal events activated by ACh in the TAG.

The compartment structures of the antennal lobe in the ant, Aphaenogaster smythiesi japonica.

Pheromones are important cues for social insects such as ants. As a first step in elucidation of pheromonal information processing mechanisms in the myrmicine ant, we investigated the morphological structure of the antennal lobe. Using autofluorescence imaging, labeling of neuronal filamentous actin, and reduced silver impregnation staining, the antennal lobe was found to consist of five compartments that, each received input from a different antennal sensory tract. Two major tracts of projection neurons, the medial and lateral antenno-cerebral tract (m- and 1-ACT), originated from a different region of the antennal lobe. The m-ACT originated from the posterior part of the antennal lobe whereas the 1-ACT originated from the anterior part. These results demonstrate a spatial segregation of function within the antennal lobe.

The dance of the honeybee: how do honeybees dance to transfer food information effectively?

A honeybee informs her nestmates of the location of a flower she has visited by a unique behavior called a "waggle dance." On a vertical comb, the direction of the waggle run relative to gravity indicates the direction to the food source relative to the sun in the field, and the duration of the waggle run indicates the distance to the food source. To determine the detailed biological features of the waggle dance, we observed worker honeybee behavior in the field. Video analysis showed that the bee does not dance in a single or random place in the hive but waggled several times in one place and then several times in another. It also showed that the information of the waggle dance contains a substantial margin of error. Angle and duration of waggle runs varied from run to run, with the range of +/-15 degrees and +/-15%, respectively, even in a series of waggle dances of a single individual. We also found that most dance followers that listen to the waggle dance left the dancer after one or two sessions of listening.

GABAergic and non-GABAergic spiking interneurons of local and intersegmental groups in the crayfish terminal abdominal ganglion.

In the first step toward identifying the neurotransmitter released from spiking interneurons of both local and intersegmental groups in the crayfish terminal abdominal ganglion, the authors examined whether spiking local interneurons and ascending intersegmental interneurons contain the transmitter gamma-aminobutyric acid (GABA). In this paper, 17 identified ascending interneurons and three spiking local interneurons were stained by intracellular injection of Lucifer yellow and subsequently treated for immunocytochemical staining against GABA. Double-labeling experiments revealed that six identified ascending interneurons are GABAergic, but no spiking local interneurons show GABA-like immunoreactivity. Four ascending interneurons with GABA-like immunoreactivity (reciprocal closing ascending neuron 5 [RC-5], reciprocal opening ascending neuron 6 [RO-6], variable-effect ascending interneuron 1 [VE-1], and no-effect ascending interneuron 4[NE-4]) had cell bodies that formed a cluster on the ventral surface of the rostral edge of the ganglion, whereas two GABAergic interneurons (coinhibiting ascending interneuron 2 [CI-2] and NE-2) had cell bodies in a caudal region around the cell body of the seventh flexor inhibitor (FI) motor neuron. Another four rostral interneurons (RC-2, RC-3, RC-4, and NE-3) and seven caudal interneurons (CI-3, RC-7, RO-1, RO-2, RO-3, RO-4, and NE-1) had no GABA-like immunoreactivity. Because VE-1 is known to make direct inhibitory connections with other ascending interneurons, whereas RC-3 and RO-1 are known to make direct excitatory connections, the immunocytochemical results from this study are consistent with previous physiological studies. Although many spiking local interneurons (including spiking local interneuron 1 of the anterior group [sp-ant1]) made direct inhibitory connections with nonspiking local interneurons, three spiking local interneurons (sp-ant1, spiking local interneuron 6 of the medial group [sp-med6], and spiking interneuron 5 of the posterior group [sp-post]) do not show GABA-like immunoreactivity. These results suggest that the inhibitory transmitter released from spiking local interneurons is not GABA but that another substance mediates the inhibitory action of these interneurons. J. Comp. Neurol. 410:677-688.

Distribution of GABAergic premotor nonspiking local interneurones in the terminal abdominal ganglion of the crayfish.

The inhibitory neurotransmitter of premotor nonspiking local interneurones in the crayfish terminal abdominal ganglion was investigated physiologically and immunocytochemically. Depolarization of a nonspiking interneurone evoked a hyperpolarization in a uropod motor neurone. The amplitude of hyperpolarization in the motor neurone was gradually decreased under low-calcium/high-magnesium saline. Local pressure injection of gamma-aminobutyric acid (GABA) into the neuropil caused a similar hyperpolarization of the motor neurone. These physiological studies suggested a GABAergic inhibitory interaction between nonspiking interneurones and the motor neurones. Premotor nonspiking interneurones are classified into two subgroups ofposterolateral (PL) and anterolateral (AL) interneurones, and AL interneurones are further divided into three subtypes. A combination of intracellular staining from nonspiking local interneurones with Lucifer yellow and immunocytochemical staining with an antiserum directed against GABA revealed that all the PL interneurones sampled in this study showed GABA-like immunoreactivity. A population of cell bodies (n = 6-11) with a small diameter (15-30 microm) packed together forming a cluster showed GABA-like immunoreactivity, and the cell bodies of most PL interneurones were found in this cluster. To compare the number and the pattern of main branches of PL interneurones, cells were classified into three identifiable sets of interneurones, called PL-1, PL-2, and PL-3. By contrast, about one-half of AL interneurones, especially the third subtype of AL interneurones, which have cell bodies located ventrolaterally in the ganglion, did not show GABA-like immunoreactivity. Furthermore, the position of cell bodies of GABA-immunoreactive AL interneurones was scattered compared to that of PL interneurones.

Morphological and physiological bases of crayfish local circuit neurones.

As the subject of neuroethological studies, arthropods offer several advantages to elucidate the neural processes that generate and control behavioural acts. The relative simplicity of their neural organization and their identifiable neurones are particularly advantageous. The crayfish, Procambarus clarkii, uropods are paired appendages of the last abdominal segment that show bilateral closing movement in response to mechanical stimulation. This movement, the avoidance "dart" response, is mediated by a cascade and parallel organization of local circuit neurones in the terminal abdominal ganglion. Thirty intersegmental ascending interneurones and 20 spiking local interneurones have been identified both morphologically and physiologically. They receive exteroceptive inputs directly from the sensory afferents. Two complementary groups of unilateral nonspiking local interneurones have also been functionally identified. They exert fine control over the wide range of activity of motor neurones in a graded and sustained manner. These nonspiking interneurones form opposing and parallel pathways that are essential in modulating the pattern of movement of the uropods.

Processing of proprioceptive signals by ascending interneurones in the terminal abdominal ganglion of the crayfish.

Intersegmental interneurones are crucial for the appropriate coordination of the activity of local circuits located in different body segments. We have analysed the synaptic inputs to ascending intersegmental interneurones from a proprioceptor in the tailfan of the crayfish. Twenty identified interneurones responded during stimulation of the exopodite-endopodite chordotonal organ. Of these 20 interneurones, three were excited phaso-tonically, nine were excited phasically and eight were inhibited. All received convergent exteroceptive inputs from water-motion- or touch-sensitive hairs on the uropods. The effects of simultaneous exteroceptive and proprioceptive stimulation depended upon the identity of an interneurone. For interneurones that were inhibited by proprioceptive stimulation, suprathreshold exteroceptive responses were reduced to a subthreshold level by simultaneous proprioceptive stimulation. In contrast, for interneurones that were excited by proprioceptive stimulation, the simultaneous application of subthreshold proprioceptive and exteroceptive stimulation elicited action potentials. Two of the interneurones that receive proprioceptive input (NE-1 and RC-8) are known to be presynaptic to giant interneurones that mediate and coordinate the tail-flip. Many of the other interneurones that receive proprioceptive inputs in the tailfan are known to excite abdominal extensor motor neurones. Thus, proprioceptive input to these intersegmental interneurones could serve two roles: first, to extend the abdomen during postural movements or prior to escape and, second, to drive the tail-flip escape response.

GABA-like immunoreactivity of an identified nonspiking local interneurone in the crayfish terminal abdominal ganglion

Using an antiserum directed against -aminobutyric acid (GABA) to label neurones with GABA-like immunoreactivity, approximately 70 central neurones (68±9; mean ± s.e.m., N=9) were labelled in the terminal abdominal ganglion of the crayfish Procambarus clarkii. This mean number of neurones with GABA-like immunoreactivity represents approximately 10 % of the total number of neurones in the terminal ganglion. A combination of intracellular staining using Lucifer Yellow and immunocytochemical staining revealed that an identified nonspiking local interneurone (the local directionally selective interneurone, LDS) showed GABA-like immunoreactivity.

Late onset hepatic failure due to hepatitis B virus with mutations in the pre-core region.

A 60-year-old man complained of severe general fatigue on October 11, 1992. Pertinent laboratory findings were: aspantate aminotransferase (AST) 1920 IU, alanine aminotransferase (ALT) 2050 IU, and total bilirubin (T. Bil) 124 micromol/l (normal range, 0-17 micromol/l). Virological assay revealed that hepatitis B surface antigen (HBsAg), anti-hepatitis B e (HBe), anti-HBc, and immunoglobulin M (IgM) anti-HBc were positive, and anti-HBs, HBeAg, and anti-delta antibody were negative. A diagnosis of acute hepatitis due to hepatitis B virus was made. Despite a decrease in transaminase, jaundice worsened and prothrombin time was prolonged. On the 60th day of hospitalization, massive ascites developed, but the patient's consciousness was not impaired. Although, albumin and diuretics were given, the ascites further increased. Paracentesis of 2000 ml of ascitic fluid was performed twice a week. On the 120th day of hospitalization, the patient passed black stools and he exhibited renal failure 3 weeks later. Although severe jaundice persisted, he was still alert. On the 150th day of hospitalization, massive gastrointestinal bleeding occurred, due to hemorrhagic gastritis. Despite receiving intensive care, the patient died. Determination of the HBV DNA sequence revealed two point mutations in the pre-core region; these have not been reported elsewhere.

Colonic and peritoneal tuberculosis associated with coloduodenal fistula.

We report a very rare case of tuberculous colitis that showed relatively long-segment involvement of the colon near the hepatic flexure with coloduodenal fistula that caused severe malnutrition. The formation of fistula in abdominal tuberculosis is very rare. This is the eighth reported case of abdominal tuberculosis with fistula and the first reported case with a coloduodenal fistula.

Retinal cell death by light damage.

PURPOSE: To determine the relationship between apoptotic photoreceptor cell death and the duration of light exposure. METHODS: Ten-week-old male albino rats (Wistar strain) were dark-adapted for 2 days and then exposed to intense light for 12 hours, and 1, 2, 3, 7, 14, 21, and 28 days. The presence of apoptosis was confirmed by electron microscopy and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method. Differences in the apoptotic processes of the photoreceptor cells in the superior, posterior pole, and inferior portions of the retina were determined. RESULTS: Photoreceptor cells showed TUNEL-positive staining, whereas the cells in the inner nuclear layer, ganglion cell layer and retinal pigment epithelia exhibited weak positive or negative TUNEL staining. By electron microscopy, photoreceptor cells showed typical apoptotic nuclear changes and formation of apoptotic bodies. CONCLUSIONS: The sensitivity to light damage and style of death differed among retinal cells by location and cell type.

Retinal light damage: protective effect of alpha-tocopherol.

We studied histologically the protective effect of alpha-tocopherol to retinal light damage. After 3-week-old albino rats were fed with an alpha-tocopherol deficient or supplemented diet and kept in a 12-hour dim light/12-hour dark environment for 8 weeks, each animal was exposed to intense light (2500 lux) for 1, 3, 6, 12, 24, and 72 hours. The eyes were enucleated and prepared for transmission electron microscopy study and image analysis of phagosomes. Before light exposure, the alpha-tocopherol content of the neural retina of the deficient and supplemented groups was 0.3 microgram and 23.34 micrograms, respectively. After 1- and 3-hour exposures, morphological changes in the retinal pigment epithelium and photoreceptor membranes were more extensive in the deficient group than in the supplemented group. After a 24-hour exposure, pyknotic photoreceptor nuclei were more numerous in the deficient group than the supplemented group. After 3-, 6-, and 12-hour exposures, large phagosomes were more numerous in the deficient group than in the supplemented group. These findings suggest that alpha-tocopherol can protect the retina from light injury for up to 24 hours of exposure.

A chorioretinal lesion in a patient with Kimura's disease.

A 47-year-old male patient with Kimura's disease showed a white and slightly elevated choroidal lesion surrounded by serous retinal detachment in his left eye. Serological examination revealed esinophilia. Swelling in his left cheek had been increasing during a period of 20 years, and diagnosis of Kimura's disease had been made earlier by biopsy. Fluorescein angiography suggested disturbance of choroidal circulation and dysfunction of retinal pigment epithelium. After corticosteroid therapy, improvement was seen in the fundus findings, the swelling in the cheek and eosinophilia.