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Differentiated mammalian somatic cell nuclei and embryonic nuclei can now be reprogrammed to develop into young if they are introduced into enucleated oocytes. The success rates for cloning are generally low, however, and peri- and postnatal death rates of the young are high. Cloning technology will be useful for the genetic improvement of farm animals, therapeutic human protein production, and organ or tissue transplantation into humans. In addition, the information obtained on nuclear reprogramming will be helpful for understanding the fundamental mechanisms of differentiation and aging.
A single proprioceptor in the tailfan of the crayfish, Procambarus clarkii (Girard), innervated by only twelve sensory neurones encodes the position and the direction and velocity of movement of the exopodite relative to the endopodite. Most of the sensory neurones project to, and terminate in, the terminal abdominal ganglion where they form a map in which projection position is based on the velocity threshold of the sensory neurone. The sensory signals from this small proprioceptor have significant effects on the neuronal circuits mediating escape swimming and activate the lateral giant interneurone directly through monosynaptic connections and indirectly via a disynaptic pathway involving a number of interposed intersegmental interneurones. The lateral giant interneurones are activated through electrical synapses whereas the ascending interneurones in the disynaptic pathway are excited through both electrical and chemical synapses. The proprioceptive signals are also responsible for evoking widespread presynaptic inhibition of exteroceptive afferents that reduces the efficacy of their outputs. This pathway therefore reduces afference caused by water movement as a result of an animals own escape movements. Movements of the chordotonal organ also lead to a delayed input to giant motor neurone that is timed to occur during flexion movements of the abdomen. Thus not only do the proprioceptive signals activate the escape pathway leading to a tail-flip, but they also protect it from unwanted sensory input, and may also prevent depression of its neuromuscular synapses.
Functional significance of an immediate early gene ZENK (zif/268) was examined in telencephalic regions (homologues of neocortex and basal ganglia) of newly-hatched quail chicks; hyperstriatum accessorium (HA), hyperstriatum ventrale (HV), neostriatum (N) and lobus parolfactorius (LPO). Chicks were trained by a green bead soaked either in a strong aversant (methylanthranilate, MeA), in a weak aversant (MeA diluted by ethanol, 1/3MeA), or in water. Chicks were then tested at 45–50 min post-training, and immediately processed for ZENK immunostaining. Neither the training condition (MeA, 1/3MeA, or water) nor the responses at test (recall or amnesia) significantly contributed to the immunopositive cell densities in all of these regions. On the other hand, single intraperitoneal injection of metrazole (CNS convulsant) induced a transient epileptiform seizure, and caused significantly enhanced ZENK expression in HV and LPO but not in HA and N. However, the metrazol-induced seizure did not interfere with the following passive avoidance training, and chicks successfully learned to avoid the aversive bead when tested at 24 hr subsequently. Among three groups of chicks (metrazol-treated, saline control, and untreated chicks), no significant differences were found in their responses at test (recall, generalized avoidance, or amnesia). These results suggest that enhanced ZENK expression may represent lasting neural activities, but may not be involved specifically in the passive avoidance memory formation.
The acoustic structures of distance calls in sexually mature Bengalese finches (Lonchura striata var. domestica) show distinct sexual differences. We conducted a neuroethological study to identify the neural mechanism in the brain by which sexually mature male and female birds produce these acoustically different distance calls. Bilateral lesions of the dorsomedial (DM) nucleus of the intercollicular complex, known as the midbrain vocal center, eliminated distance calls in sexually mature males and females, but electrical stimulation of the DM in males and females induced calls that showed sexual differences and were acoustically similar to the distance calls of males and females. These results confirm that the DM in both sexes is one of the nuclei of the vocal control system that controls calling behavior. Neural tracer was injected into the DM in order to identify sexual differences in the neural input and output connections in the DM. Anatomical tracing of DM revealed the existence of labeled somata in the ipsilateral robust nucleus of the archistriatum (RA) in males. After bilateral RA lesions, however, males produced distance calls that were acoustically similar to female distance calls. These results suggest that the DM is one of the nuclei which generate the distance call patterns of both sexes, and that the neural information from the RA to the DM change the distance call pattern of male.
The phase of locomotor activity of the onion fly, Delia antiqua, in LD12:12 advanced at a low temperature (20°C) as compared with that at a high temperature (25°C). The free-running period (τ) in constant darkness (DD) at 20°C became shorter than that at 25°C, suggesting that the phase advance of locomotor activity in LD cycles at 20°C was caused by the decrease in τ. In constant light (LL), the locomotor activity was arrhythmic at a constant temperature. In both DD and LL, the locomotor activity was entrained to a 12 hr 25°C:12 hr 20°C temperature cycle; the activity occurred in the thermophase and its peak delayed with age. However, the delay in LL was smaller than that in DD. At a cycle of 12 hr cool (20°C) light and 12 hr warm (25°C) dark, the fly showed a similar activity pattern to that in LD 12:12 at a constant temperature (20°C or 25°C); the activity occurred in the light phase. This suggests that LD cycle is a stronger zeitgeber than a temperature cycle to entrain the locomotor activity of D. antiqua.
The profile of homing behavior in chum salmon (Oncorhynchus keta) that migrate from coastal sea to their natal river was not known well. We thus investigated temporal behavioral profiles of pre-spawning chum salmon in terms of water depth and temperature in Ishikari Bay using a micro data logger in 1997 and 1998. Fish were caught by a set net, tagged and attached with a data logger under MS222 anesthesia, and were released at the points 5 and 3 km off from the mouth of the Ishikari River in 1997 and 1998, respectively. A temporal profile of water depth and ambient temperature along the migratory pathway of recaptured salmon indicated that they usually stayed near the surface where water temperature was relatively low. Conductivity-temperature-depth recorder (CTD) data and the sea surface temperature estimated with National Oceanic and Atmospheric Administration (NOAA) Advanced Very High Resolution Radiometer (AVHRR) indicated that the influent of river water formed a low-temperature brackish subsurface layer near the mouth of the Ishikari River. These results indicate that chum salmon homing to the Ishikari River prefer low water temperature, and wander in the subsurface layer of nearshore area close to the mouth of the Ishikari River until they are motivated to migrate upstream. The main factor that regulates behavioral pattern of returned chum salmon in coastal sea should be distribution of water temperature.
Here we produced a monoclonal antibody (termed as mH2) which specifically recognized an 86-kD subunit of Human DNA helicase II (p86) in HeLa cells. Immunohistochemical analysis of p86 throughout the cell cycle showed that its localization was changed from the nuclei to the cytoplasm, when the cells entered into prophase, and p86 localized diffusely in the cytoplasm during mitosis. Double staining of interphase HeLa cells with mH2 and an antibody, which recognized a component of nuclear pore complex, showed that p86 did not localize at the nuclear envelop in interphase cells.
For phototactic steering, Chlamydomonas detects environmental light conditions with a photoreceptor (eyespot) while rotating the cell body around its body axis. Because of the bodily rotation and the directionality of the eyespot sensitivity, the light signal perceived by the eyespot must alternate between a light period and a dark period. It is an interesting question how cells can correctly change its swimming direction while the light signals change periodically. In this study, we examined the timing of the change in cells' swimming direction with respect to the timing of the light/dark cycle occurring at the photoreceptor. Most of the cells that displayed positive phototaxis had the eyespot facing the outside of the helical swimming track. We found that when phototactic light was applied from the direction perpendicular to the swimming direction of a cell, a phototactic response was initiated when the eyespot faced the light source. This was constantly observed irrespective of the phase at which the phototactic light was turned on. The initial change observed after the light stimulation was a decrease in the pitch angle of the helical swimming path, which caused the cell to swim less farther away from the light source than when unstimulated. This change was followed by a large turn toward the light source, which occurred when the eyespot faced away from the light. These observations indicate that the dominance of the cis-flagellum (the flagellum nearest to the eyespot) over the trans-flagellum (the flagellum farthest from the eye-spot) decreases during the light phase and increases during the dark phase. Thus, both light reception (on response) and the cessation of light perception (off response) by the eyespot are important for producing phototactic turns.
In Xenopus, semi-xenogeneic JB skins render donor-specific tolerance to perimetamorphic JJ larvae, whereas the same grafts are never accepted by adults. To clarify the mechanisms underlying tolerance induction, we tried to find a method of inducing tolerance in adults. First, we reconstituted early-thymectomized (E-Txd) adults with larval or adult thymi. All JB skin grafts transplanted to E-Txd adults that had been reconstituted with larval thymi were rejected, while almost all of the E-Txd larvae that had been reconstituted with larval or adult thymi were rendered tolerant. Second, we transplanted tolerated JB (tol-JB) skin, i.e., JB skin that reportedly possessed a suppressive activity (Ono and Tochinai, 1995), to late-thymectomized adults and found that those adults were rendered tolerant. Third, when tol-JB skin and larval or adult thymi were simultaneously transplanted to E-Txd animals, many of the E-Txd adults were rendered tolerant. The overall results indicate that donor-specific tolerance can be induced in thymectomized JJ adults by cotransplanting either a larval or an adult thymus and a tol-JB skin graft.
Nonsteroidal anti-inflammatory drugs (NSAIDs) such as sodium salicylate, aspirin and indomethacin have been reported to activate the heat shock transcription factor (HSF) without enhancing the expression of the heat shock proteins (HSPs). We investigated the effects of NSAIDs on the heat shock-induced HSP expression in fish cell line CHSE-214. Here we reveal that in CHSE-214 cells, co-exposure to sodium salicylate/aspirin and heat shock (24°C) enhances and prolongs the heat shock-induced HSP70 expression presumably through activation of the HSF. In contrast, the heat shock-induced HSP90 expression was inhibited by these NSAIDs. Thus, sodium salicylate and aspirin are likely to exert different effects on the heat shock-induced HSP70 and HSP90 expression. Indomethacin, another cyclooxygenase inhibitor, had no stimulatory or inhibitory effects on the heat shock-induced HSP70 and 90 expression, thereby indicating that sodium salicylate and aspirin may modulate the heat shock response via pathways not involving cyclooxygenase. Since anti-oxidants could inhibit the heat shock-induced HSP70 expression, the stimulatory effects of sodium salicylate and aspirin on the HSP70 expression are not likely due to their ability to act as anti-oxidants. Additionally, sodium salicylate and aspirin could exert synergistic effects on the HSP70 expression at lower temperatures (20–22°C) that did not induce the HSP70 expression.
Microinjection of E-64 into single blastomeres of Xenopus embryos at the 2-cell stage arrested cell division. In vitro treatment of cultured individual cells at the blastular embryonic stage with E-64 c also arrested the cleavages. We examined the effects of E-64 on the proteasome and calcium-activated protease in Xenopus embryos in vitro. E-64 inhibited calpain-like protease activity. Calpain-like protease activity was stimulated during cleavage. These results indicated that a calpain-like protease might be involved in the cell division of early Xenopus embryos.
In an attempt to develop a technique of fluorescence in situ hybridization (FISH) to detect DNA in Paramecium, we examined three different DNA probes, total genomic DNA, genomic DNA encoding C5 phagosomal membrane antigen, and telomere, prepared from P. multimicronucleatum. In accordance with the conventional method, total genomic DNA probe was denatured at 75–80°C for 2 min, and the cells were denatured at 75, 80, 85, or 90°C for 5 or 10 min. The homogeneous hybridization signal with the total genomic DNA probe was obtained at 85°C for 10 min, or at 90°C for 5 min or 10 min. This condition was applied for the smaller DNA probe, C5 (1. 3 kb, the size close to detection limits), in which the expected tiny signals throughout the macronuclear nucleoplasm was observed. However, the condition was not successful for the telomeric DNA probe. The hybridization signals of telomeric DNA were only detected when both cells and probes were denatured simultaneously in a same denaturation buffer. In the case of the simultaneously denatured samples, the preservation of the nuclear morphology was relatively poor, however, the signals of the telomeric DNA probe were observed in the periphery of the macronucleus. As a negative control, an irrelevant 40 kb human cosmid probe was examined by both conventional and simultaneous denaturation methods, and none of the hybridization signal was observed with this probe. These results suggest that the current methods allow us to follow localization of the specific sequences within the macronuclear compartment.
In male Japanese eels, eel spermatogenesis-related substance (eSRS) 3 and 4, having high sequence-similarities to zona pellucida protein (ZP) 2 and ZP3, respectively, are down-regulated by gonadotropin stimulation, with their transcripts disappearing upon the initiation of spermatogenesis. Using Northern blot analysis, we investigated the expression of eSRS3 and 4 mRNA in the developing ovary and the liver of SPH (salmon pituitary homogenate)-injected female eels. Both transcripts were detected in the ovary, but not in the liver. When the eel ovary was subjected to in situ hybridization using eSRS3 and 4 cRNA probes, the cytoplasm of previtellogenic oocytes showed a strong signal in comparison with the weak signal in vitellogenic oocytes. Furthermore, stronger signals were observed in the chromatin-nucleolus and the perinucleolus stages than in the oil-droplet stage. Subsequently, we synthesized peptides that were deduced from eSRS3 and 4 cDNAs and generated specific antibodies against them. Staining of the cytoplasm of oocytes in the previtellogenic stage and of egg envelopes in the vitellogenic stage occurred when these antibodies were used in an immunohistochemical analysis. These expression patterns in the ovary suggest that eSRS3 and 4 are components of the eel egg envelope.
The estrous cycle of the Asian elephant (Elephas maximus) was monitored by analysis of exfoliative cytology in the vaginal vestibule and serum progesterone concentrations. Appearance frequency of each 5 exfoliative cells; parabasal, intermediate, superficial anuclear and nuclear cells and leukocytes; on the smear collected from two elephants was calculated, and serum progesterone concentrations were measured by radioimmunoassay. Serum progesterone concentrations changed regularly with the cycle between 14 and 17 weeks. Using spectrum analysis (Yule-Walker method) to appearance frequency of exfoliative cells, it was found that the time when a superficial cell markedly appeared in vaginal vestibule corresponded to the time when serum progesterone concentration was almost negligible. It is suggested that the time when numbers of two kinds of superficial (anuclear and nuclear) cells and parabasal and intermediate cells increase to the smear of the elephant, means the period from proestrus to estrus and from metestrus to diestrus, respectively.
The cellular localization of inhibin α, βA and βB subunits in cyclic ovaries of the guinea pig was investigated. The immunoreactivity of inhibin α, βA and βB subunits was localized to the granulosa cells of some large healthy follicles in each ovary throughout the estrous cycle. The number of follicles that stained was in accordance with the number of offspring typical in guinea pigs. Inhibin βB was also localized to the granulosa cells of small antral follicles on Day 4. There were two kinds of staining patterns for inhibin α, βA and βB subunits on Day 12: strongly stained follicles identical to those observed on Days 8 and 16, and weakly stained follicles that showed atresia in hematoxylin and eosin (HE) stained sections. Two types of ovarian cysts were found throughout the estrous cycle in this experiment: serous cysts and follicular cysts. The incidence of serous cysts and follicular cysts were 64% and 24% of animals, respectively. There was no positive reaction for inhibin α, βA and βB subunits in the corpora lutea, other follicles or any kind of ovarian cyst during the estrous cycle. These results comfirm that only dominant follicles stain positively for inhibin α and βA subunits and are in agreement with the phenomenon that the follicular development of guinea pigs shows two waves of growth. This study is also the first to describe the ovarian cysts during the estrous cycle in guinea pigs systematically.
Morphological analysis of a small freshwater goby, Rhinogobius flumineus, showed a distinct dimorphic asymmetry in the lower jaw. The mouth of each fish opened with a slight but definite distortion toward the right or left side, depending on the individual. Right-opening mouth (dextral) fish had a right lower jaw that was more protruded than the left one, and left-opening (sinistral) fish had a more protruded left lower jaw. No fish had laterally symmetric lower jaws, indicating that the asymmetry was different from ‘fluctuating asymmetry’. These fish used either the right or left side of the mouth when picking up food from the bottom, but neither dextral nor sinistral individuals used one side more frequently than the other side. The mouth asymmetry, however, was related to the stationary posture: dextral fish stayed on the bottom with the line of their bodies curved to the right more frequently than to the left, and vice versa for the sinistral fish. Genetics of the morph phenotype were investigated by observing the frequencies of morphs in F1 under captive breeding. Mouth dimorphism seems to be determined by the Mendelian one-locus-two-alleles system, in which dextrality is dominant over sinistrality and the dominant gene acts as the lethal one when in a homozygote.
We sequenced the mitochondrial cytochrome b, 12S rRNA and 16S rRNA genes from three insectivoran species (Japanese mole, shrew mole and musk shrew) and one chiropteran species(Japanese pipistrelles) and the cytochrome c oxidase subunit II (COII) gene from a chiropteran species. The phylogenetic relationship of core Insectivora or Eulipotyphla among major Eutherian orders was examined for these genes. A total evaluation of the maximum likelihood analyses of the four genes suggests that Chiroptera is the closest to Eulipotyphla among 4 eutherian groups (Primates, Eulipotyphla, Fereuungulata, Rodentia).