Sexual behavior mutants revisited: molecular and cellular basis of Drosophila mating.

The study of Drosophila melanogaster by a combination of forward genetics with specific mutants, and reverse genetics, in which a given gene is expressed in an appropriate brain area to test its effect on behavior, provides a unique opportunity to explore the causal relationship between a particular gene, its function in the cell and the behavioral outcome at the organismic level. Enhanced male-to-male courtship has been shown to occur as a result of mutations in several different genes. For example, the Voila mutant exhibits intense GAL4 reporter expression in the tarsal gustatory sensilla, suggesting the importance of tapping by a male on the female abdomen with his forelegs.

Two independent pathways of maternal cell transmission to offspring: through placenta during pregnancy and by breast-feeding after birth.

Cell transmission from mother to offspring was demonstrated using mice with green fluorescent protein (GFP) transgenic markers. GFP transgene heterozygous (+/-) females were mated with GFP (-/-) males, and GFP(+) cells in the GFP (-/-) fetuses generated between them were analysed to assess maternal blood cell transmission to conceptuses in utero. The GFP+ maternal cells were observed throughout the body of the fetuses, as shown by fluorescence stereomicroscopy.

Major sites for the differentiation of V alpha 14(+) NKT cells inferred from the V-J junctional sequences of the invariant T-cell receptor alpha chain.

CD1d-restricted mouse NK1.1(+) TCR alpha beta(+) natural killer T (NKT) cells predominantly use an invariant TCR alpha chain encoded by V alpha 14 and J alpha 281 gene segments with a one-nucleotide N region. We found that NKT cells generated in the culture of fetal liver precursors possessed V alpha 14-J alpha 281 junctions that could be produced without the action of terminal deoxyribonucleotidyl transferase (TdT), indicating that NKT cells derived from fetal liver precursors are distinguishable from those from adult precursors with TdT expression.

The calcium-independent protein kinase C participates in an early process of CD3/CD28-mediated induction of thymocyte apoptosis.

Thymocyte negative selection eliminates self-reactive clones and involves both a T-cell receptor (TCR)/CD3-mediated signal and a costimulatory signal, which can be delivered via CD28. Anti-CD3/anti-CD28-triggered apoptosis in isolated CD4+CD8+ thymocytes in vitro provides a basic model for negative selection. Effects of isoform-selective and non-isoform-selective inhibitors of protein kinase C (PKC) on this apoptotic process suggest that activation of Ca2+-independent PKC isoforms during the first 2-3 hr of culture is essential for inducing apoptosis, and that Ca2+-dependent PKC isoforms may be influential, but not essential, for apoptosis.

Three-dimensional solution structure of omega-conotoxin TxVII, an L-type calcium channel blocker.

We determined the three-dimensional structure of omega-conotoxin TxVII, a 26-residue peptide that is an L-type calcium channel blocker, by (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 411 distance constraints obtained from nuclear Overhauser effect connectivities, 20 torsion angle constraints, and 21 constraints associated with hydrogen bonds and disulfide bonds. The root-mean-square deviations about the averaged coordinates of the backbone atoms (N, C(alpha), C, and O) and all heavy atoms were 0.

Efficient cloning and engineering of giant DNAs in a novel Bacillus subtilis genome vector.

The Genome of Bacillus subtilis 168 was used for cloning and engineering of large-sized DNAs. A mouse genomic DNA of approximately 120 kb was cloned into a locus of the B. subtilis genome by ordered assembly of 20- to 50-kb mouse DNA segments.

Expression, purification and crystallization of human tau-protein kinase I/glycogen synthase kinase-3beta.

Human tau-protein kinase I (TPK-I; also known as glycogen synthase kinase-3beta, GSK-3beta) is a serine/threonine protein kinase. Full-length TPK-I/GSK-3beta was expressed in Escherichia coli as a fusion protein with a 6xHis tag at the C-terminus and was crystallized using the hanging-drop vapour-diffusion method. Prismatic crystals of dimensions 0.

Excitability increase induced by beta-adrenergic receptor-mediated activation of hyperpolarization-activated cation channels in rat cerebellar basket cells.

In the preceding paper, we showed that norepinephrine (NE) enhances the spontaneous spike firings in cerebellar interneurons, basket cells (BCs), resulting in an increase in the frequency of BC-spike-triggered inhibitory postsynaptic currents (IPSCs) in Purkinje cells (PCs), and that the effects of NE on GABAergic BCs are mediated by beta(2)-adrenergic receptors. This study aimed to further examine the ionic mechanism underlying the beta-adrenoceptor-mediated facilitation of GABAergic transmission at the BC-PC synapses. Using cerebellar slices obtained from 15- to 21-day-old rats and whole cell recordings, we investigated ionic currents in the BCs and the effects of the beta-agonist isoproterenol (ISP) as well as forskolin on the BC excitability.

beta-adrenergic receptor-mediated presynaptic facilitation of inhibitory GABAergic transmission at cerebellar interneuron-Purkinje cell synapses.

Norepinephrine (NE) has been shown to elicit long-term facilitation of GABAergic transmission to rat cerebellar Purkinje cells (PCs) through beta-adrenergic receptor activation. To further examine the locus and adrenoceptor subtypes involved in the NE-induced facilitation of GABAergic transmission, we recorded inhibitory postsynaptic currents (IPSCs) evoked by focal stimulation with paired-pulse (PP) stimuli from PCs in rat cerebellar slices by whole cell recordings and analyzed the PP ratio of the IPSC amplitude. NE increased the IPSC amplitude with a decease in the variance of the PP ratio, which was mimicked by presynaptic manipulation of the transmission caused by increasing the extracellular Ca(2+) concentration, confirming that the presynaptic adrenergic receptors are responsible for the facilitation.

Axonal growth from the habenular nucleus along the neuromere boundary region of the diencephalon is regulated by semaphorin 3F and netrin-1.

In neural development, major tracts are often formed along the neuromere boundary regions, although the molecular mechanism underlying this formation remains to be clarified. In the diencephalon, axons from the habenular nucleus extend along the neuromere boundary region between p1 and p2. At embryonic days 13-15, among members of class 3 semaphorins, only semaphorin 3F (Sema3F) was expressed in the diencephalon.

A triphasic curve characterizes the retention of lever-pressing behavior rewarded by lateral hypothalamic stimulation during the immediate-post-trial period in rats: implications for a transient-intermediate stage between short- and long-term memory.

We investigated the retention of lever-pressing behavior in rats rewarded by lateral hypothalamic (LHT) stimulation. Rats with electrodes implanted into the lateral hypothalamus were trained to press a lever to receive LHT stimulation. More than 90% of rats which had self-stimulated for longer than 30 min retained this behavior when tested 1 or 7 days later, indicating that intracranial self-stimulation (ICSS) produced a stable long-term memory lasting for at least 7 days.

Jumonji is a nuclear protein that participates in the negative regulation of cell growth.

The jumonji (jmj) gene, obtained by a gene trap strategy, is essential for mouse embryogenesis and is suggested to play important roles in cell growth during development. The amino acid sequence of the Jmj protein includes a nuclear localization signal and a DNA binding motif called the AT-rich interaction domain (ARID). To investigate the biological functions of the Jmj protein, we prepared specific antibodies.

HNF-4 plays a pivotal role in the liver-specific transcription of the chipmunk HP-25 gene.

The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5' flanking region. The gene spans approximately 7 kb and consists of three exons.

Antisense DNA against calcineurin facilitates memory in contextual fear conditioning by lowering the threshold for hippocampal long-term potentiation induction.

In the previous study, we demonstrated that the antisense oligodeoxynucleotides against calcineurin Aalpha and Abeta, catalytic subunits of Ca(2+)/calmodulin-dependent protein phosphatase, produce a facilitatory effect on long-term potentiation induction in the hippocampal CA1 region in rats anesthetized with urethane. Here, we have studied how animals, in which the hippocampal long-term potentiation induction is enhanced by antisense oligodeoxynucleotides against calcineurin, perform in learning tasks that depend on hippocampal function. The rats received antisense oligodeoxynucleotides by bilateral ventricular administration via miniosmotic pumps.

Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex.

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate.

Development of an antibody against a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein and identification of a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein in hippocampal neurons.

Brain injury-derived neurotrophic peptide is a 13-amino acid peptide derived from a 15,000 mol. wt neurotrophic factor released from sites of mechanical injury in neonatal rat brain. This peptide promotes survival of septal cholinergic neurons and mesencephalic dopaminergic neurons, and protects hippocampal neurons from glutamate-induced neurotoxicity.

Fruitless is in the regulatory pathway by which ectopic mini-white and transformer induce bisexual courtship in Drosophila.

Bisexual courtship in male Drosophila melanogaster may be induced in some circumstances. These include ectopic expression of the transformer (tra) gene, ectopic expression of the mini-white (mw) gene, and the homozygous presence of mutant alleles of the fruitless (fru) gene. Experiments were performed to determine if ectopic mw and fru, as well as ectopic tra and fru, acted in the same pathway to control courtship.

Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells.

To obtain a reliable molecular probe to trace the origin of germ cell lineages in birds, we isolated a chicken homolog (Cvh) to vasa gene (vas), which plays an essential role in germline formation in Drosophila. We demonstrate the germline-specific expression of CVH protein throughout all stages of development. Immunohistochemical analyses using specific antibody raised against CVH protein indicated that CVH protein was localized in cytoplasm of germ cells ranging from presumptive primordial germ cells (PGCs) in uterine-stage embryos to spermatids and oocytes in adult gonads.

Identification and cataloging of genes induced by long-lasting long-term potentiation in awake rats.

Maintenance of long-term potentiation (LTP) requires de novo gene expression. Here we report the direct isolation, using PCR-differential display, of genes whose expression level was altered after induction of long-lasting LTP in the hippocampus of freely moving awake rats. Differential display using 480 primer combinations revealed 17 cDNA bands that showed a reproducible change in expression level.

Synaptic activation of AMPA receptors inhibits GABA release from cerebellar interneurons.

A single neurotransmitter elicits diverse physiological responses through activation of multiple receptor subtypes and/or heterosynaptic interactions involving distinct synaptic targets. We found that a typical excitatory transmitter released from the climbing fiber (CF) in the cerebellar cortex not only excited Purkinje cells directly but also presynaptically inhibited GABAergic transmission from interneurons converging on the same Purkinje cells. Both homosynaptic and heterosynaptic actions of the CF transmitter (possibly glutamate) were mediated by activation of AMPA receptors.

Requirement of seminolipid in spermatogenesis revealed by UDP-galactose: Ceramide galactosyltransferase-deficient mice.

Although seminolipid has long been suspected to play an essential role in spermatogenesis because of its uniquely abundant and temporally regulated expression in the spermatocytes, direct experimental evidence has been lacking. We have tested the hypothesis by examining the testis of the UDP-galactose:ceramide galactosyltransferase-deficient mouse, which is incapable of synthesizing seminolipid. Spermatogenesis in homozygous affected males is arrested at the late pachytene stage and the spermatogenic cells degenerate through the apoptotic process.

Successive expression and activation of NFAT family members during thymocyte differentiation.

Differentiation of immature CD4(+)CD8(+) thymocytes to mature CD4(+) or CD8(+) T cells is induced by positive selection and appears to involve calcineurin-dependent activation of NFAT, a family of transcription factors. NFATx is predominantly expressed in CD4(+)CD8(+) thymocytes, whereas NFATp and NFATc are expressed at much lower levels in the thymus than in mature T cells. However, how or when each NFAT member is involved in the differentiation pathway is unclear.

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development.

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells.