Granule enzyme exocytosis assay for cytotoxic T lymphocyte activation.

Upon activation by specific target cells, cytotoxic T lymphocytes (CTL) release into the culture medium the content of cytoplasmic granules that contain serine esterases. The amount of enzyme released during CTL activation can be easily quantitated by spectrophotometric measurement of the colored product of the enzymatic degradation of a synthetic substrate. In the primary method presented here, CTL are activated with monoclonal antibodies prepared against the T cell receptor (TCR) complex, then activation is quantitated according to the amount of serine esterase released in the supernatant.

Expression analysis of the lingerer gene in the larval central nervous system of Drosophila melanogaster.

The lingerer (lig) gene is necessary for initiation and termination of copulatory behavior in Drosophila melanogaster. The lig gene encodes cytoplasmic proteins, and is expressed in the central nervous system (CNS) during the late third-instar larval stage when the lig function is required for normal copulation to occur after adult eclosion. To characterize the lig-expressing cells in the late third-instar larval CNS, we have isolated a genomic fragment containing the promoter/enhancer region of the lig gene, and established transgenic lines in which expression of reporter genes is controlled by the lig promoter/enhancer.

Mouse RanBPM is a partner gene to a germline specific RNA helicase, mouse vasa homolog protein.

Germ cell-specific ATP-dependent RNA helicase, the product of the mouse vasa homolog (Mvh), has been shown to play an essential role in the development of the male germ cell. In male Mvh knockout mice, premeiotic germ cells arrest at the zygotene stage. To investigate the role of MVH protein in the progression of meiosis, we searched for genes encoding partners that interact with MVH in testicular germ cells.

Expression of the mouse Aven gene during spermatogenesis, analyzed by subtraction screening using Mvh-knockout mice.

As an attempt to investigate the function of the mouse Vasa-homolog (MVH) protein, a germ cell-specific RNA helicase, we have cloned genes that fail to be expressed in homozygous Mvh-knockout testes. One of the cDNA clones obtained by selective PCR subtraction cloning matched two cDNA sequences in the mouse testis cDNA database, and was highly homologous to Aven, a human gene regulating cell death. Sequences of the two mouse testis cDNAs in the database revealed that they share an 800 bp sequence but have quite different 5'-regions; the latter are derived from different exons by alternative use of transcription initiation sites.

lingerer, a Drosophila gene involved in initiation and termination of copulation, encodes a set of novel cytoplasmic proteins.

In an effort to uncover genetic components underlying the courtship behavior of Drosophila melanogaster, we have characterized a novel gene, lingerer (lig), mutations of which result in abnormal copulation. Males carrying a hypomorphic mutation in lig fail to withdraw their genitalia upon termination of copulation, but display no overt abnormalities in their genitalia. A severe reduction in the dosage of the lig gene causes repeated attempted copulations but no successful copulations.

[Give cosmic sense to next generations through cosmic/space biology].

Life sciences are currently most important fields for every person. Each subject of life sciences is deeply correlated to cosmic events, and young generations can be more interested in life sciences if they know this correlation. In this respect, cosmic/space biology can play essential roles in fostering the science-oriented generations.

In vitro clonal analysis of rat cerebral cortical neurons expressing latexin, a subtype-specific molecular marker of glutamatergic neurons.

Latexin is expressed in a subset of glutamatergic projection neurons located in the lateral but not the dorsomedial neocortex in rat. In the present study, we performed clonal analysis of embryonic cortical neurons in vitro using latexin as a subtype-specific molecular marker of glutamatergic neurons to address whether certain precursors in early cortical anlage are already committed to a single molecular phenotype. Dissociated cells from lateral cortex at embryonic day 12 were labelled with a replication-deficient retroviral vector containing the bacterial lacZ gene, and cultured in a monolayer for 3 weeks.

[Superbugs viewed from cosmobiology].

Superbugs (microorganisms living in unfamiliar and very harsh environments) are located in the center of scientific interests in the sense that 1) most of their habitats belong to marginal regions of the biosphere, 2) clues for the elucidation of the origin of life can be deduced from them, and 3) they are deeply correlated to the extraterrestrial life. Not only for the basic scientific interests, but also for the applied fields, the spot light is shed to them. We, human beings, have been deeply dependent on other organisms through the global material flow they make.

Vasa homolog genes in mammalian germ cell development.

Many vasa homologue genes to Drosophila vasa have been isolated in various animal species. They provide specific molecular probes to analyze the establishment and the differentiation of germ cell lineage. In mammals, the expression of VASA protein becomes detectable in PGCs at the late migrating stage.

Recombinational transfer of 100-kilobase genomic DNA to plasmid in Bacillus subtilis 168.

Transformation of Bacillus subtilis by a plasmid requires a circular multimeric form. In contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. A recombinational transfer system was constructed with this intrinsic B.

Inhibition of protein phosphatase 2A overrides tau protein kinase I/glycogen synthase kinase 3 beta and cyclin-dependent kinase 5 inhibition and results in tau hyperphosphorylation in the hippocampus of starved mouse.

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease (AD) brain. Starvation of adult mice induces tau hyperphosphorylation at many paired helical filaments sites and with a similar regional selectivity as those in AD, suggesting that a common mechanism may be mobilized. Here we investigated the mechanism of starvation-induced tau hyperphosphorylation in terms of tau kinases and Ser/Thr protein phosphatases (PP), and the results were compared with those reported in AD brain.

Genomic organization, transcription start sites, and chromosomal location of the Drosophila cortactin gene.

An actin filament binding protein cortactin was initially identified as a major phosphotyrosine-containing protein in v-Src-transformed chicken embryo fibroblast cells. The mouse, human, and Drosophila homologs were independently identified as a signaling molecule involved in a mitogenic response, as a product of a putative oncogene EMS1, and as a molecule interacting with a scaffolding protein ZO-1, respectively. In this report, we describe the cloning of the Drosophila cortactin gene, which consists of four exons and three introns, covering 3 kilobases in length.

Male reproductive defects caused by puromycin-sensitive aminopeptidase deficiency in mice.

Male reproductive performance is composed of two principal elements, copulation and spermatogenesis. A wealth of literature has described the intricate web of endocrine events underlying these biological processes. In the present study we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice are infertile, lack copulatory behavior, and have impaired spermatogenesis.

Puromycin-sensitive aminopeptidase is essential for the maternal recognition of pregnancy in mice.

Maternal recognition of pregnancy in rodents requires semicircadian surges of hypophyseal PRL secretion during early gestation, which are required for the formation of the corpus luteum of pregnancy (CLP). Here we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice display female infertility that results from impaired formation of CLP. Transplantation of mutant ovaries into normal females restored fertility but not vice versa.

Gene organization of bovine BCNT that contains a portion corresponding to an endonuclease domain derived from an RTE-1 (Bov-B LINE), non-LTR retrotransposable element: duplication of an intramolecular repeat unit downstream of the truncated RTE-1.

BCNT (a protein named after Bucentaur or craniofacial development protein 1) has a unique structure in Ruminantia. Bovine BCNT contains a region of the endonuclease domain derived from a truncated RTE-1 (previously called Bov-B LINE), a non-LTR retrotransposable repetitive element, and two repeat units (intramolecular repeat, IR) each with 40 amino acids in the C-terminal region. In contrast the human and mouse BCNT proteins contain one repeat unit and lack the RTE-1-derived portion.

Yak1p, a DYRK family kinase, translocates to the nucleus and phosphorylates yeast Pop2p in response to a glucose signal.

POP2 protein of Saccharomyces cerevisiae is a component of a protein complex that regulates the transcription of many genes. We found that the 97th threonine residue (Thr 97) of Pop2p was phosphorylated upon glucose limitation. The Thr 97 phosphorylation occurred within 2 min after removing glucose and was reversed within 1 min after the readdition of glucose.

Mutations in the novel membrane protein spinster interfere with programmed cell death and cause neural degeneration in Drosophila melanogaster.

Mutations in the spin gene are characterized by an extraordinarily strong rejection behavior of female flies in response to male courtship. They are also accompanied by decreases in the viability, adult life span, and oviposition rate of the flies. In spin mutants, some oocytes and adult neural cells undergo degeneration, which is preceded by reductions in programmed cell death of nurse cells in ovaries and of neurons in the pupal nervous system, respectively.

Green marker for colonies of Bacillus subtilis.

A Bacillus subtilis plasmid encoding a green fluorescence protein gene (gfp) was constructed. The fluorescence of B. subtilis colonies having this plasmid on agar plates was so high that they could be readily discerned visually under UV light.

Cloning and characterization of a novel serine/threonine protein kinase gene expressed predominantly in developing brain.

We have isolated a rat gene, sbk, that encodes a novel serine/threonine protein kinase possessing a consensus sequence for an SH3-binding domain from developing rat brain. Rat SBK comprises 417 amino-acid residues consisting of a serine/threonine protein kinase consensus sequence followed by a C-terminal proline-rich region. Sequence comparison with other known kinases revealed that sbk belongs to a novel family of serine/threonine protein kinases structurally related to a Xenopus gastrula-specific protein kinase, Pk9.

A broad replication origin of Drosophila melanogaster, oriDalpha, consists of AT-rich multiple discrete initiation sites.

This work shows that the replication origin of Drosophila melanogaster, oriDalpha, consists of multiple discrete initiation sites. We attempted to map at high resolution the initiation sites in oriDalpha with a quantitative nascent DNA abundance assay using a competitive polymerase chain reaction (PCR) method. Nascent DNA was prepared from either cells blocked in very early S-phase and then labeled with 5-bromo-2'-deoxyuridine (BrdU), or asynchronously growing cells labeled briefly with BrdU.

A role of jumonji gene in proliferation but not differentiation of megakaryocyte lineage cells.

In this study, megakaryocytopoiesis was investigated in the recessive mutant mouse, jumonji, obtained by a gene-trap strategy. We investigated the number of megakaryocyte progenitors in the fetal liver, yolk sac, and peripheral blood of jumonji homozygous embryos by in vitro colony forming assay and monitored colony formation from single megakaryocyte progenitors. We also investigated the differentiation of jumonji-deficient megakaryocytes in terms of the expression of megakaryocyte differentiation markers PF4, CD62P, and GATA-1, proplatelet formation, cytoplasmic maturation, and endomitosis.

Tau-tubulin kinase phosphorylates tau at Ser-208 and Ser-210, sites found in paired helical filament-tau.

Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimer's patients. The kinase that phosphorylated Ser-208 and Ser-210 in PHF-tau had remained unknown. We used anti-pS208 and anti-pS210 antibodies and Western blots to confirm that the tau-tubulin kinase (TTK) phosphorylates tau at Ser-208 and at Ser-210.

Role of Thr(11) in the binding of omega-conotoxin MVIIC to N-type Ca2+ channels.

As replacement of Thr(11) of omega-conotoxin MVIIC with Ala significantly reduced the affinity for both N- and P/Q-type calcium channels, we examined the effect of substitution at this position with other residues. Binding assays using rat cerebellar P2 membranes showed that the affinity is in the order of Leu>Val, aminobutyric acid, Thr>Asn&z.Gt;Ser, Ala, Asp, Phe, Tyr for N-type channels and Thr>Leu, Val, aminobutyric acid, Asn, Ser>Ala&z.