Down-regulation of trypsinogen expression is associated with growth retardation in alpha1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity

Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8^–/–)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8^–/– embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8^–/– embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8^–/– mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8^–/– mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH₂). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8^–/–mice.     

Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue

A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.     

Saccharomyces cerevisiae Bor1p is a boron exporter and a key determinant of boron tolerance

Boron is toxic to living organisms when present in excess. Saccharomyces cerevisiae Bor1p is a plasma membrane protein that decreases the intracellular concentration of boron and confers boron tolerance in yeasts. We investigated the detailed characteristics of boron transport by Bor1p and its roles in boron tolerance. Boron transport assays showed that the bor1 deletion mutant (bor1Delta) accumulates higher intracellular concentrations of boron and has a lower rate of boron export. The bor1Delta showed greater susceptibility to high concentrations of boron than the wild-type strain, and the growth rates of both strains were negatively correlated with the intracellular concentrations of boron. With normal to toxic levels of external boron, green fluorescent protein (GFP)-tagged Bor1p localized to the plasma membrane irrespective of the concentration of boron in the medium. Taken together, these results establish Bor1p as a plasma membrane boron exporter and a key determinant of boron tolerance.     

Theste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeastSchizosaccharomyces pombe

When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13 ⁺ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13 ⁺ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13 ⁺ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13 ⁺ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.     

Isolation and characterization of psychrophiles producing cold-active beta-galactosidase

AIMS: The present study was conducted to screen for psychrophilic micro-organisms that are able to hydrolyse lactose at low temperature, and to examine the cold-active beta-galactosidase produced by the isolated psychrophilic micro-organisms. METHODS AND RESULTS: Psychrophilic bacteria, which grow on lactose as a sole carbon source, were isolated from soil from Hokkaido, Japan. The phenotype and sequence of 16S rDNA of the isolated strains indicated a taxonomic affiliation to Arthrobacter psychrolactophilus. The isolated A. psychrolactophilus strains were able to grow on lactose at below 5 degrees C, and showed cold-active beta-galactosidase activity, which was highly specific at even 0 degrees C. CONCLUSIONS: Facts in this study may indicate the possibility that the isolated strains produce novel beta-galactosidases that are able to hydrolyse lactose at low temperature, although some strains have isozymes. SIGNIFICANCE AND IMPACT OF THE STUDY: It may be possible that the cold active beta-galactosidases from the isolated strains can be applied to the food industry, e.g. processing of milk and whey below 5 degrees C.     

Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion

Bacteriophage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 Å resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.     

Molecular structure of the Japanese hepatitis C viral genome

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et. al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524–9528)indicated that this virus is a member of a new class of positive-stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin-like serine proteinase. NTPase and RNA-dependent RNA polymerase     

An Analytic Solution of the Cable Equation Predicts Frequency Preference of a Passive Shunt-End Cylindrical Cable in Response to Extracellular Oscillating Electric Fields

Under physiological and artificial conditions, the dendrites of neurons can be exposed to electric fields. Recent experimental studies suggested that the membrane resistivity of the distal apical dendrites of cortical and hippocampal pyramidal neurons may be significantly lower than that of the proximal dendrites and the soma. To understand the behavior of dendrites in time-varying extracellular electric fields, we analytically solved cable equations for finite cylindrical cables with and without a leak conductance attached to one end by employing the Green's function method. The solution for a cable with a leak at one end for direct-current step electric fields shows a reversal in polarization at the leaky end, as has been previously shown by employing the separation of variables method and Fourier series expansion. The solution for a cable with a leak at one end for alternating-current electric fields reveals that the leaky end shows frequency preference in the response amplitude. Our results predict that a passive dendrite with low resistivity at the distal end would show frequency preference in response to sinusoidal extracellular local field potentials. The Green's function obtained in our study can be used to calculate response for any extracellular electric field.     

Efficient expression of recombinant soluble human FcγRI in mammalian cells and its characterization

The extracellular domain of human FcγRI which interacts with a human IgG was expressed as recombinant soluble human FcγRI (rshFcγRI) by Chinese hamster ovary (CHO) cell. Stable CHO cell clones with efficient expression of rshFcγRI were established based on a dihydrofolate reductase (DHFR)/methotrexate (MTX) gene-amplification system. The CHO clones efficiently produced rshFcγRI under high-density continuous culture in a bioreactor. After 53 days of culture, the number of cells had reached approximately 4 × 10? cells/mL in the bioreactor and the average production of rshFcγRI had reached 7.4 mg L-medium?1 day?1. Secreted rshFcγRI was purified to a homogeneous state using cation exchange and affinity chromatographies. The binding affinities of rshFcγRI to human IgG subclasses were determined using surface plasmon resonance analysis. The binding affinities of rshFcγRI to human IgG1/κ and IgG3/κ were high (1.59 × 10?1? and 2.81 × 10?1? M, respectively), whereas that of rshFcγRI to human IgG4/κ was lower binding affinity (1.41 × 10?? M). Binding to IgG2/κ was not detectable. Examination of circular dichroism spectra indicated that rshFcγRI was rich in β-structures and loop or turn structures, but there were few α-helices. These results may be valuable for further studies of the structure and function of human FcγRI.     

Determining the role of specific signaling molecules during lymphocyte development in vivo: instant transgenesis

A common method of determining the role of specific signaling molecules during lymphocyte development is to generate a transgenic mouse. This procedure, while informative, is time consuming, expensive and ultimately does not guarantee a defined answer. Here we present a protocol in which the in vivo effects of a gene of interest on both B and T lymphocyte development may be determined simultaneously in a relatively short time period. This is achieved by introducing a defined gene, such as a wild-type or mutated signaling molecule, into a lymphoid progenitor population by retroviral infection. The retrovirus generates a bicistronic message encoding the gene of interest and GFP, thus enabling identification of retrovirally transduced cells in subsequent lymphocyte lineages. The cells are then introduced into mice deficient for recombinase activating gene 1 (Rag-/- mice), thus allowing the development of donor-derived B and T lymphocytes in vivo. Using this technique, results can be obtained within 3-8 weeks.