Tetsuo Kojima

Plat-E: an efficient and stable system for transient packaging of retroviruses

A potent retrovirus packaging cell line named Platinum-E (Plat-E) was generated based on the 293T cell line. Plat-E is superior to existing packaging cell lines regarding efficiency, stability and safety. The novel packaging constructs utilized in establishment of Plat-E ensure high and stable expression of viral structural proteins. Conventional packaging constructs made use of the promoter of MuLV-LTR for expression of viral structural genes gag-pol and env, while our packaging constructs utilized the EF1α promoter, which is 100-fold more potent than the MuLV-LTR in 293T cells in combination with the Kozaks consensus sequence upstream of the initiation codon resulting in high expression of virus structural proteins in Plat-E cells. To maintain the high titers of retroviruses under drug selection pressure, we inserted the IRES (internal ribosome entry site) sequence between the gene encoding gag-pol or env, and the gene encoding a selectable marker in the packaging constructs. Plat-E cells can stably produce retroviruses with an average titer of 1 × 107/ml for at least 4 months. In addition, as we used only the coding sequences of viral structural genes to avoid inclusion of unnecessary retrovirus sequences in the packaging constructs, the probability of generating the replication competent retroviruses (RCR) by recombination can virtually be ruled out.

TROY, a newly identified member of the tumor necrosis factor receptor superfamily, exhibits a homology with Edar and is expressed in embryonic skin and hair follicles.

In a signal sequence trap screening of the murine brain, we identified a new member of the tumor necrosis factor receptor superfamily designated TROY. TROY is a type I membrane protein of 416 amino acids with characteristic cysteine-rich motifs in the extracellular domain and a tumor necrosis factor receptor-associated factor (TRAF) 2 binding sequence in the cytoplasmic domain of 223 amino acids. In fact, activation of nuclear factor κB was induced by the overexpression of TROY and inhibited by dominant negative forms of TRAF2, TRAF5, and TRAF6, indicating that TRAFs and nuclear factor κB are involved in the signal transduction of TROY. We also cloned a cDNA for a human counterpart, which showed a 75% homology with mouse TROY at the amino acid level. The extracellular domain of TROY exhibits an extensive homology with that of Edar, a receptor that specifies hair follicle fate. TROY mRNA is strongly expressed in brain and embryo and moderately expressed in the heart, lung, and liver but not the spleen. In the embryo, the expression level is particularly strong in the skin. Interestingly, in situ hybridization analysis of the embryo showed that TROY mRNA was exclusively expressed in the epithelium of many tissues. On the other hand, in neonatal mice, TROY is expressed in hair follicles like Edar as well as in the cerebrum, suggesting pleiotropic functions of TROY in development as well as in the adult mice. The Troy gene is located near the waved coat (Wc) locus, a mutant related to abnormalities in skin and hair.

A signal sequence trap based on a constitutively active cytokine receptor

Targeting of secreted and cell-surface proteins to the cell membrane is mediated by a short hydrophobic stretch of amino acids, termed the signal sequence. We have developed a method that detects signal sequences in cDNA fragments based on their ability to redirect a constitutively active mutant of a cytokine receptor to the cell surface, thereby permitting interleukin-3 (IL-3)-independent growth of Ba/F3 cells. Retrovirus-mediated expression of the fusions in IL-3–dependent cells was followed by selection of clones for growth in the absence of IL-3. Infection of cells with 5 × 106 viral particles in a pilot experiment led to the isolation of 150 known and 48 novel cDNA clones, and all the known cDNA clones were found to encode secreted and cell-surface proteins. In addition, we isolated type II membrane proteins, which have not been detected by existing signal sequence trap strategies.

High-power red beam generation by frequency-doubling of a Nd:YAG laser

This letter reports the generation of 6.1 W of red laser radiation by frequency-doubling a Nd:YAG laser operating at a wavelength of 1.319 /spl mu/m with a KTP crystal. The conversion efficiency and the stability of the red output power were both improved through the suppression by means of a thin etalon of two-wavelength operation in the 1.3-/spl mu/m region.

Molecular cloning and characterization of a mouse homolog of human TNFSF14, a member of the TNF superfamily

A member of the tumor necrosis factor (TNF) superfamily, human TNFSF14 (hTNFSF14)/HVEM-L (herpes virus entry mediator ligand) was isolated as a cellular ligand for HVEM/TR2 and human lymphotoxin β receptor (LTβR). TNFSF14 induces apoptosis and suppresses tumor formation. We have isolated a cDNA clone for a mouse homologue of hTNFSF14 by signal sequence trap (SST) screening which we recently developed. The deduced amino acid sequence of the mouse TNFSF14 (mTNFSF14) cDNA comprised 239 amino acid residues and was 77% identical to the hTNFSF14 protein. In Northern blot analysis, 2.1 kb and 4.2kb mTNFSF14 transcripts were detected in spleen and lung, and in heart, respectively. Fluorescence in situ hybridization analysis localized the mTNFSF14 gene Tnfsf14 to chromosome 17 which is tightly linked with Tnf, Lta, and Ltb.

All-solid-state 5-kHz 0.2-TW Ti:sapphire laser system.

We have developed an all-solid-state 5-kHz Ti:sapphire Laser System, which produces 22-fs, 0.2-TW pulses. An average power of 22.2 W is the highest ever obtained in ultrashort laser sources. The serious thermal lensing due to high power pumping in a small area of the Ti:sapphire crystal is controlled successfully by a stable quasi-cavity with two concave mirrors.

20-W, 10-kHz UV beam generation by an all-solid-state laser

Highest 266-nm UV power, to our knowledge, of 20-W was generated by an all-solid-state laser with a high-quality CLBO crystal.

High-power high-efficient diode-side-pumped Nd: YAG laser

We have demonstrated high-power and high-efficient performance of a cw Nd: YAG laser with simple and scalable side-pumping coxQuration. The “um output power of 147 W was obtained with the electric efficiency of 14.8 %. Ths is to our knowledge, the highest efficiency reported for diode-side-pumped Nd: YAG lasers. High-brightness operation was also camed out by applying bifocusing compensation of the Nd: YAG md. The best beam quality of M2 = 5.9 was obtained with the output power of 107 W. The brightness is calculated to be 272 MW/cm2sr.

1-kW high-quality beam generation from a diode-side-pumped Nd:YAG rod laser

We demonstrated a power scaling of Nd: YAG rod laser by cascaded-coupling of two identical bifocusing compensation resonators. The maximum output power of 1030 W was achieved with the beam quality of M2 = 9. The corresponding electrical-to-optical efficiency was 23 %.

High brightness 127 W green beam generation by intracavity-frequency-doubling of diode-pumped Nd:YAG laser

Summary form only given. High average power green lasers based on intracavity-doubling of diode pumped solid-state-lasers are of considerable current interest because of their compact design and efficient performance for numerous applications, such as Ti sapphire laser pumping, 4th harmonic generation. Although, several papers reported over >100 W green power generation, their beam quality factors are M/sup 2/>19, thus the green beams are not adequate in particular for 4th harmonic generation. In this work, we present a high-brightness green laser system that generates up to 127 W green power, with the beam quality of M/sup 2/<9.