Periplasmic secretion of native ovalbumin without signal cleavage in Escherichia coli

In Escherichia coli cells carrying wild-type ovalbumin cDNA, some of the recombinant protein was secreted into the periplasmic space. In contrast, a signal-region mutant form of ovalbumin (deletion, Gly1 to Ala39) was not detected in the periplasm despite being synthesized at the same level as the wild-type protein. Chemical and spectroscopic analyses showed that periplasmic ovalbumin assumes a conformation indistinguishable from that of native egg white ovalbumin. We concluded that a process resembling the secretion of ovalbumin process in the oviduct occurs also in bacteria.     

Requirement of negative residues,Asp 95 and Asp 105, in S2 on membrane integration of a voltage-dependent K+ channel,KAT1

Voltage-dependent K+ channels consist of a voltage-sensing region and a pore-forming region. Here we have identified the negative residues of the second transmembrane segment in the plant voltage-dependent K+ channel, KAT1, which involves the function of voltage sensing. Point mutations at D95 and D105 but not D89 and D116 failed to complement the K+ uptake deficient properties of the mutant yeast. In vitro translation and translocation experiments showed that the membrane integration of the third and fourth segments involving voltage sensor were impaired by the replacement of D95 or D105 by serine. These data show that both the residues play a crucial role in the membrane topogenesis of the voltage sensor in KAT1.     

Two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis sp. strain PCC6803

The ndhD gene encodes a membrane protein component of NAD(P)H dehydrogenase. The genome of Synechocystis sp. PCC6803 contains 6 ndhD genes. Three mutants were constructed by disrupting highly homologous ndhD genes in pairs. Only the DeltandhD1/DeltandhD2 (DeltandhD1/D2) mutant was unable to grow under photoheterotrophic conditions and exhibited low respiration rate, although the mutant grew normally under photoautotrophic conditions in air. The DeltandhD3/DeltandhD4 (DeltandhD3/D4) mutant grew very slowly in air and did not take up CO(2). The results demonstrated the presence of two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis PCC6803 cells. TheDeltandhD5/DeltandhD6 (DeltandhD5/D6) mutant grew like the wild-type strain. Under far-red light (>710 nm), the level of P700(+) was high in DeltandhD1/D2 and M55 (ndhB-less mutant) at low intensities. The capacity of Q(A) (tightly bound plastoquinone) reduction by plastoquinone pool, as measured by the fluorescence increase in darkness upon addition of KCN, was much less in DeltandhD1/D2 and M55 than in DeltandhD3/D4 and DeltandhD5/D6. We conclude that electrons from NADPH are transferred to the plastoquinone pool mainly by the NdhD1.NdhD2 type of NAD(P)H dehydrogenases.     

Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN-gamma production

Although CD8+ IL-2Rbeta (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rbeta-negative or -low (CD122-) T cells conversely decreased. The IFN-gamma production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-gamma than did CD8+ CD122- T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-gamma production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-gamma production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-gamma. The CD3-stimulated IFN-gamma production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-gamma than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-gamma production and are suggested to be involved in the immunological changes with aging.     

Inhibition of hyaluronan synthesis by vesnarinone in cultured human myofibroblasts

Hyaluronan (HA), which is a major component of the extracellular matrix (ECM), is regulated during myofibroproliferative responses to numerous forms of inflammatory stimuli. It is a key factor involved in cellular migration and adherence. The development of a potent and non-toxic inhibitor of HA synthesis would open up a new avenue for the treatment of fibrocontractive diseases such as pulmonary fibrosis and liver cirrhosis. In this study, the effects of vesnarinone (OPC-8212: 3,4-dihydro-6-[4-(3, 4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone) on the secretion of HA in human myofibroblast cell lines (MRC-5 and LI90 cells, referred to as pulmonary and hepatic myofibroblasts, respectively) were examined. Vesnarinone specifically and dose-dependently inhibited HA secretion by myofibroblasts up-regulated by fetal calf serum (FCS). The treatment of vesnarinone did not modify the phenotype of myofibroblast cells in culture. Vesnarinone also potently inhibited the HA secretion by the two myofibroblast cell lines up-regulated by transforming growth factor-beta1 (TGF-beta1) or tumor necrosis factor-alpha (TNF-alpha). The addition of vesnarinone to myofibroblasts resulted in a significant decrease of HA synthase (HAS) activity, with or without the addition of FCS or either cytokine. These findings suggest that vesnarinone inhibits the secretion of HA in myofibroblasts by specifically suppressing HAS activity, and may therefore prove useful for the treatment of chronic inflammation and tissue fibrosis.     

Direct gene delivery into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants

A procedure for direct gene transfer into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants is presented. By modifying the microspore culture method and adopting the firefly luciferase (Luc) gene as a non-destructive marker, we could obtain stably transformed androgenetic embryos from bombarded microspores. Luc-positive embryos were easily isolated from the large non-transformed population using a high-sensitivity bioluminescent image analyzer. PCR and Southern blot analyses confirmed that the introduced transgene was integrated stably into the genome of the selected embryos. Diploidized plants obtained from the haploid embryos were self-pollinated, and all of the offspring tested were Luc-positive, indicating rapid fixation of the transgene which is characteristic of doubled haploids. Received: 14 May 1997 / Revision received: 15 July 1997 / Accepted: 28 July 1997     

MACROSCOPIC OBSERVATIONS OF THE FACIAL MUSCLES IN THE BAIKAL SEAL (PHOCA SIBIRICA)

This study was performed to determine if, as expected, the enlarged eye of the Baikal seal ( Phoca sibirica ) has an influence on the form and function of the skull and facial muscles. Macroscopic observation of these muscles demonstrated that the M. orbicularis oculi expands around the palpebral fissure and that some facial muscles attach and insert in the M. orbicularis oculi , possibly supporting M. orbicularis oculi function. We suggest that these muscles move the eye and palpebral area and constitute a morphological and synergistic facial muscle complex system. Further, the development of the M. rectus lateralis around the sclera of the eye indicates that this muscle is also involved in eye movement.     

Induction of Conjugation by Methyl Cellulose in Paramecium

ABSTRACT An improved method has been developed for the induction of selfing conjugation in Paramecium. Methyl cellulose induces selfing conjugation simply and efficiently in all species examined. Induction of conjugation by methyl cellulose was characterized in P. caudatum , where it occurred only in sexually mature, mating reactive cells. Conjugation produced sexual offspring and the time course of nuclear processes was substantially the same as that in natural conjugation between cells of complementary mating types. The method is useful for genetic studies in a wide variety of Paramecium species including P. caudatum, P. tetraurelia, P. multimicronucleatum and P. bursaria.     

Structural Analysis ofArabidopsis thaliana Chromosome 5. VI. Sequence Features of the Regions of 1,367,185 bp Covered by 19 Physically Assigned P1 and TAC Clones

Nineteen Pl and TAC clones, which have been mapped on the finephysical map of the Arabidopsis thaliana chromosome 5, weresequenced according to the shotgun-based strategy, and theirstructural features were analysed. The total length of the regionssequenced in this study was 1,367,185 bp. Combining this withthe regions covered by 90 P1 and TAC clones proviously reported,the total length of chromosome 5 sequenced to date becomes 8,058,855bp. On the basis of similarity search against protein and ESTdatabases and gene modeling with computer programs, a totalof 330 potential protein-coding regions were identified, bringingan average density of the genes to approximately one gene per4.1 kb. Introns were identified in 81.0% of the potential proteingenes for which the entire gene structure was predicted, withan average number per gene of 4.2 and an average length of theintrons of 180 bp. The RNA-coding genes identified were 9 tRNAgenes corresponding to 8 amino acid species and 2 genes forU2 nuclear RNA. These sequence features are essentially identicalto those in the previously reported sequences. The sequencedata and gene information are available on the World Wide Webdatabase KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.