Unresponsiveness of peripheral T cells induced by apoptotic bodies derived from autologous T cells

Several reports described the dose-dependent effect of Staphylococcus aureus enterotoxin B (SEB) regarding both levels of apoptosis and anergy of T cells. We investigated here whether T-cell apoptosis induced with SEB causes unresponsiveness of naive T cells. Apoptotic bodies were isolated from human T cells stimulated with antigen-presenting cells (APCs) and SEB by the continuous density gradient centrifugation method. When naive T cells were stimulated with APCs and SEB in the presence of apoptotic bodies, their proliferation was dose dependently suppressed and their TCRs were less downregulated than those of T cells stimulated without apoptotic bodies. Furthermore, those T cells were predisposed not to respond to restimulation with fresh APCs and SEB in the absence of apoptotic bodies. These results, taken together with the observation of tight binding of apoptotic bodies to APCs, imply that T cells stimulated in the presence of apoptotic bodies may undergo unresponsiveness due to interruption of contact with APCs.     

Isolation and characterization of novel peroxisome biogenesis-defective Chinese hamster ovary cell mutants using green fluorescent protein

We developed an improved method for isolation of peroxisome biogenesis-defective somatic animal cell mutants, using a combination of green fluorescent protein (GFP) expression and the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) selection method. We used TKaG1 and TKaG2 cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNAs each encoding rat Pex2p as well as GFP tagged at the C-terminus with peroxisome targeting signal type 1 (PTS1) or N-terminally PTS2-tagged GFP. P9OH/UV-resistant cell colonies were examined for intracellular location of GFP on unfixed cells, by fluorescence microscopy. Seven each of the mutant cell clones isolated from TKaG1 and TKaG2 showed cytosolic GFP-PTS1 and PTS2-GFP, respectively, indicating the defect in peroxisome assembly. By transfection of PEX2, PEX5, PEX6, and PEX12 cDNAs and cell fusion analysis between the CHO cell mutants, five different complementation groups (CGs) were identified. Two mutant clones, ZPG207 and ZPG208, belonged to novel CGs. Further CG analysis using fibroblasts from patients with peroxisome biogenesis disorders, including rhizomelic chondrodysplasia punctata (RCDP), revealed that ZPG208 belonged to none of human CGs. ZPG207 was classified into the same CG as RCDP. Taken together, ZPG208 is in a newly identified, the 12th, CG in peroxisome-deficient CHO mutants reported to date and represents a novel mammalian CG.     

Halobacterial rhodopsins

Following the discovery of the bacteriorhodopsin proton pump in Halobacterium halobium (salinarum), not only the halorhodopsin halide pump and two photosensor rhodopsins (sensory rhodopsin and phoborhodopsin) in the same species, but also homologs of these four rhodopsins in strains of other genera of Halobacteriaceae have been reported. Twenty-eight full (and partial) sequences of the genomic DNA of these rhodopsins have been analyzed. The deduced amino acid sequences have led to new strategies and tactics for understanding bacterial rhodopsins on a comparative basis, as summarized briefly in this article. The data discussed include (i) alignment of the sequences to qualify/characterize the conserved residues; (ii) assignment of residues that cause differences in function(s)/properties; and (iii) phylogeny of the halobacterial rhodopsins to suggest their evolutionary paths. The four kinds of rhodopsin in each strain are assumed, on the basis of their genera-specific distributions, to have arisen by at least two gene-duplication processes during evolution prior to generic speciation. The first duplication of the rhodopsin ancestor gene yielded two genes, each of which was duplicated again to give four genes in the ancestor halobacterium. The bacterium carrying four rhodopsin genes, after accumulating mutations, became ready for generic speciation and the delivery of four rhodopsins to each species. The original rhodopsin ancestor is speculated to be closest to the proton pump (bacteriorhodopsin).     

Genetic relatedness among species in Aspergillus section Clavati as measured by electrophoretic comparison of enzymes,DNA base composition,and DNA-DNA hybridization

Aspergillus subgenus Clavati has four recognized species: A. clavatus (the type species), A. clavatonanicus, A. giganteus, and A. longivesica. These species are strictly anamorphic (mitotic) and defined by the morphological species concept. However, their genealogical relationships remain uncertain. In this study, we examined the genetic relatedness among the four species in this section, using electrophoretic comparison of enzymes, DNA base composition, and DNA-DNA hybridization. In a dendrogram based on the calculated similarity values of four enzymes, 10 strains in section Clavati, 3 strains in the xerophilic species, a strain in section Ornati, and a strain in section Cremei were separated into nine major clusters at a 60% similarity level. A. longivesica JCM 10186(T) had Q-10 in our analysis, but Kuraishi et al. (1990) reported A. longivesica JCM 1720(T) had Q-9 (49%) and Q-10 (46%). The G+C contents of the four species of section Clavati ranged from 48 to 50 mol%. The degree of the intraspecific reassociation among the DNAs from the strains of these species ranged from 77 to 99%, whereas the degrees of interspecific relative binding among strains of the four species ranged from 30 to 59%. Our data from enzyme patterns and DNA relatedness support the validity of the three species in section Clavati, except for A. longivesica.     

Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai)

The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0∼300 μl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (≧10 μ1/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 μl/kg f.w./h–10 μl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear. Received: 1 July 1998 / Accepted: 6 October 1998     

Bioactivity of orally administered unnatural isomers, [6R]-5-formyltetrahydrofolate and [6S]-5,10-methenyltetrahydrofolate, in humans

It has been assumed that humans cannot utilize 5,6,7,8-tetrahydrofolates with the unnatural configuration at carbon 6, since these folates are enzymatically and microbiologically inactive. We hypothesized that orally administered unnatural [6R]-5-formyltetrahydrofolate or [6S]-5,10-methenyltetrahydrofolate is bioactive in humans. Subjects were given independent oral doses of these unnatural folates and of a natural [6S]-5-formyltetrahydrofolate. Plasma, before and after the dose for 4 h, and 2 h urine were collected. Areas under the curve for the change in plasma folate concentrations were measured microbiologically and urinary folates were measured using HPLC. Based on findings of plasma and urinary folates, the unnatural folates were estimated to be 14-50% active as compared to [6S]-5-formyltetrahydrofolate. The major plasma and urinary folate was [6S]-5-methyltetrahydrofolate in all experiments. In urine, a [6S]-5-formyltetrahydrofolate peak was observed only after a [6S]-5-HCO-H4folate dose and peaks of unnatural [6S]-10-formyltetrahydrofolate and 5-formyltetrahydrofolate were identified after a [6R]-5-formyltetrahydrofolate dose. A possible pathway that explains our findings is discussed. This pathway includes the oxidation of the unnatural [6S]-10-formyltetrahydrofolate to 10-formyl-7,8-dihydrofolate which can be further metabolized by 5-amino-4-imidazolecarboxamide-ribotide transformylase producing dihydrofolate. Dihydrofolate can then be metabolized to [6S]-5-methyltetrahydrofolate by well-established metabolism.     

Expression of inducible nitric oxide (NO) synthase but not prevention by its gene ablation of hepatocarcinogenesis with fibrosis caused by a choline-deficient, L-amino acid-defined diet in rats and mice.

Expression of inducible nitric oxide synthase (iNOS) and effects of iNOS gene ablation on the hepatocarcinogenesis associated with fibrosis caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male F344 rats and C57BL/6J wild-type and iNOS-/- mice. Western blot, RT-PCR and immunohistochemical analyses revealed increased expression of iNOS protein and mRNA in the livers of rats and wild-type mice fed a CDAA diet for 12-80 weeks, associated with elevated serum NO(x) and liver nitrotyrosine levels. iNOS-/- mice demonstrated greater liver injury and fibrosis in the early stage than their wild-type counterparts, but this did not significantly affect the incidence and multiplicity of altered foci, adenomas and hepatocellular carcinomas in spite of immunohistochemical iNOS expression in these lesions. Results suggested no major determinant roles of the expressed iNOS in the development of liver tumors caused by the CDAA diet.