New clade of silicified bolidophytes that belong to Triparma (Bolidophyceae,Stramenopiles)

The small phytoplankton genus Triparma belongs to the class Bolidophyceae and contains two distinct forms: silicified species and naked flagellated species (formerly Bolidomonas). Recent studies showed that four silicified species/strains (Triparma laevis f. inornata, T. laevis f. longispina, T. strigata, and T. aff. verrucosa) belong to a single clade that is paraphyletic, because it also contains an unclassified flagellated strain, and is sister to a flagellated species, T. eleuthera. In this study, we isolated and characterized two new strains of silicified species to test the phylogenetic unity of silicified bolidophytes. The isolates were identified as T. retinervis strains because they possessed fine areolation on the cell wall. 18S rDNA and rbcL phylogenetic analyses demonstrated that T. retinervis formed a new silicified clade that is sister to the flagellated species T. pacifica. This reveals that there are at least two distinct clades including both silicified and flagellated Triparma species.     

Down-regulation of prostaglandin E2 receptors in regenerating rat liver and its physiological significance.

The properties of prostaglandin (PG) E2 receptors in regenerating liver were studied using rat hepatocytes in primary culture. The control cells possessed stereo-specific PGE2 receptors with Bmax and Kd values, at 4 degrees C, of 526 fmol/mg protein and 6.5 nM respectively. In cells from regenerating liver after 70% hepatectomy, Bmax was reduced to 42-43% that of the controls; Kd did not change. Administration of indomethacin before surgery prevented Bmax reduction. These results indicate that PGE2, produced during the regeneration process, evoked cellular events and regulated the density of its receptors.     

Characterization of the cupin-type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralis

The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The purified enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe(2+), but inhibited by Zn(2+) and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k(cat). The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight differences.     

Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast

An intrinsic ATPase inhibitor and 9-kDa protein are regulatory factors of mitochondrial ATP synthase in Saccharomyces cerevisiae. A gene encoding the ATPase inhibitor was isolated from a yeast genomic library with synthetic oligonucleotides as hybridization probes and was sequenced. The deduced amino acid sequence showed that the precursor protein contains an amino-terminal presequence of 22 amino acid residues. Mutant strains that did not contain the inhibitor and/or the 9-kDa protein were constructed by transformation of cells with their in vitro disrupted genes. The disruption of the chromosomal copy in recombinant cells was verified by Southern blot analysis, and the absence of the proteins in the mutant cells was confirmed by Western blot analysis. All the mutants could grow on a nonfermentable carbon source and the oxidative phosphorylation activities of their isolated mitochondria were the same as that of normal mitochondria. However, an uncoupler, carbonylcyanide-m-chlorophenylhydrazone, induced marked ATP hydrolysis in the inhibitor-deficient mitochondria, but not in normal mitochondria. These observations suggest that the ATPase inhibitor inhibits ATP hydrolysis by F1F0-ATPase only when the membrane potential is lost.     

Fluorometric determination of N-terminal prolyl dipeptides, proline and hydroxyproline in human serum by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive HPLC method for the determination of prolyl dipeptides, Pro and Hyp in serum was developed. After deproteinization of serum and pretreatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp, were separated on tandem reversed-phase columns by a gradient elution at 55°C and detected by fluorescence measured at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides were 2–5 fmol/injection (S/N=3). Pro–Hyp, Pro–Gly and Pro–Pro were identified as serum prolyl dipeptides. The within-day and between-day relative standard deviations were 1.5–7.9 and 2.4–10.8%, respectively. The recoveries were in the range of 90.8–97.3%. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human serum (n=10) were 0.64±0.35, 0.078±0.047, 0.022±0.016, 177.0±43.0 and 11.1±3.5 μM, respectively. The concentrations of Pro–Hyp and Pro–Pro in serum of a patient with bone metastases of prostatic cancer were about three times and 50 times, respectively, higher than those in normal human serum.     

Common Neutralization Epitope in Minor Capsid Protein L2 of Human Papillomavirus Types 16 and 6

Studies of virus neutralization by antibody are a prerequisite for development of a prophylactic vaccine strategy against human papillomaviruses (HPVs). Using HPV16 and -6 pseudovirions capable of inducing beta-galactosidase in infected monkey COS-1 cells, we examined the neutralizing activity of mouse monoclonal antibodies (MAbs) that recognize surface epitopes in HPV16 minor capsid protein L2. Two MAbs binding to a synthetic peptide with the HPV16 L2 sequence of amino acids (aa) 108 to 120 were found to inhibit pseudoinfections with HPV16 as well as HPV6. Antisera raised by immunizing BALB/c mice with the synthetic peptide had a cross-neutralizing activity similar to that of the MAb. The data indicate that HPV16 and -6 have a common cross-neutralization epitope (located within aa 108 to 120 of L2 in HPV16), suggesting that this epitope may be shared by other genital HPVs.     

Differential and collaborative actions of Rad51 paralog proteins in cellular response to DNA damage

Metazoan Rad51 plays a central role in homologous DNA recombination, and its activity is controlled by a number of Rad51 cofactors. These include five Rad51 paralogs, Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3. We previously hypothesized that all five paralogs participate collaboratively in repair. However, this idea was challenged by the biochemical identification of two independent complexes composed of either Rad51B/C/D/XRCC2 or Rad51C/XRCC3. To investigate if this biochemical finding is matched by genetic interactions, we made double mutants in either the same complex (rad51b/rad51d) or in both complexes (xrcc3/rad51d). In agreement with the biochemical findings the double deletion involving both complexes had an additive effect on the sensitivity to camptothecin and cisplatin. The double deletion of genes in the same complex, on the other hand, did not further increase the sensitivity to these agents. Conversely, all mutants tested displayed comparatively mild sensitivity to γ-irradiation and attenuated γ-irradiation-induced Rad51 foci formation. Thus, in accord with our previous conclusion, all paralogs appear to collaboratively facilitate Rad51 action. In conclusion, our detailed genetic study reveals a complex interplay between the five Rad51 paralogs and suggests that some of the Rad51 paralogs can separately operate in later step of homologous recombination.     

Induction of permanent mixed chimerism and skin allograft tolerance across fully MHC-mismatched barriers by the additional myelosuppressive treatments in mice primed with allogeneic spleen cells followed by cyclophosphamide

A pure method of drug (cyclophosphamide plus busulfan)-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers in mice has been established. The components of the method are i.v. administration of 1 x 108 allogeneic spleen cells on day 0, i.p. injection of 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell-depleted 1 x 107 bone marrow cells from the same donor on day 3. Recipient B10 (H-2b; IE-) mice prepared with this conditioning developed donor-specific tolerance, and long-lasting survival of skin allografts was shown in almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive Vbeta11+ T cells were reduced in periphery as seen in untreated B10.D2 (H-2d; IE+) mice. The specific tolerant state was confirmed by the specific abrogation against donor Ag in the assays of CTL activity and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophosphamide-induced tolerance, which have been reported by us since 1984, can be overcome by the additional treatments with the myelosuppressive drug busulfan, followed by 1 x 107 T cell-depleted bone marrow cells. To our knowledge, this is the first report to induce allograft tolerance with a short course of the Ag plus immunosuppressive drug treatment without any kind of mAbs (pure drug-induced tolerance).     

Dimeric sesquiterpene thioalkaloids with potent immunosuppressive activity from the rhizome of Nuphar pumilum: structural requirements of nuphar alkaloids for immunosuppressive activity

Potent immunosuppressive dimeric sesquiterpene thioalkaloids, 6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B and 6'-hydroxythionuphlutine B, were isolated from the rhizome of Nuphar pumilum together with five inactive quinolizidine alkaloids, neothiobinupharidine, nupharidine, deoxynupharidine, 7-epideoxynupharidine and nupharolutine. These dimeric sesquiterpene thioalkaloids were found to significantly inhibit anti-sheep erythrocyte plaque forming cell formation in mouse splenocytes at 1 microM. At this concentration. 6-hydroxythiobinupharidine, 6-hydroxythionuphlutine B and 6'-hydroxythionuphlutine B did not show cytotoxic effects to mouse splenocytes, and 6,6'-dihydroxythiobinupharidine also showed only minor or minimal cytotoxicity. By comparison of the inhibitory activity of several Nuphar alkaloids on anti-sheep erythrocyte plaque forming cell formation, some structural requirements of Nuphar alkaloids for immunosuppressive activity were obtained. Namely, the 6- or 6'-hydroxyl group at the quinolizidine ring of dimeric sesquiterpene thioalkaloids is essential for the immunosuppressive effect. The number of hydroxyl groups appears to be related to the cytotoxicity, and the influence on splenocytes is greater with increasing numbers of hydroxyl groups.