The mitochondrial plasmid of the true slime mold <Emphasis Type="Italic">Physarum polycephalum</Emphasis> bypasses uniparental inheritance by promoting mitochondrial fusion

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF⁺ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF^– strain KM88. Of the three mF^– × mF⁺ crosses, only KM88 × JE8 displayed complete uniparental inheritance. However, in KM88 × TU111 and KM88 × NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.The first two authors have equally contributed to this work     

Isolation,characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae

Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes. However, Southern hybridization of C. merolae cell-nuclear DNA with a yeast actin-gene probe has suggested the presence of an actin gene in the C. merolae genome. In the present study, an actin gene was isolated from a C. merolae genomic library using a yeast actin-gene probe. The C. merolae actin gene has no intron. The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42003 Da. Southern hybridization indicated that the C. merolae genome contains only one actin gene. This gene is transcribed at a size of 2.4 kb. When Southern hybridization was performed with C. merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII. A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C. merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.     

Intracellular localization and translocation of 1 alpha, 25-dihydroxyvitamin D3 receptor in osteoblasts.

The intracellular localization and translocation of the 1 alpha,25-dihydroxyvitamin D3 receptor (1 alpha,25(OH)2D3 receptor) in osteoblasts of mouse parietal bone and MC3T3-E1 cells were examined immunocytochemically using a rat monoclonal antibody to 1 alpha,25(OH)2D3 receptor. In osteoblasts of parietal bones in vivo, immunoreactivity for 1 alpha,25(OH)2D3 receptor was detected not only in the nuclei but also in lysosomal structures, and also sparsely in the cytoplasmic matrix. The transport of 1 alpha,25(OH)2D3 receptor was investigated immunocytochemically after incubation with 1 alpha,25(OH)2D3. In osteoblasts of parietal bones, after 1 min incubation with 10(-8) M 1 alpha,25(OH)2D3, the perinuclear cytoplasm showed intense immunoreactivity for 1 alpha,25(OH)2D3 receptor. After 10 min incubation, immunoreactivity was intense in the nuclei, especially in the heterochromatin. In MC3T3-E1 cells, after 1 min incubation with 1 alpha,25(OH)2D3, immunoreactivity for 1 alpha,25(OH)2D3 receptor was found in the form of a fibrillar structure extending radially to the periphery of the cells. The immunostaining pattern of anti-1 alpha,25(OH)2D3 receptor was similar to the distribution of microtubules stained with anti-beta-tubulin antibody. After 10 min incubation, the nuclei showed intense immunoreactivity for 1 alpha,25(OH)2D3 receptor. Incubation with colchicine dissociated the fibrillar structures and inhibited the intranuclear localization of the 1 alpha,25(OH)2D3 receptor. These findings suggest that the 1 alpha,25(OH)2D3 receptor is located in the nuclei, in lysosomal structures and also sparsely in the cytoplasmic matrix of osteoblasts in vivo, and that the receptor is transported to the perinuclear cytoplasm via microtubules to be then translocated into the nucleus, especially into the heterochromatin.     

Biological activities of glycoproteins of HVJ (Sendai virus) studied by reconstitution of hybrid envelope and by concanavalin A-mediated binding: a new function of HANA protein and structural requirement of F protein in hemolysis.

When HANA protein of HVJ (Sendai virus) was denatured by dithiothreitol treatment, concanavalin A added to the system or influenza virus hemagglutinin reconstituted with treated HVJ-envelope to form hybrid by SM-2 dialysis method could not replace a role of HANA for hemolysis. Binding of the treated samples to erythrocytes was restored by them, however. Thus, a hypothesis is proposed that intactness of not only F but also HANA protein of HVJ is required for envelope-cell fusion, in other words, HANA protein of HVJ may play some positive role other than binding in the process of the virus-induced hemolysis.     

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.     

Zonation of the adrenal cortex. III. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex.

A microsomal fraction was obtained from the zona glomerulosa of the bovine adrenal cortex. Glucose-6-phosphate activity of the fraction was found to be much lower than that of the liver. Contents of RNA and phospholipids, besides electron microscopic findings, of the fraction also indicate that it is rich in smooth-surfaced endoplasmic reticulum. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including the microsomal fraction described above. The amount of cytochrome P-450 in mitochondria and that in microsomes were determined to be 0.73 and 0.32 nmoles/mg protein, respectively. The CO-difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40-50% of cytochrome P-450 in the samples were converted to cytochrome P-420 within 20-30 seconds of incubation with deoxycholate.     

Phase I clinical and pharmacokinetic study ofN 4-behenoyl-1-β-d-arabinofuranosylcytosine

A phase I study ofN ⁴-behenoyl-1-β-d-arabinofuranosylcytosine (BHAC) was conducted in 66 patients, 41 with solid tumors and 25 with hematological malignancies. The patients received either a 2-h single intravenous (i.v.) drip infusion (Schedule 1) or consecutive daily 2-h i.v. infusions (Schedule 2). In Schedule 1 the daily dose was initiated with 1.5 mg kg^?1 which was escalated up to 7 mg kg^?1. Side-effects were mild, and included nausea, vomiting, epilation, and hot flushes. Because of the presence of the solvent vehicle, HCO-60 and in consideration of the mechanism of action of BHAC, the dose escalation was stopped at 7 mg kg^?1. In Schedule 2, the daily dose was started with 1.5 mg kg^?1 which was escalated up to 8 mg kg^?1 and given for 2–16 days. Myelosuppression was found to be dose-limiting toxicity. The maximum tolerated dose (MTD) in patients with non-hematological solid tumors was assumed to be 5 mg kg^?1 daily × 5 days. The plasma disappearance curve of BHAC looked biphasic, and when 4 mg kg^?1 of BHAC were administered the half-lives of the initial phase (t _1/2α) and the second phase (t _1/2β) were calculated as 0.798 and 5.76 h respectively. In Schedule 2 complete remission was observed in 5 out of 21 patients with acute leukemia, one partial remission in Hodgkin’s disease, and one 1-B response (Karnofsky) in thyroid papillary adenocarcinoma.     

Enhanced osteoinduction by intramuscular grafting of BMP-beta-TCP compound pellets into murine models.

The osteoinductive effects of bone morphogenetic protein (BMP, derived from murine osteosarcoma) were studied with regard to its use combined with beta-tricalcium phosphate (beta-TCP). BMP and beta-TCP were molded into pellets by the "pressure method", originated by us and transplanted to ddY mice. Control mice received interdorsal muscular implantations of either the BMP or beta-TCP pellets. The animals were sacrificed 1, 2 and 3 weeks after grafting, for radiological, histochemical, and ultrastructural observations. The BMP-beta-TCP compound pellets induced faster cartilage and bone formation, whereas these activities were slower when pellets made solely of BMP were used. The beta-TCP pellets demonstrated no osteoinductive properties. Observations revealed two types of beta-TCP resorbing multinuclear giant cells. One was osteoclastic, expressing calcitonin receptors, having numerous mitochondria and ruffled border-like structures; the other was not osteoclastic in nature. In animals grafted with the compound pellets, a great number of osteoclastic cells gathered on the pellets, much earlier than those grafted with the pellets made of BMP alone. Then, osteoblastic bone formation over the cement lines followed an osteoclastic resorption of both beta-TCP and newly formed bone. In contrast, BMP induced few osteoclastic cells, resulting in slower bone coupling. Furthermore, the faster bone formation induced by the compound pellets seemed to be associated with the presence of beta-TCP. Porous by nature, beta-TCP would entrap BMP within its micropores, and thus, the intrinsically diffusible BMP is retained and its action consequently prolonged. In addition, the compound pellet offered increased surface contact between BMP and mesenchymal cells. Therefore, BMP-beta-TCP compound pellets induce cartilage and bone formation more rapidly than does BMP alone.     

Effect of prostaglandin E1 on preservation injury of canine liver grafts preserved in UW solution

This study investigated whether prostaglandin E₁ (PGE₁) could reduce hepatic injury to the liver graft caused by harvesting and 24-h preservation in University of Wisconsin (UW) solution in a canine model. The PGE₁-treated group was intravenously administered 0.5 g/kg per minute of PGE₁ for 30 min before harvesting, as well as a concentration of 1 mg/l PGE₁ in the washout and UW solutions. In both the PGE₁-treated and the control group, all recipients survived for 1 week or more after transplantation. Arterial ketone body ratio (AKBR) remained over 1.0 in the early postoperative period. The PGE₁ group showed significant reductions in guanase, GOT, and LDH during the early postoperative period compared to the untreated control group. Histological examination disclosed partial mitochondrial swelling, hepatocyte vacuolation, and necrosis in the control group, while such abnormalities were rarely seen in the PGE₁ group. These results suggest that PGE₁ can effectively reduce hepatic injury to liver grafts preserved in UW solution prior to transplantation.     

Prediction of ACNU plasma concentration-time profiles in humans by animal scale-up

Summary Plasma concentration-time profiles of nimustine hydrochloride, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), in the mouse, rat, rabbit, and dog were determined by high-performance liquid chromatographic analysis. The pharmacokinetic parameters for these four animal species and previously reported clinical data were analyzed for investigation of interspecies correlation. Loglog plots of body weight (W; kg) vs total plasma clearance (CLtot, p; ml/min) and steady-state distribution volume (Vd, ss; 1) for the four animal species were linear, with high correlation coefficients (r 0.996 for both parameters), despite the fact that the nonrenal clearance was >97% in these species. Linear regression on the plots excluding human data yielded allometric equations (CL_tot,p=50.6 W^0.957; Bd, ss=1.29 W^1.03) that were extrapolated to predict ACNU pharmacokinetic parameters in humans. For both parameters, however, there were 3-fold differences between the predicted and observed parametric values. To investigate these discrepancies, we measured serum protein binding of ACNU in these animal species and in humans. The values of CLtot,p and Vd,ss were converted into those of CL^u _tot,p and Vd,^u _ss, which correspond to the parameters for unbound ACNU. In this case, correlation coefficients of the log-log plots excluding human data (CL^u _tot,p=71.7 W^0.891; Bd,^u _ss=1.82 W^0.966) were also high (r0.991). The extrapolated values vs those observed in a 70-kg human were the following: CL^u _tot,p, 3,160 vs 2,290 ml/min; Vd,^u _ss, 110 vs 1061. Thus, the animal data were successfully extrapolated to yield better predictions of human pharmacokinetic parameters if the analysis was based on the unbound plasma concentration of ACNU. In addition, the predicted plasma concentration-time profile for humans also showed good agreement with the observed ones. These results suggest the importance of measuring unbound fractions of drugs for more accurate prediction of human pharmacokinetic parameters by extrapolation of animal data to the human situation.