Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.     

Contribution of microsomal cytochrome b5 electron transport system coupled with Δ5-3β-hydroxysteroid dehydrogenase to androgen synthesis from progesterone

Cytochromes P-450 and $\text{b}$₅ were observed in the microsomal fraction of interstitial tissue of rat testes. Microsomal cytochrome $\text{b}$₅ was reduced by the NADH coupled with the activities of Δ⁵-3β-hydroxysteroid dehydrogenase with Δ⁵-Δ⁴ isomerase through conversion of pregnenolone to progesterone. Activities of NADPH-supported 17α-hydroxylase and C-17-C-20 lyase which converted progesterone to androstenedione were stimulated by either the presence of NADH or the oxidative reaction by the dehydrogenase upon Δ⁵-3β-hydroxysteroids. Androstenedione production enhanced by the reaction of the dehydrogenase was decreased by addition of the antibody against NADH-cytochrome $\text{b}$₅ reductase which was purified from rat hepatic microsomes, suggesting the active participation of cytochrome $\text{b}$₅ in the androgen synthesis.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Fertilizability of cryopreserved zona-free hamster ova

Mature unfertilized ova from superovulated hamsters were freed from all investments and frozen at ?50°C. They were cooled at about 1°C/min to 0°C then at 0.8° to 0.6°C/min to ?50°C. At 0°C, dimethyl sulfoxide was added to a final concentration of 1.25 M. The ova were stored at ?50°C for up to four months. Thawing was performed at 2–4°C/min and followed by several washes with insemination medium. Approximately 90% of the ova were normal in appearance after thawing. The frozen and thawed ova with normal appearance could be penetrated by hamster or human spermatozoa at a rate comparable to unfrozen controls. The ability of hamster ova to tolerate storage at a relatively convenient temperature (?50°C) for long periods (tested for up to four months) makes possible their shipment at low cost to institutions lacking this resource. There they can be used for basic biological studies of sperm–egg interaction or in the clinical assessment of human sperm quality.     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate     

Nucleic acid metabolism in cold-treated wheat embryos

The incorporation of ₃₂P into nucleic acid fractions separatedon a MAK column was compared for normally germinated and cold-treatedwheat embryos. ₃₂P accumulation in DNA fraction was decreasedby cold treatment, although that in the RNA fractions was slightlypromoted. The synthesis of the fraction, probably mRNA, elutedafter the peak of heavy rRNA was enhanced in cold-treated embryosand suppressed when the embryos were cold-treated in the presenceof 8-azaguanine, an inhibitor of vernalization. (Received May 2, 1975; )     

Fatty acid and sterol compositions in mushrooms of ten species of polyporaceae

The simple lipids present in ten species of Polyporaceae (Piptororus betulinus, Coriolus pargamenus, C. versicolor, C. heteromorphus, Formitopsis cytisina, F. pinicola, Microporus flabelliformis, Gloephyllum saepiarium, Crytoderma citrinum and Grifola frondosa) were investigated. The fatty acids that these species had in common were C₁₆-saturated acids (except in P. betulinus) and C₁₈-unsaturated acids. Ergosterol and ergosta-7,22-dien-3β-ol were isolated from these mushrooms. Lupeol was obtained from G. saepiarium. Ergost-7-en-3β-ol, lanosterol and 24-methylene-24,25-dihydrolanosterol were tentatively identified.     

Studies on Flavor Components of Foodstuffs

Distribution of tetramethylpyrazine (T.M.P.) in Japanese fermented foodstuffs was investigated by more accurate analytical method.

Namely, the method was successful when trapping T.M.P. with picric acid after flash evaporation of the foodstuffs, followed by analysis with gas chromatography.

T.M.P. was detected in many Japanese fermented foodstuffs, especially in Miso, Soy sause and Natto, which suggests that alkylpyrazines may play an important role as flavor of those foodstuffs.     

DNA content contributes to nuclear size control in Xenopus laevis

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.     

Induced pluripotent stem cell‐derived myeloid cells expressing OX40 ligand amplify antigen‐specific T cells in advanced melanoma

Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune‐related adverse events have been reported. Therefore, more effective and antigen‐specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS‐ML). In this study, we generated OX40L‐overexpressing iPS‐ML (iPS‐ML‐Zsgreen‐OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS‐ML‐Zsgreen‐OX40L suppressed the progression of B16‐BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)‐expressing B16 melanoma (MO4). The number of antigen‐specific CD8⁺ T cells was higher in spleen cells treated with OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L than in those without OX40L. The OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L significantly increased the number of tumor‐infiltrating T lymphocytes (TILs) in MO4 tumor. Flow cytometry showed decreased regulatory T cells but increased effector and effector memory T cells among the TILs. Although we plan to use allogeneic iPS‐ML in the clinical applications, iPS‐ML showed the tumorgenicity in the syngeneic mice model. Incorporating the suicide gene is necessary to ensure the safety in the future study. Collectively, these results indicate that iPS‐ML‐Zsgreen‐OX40L therapy might be a new method for antigen‐specific cancer immunotherapy.