Oxygen evolution in dark-developed spruce chloroplasts

Chloroplasts isolated from dark-grown seedlings of Picea abiesshowed activities of DCIP and Fecy photoreductions widiout additionof an electron donor to PS-II. In addition to this, when light-inducedoxygen evolution was measured with an oxygen electrode, a significantamount of oxygen was found. These results indicate that thephotoreductions are coupled to the oxygen-evolving reaction.Furthermore, thylakoid membranes were functional in the protonuptake and the 515-nm absorbance change as parameters of theirphysicochemical functions. Electron microscopy showed that thylakoidswere well-developed with prolamellar bodies and partially stackedto from grana. We conclude that oxygen-evolving ability and the physicochemicalfunction of thylakoid membranes develop in chloroplasts of dark-grownspruce seedlings. (Received September 21, 1974; )     

Oxygenation-linked changes in the ultraviolet absorption and circular dichroism spectra of spiny lobster hemocyanin

As an approach to elucidate the mechanism of the protein structure change in the cooperative ligand binding, the UV difference and CD spectra of aromatic residues in Panulirus japonicus (spiny lobster) hemocyanin were examined. The native hemocyanin showed an O2-induced narrow-banded change in the absorption spectrum around 290 nm, which was not affected by pH in the range of 7.5 to 9.5. When the native hexameric protein was stripped of divalent cations with EDTA (at pH 7.5), the magnitude of the narrow-banded difference was reduced to about half, whereas it was almost completely abolished on dissociation into subunits (stripped at pH 9.5). The magnitude of the absorption change was found to be proportional to the degree of O2 saturation in the native and stripped hemocyanins. It was inferred that the spectral difference reflects a tertiary structure change directly linked to the oxygenation, though it depends greatly on the subunit association. Panulirus hemocyanin showed negative CD bands in the region of 260 to 300 nm, the intensities of which were considerably reduced by oxygenation and also by dissociation into subunits.     

A cytological study of the yoshida sarcoma. An ascites tumor op white rats

Summary In the Yoshida sarcoma a strain of tumor cells is present which have their own characteristic chromosome constitution and multiply by regular mitosis. The well-balanced complement of ±40 chromosomes consists of two distinct groups: one is represented by 22 to 24 rodshaped elements which probably come directly and without change from the original normal cell, the other group comprises 16 to 18 Vand J-shaped chromosomes which are specific for the tumor cells. Their exact origin is unknown, but must be mutational in character. Because of this morphological peculiarity, the chromosomes of the tumor cells are markedly different from those of the host cells for which 42 rodshaped elements are typical. No transitional types bridging the gap between ordinary and tumor cells occur. The individuality of the chromosomes in the strain cells remains unchanged during successive transplant generations from rat to rat. The growth of the tumor is primarily caused by the proliferation of these strain cells. In the course of multiplication part of the proliferating cells become abnormal and undergo aberrant mitotic processes owing probably to an alteration of the spindle mechanism, structural changes of the chromosomes, and some other causes. The frequently occurring tumor cells showing mitotic abnormalities are, therefore, derivatives of the sub-diploid strain cells. Destruction of the derivative cells by chemical treatment (podophyllin, CaCl₂) is followed by multiplication of the resistant strain cells.Comparable evidence has been found in two new strains of ascites tumor similar to the Yoshida sarcoma. Their strain cells have the same total chromosome number as the Yoshida strain cells and, within the set, the same two groups of rod-shaped and of Vor J-shaped chromosomes, but differ from each other as well as from the Yoshida sarcoma in the number of Vand J-shaped chromosomes.Contribution No. 263 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan.     

Interaction of antisense DNA with nucleic acids/proteins.

In order to study interaction of various types of labeled antisense DNAs were prepared. Fluorescein and 2,2,6,6-tetramethypiperidine-N-oxyl were the label molecules, which were introduced to 5'-end of oligonucleotides and their analogs. Interactions of labeled antisense DNAs with nucleic acids or proteins such as HSA, HIG and TF, were studied by UV, fluorescence depolarization spectroscopy, and ESR spectroscopy. Hybrid formation of antisense DNAs with oligonucleotides in solution could be monitored by the increase in fluorescence anisotropy (r) and by intensity change in ESR spectra. When phosphorothioate type antisense molecules anchoring fluorescein (F-OPT) were mixed with proteins, r drastically increased, whereas ODN slightly increased. These results suggest that OPTs have much more affinity for proteins than ODNs.     

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.     

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp⁰ strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp⁰ strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp⁰ suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp⁰ backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.