Nontranslation of foreign genetic information for fraction 1 protein under circumstances favorable for direct transfer ofNicotiana gossei isolated chloroplasts intoN. Tabacum protoplasts

Summary The nuclei and cytoplasm ofN. gossei andN. tabacum are compatible to the extent that reciprocal, interspecific F₁ hybrids can be produced by conventional breeding techniques. Conditions were established in which manyN. gossei isolated chloroplasts could be seen by phase and fluorescence microscopy to adhere to 40% of the population of protoplasts obtained from white tissue of variegatedN. tabacum plants and to remain attached after washing the protoplasts. Chloroplasts also could be seen to enter the interior of the protoplasts. After treating albino protoplasts withN. gossei chloroplasts, the protoplasts were subjected to further conditions whereby 65 calluses containing shoots developed. TwentyN. tabacum protoplasts not treated with foreign chloroplasts also produced calluses with shoots to serve as a control. All calluses developed chlorophyll irrespective of whether or not the albino protoplasts had been treated with isolatedN. gossei chloroplasts. The Fraction 1 protein ofN. tabacum has a different electrophoretic mobility from the protein ofN. gossei or anN. gossei xN. tabacum F₁ hybrid. The Fraction 1 protein large subunit is coded by chloroplast DNA, whereas the small subunit is coded by nuclear DNA. Fraction 1 protein was isolated from the variegated shoots of the 65 calluses obtained after treating albino protoplasts with foreign chloroplasts. Immunoelectrophoresis demonstrated the protein from each callus to have a mobility identical toN. tabacum protein. Therefore, under circumstances highly favorable for the direct transfer ofN. gossei isolated chloroplasts (and possibly nuclei also) intoN. tabacum protoplasts, no evidence was obtained to suggest that genetic information contained in the isolated foreign organelles was being translated into the polypeptides of either the large or small subunits of Fraction 1 protein contained in newly differentiated leaves derived from the protoplasts. Supported by Research Grant PCM-75-07368 from the National Science Foundation.     

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; )     

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.     

MicroRNA-30c attenuates fibrosis progression and vascular dysfunction in systemic sclerosis model mice

Molecular Biology Reports - Systemic sclerosis (SSc) is characterized by peripheral circulatory disturbance and fibrosis in skin and visceral organs. We recently demonstrated that...     

Stem‐cell‐based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies

Various advances have been made in the treatment of retinal diseases, including new treatment strategies and innovations in surgical devices. However, the treatment of degenerative retinal diseases, such as retinitis pigmentosa (RP) and age‐related macular degeneration (AMD), continues to pose a significant challenge. In this review, we focus on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to treat retinal diseases by harnessing the ability of stem cells to differentiate into different body tissues. The retina is a tissue specialized for light sensing, and its degradation leads to vision loss. As part of the central nervous system, the retina has very low regenerative capability, and therefore, treatment options are limited once it degenerates. Nevertheless, innovations in methods to induce the generation of retinal cells and tissues from ESCs/iPSCs enable the development of novel approaches for these irreversible diseases. Here we review some historical background and current clinical trials involving the use of stem‐cell‐derived retinal pigment epithelial cells for AMD treatment and stem cell‐derived retinal cells/tissues for RP therapy. Finally, we discuss our future vision of regenerative treatment for retinal diseases with a partial focus on our studies and introduce other interesting approaches for restoring vision.     

Cysteine Residues in the Major Capsid Protein,Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation

The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.