The effects of guanine and cytosine variation on dinucleotide frequency and amino acid composition in the human genome

Summary One hundred twelve human DNA sequences were analyzed with respect to dinucleotide frequency and amino acid composition. The variation in guanine and cytosine (G+C) content revealed: (1) at 2–3 and 3-1 doublet positions CG discrimination is attenuated at high G+C, but TA disfavor is enhanced, and (2) several amino acids are subject to G+C change. These findings have been reported in part for collections of sequences from various species. The present study confirms that in a single organism-the human-the G+C effects do exist. Aspects of the argument that connects G+C with protein thermal stability are also discussed.     

The Lipid Phase of Thylakoid and Cytoplasmic Membranes from the Blue-Green Algae (Cyanobacteria), Anacystis nidulans and Anabaena variabilis

The lipid phases of the thylakoid and cytoplasmic membranesfrom the blue-green alga, Anacystis nidulans, were studied bya spin-probe method using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl.The thylakoid and cytoplasmic membranes of this alga were bothin the liquid crystalline state at growth temperature, and inthe phase separation state at about 0?C. The thylakoid membranesentered the phase separation state at a temperature higher thanthe cytoplasmic membranes. The lipid phase of the thylakoidmembranes from Anabaena variabilis was studied in a similarway, and these membranes were found also to undergo the phasetransition. The temperature for the onset of the phase separationand the fluidity of the membrane lipids of both algae dependedon the growth temperature of the culture. (Received April 9, 1984; Accepted June 1, 1984)     

Disappearance of a Novel Protein Component of the 26S Proteasome duringXenopusOocyte Maturation

We have prepared polyclonal antibodies againstXenopus20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development ofXenopus laevis.A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

The Neuroprotective Effects of Coccomyxa Gloeobotrydiformis on the Ischemic Stroke in a Rat Model

Stroke is a major cause of mortality and the leading cause of permanent disability. In this study, we adopted the classic middle cerebral artery occlusion(MCAO) stroke model to observe the therapeutic effects of coccomyxa gloeobotrydiformis(CGD) on ischemic stroke, and discuss the underlying mechanisms. Low dose (50 mg/kg.day) and high dose (100 mg/kg.day) concentrations of the drug CGD were intragastrically administrated separately for 8 weeks. Infarct volumes, neurologic deficits and degree of stroke-induced brain edema were measured 24 hours after reperfusion. Furthermore, oxidative stress related factors (SOD and MDA), mitochondrial membrane potential, and apoptosis regulatory factors (mitochondrial Cyt-C, Bcl-2, Bax, and caspase-3) were all investigated in this research. We found that CGD attenuated cerebral infarction, brain edema and neurologic deficits; CGD maintained the mitochondrial membrane potential and decreased mitochondrial swelling. It also prevented oxidative damage by reducing MDA and increasing SOD. In addition, CGD could effectively attenuate apoptosis by restoring the level of mitochondrial Cyt C and regulating the expression of Bcl-2, Bax and caspase 3. These results revealed that CGD has a therapeutic effect on ischemic stroke, possibly by inducing mitochondrial protection and anti-apoptotic mechanisms.     

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7(+) MyoD(-) undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis.     

Redox-state dependent blinking of single photosystem I trimers at around liquid-nitrogen temperature

Efficient light harvesting in a photosynthetic antenna system is disturbed by a ragged and fluctuating energy landscape of the antenna pigments in response to the conformation dynamics of the protein. This situation is especially pronounced in Photosystem I (PSI) containing red shifted chlorophylls (red Chls) with the excitation energy much lower than the primary donor. The present study was conducted to clarify light-harvesting dynamics of PSI isolated from Synechocystis sp. PCC6803 by using single-molecule spectroscopy at liquid?nitrogen temperatures. Fluorescence emission at around 720?nm from the red Chls in single PSI trimers was monitored at 80–100?K. Intermittent variations in the emission intensities, so-called blinking, were frequently observed. Its time scale lay in several tens of seconds. The blinking amplitude depended on the redox state of the phylloquinone (A₁). Electrochromic shifts of Chls induced by the negative charge on A₁ were calculated based on the X-ray crystallographic structure. A Chl molecule, Chl-A839 (numbering according to PDB 5OY0), bound near A₁ was found to have a large electrochromic shift. This Chl has strong exciton coupling with neighboring Chl (A838) whose site energy was predicted to be determined by interaction with an arginine residue (ArgF84) [Adolphs et al., 2010]. A possible scenario of the blinking was proposed. Conformational fluctuations of ArgF84 seesaw the excitation-energy of Chl-A838, which perturbs the branching ratio of excitation-energy between the red Chl and the cationic form of P700 as a quencher. The electrochromic shift of Chl-A839 enhances the effect of the conformation dynamics of ArgF84.     

Isolation and Characterization of Multipotential Mesenchymal Cells from the Mouse Synovium

The human synovium contains mesenchymal stem cells (MSCs), which are multipotential non-hematopoietic progenitor cells that can differentiate into a variety of mesenchymal lineages and they may therefore be a candidate cell source for tissue repair. However, the molecular mechanisms by which this can occur are still largely unknown. Mouse primary cell culture enables us to investigate the molecular mechanisms underlying various phenomena because it allows for relatively easy gene manipulation, which is indispensable for the molecular analysis. However, mouse synovial mesenchymal cells (SMCs) have not been established, although rabbit, cow, and rat SMCs are available, in addition to human MSCs. The aim of this study was to establish methods to harvest the synovium and to isolate and culture primary SMCs from mice. As the mouse SMCs were not able to be harvested and isolated using the same protocol for human, rat and rabbit SMCs, the protocol for humans was modified for SMCs from the Balb/c mouse knee joint. The mouse SMCs obtained showed superior proliferative potential, growth kinetics and colony formation compared to cells derived from muscle and bone marrow. They expressed PDGFRá and Sca-1 detected by flow cytometry, and showed an osteogenic, adipogenic and chondrogenic potential similar or superior to the cells derived from muscle and bone marrow by demonstrating in vitro osteogenesis, adipogenesis and chondrogenesis. In conclusion, we established a primary mouse synovial cell culture method. The cells derived from the mouse synovium demonstrated both the ability to proliferate and multipotentiality similar or superior to the cells derived from muscle and bone marrow.