Dynamics of Mitochondrial DNA Nucleoids Regulated by Mitochondrial Fission Is Essential for Maintenance of Homogeneously Active Mitochondria during Neonatal Heart Development

Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis, including mitochondrial DNA (mtDNA) distribution. Cardiac myocytes have a specialized cytoplasmic structure where large mitochondria are aligned into tightly packed myofibril bundles; however, recent studies have revealed that mitochondrial dynamics also plays an important role in the formation and maintenance of cardiomyocytes. Here, we precisely analyzed the role of mitochondrial fission in vivo. The mitochondrial fission GTPase, Drp1, is highly expressed in the developing neonatal heart, and muscle-specific Drp1 knockout (Drp1-KO) mice showed neonatal lethality due to dilated cardiomyopathy. The Drp1 ablation in heart and primary cultured cardiomyocytes resulted in severe mtDNA nucleoid clustering and led to mosaic deficiency of mitochondrial respiration. The functional and structural alteration of mitochondria also led to immature myofibril assembly and defective cardiomyocyte hypertrophy. Thus, the dynamics of mtDNA nucleoids regulated by mitochondrial fission is required for neonatal cardiomyocyte development by promoting homogeneous distribution of active mitochondria throughout the cardiomyocytes.     

Ca2+ enrichment in culture medium potentiates effect of oligonucleotides

Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca²⁺ enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ∼100 nm in size are found in Ca²⁺-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory.     

Comparative evaluation of obesity-related parameters in junior sumo wrestlers and children with obesity

[Purpose]Exercise is a key factor in preventing obesity and metabolic syndrome. Sumo wrestlers increase their body size from childhood for athletic advantage; however, the risk of metabolic syndrome in junior sumo wrestlers is undetermined. Preventive measures against pediatric obesity should be initiated during childhood to prevent obesity in adulthood, considering its high global incidence. We comparatively evaluated the risk factors for metabolic syndrome in junior sumo wrestlers and children with obesity.[Methods]We enrolled 70 male children (age 9–17 years [sumo group, n = 14] and 9–14 years [other sports and non-exercise groups, n = 28 each]) and evaluated their anthropometric parameters (height, weight, body mass index z-score, obesity rate, waist circumference, waist to height ratio) and hematological parameters (total, low-density, high-density, and non-high-density lipoprotein-cholesterol; triglycerides; plasma glucose, and glycated hemoglobin levels).[Results]The BMI z-score, obesity rate, waist circumference (p < 0.05, along with the non-exercise group), and systolic blood pressure were significantly higher and the high-density cholesterol level was lower in the sumo group than in the other sports group (p < 0.05). The waist to height ratio was significantly higher in the non-exercise group than in the other sports group (p < 0.05). No significant difference was found in other blood lipid, plasma glucose (significantly lower level than the reference range in the sumo group, p < 0.05), and glycated hemoglobin (within the reference range in all groups) levels among the three groups.[Conclusion]Junior sumo wrestlers had a larger body size and higher blood pressure than children with obesity who exercised regularly. This provides direction for future research into targeted preventive interventions against metabolic syndrome for junior sumo wrestlers with large body size.     

Adaptation of Restriction Landmark Genomic Scanning (RLGS) to plant genome analysis

Restriction Landmark Genomic Scanning (RLGS) profiles, which are based on the concept of using restriction enzyme sites as landmarks and are generated by two-dimensional gel electrophoresis, were applied to the analysis of plant genomes. This study identified landmark enzymes in RLGS profiles of rice, tobacco, and arabidopsis, because the success of RLGS analysis depends on finding useful landmarks and these are the most frequently studied higher plants. As the results demonstrate, in each plant RLGS analysis various landmark enzymes were identified as useful landmark enzymes. However, the number of spots in a single profile was smaller than in mouse RLGS profiles and differed remarkably in the various plants. In addition, we demonstrate the optimal electrophoresis conditions and a convenient spot-cloning method.     

Detection of 6-nitrotryptophan in proteins by Western blot analysis and its application for peroxynitrite-treated PC12 cells.

We have previously reported on the formation of 6-nitrotryptophan by the reaction of reactive nitrogen species with a tryptophan residue in human Cu, Zn-superoxide dismutase (SOD) (F. Yamakura et al., J. Biochem. 138 (2005) 57-69). Here, we report on the preparation of anti-6-nitrotryptophan antiserum by using synthesized 6-nitrotryptophan-conjugated keyhole limpet hemocyanin as an antigen and the purification of the antibody by using a 6-nitrotryptophan-conjugated affinity column. The purified antibody was immunoreactive with 6-nitrotryptophan residue containing Cu, Zn-SOD but not immunoreactive with Cu, Zn-SOD, Mn-SOD, bovine serum albumin, and 3-nitrotyrosine residue containing Mn-SOD. Nitro group of 6-nitrotryptophan was reduced by sodium hydrosulfite to form 6-aminotryptophan as a major product. The reduced 6-nitrotryptophan residues lost its immunoreactivity with the antibody. We detected different immunoreactive bands between using antibody for 6-nitrotryptophan residues and that for 3-nitrotyrosine residues in crude extracts of neuron-like PC12 cells treated with peroxynitrite by a Western blot analysis. Western blot analysis for two-dimensional gel electrophoresis showed nine intensively stained immunoreactive spots for 6-nitrotryptophan residues in the peroxynitrite-treated PC12 cells, which were subjected to trypsin digestion and LC-ESI-MS/MS analysis. We identified M2 pyruvate kinase, elongation factor 2, mitochondrial aconitase, pyruvate carboxylase, and heat shock protein HSP90alpha as candidates for 6-nitrotryptophan residues containing proteins, with peptide coverage over 10%, in crude extracts of peroxynitrite-treated PC12 cells.     

Improved RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens.

Recently, in addition to DNA, RNA extracted from archival tissue specimens has become an invaluable source of material for molecular biological analysis. Successful amplification with PCR/RT-PCR is problematic when using amplicons of short size due to degradation of DNA or RNA. We established an improved method for efficient RT-PCR amplification of RNA extracted from archival formalin-fixed, paraffin-embedded tissue by the elimination of RNA modification and the restoration of RNA template activity. Namely, the preheating in citrate buffer (pH 4.0) of RNA extracted from long-term preserved tissue specimens resulted in significantly increased efficiency of RT-PCR.     

Phosphorylated 350 kD protein in the nucleus as it is associated with cell transformation

Protein kinases are thought to play a key role in signal transduction and oncogenesis, but little is known about the intranuclear phosphorylation events associated with transformation. Here we report on cell cycle-dependent phosphorylation of cytoskeleton-associated 350 kD protein and the regular interchange in its location between the nucleus and cytoplasm of normal cells. Persistent intranuclear location of the phosphorylated 350 kD protein was also found throughout the cell cycle in transformed cells, as detected by immunoprecipitation of 32P-phosphorylated 350 kD protein from isolated nuclei and immunofluorescent staining with a monoclonal antibody that recognized phosphorylated site of 350 kD protein. A conditional transformed phenotype induced by a temperature-sensitive (ts) viral oncogene or a transforming growth factor was also associated with the intranuclear presence of the phosphorylated 350 kD protein. Thus the 350 kD protein seems to be a target molecule of protein kinases that are stimulated directly or indirectly by growth factors or by oncogene products in the nucleus, and appears to be a new transformation-related nuclear antigen.     

Intracellular Ca2+ Concentration and Cytoplasmic Streaming in Vallisneria Mesophyll Cells

Intracellular Ca²⁺ concentration regulating the cytoplasmicstreaming in Vallisneria mesophyll cells was estimated. Theleaf segment was cut open at the middle of the mesophyll celllayers and the exposed mesophyll cells were treated with testsolutions of various Ca²⁺ concentrations in the dark. This allowedA23187 [GenBank] , a calcium ionophore, to exert its full effect on thecell membrane. The streaming was induced or maintained in solutions which containedCa²⁺ at lower than 10^–6M. However, Ca²⁺ at concentrationshigher than 10^–5M had a definite, inhibitory effect. Theinduction and cessation of streaming could be repeated by alternatelychanging the solutions. (Received March 14, 1986; Accepted May 15, 1986)     

Aberrancy in immunogenicity and cell-surface expression of H-2 antigens on erythrocytes

Immunogenicity for T cell-independent B-cell response assessed by splenic plaque-forming cell (PFC) response and cell-surface expression measured by laser flow cytometry of various class I H-2 antigens on mouse red blood cells (RBC) were compared. It was found that the order of magnitude of both immunogenicity and cell-surface expression on RBC is H-2D^d H-2D^b > H-2K^d, H-2K^b. Furthermore, H-2^d public antigens and H-2L^d antigens were neither immunogenic nor easily demonstrable on RBC. These findings contrasted with poor immunogenicity for PFC response (Nakashima et al. 1982, 1983) and proportionally strong expression of H-2 antigens on lymphoid cells. Immunogenicity and cell-surface expression of H-2D^d antigen on RBC were not shown to be controlled by the action of genes outside H-2D. It was therefore suggested that a number of H-2 antigens, including H-2K^d private, H-2K^b private, and H-2^d public specificities are at least functionally defective on RBC. This is possibly due to the structural characteristics of the antigens. Since immunogenicity and cell-surface expression were in parallel, the expression of H-2 antigens on RBC must be dictated by a subset of B cells whose activity was assessed by PFC response. This finding supports the view that the H-2 molecules display a new category of activity which is different from their ability to activate T cells and depends on their expression on RBC.     

Application of flow cytometry to differentiating Exophiala dermatitidis E. moniliae and E. jeanselmei from each other

We applied a flow cytometry apparatus (FCM) to differenciating Exophiala dermatitidis, E. moniliae and E. jeanselmei from each other. The wavelength of the argon laser emitted from the FCM was 488 nm and the aperture of nozzle from which the stream of fluid containing single cells was blown out was 100 m. By irradiating the stream with laser by either the forward light scatter (FLS) or by the perpendicular light scattr (PLS), we were able to get two pieces of informations. Histograms displayed by the FLS indicate the cell size, while dot displays by the PLS reflect the cell structure. As a result, E. dermatitidis was clearly differenciated from either E. moniliae or E. jeanselmei by their histograms by FLS. In addition, dot displays by the PLS differenciated E. moniliae from E. jeanselmei.In conclusion, flow cytometry is available for differenciating E. dermatitidis, E. moniliae and E. jeanselmei from each other.