Development of internal standard for lipoprotein subclass analysis using dual detection gel-permeation high-performance liquid chromatography system

The LipoSEARCH® System is an innovative lipoprotein class analysis method based on gel-permeation high-performance liquid chromatography (HPLC). This system uses a gel permeation column to separate the major lipoprotein subclasses (chylomicron, very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein) in serum according to particle size and splits them into two pathways to measure total cholesterol (TC; esterified + unesterified cholesterol) and triglyceride (TG) concentrations simultaneously to obtain chromatograms for each.These chromatograms were analyzed based on the results of the calibration serum by fitting Gaussian curves to profile the 20 lipoprotein subclasses defined in detail. An important assumption of this HPLC system is its simultaneous detection of two pathways to guarantee the accuracy of each analysis. Therefore, in the present study, we investigated the development of an internal standard that can guarantee the simultaneous detection of this system by adding a pigment to the serum. We focused on quinone pigments with absorption at 550 nm, which is the wavelength used for the enzymatic assay of TC and TG concentrations in the system. As a result, we succeeded in producing overlapping pigment peaks that appeared after the analytical chromatograms in two pathways. It is also suggested that the pigment solution as an internal standard is stable in freezing storage and has little effect on the analysis. The developed internal standard is expected to contribute to the accuracy assurance of lipoprotein analysis by this dual-detection HPLC system.     

A specific inhibitor to δ-aminolevulinate dehydratase in rat bone marrow cells

A factor that specifically inhibited δ-aminolevulinate dehydratase was found in rat bone marrow cells. The inhibitor, which was located in the supernatant fraction of the bone marrow hemolysate, was purified about 12-fold by ammonium sulfate fractionation and column chromatography on Sephadex G-75. The partially purified inhibitor was heat labile and sensitive to trypsin and was denatured by urea. It had a pH optimum of 7.5–8.0, and a molecular weight of 28,000. It inhibited the activity of δ-aminolevulinate dehydratase noncompetitively.     

Fusion of cell ghosts with sexually opposite type cells in Dictyostelium discoideum

In the sexual cycle of Dictyostelium discoideum, haploid cells of two opposite mating types, strains HM1 and NC4, acquire fusion-competence under certain conditions, such as suspension culture in the dark, and fuse specifically to form giant zygote cells. Each giant cell engulfs the surrounding cells, gradually increases in size, and finally develops into a macrocyst that is a sexual structure in D. discoideum. Fusion-competent HM1 cells suspended in a solution were frozen and thawed to make cell ghosts. When cell ghosts were introduced into fusion-competent and -incompetent intact NC4 cells, the cell ghosts killed them in a short time, but the fusion-competent cells were killed in preference to the fusion-incompetent cells. This killing occurred through the fusion of the cell ghosts directly to intact cell membranes. Since the fusion was specific, the fusion between ghosts and cells appears to be essentially the same as that between intact cells during the sexual cycle in molecular mechanisms.     

Spatial expression patterns of genes involved in cyclic AMP responses in Dictyostelium discoideum development

The spatial expression patterns of genes involved in cyclic adenosine monophosphate (cAMP) responses during morphogenesis in Dictyostelium discoideum were analyzed by in situ hybridization. Genes encoding adenylyl cyclase A (ACA), cAMP receptor 1, G-protein alpha2 and beta subunits, cytosolic activator of ACA (CRAC and Aimless), catalytic subunit of protein kinase A (PKA-C) and cAMP phosphodiesterases (PDE and REG-A) were preferentially expressed in the anterior prestalk (tip) region of slugs, which acts as an organizing center. MAP kinase ERK2 (extracellular signal-regulated kinase-2) mRNA, however, was enriched in the posterior prespore region. At the culmination stage, the expression of ACA, CRAC and PKA-C mRNA increased in prespore cells in contrast with the previous stage. However, no alteration in the site of expression was observed for the other mRNA analyzed. Based on these findings, two and four classes of expression patterns were catalogued for these genes during the slug and culmination stages, respectively. Promoter analyses of genes in particular classes should enhance understanding of the regulation of dynamic and coordinated gene expression during morphogenesis.     

Sexual Development of Cellular Slime Molds

Macrocyst formation represents sexual development of cellular slime molds and begins with fusion between cells of compatible mating types. Homothallic and heterothallic strains as well as bisexual and asexual ones have been described. Macrocyst development requires certain environmental conditions such as darkness and excessive humidity. Sexual cell fusion has been analyzed at a molecular level in Dictyostelium discoideum , and several cell surface proteins related to it have been identified. Some of them are common to both mating types, while others are specific to one or other type. The involvement of cell-surface carbohydrates has also been suggested, though direct evidence for this is still lacking. Macrocyst formation is regulated by diffusible, pheromone like substances. Genetic studies on sexual development are scarce, probably because no suitable mutants have been available. However, several asexual mutants, as well as antibody and nucleotide probes, have recently been obtained, so mechanisms of sexual cell fusion may be understood in the near future. Considering the unique phylogenical position of cellular slime molds, analysis of sexual development in these organisms should contribute to the understanding of the mechanism and evolution of sexual reproduction systems.     

Measurement of Technetium-99m Sestamibi Signals in Rats Administered a Mitochondrial Uncoupler and in a Rat Model of Heart Failure

Background

Many methods have been used to assess mitochondrial function. Technetium-99m sestamibi (^99mTc-MIBI), a lipophilic cation, is rapidly incorporated into myocardial cells by diffusion and mainly localizes to the mitochondria. The purpose of this study was to investigate whether measurement of ^99mTc-MIBI signals in animal models could be used as a tool to quantify mitochondrial membrane potential at the organ level.

Methods and Results

We analyzed ^99mTc-MIBI signals in Sprague-Dawley (SD) rat hearts perfused with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler known to reduce the mitochondrial membrane potential. ^99mTc-MIBI signals could be used to detect changes in the mitochondrial membrane potential with sensitivity comparable to that obtained by two-photon laser microscopy with the cationic probe tetramethylrhodamine ethyl ester (TMRE). We also measured ^99mTc-MIBI signals in the hearts of SD rats administered CCCP (4 mg/kg intraperitoneally) or vehicle. ^99mTc-MIBI signals decreased in rat hearts administered CCCP, and the ATP content, as measured by ³¹P magnetic resonance spectroscopy, decreased simultaneously. Next, we administered ^99mTc-MIBI to Dahl salt-sensitive rats fed a high-salt diet, which leads to hypertension and heart failure. The ^99mTc-MIBI signal per heart tissue weight was inversely correlated with heart weight, cardiac function, and the expression of atrial natriuretic factor, a marker of heart failure, and positively correlated with the accumulation of labeled fatty acid analog. The ^99mTc-MIBI signal per liver tissue weight was lower than that per heart tissue weight.

Conclusion

Measurement of ^99mTc-MIBI signals can be an effective tool for semiquantitative investigation of cardiac mitochondrial membrane potential in the SD rat model by using a chemical to decrease the mitochondrial membrane potential. The ^99mTc-MIBI signal per heart tissue weight was inversely correlated with the severity of heart failure in the Dahl rat model.     

Epigenetic response of endothelial cells to different wall shear stress magnitudes: A report of new mechano-miRNAs

Endothelial cells (ECs) respond to flow stress via a variety of mechanisms, leading to various intracellular responses that can modulate the vessel wall and lead to diseases if the flow is disturbed. Mechano-microRNAs (miRNAs) are a subset of miRNAs in the ECs that are flow responsive. Mechano-miRNAs were shown to be related to atherosclerosis pathophysiology, and a number of them were identified as pathologic. Here, we exposed human carotid ECs to different wall shear stresses (WSS), high and low, and evaluated the response of miRNAs by microarray and quantitative polymerase chain reaction analysis. We discovered five new mechano-miRNAs that were not reported in that context previously to the best of our knowledge. Moreover, functional pathway analysis revealed that under low WSS conditions, several pathways regulating apoptosis are affected. In addition, KLF2 and KLF4, known atheroprotective genes, were downregulated under low WSS and upregulated under high WSS. KLF2 and VCAM1, both angiogenic, were upregulated under high WSS. NOS3, which is vascular protective, was also upregulated with higher WSS. On the contrary, ICAM-1 and E-selectin, both atherogenic and proinflammatory, were upregulated with high WSS. Collectively, the epigenetic landscape with the gene expression analysis reveals that low WSS is associated with a proapoptotic state, while high WSS is associated with a proliferative and proinflammatory state.     

An improved pulsed-field polyacrylamide gel electrophoresis system for physical selection of linking clones: Isolation of SfiI linking clones from a chromosome 21-specific library

We had previously developed an efficient procedure for selective cloning of rare-cutter linking fragments that is based on physical separation of linking clone DNAs by pulsed-field polyacrylamide gel electrophoresis (PF-PAGE). An advantage of the physical selection procedure over the conventional cloning-based ones utilizing the insertion of selection marker or vector sequences into the rare-cutter sites is that it can be readily applied to the selection of linking gragments for rare-cutters, generating ambiguous cohesive end sequences such as SfiI (GGCCNNNN/NGGCC). In the present work, the physical separation procedure was improved by introducing a discontinuous buffer system into PF-PAGE, and its feasibility was exemplified by the selective isolation of SfiI linking clones from a human chromosome 21-specific library. This simple and efficient procedure will provide a useful tool for genome analysis.     

Expression of vascular endothelial growth factor receptor 1 in bone marrow-derived mesenchymal cells is dependent on hypoxia-inducible factor 1

Bone marrow-derived cells are recruited to sites of ischemia, where they promote tissue vascularization. This response is dependent upon the expression of vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1), which mediates cell migration in response to VEGF or placental growth factor (PLGF). In this study, we found that exposure of cultured mouse bone marrow-derived mesenchymal stromal cells (MSC) to hypoxia or an adenovirus encoding a constitutively active form of hypoxia-inducible factor 1 (HIF-1) induced VEGFR1 mRNA and protein expression and promoted ex vivo migration in response to VEGF or PLGF. MSC in which HIF-1 activity was inhibited by a dominant negative or RNA interference approach expressed markedly reduced levels of VEGFR1 and failed to migrate or activate AKT in response to VEGF or PLGF. Thus, loss-of-function and gain-of-function approaches demonstrated that HIF-1 activity is necessary and sufficient for basal and hypoxia-induced VEGFR1 expression in bone marrow-derived MSC.