Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp), which has been designed based on the structure of ovokinin (2-7), significantly reduces the systolic blood pressure at a dose of 100 microg/kg after oral administration in spontaneously hypertensive rats (SHRs). In this study, we generated a transgenic soybean which accumulates novokinin. A vector encoding a modified beta-conglycinin alpha' subunit (4novokinin-alpha') in which four novokinin sequences have been incorporated by site-directed mutagenesis was introduced into somatic embryos by whisker-mediated gene transformation to produce a transgenic soybean. The 4novokinin-alpha' occupied 0.5% of total soluble protein and 5% of the beta-conglycinin alpha' subunit in the transgenic soybean seeds. Protein extracted from the transgenic soybean reduced systolic blood pressure after single oral administration in SHRs at a dose of 0.15 g/kg. Defatted flour from the transgenic soybean also reduced the systolic blood pressure at a dose of 0.25 g/kg. Thus, the 4novokinin-alpha' produced in soybean exhibited an anti-hypertensive activity in SHRs after oral administration.     

Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activity

Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near −40 °C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree ( Cercidiphyllum japonicum ), of which XPCs exhibited deep supercooling to −40 °C, were prepared by methanol extraction. The crude extracts were purified by liquid–liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8–9.0 °C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-3- O - β -glucoside (Q3G), kaempferol-7- O - β -glucoside (K7G), 8-methoxykaempferol-3- O - β -glucoside (8MK3G) and kaempferol-3- O - β -glucoside (K3G). Microscopic observations confirmed the presence of flavonoids in cytoplasms of XPCs. These results suggest that diverse kinds of anti-ice nucleation substances, including flavonol glycosides, may have important roles in deep supercooling of XPCs.     

Human prohibitin 1 maintains the organization and stability of the mitochondrial nucleoids

Mitochondrial prohibitin (PHB) proteins have diverse functions, such as the regulation of apoptosis and the maintenance of mitochondrial morphology. In this study, we clarified a novel mitochondrial function of PHB1 that regulates the organization and maintenance of mitochondrial DNA (mtDNA). In PHB1-knockdown cells, we found that mtDNA is not stained by fluorescent dyes, such as ethidium bromide and PicoGreen, although the mitochondrial membrane potential still maintains. We also demonstrated that mtDNA, which is predominantly found in the NP-40-insoluble fraction when isolated from normal mitochondria, is partially released into the soluble fraction when isolated from PHB1-knockdown cells, indicating that the organization of the mitochondrial nucleoids has been altered. Furthermore, we found that PHB1 regulates copy number of mtDNA by stabilizing TFAM protein, a known protein component of the mitochondrial nucleoids. However, TFAM does not affect the organization of mtDNA as observed in PHB1-knockdown cells. Taken together, these results demonstrate that PHB1 maintains the organization and copy number of the mtDNA through both TFAM-independent and -dependent pathways.     

Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.     

Glycoform analysis of Japanese cypress pollen allergen, Cha o 1: a comparison of the glycoforms of cedar and cypress pollen allergens

A Japanese cypress (Chamaecyparis obtusa) pollen allergen, Cha o 1, is one of the major allergens that cause allergic pollinosis in Japan. Although it has been found that Cha o 1 is glycosylated and that the amino acid sequence is highly homologous with that of Japanese cedar pollen allergen (Cry j 1), the structure of N-glycans linked to Cha o 1 remains to be determined. In this study, therefore, we analyzed the structures of the N-glycans of Cha o1. The N-glycans were liberated by hydrazinolysis from purified Cha o 1, and the resulting sugar chains were N-acetylated and pyridylaminated. The structures of pyridylaminated N-glycans were analyzed by a combination of exoglycosidase digestion, two dimensional (2D-) sugar chain mapping, and electrospray ionization mass spectrometry analysis. Structural analysis indicated that the major N-glycan structure of Cha o1 is GlcNAc2Man3Xyl1Fuc1GlcNAc2 (89%), and that high-mannose type structures (Man9GlcNAc2, Man7GlcNAc2) occur as minor components (11%).     

Voluntary running exercise alters microbiota composition and increases n-butyrate concentration in the rat cecum

The effects of voluntary wheel-running exercise on cecal microbiota and short-chain fatty acid production were investigated in rats. The microbiota composition was notably different between the exercised and sedentary rats. Furthermore, the exercised rats showed a significantly higher n-butyrate concentration than the sedentary rats. This alteration of the cecal microbial environment may contribute to the beneficial effect of exercise on gastrointestinal disorders.     

Mechanism of asymmetric ovarian development in chick embryos

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor alpha in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor alpha and cyclin D1.     

Power spectral analysis of heart rate variability for assessment of diurnal variation of autonomic nervous activity in guinea pigs.

We established characteristics of power spectral analysis of heart rate variability, and assessed the diurnal variations of autonomic nervous function in guinea pigs. For this purpose, an electrocardiogram (ECG) was recorded for 24 hr from conscious and unrestrained guinea pigs using a telemetry system. There were two major spectral components, at low frequency (LF) and high frequency (HF) bands, in the power spectrum of HR variability. On the basis of these data, we defined two frequency bands of interest: LF (0.07-0.7 Hz) and HF (0.7-3.0 Hz). The power of LF was higher than that of HF in the normal guinea pigs. Atropine significantly reduced power at HF. Propranolol also significantly reduced power at LF. Furthermore, the decrease in the parasympathetic mechanism produced by atropine was reflected in a slight increase in the LF/HF ratio. The LF/HF ratio appeared to follow the reductions of sympathetic activity produced by propranolol. Autonomic blockade studies indicated that the HF component reflected parasympathetic activity and the LF/HF ratio seemed to be a convenient index of autonomic balance. Nocturnal patterns, in which the values of heart rate in the dark phase (20:00-06:00) were higher than those in the light phase (06:00-20:00), were observed. However, the HF, LF and the LF/HF ratio showed no daily pattern. These results suggest that the autonomic nervous function in guinea pigs has no clear circadian rhythmicity. Therefore, this information may be useful for future studies concerning the autonomic nervous function in this species.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside