Hydroxymethylglutaryl--CoA reductase inhibitor inhibits induction of nitric oxide synthase in 3T3--L1 preadipocytes

Preadipocytes are considered to play a role in adipose tissue inflammation in obesity. The purpose of this study was to determine whether hydroxymethylglutaryl-CoA reductase inhibitor (statin) modulates the nitric oxide (NO) production via inducible NO synthase (iNOS) in preadipocytes. Undifferentiated 3T3-L1 cells, a model of preadipocytes, significantly produced NO by the treatment with the combination of lipopolysaccharide (L), tumor necrosis factor-alpha (T) and interferon-gamma (I). Pre-incubation with simvastatin, a lipophilic statin, or pravastatin, a hydrophilic one, dose-dependently inhibited the NO production in the LTI-treated cells. The effect of simvastatin was offset by mevalonate or geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS paralleled the NO production. The nuclear factor-kappaB (NF-kappaB) was activated by the LTI-treatment, and was inhibited by addition of simvastatin or pravastatin. Mevalonate or GGPP completely offset the effect of simvastatin. Simvastatin or pravastatin also decreased the LTI-stimulated interleukin-6 (IL-6) secretion. These effects of pravastatin were relatively weak compared with those of simvastatin. Y27632, an inhibitor of Rho kinase, also inhibited the LTI-induced NF-kappaB activation and iNOS expression, and decreased the production of NO and IL-6 in 3T3-L1 preadipocytes. These results suggest that statins, especially lipophilic types, inhibit induction of iNOS by inhibiting the small GTP-binding protein signal in preadipocytes.     

Pre‐initiation complex assembly functions as a molecular switch that splits the Mcm2‐7 double hexamer

Initiation of chromosomal DNA replication in eukaryotes involves two steps: licensing and firing. In licensing, a core component of the replicative helicase, the Mcm2–7 complex, is loaded onto replication origins as an inactive double hexamer, which is activated in the firing step by firing factors. A reaction intermediate called the pre‐initiation complex (pre‐IC) has been proposed to assemble transiently during firing, but the existence of the pre‐IC has not yet been confirmed. Here, we show, by systematic chromatin immunoprecipitation, that a distinct intermediate that fits the definition of the pre‐IC assembles during firing in the budding yeast Saccharomyces cerevisiae. Pre‐IC assembly is observed in the absence of Mcm10, one of the firing factors, and is mutually dependent on all the firing factors whose association to replication origins is triggered by cyclin‐dependent kinase. In the pre‐IC, the Mcm2–7 double hexamer is separated into single hexamers, as in the active helicase. Our data indicate that pre‐IC assembly functions as an all‐or‐nothing molecular switch that splits the Mcm2–7 double hexamer.     

Involvement of the visual change detection process in facilitating perceptual alternation in the bistable image

A bistable image induces one of two perceptual alternatives. When the bistable visual image is continuously viewed, the percept of the image alternates from one possible percept to the other. Perceptual alternation was previously reported to be induced by an exogenous perturbation in the bistable image, and this perturbation was theoretically interpreted to cause neural noise, prompting a transition between two stable perceptual states. However, little is known experimentally about the visual processing of exogenously driven perceptual alternation. Based on the findings of a previous behavioral study (Urakawa et al. in Perception 45:474–482, 2016), the present study hypothesized that the automatic visual change detection process, which is relevant to the detection of a visual change in a sequence of visual events, has an enhancing effect on the induction of perceptual alternation, similar to neural noise. In order to clarify this issue, we developed a novel experimental paradigm in which visual mismatch negativity (vMMN), an electroencephalographic brain response that reflects visual change detection, was evoked while participants continuously viewed the bistable image. In terms of inter-individual differences in neural and behavioral data, we found that enhancements in the peak amplitude of vMMN1, early vMMN at a latency of approximately 150 ms, correlated with increases in the proportion of perceptual alternation across participants. Our results indicate the involvement of automatic visual change detection in the induction of perceptual alternation, similar to neural noise, thereby providing a deeper insight into the neural mechanisms underlying exogenously driven perceptual alternation in the bistable image.     

Binding of a highly de-N-acetylated chitosan to Japanese pheasan lysozyme as measured by 1H-NMR spectroscopy

Binding of a highly de-N-acetylated chitosan to Japanese pheasant lysozyme (JPL), which differs from hen egg white lysozyme (HEWL) by nine amino acid substitutions (including Arg114-->His), was investigated by 1H-NMR spectroscopy. The profile of the one-dimensional spectrum of JPL is essentially identical to that of HEWL. Using two-dimensional spectra of JPL and HEWL, several aromatic and aliphatic proton resonances of JPL were assigned by comparison. When a highly de-N-acetylated chitosan (number-average degree of polymerization, about 18; degree of acetylation, 0.04), where the N-acetylated units are predominantly surrounded by de-N-acetylated units (a monoacetylated chitosan), was added to the JPL solution, the NMR signals were clearly affected in Trp28 C5H and Ile98 gammaCH, as in the case of binding to HEWL. The dissociation constant of the monoacetylated chitosan evaluated from the NMR signal responses was calculated to be 0.23+/-0.05 mm (-31.5 kJ/mol), which is similar to that of HEWL (0.11+/-0.02 mm, -33.3 kJ/mol). Thus, the Arg-->His substitution of the 114th amino acid, which participates in sugar residue binding at the right-sided subsite F, did not significantly affect the chitosan binding. In addition, the C2H signal of His114 of JPL was not affected by the chitosan binding. These results suggest that the monoacetylated chitosan binds to subsites E and F through the left-sided binding mode.     

Inductive signal and tissue responsiveness defining the tectum and the cerebellum

The mes/metencephalic boundary (isthmus) has an organizing activity for mesencephalon and metencephalon. The candidate signaling molecule is Fgf8 whose mRNA is localized in the region where the cerebellum differentiates. Responding to this signal, the cerebellum differentiates in the metencephalon and the tectum differentiates in the mesencephalon. Based on the assumption that strong Fgf8 signal induces the cerebellum and that the Fgf8b signal is stronger than that of Fgf8a, we carried out experiments to misexpress Fgf8b and Fgf8a in chick embryos. Fgf8a did not affect the expression pattern of Otx2, Gbx2 or Irx2. En2 expression was upregulated in the mesencephalon and in the diencephalon by Fgf8a. Consequently, Fgf8a misexpression resulted in the transformation of the presumptive diencephalon to the fate of the mesencephalon. In contrast, Fgf8b repressed Otx2 expression, but upregulated Gbx2 and Irx2 expression in the mesencephalon. As a result, Fgf8b completely changed the fate of the mesencephalic alar plate to cerebellum. Quantitative analysis showed that Fgf8b signal is 100 times stronger than Fgf8a signal. Co-transfection of Fgf8b with Otx2 indicates that Otx2 is a key molecule in mesencephalic generation. We have shown by RT-PCR that both Fgf8a and Fgf8b are expressed, Fgf8b expression prevailing in the isthmic region. The results all support our working hypothesis that the strong Fgf8 signal induces the neural tissue around the isthmus to differentiate into the cerebellum.     

Expression of an ethylene-forming enzyme from Pseudomonas syringae under the control of alcohol dehydrogenase gene promoter in tobacco roots

Several lines of transgenic tobacco that expressed an ethylene-forming enzyme from Pseudomonas syringae fused with -glucuronidase as a histochemical marker under the control of tobacco alcohol dehydrogenase gene (NtADH) promoter were constructed. The NtADH promoter was previously shown to be active in late growth stage when expressed in BY2 cultured tobacco cells (Nicotiana tabacum). Ethylene production and expression of the marker gene in transgenic tobacco took place only in roots, and the root-limited expression was explicable by induction of NtADH promoter under anaerobic condition.     

Visual demonstration of calcium accumulation in human arteries of upper and lower limbs

To elucidate the calcium content of the arteries in the upper and lower limbs, the authors determined the calcium content of all the arteries in the upper and lower limbs continuously by microwave-induced plasma-atomic emission spectrometry. The subjects were an 87-yr-old man and a 72-yr-old woman. The calcium content was determined both in the arteries of the upper limbs continuously, such as the subclavian arteries and its distal arteries, and in the arteries of the lower limbs, such as the common iliac arteries and its distal arteries. The common finding that the higher accumulation of calcium occurred in the arteries of the lower limbs in comparison to the arteries of the upper limbs and extremely high accumulation of calcium occurred in the common, external, and internal iliac arteries was obtained in the two subjects. The calcium content of the arteries in the upper and lower limbs was visually demonstrated.     

Total Ca2+ handling for E-C coupling in the whole heart: an integrative analysis

We assessed total Ca2+ handling (transport, flux) in excitation-contraction (E-C) coupling in a beating left ventricle (LV). We developed a new integrative analysis method that utilizes the internal Ca2+ recirculation fraction (RF), O2 consumption (V(O2)) for Ca2+ handling, and O2 cost of Emax (contractility index) of the LV. We obtained the RF from the beat constant of the exponential decay component of the postextrasystolic potentiation, and the O2 cost of Emax from V(O2) measured at different Emax. Our equation calculated the unknown total Ca2+ handling, futile Ca2+ cycling, and Ca2+ reactivity of Emax from the RF and Ca2+ handling V(O2). The calculated total Ca2+ handling fell between 30 and 110 micromol/kg, depending on Emax and pathological conditions. Our method also allowed an assessment of futile Ca2+ cycling and Ca2+ reactivity of Emax in a beating LV. These data are not available using conventional methods. Our method can be used to better understand the pathophysiology of total Ca2+ handling in a beating heart.     

Identification, cloning, and initial characterization of a novel mouse testicular germ cell-specific antigen

A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.