Acutely administered melatonin restores hepatic mitochondrial physiology in old mice

Damage to mitochondria as a result of the intrinsic generation of free radicals is theoretically involved in the processes of cellular aging. Herein, we investigated whether acutely administered melatonin, due to its free radical scavenging activity, would influence mitochondrial metabolism. Mitochondrial respiratory activity and respiratory chain complex I and IV activities in liver mitochondria from a strain of senescence-accelerated-prone mice (SAMP8) and a strain of senescence-accelerated-resistant mice (SAMR1) were measured when the animals were 12 months of age. Respiratory control index (RCI), ADP/O ratio, State 3 respiration and dinitrophenol (DNP)-dependent uncoupled respiration were significantly lower in SAMP8 than in SAMR1. In contrast, State 4 respiration was significantly higher in SAMP8 than in SAMR1. Activities of complexes I and IV in SAMP8 were significantly lower than in SAMR1. Melatonin administration (10mg/kg body weight, intraperitoneally) 1h prior to sacrifice significantly increased RCI, ADP/O ratio, State 3 respiration and DNP-induced uncoupled respiration in SAMP8 while also significantly reducing State 4 respiration in SAMP8. The injection of melatonin also significantly increased complex I activity in both mouse strains and complex IV activity in the liver of SAMP8 mice. These results document an age-related decrease in hepatic mitochondrial function in SAM which can be modified by an acute pharmacological injection of melatonin; the indole stimulated mitochondrial respiratory chain activity which would likely reduce deteriorative oxidative changes in mitochondria that normally occur in advanced age.     

CD8+ T cell-mediated airway hyperresponsiveness and inflammation is dependent on CD4+IL-4+ T cells

CD4+ T cells, particularly Th2 cells, play a pivotal role in allergic airway inflammation. However, the requirements for interactions between CD4+ and CD8+ T cells in airway allergic inflammation have not been delineated. Sensitized and challenged OT-1 mice in which CD8+ T cells expressing the transgene for the OVA(257-264) peptide (SIINFEKL) failed to develop airway hyperresponsiveness (AHR), airway eosinophilia, Th2 cytokine elevation, or goblet cell metaplasia. OT-1 mice that received naive CD4+IL-4+ T cells but not CD4+IL-4- T cells before sensitization developed all of these responses to the same degree as wild-type mice. Moreover, recipients of CD4+IL-4+ T cells developed significant increases in the number of CD8+IL-13+ T cells in the lung, whereas sensitized OT-1 mice that received primed CD4+ T cells just before challenge failed to develop these responses. Sensitized CD8-deficient mice that received CD8+ T cells from OT-1 mice that received naive CD4+ T cells before sensitization increased AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged with allergen. In contrast, sensitized CD8-deficient mice receiving CD8+ T cells from OT-1 mice without CD4+ T cells developed reduced AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged. These data suggest that interactions between CD4+ and CD8+ T cells, in part through IL-4 during the sensitization phase, are essential to the development of CD8+IL-13+ T cell-dependent AHR and airway allergic inflammation.     

The Importance of cGMP Signaling in Sensory Cilia for Body Size Regulation in Caenorhabditis elegans

The body size of Caenorhabditis elegans is thought to be controlled by sensory inputs because many mutants with sensory cilium structure defects exhibit small body size. The EGL-4 cGMP-dependent protein kinase acts in sensory neurons to reduce body size when animals fail to perceive sensory signals. In addition to body size control, EGL-4 regulates various other behavioral and developmental pathways, including those involved in the regulation of egg laying and chemotaxis behavior. Here we have identified gcy-12, which encodes a receptor-type guanylyl cyclase, as a gene involved in the sensory regulation of body size. Analyses with GFP fusion constructs showed that gcy-12 is expressed in several sensory neurons and localizes to sensory cilia. Genetic analyses indicated that GCY-12 acts upstream of EGL-4 in body size control but does not affect other EGL-4 functions. Our studies indicate that the function of the GCY-12 guanylyl cyclase is to provide cGMP to the EGL-4 cGMP-dependent kinase only for limited tasks including body size regulation. We also found that the PDE-2 cyclic nucleotide phosphodiesterase negatively regulates EGL-4 in controlling body size. Thus, the cGMP level is precisely controlled by GCY-12 and PDE-2 to determine body size through EGL-4, and the defects in the sensory cilium structure may disturb the balanced control of the cGMP level. The large number of guanylyl cyclases encoded in the C. elegans genome suggests that EGL-4 exerts pleiotropic effects by partnering with different guanylyl cyclases for different downstream functions.     

A method for Pmo25-associated kinase assay in fission yeast: the activity is dependent on two gC kinases Nak1 and Sid1

In fission yeast, the conserved proteins, MO25/Pmo25, GC kinase/Nak1, Furry/Mor2, NDR kinase/Orb6, and Mob2, constitute the morphogenesis Orb6 network (MOR). Previously we showed that Pmo25 functions as an upstream component of MOR and that it plays a connecting role between the septation initiation network (SIN) and MOR. Here we establish a Pmo25-associated kinase assay and show that the activity is dependent on Nak1/MOR and Sid1/SIN.     

Cellular distribution of Mr 25,000 protein, a protein partially overlapping phosvitin and lipovitellin 2 in vitellogenin B1, and yolk proteins in Xenopus laevis oocytes and embryos

A phosphorylated protein with molecular mass of 25,000 (pp25) can be derived from Xenopus laevis vitellogenin B1. In order to clarify the distribution of pp25, the changes in the concentration and localization of this protein in oocytes and embryos were examined by immunoblotting and immunohistochemistry using anti-pp25 antibodies, and compared with those of yolk proteins. In oocytes, pp25 was shown to localize characteristically at the surface just below the plasma membrane by immunohistochemical analysis. Interestingly, during embryogenesis, immunocytochemical staining revealed a transition of the pp25 distribution from beneath the outer surface of each germ layers to endoderm during tailbudding. In contrast, yolk proteins were localized in endoderm constantly throughout the developmental stages. However, the level of pp25 in the cytoplasm gradually decreased following the growth of embryos at the tailbud stage and disappeared at the tadpole stage, as shown by immunoblot analysis. These results suggest that pp25 could play different roles from those of yolk proteins such as lipovitellin and phosvitin in X. laevis oocytes and developing embryos.     

Adrenomedullin enhances therapeutic potency of bone marrow transplantation for myocardial infarction in rats

Adrenomedullin (AM), a potent vasodilator, induces angiogenesis and inhibits cell apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. Transplantation of bone marrow-derived mononuclear cells (MNC) induces angiogenesis. We investigated whether infusion of AM enhances the therapeutic potency of MNC transplantation in a rat model of myocardial infarction. Immediately after coronary ligation, bone marrow-derived MNC (5 x 10(6) cells) were injected into the ischemic myocardium, followed by subcutaneous administration of 0.05 microg x kg(-1) x min(-1) AM (AM-MNC group) or saline (MNC group) for 3 days. Another two groups of rats received subcutaneous administration of AM alone (AM group) or saline (control group). Hemodynamic and histological analyses were performed 4 wk after treatment. Cardiac infarct size was significantly smaller in the MNC and AM groups than in the control group. A combination of AM infusion and MNC transplantation demonstrated a further decrease in infarct size. Left ventricular (LV) maximum change in pressure over time and LV fractional shortening were significantly improved only in the AM-MNC group. AM significantly increased capillary density in ischemic myocardium, suggesting the angiogenic potency of AM. AM infusion plus MNC transplantation demonstrated a further increase in capillary density compared with AM or MNC alone. Although MNC apoptosis was frequently observed 72 h after transplantation, AM markedly decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells among the transplanted MNC. In conclusion, AM enhanced the angiogenic potency of MNC transplantation and improved cardiac function in rats with myocardial infarction. This beneficial effect may be mediated partly by the angiogenic property of AM itself and by its antiapoptotic effect on MNC.     

Effects of divalent cations on thermophilic inorganic pyrophosphatase.

Divalent cations were shown to affect the structure and thermostability of thermophilic inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] purified from Bacillus stearothermophilus and thermophilic bacterium PS-3. The properties of the enzymes from the two sources were found to be very similar. The enzymes were very unstable to heart in the absence of divalent cations, being inactivated gradually even at 40 degrees C. However, they became stable to heat denaturation in the presence of Mg2+, between pH 7.8 and 9.0. Similar induced thermostability was detected when Mn2+, Co2+, Ca2+, Cd2+, and ZN2+ were added, though the latter three cations were not essential for enzyme activity. On adding divalent cations, the optical properties such as absorption spectra, fluorescence spectra, and circular dichroism (CD) were changed. Gel filtration and disc electrophoresis revealed that the molecular weight of both enzymes was 5.4 x 10(4) in Tris-SO4 buffer and 11 x 10(4) in Tris-HCL buffer, suggesting monomer-dimer transformation. In the presence of divalent cations in Tris-SO4 fuffer, the enzymes dimerized; this was confirmed by sedimentation velocity measurements. The enzymes in Tris-HCL buffer did not show thermostability unless divalent cations were added. The results in the present study indicate that binding of divalent cations to each enzyme caused some conformational change in the vicinity of aromatic amino acid residues leading to dimerization of the enzyme molecule so that it became thermostable. It was also suggested that histidyl residues play an important role in the thermostability induced by divalent cations on the basis of the pH dependencies of thermostability and CD spectra.     

Ssh AI restriction endonuclease from Salmonella shikmonah

A new type II restriction endonuclease, SshAI, was purified from Salmonella shikmonah TK139 of kangaroo origin. The recognition and cleavage specificity of Ssh AI was determined to be 5'-CC/TNAGG-3', identical to that of SauI from Streptomyces aureofaciens and Bsu36I from Bacillus subtilis. Based on closely related and in part overlapping recognition specificities of Ssh AI and other restriction endonucleases, a close evolutionary relationship is proposed for all known Salmonella restriction endonucleases.     

A hairpin DNA aptamer coupled with groove binders as a smart switch for a field-effect transistor biosensor

We report here that a hairpin-structured DNA that possesses an anti-ATP aptamer sequence successfully detected target ATP or adenosine in a temperature-dependent manner by nanoscale intramolecular displacement on the surface of a gold electrode as an extended gate of a field-effect transistor (FET). The structural switching of the hairpin aptamer from closed loop to open-loop conformations was accompanied by the release of the preloaded DNA binder (DAPI) from the stem part of the hairpin aptamer into the solution phase. The loss of intrinsic positive charges of DAPI (2+) from the diffusion layer at the gate/solution nano-interface as a result of target capturing was responsible for generating a specific signal by the field-effect. We emphasize a new aspect of the structured DNA aptamer in combination with FET: the DAPI-loaded hairpin aptamer successfully detected even uncharged adenosine, which remains a major challenge for FET-based biosensors. Given the simplicity in design of the primary and secondary structures of oligonucleotide aptamers, it is easy to apply this technology to a wide variety of bio-analytes, irrespective of their electric charges. In view of these advantages, our findings may offer a new trend in the design of stimuli-responsive "smart" biomolecular switches for semiconductor-based biosensors.