Spectroelectrochemical evaluation of redox potentials of cysteine tryptophylquinone and two hemes c in quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans has a novel cofactor cysteine tryptophylquinone (CTQ) in the smallest gamma subunit and two hemes c in the largest alpha subunit [Datta, S., Mori, Y., Takagi, K., Kawaguchi, K., Chen, Z., Okajima, T., Kuroda, S., Ikeda, T., Kano, K., Tanizawa, K., and Mathews, F. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14268-14273]. The spectral change of QH-AmDH was assigned to the redox reaction of the hemes c alone. The redox potentials of the two hemes c with His and Met as the second axial ligands, respectively, were determined to be 0.149 and 0.235 V versus SHE at pH 7.0 by a mediator-assisted continuous-flow column electrolytic spectroelectrochemistry (MCES). The monomeric gamma subunit of QH-AmDH was isolated from urea-treated QH-AmDH. The fully oxidized and reduced forms of the gamma subunit exhibited a unique absorption band centered at 380 nm and a shoulder band around 315 nm, respectively, at neutral pH. The two-electron redox potential of CTQ in the isolated gamma subunit was evaluated to be 65 mV at pH 7.0 by MCES. The redox reaction was linked to the two-proton transfer at pH <8.6 and to a single-proton transfer at pH >8.6. The pK(a) value (K(a) being the acid dissociation constant) of 8.6 was assigned to one of the phenolic OH groups of the quinol form. Upon deprotonation, the red shift of the shoulder band was observed. The gamma subunit adsorbed on a glassy carbon electrode, and gave a direct but quasi-reversible electrochemical signal. Intra- and interprotein electron transfers of QH-AmDH are discussed from thermodynamic and structural points of view.     

MAP2 is required for dendrite elongation,PKA anchoring in dendrites,and proper PKA signal transduction

Microtubule-associated protein 2 (MAP2) is a major component of cross-bridges between microtubules in dendrites, and is known to stabilize microtubules. MAP2 also has a binding domain for the regulatory subunit II of cAMP-dependent protein kinase (PKA). We found that there is reduction in microtubule density in dendrites and a reduction of dendritic length in MAP2-deficient mice. Moreover, there is a significant reduction of various subunits of PKA in dendrites and total amounts of various PKA subunits in hippocampal tissue and cultured neurons. In MAP2-deficient cultured neurons, the induction rate of phosphorylated CREB after forskolin stimulation was much lower than in wild-type neurons. Therefore, MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.     

Role of KIFC3 motor protein in Golgi positioning and integration

KIFC3, a microtubule (MT) minus end-directed kinesin superfamily protein, is expressed abundantly and is associated with the Golgi apparatus in adrenocortical cells. We report here that disruption of the kifC3 gene induced fragmentation of the Golgi apparatus when cholesterol was depleted. Analysis of the reassembly process of the Golgi apparatus revealed bidirectional movement of the Golgi fragments in both wild-type and kifC3-/- cells. However, we observed a markedly reduced inwardly directed motility of the Golgi fragments in cholesterol-depleted kifC3-/- cells compared with either cholesterol-depleted wild-type cells or cholesterol-replenished kifC3-/- cells. These results suggest that (a) under the cholesterol-depleted condition, reduced inwardly directed motility of the Golgi apparatus results in the observed Golgi scattering phenotype in kifC3-/- cells, and (b) cholesterol is necessary for the Golgi fragments to attain sufficient inwardly directed motility by MT minus end-directed motors other than KIFC3, such as dynein, in kifC3-/- cells. Furthermore, we showed that Golgi scattering was much more drastic in kifC3-/- cells than in wild-type cells to the exogenous dynamitin expression even in the presence of cholesterol. These results collectively demonstrate that KIFC3 plays a complementary role in Golgi positioning and integration with cytoplasmic dynein.     

Dietary Zn deficiency does not influence systemic blood pressure and vascular nitric oxide signaling in normotensive rats

Because zinc (Zn) is an important component for cell protection against certain oxygen species, it has been suggested that Zn deficiency impairs the potent oxidant defense capacity, which is constitutively provided in the vascular system. However, the influence of dietary Zn deficiency on systemic blood pressure and vascular system is controversial and unclear. We therefore examine the effect of dietary Zn deficiency on systemic blood pressure, a potent superoxide scavenger, aortic Cu/Zn superoxide dismutase (SOD) activity, a most representative synthase of the endothelium-derived relaxing factor, and aortic endothelial nitric oxide synthase (eNOS) expression. Furthermore, the direct effects of intravenous administration of NOS inhibitor, N ^ω-nitro-l-arginine methyl ester (l-NAME), and a SOD mimetic compound, tempol, in normotensives were tested in Wistar-Kyoto (WKY) rats. A Zn-deficient diet (4 wk) contributed to growth retardation, the decrease in thymus weight, and the lower levels of serum Zn compared with the standard diet group. However, no significant difference in conscious systolic and diastolic blood pressure was found in the Zn-deficiency group. The administration of l-NAME caused an increase in the mean arterial pressure (MAP) levels in the two groups of rats and the involvement of the vasodilator nitric oxide (NO) in the regulation of systemic BP in the normotensive state. On the other hand, administration of the superoxide scavenger, tempol, led to a decrease in MAP levels in the two groups of rats, indicating the participation of the oxygen free radical, superoxide, in the maintenance of the systemic BP in a normotensive state. There were no significant differences between the Zn-deficient diet group and the standard diet group in the normotensive state. eNOS expression and Cu/Zn SOD activity in the aorta were also intact in Zn-deficient normotensive rats. These findings suggest that the 4 wk of Zn deficiency was inadequate to alter systemic blood pressure and focal NO signaling in the normotensive state. Long-term Zn deficiency affects the neuronal, immune, and hematopoietic systems, which contribute to systemic and/or local circulation. However, Zn deficiency alone does not cause hypertension and local vascular dysfunction in the normotensive state.     

Role of SODD in regulation of tumor necrosis factor responses

Signaling from tumor necrosis factor receptor type 1 (TNFR1) can elicit potent inflammatory and cytotoxic responses that need to be properly regulated. It was suggested that the silencer of death domains (SODD) protein constitutively associates intracellularly with TNFR1 and inhibits the recruitment of cytoplasmic signaling proteins to TNFR1 to prevent spontaneous aggregation of the cytoplasmic death domains of TNFR1 molecules that are juxtaposed in the absence of ligand stimulation. In this study, we demonstrate that mice lacking SODD produce larger amounts of cytokines in response to in vivo TNF challenge. SODD-deficient macrophages and embryonic fibroblasts also show altered responses to TNF. TNF-induced activation of NF-kappaB is accelerated in SODD-deficient cells, but TNF-induced c-Jun N-terminal kinase activity is slightly repressed. Interestingly, the apoptotic arm of TNF signaling is not hyperresponsive in the SODD-deficient cells. Together, these results suggest that SODD is critical for the regulation of TNF signaling.     

Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.     

Induction of Cross-Reactivity in an Endogenous Viral Peptide Non-Reactive to FBL-3 Tumor-Specific Helper T-Cell Clones

We previously reported a helper T-cell (Th) epitope (peptide i) which corresponded to the sequence ranging from positions 462 to 479 from the N-terminus of the Friend-murine leukemia virus (F-MuLV) envelope protein (env_462-479). Homologous sequences exist in both Moloney-murine leukemia (M-MuLV env_452-469) and endogenous AKV (AKV env_453-470) viruses, which differ from F-MuLV env_462-479 in 5 and 7 amino acids, respectively. However, peptide i-specific Th clones did not respond to either of the corresponding exogenous or endogenous peptides. One amino acid substitution in M-MuLV env_452-469 (Asn to Tyr at position 465: N465Y) and three amino acids in AKV env_453-470 (H460S, A466Y and Y468H) endowed both peptides with the reactivity to one of the Th clones, F5-5, almost to the same degree as peptide i. However, the other Th clones responded differently to each of the modified endogenous peptides substituted by one to three amino acids. The cells responsive to the cross-reactive peptides occupied only a minor portion, if any, of the bulk cultured lymph node cells from peptide i-immune mice, and in particular, no significant response to the modified endogenous peptides was observed in repeated experiments. The exchange of at least 3 residues was necessary for the endogenous peptide to acquire sufficient cross-reactivity to two of the three Th clones. However, it was noticeable that a single substitution of alanine by tyrosine at the dominant T-cell receptor (TCR) contact position of the peptide i_e generated a weak but significant cross-reactivity to one of the three Th clones in this study. Thus, peptides of endogenous retroviral origin that would be modified by mutational events might become ‘non-self’ and prime Th cells leading to auto-antibody production and resulting in autoimmune disease.     

Effects of Aging and Idiopathic Parkinson’s Disease on Tactile Temporal Order Judgment

It is generally accepted that the basal ganglia play an important role in interval timing that requires the measurement of temporal durations. By contrast, it remains controversial whether the basal ganglia play an essential role in temporal order judgment (TOJ) of successive stimuli, a behavior that does not necessarily require the measurement of durations in time. To address this issue, we compared the effects of idiopathic Parkinson’s disease (PD) on the TOJ of two successive taps delivered to each hand, with the arms uncrossed in one condition and crossed in another. In addition to age-matched elderly participants without PD (non-PD), we examined young healthy participants so that the effect of aging could serve as a control for evaluating the effects of PD. There was no significant difference between PD and non-PD participants in any parameter of TOJ under either arm posture, although reaction time was significantly longer in PD compared with non-PD participants. By contrast, the effect of aging was apparent in both conditions. With their arms uncrossed, the temporal resolution (the interstimulus interval that yielded 84% correct responses) in elderly participants was significantly worse compared with young participants. With their arms crossed, elderly participants made more errors at longer intervals (~1 s) than young participants, although both age groups showed similar judgment reversal at moderately short intervals (~200 ms). These results indicate that the basal ganglia and dopaminergic systems do not play essential roles in tactile TOJ involving both hands and that the effect of aging on TOJ is mostly independent of the dopaminergic systems.     

Identification of 4-Trimethylaminobutyraldehyde Dehydrogenase (TMABA-DH) as a Candidate Serum Autoantibody Target for Kawasaki Disease

Kawasaki disease (KD), an acute vasculitis that preferentially affects coronary arteries, is still the leading cause of acquired heart disease in children. Although the involvement of immune system malfunction in the onset of KD is suggested, its etiology still remains to be clarified. We investigated autoantibodies in KD patients, which are frequently found in sera from patients with autoimmune diseases, vasculitides and arteritides. We performed two-dimensional western blotting and LC-MS/MS to analyze the antigens of autoantibodies, detected two protein spots with 4 out of 24 sera from KD patients but not with 6 control sera, and identified the antigens as 4-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH). A slot blot analysis with TMABA-DH as an antigen also revealed higher reactivities of patients'' sera than control sera (positive rates: 18/43 vs 3/41). Using an enzyme-linked immunosorbent assay (ELISA), we found that the reactivity of anti-TMABA-DH antibodies in sera from KD patients was significantly higher than that in sera from age-matched controls. The optimal cut-off value of 0.043 had a sensitivity of 83.7% and a specificity of 80.0% in detecting KD patients (positive rates: 37/43 for KD patients, 9/41 for controls). Immunohistochemistry performed on thin sections of rat heart revealed that TMABA-DH colocalized with myosin light chains in cardiac myocytes. Patient sera with high reactivity gave similar immunostaining pattern. These results suggest that the detection of anti-TMABA-DH autoantibody could be a potential strategy for a diagnosis of KD.