Alterations in vascular endothelial function in the aorta and mesenteric artery in type II diabetic rats

We used the partial protection exerted by suitable dosages of nicotinamide against the beta-cytotoxic effect of streptozotocin (STZ) to create an experimental diabetic syndrome in adult rats that appears closer to type II diabetes mellitus than other available animal models. The dosage of 230 mg/kg of nicotinamide given intraperitoneally 15 min before STZ administration (65 mg/kg i.v.) yielded animals with hyperglycemia (187.8 +/- 17.8 vs. 103.8 +/- 2.8 mg/dL in controls; P < 0.001) and preservation of plasma insulin levels. This study assessed the relationship between endothelial dysfunction and agonist-induced contractile responses in such rats. In the thoracic aorta, the acetylcholine (ACh) induced relaxation was significantly reduced and the noradrenaline (NA) induced contractile response was significantly increased in diabetic rats compared with age-matched control rats. In the superior mesenteric artery, the ACh-induced relaxation was similar in magnitude between diabetic and age-matched control rats; however, the ACh-induced endothelium-derived hyperpolarizing factor (EDHF) type relaxation was significantly weaker in diabetic rats than in the controls. The phenylephrine (PE) induced contractile response was not different between the two groups. The plasma concentration of NOx (NO2- + NO3-) was significantly lower in diabetic rats than in control rats. We conclude that vasomotor activities in conduit arteries are impaired in this type II diabetes model.     

Fluorescence resonance energy transfer between points on actin and the C-terminal region of tropomyosin in skeletal muscle thin filaments

Fluorescence resonance energy transfer between points on tropomyosin (positions 87 and 190) and actin (Gln-41, Lys-61, Cys-374, and the ATP-binding site) showed no positional change of tropomyosin relative to actin on the thin filament in response to changes in Ca2+ concentration (Miki et al. (1998) J. Biochem. 123, 1104-1111). This is consistent with recent electron cryo-microscopy analysis, which showed that the C-terminal one-third of tropomyosin shifted significantly towards the outer domain of actin, while the N-terminal half of tropomyosin shifted only a little (Narita et al. (2001) J. Mol. Biol. 308, 241-261). In order to detect any significant positional change of the C-terminal region of tropomyosin relative to actin, we generated mutant tropomyosin molecules with a unique cysteine residue at position 237, 245, 247, or 252 in the C-terminal region. The energy donor probe was attached to these positions on tropomyosin and the acceptor probe was attached to Cys-374 or Gln-41 of actin. These probe-labeled mutant tropomyosin molecules retain the ability to regulate the acto-S1 ATPase activity in conjunction with troponin and Ca2+. Fluorescence resonance energy transfer between these points of tropomyosin and actin showed a high transfer efficiency, which should be very sensitive to changes in distance between probes attached to actin and tropomyosin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that the C-terminal region of tropomyosin did not shift significantly relative to actin on the reconstituted thin filament in response to the change of Ca2+ concentration.     

Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP-ribosyltransferase from the cabbage butterfly

The cabbage butterfly, Pieris rapae, produces an ADP-ribosylating cytotoxic protein, pierisin-1. Unlike other ADP-ribosylating toxins, the acceptor site for ADP-ribosylation by pierisin-1 is the N-2 position of guanine bases in DNA. The present study was designed to characterize this novel guanine-specific ADP-ribosyltransferase, pierisin-1. The N-terminal polypeptide from Met-1 to Arg-233, but not the C-terminal Ser-234-Met-850 polypeptide, was found to exhibit guanine ADP-ribosyltransferase activity. Trypsin-treated pierisin-1, which is considered to be a "nicked" full-length form composed of associated N- and C-terminal fragments, also demonstrated such activity. Optimum conditions for the N-terminal polypeptide of pierisin-1 were pH 8-10, 37-40 degrees C, in the presence of 100-200 mM NaCl or KCl. Other metal ions such as Ca(2+) or Mg(2+) were not required. Kinetic studies demonstrated potent ADP-ribosyltransferase activity with a K(M) value for NAD of 0.17 mM and k(cat) of 55 per second. Under these optimum conditions, the specific activity of trypsin-treated pierisin-1 was about half (k(cat) = 25 per second). When the conditions were changed to pH 5-7 or 10-20 degrees C, some activity (6-55% or 5-20%, respectively, of that under optimal conditions) of the N-terminal polypeptide was still evident; however, almost all of the trypsin-treated enzyme activity disappeared. This implies the inhibition of the N-terminal enzyme domain by the associated C-terminal fragment. Long-term reactions indicated that a single molecule of pierisin-1 has the capacity to generate more than 10(6) ADP-ribosylated DNA adducts, which could cause the death of a mammalian cell.     

Identification of a factor Xa-interactive site within residues 337-372 of the factor VIII heavy chain

We recently demonstrated that the residues 337-372, comprising the acidic C-terminal region in A1 subunit, interact with factor Xa during the proteolytic inactivation of factor VIIIa (Nogami, K., Wakabayashi, H., and Fay, P. J. (2003) J. Biol. Chem. 278, 16502-16509). We now show this sequence is important for factor Xa-catalyzed activation of factor VIII. Peptide 337-372 markedly inhibited cofactor activation, consistent with a delay in the rate of cleavage at the A1-A2 junction. Studies using the isolated factor VIII heavy chain indicated that the peptide completely blocked cleavage at the A1-A2 junction (IC50 = 11 microm) and partially blocked cleavage at the A2-B junction (IC50 = 100 microm). Covalent cross-linking was observed between the 337-372 peptide and factor Xa following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the peptide quenched the fluorescence of dansyl-Glu-Gly-Arg active site-modified factor Xa, suggesting that residues 337-372 directly interact with factor Xa. Studies using a monoclonal antibody recognizing residues 351-365 as well as the peptide to this sequence further restricted the interactive region. Mutant factor VIII molecules in which clustered acidic residues in the 337-372 segment were converted to alanine were evaluated for activation by factor Xa. Of the mutants tested, only factor Xa-catalyzed activation of the D361A/D362A/D363A mutant was inhibited with peak activity of approximately 50% and an activation rate constant of approximately 30% of the wild type values. These results indicate that the 337-372 acidic region separating A1 and A2 domains and, in particular, a cluster of acidic residues at position 361-363 contribute to a unique factor Xa-interactive site within the factor VIII heavy chain that promotes factor Xa docking during cofactor activation.     

Mono(ADP-ribosyl)ation of the N2 amino groups of guanine residues in DNA by pierisin-2, from the cabbage butterfly, Pieris brassicae

Pierisin-2 is a cytotoxic and apoptosis-inducing protein present in Pieris brassicae with a 91% homology in the deduced amino acid sequences to pierisin-1 from Pieris rapae. We earlier showed pierisin-1 to catalyze mono(ADP-ribosyl)ation of 2'-deoxyguanosine (dG) in DNA to form N2-(ADP-ribos-1-yl)-2'-deoxyguanosine, this DNA modification appearing linked to its cytotoxicity and ability to induce apoptosis in mammalian cell lines. In this paper, we documented evidence that pierisin-2 also catalyzed ADP-ribosylation of dG in DNA to give the same reaction product as demonstrated for pierisin-1, with similar efficiency. With oligonucleotides as substrates, ADP-ribosylation by pierisin-2 was suggested to occur by one-side attack of the carbon atom at 1 position of the ribose moiety in NAD toward N2 of dG. The presence of a unique ADP-ribosylation toxin targeting dG in DNA in two distinct species in a Pieris genus could be a quite important finding to better understand biological functions of pierisin-1 and -2 in Pieris butterflies and the generic evolution of these cabbage butterflies.     

Methyl-beta-cyclodextrin induces mitochondrial cholesterol depletion and alters the mitochondrial structure and bioenergetics

There is growing evidence of mitochondrial membrane raft-like microdomains that are involved in the apoptotic pathway. The aim of this study was to investigate the effect of methyl-beta-cyclodextrin (MβCD), being a well-known lipid microdomain disrupting agent and cholesterol chelator, on the structure and bioenergetics of rat liver mitochondria (RLM). We observed that MβCD decreases the function of RLM, induces changes in the mitochondrial configuration state and decreases the calcium chloride-induced swelling. These data suggest that disruption of mitochondrial raft-like microdomains by cholesterol efflux on one hand impairs mitochondrial bioenergetics, but on the other hand it protects the mitochondria from swelling.     

Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons

A molecular basis of survival from neuronal injury is essential for the development of therapeutic strategy to remedy neurodegenerative disorders. In this study, we demonstrate that an EF-hand Ca2+-binding protein neuronal Ca2+ sensor-1 (NCS-1), one of the key proteins for various neuronal functions, also acts as an important survival factor. Overexpression of NCS-1 rendered cultured neurons more tolerant to cell death caused by several kinds of stressors, whereas the dominant-negative mutant (E120Q) accelerated it. In addition, NCS-1 proteins increased upon treatment with glial cell line-derived neurotrophic factor (GDNF) and mediated GDNF survival signal in an Akt (but not MAPK)-dependent manner. Furthermore, NCS-1 is significantly up-regulated in response to axotomy-induced injury in the dorsal motor nucleus of the vagus neurons of adult rats in vivo, and adenoviral overexpression of E120Q resulted in a significant loss of surviving neurons, suggesting that NCS-1 is involved in an antiapoptotic mechanism in adult motor neurons. We propose that NCS-1 is a novel survival-promoting factor up-regulated in injured neurons that mediates the GDNF survival signal via the phosphatidylinositol 3-kinase-Akt pathway.     

A protein associated with toll-like receptor 4 (PRAT4A) regulates cell surface expression of TLR4

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.     

Identification and characterization of GSRP-56, a novel Golgi-localized spectrin repeat-containing protein

Spectrin repeat (SR)-containing proteins are important for regulation of integrity of biomembranes, not only the plasma membrane but also those of intracellular organelles, such as the Golgi, nucleus, endo/lysosomes, and synaptic vesicles. We identified a novel SR-containing protein, named GSRP-56 (Golgi-localized SR-containing protein-56), by a yeast two-hybrid method, using a member of the transient receptor potential channel family, TRPV2, as bait. GSRP-56 is an isoform derived from a giant SR-containing protein, Syne-1 (synaptic nuclear envelope protein-1, also referred to as Nesprin-1 or Enaptin), predicted to be produced by alternative splicing. Immunological analysis demonstrated that this isoform is a 56-kDa protein, which is localized predominantly in the Golgi apparatus in cardiomyocytes and C2C12 myoblasts/myotubes, and we found that two SR domains were required both for Golgi targeting and for interaction with TRPV2. Interestingly, overexpression of GSRP-56 resulted in a morphological change in the Golgi structure, characterized by its enlargement of cis-Golgi marker antibody-staining area, which would result partly from fragmentation of Golgi membranes. Our findings indicate that GSRP-56 is a novel, particularly small Golgi-localized member of the spectrin family, which possibly play a role in maintenance of the Golgi structure.     

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Mitochondria divide and fuse continuously, and the balance between these two processes regulates mitochondrial shape. Alterations in mitochondrial dynamics are associated with neurodegenerative diseases. Here we investigate the physiological and cellular functions of mitochondrial division in postmitotic neurons using in vivo and in vitro gene knockout for the mitochondrial division protein Drp1. When mouse Drp1 was deleted in postmitotic Purkinje cells in the cerebellum, mitochondrial tubules elongated due to excess fusion, became large spheres due to oxidative damage, accumulated ubiquitin and mitophagy markers, and lost respiratory function, leading to neurodegeneration. Ubiquitination of mitochondria was independent of the E3 ubiquitin ligase parkin in Purkinje cells lacking Drp1. Treatment with antioxidants rescued mitochondrial swelling and cell death in Drp1KO Purkinje cells. Moreover, hydrogen peroxide converted elongated tubules into large spheres in Drp1KO fibroblasts. Our findings suggest that mitochondrial division serves as a quality control mechanism to suppress oxidative damage and thus promote neuronal survival.