Selective Cytotoxicity of Staphylococcal α-Hemolysin (α-Toxin) against Human Leukocyte Populations.

Staphylococcus aureus produces a variety of exoproteins that interfere with host immune systems. We attempted to purify cytotoxins against human leukocytic cells from the culture supernatant of S. aureus by a combination of ammonium sulfate precipitation, ion-exchange chromatography on a CM-cellulose column and HPLC on a Mono S 5/50 column. A major protein possessing cytotoxicity to HL60 human promyelocytic leukemia cells was purified, and the protein was identified as α-hemolysin (Hla, α-toxin) based on its molecular weight (34 kDa) and N-terminal amino acid sequence. Flow cytometric analysis suggested differential cytotoxicity of Hla against different human peripheral blood leukocyte populations. After cell fractionation with density-gradient centrifugation, we found that peripheral blood mononuclear cells (PBMCs) were more susceptible to Hla than polymorphonuclear leukocytes. Moreover, cell surface marker analysis suggested that Hla exhibited slightly higher cytotoxicity against CD14-positive PBMCs (mainly monocytes) than CD3- or CD19-positive cells (T or B lymphocytes). From these results, we conclude that human leukocytes have different susceptibility to Hla depending on their cell lineages, and thereby the toxin may modulate the host immune response.

Constitutive turnover of phosphorylation at Thr-412 of human p57/coronin-1 regulates the interaction with actin.

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/coronin-1 antibodies and HL60 cell lysates revealed that ß-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/coronin-1 regulates its interaction with actin.

Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation.

Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.

Vascular NAD(P)H oxidase mediates endothelial dysfunction in basilar arteries from Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

We examined the responses of basilar arteries taken from Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type 2 diabetes model. Both the nitric oxide (NO)-mediated relaxation and the cyclic 3',5'-guanosine monophosphate (cGMP) production elicited by acetylcholine (ACh) were much weaker in OLETF rats than in age-matched control Long Evans Tokushima Otsuka (LETO) rats. The contraction induced by an NO synthase (NOS) inhibitor [N(G)-nitro-L-arginine (L-NNA)] was weaker in the OLETF group. In that group, application of apocynin, an NAD(P)H oxidase inhibitor, normalized (i) ACh-induced relaxation, (ii) L-NNA-induced contraction, and (iii) ACh-induced cGMP production to the LETO levels. Superoxide anion production was greater in basilar arteries from OLETF rats than in those from LETO rats. The protein expression of gp91(phox), an NAD(P)H oxidase subunit, was upregulated in the OLETF arteries (versus LETO ones). These results suggest that the existence of endothelial dysfunction in basilar arteries in type 2 diabetes is related to increased oxidative stress mediated via NAD(P)H oxidase. Possibly, an impairment of NO-dependent relaxation responses and a basal impairment of NO signaling may be responsible for the increased risk of adverse cerebrovascular events in type 2 diabetes.

Treatment with vitamin k(2) combined with bisphosphonates synergistically inhibits calcification in cultured smooth muscle cells.

AIM: Vascular calcification is a common feature in patients with advanced atherosclerosis, postmenopausal women and patients with renal failure, which results in reduced elasticity of arteries. Pamidronate, a bisphosphonate, is used as a therapeutic agent for anti-osteoporosity, although there are adverse side effects, such as renal damage and aortic inflamed plaque rupture. In the present study, we demonstrated the effects of vitamin K(2) alone or in combination with pamidronate in an arterial calcification model induced using inorganic phosphate in cultured bovine aortic smooth muscle cells (BASMCs). METHODS: Calcification was induced by the addition of Pi (3 mM) in BASMCs. Calcium deposition was determined by Calcium C-test Wako and von Kossa staining. mRNA expression was assessed by semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: Calcium deposition assay and von Kossa staining showed that calcification could be inhibited in a dose-dependent manner by treatment with vitamin K(2) alone, and that its inhibitory effect was enhanced when combined with pamidronate. It was found that the expression of tropoelastin mRNA was synergistically enhanced by combined treatment with vitamin K(2) and pamidronate, and the expression matrix Gla protein mRNA and osteopontin mRNA expression were also enhanced and suppressed, respectively, by treatment with vitamin K(2) or pamidronate. Moreover, our data showed that the suppression of TE expression by siRNA significantly increased Pi-induced vascular calcification. CONCLUSION: Taken together, our study suggests that vitamin K(2) in combination with pamidronate synergistically inhibits arterial calcification via the increased expression of tropoelastin, which would be a useful marker for developing effective therapeutic or prophylactic agents for arterial calcification.

Chronic pain-induced emotional dysfunction is associated with astrogliosis due to cortical delta-opioid receptor dysfunction.

It has been widely recognized that chronic pain could cause physiological changes at supraspinal levels. The delta-opioidergic system is involved in antinociception, emotionality, immune response and neuron-glia communication. In this study, we show that mice with chronic pain exhibit anxiety-like behavior and an increase of astrocytes in the cingulate cortex due to the dysfunction of cortical delta-opioid receptor systems. Using neural stem cells cultured from the mouse embryonic forebrain, astrocyte differentiation was clearly observed following long-term exposure to the selective delta-opioid receptor antagonist, naltrindole. We also found that micro-injection of either activated astrocyte or astrocyte-conditioned medium into the cingulate cortex of mice aggravated the expression of anxiety-like behavior. Our results indicate that the chronic pain process promotes astrogliosis in the cingulate cortex through the dysfunction of cortical delta-opioid receptors. This phenomenon may lead to emotional disorders including aggravated anxiety under chronic pain-like state.

Role of delta-opioid receptor function in neurogenesis and neuroprotection.

The present study was undertaken to evaluate the implication of delta-opioid receptor function in neurogenesis and neuroprotection. We found that the stimulation of delta-opioid receptors by the selective delta-opioid receptor agonist SNC80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide] (10 nm) promoted neural differentiation from multipotent neural stem cells obtained from embryonic C3H mouse forebrains. In contrast, either a selective micro-opioid receptor agonist, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), or a specific kappa-opioid receptor agonist, (-)-trans-(1S,2S)-U-50488 hydrochloride (U50,488H), had no such effect. In addition to neural differentiation, the increase in cleaved caspase 3-like immunoreactivity induced by H2O2 (3 microm) was suppressed by treatment with SNC80 in cortical neuron/glia co-cultures. These effects of SNC80 were abolished by a Trk-dependent tyrosine kinase inhibitor: (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(cde)trinden-1-one (K-252a). The SNC80-induced neural differentiation was also inhibited by treatment with the protein kinase C (PKC) inhibitor, phosphatidylinositol 3-kinase (PI3K) inhibitor, mitogen-activated protein kinase kinase (MEK) inhibitor or Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These findings raise the possibility that delta-opioid receptors play a crucial role in neurogenesis and neuroprotection, mainly through the activation of Trk-dependent tyrosine kinase, which could be linked to PI3K, PKC, CaMKII and MEK.

Tropoelastin inhibits vascular calcification via 67-kDa elastin binding protein in cultured bovine aortic smooth muscle cells.

In cases of vascular calcification, the expression of tropoelastin is down-regulated, which most likely decreases elastic fiber formation. However, the function of tropoelastin in vascular calcification remains unknown. We investigated whether tropoelastin affects the induction of vascular calcification. Calcification was induced using inorganic phosphate in cultured bovine aortic smooth muscle cells. The increase in tropoelastin due to the addition of recombinant bovine tropoelastin (ReBTE; 1 or 10 microg/ml) or beta-aminopropionitrile (25 microg/ml) significantly inhibited calcification at day 6, as assessed by the o-cresolphthalein complexone method. The addition of an elastin-derived peptide, VGVAPG peptide (0.1-1,000 nM), inhibited calcification at day 6 in a dose-dependent manner. In addition, these responses of beta-aminopropionitrile, ReBTE, and VGVAPG peptide were confirmed using von Kossa staining. To examine whether ReBTE inhibited calcium deposition via the elastin binding protein, lactose and elastin-specific antibody were used. The combination of lactose (20 mM) or this antibody (50 microg/ml) with ReBTE (10 microg/ml) attenuated the inhibition of calcification. These results suggest that increased tropoelastin inhibits vascular calcification in this model via the interaction between tropoelastin and elastin binding protein.

Characterization of an in vitro model of calcification in retinal pigmented epithelial cells.

Little is known about the relationship at the molecular and cellular levels between vascular calcification and elastic fibers essential for elasticity. To gain a better understanding of the physiological function of elastin in vascular calcification, we developed a calcification model on cultured bovine retinal-pigmented-epithelial cells (RPEs) that do not express endogenous tropoelastin. The addition of inorganic phosphate (NaH2PO4; Pi) induced calcium deposition in RPEs. The Pi-induced calcification, as assessed by the o-cresolphthalein complexone method, Goldenbergs method, and von Kossa staining, was completely inhibited by treatment with clodronate (DMDP) and phosphonoformic acid (PFA) and was weakly suppressed by treatment with levamisole. Moreover, the osteopontin mRNA expression was upregulated in the Pi-induced calcification of RPEs. These reactions in RPEs were characteristically consistent with those already established in cultured bovine aortic smooth muscle cells (BASMCs). Furthermore, bacterially expressed tropoelastin inhibited calcium deposition in RPEs as well as in BASMCs. Finally, Pi-induced calcification was partially suppressed after the addition of tropoelastin due to elastic fiber formation. In conclusion, we suggest that this calcification model in RPEs is useful for analyzing the relation between elastic fibers and vascular calcification, and that tropoelastin and elastic fibers may contribute to the inhibition of vascular calcification.

Separation of stereoisomers of several furan derivatives by capillary gas chromatography-mass spectrometry, supercritical fluid chromatography, and liquid chromatography using chiral stationary phases.

The direct separation of several stereoisomers (enantiomers and geometrical isomers) of furan derivatives, important intermediates for the synthesis of physiologically active natural products, was achieved using capillary gas chromatography/mass spectrometry with a per-O-methyl-beta-cyclodextrin, supercritical fluid chromatography and high-performance liquid chromatography with a tris(3,5-dimethylphenylcarbamate) of cellulose or amylose for the chiral stational phases, respectively. The temperature dependence of the peak resolution (Rs) and the retention factor (k) over the range of 110-130 degrees was studied using crotyl furfuryl ether in gas chromatography. Successive increases in the Rs value and of the difference between the k value of the E-isomer and the k value of the Z-isomer were observed when the gradient temperature was decreased. The per-O-methyl-beta-cyclodextrin column was suitable for use with volatile furan ethers whose molecular masses are between 150 and 180. In conclusion, the separation of thermally unstable furan derivatives was accomplished using supercritical fluid chromatography and high-performance liquid chromatography.

Advanced glycation end product-modified beta2-microglobulin is a component of amyloid fibrils of primary localized cutaneous nodular amyloidosis.

Primary localized cutaneous nodular amyloidosis is a rare form of cutaneous amyloidosis. Amyloid fibrils in primary localized cutaneous nodular amyloidosis have been reported to be originated from immunoglobulin light chains. Immunohistochemical studies on the lesional skins of four patients with primary localized cutaneous nodular amyloidosis demonstrated that amyloid deposits of all cases showed a positive reaction with the antibodies for beta2-microglobulin and advanced glycation end products as well as immunoglobulin light chain (kappa or lambda). No beta2-microglobulin and advanced glycation end product immunoreactivity was found in the amyloid deposits of other primary localized cutaneous amyloidosis (lichen amyloidosis and macular amyloidosis). Double immunofluorescence study of the lesional skin of primary localized cutaneous nodular amyloidosis showed that anti-kappa light chain, anti-beta2-microglobulin and anti-advanced glycation end product antibodies mostly reacted with the same area of amyloid deposit. Amyloid proteins were sequentially extracted with distilled water from one case of primary localized cutaneous nodular amyloidosis and recovered in the five water-soluble fractions (fractions I-V). Immunoblot assay of amyloid fibril proteins demonstrated that immunoreactive polypeptides with anti-kappa light chain antibody (29 kDa) and with anti-beta2-microglobulin antibody (12 kDa) were detected in fractions I-V, whereas immunoreactive polypeptide with anti-advanced glycation end product antibody (12 kDa) was detected exclusively in fractions III-V but not in fractions I and II. Two-dimensional polyacrylamide gel electrophoresis revealed that 12 kDa polypeptide in fractions I and II was electrophoretically identical with authentic beta2-microglobulin and that beta2-microglobulin in fractions III-V was advanced glycation end product-modified beta2-microglobulin with more acidic pI value. These results indicate that beta2-microglobulin is another major component of amyloid fibrils in primary localized cutaneous nodular amyloidosis and that beta2-microglobulin in primary localized cutaneous nodular amyloidosis is partly subjected to the modification of advanced glycation end product.

Accelerated calcification represses the expression of elastic fiber components and lysyl oxidase in cultured bovine aortic smooth muscle cells.

Vascular calcification is a common feature of advanced atherosclerosis resulting in reduced elasticity of elastic arteries. However, the relationship between elastic fibers and vascular calcification at the molecular and cellular levels remains unknown. We investigated the expression of major elastic fiber components such as tropoelastin (TE) and fibrillin-1 (FBN1) and elastin-related enzyme, lysyl oxidase (LO), in a calcification model using beta-glycerophosphate (beta-GP) in cultured bovine aortic smooth muscle cells (BASMCs). Ten mM of beta-GP stimulated calcium deposition in a time-dependent manner. As determined by Western blot analysis, 10 mM of beta-GP time-dependently decreased TE and FBN1 protein levels. TE, FBN1, and LO mRNA levels, assessed by reverse transcription-polymerase chain reaction, were also decreased by exposure to 10 mM beta-GP. Furthermore, we investigated whether the processes of calcification in BASMCs directly control these regulations. In experiments using levamisole, an alkaline phosphatase inhibitor, and DMDP, a bisphosphonate, both inhibitors inhibited down-regulation during beta-GP-induced calcification, suggesting that the down-regulation of TE, FBN1, and LO directly relates to calcium deposition. In cases of vascular calcification, the decreased expression of TE, FBN1, and LO may be partially responsible for decreased vascular elasticity and also for the decreased formation of new elastic fibers.