Periodontal pathogen accelerates lipid peroxidation and atherosclerosis.

Recent studies have shown an association between periodontal disease and cardiovascular disease. We previously reported that intravenous challenge with Aggregatibacter actinomycetemcomitans (Aa) accelerated atherosclerosis in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice. In this study, we investigated whether live cells were required for atherosclerosis induction or whether lipopolysaccharide (LPS) alone was sufficient to increase atherosclerotic damage. Mice were injected intravenously with live Aa HK1651, heat-killed (H.K.) Aa, or Aa LPS 3 times a week for 3 weeks and were sacrificed at 15 weeks of age. The areas of the aortic sinus that were covered with atherosclerotic plaques were significantly larger in mice treated with live Aa, H.K. Aa, or Aa LPS compared with vehicle-challenged mice. The order of the extent of atherosclerosis was live Aa > H.K. Aa > Aa LPS > sham. Toll and nucleotide oligomerization domain (NOD)-like receptor mRNA expression significantly increased in the live Aa, H.K. Aa, and Aa LPS treatment groups. Aa challenge markedly promoted the oxidation of LDL through oxidative stress involving NADPH oxidase- and myeloperoxidase-derived reactive oxygen species. These results suggested that Aa promoted innate immune signaling and low-density lipoprotein (LDL) oxidation and may facilitate atheroma development.

Clinical course of drug-induced hypersensitivity syndrome treated without systemic corticosteroids.

BACKGROUND/AIM: Drug-induced hypersensitivity syndrome (DIHS) is a severe reaction to drugs which characteristically occurs after a long latency period. In addition, human herpes virus 6 (HHV-6) reactivation is a characteristic finding in DIHS, which has been known to be related to disease severity. Because DIHS has generally been treated by systemic corticosteroids, the natural clinical course is not clear. METHODS: Data for patients with both DIHS and HHV-6 reactivation were retrospectively collected from four hospitals. RESULTS: Data were collected on 12 patients ranging in age from 21 to 76 years (median, 65.5). All cases had been suspected of DIHS at their initial visit, and the elevation of serum anti-HHV-6 antibody had been confirmed (4-256 times: median; 32). The culprit drugs were carbamazepine (6), salazosulfapyridine (4), mexiletine (1) and zonisamide (1). The period of latency from the first administration of the drug ranged from 15 to 50 days (median, 30). All patients were treated conservatively for DIHS without systemic corticosteroids. The peaks of the patients' symptoms and laboratory findings were as follows (days from the onset of skin lesions): fever, 4-16 (median, 10.5); liver abnormality, 3-22 (median, 7.5); leukocytosis, 7-20 (median, 9). All patients recovered without pneumonia, myocarditis, nephritis or other systemic disease, from 7 to 37 days (median, 18) after withdrawal of the drug and from 11 to 44 days (median, 21) after the onset of skin lesions. CONCLUSION: It might be unnecessary to give systemic corticosteroids immediately to all patients suspected of having DIHS.

Nasal immunization with Porphyromonas gingivalis outer membrane protein decreases P. gingivalis-induced atherosclerosis and inflammation in spontaneously hyperlipidemic mice.

Porphyromonas gingivalis has been shown to accelerate atherosclerotic lesion development in hyperlipidemic animals. We assessed the potential of a nasal vaccine against P. gingivalis infection for the prevention of atherosclerosis. Apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice were nasally immunized with the 40-kDa outer membrane protein (OMP) of P. gingivalis plus cholera toxin (CT) as adjuvant and then challenged intravenously with P. gingivalis strain 381. The animals were euthanized 11 or 14 weeks later. Atheromatous lesions in the proximal aorta of each animal were analyzed histomorphometrically, and the serum concentrations of 40-kDa OMP-specific antibodies and cytokines were determined. The areas of the aortic sinus that were covered with atherosclerotic plaque and the serum levels of inflammatory cytokines and chemokines were increased in Apoe(shl) mice challenged with P. gingivalis compared to nonchallenged mice. In comparison, nasal immunization with 40-kDa OMP plus CT significantly reduced atherosclerotic plaque accumulation in the aortic sinus and lowered the serum levels of cytokines and chemokines compared to nonimmunized animals. Nasal immunization also induced 40-kDa OMP-specific serum immunoglobulin G (IgG) and saliva IgA antibody responses. These findings suggest that systemic infection with P. gingivalis accelerates atherosclerosis in Apoe(shl) mice, and 40-kDa OMP plus CT may be an effective nasal vaccine for the reduction of atherosclerosis accelerated by P. gingivalis in the hyperlipidemic mouse model.

Novel resistin promoter polymorphisms: association with serum resistin level in Japanese obese individuals.

OBJECTIVES: Resistin, an adipocyte-secreted cytokine recently discovered in mice, has been proposed as a link between obesity and diabetes. We analyzed resistin gene polymorphisms and examined their association with serum resistin level and obesity phenotypes in humans. SUBJECTS AND METHODS: Sixty young, obese, non-diabetic subjects taking no medication were studied. DNA sequencing and genotyping of identified single nucleotide polymorphisms were performed. Associations between polymorphisms and serum resistin level, BMI, body composition, fat distribution, and several indices of insulin sensitivity were examined. Moreover, single nucleotide polymorphisms in the promoter region were examined for their influence on resistin gene transcriptional activity using luciferase reporter vectors. RESULTS: Ten non-coding single nucleotide polymorphisms were found. The -638G>A, -420C>G, and -358G>A polymorphisms in the promoter region showed marked linkage disequilibrium with each other, and were associated with serum resistin level; however, there was no association between these polymorphisms and parameters related to adiposity or insulin resistance. The results of luciferase assay revealed that -638G>A together with the -420C>G polymorphism influenced resistin gene transcriptional activity. CONCLUSION: We found that variability in the serum resistin level might be related to polymorphic variants of the promoter region of the gene.

Suppressive effects of breast milk on oxidative DNA damage in very low birthweight infants.

BACKGROUND: Human milk contains many kinds of antioxidant and is considered to prevent diseases mediated by oxygen free radicals in very low birthweight (VLBW) infants. AIMS: To examine the antioxidant effects of breast milk in VLBW infants by determining urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion, which is known to be a non-invasive marker for in vivo oxidative DNA damage. METHODS: Urinary 8-OHdG concentrations were measured in 15 breast fed and 14 formula fed VLBW infants at 2, 7, 14, and 28 days of age. RESULTS: Urinary 8-OHdG excretion at 14 and 28 days of age was significantly lower than at 2 and 7 days of age in the breast fed group, and significantly lower than in the formula fed group. CONCLUSION: This is the first direct evidence of the antioxidant action of human milk in VLBW infants.

Development of cDNA microarray for expression profiling of estrogen-responsive genes.

Estrogen plays an important role in many physiological events including carcinogenesis and the development of human breast cancer. However, the molecular mechanisms of estrogen signaling in cancers have not been clarified hitherto and accurate therapeutic prediction of breast cancer is earnestly desired. We first carried out estrogen-responsive expression profiling of approximately 9000 genes in estrogen receptor-positive human MCF-7 breast cancer cells. Based on the results, estrogen-responsive genes were selected for production of a custom-made cDNA microarray. Using a microarray consisting of the narrowed-down gene subset, we first analyzed the time course of the estrogen-responsive gene expression profiles in MCF-7 cells, resulting in subdivision of the genes up-regulated by estrogen into early-responsive and late-responsive genes. The expression patterns of several genes were confirmed by Northern blot analysis. We also analyzed the effects of the estrogen antagonists ICI 182780 and 4-hydroxytamoxifen (OHT) on the estrogen-responsive gene expression profiles in MCF-7 cells. While the regulation of most of the genes by estrogen was completely abolished by ICI 182780, some genes were partially regulated by estrogen even in the presence of OHT. Furthermore, the estrogen-responsive gene expression profiles of twelve cancer cell lines derived from the breast, ovary, stomach and other tissues were obtained and analyzed by hierarchical clustering including the profiles in MCF-7 cells. Several genes also showed up-regulation or down-regulation by estrogen in cell lines other than MCF-7 cells. The significance of the estrogen-responsive genes identified in these analyses concerning the nature of cancer is discussed.

OsPAA2, a distinct alpha 1 subunit gene for the 20S proteasome in rice (Oryza sativa L.).

The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins and other diverse biological functions. The 20S proteasome is constituted of 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and the two inner rings are composed of seven different alpha and beta type subunits, respectively, giving an alpha 7 beta 7 beta 7 alpha 7 structure. We previously reported the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all the subfamilies from monocot. In this study, a distinct cDNA sequence encoding the alpha1 subunit, OsPAA2, was identified. The amino acid sequence similarity between the two rice alpha1 subunits was as low as 59.6%, contrasting with those between paralogs of Arabidopsis proteasome subunit genes. The expression pattern of the OsPAA2 gene was different from that of another alpha1 gene, OsPAA1. These data suggest that OsPAA2 might play a distinct role from that of OsPAA1 in the 20S proteasome complex.

Cholesteryl ester transfer protein polymorphism associated with macroangiopathy in Japanese patients with type 2 diabetes.

A polymorphism in the gene for cholesteryl ester transfer protein (CETP) has been reported to be associated with serum cholesterol levels and risk for atherosclerotic vascular diseases, and to clarify the relationship between the gene polymorphism for CETP and macroangiopathy in diabetes mellitus, a cross-sectional study was performed. The subjects of the study were182 Japanese (age: 59.6+/-8.6 years) with type 2 diabetes and no signs of renal dysfunction, 24 of whom had macroangiopathy, and 158 of whom did not. The genotype of the subjects for the TaqIB polymorphism of CETP in intron one was analyzed by using polymerase chain reaction - restriction fragment length polymorphism. Serum CETP levels were significantly higher in the B1/B1 genotype than in the other genotypes (P<0.05). The serum CETP levels were correlated with the serum LDL cholesterol levels (P<0.01), but not with the HDL cholesterol levels. Macroangiopathy was more frequently observed in subjects with the B1/B1 genotype than in the other genotypes (odds ratio=2.953, 95% confidence interval=1.250-6.977, P=0.0136). Logistic regression analysis revealed that the CETP genotype was independently associated with macroangiopathy. The exact mechanism underlying the association remains unknown, but differences in serum CETP levels may be involved.

Effects of n-3 polyunsaturated fatty acids and vitamin E on colonic mucosal leukotriene generation, lipid peroxidation, and microcirculation in rats with experimental colitis.

AIMS: We investigated the effect of n-3 polyunsaturated fatty acids (PUFAs) on mucosal levels of leukotrienes (LTs) and lipid peroxide (LPO), and on mucosal microcirculation, in rats with experimental colitis induced by dextran sulfate sodium (DSS). METHODS: We fed Wistar rats a perilla oil-enriched diet containing alpha-linolenic acid (63.2% of total fatty acids) with various doses of vitamin E for 4 weeks, with 4% DSS added to the drinking water during the last week. Control rats were fed a diet produced from soybean oil containing alpha-linolenic acid (5.1% of total fatty acids). Colonic mucosal blood flow was measured with a laser Doppler flowmeter. RESULTS: The mucosal level of arachidonic acid was significantly lower and that of eicosapentaenoic acid was significantly higher in the experimental group. The mucosal level of LPO in the experimental group fed a trace or ordinary dose of vitamin E was significantly higher than that of the controls. The production of LTB(4) and LTC(4) from the colonic mucosa in the experimental group was significantly lower than that in controls. However, only the experimental group fed a vitamin E dose 4-fold higher than that given to the controls showed a significant increase in mucosal blood flow. CONCLUSION: These results suggest that n-3 PUFAs increase mucosal blood flow by inhibiting LT production when there is sufficient vitamin E to inhibit lipid peroxidation in rats with experimental colitis.

Monoclonal antibody against vascular cell adhesion molecule-1 inhibits neointimal formation after periadventitial carotid artery injury in genetically hypercholesterolemic mice.

Vascular cell adhesion molecule (VCAM)-1 is induced in smooth muscle cells after arterial injury, in which it has been implicated in the recruitment of inflammatory cells to the site of injury. To investigate the effect of hypercholesterolemia on VCAM-1 induction after injury and the role of VCAM-1 in neointimal response to injury, we injured the carotid artery of wild-type and apolipoprotein E null (KO) mice fed normal and high cholesterol chow. We demonstrate a graded response of VCAM-1 induction as well as monocyte/macrophage infiltration by immunohistochemistry 3 days after injury that correlated with increasing circulating cholesterol levels. Three weeks after injury, KO mice fed high cholesterol chow (KO HC group) had a significantly greater neointimal formation compared with wild-type and KO mice fed normal chow (P<0.05). Inhibition of VCAM-1 function in the KO HC group by monoclonal antibody treatment significantly reduced monocyte/macrophage infiltration and neointimal formation. There was reduced alpha-actin expression in KO HC mice 7 days after injury that was partially inhibited by VCAM-1 antibody treatment. Cell migration in an in vitro injury model was partially inhibited by monoclonal VCAM-1 antibody treatment. We propose an additional role for VCAM-1 in smooth muscle cell activation and neointimal formation after injury.

Primary structural features of the 20S proteasome subunits of rice (Oryza sativa).

The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins, and it also has other diverse biological functions. Its structure comprises 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and two inner rings form an alpha7beta7beta7alpha7 structure, and each subunit, alpha and beta, exists as seven different types, thus giving 14 kinds of subunits. In this study, we report the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all of the subunits from monocots. Amino acid sequence homology within the rice family (alpha-type: 28.9-42.1%; beta-type: 17.2-31. 9%) were lower than those between rice subunits and corresponding orthologs from Arabidopsis and yeast (alpha-type: 49.2-94.5%; beta-type: 34.8-87.7%). Structural features observed in eukaryotic proteasome subunits, i.e., alpha- or beta-type signature at the N-termini, Thr active sites in beta1, beta2 and beta5 subunits, and nuclear localization signal-like sequences in some alpha-type subunits, were shown to be conserved in rice.

Activated factor XII levels are dependent on factor XII 46C/T genotypes and factor XII zymogen levels, and are associated with vascular risk factors in patients and healthy subjects.

Coagulation factor XII (FXII) is activated on contact with various biologic surfaces, including subendothelial tissues and lipoprotein particles. Thus, the plasma level of activated FXII (XIIa) might represent vascular lesions or be a marker of abnormal lipid metabolism. A 46C/T polymorphism was recently described in the FXII gene close to the ATG translation initiation codon, which was associated with inter-individual variation of plasma FXII zymogen levels. The present paper reports the association of the 46C/T polymorphism with plasma XIIa levels in apparently healthy subjects, and in patients with ischemic cerebrovascular disease (CVD) and arteriosclerosis obliterans (ASO). XIIa levels were not significantly different between patients and controls, but were strongly dependent on XII 46C/T genotypes (2.07 +/- 0.81, 1.65 +/- 0.63, and 0.93 +/- 0.41 ng/ml for C/C, C/T, and T/T genotypes, respectively; P < 0.0001). This association was evident for each group studied (P < 0.0001 for CVD and controls; P= 0.0007 for ASO). There were positive correlations between plasma FXII clotting activity and XIIa levels. In a univariate analysis, XIIa correlated with total cholesterol, triglycerides, plasminogen activator inhibitor-1, and C-reactive protein (CRP), although the presence of conventional cardiovascular risk factors (male sex, smoking, hypertension, hypercholesterolemia, diabetes) did not significantly increase XIIa. Stepwise regression analyses revealed that the XII clotting activity had the strongest association with XIIa. In conclusion, XIIa levels depended on XII 46C/T genotype and correlated with some cardiovascular risk factors. Thus, the FXII genotype should be taken into consideration for interpretation of plasma XIIa levels.

Kaposi's varicelliform eruption of an elderly patient with multiple myeloma.

Kaposi's varicelliform eruption (KVE) is characterized by disseminated vesiculopustules and erosions due to a herpes virus infection superimposed on a preexisting dermatosis such as atopic dermatitis. However, the occurrence of KVE in patients with multiple myeloma seems to be very rare; to our knowledge, only one such case has been reported. This report documents a second case of KVE in a patient with multiple myeloma.

Phosphorylation of Amyloid Precursor Protein (APP) Family Proteins.

It has been well established that ß-amyloid peptide is the principal protein component of extracellular cerebral amyloid deposits in patients with Alzheimer's disease (1,2). ß-Amyloid is derived from a large precursor protein, amyloid precursor protein (APP), which is an integral membrane protein, with a receptor-like structure (3). APP is a member of a gene family which encodes extremely well-conserved membrane proteins. APP/APP-like genes have been isolated from various species including fly (4), nematode (5), and fish (6). In mammals, two APP-like genes, amyloid precursor-like protein 1 (APLP1) and 2 (APLP2), have been isolated (7,8). The amino acid sequences of these APP family proteins are highly conserved, especially in the cytoplasmic domain, except that unlike APP, APP-like proteins lack the ß-amyloid sequence. It has been thought that APP and APLP2 have a similar physiological function (9). In contrast, APLP1 is believed to differ functionally from APP and APLP2, although the physiological functions of these APP family proteins have not yet been well analyzed.

Reconstituted HDL containing human apolipoprotein A-1 reduces VCAM-1 expression and neointima formation following periadventitial cuff-induced carotid injury in apoE null mice.

Arterial injury triggers an inflammatory response in part mediated by induction of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and is implicated in neointimal thickening. Since HDL is known to reduce cytokine-activated VCAM-1 expression, we tested the hypothesis that VCAM-1 expression and neontimal thickening following arterial injury are inhibited by reconstituted human HDL containing plasma-derived apoA-1 (rHDL). We used the carotid cuff injury in apoE (-/-) mice fed high cholesterol. Mice received rHDL (40 mg/kg) intravenously every other day for 3 weeks. Compared to control, rHDL treatment inhibited neointima formation (0. 008 +/- 0.004 mm(2) vs. 0.037 +/- 0.019 mm(2); P < 0.01) 21 days after injury, reduced VCAM-1 expression, and decreased monocyte/macrophage infiltration as assessed by histomorphometric analysis within the first week after injury. These changes occurred without any effect on plasma total and HDL cholesterol levels as well as the arterial tissue cholesterol levels. rHDL treatment also reduced the formation of modified lipoprotein in the arterial wall compared to control within the first week after injury. This finding suggests an antioxidant effect of rHDL associated with reduced VCAM-1 expression and neointimal formation after arterial injury.

Effects of troglitazone on fat distribution in the treatment of male type 2 diabetes.

We investigated the efficacy of additional administration of 400 mg troglitazone (+T), which became available as a treatment for type 2 diabetes following the demonstration of its ability to reduce insulin resistance, in combination with diet (D + T) or sulfonylurea (S + T) therapy. Body fat area as determined by computed tomographic (CT) scanning at the umbilical level, as well as several clinical and biochemical parameters of glycemic control and lipid metabolism, were compared before and after 3 months of additional treatment with troglitazone. The body mass index (BMI) tended to increase in both groups (22.7 +/- 0.6 v 23.2 +/- 0.6 kg/m2 in D + T, nonsignificant [NS]; 22.2 +/- 0.5 v 22.3 +/- 0.5 kg/m2 in S + T, NS), while it tended to decrease in the control group (only diet therapy, 23.6 +/- 0.6 v 23.1 +/- 0.8 kg/m2, NS). Mean blood pressure ([BP] 96 +/- 3 v 89 +/- 4 mm Hg, P < .05) decreased significantly in the D + T group. Changes in the glycemic and lipid profile and leptin did not reach statistical significance. The D + T group showed a significant decline in immunoreactive insulin ([IRI] 12.4 +/- 1.2 v 8.0 +/- 1.0 microU/mL, P < .05), reflecting markedly reduced insulin resistance, as well as a significant increase in plasma insulin-like growth factor-1 ([IGF-1] 175.7 +/- 14.2 v 189.8 +/- 12.6 ng/mL, P < .05). A slight weight gain was associated with a tendency for subcutaneous fat to increase, while visceral fat decreased in both troglitazone-treated groups. The decrease in the visceral to subcutaneous fat ratio (V/S ratio) was statistically significant in the D + T group (1.09 +/- 0.11 v 0.94 +/- 0.09, P < .05), while the V/S ratio in the control group did not change. A notable finding of this study is the difference in the response to troglitazone between subcutaneous and visceral adipose tissue. It is suggested that troglitazone may exert beneficial effects by reducing visceral fat.

[Effect of lipoprotein metabolism abnormality for intimal thickening].

Recent advances of lipoprotein metabolism abnormality were reviewed. Recombinant apolipoprotein and transgenic mice expressing high levels of apolipoproteins or apolipoprotein deficient mice produced by gene targetting technique are providing important information on effect of lipoprotein metabolism. We made novel mouse cuff injury model for transgenic mice and knock-out mice. This injury model will also provide important information on intimal thickening using transgenic mice and knock-out mouse.

A 127-kDa protein (UV-DDB) binds to the cytoplasmic domain of the Alzheimer's amyloid precursor protein.

Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP.