Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) increases serum testosterone concentration and enhances steroidogenic ability of Leydig cells in male rats.

Although Lepidium meyenii (maca), a plant growing in Peru's central Andes, has been traditionally used for enhancing fertility and reproductive performance in domestic animals and human beings, effects of maca on reproductive organs are still unclear. This study examined whether feeding the hydroalcoholic extract powder of maca for 6 weeks affects weight of the reproductive organs, serum concentrations of testosterone and luteinising hormone (LH), number and cytoplasmic area of immunohistochemically stained Leydig cells, and steroidogenesis of cultured Leydig cells in 8-week-old male rats. Feeding the extract powder increased weight of seminal vesicles, serum testosterone level and cytoplasmic area of Leydig cells when compared with controls. Weight of prostate gland, serum LH concentration and number of Leydig cells were not affected by the maca treatment. The testosterone production by Leydig cells significantly increased when cultured with 22R-hydroxycholesterol or pregnenolone and tended to increase when cultured with hCG by feeding the extract powder. The results show that feeding the hydroalcoholic extract powder of maca for 6 weeks increases serum testosterone concentration associated with seminal vesicle stimulation in male rats, and this increase in testosterone level may be related to the enhanced ability of testosterone production by Leydig cells especially in the metabolic process following cholesterol.

Effects of instant coffee consumption on oxidative DNA damage, DNA repair, and redox system in mouse liver.

To examine the effects of instant coffee consumption on cancer risk, we analyzed the oxidative DNA damage levels and the DNA repair and redox systems in the livers of coffee-fed mice. Three-week-old male ICR mice were fed with/without 0.1% (w/v) instant coffee solution. At 2, 4, and 8 mo, the levels of 8-hydroxydeoxyguanosine (8-OH-dG), a major form of oxidative DNA damage, and the expression of mouse 8-OH-dG repair-associated genes and redox system-associated genes, the SOD activity, and the LPO level were analyzed. Simultaneously, half of the mice were fed a low vitamin (LV) diet (autoclaved diet) to disturb the defense system against oxidative stresses. As a result, the 8-OH-dG level was increased in the livers of LV diet (+ water)-fed mice for 8 mo, in comparison to those of the 0 M control mice and normal diet (+ water)-fed mice. However, no significant differences between water drinking and coffee drinking were observed, in terms of the 8-OH-dG level. In addition, the 8-OH-dG repair-associated gene expression, the SOD activity, and the LPO level also showed no significant differences between water drinking and coffee drinking in all mouse groups. On the other hand, among the redox system-associated genes, only the expression of GPx1 was changed. These results suggest that instant coffee consumption has little, if any, effect on the risk of liver cancer due to oxidative stresses.

Effect of raloxifene on bone mineral density and biochemical markers of bone turnover in Japanese postmenopausal women with osteoporosis: results from a randomized placebo-controlled trial.

The safety and efficacy of raloxifene, a selective estrogen receptor modulator (SERM), has been studied extensively in large, global clinical trials. However, the effect of raloxifene on bone mineral density (BMD) and on biochemical markers of bone turnover in Japanese postmenopausal women with osteoporosis has not been rigorously evaluated. This study was designed to assess the safety and efficacy of raloxifene in Japanese postmenopausal women with osteoporosis following 1 year of therapy. Participants in this multicenter trial were randomly assigned to receive placebo, raloxifene 60 mg/day (RLX60), or raloxifene 120 mg/day (RLX120). Lumbar spine BMD was measured at baseline, 24, 40, and 52 weeks, and biochemical markers of bone turnover were assessed at baseline, 12, 24, and 52 weeks. Serum lipids were assessed at baseline, 12, 24, 40, and 52 weeks, and breast examinations and transvaginal ultrasound of the endometrium were performed at enrollment and 52 weeks. Compared with baseline, women taking RLX60 had significant increases in lumbar spine (L2-L4) BMD at 24 weeks (+3.3%, p<0.001) through 52 weeks (+3.5%, p<0.001) of therapy, and similar results were observed in the RLX120 group. Markers of bone turnover and total cholesterol and LDL-C were significantly reduced, and no significant treatment-group difference was observed for patients reporting at least one adverse event following randomization. In addition, there were no reported venous thromboembolic events (VTE) in any treatment group. The results of this study demonstrate that raloxifene is associated with early increases in lumbar spine BMD, has favorable effects on biochemical markers of bone turnover and lipid profile, and is well tolerated in postmenopausal Japanese women.

Filling a cavity dramatically increases pressure stability of the c-Myb R2 subdomain.

Cavities or packing defects in proteins may generally be related with the dynamics and function of a protein. In the c-Myb R2 subdomain, its single cavity has been shown to be crucial for its DNA recognition. Cavities are also considered important in determining the pressure stability of a protein. In the present work, high-pressure proton nuclear magnetic resonance ((1)H NMR) spectroscopy at 750 MHz is used to study the effect of a cavity-filling mutation (V103L) on the stability of the c-Myb R2 subdomain in the pressure range between 1 and 3,700 bar at 5 degrees C. A dramatic increase in the pressure stability of the c-Myb R2 subdomain is attained, from which we estimate the cavity size to be 35.3 A(3), in good agreement with literature values. We also evaluated the increase in thermodynamic stability DeltaG(0)(1bar) from 5.35 kJ/mol to 7.34 kJ/mol by the mutation, giving a clear example of the effect of a cavity on the global stability of a globular protein.

Vascular calcification and inorganic phosphate.

Vascular calcification is highly correlated with elevated serum phosphate levels in uremic patients. To shed light on this process, we examined the ability of extracellular inorganic phosphate (Pi) levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro. When cultured in media containing normal physiological levels of Pi (1.4 mmol/L Pi), HSMC grew in monolayers and did not mineralize. In contrast, HSMC cultured in media containing Pi levels comparable to those seen in hyperphosphatemic individuals (>1.4 mmol/L), showed dose-dependent increases in cell culture calcium deposition. Mechanistic studies showed that elevated Pi treatment of HSMC also enhanced the expression of the osteogenic markers, osteocalcin and Cbfa-1. The effects of elevated Pi on HSMC were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor, phosphonoformic acid (PFA), to dose-dependently inhibit Pi-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. Using polymerase chain reaction and Northern blot analyses, the NPC in HSMC was identified as Pit-1 (Glvr-1), a member of the type III NPCs. Interestingly, platelet-derived growth factor-BB (PDGF-BB), a potent atherogenic stimulus, increased the maximum velocity (Vmax) but not the affinity (Km) of phosphate uptake, enhanced the expression of Pit-1 mRNA, and induced HSMC culture calcification in a time- and dose-dependent manner. Importantly, in the presence of PDGF, HSMC culture calcification occurred under normophosphatemic conditions. These data suggest that elevated Pi may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and may help explain both the phenomena of human metastatic calcification under hyperphosphatemic conditions as well as increased calcification in PDGF-rich atherosclerotic lesions.

[A system of health education using dietary assessment].

We decided to focus attention on healthy young people, and to conduct a survey of their diet, as well as their physical activity and health condition, so that we made diagnoses and also give instructions on each subject using a computer program to promote their health. We carried out this experiment using this method with 836 young female subjects in order to find out whether it can be applied as a useful health education approach. 1. According to the survey, when the subjects made efforts in making the instructions as convenient as possible, the percentage of subjects who could carry out advice even 4 months later was seen to be higher. 2. The advice for which a significantly high percentage of subjects said "want to improve their daily action" and could actually carry out and improve them, was as follows: they should eat vegetables, have a substantial breakfast, drink milk every day, refrain from too much salt, not go without meals, increase the frequency of eating seaweed, and refrain from snacks. 3. The two kinds of scores used to evaluate diets showed that assessment after nine months gave lower values than those obtained when this survey started. Using these two evaluating scores, changes in diets were divided into five patterns, and their relation to the question "whether they used the advice or not" was examined. As a result, the percentage showing favorable diet changes in subjects who "took advice" was showed to be significantly high. 4. Subjects who received advice on physical activity exhibited improvement in carrying out routine exercises as well as making their daily life more active. 5. With subjects who took advice on both diet and physical activity, the number who complained about fatigue was significantly reduced. Although it is difficult to improve diet and exercise habits, the results of the survey suggest that our approach should be useful for improving health.

Structural analyses of DNA recognition by the AML1/Runx-1 Runt domain and its allosteric control by CBFbeta.

The core binding factor (CBF) heterodimeric transcription factors comprised of AML/CBFA/PEBP2alpha/Runx and CBFbeta/PEBP2beta subunits are essential for differentiation of hematopoietic and bone cells, and their mutation is intimately related to the development of acute leukemias and cleidocranial dysplasia. Here, we present the crystal structures of the AML1/Runx-1/CBFalpha(Runt domain)-CBFbeta(core domain)-C/EBPbeta(bZip)-DNA, AML1/Runx-1/CBFalpha(Runt domain)-C/EBPbeta(bZip)-DNA, and AML1/Runx-1/CBFalpha(Runt domain)-DNA complexes. The hydrogen bonding network formed among CBFalpha(Runt domain) and CBFbeta, and CBFalpha(Runt domain) and DNA revealed the allosteric regulation mechanism of CBFalpha(Runt domain)-DNA binding by CBFbeta. The point mutations of CBFalpha related to the aforementioned diseases were also mapped and their effect on DNA binding is discussed.

Increased retinal blood flow in patients with Graves' disease: influence of thyroid function and ophthalmopathy.

OBJECTIVE: Graves' ophthalmopathy (GO), resulting from the inflammation of retro-orbital tissue, is one of the major complications of Graves' disease (GD). We investigated the clinical usefulness of the measurement of retinal blood flow (RBF) in the evaluation of GO and its activity. MEASUREMENT: RBF was quantitated by pulsed Doppler mode at just below the branch of central retinal artery, from which the resistance index (RI) was calculated. PATIENTS: Forty-seven euthyroid GD patients and 70 gender- and age-matched normal controls were measured for RI to investigate the effect of GO on RBF. To investigate the effect of hyperthyroidism, 20 GD patients were measured for RI changes during antithyroid drug (ATD) therapy. Furthermore, 17 GD patients with clinically overt GO were measured for RI changes during treatment with glucocorticoid plus retro-orbital radiation. RESULTS: RI and exophthalmos showed a significant positive correlation in 47 treated euthyroid GD patients without clinically overt GO (r=0.307, P<0.05), but not in 70 age- and sex-matched normal subjects (r=0.185, P=0.161). Furthermore, RI, but not exophthalmos, significantly correlated with serum TSH receptor antibodies, an indicator for the disease activity of GO. ATD therapy significantly reduced RI in GD patients from 0.719+/-0.041 in the hyperthyroid state to 0.661+/-0.051 in the euthyroid state, but not to the levels observed in normal subjects having the similar exophthalmos (0.640+/-0.049). The fractional reduction of RI during ATD therapy significantly correlated with those of pulse pressure and ultrasonographic distensibility in carotid artery, but not with those of serum vascular injury markers. In 17 GD patients with clinically overt GO, all four patients having adipose tissue enlargement but not extraocular muscle hypertrophy (inactive GO) showed RI within the mean +/- 1 s.d. for treated GD patients without GO. In the other 13 GD patients having extraocular muscle hypertrophy (active GO), four and eight patients showed RI outside mean +/- 2 s.d. and mean +/- 1 s.d. respectively. Treatment with glucocorticoid plus radiation moved RI in 8 out of 10 patients toward the mean values of GD patients without GO, in spite of little improvement of exophthalmos. CONCLUSIONS: It was suggested that GD patients showed altered retinal hemodynamics, possibly resulting either from the cardiovascular effect of hyperthyroidism or from retro-orbital inflammation, particularly in extraocular muscle.

Rare somatic inactivation of the multiple endocrine neoplasia type 1 gene in secondary hyperparathyroidism of uremia.

The molecular pathway of autonomous growth of the parathyroid glands in uremic patients is poorly understood. Loss of heterozygosity at the recently identified multiple endocrine neoplasia type 1 (MEN1) gene locus on chromosome 11q13 has been found in a subset of parathyroid glands from patients with refractory hyperparathyroidism. To clarify the role of the MEN1 gene in parathyroid tumorigenesis, we analyzed 81 parathyroid glands from 22 Japanese uremic patients for allelic loss on chromosomal arm 11q13 DNA using 3 flanking markers (PYGM, D11S4946, and D11S449) and for mutations of the MEN1-coding exons by PCR-based single strand conformation polymorphism analysis and sequencing. Allelic loss on 11q13 was observed in 6 glands (7%), and 1 of 6 demonstrated a previously unrecognized somatic frameshift deletion (331delG) of the MEN1 gene. This mutation would probably result in a nonfunctional menin protein, consistent with a tumor suppressor mechanism. Clinical and pathological characteristics of hyperparathyroidism were unrelated to the presence or absence of loss of heterozygosity on 11q13 and MEN1 gene mutations. These observations indicate that somatic inactivation of the MEN1 gene contributes to the pathogenesis of uremia-associated parathyroid tumors, but its role in this disease appears to be very limited.

Phosphate regulation of vascular smooth muscle cell calcification.

Vascular calcification is a common finding in atherosclerosis and a serious problem in diabetic and uremic patients. Because of the correlation of hyperphosphatemia and vascular calcification, the ability of extracellular inorganic phosphate levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro was examined. HSMCs cultured in media containing normal physiological levels of inorganic phosphate (1.4 mmol/L) did not mineralize. In contrast, HSMCs cultured in media containing phosphate levels comparable to those seen in hyperphosphatemic individuals (>1.4 mmol/L) showed dose-dependent increases in mineral deposition. Mechanistic studies revealed that elevated phosphate treatment of HSMCs also enhanced the expression of the osteoblastic differentiation markers osteocalcin and Cbfa-1. The effects of elevated phosphate on HSMCs were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor phosphonoformic acid, to dose dependently inhibit phosphate-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. With the use of polymerase chain reaction and Northern blot analyses, the NPC in HSMCs was identified as Pit-1 (Glvr-1), a member of the novel type III NPCs. These data suggest that elevated phosphate may directly stimulate HSMCs to undergo phenotypic changes that predispose to calcification and offer a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions. The full text of this article is available at http://www.circresaha.org.

Chemically prepared hevein domains: effect of C-terminal truncation and the mutagenesis of aromatic residues on the affinity for chitin.

Chemically prepared hevein domains (HDs), N-terminal domain of an antifungal protein from Nicotiana tabacum (CBP20-N) and an antimicrobial peptide from Amaranthus caudatus (Ac-AMP2), were examined for their affinity for chitin, a beta-1,4-linked polymer of N-acetylglucosamine. An intact binding domain, CBP20-N, showed a higher affinity than a C-terminal truncated domain, Ac-AMP2. The formation of a pyroglutamate residue from N-terminal Gln of CBP20-N increased the affinity. The single replacement of any aromatic residue of Ac-AMP2 with Ala resulted in a significant reduction in affinity, suggesting the importance of the complete set of three aromatic residues in the ligand binding site. The mutations of Phe18 of Ac-AMP2 to the residues with larger aromatic rings, i.e. Trp, beta-(1-naphthyl)alanine or beta-(2-naphthyl)alanine, enhanced the affinity, whereas the mutation of Tyr20 to Trp reduced the affinity. The affinity of an HD for chitin might be improved by adjusting the size and substituent group of stacking aromatic rings.

Peroxisome proliferator-activated receptor-gamma agonists increase vascular endothelial growth factor expression in human vascular smooth muscle cells.

Vascular endothelial growth factor (VEGF), expressed in a variety of mesenchymal cells including vascular smooth muscle cells (VSMC), is a potent mitogen for endothelial cells, and is used clinically applied for ischemic disease of peripheral vessels. To determine whether peroxisome proliferator-activated receptor gamma (PPARgamma) regulates VEGF production in VSMC, we examined VEGF secretion from VSMC treated with PPAR agonists. Troglitazone increased VEGF secretion in a time- and dose-dependent manner (261 +/- 35% with 25 mM of troglitazone for 24 h), and also increased levels of VEGF mRNA. VEGF secretion was also increased by other PPARgamma agonists, pioglitazone, LY171883, and 15d-PGJ2 (224 +/- 17.1%, 247 +/- 36.8% and 171 +/- 7.8%, respectively), but not the PPARgamma agonists bezafibrate and Wy14643 (85.2 +/- 1.5%, 94.6 +/- 3.2, respectively). Our findings suggest that thiazolidinediones might be useful for the therapeutic angiogenesis for ischemic artery disease.

Mechanism of atherosclerotic calcification.

Calcification is almost invariably associated with atherosclerotic plaque lesions. Recent data suggest that plaque calcification is an active, regulated process similar to osteogenesis. In order to clarify the mechanism of plaque calcification, we developed an in vitro model of vascular calcification by utilizing bovine vascular smooth muscle cells (BVSMCs). This model is useful in that diffuse and massive calcification can be induced within 2 weeks and thereby biochemical analyses of vascular calcification can be performed. We have analyzed several aspects of vascular calcification by using this model and demonstrated as follows: 1) in vitro calcification of BVSMCs is regulated by calciotropic hormones and BVSMCs are equipped with a unique autocrine and/or paracrine system regulating calcium metabolism. 2) Sodium-dependent phosphate cotransport plays a crucial role in BVSMC calcification as well as in mineralization of skeletal tissues. 3) BVSMCs acquire osteoblastic phenotype under certain conditions. Finally, we discuss the roles of macrophages in the development of atherosclerotic calcification. Interferon-gamma (IFN-gamma) induces gene expression of 25-hydrovitamin D-1 alpha-hydroxylase (1 alpha OHase) and its activity in macrophages. Since 1 alpha OHase can locally convert 25-hydroxyvitamin D into 1 alpha, 25-dihydroxyvitamin D (1,25(OH)2D), an active metabolite of vitamin D, it is suggested that local production of 1,25(OH)2D by macrophages may promote atherosclerotic calcification. Moreover, macrophages may be involved in the phenotypic changes of vascular smooth muscle cells (VSMCs) to acquire calcifying capacity. Therefore, the phenotypic changes of VSMCs in atherosclerotic plaque may contribute to the development of atherosclerotic calcification.

The SCG10-related gene family in the developing rat retina: persistent expression of SCLIP and stathmin in mature ganglion cell layer.

Neuronal growth-associated proteins (GAPs), such as GAP-43 and SCG10, are thought to play crucial roles in both axonal and dendritic outgrowth during neural development and regeneration, although the underlying mechanisms remain largely unknown. The recent finding that SCG10 is a microtubule regulator and also the identification of RB3 and SCLIP as two new SCG10-related members prompted us to investigate the roles of SCG10-related family in neural development, using the retina as a model system. We determined the temporal expression and the spatial distribution of SCG10-related mRNAs in the developing rat retina. Semiquantitative analysis by RT-PCR revealed that in prenatal retina, levels of SCG10 and stathmin mRNAs were higher than those of RB3 and SCLIP. In the postnatal retina, the level of SCLIP increased, whereas the level of RB3 remained low. In situ hybridization revealed that GAP-43 and all of the SCG10-related family mRNAs were present in the retinal ganglion cells (RGCs) at all stages of retinal development, and that stathmin mRNA was present in mitotic neuroblastic cells. Differential expression of SCG10 and other members of the family became more evident as retinal development proceeded; SCG10 and RB3 expression were relatively specific in the RGCs and amacrine cells, whereas SCLIP was also evident in bipolar and horizontal cells. Stathmin mRNA was highly expressed both in the RGCs and other interneurons. These results indicate that multiple SCG10-related proteins are expressed in single neurons including RGCs, and suggest that these nGAPs play similar but distinct roles in differentiation and functional maintenance of retinal neurons.

Chemopreventive effect of 24R,25-dihydroxyvitamin D(3) in N, N'-dimethylhydrazine-induced rat colon carcinogenesis.

In this study we investigated the effects of 24R,25-dihydroxyvitamin D(3) [24R,25(OH)(2)D(3)] on N,N'-dimethylhydrazine (DMH)-induced rat colon carcinogenesis. For experiments 1 and 2, 50 F344 male, 6-week-old rats were divided into five groups in each experiment. Animals were given s.c. injections of DMH once a week for 4 weeks. Those in groups 1-5 were given 24R,25(OH)(2)D(3) in the diet (10, 5, 2.5, 1.25 or 0 p.p.m., respectively) during the post-initiation stage in experiment 1 and during the initiation stage in experiment 2. At termination, the numbers of aberrant crypt foci (ACF) in the rat colonic mucosa were decreased dose-dependently in rats treated with 24R,25(OH)(2)D(3) during the post-initiation stage, but not in the initiation stage. For experiment 3, 15 male, 9-week-old rats were divided into three groups and given 24R,25(OH)(2)D(3) in the diet (10, 5 or 0 p.p.m.). Animals were injected with 5-bromo-2'-deoxyuridine (BrdU) i.p. 1 h before death to examine DNA synthesis in the colon mucosa. BrdU labeling indices were decreased dose-dependently in colonic crypts of rats treated with 24R, 25(OH)(2)D(3). In experiment 4, using the multicarcinogenic protocol we could analyze our data with respect to not only one separate organ, but at the organism level. Sixty-eight male, 6-week-old rats were treated with DMH, N-methylnitrosourea, 2, 2'-dihydroxy-di-n-propylnitrosamine, diethylnitrosamine and N-butyl-N-(4-hydroxybutyl)nitrosamine in weeks 1-4 and were then given 24R,25(OH)(2)D(3) in the diet (5, 1 or 0 p.p.m.) throughout weeks 5-30. Examination of the development of tumors and preneoplastic lesions in various organs revealed that 24R, 25(OH)(2)D(3) inhibited colonic tumor development significantly but exerted no effects on tumor induction in other organs. In conclusion, these results strongly indicate that 24R,25(OH)(2)D(3) inhibits colon carcinogenesis specifically, without any enhancement of carcinogenesis in other organs, when administered in the post-initiation phase.

Inverse relationship between circulating oxidized low density lipoprotein (oxLDL) and anti-oxLDL antibody levels in healthy subjects.

Oxidized low density lipoprotein (oxLDL) has been implicated in the pathogenesis of atherosclerosis. Recent studies have shown that immunization of animals with oxLDL results in suppression of atherogenesis. Antibody against oxLDL (oxLDL Ab) is detectable in human sera, although its biological significance is not well established. We examined the relationship between oxLDL Ab titer and circulating oxLDL level in 130 healthy Japanese subjects. OxLDL was measured as apolipoprotein (apo) B-containing lipoproteins carrying oxidized phosphatidylcholines by a sensitive ELISA. IgG class oxLDL Ab titer was measured by ELISA. Plasma oxLDL concentration was very low and it corresponded on average to one to two out of 1000 apoB-containing lipoproteins in plasma. Plasma oxLDL correlated positively with LDL cholesterol and inversely with oxLDL Ab titer. These associations remained significant and independent in multiple regression analysis including age, gender, smoking, and high-density lipoprotein cholesterol. These data indicate that healthy subjects have a very low concentration of oxLDL in the circulation, and that oxLDL Ab titer is in an inverse relationship with plasma oxLDL concentration in this population. Although these results suggest that oxLDL Ab may play a role in maintaining the low level of plasma oxLDL, its role in atherogenesis awaits further studies.

Correlation between the intima-media thickness of the carotid artery and aortic pulse-wave velocity in patients with type 2 diabetes. Vessel wall properties in type 2 diabetes.

OBJECTIVE: The aim of this study was to assess the relationship between atherotic (structural) and sclerotic (functional) changes in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: Aortic distensibility and carotid intimal-media thickness (IMT) were evaluated using carotid-femoral aortic pulse-wave velocity (a-PWV) and high-resolution B-mode ultrasonography in 271 patients with type 2 diabetes and 285 age-matched control subjects. RESULTS: a-PWV and carotid IMT were significantly higher in the patients than in the control subjects in all age-groups (P < 0.0001, respectively). The carotid IMT and a-PWV were significantly correlated with age in both the patients with type 2 diabetes and control subjects. There was a significant positive relationship between the carotid IMT and a-PWV in both the patients (r = 0.482, P < 0.0001) and control subjects (r = 0.424, P < 0.0001). The slope of the regression line for the carotid IMT to the a-PWV was significantly steeper in the diabetic patients than in the control subjects (P < 0.05). Multiple regression analysis in all subjects showed that age, diabetic state, and cigarette smoking were independently common risk factors for the increase in carotid IMT and a-PWV. In the diabetic patients, the independent risk factors associated with the carotid IMT were age, hyperlipidemia, and duration of diabetes (R2 = 0.232, P < 0.0001), while those associated with a-PWV were age and duration of diabetes (R2 = 0.334, P < 0.0001). CONCLUSIONS: The results indicated that diabetic patients showed more advanced changes in atherosis than that in sclerosis as compared with age- and sex-matched control subjects. Such atherotic changes in diabetic patients may be associated with hyperlipidemia.

Distinct role of cAMP and cGMP in the cell cycle control of vascular smooth muscle cells: cGMP delays cell cycle transition through suppression of cyclin D1 and cyclin-dependent kinase 4 activation.

cAMP and cGMP are known to suppress vascular smooth muscle cell (SMC) proliferation. In this study, our aim was to delineate the molecular mechanism underlying cAMP and cGMP suppression of cell cycle transition in human SMCs. cAMP inhibits both platelet-derived growth factor-stimulated cyclin-dependent kinase (cdk) 2 and cdk4 activation through upregulation of the cdk2 inhibitor p27(Kip1) and downregulation of cyclin D1 expression, which leads to a complete arrest of the cells in phase G(1). In contrast, cGMP inhibits cyclin D1 expression, inhibits cdk4 activation, and delays platelet-derived growth factor-mediated cdk2 activation, resulting in a delay in G(1)/S transition. A transient increase in p27(Kip1) in cdk2 immunoprecipitates, without changes in total cellular p27(Kip1) levels, correlates with the delay in cdk2 activation caused by cGMP. Thus, cAMP and cGMP differentially affect cell cycle through distinct regulation of cell cycle molecules in human SMCs.

Shape and energetics of a cavity in c-Myb probed by natural and non-natural amino acid mutations.

The shape and the energetics of a functional cavity in the R2 subdomain (90-141) of the c-Myb DNA-binding domain were investigated by spectroscopy and thermodynamic analysis. We focused on the valine 103 residue located in front of the cavity. Nine mutants, in which valine 103 was substituted with alanine, 2-aminobutyric acid, norvaline, norleucine, leucine, isoleucine, allo -isoleucine, cyclohexylglycine, and cyclohexylalanine, were chemically synthesized and analyzed. These mutants provided a wide distribution of sizes which ranged from forming additional cavity space to filling and overflowing the cavity space. Temperature-scanning circular dichroism measurements and differential scanning calorimetry revealed a linear relationship between the van't Hoff enthalpy and the thermal transition temperature for the cavity-filling mutations. On the other hand, the mutants with side-chains larger than the side-chain of leucine resulted in a relatively low transition enthalpy and temperature, most likely due to the exposure of the side-chain to solvent and the increase in the entropy of the folded states. Branching at the beta-carbon atom reduced the unfolding free energy due to the steric constraint in the cavity. In particular, the mutational elongation of the side-chain from beta-carbon to the trans -to-CO direction proved to be more hindered than that from beta-carbon to the trans -to-NH. The unfolding free energy versus side-chain volume formed a bell-shaped plot with a maximum free energy for the leucine mutant. The difference in the transition free energy for cavity-filling mutants with beta-unbranched side-chains were two to four times larger than the difference in the transfer energy from organic solvent to water. Therefore, the increase in unfolding free energy would most likely be attributed to van der Waals interactions in the cavity wall, which would be a origin of stabilization by the sliding of tryptophan 95 into the cavity upon DNA binding.

Dexamethasone enhances In vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells.

Vascular calcification is often associated with atherosclerotic lesions. Moreover, the process of atherosclerotic calcification has several features similar to the mineralization of skeletal tissue. Therefore, we hypothesized that vascular smooth muscle cells might acquire osteoblastic characteristics during the development of atherosclerotic lesions. In the present study, we investigated the effect of dexamethasone (Dex), which is well known to be a potent stimulator of osteoblastic differentiation in vitro, on vascular calcification by using an in vitro calcification model. We demonstrated that Dex increased bovine vascular smooth muscle cell (BVSMC) calcification in a dose- and time-dependent manner. Dex also enhanced several phenotypic markers of osteoblasts, such as alkaline phosphatase activity, procollagen type I carboxy-terminal peptide production, and cAMP responses to parathyroid hormone in BVSMCs. We also examined the effects of Dex on human osteoblast-like (Saos-2) cells and compared its effects on BVSMCs and Saos-2 cells. The effects of Dex on alkaline phosphatase activity and the cAMP response to parathyroid hormone in BVSMCs were less prominent than those in Saos-2 cells. Interestingly, we detected that Osf2/Cbfa1, a key transcription factor in osteoblastic differentiation, was expressed in both BVSMCs and Saos-2 cells and that Dex increased the gene expression of both transcription factors. These findings suggest that Dex may enhance osteoblastic differentiation of BVSMCs in vitro.