Natural polyamines, spermine, spermidine, and putrescine, play a pivotal role in the regulation of gene expression; therefore, the age-dependent decreases and the disease-dependent increases in polyamine synthesis suggest a possible contribution of polyamines to the age-related and disease-associated changes in cellular function. In this study, we examined the effects of polyamines on the cellular function and the expression of adhesion molecules on human PBMCs from healthy volunteers. Flow cytometry revealed that PBMCs cultured with spermine decreased mean fluorescent intensities (MFIs) of CD11a and CD18 in the lymphocyte light-scattered region, but not in the monocyte region. This suppression was observed in a dose- and time-dependent manner and found nonspecifically on all cell subsets we tested (CD3+, CD4+, CD8+, CD19+, CD45RA+, CD45RO+, CD4+CD45RA+, CD4+CD45RO+, CD8+CD45RA+, CD8+CD45RO+). The decreases of CD11a and CD18 MFIs were accompanied by the decrease in adherent capacity of PBMCs to HUVECs. Spermine did not hinder cell activities or cell viability. Among 42 healthy volunteers (mean, 49.5 years old; from 26 to 69), blood spermine levels inversely correlated with the CD11a MFIs of cells in the lymphocyte region (r = −0.48; p = 0.001), but not with those in the monocyte region. The effects of spermidine seemed weaker than those of spermine, and blood spermidine levels had no correlation with CD11a MFIs of the lymphocyte region. Putrescine had no effect on the expressions of membrane molecules. Polyamines, especially spermine, decrease LFA-1 (CD11a/CD18) expression on human lymphocyte and adhesion capacity of PBMCs to HUVECs.

Background— Subclinical mutations in genes associated with the congenital long-QT syndromes (LQTS) have been suggested as a risk factor for drug-induced LQTS and accompanying life-threatening arrhythmias. Recent studies have identified genetic variants of the cardiac K + channel genes predisposing affected individuals to acquired LQTS. We have identified a novel Na + channel mutation in an individual who exhibited drug-induced LQTS. Methods and Results— An elderly Japanese woman with documented QT prolongation and torsade de pointes during treatment with the prokinetic drug cisapride underwent mutational analysis of LQTS-related genes. A novel missense mutation (L1825P) was identified within the C-terminus region of the cardiac Na + channel ( SCN5A ). The L1825P channel heterologously expressed in tsA-201 cells showed Na + current with slow decay and a prominent tetrodotoxin-sensitive noninactivating component, similar to the gain-of-function phenotype most commonly observed for SCN5A -associated congenital LQTS (LQT3). In addition, L1825P exhibited loss of function Na + channel features characteristic of Brugada syndrome. Peak Na + current density observed in cells expressing L1825P was significantly diminished, and the voltage dependence of activation and inactivation was shifted toward more positive and negative potentials, respectively. Conclusions— This study demonstrates that subclinical mutations in the LQTS-related gene SCN5A may predispose certain individuals to drug-induced cardiac arrhythmias.

Well-ordered architectures of self-assembling porphyrins [1] have been attracting considerable interest in light of potential applications in material science, [2] template-directed synthesis, [3] reaction catalysis, [4] and duplication of photosynthetic functions of light harvesting and charge separation. [5] Among these, the coordination interaction between zinc porphyrin and pyridine groups have been often used to construct selfassembled porphyrin architectures both in infinite and discrete forms. [1±6] Fleischer and Shachter have reported a linear infinite polymeric aggregate from 5-p-pyridyl-10,15,20-triphenylporphyrinato zinc(ii), [6] whereas Hunter and co-workers reported the formation of a cyclic tetramer from a zinc(ii) porphyrin bearing a p-(iso-nicotinamide)phenyl group [7] and Imamura and co-workers reported a similar cyclic tetramer from 5-p-pyridyl-10,15,20triaryl porphyrinato ruthenium(ii), [8] both without X-ray crystal structures. Herein, we report a self-assembling cyclic tetramer of 5-p-pyridyl-15-(3,5-di-tert-butylphenyl)porphyrinato zinc(ii) (Z1) with its X-ray structure and a self-assembling box-shape tetramer of meso ± meso-linked bis{5-p-pyridyl-15-(3,5-di-octyloxyphenyl)porphyrinato zinc(ii)} (Z2; Scheme 1). In the latter case, a very stable supramoleuclar aggregate is constructed as a result of many cooperative interactions with simultaneous structural rigidification, as suggested by its unique absorption and fluorescence spectra. 5-p-Pyridyl-15-(3,5-di-tert-butylphenyl)porphyrin (H1) was prepared by condensation of 4-pyridinecarboxaldehyde and 3,5-di-tert-butylbenzaldehyde with 2,2'-dipyrrylmethane in 8 % yield, and subsequent zinc insertion with Zn(OAc) 2 afforded Z1 quantitatively. The 1 H NMR spectrum of Z1 in CDCl 3 is concentration independent at > 3 mm and exhibits large upfield shifts for the pyridyl protons at d 6.18 and 2.17 ppm in comparison to those of H1 at d 8.29 and 7.81 ppm, which indicates the coordination of the pyridyl group to zinc(ii) porphyrin. As judged from the chemical shifts of the pyridyl group, Z1 forms a similar aggregate in C 6 D 6 (d 5.94 and 2.59 ppm) but exists in a monomeric form in [D 8 ]THF (d 8.97 and 8.31 ppm). The latter result suggested the coordination of THF to the zinc center, thus preventing self-assembling. Vapor pressure osmometry (VPO) in CHCl 3 afforded average molecular weights of 2760 AE 370 g mol À1 for Z1 and 550 AE 20 gmol À1 for H1 in a range of 3.0 ± 13.0 Â 10 À3 m, which correspond to (Z1) 4 and monomeric H1, respectively. The absorption spectrum of Z1 in CHCl 3 is concentration dependent in an range of roughly 10 À6 ± 10 À5 m and shows the Q-band at 540 nm at < 10 À6 m and at 549 nm at > about 10 À5 m (Figure 1), which correspond, respectively, to four-coordinate and five-coordinate zinc porphyrin units. A good fit for the observed sigmoidal curve is obtained by assuming porphyrin tetramer formation ((Z1) 4 ), which gives an association constant of K 4 1.4 Â 10 15 m À3 . [9]

This paper is available online at http://dmd.aspetjournals.org ABSTRACT:The expression levels of mRNAs for MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), and cytochrome P450 3A (CYP3A) in Caco-2 cells were quantitatively compared with those in human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas. Caco-2 cells (passages 36-88) were kindly supplied by several laboratories in Japan. Human duodenal enterocytes were obtained from five healthy male volunteers. Normal colorectal tissues and colorectal adenocarcinomas were simultaneously obtained from seven patients with primary colorectal adenocarcinoma. MDR1, MRP1, MRP2, and CYP3A mRNA levels were determined by real-time quantitative polymerase chain reactions (PCR). Relative concentrations of mRNAs for target proteins (MDR1, MRP1, MRP2, and CYP3A) and glyceraldehyde-3-phosphate dehydrogenase in Caco-2 cells were 1.00 ؎ 0.15, 1.02 ؎ 0.06, 0.94 ؎ 0.10, and 0.68 ؎0.60, respectively, and those in human enterocytes were about 12-, 3-, 7-, and 8000-fold higher than in the Caco-2 cells, respectively. In contrast, MDR1, MRP1, and CYP3A mRNA levels in Caco-2 cells were comparable to those in normal colorectal tissue and colorectal adenocarcinoma.

3D-OCT confirmation of the recrossing into the jailed side branch is feasible during PCI and may help to achieve distal rewiring and favourable stent positioning against the side branch ostium, leading to reduction in ISA and potentially better clinical outcomes.

Purpose: RUNX3 is a known tumor suppressor gene in several carcinomas. Aberration in RUNX3 expression has not been described for cutaneous melanoma. Therefore, we assessed the expression of RUNX3 in cutaneous melanoma and its regulatory mechanisms relative to tumor progression. Experimental Design: The expression of RUNX3 mRNA and miR-532-5p (microRNA) was assessed in melanoma lines and in primary and metastatic melanoma tumors. Results: RUNX3 mRNA expression was down-regulated in 11 of 11 (100 %) metastatic melanoma lines relative to normal melanocytes (P < 0.001). Among 123 primary and metastatic melanoma tumors and 12 normal skin samples, RUNX3 expression was significantly down-regulated in primary melanomas (n = 82; P = 0.02) and in melanoma metastasis (n = 41; P < 0.0001) versus normal skin (n = 12). This suggested that RUNX3 down-regulation may play a role in the development and progression of melanoma. RUNX3 promoter region hypermethylation was assessed as a possible regulator of RUNX3 expression using methylation-specific PCR. Assessment of RUNX3 promoter region methylation showed that only 5 of 17 (29%) melanoma lines, 2 of 52 (4%) primary melanomas, and 5 of 30 (17%) metastatic melanomas had hypermethylation of the promoter region. A microRNA (miR-532-5p) was identified as a target of RUNX3 mRNA sequences. miR-532-5p expression was shown to be significantly up-regulated in melanoma lines and metastatic melanoma tumors relative to normal melanocytes and primary melanomas, respectively. To investigate the relation between RUNX3 and miR-532-5p, anti^miR-532-5p was transfected into melanoma lines. Inhibition of miR-532-5p up-regulated both RUNX3 mRNA and protein expression. Conclusions: RUNX3 is down-regulated during melanoma progression and miR-532-5p is a regulatory factor of RUNX3 expression.The prognosis for patients with American Joint Committee on Cancer (AJCC) stage I/II melanoma is excellent, with an average 10-year survival rate of 85% (1). However, as melanoma progresses from localized to metastatic disease, survival drops significantly. The 10-year survival rate for AJCC stage IV disease is less than 10% (1). A better understanding of the regulating factors contributing to melanoma tumor growth, progression, and metastases is needed.Three members of the Runt-related (RUNX) family of genes, RUNX1, RUNX2, and RUNX3 transcription factors, are known as developmental regulators important in the inception and progression of a variety of human cancers and experimentally induced mouse tumors (2 -8). RUNX are transcription factors that are known to function as scaffolds and interact with coregulatory factors often involved in tissue differentiation (9). RUNX proteins are located in the nucleus, whereby downregulation of function has been linked to various cancers (9). Studies have also shown RUNX proteins to regulate gene expression by interacting with chromatin remodeling enzymes (10). RUNX3, in particular, has been shown to be involved in gastric tumor progression. In gastric cancer and other ...

Abstract-Experiments were carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (50 nL) of N-methyl-D-aspartic acid (1, 5, and 10 mmol/L), but not artificial cerebrospinal fluid, into the hypothalamic arcuate nucleus (ARCN) elicited increases in mean arterial pressure (5.7Ϯ0.5, 13.2Ϯ1.4, and 17.3Ϯ1.1 mm Hg, respectively) and heart rate (24.3Ϯ4. Key Words: blood pressure Ⅲ heart rate Ⅲ intrathecal injection Ⅲ microinjection Ⅲ N-methyl-D-aspartic acid Ⅲ sympathetic nerve activity T he hypothalamic arcuate nucleus (ARCN) is located bilaterally at the base of the third ventricle. Direct projections from the ARCN to the intermediolateral cell column of the spinal cord (IML), rostral ventrolateral medullary pressor area (RVLM), nucleus tractus solitarius, dorsal motor nucleus of the vagus, parabrachial nucleus, raphe nuclei, periaqueductal gray, and hypothalamic paraventricular nucleus (PVN) have been identified. 1-3 These reports suggest that the ARCN may be involved in the central regulation of cardiovascular function. There are very few studies in which the ARCN has been stimulated chemically to evaluate its role in cardiovascular or sympathetic nerve regulation. 4 -6 Therefore, a systematic study was carried out to investigate the cardiovascular effects of ARCN stimulation. Methods General ProceduresExperiments were done in adult male Wistar rats (Charles River Laboratories, Wilmington, MA) weighing 300 to 360 g (nϭ91). All of the animals were housed under controlled conditions with a 12:12-hour light-dark cycle. Food and water were available to the animals ad libitum. The experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the approval of the institutional animal care and use committee of the university.The general procedures have been described in detail elsewhere. 7 Briefly, the rats were anesthetized with urethane (1.2 to 1.4 g/kg; injected IV in divided doses). The absence of a pressor response and/or withdrawal of the limb in response to pinching of a hind paw indicated that the rats were properly anesthetized. The rats were artificially ventilated, and end-tidal CO 2 was maintained at 3.5% to 4.5%. Rectal temperature was maintained at 37.0Ϯ0.5°C. Blood pressure and heart rate (HR) were recorded by standard techniques. VagotomySilk sutures were placed loosely around the vagus nerves bilaterally for subsequent identification and sectioning of the nerves. Microinjections Into the ARCNDetails of microinjection procedure are mentioned elsewhere. 7 The coordinates for the ARCN were as follows: 3.6 to 3.8 mm caudal to the bregma, 0.1 to 0.2 mm lateral to the midline, and 9.8 to 10.2 mm deep from the dura. The same coordinates for the ARCN were used

Demyelinating Charcot-Marie-Tooth disease (CMT) and chronic inflammatory demyelinating polyneuropathy (CIDP) are both demyelinating polyneuropathies. The differences in nerve enlargement degree and pattern at multiple evaluation sites/levels are not well known. We investigated the differences in nerve enlargement degree and the distribution pattern of nerve enlargement in patients with demyelinating CMT and CIDP, and verified the appropriate combination of sites/levels to differentiate between these diseases. Ten patients (aged 23-84 years, three females) with demyelinating CMT and 16 patients (aged 30-85 years, five females) with CIDP were evaluated in this study. The nerve sizes were measured at 24 predetermined sites/levels from the median and ulnar nerves and the cervical nerve roots (CNR) using ultrasonography. The evaluation sites/levels were classified into three regions: distal, intermediate and cervical. The number of sites/levels that exhibited nerve enlargement (enlargement site number, ESN) in each region was determined from the 24 sites/levels and from the selected eight screening sites/levels, respectively. The cross-sectional areas of the peripheral nerves were markedly larger at all evaluation sites in patients with demyelinating CMT than in patients with CIDP (p < 0.01). However, the nerve sizes of CNR were not significantly different between patients with either disease. When we evaluated ESN of four selected sites for screening from the intermediate region, the sensitivity and specificity to distinguish between demyelinating CMT and CIDP were 0.90 and 0.94, respectively, with the cut-off value set at four. Nerve ultrasonography is useful to detect nerve enlargement and can clarify morphological differences in nerves between patients with demyelinating CMT and CIDP.