The Biomarker Salivary SP-D May Indicate Small Airway Inflammation and Asthma Exacerbation.

BACKGROUND: Noninvasive and child-friendly biomarkers are important tools for understanding the various phenotypes of childhood asthma. Objective: The aim of this study was to examine the usefulness of salivary surfactant protein (SP) D in assessing the pathophysiology of childhood asthma. METHODS: We measured salivary concentrations of SP-D and forced oscillation technique (FOT) indexes in 19 healthy controls and 21 asthmatic children. Regression equations for the predictive values of FOT indexes were generated from healthy controls. We analyzed the correlations between salivary SP-D concentration and percentages of the predictive values of FOT indexes, as well as the severity of exacerbation. RESULTS: We found that salivary SP-D levels were higher in asthmatic children than in healthy controls. In the asthmatic children, salivary SP-D levels correlated with the percentages of predicted differences in resistance between 5 Hz and 20 Hz (%R5-R20), which represented the resistance of peripheral airways, and with the severity of asthma exacerbation. CONCLUSIONS: Salivary SP-D may reflect asthmatic inflammation in peripheral small airways and may be a useful marker for monitoring the degree of exacerbation in childhood asthma.

HLA-B is the best candidate of susceptibility genes in HLA for Japanese ulcerative colitis.

Recently, a genome-wide association study for ulcerative colitis (UC) in the UK population was reported, and several susceptibility loci including the human leukocyte antigen (HLA) region were identified. The strongest association in the HLA region was found at a 400 kb haplotype block containing HLA-DRB1. In Japanese population, previous study suggested the association between UC and HLA-B*52; however, HLA typing was determined using serotyping with the small sample size. The purpose of this study was to perform an association study in HLA-B by genotyping. A total of 320 patients with UC and 322 healthy controls were recruited in this case-control study. All subjects were Japanese. Genotyping of HLA-B was performed by polymerase chain reaction using a sequence-specific primer. When the allele frequencies were compared, significant associations were found with B*52 [odds ratio (OR) = 3.65, P = 1.6 x 10(-17), P(c) = 3.7 x 10(-16)] and B*4002 (OR = 0.52, P = 0.00030, P(c) = 0.0068). The allele frequency of B*52 was significantly higher in patients diagnosed before 40 years of age than in those diagnosed after 40 years (OR = 1.79, P = 0.010, P(c) = 0.020). A combination association map of Japanese UC using our current and previous studies showed two equal peaks of association on HLA-DRB1 and HLA-B, indicating the possible existence of two casual variants in the HLA region inside and outside the 400 kb block found in UK. We conclude that HLA-B contributes to the susceptibility to Japanese UC, especially cases with younger age of onset. The strength of association for HLA-B was equal to that for HLA-DRB1 in Japanese UC, in contrast to the UK population.

A review of medial temporal lobe and caudate contributions to visual category learning.

Here we review recent functional neuroimaging, neuropsychological and behavioral studies examining the role of the medial temporal lobe (MTL) and the caudate in learning visual categories either by verbalizeable rules or without awareness. The MTL and caudate are found to play dissociable roles in different types of category learning with successful rule-based (RB) categorization depending selectively on the MTL and non-verbalizeable information-integration (II) category learning depending on the posterior caudate. These studies utilize a combination of experimental cognitive psychology, mathematical modeling (Decision Bound Theory (DBT)) and cognitive computational modeling (the COVIS model of Ashby et al. [1998. A neuropsychological theory of multiple systems in category learning. Psychological Review 105, 442-481]) to enhance the understanding of data obtained via functional magnetic resonance imaging (fMRI). The combination of approaches is used to both test hypotheses of the cognitive model and also to incorporate hypotheses about the strategies used by participants to direct analysis of fMRI data. Examination of the roles of the MTL and caudate in visual category learning holds the promise of bridging between abstract cognitive models of behavior, systems neuroscience, neuropsychology, and the underlying neurophysiology of these brain regions.

Both N-terminal and C-terminal regions of steroid sulfatase are important for enzyme activity.

Steroid sulfatase (STS) is localized in the endoplasmic reticulum and catalyzes desulfation of 3beta-hydroxysteroid sulfates. X-linked ichthyosis (XLI) is an inherited skin disorder caused by deficiency of STS enzyme activity. We previously reported a case in which XLI with a one-base change in the STS gene and variation in amino acid Q560P developed. In this study, we performed molecular analysis to determine the importance of terminal regions of STS and the effect of mutant STS on STS enzyme activity. To examine the effect of terminal truncated STS on the enzyme activity, N- and C-terminal truncated STS expression vectors were transfected into COS-1 cells. The activity of truncated STS lacking the N-terminal regions declined, and the activity of C-terminal-truncated STS declined with extension of the truncated C-terminal region. Although the results of pulse-chase experiments showed that a one-base mutant STS (Q560P) and C-terminal-truncated STS (deltaC2 (1-559)) had no effects on protein synthesis and degradation, the mutant STS and C-terminal-truncated STS have dominant negative effect on STS enzyme activity when the STS mutant or truncated STS protein and a wild-type STS protein coexist in cells. Results of coprecipitation of the truncated STS with an STS-FLAG fusion protein showed that STS formed a dimer conformation in cells. In this study, we have shown that both the N-terminal region and C-terminal region are important for STS enzyme activity. The C-terminal mutant has a dominant negative effect on wild-type STS.

Combinations of the presence or absence of cerebral microbleeds and advanced white matter hyperintensity as predictors of subsequent stroke types.

BACKGROUND AND PURPOSE: Previous studies have shown microbleeds to be a risk factor for intracerebral hemorrhage and white matter hyperintensity (WMH) to be a risk factor for ischemic stroke. This study was performed to determine whether combinations of the presence or absence of microbleeds and advanced WMH are risk factors for subsequent recurrent stroke types. METHODS: In 266 patients with stroke, microbleeds on T2*-weighted MR images were counted, and WMH on T2-weighted images was graded. Patients were divided into 4 groups by the combinations of the presence or absence of microbleeds and advanced WMH and were followed up for stroke recurrence. RESULTS: During a mean follow-up period of 564.8 +/- 220.5 days, 26 patients developed recurrent strokes, including 10 intracerebral hemorrhages and 16 ischemic strokes. Patients with microbleeds without advanced WMH (n = 42) developed only intracerebral hemorrhages (n = 8), and the recurrence rate of intracerebral hemorrhage in those patients estimated by the Kaplan-Meier method was the highest in the 4 groups (14.3% in 1 year and 21.2% in 2 years). In contrast, patients with advanced WMH without microbleeds (n = 39) developed only ischemic strokes (n = 6), and the estimated recurrent rate of ischemic stroke in those patients was the highest in the 4 groups (10.5% in 1 year and 17.4% in 2 years). Cox proportional hazards regression analysis revealed that microbleeds were associated with intracerebral hemorrhage (hazard ratio [HR], 85.626; 95% confidence interval [CI], 6.344-1155.649) and that advanced WMH was negatively associated with intracerebral hemorrhage (HR, 0.016; 95% CI, 0.001-0.258). Advanced WMH was associated with ischemic stroke (HR, 10.659; 95% CI, 2.601-43.678). CONCLUSION: It appears that patients at high risk of subsequent intracerebral hemorrhage or ischemic stroke can be identified by combinations of the presence or absence of microbleeds and advanced WMH.

Development of a recombinant yeast assay to detect ah-receptor ligands.

Endocrine systems of humans and animals are disturbed by dioxin-like compounds, which are ligands of the aryl hydrocarbon receptor (AhR). It is important to determine the accumulation of dioxin-like compounds in the environment for maintenance of human health. In this study, we developed a new method for screening ligands of the AhR using a yeast hybrid system. Reporter genes constructed by the insertion of dioxin response elements were integrated into HIS and lacZ yeast genomes. Then yeast was transformed with GAL4-activated domain-fused AhR and aryl hydrocarbon receptor nuclear translocator expression constructs. At 10(-4) M of beta-naphthoflavone, which is an AhR ligand, the absorbance of optical density at 600 nm (OD 600) and beta-galactosidase activity was significantly increased. beta-galactosidase activity was increased when the concentration of 3-methylcholanthrene (MC) was increased. ATP concentration increased as concentration of MC increased up to 10(-10) M but decreased at higher concentrations. The concentration of ATP in the cell suspensions increased linearly with OD 600, used as an index of cell density (r(2) = 0.8366, F = 209.9, p < 0.0001, n = 44). The established yeast assay could possibly be used in the future to detect dioxin-like compounds in environmental samples.

Ultrastructural study of the extra-articular Leeds-Keio ligament prosthesis.

BACKGROUND: There have been several histological studies of the Leeds-Keio ligament in anterior cruciate ligament reconstruction, but there have been few of the Leeds-Keio ligament in the extra-articular portion. AIMS/METHODS: To report the histological and ultrastructural findings of two cases of medial patellofemoral ligament reconstruction using the Leeds-Keio ligament, removed 6.1 years and 8.7 years after implantation. RESULTS: In both cases, the tissue over the Leeds-Keio ligament was a ligament-like tissue. Electron microscopy showed that the diameter of the collagen fibrils in the tissue over the Leeds-Keio ligament was unimodal in the case investigated 6.1 years after implantation but bimodal in the case investigated after 8.7 years. CONCLUSIONS: The tissue over the Leeds-Keio ligament may continue to grow with prolonged periods of mechanical stress.

RIP 140 modulates transcription of the steroidogenic acute regulatory protein gene through interactions with both SF-1 and DAX-1.

Coregulators have been suggested to act as a bridging apparatus between nuclear receptors and the transcriptional machinery. The orphan receptor SF-1 plays a role in controlling the basal and cAMP-stimulated expression of the human steroidogenic acute regulatory protein gene. DAX-1 is the gene responsible for X-linked adrenal hypoplasia congenita and blocks steroid biosynthesis by impairing the expression of steroidogenic acute regulatory protein. In the present study we examined the role of coregulators in the actions of SF-1 and DAX-1 on the human steroidogenic acute regulatory protein promoter. We found that the coregulator RIP 140 interacts with SF-1 in the yeast two-hybrid system. Glutathione-S-transferase pull-down assays and coimmunoprecipitations confirmed the interaction between RIP 140 and SF-1. RIP 140 was also shown to interact with DAX-1. When an RIP 140 expression vector was introduced into Y-1 cells, basal and cAMP-stimulated human steroidogenic acute regulatory protein promoter activities decreased. The inhibitory effect of RIP 140 on human steroidogenic acute regulatory protein promoter activity was dependent upon the presence of SF-1. The cAMP response of an SF-1 response element was inhibited by both RIP 140 and DAX-1 expression vectors at low concentrations of plasmids. We conclude that RIP 140 binds to the orphan nuclear receptor SF-1 and DAX-1 and modulates their actions on the human steroidogenic acute regulatory protein promoter.

Triplet repeat polymorphism in the MICA gene in HLA-B27 positive and negative caucasian patients with ankylosing spondylitis.

Previously, we reported a triplet repeat polymorphism in the transmembrane region within the MICA gene closely linked to HLA-B in a limited number of B27-positive Caucasian patients with ankylosing spondylitis (AS) (N = 48). In this study, we enrolled much more patients including some negative for B27, 162 AS subjects consisting of 140 B27-positive, and 22 B27-negative patients. The microsatellite allele consisting of 4 repetitions of (GCT/AGC) (A4 allele) was present at a significantly higher phenotype frequency in the patient group than in the ethnically matched control group (Pc < 0.000001). However, the frequency of the A4 allele was not significantly higher in the B27-positive and B27-negative patient groups, as compared to the B27-positive and B27-negative control groups, respectively. The higher phenotype frequency of the A4 allele in the patient group was supposed to be due to a strong linkage disequilibrium between the MICA and HLA-B genes. Thus, the possibility that the MICA gene is involved in the pathogenesis of AS can be excluded, supporting the hypothesis of a primary association of AS with HLA-B27.

Suppressive effects of novel ferulic acid derivatives on cellular responses induced by phorbol ester, and by combined lipopolysaccharide and interferon-gamma.

A total of 23 ferulic acid (FA) derivatives were synthesized, and investigated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus (EBV) activation and superoxide (O(2)(-)) generation. Most of the derivatives showed significant EBV activation suppression or cytotoxicity at a concentration of 100 microM, with FA15 as the most potent suppressor. In both assays, FA6-FA17, bearing straight- or branched-alkyl side chains, exhibited marked suppression of O(2)(-) generation, with both FA16 and FA17 being highly active, while FA itself was virtually inactive. The activity differences seen between FA16/FA17 and FA are attributable, at least in part, to their cellular incorporating efficiencies. Further, both FA15 and FA21 attenuated the expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins, while FA did not. Our results suggest that these novel FA derivatives are effective chemopreventive agents.

Expression of enzyme associated with steroid hormone synthesis and local production of steroid hormone in endometrial carcinoma cells.

Endometrial carcinoma is a common malignancy of the genital tract. In the present study, we examined the expression of human steroidogenic acute regulatory (StAR) protein, P450 side-chain cleavage enzyme (P450 scc), 17alpha-hydroxylase/17,20-desmolase (P45017alpha) and aromatase (P450 arom) in endometrial carcinoma cells to clarify the ability of these cells to produce steroid hormones. The results of RT-PCR analysis showed that StAR, P450 scc and P45017alpha genes were expressed in endometrial carcinoma cells. To examine the protein expression of StAR and P450 scc, we performed Western blotting using extracts from carcinoma cells. StAR protein and P450 scc were detected in both HHUA and HOUA-1 cells. The production of pregnenolone in HHUA cells increased 2.4-fold with transfection of a StAR expression vector and 4.3-fold with transfection of an F2 side-chain cleavage system. The RT-PCR product of 3beta-hydroxysteroid dehydrogenase was present in endometrial carcinoma cells. In endometrial carcinoma cells, the production of progesterone also increased with over-expression of StAR and the F2 system. Results of steroid metabolic assays showed that endometrial carcinoma cells produced not only 17alpha-hydroxyprogesterone but also androstenedione. Endometrial carcinoma cells express enzymes that are associated with the production of steroid hormones. Locally produced steroid hormones may have effects on tumor proliferation and tumorigenesis.

Prognostic value of tumor cell proliferation and intratumor microvessel density in advanced gastric cancer treated with curative surgery.

To clarify the prognostic value of tumor cell proliferation [proliferating cell nuclear antigen (PCNA)-positive rate assessed by anti-PCNA immunostaining] and intratumor microvessel density (/0.74 mm2, assessed by anti-CD34 immunostaining) in gastric cancer, 192 advanced gastric cancer patients who underwent curative surgery were investigated. Multivariate analysis showed that the following variables were significantly related to prognosis: age (P=0.029), depth of invasion (P=0.005), lymph node metastasis (P=0.006), lymphatic invasion (P=0.038), venous invasion (P=0.0005), PCNA-positive rate (P=0.049) and intratumor microvessel density (P=0. 011). In conclusion, tumor cell proliferation and intratumor microvessel density were independent prognostic factors in patients with advanced gastric cancer undergoing curative surgery.

Bone metabolic markers in bone metastases.

The efficacy and cost/performance benefit of radionuclide bone scintigraphy in monitoring metastatic bone activity remain controversial. Recently developed bone metabolic markers are expected to play an additional role in the diagnosis of bone metastasis. We measured osteoclastic and osteoblastic markers in 267 patients with breast cancer (100 with bone metastasis), 38 patients with prostatic cancer (25 with bone metastasis), 50 patients with lung cancer (12 with bone metastasis) and 33 patients with miscellaneous cancers (13 with bone metastasis) and compared the values in the presence and absence of bone metastasis. Bone metabolic markers, both osteoclastic and osteoblastic, increased significantly in patients with bone metastasis. In breast cancer (bone metastasis is mostly of the mixed type), osteoclastic markers were good in detecting bone metastasis. In prostatic cancer (bone metastasis is mostly osteoblastic), osteoclastic and osteoblastic markers were equally effective in detecting bone metastasis. In lung cancer (bone metastasis is mostly osteolytic), osteoclastic markers were elevated preferentially in bone metastasis. Over all, osteoclastic markers were more sensitive in the diagnosis of bone metastasis, and among osteoclastic markers, serum pyridionoline-cross-linked carboxyterminal telopeptide was the most efficient in both specificity (91.0%) and sensitivity (48.6%) for detecting bone metastasis.

The effect of the arylhydrocarbon receptor on the human steroidogenic acute regulatory gene promoter activity.

The steroidogenic acute regulatory (StAR) protein is a rate-limiting factor in steroid hormone production. The StAR protein plays a role in the movement of cholesterol from the outer membrane to the inner membrane, where cholesterol side chain cleavage enzyme exists. Dioxins, which may act as 'endocrine disruptors', mimic and antagonize endogenous hormone actions in vivo. Although the mechanism of endocrine disruption is not clear, the actions of dioxins are known to be mediated by binding to the arylhydrocarbon receptor (AhR), and it is known that dioxins act as transcription factors to endocrine-associated gene expression. In the present study, we examined the effect of the AhR on the human StAR gene promoter, and we clarified the action mechanisms of environmental endocrine disruptors. We transfected constructs containing the human StAR gene promoter sequences pGL(2) 1.3-kb StAR (nt -1293 to +39) into mouse Y-1 adrenal tumor cells and measured the promoter activity of the StAR gene. With the addition of beta-napthoflavone (betaNF), which is a ligand of AhR, to the culture medium, the activity of the StAR gene promoter increased significantly (P<0.05), and with the addition of 1 microM of betaNF, it became maximum (3.1+/-0.6-fold higher than the control value). When the AhR and ARNT were co-transfected together in Y-1 cells or human adrenocortical carcinoma H295R cells, the promoter activity of the StAR gene significantly (P<0.05) increased, to a level 1.4+/-0.01-fold higher in Y-1 cells and to a level 1.6+/-0.04-fold higher in H295R cells than the control level, when 1 microM of betaNF was added. We examined the effect of induction of cAMP with transfection with AhR or ARNT. With the addition of 1 mM 8-Br-cAMP, there were no differences between the StAR gene promoter activities in the group in which AhR and ARNT was introduced and in the group in which they were not introduced. The results suggest that AhR plays a role in the promoter activity of the human StAR gene and that the effect of AhR on StAR gene expression may cause a disturbance to the human endocrine system.

The configuration of methyl jasmonate affects paclitaxel and baccatin III production in Taxus cells.

All stereoisomers of methyl jasmonate (MJA) were prepared, and their effects on cell yield and promotion of paclitaxel (Taxol) and baccatin III production investigated in cell suspension cultures of Taxus media. (3R,7S)-MJA showed the strongest cell growth inhibition, followed by (3R,7R)-MJA. In contrast, (3S,7R)- and (3S,7S)-MJA had very low inhibitory effects, indicating that this inhibition depends largely on the (3R)-configuration. In terms of the promotion of paclitaxel and baccatin III production, (3R,7R)-MJA had the highest activity. Although it showed considerable activity at low concentration, at higher concentrations the activity was decreased due to strong inhibition of cell growth. Interestingly, paclitaxel and baccatin III contents increased even at a high (3S,7R)-MJA concentration, whereas the other isomers had the opposite effects. These findings are interpreted to suggest that the optimum configuration is (3R,7R), the (3R)-configuration not being indispensable, and that the (7R)-configuration is suitable for the promotion of paclitaxel and baccatin III production.

Thermal polymorphic transformation of p-tert-Butylcalix

Studies were made on the polymorphism of p-tert-butylcalix[4]arene derivatives having amino acid groups on the lower rim to stabilize their hydrophilic pseudocavity by circular intramolecular hydrogen bonding. The calixarenes exhibit a polymorphic transformation upon heating in the solid state. This transformation is controlled by the thermal history of solids, accompanying the change on the conformation of the calixarene skeleton and also the change of hydrogen bonding in the hydrophilic pseudocavity.

Perifoveal microcirculation in eyes with epiretinal membranes.

BACKGROUND/AIMS: Eyes with epiretinal membranes (ERMs) often have alterations of retinal vessels. The authors studied perifoveal microcirculation in eyes with epiretinal membranes (ERMs) using scanning laser ophthalmoscope (SLO) fluorescein angiography. METHODS: Mean capillary blood flow velocity (CFV) was measured as an index of perifoveal microcirculation by SLO fluorescein angiography in 26 eyes with ERMs (19 eyes with idiopathic epiretinal membranes, seven eyes with epiretinal membranes after retinal detachment surgery) before and 6 months after vitreous surgery, and in 23 healthy control subjects. RESULTS: The mean CFV was significantly reduced in eyes with ERMs compared with healthy controls (p=0.012), and the postoperative mean CFV was significantly increased compared with the preoperative mean CFV (p=0.041). CONCLUSION: Significant changes of capillary blood flow velocity in the perifoveal areas were observed between normal subjects and eyes with epiretinal membranes. This indicates that eyes with ERMs show abnormal haemodynamics in the perifoveal capillaries.

Preparation of ferulic acid and its application for the synthesis of cancer chemopreventive agents.

We suggest that chemical raw materials can best be obtained from natural resources. Ferulic acid is easily prepared in large quantities from rice bran pitch, a blackish brown waste oil with high viscosity, discharged in the process of the rice bran oil production. As an application of ferulic acid, potential cancer chemopreventive agents could be synthesized using organic synthetic methods.