Baseline MRA predicts the treatment response to vasodilator udenafil in patients with secondary Raynaud's phenomenon.

OBJECTIVES: High-resolution MR angiography (HR-MRA) demonstrates blood flow in the digital arteries, which correlates with the severity of Raynaud's phenomenon (RP). This study investigates whether baseline HR-MRA of the hand can predict the treatment response to udenafil, a new PDE5-inhibitor, in patients with secondary RP. METHODS: Baseline MRA and Doppler ultrasound were obtained in 12 patients with secondary RP. The patients were treated with udenafil 100 mg/day for 4 weeks and changes in blood flow were measured. Blood flow on MRA was scored on a 4-point scale: 0, no visible flow; 1, visible flow to the proximal phalanx; 2, to the middle phalanx; and 3, to the distal phalanx. Peak systolic velocity (PSV) was measured to determine blood flow. Paired t-test and ANOVA were used to determine the treatment response of the different MRA scores. RESULTS: On baseline MRA, 53.3% of digital arteries had an MRA score of 0, 25.8% MRA score of 1, 9.2% MRA score of 2, and 11.6% MRA score of 3. Overall, 4-week udenafil treatment improved digital flow (p<0.05) in all MRA scores. Digital arteries with MRA score 2 showed the best response with improvement in PSV by 14.5 mm/sec (p<0.01), whereas improvement in arteries of MRA scores 1 and 3 were not better than an MRA score of 0 (all, p>0.05). CONCLUSIONS: Digital arteries with moderate blood flow observed on MRA respond best to treatment with udenalfil. Therefore, baseline MRA may help predict treatment response in patients with secondary RP.

Intramyocardial transfer of hepatocyte growth factor as an adjunct to CABG: phase I clinical study.

The purpose of this phase I clinical trial was to evaluate the safety, tolerability and potential efficacy of VM202, naked DNA expressing two isoforms of hepatocyte growth factor, as an adjunct therapy to coronary artery bypass grafting (CABG) in patients with ischemic heart disease (IHD). Nine patients were assigned to receive increasing doses (0.5 to 2.0 mg) of VM202 injected into the right coronary artery (RCA) territory following completion of CABG for the left coronary artery territory. Patients were evaluated for safety and tolerability, and changes in myocardial functions were monitored via echocardiography, cardiac magnetic resonance imaging and myocardial single photon emission computed tomography throughout 6-month follow-up period. No serious complication related to VM202 was observed throughout the 6-month follow-up period. Global myocardial functions (wall motion score index, P=0.0084; stress perfusion, P=0.0002) improved during the follow-up period. In the RCA region, there was an increase in the stress perfusion (baseline vs 3-month, P=0.024; baseline vs 6-month, P=0.024) and also in the wall thickness of the diastolic and systolic phases. Intramyocardial injection of VM202 can be safely used in IHD patients with the tolerable dose of 2.0 mg. In addition, VM202 might appear to have improved regional myocardial perfusion and wall thickness in the injected region.

Oligomerization of the heptahelical G protein coupling receptors: a case for association using transmembrane helices.

The heptahelical G protein coupling receptors oligomerize extensively via transmembrane domains, in association with heterotrimeric G proteins. This provides higher affinity for agonists, conformational stability necessary for signal transduction, and protection from intracellular proteinases. The oligomerization is relevant to organismic pathophysiology and could be targeted by natural or modified agonists.

Usefulness of concurrent reading using thin-section and thick-section CT images in subcentimetre solitary pulmonary nodules.

AIM: To evaluate the differences in the characterization and recommendation for follow-up of subcentimetre solitary pulmonary nodules (SSPNs) between 5 and 1mm section CT, and to compare the assessments generated by four radiologists MATERIALS AND METHODS: Five hundred and twenty-nine patients who had SSPNs on chest CT reconstructed using both 5 and 1mm sections were enrolled. Two image subsets of 5 and 1mm CT images of each nodule were interpreted independently by four radiologists. Nodule size, consistency (solid, partly solid, non-solid), the presence of calcification, and recommendations for follow-up were evaluated. If a non-calcified solid nodule was confirmed using CT, recommendation for follow-up was based on Fleischner Society guidelines. Data assessed by each radiologist were compared, and interobserver agreements were determined using the intraclass correlation coefficients and kappa value. RESULTS: Using 1mm CT images, the nodule sizes were significantly larger than on 5mm CT images (paired t-test, p<0.01). The presence of calcification and nodule consistency were significantly different between 5 and 1mm CT images (McNemar test for the presence of calcification, p<0.01; Wilcoxon signed test for nodule consistency, p<0.01). On 1mm CT images there was significantly higher agreement regarding nodule consistency than on 5mm CT (kappa=0.78 and 0.67, respectively). CONCLUSIONS: Concurrent use of thin-section and thick-section CT can provide more accurate nodule assessment and higher interobserver agreement in SSPN.

Dimers of the neuropeptide Y (NPY) Y2 receptor show asymmetry in agonist affinity and association with G proteins.

In conditions precluding activation of G proteins, the binding of agonists to dimers of the neuropeptide Y (NPY) Y2 receptor shows two components of similar size, but differing in affinity. The dimers of all NPY receptors are solubilized as approximately 180-kDa complexes containing one G protein alpha beta gamma trimer. These heteropentamers are stable to excess agonists, chelators, and alkylators. However, dispersion in the weak surfactant cholate releases approximately 300-kDa complexes. These findings indicate that both protomers in the Y2 dimer are associated with G protein heterotrimers, but the extent of interaction depends on affinity for the agonist peptide. The G protein in contact with the first-liganded, higher-affinity protomer should have a stronger interaction with the receptor and a larger probability of activation.

In vitro analysis of promoter elements regulating transcription of the phosphoenolpyruvate carboxykinase (GTP) gene.

A cell-free system for the study of transcription from the promoter of the phosphoenolpyruvate carboxykinase (GTP) gene by using nuclear extracts from rat tissues was developed. The level of basal transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter between -490 and +73 was highest when extracts from liver nuclei, rather than kidney, spleen, and HeLa nuclear extracts, were used. A series of 5' deletions and block mutations were also tested for their effects on basal transcription in vitro. The promoter truncated to -355 had the highest rate of basal transcription, while subsequent deletion to -277 markedly decreased the rate of transcription. Further deletion of the promoter to -134 resulted in a twofold increase in the basal level of transcription compared with that of the promoter deleted to -277. However, subsequent deletion of the NF-1-CCAAT-binding transcription factor binding site or the proximal cyclic AMP (cAMP) regulatory element caused a decrease in basal transcription. Block mutations were inserted into nine specific protein-binding regions of the PEPCK promoter previously shown to be of functional significance or to bind nuclear proteins. Mutation of the TATA box resulted in a 94% decrease in the level of transcription noted with the intact promoter, while sequence substitutions within the proximal cAMP regulatory element decreased the transcription rate to 25%. The addition of the catalytic subunit of cAMP-dependent protein kinase to the in vitro system stimulated transcription from the intact promoter or from a promoter deletion to -109. However, a promoter deletion to -68, which removes the proximal cAMP regulatory element, was unresponsive to added protein kinase catalytic subunit. These findings indicate that the PEPCK promoter between -490 and +73 contains sequences responsive to hormonal and tissue-specific factors in nuclei from rat tissues. The sensitivity of this in vitro transcription system closely mimics the process regulating PEPCK transcription in rat tissues and should make it ideal for testing the function of purified transcription factors.

The role of the CCAAT/enhancer-binding protein in the transcriptional regulation of the gene for phosphoenolpyruvate carboxykinase (GTP).

Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue- and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient contransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.

Effect of postnatal catch-up growth on blood pressure in children at 3 years of age.

Size at birth and early postnatal growth rates appear to be important determinants of cardiovascular diseases. We examined whether intrauterine growth restriction or the subsequent catch-up postnatal weight gain leads to higher blood pressure in early life to confirm that size at birth and early postnatal growth rates appear to be important determinants of blood pressure changes in early life. Of 407 children born between December 2001 and November 2002 in hospital based-birth cohorts, 102 were followed up at 3 years of age (24.2%) at Ewha Womans University Hospital in Seoul, Korea. At 3 years of age, those who had a low birth weight still belonged in the lower-weight group than the others. The subjects' systolic blood pressure was correlated with their current weight (r=0.41) and weight gain (r=0.39), but not with their birth weight. Those with a higher current weight and higher weight gain based on birth weight (conditional weight gain) had the highest blood pressure. Systolic blood pressure increased by 0.2 mm Hg for every 100-g increase in weight at 3 years and, independently, by 1.5 mm Hg for every 100-unit increase in conditional weight gain. This study suggests that birth weight is not directly associated with blood pressure, but accelerated growth, which occurs mostly in those born with a low birth weight, seems to affect blood pressure in early life.

Modulation of hormone response elements by promoter environment.

Hormone response elements (HREs) are nucleotide sequences that confer onto promoters the ability to alter their transcriptional pace in response to hormones. Growing evidence indicates that the functional activity of HREs can be significantly modulated by their promoter environment, making it possible for genes containing the same HRE to display diversity in their responsiveness to a given hormone signal.

Characterization of the 5' flanking region of the gene encoding rat liver glycogen phosphorylase.

A genomic region encompassing 800 bp of the promoter-regulatory region and exon 1 of the gene (LGP) encoding rat liver glycogen phosphorylase has been isolated and characterized. Transcripts of the LGP gene initiate predominantly within an 8-bp region 48-bp upstream from the start codon. Additional transcripts were detected that initiate as far as 95 bp upstream from the start codon. To identify cis-acting sequences involved in regulating transcription, HepG2 cells were transfected with vectors containing serial deletions of the promoter-regulatory region of LGP ligated to the cat reporter gene. Two upstream regions were found to enhance transcription. One of these regions contains an alternating purine-pyrimidine sequence. LGP, which lacks a consensus TATA sequence, is like TATA-less and CAAT-less housekeeping genes in that it contains G + C-rich domains upstream from multiple transcription start points. Nuclear proteins from adult rat tissues bound in a tissue-specific fashion to one of these G + C-rich regions.

Identification of a retinoic acid response domain involved in the activation of the beta 1-adrenergic receptor gene by retinoic acid in F9 teratocarcinoma cells.

The density of beta 1-adrenergic receptors (beta 1-AR) is up-regulated upon differentiation of embryonic F9 teratocarcinoma cells by retinoic acid (RA) to the primitive endodermal phenotype. To identify the domains involved in RA-mediated activation of beta 1-AR gene transcription, three kb of 5'-flanking sequence of the beta 1-AR gene were ligated to a luciferase reporter gene and transiently transfected into F9 cells that were pre-exposed to 100 nM RA for 2 days. By generating deletions in the beta 1-AR promoter, a region between -125 and -100 was found to mediate a 3-fold induction in cells exposed to RA for an additional 2 days. Through site-directed mutagenesis of this region, it was determined that the RA responsive element (RARE) was organized as a direct repeat separated by 5 nucleotides in which the 5'-most AGGTCG half-site was between nucleotides -106 and -101 and the 3'-most AGGTCA half-site was between nucleotides -117 and -112. The RA receptor alpha (RAR alpha) isoform bound to the oligomer representing the sequences between -125 and -100 as a heterodimer complex with the retinoid X receptor alpha (RXR alpha). In a separate study, it was determined that the nucleotides between -125 and -100 are involved in thyroid hormone-mediated activation of the beta 1-AR gene in ventricular myocytes. Therefore, transcriptional activation of the beta 1-AR gene by thyroid hormone or RA involves a single binding site in the promoter.

Influence of maternal serum levels of vitamins C and E during the second trimester on birth weight and length.

OBJECTIVE: It has been known that maternal oxidative stress during pregnancy plays an important role in fetal growth. However, the association between antioxidant vitamin levels and birth outcomes is not conclusive. We investigated the relationship between maternal serum levels of vitamins C and E during the second trimester and birth weight and length. DESIGN: Prospective cohort study. SETTING: Outpatient-clinic of obstetrics, Ewha Womans University Hospital, South Korea. SUBJECTS AND METHODS: The study subjects were 239 healthy, pregnant women who visited an obstetric clinic for antenatal care, and their singleton live births, in Seoul, Korea, between August 2001 and March 2003. We measured the levels of vitamins C and E in maternal serum during the period 24-28 gestational weeks. Each woman was interviewed for dietary intake by trained interviewers during the second trimester. RESULTS: The serum concentration of maternal vitamin C during the second trimester was significantly associated with birth weight and length in the group of full-term deliveries. An increase of 1 microg/ml in the serum vitamin C level increased the birth weight by 27.2 g and the birth length by 0.17 cm. When we considered the levels of vitamins C and E together in the relationship with birth weight and length, we found that the heaviest birth weight and the longest birth length belonged to the group of upper vitamin C/upper vitamin E. However, dietary intake estimated by 24-h recall method was not a predictor of the levels of serum vitamins C and E. CONCLUSION: We found that maternal serum vitamin C levels during the second trimester were positively correlated with birth weight and length in full-term babies. We also found that birth weight and length were highest when the levels of both vitamins C and E were high. Our results indicate the importance of antioxidant nutrient balance for pregnant women who are exposed to various oxidants through food, drinking water, or inhaled air.

Expression and regulation of carnitine palmitoyltransferase-Ialpha and -Ibeta genes.

Two genes control expression of mitochondrial carnitine palmitoyltransferase-I (CPT-I), the enzyme that catalyzes the primary rate-controlling step in fatty acid oxidation. Two CPT-I isoforms have been found--a "liver" isoform (CPT-Ialpha) expressed in most tissues, but not in skeletal muscles, and a "muscle" isoform (CPT-Ibeta) expressed in muscles and adipocytes. Liver CPT-Ialpha increases dramatically at birth, but heart CPT-Ialpha is abundant in the fetus and diminishes at birth. Insulin, thyroid hormone, and fatty acids regulate expression of CPT-Ialpha in liver, whereas electrical stimulation increases CPT-Ibeta and decreases CPT-Ialpha in cardiac myocytes. Both genes are TATA-less and contain Sp1 transcription factor binding sites upstream of the start site of transcription. Multiple transcripts of both CPT-Ialpha and CPT-Ibeta exist, some of which may have roles in regulating fatty acid oxidation.

Preoperative determination of anatomic variations of the small saphenous vein for varicose vein surgery by three-dimensional computed tomography venography.

OBJECTIVE: To define the anatomical variations of small saphenous vein (SSV) for varicose vein (VV) surgery by three-dimensional computed tomography venography (3D-CTV) and to analyse the impact of this preoperative evaluation on surgical outcomes. METHODS: A total of 120 consecutive limbs with SSV insufficiency having undergone VV surgery from January 2005 until December 2007 were enrolled. The medical records and images were analysed retrospectively. RESULTS: The relationship between SSV and gastrocnemial vein (GNV) were categorized into two: (a) SSV and GNV drained to popliteal vein (PV) separately (100 limbs, 87%) and (b) SSV and GNV made common channel which drained to PV (15 limbs, 13%). Saphenopopliteal junction morphology was normal (75 limbs), severe tortuosity near PV (19 limbs), ampullary ectasia (4 limbs) and duplicated drainage to PV (2 limbs). No recurrence of VV was noted. CONCLUSIONS: CTV can provide thorough preoperative anatomic information of the SSV variations and reduce the recurrence of VV.

Finding ancient parasite larvae in a sample from a male living in late 17th century Korea.

Parasitological examination of samples from tombs of the Korean Joseon Dynasty (1392-1910) could be helpful to researchers in understanding parasitic infection prevalence in pre-industrial Korean society. Whereas most of our previous parasitological studies revealed the presence of ancient parasite eggs in coprolites of Korean mummies, a sample from a man living in late 17th century Korea proved to be relatively unique in possessing what appeared to be several species of parasite larvae. The larvae identified included Strongyloides stercoralis and Trichostrongylus spp., along with eggs of Ascaris lumbricoides, Trichuris trichiura, and Paragonimus westermani. Since ancient parasite larvae retain enough morphology to make proper species identification possible, even after long burial times, the examination of parasite larvae within ancient samples will be conducted more carefully in our future work.

The neuropeptide Y (NPY) Y2 receptors are largely dimeric in the kidney, but monomeric in the forebrain.

The neuropeptide Y(NPY) Y2 receptors are detected largely as dimers in the clonal expressions in CHO cells and in particulates from rabbit kidney cortex. However, in two areas of the forebrain (rat or rabbit piriform cortex and hypothalamus), these receptors are found mainly as monomers. Evidence is presented that this difference relates to large levels of G proteins containing the Gi alpha -subunit in the forebrain areas. The predominant monomeric status of these Y2 receptors should also be physiologically linked to large synaptic inputs of the agonist NPY. The rabbit kidney and the human CHO cell-expressed Y2 dimers are converted by agonists to monomers in vitro at a similar rate in the presence of divalent cations.

Energy dependence of RNA degradation in rabbit reticulocytes.

RNA degradation in rabbit reticulocytes was partially energy dependent. Reticulocytes were incubated for 10 h in Krebs-Henseleit bicarbonate buffer that was gassed with 95% O2-5% CO2 and contained glucose (30 mM) or 2-deoxyglucose (20 mM) and 2,4-dinitrophenol (0.2 mM). The rate of RNA degradation was reduced 41% in the presence of the metabolic inhibitors. When the buffer was gassed with 95% N2-5% CO2 and no substrate was added, the disappearance of RNA was decreased 55%. The cellular ATP content was depleted either by addition of the metabolic inhibitors or by incubation under anoxic conditions. ATP depletion did not modify the ratio of ribosomal subunits + monomers to polysomes. Puromycin and cycloheximide, which increased or decreased, respectively, the proportion of ribosomal RNA in subunits + monomers, blocked protein synthesis but did not alter the rate of RNA degradation. These experiments indicated that energy depletion inhibited RNA degradation by a mechanism that did not depend on the inhibition of protein synthesis. No evidence was obtained to indicate that the ratio of subunits + monomers to polysomes affected RNA degradation.

Hormone response units: one plus one equals more than two.

The transcription rate of many genes, and particularly those which code for metabolically important proteins, is regulated by various hormones. Detailed analysis of the promoters of these genes has shown that, while functional 'Hormone response elements' exist, the hormonal responsiveness of many promoters is often synergistically mediated by several cis-elements, collectively referred to as a hormone response unit. The utilization of a hormone response unit to mediate a response offers several regulatory advantages, including an expansion of the range of transcriptional responses and modulation of the response by tissue- and developmental-specific cues. Furthermore, the presence of Hormone Response Units may provide a mechanism for the coordination of information from two or more signaling pathways into a single, integrated and exquisitely controlled transcriptional response. The protein-protein interactions that likely mediate many of the synergistic functional characteristics of Hormone Response Units may provide unique targets for therapeutic intervention.

Differential regulation in the heart of mitochondrial carnitine palmitoyltransferase-I muscle and liver isoforms.

Carnitine palmitoyltransferase-I (CPT-I) plays a crucial role in regulating cardiac fatty acid oxidation which provides the primary source of energy for cardiac muscle contraction. CPT-I catalyzes the transfer of long chain fatty acids into mitochondria and is recognized as the primary rate controlling step in fatty acid oxidation. Molecular cloning techniques have demonstrated that two CPT-I isoforms exist as genes encoding the 'muscle' and 'liver' enzymes. Regulation of fatty acid oxidation rates depends on both short-term regulation of enzyme activity and long-term regulation of enzyme synthesis. Most early investigations into metabolic control of fatty acid oxidation at the CPT-I step concentrated on the hepatic enzyme which can be inhibited by malonyl-CoA and can undergo dramatic amplification or reduction of its sensitivity to inhibition by malonyl-CoA. The muscle CPT-I is inherently more sensitive to malonyl-CoA inhibition but has not been found to undergo any alteration of its sensitivity. Short-term control of activity of muscle CPT-I is apparently regulated by malonyl-CoA concentration in response to fuel supply (glucose, lactate, pyruvate and ketone bodies). The liver isoform is the only CPT-I enzyme present in the mitochondria of liver, kidney, brain and most other tissues while muscle CPT-I is the sole isoform expressed in skeletal muscle as well as white and brown adipocytes. The heart is unique in that it contains both muscle and liver isoforms. Liver CPT-I is highly expressed in the fetal heart, but at birth its activity begins to decline whereas the muscle isoform, which is very low at birth, becomes the predominant enzyme during postnatal development. In this paper, the differential regulation of the two CPT-I isoforms at the protein and the gene level will be discussed.

Characterization of CCAAT/enhancer-binding protein alpha as a cyclic AMP-responsive nuclear regulator.

The alpha isoform of CCAAT/enhancer-binding protein (C/EBPalpha) is a transcription factor that regulates expression of genes linked to adipose differentiation and hepatic nutrient metabolism. Recently, our laboratory has characterized a role for C/EBPalpha in mediating hormonal responsiveness. For example, the cAMP responsiveness of the phosphoenolpyruvate carboxykinase gene promoter in liver requires synergism among the cAMP response element-binding protein (CREB), C/EBPalpha, and activator protein-1. In the present study, we show that C/EBPalpha can functionally substitute for CREB in this cAMP response unit, i.e. cAMP responsiveness can occur in the absence of CREB. This observation is physiologically relevant since both CREB and C/EBPalpha have been shown to bind with high affinity to the cAMP response element in this particular promoter. Structure/function analysis of C/EBPalpha identified specific mutations that differentially affected its constitutive and protein kinase A-inducible activities. This finding suggests that the mechanism whereby C/EBPalpha mediates constitutive transactivation is distinct from that whereby it mediates cAMP responsiveness. These data support the hypothesis that C/EBPalpha plays a critical role in metabolism, in part by participating in the hormonal regulation of expression of metabolically important genes.