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Primary aldosteronism (PA) is the most common cause of secondary hypertension, and a simpler non-invasive method for identification of aldosterone-producing adenoma (APA) is required to improve the standard of medical treatment for PA patients. We retrospectively analyzed the clinical data of hypertensive patients with an aldosterone/renin ratio (ARR) ≥30 (ng/dL)/(ng/mL/h), and surgical and/or adrenal venous sampling (AVS) results served as the gold standard for APA diagnosis. The study aimed to determine whether positive CCT and SIT results plus a unilateral adrenal nodule found by CT allow unambiguous identification of an APA with high diagnostic specificity. Clinical data from 71 APA and 47 non-APA patients were collected, and logistic regression analysis was performed to construct models. Receiver operating characteristic (ROC) curves were used to analyze the efficacy of diagnostic tests. The areas under the ROC curves (AUCs) were similar between the post-SIT plasma aldosterone concentration (PAC) and post-CCT PAC (p > 0.05). The optimal post-SIT and post-CCT PAC cutoff values were 17.2 and 21.2 ng/dL, respectively. Positive CT findings combined with a post-SIT PAC >17.2 ng/dL or post-CCT PAC >21.2 ng/dL provided specificities of 97.8% and 95.7% for predicting APA, respectively. Logistic diagnostic models 1 (M1, CT finding + post-SIT PAC) and 2 (M2, CT finding + post-CCT PAC) were built, which showed equivalent diagnostic value (AUC = 0.959 and 0.932, respectively) (p > 0.05). The models combining CT findings with post-SIT PACs or post-CCT PACs represent an easier method to distinguish APA patients from other hypertensive patients with positive upright ARR results, especially in primary care where AVS may be unavailable.

To investigate the diagnostic value of different captopril challenge test (CCT) diagnostic criteria for diagnosing primary aldosteronism (PA).We collected the clinical data of 184 patients with hypertension retrospectively in West China Hospital of Sichuan University. Receiver operating characteristic (ROC) curves were used to analyze the post-CCT efficacy of aldosterone renin activity ratio (ARR), plasma aldosterone concentration (PAC), plasma renin activity (PRA) and PAC suppression rate for PA diagnosis.This study included 125 cases of primary aldehyde (PA group) and 59 cases of essential hypertension (EH group), and there were 38 normal renin primary hypertension (NREH group) and 21 low renin primary hypertension (LREH group) in EH group. The post-CCT PAC suppression rate (median (PBoth ARR and PAC have higher diagnostic value than the post-CCT PAC suppression rate, post-CCT PAC is especially suitable as a confirmatory testing criterion of PA.

Norman E Buroker,1 Xue-Han Ning,1,2,† Zhao-Nian Zhou,3 Kui Li,4 Wei-Jun Cen,4 Xiu-Feng Wu,3 Wei-Zhong Zhu,5 C Ronald Scott,1 Shi-Han Chen1 1Department of Pediatrics, University of Washington, 2Division of Cardiology, Seattle Children’s Hospital Research Foundation, Seattle, WA, USA; 3Laboratory of Hypoxia Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; 4Lhasa People Hospital, Lhasa, Tibet; 5Center for Cardiovascular Biology and Regenerative Medicine, University of Washington, Seattle, WA, USA †Xue-Han Ning passed away on April20, 2015 Abstract: Chronic mountain sickness (CMS) is estimated at 1.2% in Tibetans living at the Qinghai–Tibetan Plateau. Eighteen single-nucleotide polymorphisms (SNPs) from nine nuclear genes that have an association with CMS in Tibetans have been analyzed by using pairwise linkage disequilibrium (LD). The SNPs included are the angiotensin-converting enzyme (rs4340), the angiotensinogen (rs699), and the angiotensin II type 1 receptor (AGTR1) (rs5186) from the renin–angiotensin system. A low-density lipoprotein apolipoprotein B (rs693) SNP was also included. From the hypoxia-inducible factor oxygen signaling pathway, the endothetal Per-Arnt-Sim domain protein 1 (EPAS1) and the egl nine homolog 1 (ENGL1) (rs480902) SNPs were included in the study. SNPs from the vascular endothelial growth factor (VEGF) signaling pathway included are the v-akt murine thymoma viral oncogene homolog 3 (rs4590656 and rs2291409), the endothelial cell nitric oxide synthase 3 (rs1007311 and rs1799983), and the (VEGFA) (rs699947, rs34357231, rs79469752, rs13207351, rs28357093, rs1570360, rs2010963, and rs3025039). An increase in LD occurred in 40 pairwise comparisons, whereas a decrease in LD was found in 55 pairwise comparisons between the controls and CMS patients. These changes were found to occur within and between signaling pathways, which suggests that there is an interaction between SNP alleles from different areas of the genome that affect CMS. Keywords: HAS, Qinghai-Tibetan Plateau, RAS, HIF, VEGF, pathways, miRNA

Adrenal vein sampling (AVS) and pathological report were selected as gold standard to assess the value of adrenal CT scan combined with postural stimulation test in diagnosing aldosterone-producing adenoma.The clinical data of primary aldosteronism (PA) patients including aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA) were retrospectively collected in the West China Hospital of Sichuan University. The sensitivity and specificity of adrenal CT scan combined with postural stimulation test in diagnosing APA were studied.A total of 83 APA patients and 42 IHA patients were enrolled in this study. ROC curve was plotted with increase percentage of serum aldosterone in upright position compared with recumbent position. The optimal cutoff point for APA diagnosis by postural stimulation test was 11%, and the percentage less than 11% was taken as a positive result. The patients were diagnosed by postural stimulation test, with 60 cases positive and 23 cases negative in APA patients and 12 cases positive and 30 cases negative in IHA patients. When AVS and pathological report were selected as the gold standard, the sensitivity and specificity of postural stimulation test in diagnosing APA were 72.3% and 71.4% respectively. Among the 83 APA patients, 65 patients with unilateral nodules and 18 patients with bilateral nodules were diagnosed by CT scan. The coincidence rate in APA diagnosis between CT scan and AVS or pathological report was 78.3% (65/83). Among the 60 patients with positive result in postural stimulation test, who were diagnosed bilateral nodules by CT scan, 51 patients were diagnosed as APA by AVS or pathological report (51/60).Adrenal CT combined with postural test can increase the diagnostic accuracy of APA patients.

Many therapeutic hypothermia recommendations have been reported, but the information supporting them is sparse, and reveals a need for the data of target therapeutic hypothermia (TTH) from well-controlled experiments. The core temperature ≤35°C is considered as hypothermia, and 29°C is a cooling injury threshold in pig heart in vivo. Thus, an optimal protective hypothermia (OPH) should be in the range 29–35°C. This study was conducted with a pig cardiopulmonary bypass preparation to decrease the core temperature to 29–35°C range at 20 minutes before and 60 minutes during heart arrest. The left ventricular (LV) developed pressure, maximum of the first derivative of LV (dP/dtmax), cardiac power, heart rate, cardiac output, and myocardial velocity (Vmax) were recorded continuously via an LV pressure catheter and an aortic flow probe. At 20 minutes of off-pump during reperfusion after 60 minutes arrest, 17 hypothermic hearts showed that the recovery of Vmax and dP/dtmax established sigmoid curves that consisted of two plateaus: a good recovery plateau at 29–30.5°C, the function recovered to baseline level (BL) (Vmax=118.4%±3.9% of BL, LV dP/dtmax=120.7%±3.1% of BL, n=6); another poor recovery plateau at 34–35°C (Vmax=60.2%±2.8% of BL, LV dP/dtmax=28.0%±5.9% of BL, p

The rSNPs for the genes AKT3 (rs4590656), EGLN1 (rs480902), eNOS3 (rs1007311), and VEGFA (rs699947, rs13207311, rs1570360, rs2010963) have been significantly associated with the physiological parameters in high altitude sickness Han or Tibetan Chinese patients at the Qinghai-Tibetan plateau. The alleles of each rSNP have been found to create unique transcriptional factor binding sites for transcription factors that affect the process of hypoxia gene expression in this high altitude hypoxia environment.

High altitude sickness (HAS) occurs among humans visiting or inhabiting high altitude environments. Genetic differences in the EPAS1 and EGLN1 genes have been found between lowland (Han) and highland (Tibetan) Chinese. Three SNPs within EPAS1 and EGLN1 were evaluated in Han and Tibetan patients with acute mountain sickness (AMS) and chronic mountain sickness (CMS). We compared 85 patients with AMS to 79 Han unaffected with mountain sickness (MS) as well as 45 CMS patients to 34 unaffected Tibetan subjects. The three SNPs studied were EPAS1 [ch2: 46441523 (hg18], EGLN1 (rs480902) and (rs516651). Direct sequencing was used to identify individual genotypes for the three SNPs. Age was found to be significantly associated with the EPAS1 SNP in the CMS patients while heart rate (HR) and oxygen saturation level of hemoglobin (SaO 2 ) were found to be significantly associated with the EGLN1 (rs480902) SNP in the Han patients with AMS. The individuals with CMS were found to diverge significantly for the EPAS1 SNP compared to their Tibetan control group as measured by genetic distance (0.123) indicating positive selection of the EPAS-G allele with age and illness. The EGLN1 (rs480902) SNP had a significant correlation with hematocrit (HCT), HR and SaO 2 in AMS patients. AMS and CMS were found to be significantly associated with the EPAS1 and EGLN1 SNPs compared to their Han and Tibetan control groups, respectively, indicating these nucleotide alterations have a physiological effect for the development of high altitude sickness.

Mountain sickness (MS) occurs among humans visiting or inhabiting high altitude environments. We conducted genetic analyses of the AKT3, ANGPTL4, eNOS3 and VEGFA genes in lowland (Han) and highland (Tibetan) Chinese. Ten single nucleotide polymorphisms (SNPs) were evaluated in Han and Tibetan patients with acute (A) and chronic (C) MS. We compared 74 patients with AMS to 79 Han unaffected with MS, as well as 48 CMS patients to 31 unaffected Tibetans. The ten SNPs studied are AKT3 (rs4590656, rs2291409), ANGPTL4 (rs1044250), eNOS3 (rs1007311, rs1799983) and VEGFA (rs79469752, rs13207351, rs28357093, rs1570360, rs3025039). Direct sequencing was used to identify individual genotypes for these SNPs. Hemoglobin (Hb), hematocrit (Hct), and red blood cell count (RBC) were found to be significantly associated with the AKT3 SNP (rs4590656), Hb was found to be associated with the eNOS3 SNP (rs1007311), and RBC was found to be significantly associated with the VEGFA SNP (rs1570360) in Tibetan patients with CMS. CMS patients were found to diverge significantly for both eNOS3 SNPs as measured by genetic distance (0.042, 0.047) and for the VEGFA SNP (rs28357093) with a genetic distance of 0.078 compared to their Tibetan control group. Heart rate (HR) was found to be significantly associated with the eNOS3 SNP (rs1799983) and arterial oxygen saturation of hemoglobin (SaO2) was found to be significantly associated with the VEGFA SNPs (rs13207351, rs1570360) in Han patients with AMS. The Han and Tibetan control groups were found to diverge significantly for the ANGPTL4 SNP and VEGFA SNP (rs28357093), as measured by genetic distances of 0.049 and 0.073, respectively. Seven of the SNPs from non-coding regions are found in the transcriptional factor response elements and their possible role in gene regulation was evaluated with regard to MS. AMS and CMS were found to be significantly associated with the four genes compared to their Han and Tibetan control groups, respectively, indicating that these nucleotide alterations have a physiological effect for the development of high altitude sickness.

Hypothermia preserves myocardial function, promotes signaling for cell survival, and inhibits apoptotic pathways during 45-min reperfusion. We tested the hypothesis that signaling at the transcriptional level is followed by corresponding proteomic response and maintenance of structural integrity after 3-h reperfusion. Isolated hearts were Langendorff perfused and exposed to mild (I group; n = 6, 34 degrees C) or moderate (H group; n = 6, 30 degrees C) hypothermia during 120-min total ischemia with cardioplegic arrest and 180-min 37 degrees C reperfusion. Moderate hypothermia suppressed anaerobic metabolism during ischemia and significantly diminished left ventricular end-diastolic pressure at the end of ischemia from 52.7 +/- 3.3 (I group) to 1.8 +/- 0.9 (H group) mmHg. Unlike the I group, which showed poor cardiac function and high left ventricular pressure, the H group showed preservation of myocardial function, coronary flow, and oxygen consumption. Compared with normal control hearts without ischemia (n = 5), histological staining in the I group showed marked disarray and fragmentation of collagen network (score 4-5), while the H group showed preserved collagen integrity (score 0-1). The apoptosis-linked tumor suppressor protein p53 was expressed throughout the I group only (score 4-5). The H group produced elevated expression for hypoxia-inducible factor 1alpha and heme oxygenase 1, but minimally affected vascular endothelial growth factor expression. The H group also elevated expression for survival proteins peroxisomal proliferator-activated receptor-beta and Akt-1. These results show in a constant left ventricular volume model that moderate hypothermia (30 degrees C) decreases myocardial energy utilization during ischemia and subsequently promotes expression of proteins involved in cell survival, while inhibiting induction of p53 protein. These data also show that 34 degrees C proffers less protection and loss of myocardial integrity.

Hypothermia before and/or during no-flow ischemia promotes cardiac functional recovery and maintains mRNA expression for stress proteins and mitochondrial membrane proteins (MMP) during reperfusion. Adaptation and protection may occur through cold-induced change in anaerobic metabolism. Accordingly, the principal objective of this study was to test the hypothesis that hypothermia preserves myocardial function during hypoxia and reoxygenation. Hypoxic conditions in these experiments were created by reducing O2concentration in perfusate, thereby maintaining or elevating coronary flow (CF). Isolated Langendorff-perfused rabbit hearts were subjected to perfusate (Po2= 38 mmHg) with glucose (11.5 mM) and perfusion pressure (90 mmHg). The control (C) group was at 37°C for 30 min before and 45 min during hypoxia, whereas the hypothermia (H) group was at 29.5°C for 30 min before and 45 min during hypoxia. Reoxygenation occurred at 37°C for 45 min for both groups. CF increased during hypoxia. The H group markedly improved functional recovery during reoxygenation, including left ventricular developed pressure (DP), the product of DP and heart rate, dP/d tmax, and O2consumption (MVo2) ( P < 0.05 vs. control). MVo2decreased during hypothermia. Lactate and CO2gradients across the coronary bed were the same in C and H groups during hypoxia, implying similar anaerobic metabolic rates. Hypothermia preserved MMP βF1-ATPase mRNA levels but did not alter adenine nucleotide translocator-1 or heat shock protein-70 mRNA levels. In conclusion, hypothermia preserves cardiac function after hypoxia in the hypoxic high-CF model. Thus hypothermic protection does not occur exclusively through cold-induced alterations in anaerobic metabolism.

Acute mountain sickness (AMS) occurs in up to 50% of individuals ascending to high altitudes greater than 2600 meters. An AMS Han Chinese and a normal Han group were compared for 17 single nucleotide polymorphisms (SNPs) within 9 genes that have been associated with AMS. The SNPs were analyzed with respect to linkage disequilibrium (LD) between intra- and intergenic SNP alleles and alterations in transcriptional factor binding sites (TFBS). Included in the study was the angiotensin-converting enzyme (ACE) (rs4340), the angiotensinogen (AGT) (rs699) and the angotensin II type 1 receptor (AGTR1) (rs5186) SNPs from the renin-angiotension system (RAS) as well as the GNB3 (rs2071057) SNP from G-protein signaling and a LDL apolipoprotein B (APOB) (rs693) SNP. The endothetal Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) SNP and two egl nine homolog 1 (EGLN1) SNPS (rs480902 and rs516651) from the hypoxia-inducible factor (HIF) oxygen signaling pathway were included. SNPs analyzed in the vascular endothelial growth factor (VEGF) signaling pathway are the v-akt murine thymoma viral oncogene homolog 3 (AKT3) (rs4590656 and rs2291409), the endothelial cell nitric oxide synthase 3 (eNOS3) (rs1007311 and rs1799983) and the (VEGFA) (rs79469752, rs13207351, rs28357093, rs1570360 and rs3025039). These SNP alleles alter the TFBS for TF binding. Pair-wise LD was computed between SNPs. An increase in LD occurred in 32 pair-wise comparisons while a decrease was found in 22 pair-wise comparisons between the AMS and controls. Increases and decreases in LD pairs were found within and between signaling pathways and systems indicating the interaction of SNP alleles or potential TFBS from different areas of the genome. The most drastic change in TFBS occurs with ACE (I/D) SNP (rs4340) where the ACE-I allele generates 84 potential TFBS while the ACE-D allele generates only four binding sites. The alteration in TFBS generated by the 17 SNPs is discussed with respect to AMS.

Hypothermia improves resistance to ischemia in the cardioplegia-arrested heart. This adaptive process produces changes in specific signaling pathways for mitochondrial proteins and heat-shock response. To further test for hypothermic modulation of other signaling pathways such as apoptosis, we used various molecular techniques, including cDNA arrays. Isolated rabbit hearts were perfused and exposed to ischemic cardioplegic arrest for 2 h at 34°C [ischemic group (I); n = 13] or at 30°C before and during ischemia [hypothermic group (H); n = 12]. Developed pressure, the maximum first derivative of left ventricular pressure, oxygen consumption, and pressure-rate product ( P < 0.05) recovery were superior in H compared with in I during reperfusion. mRNA expression for the mitochondrial proteins, adenine translocase and the β-subunit of F1-ATPase, was preserved by hypothermia. cDNA arrays revealed that ischemia altered expression of 13 genes. Hypothermia modified this response to ischemia for eight genes, six related to apoptosis. A marked, near fivefold increase in transformation-related protein 53 in I was virtually abrogated in H. Hypothermia also increased expression for the anti-apoptotic Bcl-2 homologue Bcl-x relative to I but decreased expression for the proapoptotic Bcl-2 homologue bak. These data imply that hypothermia modifies signaling pathways for apoptosis and suggest possible mechanisms for hypothermia-induced myocardial protection.

OBJECTIVESThis study was conducted to test hypotheses stating that: 1) altered signaling for mitochondrial membrane proteins occurs during postinfarction remodeling, and 2) successful myocardial adaptation relates to promotion of specific mitochondrial membrane components.BACKGROUNDAbnormalities in high-energy phosphate content and limitations in adenosine 5′-triphosphate (ATP) synthesis rate occur during the transition to contractile failure from compensatory remodeling after left ventricular infarction. The adenine nucleotide translocator (ANT) and F1-ATPase respectively regulate mitochondrial adenosine 5′-diphosphate (ADP)/ATP exchange and ADP-phosphorylation, which are key components of high-energy phosphate metabolism.METHODSSteady-state mRNA and protein expression for ANT isoform1 and the beta subunit of the F1-ATPase (betaF1) were analyzed in myocardium remote from the infarction zone eight weeks after left circumflex coronary artery ligation in pigs, demonstrating either successful left ventricular remodeling (LVR, n = 8) or congestive heart failure (CHF, n = 4) as determined by clinical and contractile performance parameters.RESULTSSubstantial reductions in steady-state mRNA expression for ANT1 and betaF1 relative to normal (n = 8) occur in CHF, p < 0.01, but not in LVR. Relative expression for both proteins coordinated with their respective steady-state mRNA levels; CHF at 40% normal, p < 0.05 for ANT and 70% normal for betaF1, p < 0.05.CONCLUSIONSMaintained signaling for major mitochondrial membrane proteins occurs in association with successful remodeling and adaptation after infarction. Reduced expression of these proteins relates to limited ATP synthesis capacity and high energy phosphate kinetic abnormalities previously demonstrated in CHF. These findings imply that mitochondrial processes participate in myocardial remodeling after infarction.

Severe infantile Gaucher disease associated with ichthyosis and neonatal death is a rare subgroup of Type II Gaucher disease. This group of infants has little, if any, detectable beta-glucocerebrosidase activity, and prior genetic analyses have been limited in detecting the mutations responsible for this phenotype. We document an Hispanic infant succumbing with arthrogryposis and collodion membrane covering the skin who had no detectable beta-glucocerebrosidase activity in tissue samples and who was homozygous for a rare recombinant allele, RecNciI. Microscopic evaluation demonstrated accumulation of Gaucher cells in visceral organs and extensive loss of neurons in the anterior horns, brainstem, and cortex of the nervous system. The apoptosis of neuronal cells from the anterior horns and brainstem are a reasonable explanation for the arthrogryposis and neonatal death, respectively.

Fragile X syndrome is recognized as the most common inherited cause of mental retardation in western countries. The prevalence of the fragile X syndrome in Asian populations is uncertain. We report a multi-institutional collaborative study of molecular screening for the fragile X syndrome from 1,127 Chinese mentally retarded (MR) individuals. We found that 2.8% of the Chinese MR population screened by DNA analysis had the fragile X full mutation. Our screening indicated that the fragile X syndrome prevalence was very close to that of Caucasian subjects. In addition, we found that 62.5% of fragile X chromosomes had a single haplotype for DXS548-FRAXAC1 (21-18 repeats) which was present in only 9.7% of controls. This unique distribution of microsatellite markers flanking the FMR1 CGG repeats suggests that the fragile X syndrome in Chinese populations, as in the Caucasian, may also be derived from founder chromosomes.

X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B-cell differentiation; this incapacitates antibody production in XLA patients, who suffer from recurrent, sometimes lethal, bacterial infections. BTK plays an important role in B-cell development; it interacts with several proteins in the context of signal transduction. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in a patient family. To understand the role of BTK, we studied binding of BTK SH3 domain (aa 216-273, 58 residues) and truncated SH3 domain (216-259, 44 residues) with proline-rich peptides; the first peptide constitutes the SH3 domain of BTK, while the latter peptide lacks 14 amino acid residues of the C terminal. Proline-rich peptides selected from TH domain of BTK and p120cbl were studied. It is known that BTK TH domain binds to SH3 domains of various proteins. We found that BTK SH3 domain binds to peptides of BTK TH domain. This suggests that BTK SH3 and TH domains may associate in inter- or intramolecular fashion, which raises the possibility that the kinase may be regulating its own activity by restricting the availability of both its ligand-binding modules. We also found that truncated SH3 domain binds to BTK TH domain peptide less avidly than does normal SH3 domain. Also, we show that the SH3 and truncated SH3 domains bind to peptide of p120cbl, but the latter domain binds weakly. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context, hence the weaker binding. These results delineate the importance of C-terminal in binding of SH3 domains and indicate also that improper folding and the altered binding behavior of mutant BTK SH3 domain likely leads to XLA.

Circulating miRNAs isolated from dried blood spots (DBS) were found to be associated with high altitude sickness (HAS) patients in Tibet. HAS arises from two different diseases which are acute (AMS) and chronic (CMS) mountain sickness. Circulating miRNAs differences were found between AMS Han Chinese patients and normal Han controls and between CMS Tibetan Chinese patients and normal Tibetan controls. HAS arises from hypoxia which afflicts some high altitude inhabitants or visitors and not others. The difference results from each individual’s genetic makeup where hypoxia related genes have been shown to be a major contributor to these sicknesses. Several fold changes increases (up regulation) were found in the hypoxia associated miRNAs let-7f-5p, miR-9-5p, miR-19a-3p, miR-23a-3p, miR-98-5p, miR-125a-5p, miR-181b-5p, mir-202-3p, miR-372, miR-381-3p, miR-519d, miR-520d-3p, and miR-656 for both HAS groups compared to their controls. Other miRNAs (miR-19a-3p, 302c-3p and 875-3p) were found to be up regulated in one HAS group and down regulated in the other HAS group indicating the genetic differences between the two sickness groups.

Mountain sickness (MS) occurs among humans visiting or inhabiting high altitude environments. We conducted genetic analyses of seven single nucleotide polymorphisms (SNPs) in the promoter region of VEGFA gene for lowland (Han) and highland (Tibetan) Chinese. The seven SNPs were evaluated in Han and Tibetan patients with acute (A) and chronic (C) MS. We compared 64 patients with AMS with 64 Han unaffected with MS, as well as 48 CMS patients with 32 unaffected Tibetans. The SNPs studied are rs699947, rs34357231, rs79469752, rs13207351, rs28357093, rs1570360, and rs2010963 which are found in the promoter ranging from −2,578 to −634 bp from the transcriptional start site (TSS), respectively. Direct sequencing was used to identify individual genotypes for these SNPs. Arterial oxygen saturation of hemoglobin (SaO2) was found to be significantly associated with the rs699947, rs34357231, rs13207351, and rs1570360 SNPs in Han patients with AMS, while the rs2010963 SNP was found to approach significance in the AMS study group, but found to be significantly associated in the normal Tibetan study group. The Han and Tibetan control groups were found to diverge significantly for the rs28357093 and rs2010963 SNPs, as measured by genetic distances of 0.073 and 0.054, respectively. All the SNPs are found in transcriptional factor binding sites (TFBS), and their possible role in gene regulation was evaluated with regard to MS. MS was found to be significantly associated with these SNPs compared with their Han and Tibetan control groups, indicating that these nucleotide substitutions result in TFBS changes which apparently have a physiological effect on the development of high altitude sickness.

Background Acute (AMS) and chronic (CMS) mountain sicknesses are illnesses that occur among humans visiting or inhabiting high-altitude environments, respectively. Some individuals are genetically less fit than others when stressed by an extreme high-altitude environment. Seven blood physiological parameters and five genetic polymorphisms were studied in Han patients with AMS and Tibetan patients with CMS. Methods We compared 98 AMS patients with 60 Han controls as well as 50 CMS patients with 36 Tibetan controls. The genetic loci studied are ACE I/D (rs4340), AGT M235T (rs699), AGTR1 A1166C (rs5186), GNB3 A(− 350)G (rs2071057) and APOB A/G (rs693). Results All physiological parameters (RBC, HCT, Hb, SaO2, HR, and BPs/d) studied significantly changed in the CMS patients while SaO2 and HR changed in the AMS Han patients compared to their controls. The ACE D and AGT 235 M alleles were found to be significantly associated with AMS and CMS, respectively, while a significantly high incidence of the G-protein (GNB3) (− 350)A allele was found in the AMS patients. ACE (I/D) was significantly associated with HR in CMS patients while the AGT M235T was significantly associated with SaO2 and BPs/d in AMS patients. APOB A/G was significantly associated with BPs/d in AMS and HR in CMS patients. Conclusion AMS and CMS share very similar genetic results for the ACE I/D and AGT M235T polymorphisms indicating that these mutations have an effect on both illnesses.

The fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats present in the FMR1 gene. The repeat region is within sequences characteristic of a CpG island. Methylation of CpG dinucleotides that are 5' to the CGG repeat has been shown to occur on the inactive X chromosome of normal females and on the X chromosome of affected fragile-X males, and is correlated with silencing of the FMR1 gene. The methylation status of CpG sites 3' to the repeat and within the repeat itself has not previously been reported. We have used two methylation-sensitive restriction enzymes, AciI and Fnu4HI, to further characterize the methylation pattern of the FMR1 CpG island in normal individuals and in those carrying fragile-X mutations. Our results indicate that: (i) CpG dinucleotides on the 3' side of the CGG repeat are part of the CpG island that is methylated during inactivation of a normal X chromosome in females; (ii) the CGG repeats are also part of the CpG island and are extensively methylated as a result of normal X-chromosome inactivation; (iii) similar to normal males, unaffected fragile-X males with small CGG expansions are unmethylated in the CpG island; for affected males, the patterns of methylation are similar to those of a normal, inactive X chromosome; (iv) in contrast to the partial methylation observed for certain sites in lymphocyte DNA, complete methylation was observed in DNA from cell lines containing either a normal inactive X chromosome or a fragile-X chromosome from an affected male.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemophilia B is due to multiple molecular defects in the factor IX gene. Over 80% of mutations are single base substitutions. By amplification and direct sequencing, 51 single base substitutions were found in the transcribed sequence of the factor IX genes of patients from 50 distinct families with hemophilia B. These include 30 mutations in 29 families not previously reported by us; of these, 12 are novel, i.e., not previously published in other series. Of the 51 substitutions in our overall series 23 (45%) occurred as C-to-T or G-to-A transitions at 11 sites within CG dinucleotides. It is estimated that CG transitions occur from one to two orders of magnitude more frequently than mutations in nucleotides that are not within a CG pair. More than one family had identical defects for 6 of the CG mutations. At 4 of these sites, most patients had different haplotypes compatible with distinct mutations. Non-CG-type mutations occurred throughout the coding regions with only one mutation in more than one family. The latter included 7 families with a 397 Ile-to-Thr defect that all share a rare haplotype, suggesting a common ancestor.

Severe cardiac hypoxia is responsible for significant morbidity and mortality in an emergency setting. Most cardiac hypoxia relates to ischemia and surgical events. Although the ischemic mortality rate and the risks of cardiac surgery have significantly decreased in past decades, myocardial protection still plays a major role in survival of hypoxic injury. Cross adaptation as a physiological regulation for homeostasis can resist injury caused by harmful environmental effects and diseases, including hypothermic adaptation. Treatment with hypothermia has been used for fifty years as a protective mechanism to avoid hypoxic injury. Since cold temperatures can cause damage, it is important to gather physiological data to distinguish protective from injurious temperatures. Although results of temperature trials in clinical practice vary, a critical temperature to resist hypoxic/ischemic injury in heart was found to be around 30 degrees C, suggesting a hypothermia protective threshold. Pretreatment with mild hypothermia can resist subsequent hypoxia/ischemia, implying involvement of cross adaptation in protection. Safeguard hypothermia can directly reduce the build up of harmful metabolites and energy demand in hypoxic tissues, as well as preserve mitochondrial membrane specific proteins beta subunit of F1-ATPase and adenine nucleotide translocase isoform 1. Mechanisms of preservation include inactivation of the p53 related pathways, representing anti-apoptosis, and modification of the mRNA level of succinodehydrogenease, indicating a beneficial effect on the aerobic pathway. Stress proteins are also induced. Resultant cellular adaptations serve to maintain myocardial integrity and improve functional recovery during reoxygenation or reperfusion.

The deflection of a disk brake is affected by its configuration and boundary conditions. The three-dimensional finite element software ANSYS is used to analyze deflection of the disk in this study. The calculated maximum deflection occurs at the outside diameter of the disk inner board. The disk bends in the direction from the inner board toward the outer board, and the deflection coning angle of the disk is 0.4°. The calculated deflections and the coning angles of the disk are comparable with the existing data. Deflections of various disks are compared. If the thickness of the disk support region increases 1 mm, the maximum deflection decreases 0.001 mm that is about 0.2% only and it is insignificant. If the fillet radius of the support portion changes from 1 mm to 3 mm, the maximum deflection decreases 0.076 mm that it is about 15.3%. Therefore, the effect of changing the fillet radius on the deflection is more significant than that of changing the thickness of the disk support region. The mathematical model and the calculated results can be used for designing disk brakes, and a modified disk brake is recommended.

Hypoxia-inducible factor 1α (HIF-1α) transcriptionally activates multiple genes, which regulate metabolic cardioprotective and cross-adaptive mechanisms. Hypoxia and several other stimuli induce the HIF-1α signaling cascade, although little data exist regarding the stress threshold for activation in heart. We tested the hypothesis that relatively mild short-cycle hypoxia, which produces minimal cardiac dysfunction and no sustained or major disruption in energy state, can induce HIF-1α activation. We developed a short-cycle hypoxia protocol in isolated perfused rabbit heart to test this hypothesis. By altering cycling conditions, we identified a specific cycle with O2 content and duration that operated near a threshold for causing functional injury in these rabbit hearts. Mild short-cycle hypoxia for 46 min elevated HIF-1α mRNA and protein within 45 min after reoxygenation. Expression also increased for multiple HIF-1α target genes, such as VEGF and heme oxygenase 1. After mild hypoxia, VEGF protein accumulation occurred, although HIF-1α and VEGF protein accumulation were suppressed after more severe hypoxia, which also caused depletion of ATP and nondiffusible nucleotides. In summary, these results indicate that mild near-threshold hypoxia induces HIF-1α cascade, but more severe hypoxia suppresses protein accumulation for this transcription factor and the target genes. Posttranscriptional suppression of these proteins occurs under conditions of altered energy state, exemplified by ATP depletion.

We have evaluated the structure of the CGG repeat within the FMR1 gene of an Asian population and found the most common size of the repeat to be 29 and 30 with a minor population of 36 repeats. We have isolated and sequenced DNA containing the 36 repeats and found the basis sequence to be (CGG)9AGG(CGG)9AGG-(CGG)6AGG(CGG)9; with a (CGG)6)AGG insertion, designated as 9A9A6A9. Of 144 Asian chromosomes, 11 (8%) had sequences with this insertion. Six different variations of the basic sequence were observed in the population: 9A9A6A2A9, 9A9A6A11, 9A9A16, 9A9A15, 8A9A6A6A9, and 11A6A6A9. All but one of the chromosomes with the insertion had the haplotype of DXS548/ FRAXAC1: 194/D suggesting that the sequences with the 6A insertion arose from a single ancestral allele. We have not observed the insertion in the FMR1 gene of Caucasians or Native Americans. The (CGG)6AGG insertion may be unique to Asians.

To detect gene mutations associated with autosomal dominant congenital stationary night blindness(ADCSNB) in a large Chinese family.Genomic DNAs were extracted from peripheral blood samples of 16 affected and 14 unaffected family members. According to 5 missense mutations in 3 genes reported previously, 4 pairs of primers were designed and corresponding exons containing the five mutation sites were amplified by polymerase chain reaction. Amplified products were purified and sequenced by MegaBACE1000 capillary array electrophoresis DNA sequencer. Full field electroretinogram (ERG, ISCEV) of patients was recorded and analyzed by Roland Consult System.Dark-adapted ERG showed a-wave was normal, but b-wave of the patients was markedly decreased. None of the five missense mutations were detected in 16 affected and 14 unaffected family members.The molecular pathogenesis of ADCSNB in this family does not involve point mutations or deletions of these five sites, which indicates the heterogeneity of ADCSNB.

When a speech signal is encoded in some low bit-rate coding formats, it becomes more difficult to distinguish speaker identities. The paper investigates the codec effect on acoustic and prosodic features. A new representation of prosodic features based on the piecewise fitting of the pitch contour is introduced. A method for including residual features based on the LDA (linear discriminant analysis) algorithm is suggested. By combining prosodic features with acoustic features, we can improve the performance of a speaker recognition system. A series of experiments is performed with coded speech affected by G.729A and GSM codec processes to demonstrate the effectiveness of our proposed method.

Hypothermia improves resistance to ischemia in the cardioplegia-arrested heart. This adaptive process produces changes in specific signaling pathways for mitochondrial proteins and heat-shock response. To further test for hypothermic modulation of other signaling pathways such as apoptosis, we used various molecular techniques, including cDNA arrays. Isolated rabbit hearts were perfused and exposed to ischemic cardioplegic arrest for 2 h at 34 degrees C [ischemic group (I); n = 13] or at 30 degrees C before and during ischemia [hypothermic group (H); n = 12]. Developed pressure, the maximum first derivative of left ventricular pressure, oxygen consumption, and pressure-rate product (P0.05) recovery were superior in H compared with in I during reperfusion. mRNA expression for the mitochondrial proteins, adenine translocase and the beta-subunit of F1-ATPase, was preserved by hypothermia. cDNA arrays revealed that ischemia altered expression of 13 genes. Hypothermia modified this response to ischemia for eight genes, six related to apoptosis. A marked, near fivefold increase in transformation-related protein 53 in I was virtually abrogated in H. Hypothermia also increased expression for the anti-apoptotic Bcl-2 homologue Bcl-x relative to I but decreased expression for the proapoptotic Bcl-2 homologue bak. These data imply that hypothermia modifies signaling pathways for apoptosis and suggest possible mechanisms for hypothermia-induced myocardial protection.

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.

The propeptide domain in the precursor forms of blood clotting proteins contains the recognition sequences for gamma-carboxylase. In hemophilia B, several point mutations in this propeptide domain are responsible for the inherited disease. A peptide containing the propeptide sequence of factor IX was synthesized by solid phase methods. Two dimensional 1H-NMR and CD studies indicate that this peptide motif adopts an alpha-helical structure in a 40% trifluoroethanol-containing aqueous solution. The results suggest that the amphipathic alpha-helix within the propeptide domain of factor IX could create a recognition surface for gamma-carboxylase. The influences of mutations and their relationship with the alpha-helical structure are discussed.

Heteroduplex screening of amplified fragments containing sequences of all known small haemophilic mutations in the factor IX gene localized mutations in 18 new families: 12 were at common recurrent sites; three were novel. Carriers and/or patients from each of 41 families with mutations in 7 exons and 5' and 3' non-coding regions were positive.

The gene frequencies of ADH 2 2 and ALDH 2 2 were lower in Tibetan and Mongolian populations than in Vietnamese, Han Chinese, and three Chinese minority populations.

The germ line of origin for 13 of 14 de novo hemophilia B mutations has been determined. When added to previous reports, the origin, assuming no mosaicism, occurred in 43 female and 33 male gametes. Mutation rate estimates are twofold higher in males than in females. The pooled data also indicate that male and female germ lines have different mutation rates depending upon the type of mutation.

The gene responsible for X-linked agammaglobulinemia (XLA) has been recently identified to code for a cytoplasmic tyrosine kinase (Bruton's agammaglobulinemia tyrosine kinase, BTK), required for normal B cell development. BTK, like many other cytoplasmic tyrosine kinases, contains Src homology domains (SH2 and SH3), and catalytic kinase domain. SH3 domains are important for the targeting of signaling molecules to specific subcellular locations. We have identified a family with XLA whose affected members have a point mutation (g-->a) at the 5' splice site of intron 8, resulting in the skipping of coding exon 8 and loss of 21 amino acids forming the COOH-terminal portion of the BTK SH3 domain. The study of three generations within this kinship, using restriction fragment length polymorphism and DNA analysis, allowed identification of the mutant X chromosome responsible for XLA and the carrier status in this family. BTK mRNA was present in normal amounts in Epstein-Barr virus-induced B lymphoblastoid cell lines established from affected family members. Although the SH3 deletion did not alter BTK protein stability and kinase activity of the truncated BTK protein was normal, the affected patients nevertheless have a severe B cell defect characteristic for XLA. The mutant protein was modeled using the normal BTK SH3 domain. The deletion results in loss of two COOH-terminal beta strands containing several residues critical for the formation of the putative SH3 ligand-binding pocket. We predict that, as a result, one or more crucial SH3 binding proteins fail to interact with BTK, interrupting the cytoplasmic signal transduction process required for B cell differentiation.

Publisher Summary This chapter focuses on the characterization of factor IX defects in hemophilia B patients. The procoagulant activity of factor IX is determined by the ability of dilutions of a patient's plasma to correct the prolonged clotting time of a severely factor IX-deficient plasma in a partial thromboplastin-like assay. With cloning and sequencing of the complementary DNA (cDNA) of factor IX and the entire gene, it is now possible to analyze patient DNA directly for mutations. Altered restriction patterns on Southern blots of DNA digests hybridized to radiolabeled cDNAs or genomic probes indicate gross gene deletions in several patients with severe hemophilia B. The determination of factor IX dotting activity and antigen level is, thus, important to define mutations in patients with hemophilia B efficiently.

We have developed a technique to detect accurately heterozygous carriers of a deletion. Specific target sequences were amplified by the polymerase chain reaction (PCR), and the products subsequently were analyzed by high-performance liquid chromatography. Examples from four loci demonstrated that 24-27 cycles of amplification for a single-copy DNA, based on 50 ng of genomic DNA, results in excellent quantitation that readily permits the detection of heterozygous carriers of a deletion. We have demonstrated that triplex PCR (three targets in a single PCR) entails no loss of precision. We also have demonstrated that this method can accurately differentiate the heterozygous carriers of a deletion from normal individuals in four family studies, three for Duchenne muscular dystrophy patients and one for a hemophilia B patient.

Adults with CF followed in a university center were assessed for the presence of the most common CF gene mutation, delta-F508. Excluding one member of a sibling pair, 29 of 55 subjects had two copies of delta-F508 (homozygotes), 23 had one copy of delta-F508 with the other CF mutation not identified (complex heterozygotes) and three were lacking delta-F508. A wide range of clinical severity was seen among individuals carrying two copies of the delta-F508 gene, who are genetically identical at the CF gene locus. The number of individuals diagnosed with CF as adults was significantly lower in the homozygote group (1 of 29) as compared with the heterozygote group (7 of 24). No differences were detected between groups in pulmonary function, non-pulmonary complications or overall clinical severity. These results suggest that environmental or background genetic factors contribute significantly to the variability in pulmonary and other complications seen among individuals with CF.

The frequencies of the alleles encoding isozymes of alcohol dehydrogenase and aldehyde dehydrogenase were low in Northwest Coast Amerindians compared to Chinese subjects.

Direct sequencing of the regions of the factor IX gene of likely functional significance was performed in four patients with severe hemophilia B. In two of the individuals, a transition at the dinucleotide CpG caused a nonsense mutation at arginine 333. In the other two individuals, a transition at CpG caused a nonsense mutation at arginine 29. Since these patients are all unrelated, as shown by differing alleles of the TaqI polymorphism in intron four or extensive nonoverlapping pedigrees, the mutations arose independently. In addition, the origin of one arginine 333 mutation in one family has been traced to the germline of the maternal grandfather. The frequent occurrence of mutations at arginine codons that contain the sequence CGN can be explained by the dramatic elevation of transitions at CpG. As a result, approximately one in four individuals with hemophilia B is expected to have a mutation at arginine and nonsense mutations at one of six arginine residues should be common causes of severe hemophilia.

with a monoclonal antibody (A-1) detect a prevalent dimorphism in plasma coagulation factor IX. The antibody was shown to react with a dimorphic segment of the normal factor IX sequence as follows. First, A-1 bound to isolated activation peptide (residues 146 through 180) prepared from activated factor IX from a normal plasma pool. Second, binding of recombinant factor IXs with A-1 or factor IX from normal individuals was strong only when they had Threonine (Thr) at position 148; factor IXs with the Alanine (Ala) allele at that position were far less reactive. Third, immunoblot reactivity of Escherichia coli fusion proteins containing known segments of the factor IX sequence restricted the epitope to residues 147 through 153. In 120 hemophilia B pedigrees, the Ala immunoassay allele frequency was 0.19 and did not differ from the Ala frequency in normal males. In 22 of 49 families, immunoassay testing was informative for classification of obligate or possible carriers. In one large family, 4 obligate carriers were heterozygous for the dimorphism and 3 of their 7 daughters were classified as carriers. In other families, when the affected member had less than 1 nmol/L factor IX antigen, heterozygosity for Thr/Ala alleles excluded the carrier state even when DNA studies were not informative. Strong linkage disequilibrium of Thr/Ala alleles with the common TaqI DNA polymorphism was found. Nineteen of 75 normal and hemophilic factor IX genes had the 1.3- kilobase (kb) fragment and coded for the Ala allele; the rest had the 1.8-kb fragment and coded for Thr. In selected families, the A-1 immunoassay is an inexpensive and rapid method to confirm and supplement restriction fragment length polymorphism analyses of DNA for carrier testing.

Absence of purine nucleoside phosphorylase (NP) is associated with severe T cell immune deficiency and normal B cell function. Patients with this enzyme defect accumulate inosine, guanosine, and their respective deoxycompounds, all of which are substrates for NP. We have evaluated the effect of these four NP substrates on PHA-stimulated lymphocytes and lymphoblastoid cell lines with B and T cell characteristics. Inosine and deoxyinosine had little inhibitory effect on human lymphocytes, whereas guanosine and deoxyguanosine inhibited DNA and protein synthesis in both PHA-stimulated human lymphocytes and hyman lymphoblastoid cells. In all experiments, deoxyguanosine was more toxic than guanosine. Human lymphoblastoid cells with T cell characteristics (T-LCL) were found to be particularly sensitive to the presence of deoxyguanosine. At low µM concentrations 3H-thymidine and 3H-leucine incorporation into the T-LCL was markedly decreased. At a concentration of 10 µM, no cell growth occurred and 50% of the cells were killed. Partial reversal of the toxic effect of deoxyguanosine on the T-LCL could be achieved with simultaneous addition of deoxycytidine. Human lymphoblastoid cells with B cell characteristics (B-LCL) were not inhibited at these low deoxyguanosine levels but cell growth and incorporation of 3H-thymidine and 3H-leucine were moderately decreased at higher deoxyguanosine concentrations. In a B-LCL established from a patient with NP deficiency, neither DNA nor protein synthesis was inhibited by guanosine or deoxyguanosine at concentrations that affected other B cell lines. The extreme sensitivity of the T lymphoblasts and relative resistance of B lymphoblasts to the toxicity of deoxyguanosine or one of its metabolic products may explain the selective loss of T cell function in patients with NP deficiency.