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PubMed2003

Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells.

This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca(v)) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between -60 and -10 mV. Blockade of K+ channels with TEA inhibited 83% of the current at +105 mV. Inhibition of KV channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K+ channels (BKCa) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was -32 mV and activation of BKCa with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca(v) blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca(v) 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K+-blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels.

Alternative Splicing↗
PubMed1976

Patterns of phosphoribosylpyrophosphate and ribose-5-phosphate concentration and generation in fibroblasts from patients with gout and purine overproduction.

In the majority of patients with gout and excessive uric acid production, underlying enzyme abnormalities have not been identified. In the present study, measurement of both the rate of generation and concentration of phosphoribosylpyrophosphate (PP-ribose-P) and the concentration of ribose-5-phosphate in cultured cells were undertaken to establish a classification of purine overproducers to direct study of additional enzyme defects. Fibroblasts were cultured from 24 individuals assigned to 4 groups: group 1, 5 normal controls; group 2, 5 patients with gout and normal dialy urinary uric acid excretion (gouty controls); group 3, 7 patients with well-defined enzyme abnormalities and excessive urinary acid excretion (4 with hypoxanthine-guanine phosphoribosyltransferase deficiency and 3 with excessive PP-ribose-P synthetase activity); and group 4, 7 patients with gout and excessive uric acid excretion but without grossly abnormal activities of the above enzymes in erythrocyte lysates. In all 14 fibroblast strains from patients showing excessive production of uric acid (groups 3 and 4), rates of purine synthesis de novo and PP-ribose-P concentrations exceeded values for cells from control groups. Cells from group 3 patients with hypoxanthine-guanine phosphoribosyltransferase deficiency showed normal PP-ribose-P generation, while those with excessive PP-ribose-P synthetase activity demonstrated increased generation of this regulatory substrate. All strains from group 3 patients had normal ribose-5-phosphate concentrations. Five cell strains from group 4 patients showed one of the two patterns of abnormalities in these measurements seen in strains from group 3 patients: two resembled hypoxanthine-guanine phosphoribosyltransferase-deficient cells, and three resembled cells with excessive PP-ribose-P synthetase activity. Analyses of erythrocyte enzyme preparations from two of these patients in group 4 have led to identification of a kinetic variant of each enzyme as predicted from the foregoing patterns. Two additional group 4 cell lines that showed increased ribose-5-phosphate concentrations in addition to increased PP-ribose-P concentrations and generation were classified in a separate subgroup, since in the individuals excessive purine synthesis appeared to result from increases ribose-5-phosphate concentration, leading to increased availability of PP-ribose-P. No abnormality in either hypoxanthine-guanine phosphoribosyltransferase or PP-ribose-P synthetase has been found in erythrocyte preparations from one patient so classified.

Adenine Phosphoribosyltransferase↗
PubMed2006

Dissecting the role of endothelial SURVIVIN DeltaEx3 in angiogenesis.

The identification of alternative splice variants of Survivin that possess distinct functions from those originally identified for the main Survivin isoform has greatly increased the complexity of our understanding of the role of Survivin in different cells. Previous functional studies of the Survivin splice variants have been performed almost exclusively in cancer cells. However, Survivin has increasingly been implicated in other normal physiologic and pathophysiologic processes, including angiogenesis. In this study, we dissect the involvement of Survivin DeltaEx3 in angiogenesis. We show by confocal microscopy that a pool of endothelial Survivin DeltaEx3 is localized to membrane ruffles. We also demonstrate that Survivin DeltaEx3 is the Survivin splice variant responsible for modulating angiogenesis in vitro, in tube formation assays, and in vivo, in an in vivo angiogenesis assay. Our data indicate that Survivin DeltaEx3 may regulate angiogenesis via several mechanisms including cell invasion, migration, and Rac1 activation. Our findings identify a novel pathway regulating angiogenesis through Survivin DeltaEx3 and a novel mechanism for Rac1 activation during angiogenesis. In conclusion, our results provide new insights into the regulation of endothelial cell homeostasis and angiogenesis by the Survivin proteins.

Alternative Splicing↗
PubMed2005

Identification of polymorphisms explaining a linkage signal: application to the GAW14 simulated data.

We applied three approaches for the identification of polymorphisms explaining the linkage evidence to the Genetic Analysis Workshop 14 simulated data: 1) the genotype-IBD sharing test (GIST); 2) an approach suggested by Horikawa and colleagues; and 3) the homozygote sharing test (HST). These tests were compared with a family-based association test. Two linked regions with highest nonparametric linkage scores were selected to apply these methods. In the first region, Horikawa's method identified the most SNPs within the region containing the disease susceptibility locus, while HST performed best in the second region. However, Horikawa's method also had the most type I errors. These methods show potential as additional tools to complement family-based association tests for the identification of disease susceptibility variants.

Chromosomes, Human, Pair 1↗
PubMed2006

Gene by environment QTL mapping through multiple trait analyses in blood pressure salt-sensitivity: identification of a novel QTL in rat chromosome 5.

BACKGROUND: The genetic mechanisms underlying interindividual blood pressure variation reflect the complex interplay of both genetic and environmental variables. The current standard statistical methods for detecting genes involved in the regulation mechanisms of complex traits are based on univariate analysis. Few studies have focused on the search for and understanding of quantitative trait loci responsible for gene x environmental interactions or multiple trait analysis. Composite interval mapping has been extended to multiple traits and may be an interesting approach to such a problem. METHODS: We used multiple-trait analysis for quantitative trait locus mapping of loci having different effects on systolic blood pressure with NaCl exposure. Animals studied were 188 rats, the progenies of an F2 rat intercross between the hypertensive and normotensive strain, genotyped in 179 polymorphic markers across the rat genome. To accommodate the correlational structure from measurements taken in the same animals, we applied univariate and multivariate strategies for analyzing the data. RESULTS: We detected a new quantitative train locus on a region close to marker R589 in chromosome 5 of the rat genome, not previously identified through serial analysis of individual traits. In addition, we were able to justify analytically the parametric restrictions in terms of regression coefficients responsible for the gain in precision with the adopted analytical approach. CONCLUSION: Future work should focus on fine mapping and the identification of the causative variant responsible for this quantitative trait locus signal. The multivariable strategy might be valuable in the study of genetic determinants of interindividual variation of antihypertensive drug effectiveness.

Animals↗
PubMed1998

Rat G protein-coupled receptor kinase GRK4: identification, functional expression, and differential tissue distribution of two splice variants.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G protein-coupled receptors, leading to the homologous mode of desensitization. We report here on the cloning of complementary DNAs that encode two rat GRK4 variants. Rat GRK4A (575 amino acids) displays 76% identity with the long human GRK4 splice variant. Rat GRK4B (545 amino acids) delineates a new variant that is identical to GRK4A except for a 31-amino acid deletion in the N-terminal domain, corresponding to exon VI in the human GRK4 gene. GRKs4A and B are likely produced by alternative splicing from a single gene, the partial characterization of which revealed a structural organization similar to that of the human GRK4 gene. GRK4A messenger RNA (mRNA) is abundant only in testis. A combination of in situ hybridization and quantitative RT-PCR studies demonstrated that GRK4A mRNA level increases during testicular development and predominates in leptotene to late pachytene primary spermatocytes and round spermatids. GRK4B mRNA is poorly expressed in testis and most rat tissues but is heterogeneously distributed in the kidney, with 20-fold enrichment in the outer medulla. GRKs4A and B are both functional protein kinases, as demonstrated in a rhodopsin phosphorylation assay. The differential tissue distribution of GRKA4 and GRK4B suggests that individual GRK4 variants may serve distinct physiological functions.

Amino Acid Sequence↗
PubMed2003

Genetic variations in calpain-10 gene are not a major factor in the occurrence of type 2 diabetes in Japanese.

The 112/121 haplotype combination defined by the UCSNP-43, -19, and -63 alleles in the calpain-10 gene is associated with type 2 diabetes in Mexican Americans. To determine whether this genetic variation constitutes risk of type 2 diabetes in Japanese, we investigated its frequency in 177 patients with type 2 diabetes and 172 controls. Though this variation occurs in Japanese more frequently than in Mexican Americans, there is no significant difference in frequency between diabetic (29.9%) and control (31.9%) subjects. We also screened all exons and the putative promoter of the calpain-10 gene for mutations in 96 of the genotyped patients, resulting in the identification of 7 coding variants, including 3 missense mutations and 5 nucleotide alterations in the promoter. However, their frequencies all are similar in patients and controls, suggesting that these genetic variations are not a major factor in the occurrence of type 2 diabetes in Japanese, although they could yet be associated with various phenotypes of the disease.

Aged↗
PubMed2006

Identification of distinct quantitative trait Loci affecting length or weight variability at birth in humans.

CONTEXT: The variability of human fetal growth is multifactorial. Twin and family studies demonstrate that genetic determinants influence normal fetal growth, but the responsible genetic polymorphisms are unknown. OBJECTIVE: The objective of the study was the mapping of quantitative trait loci (QTLs) for birth length and weight. DESIGN AND METHODS: To approach the genetic factors implicated in the normal variation of birth length and weight, we conducted a genome-wide approach of these two quantitative traits in 220 French Caucasian pedigrees (412 sibling pairs) using a variance components method. RESULTS: We observed evidence for several QTLs influencing birth length or birth weight independently. Whereas birth length and weight showed a close correlation (r = 0.76, P < 0.0001), their genetic variability appeared largely determined by distinct genomic loci. Birth length was influenced by two major QTLs located in 2p21 and 2q11 (LOD scores 2.69 and 3.57). The variability of birth weight was linked to another QTL on 7q35 (LOD score 3.1). Several other regions showed more modest evidence for linkage with LOD score values of 1-2 on chromosomes 7, 8, 10, 13, and 17 for birth length and chromosomes 1, 2, 6, 8, 10, 13, 14, 15, 17, and 20 for birth weight. CONCLUSION: These preliminary QTLs provide a first step toward the identification of the genomic variants involved in the variability of human fetal growth. Our results should, however, be considered preliminary until they are replicated in other studies.

Body Height↗
PubMed1995

Identification of human growth hormone-releasing hormone receptor splicing variants.

The expression pattern of the receptor for human GHRH (hGHRHr) was investigated in normal human tissues and in pituitary and ovarian tumors by reverse transcription-polymerase chain reaction. Three transcriptional variant forms of the hGHRHr were found to be expressed in the normal pituitary and in GH-secreting pituitary tumors. Form a is identical to the previously reported hGHRHr, whereas forms b and c are not described in the literature. Form b and c are predicted to be transcribed into two truncated proteins. We amplified genomic DNA using primers designed from the complementary DNA of hGHRHr. A 2-kilobase genomic DNA fragment was cloned that contains part of the hGHRHr gene consisting of two exons, e864 and e934, separated by three introns, i863, i933, and i1025. Alternative splicing of i1025 was responsible for three variant forms of hGHRHr. Nonsecreting pituitary tumors showed an abnormal expression of the hGHRHr, probably due to alternative usage of exons at the 5'-end of the gene, although they also expressed the three variant forms. No hGHRHr expression was identified in a human mammosomatotroph cell line insensitive to GHRH, in normal human liver or ovary, or in various human ovarian tumors.

Alternative Splicing↗
PubMed2003

Identification of the lantibiotic nisin Q, a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan.

Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence.

Amino Acid Sequence↗
PubMed2003

Candidate gene association study in type 2 diabetes indicates a role for genes involved in beta-cell function as well as insulin action.

Type 2 diabetes is an increasingly common, serious metabolic disorder with a substantial inherited component. It is characterised by defects in both insulin secretion and action. Progress in identification of specific genetic variants predisposing to the disease has been limited. To complement ongoing positional cloning efforts, we have undertaken a large-scale candidate gene association study. We examined 152 SNPs in 71 candidate genes for association with diabetes status and related phenotypes in 2,134 Caucasians in a case-control study and an independent quantitative trait (QT) cohort in the United Kingdom. Polymorphisms in five of 15 genes (33%) encoding molecules known to primarily influence pancreatic beta-cell function-ABCC8 (sulphonylurea receptor), KCNJ11 (KIR6.2), SLC2A2 (GLUT2), HNF4A (HNF4alpha), and INS (insulin)-significantly altered disease risk, and in three genes, the risk allele, haplotype, or both had a biologically consistent effect on a relevant physiological trait in the QT study. We examined 35 genes predicted to have their major influence on insulin action, and three (9%)-INSR, PIK3R1, and SOS1-showed significant associations with diabetes. These results confirm the genetic complexity of Type 2 diabetes and provide evidence that common variants in genes influencing pancreatic beta-cell function may make a significant contribution to the inherited component of this disease. This study additionally demonstrates that the systematic examination of panels of biological candidate genes in large, well-characterised populations can be an effective complement to positional cloning approaches. The absence of large single-gene effects and the detection of multiple small effects accentuate the need for the study of larger populations in order to reliably identify the size of effect we now expect for complex diseases.

ATP-Binding Cassette Transporters↗
PubMed2003

Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes.

BACKGROUND: Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR. METHODS: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (T(m)). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required approximately 1 min and no sample processing was needed after PCR. RESULTS: Polymorphisms in the HTR2A (T102C), beta-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 degrees C/s) of denatured products, followed by rapid heating during melting analysis (0.2-0.4 degrees C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in T(m) by <0.2 degrees C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5' tail of identical sequence was added to one of the two unlabeled primers. CONCLUSION: High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.

Base Sequence↗
PubMed1992

Resection of pulmonary metastasis to the azygous lobe from a malignant fibrous histiocytoma.

A 55-year-old man developed a pulmonary metastasis to the azygous lobe from a malignant fibrous histiocytoma of the thigh. The azygous lobe was not identified at the initial resection. A simple technique for the identification and mobilization of the azygous lobe is presented. Preoperative identification of this anatomic variant may assist in resection of parenchymal neoplasms.

Histiocytoma, Benign Fibrous↗
PubMed1998

Tracheal bronchus: a cause of prolonged atelectasis in intubated children.

Tracheal bronchus is a common anomaly that occurs in approximately 2% of people. Two children with multiple medical problems which led to endotracheal intubation are described. The hospital course for each child was complicated by persistent right upper lobe atelectasis. The presence of a tracheal bronchus was not recognized in either case initially; identification of this anatomic variant allowed appropriate changes in airway management. The potential for tracheal bronchus to cause, or be associated with, localized pulmonary problems is reviewed. The diagnosis of tracheal bronchus should be considered early in the course of intubated patients with right upper lobe complications.

Bronchi↗
PubMed2001

Advanced 3-D imaging for the evaluation of pancreatic cancer with multidetector CT.

BACKGROUND: Pancreatic cancer remains the fourth leading cause of cancer-related death. Surgery is the only option for cure. Multidetector computed tomography (CT) and advanced postprocessing techniques, can provide solutions for difficult problems in diagnosing and staging disease, and can aid radiologists in communicating findings to surgeons and oncologists. Multiplanar reformats can provide additional information on involvement of the portal vein, hepatic artery, and common bile duct. Maximum intensity projection (MIP) images and volume rendered images can aid in identification of important vascular variants. MIP images can show the relationship of tumor to the pancreatic duct or biliary tree. Curved reformatted images can show the relationship of tumor to the pancreatic duct or vascular structures.

Humans↗
PubMed2005

COMBINE genetics study: the pharmacogenetics of alcoholism treatment response: genes and mechanisms.

OBJECTIVE: Partial efficacy of treatment and differences in adverse events across individuals are a challenge and an opportunity in the treatment of alcoholism. Individuation of therapy and understanding origins of differential treatment response may require identification of inherited functional variants of genes. The neurobiology of reward, executive cognitive function, anxiety and dysphoria have been identified as critical domains that may have a genetic basis that could predict treatment response. METHOD: The COMBINE Study presents a unique opportunity to evaluate specific genetic loci (markers) that affect neurobiology central to addiction and extended withdrawal. The study also addresses variation in drug metabolism and action. Candidate genetic markers are selected for study based on functionality and abundance. RESULTS: COMT Vall58Met is a common (minor allele frequency 0.42), functional, catecholamine-metabolizing enzyme polymorphism with threefold relevance. Vall58Met alters executive cognitive function, stress and anxiety responses and brain endogenous opioid function. OPRM1 Asn40Asp is a common (minor allele frequency 0.10), functional polymorphism of the mu-opioid receptor, which may serve as a gatekeeper molecule in naltrexone's actions and was recently reported to affect naltrexone response. HTTLPR (minor allele frequency 0.40) alters serotonin transporter function to affect anxiety, dysphoria and obsessional behavior, which are assessed in COMBINE and may be related to relapse and addictive behavior. CONCLUSIONS: All genetic testing is consented through a separate human research protocol, and the testing is conducted nonclinically, confidentially and apart from the clinical record to protect human research participants who have volunteered for this aspect of COMBINE.

Acamprosate↗
PubMed2003

Molecular mechanisms of migraine: prospects for pharmacogenomics.

Migraine is a common complex disorder that affects a large portion of the population and thus incurs a substantial economic burden on society. The disorder is characterized by recurrent headaches that are unilateral and usually accompanied by nausea, vomiting, photophobia, and phonophobia. The range of clinical characteristics is broad and there is evidence of comorbidity with other neurological diseases, complicating both the diagnosis and management of the disorder. Although the class of drugs known as the triptans (serotonin 5-HT(1B/1D) agonists) has been shown to be effective in treating a significant number of patients with migraine, treatment may in the future be further enhanced by identifying drugs that selectively target molecular mechanisms causing susceptibility to the disease.Genetically, migraine is a complex familial disorder in which the severity and susceptibility of individuals is most likely governed by several genes that may be different among families. Identification of the genomic variants involved in genetic predisposition to migraine should facilitate the development of more effective diagnostic and therapeutic applications. Genetic profiling, combined with our knowledge of therapeutic response to drugs, should enable the development of specific, individually-tailored treatment.

Animals↗
PubMed2006

Biological applications of the receptor mimetic peptide mastoparan.

The receptor mimetic and mast cell degranulating peptide mastoparan (MP) translocates cell membranes as an amphipathic alpha-helix, a feature that is undoubtedly a major determinant of bioactivity through the activation of heterotrimeric G proteins. Chimeric combinations of MP with G protein-coupled receptor (GPCR) ligands has produced peptides that exhibit biological activities distinct from their composite components. Thus, chimeric peptides such as galparan and M391 differentially modulate GTPase activity, display altered binding affinities for appropriate GPCRs and possess disparate secretory properties. MP and MP-containing chimerae also bind and modulate the activities of various other intracellular protein targets and are valuable tools to manipulate and study enzymatic activity, calcium homeostasis and apoptotic signalling pathways. In addition, charge delocalisation within the hydrophilic face of MP has produced analogues, including [Lys5, Lys8,Aib10]MP, that differentially regulate mast cell secretion and/or cytotoxicity. Finally, the identification of cell penetrant variants of MP chimerae has enabled the effective intracellular delivery of non-permeable biomolecules and presents an opportunity to target novel intracellular therapeutic loci.

Amino Acid Sequence↗