bluuebunny/crossref_metadata_2025 huggingface dataset:
Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging
Authors: Van Remmen Holly , Ikeno Yuji , Hamilton Michelle , Pahlavani Mohammad , Wolf Norman , Thorpe Suzanne R. , Alderson Nathan L. , Baynes John W. , Epstein Charles J. , Huang Ting-Ting , Nelson James , Strong Randy , Richardson Arlan
Published: February 2004

Mice heterozygous for the Sod2 gene ( Sod2+/− mice) have been used to study the phenotype of life-long reduced Mn-superoxide dismutase (MnSOD) activity. The Sod2+/− mice have reduced MnSOD activity (∼50%) in all tissues throughout life. The Sod2+/− mice have increased oxidative damage as demonstrated by significantly elevated levels of 8-oxo-2-deoxyguanosine (8oxodG) in nuclear DNA in all tissues of Sod2+/− mice studied. The levels of 8oxodG in nuclear DNA increased with age in all tissues of Sod2+/− and wild-type (WT) mice, and at 26 mo of age, the levels of 8oxodG in nuclear DNA were significantly higher (from 15% in heart to over 60% in liver) in the Sod2+/− mice compared with WT mice. The level of 8oxodG was also higher in mitochondrial DNA isolated from liver and brain in Sod2+/− mice compared with WT mice. The increased oxidative damage to DNA in the Sod2+/− mice is associated with a 100% increase in tumor incidence (the number of mice with tumors) in old Sod2+/− mice compared with the old WT mice. However, the life spans (mean and maximum survival) of the Sod2+/− and WT mice were identical. In addition, biomarkers of aging, such as cataract formation, immune response, and formation of glycoxidation products carboxymethyl lysine and pentosidine in skin collagen changed with age to the same extent in both WT and Sod2+/− mice. Thus life-long reduction of MnSOD activity leads to increased levels of oxidative damage to DNA and increased cancer incidence but does not appear to affect aging.
Selective Increase in Renal Arcuate Innervation Density and Neurogenic Constriction in Chronic Angiotensin II-Infused Rats
Authors: Parkington Helena C. , Dodd Jonathan , Luff Susan E. , Worthy Katrina , Coleman Harold A. , Tare Marianne , Anderson Warwick P. , Edgley Amanda J.
Published: February 2004

This study investigated the effects of angiotensin II “slow pressor” hypertension on structure and function of nerves supplying the renal vasculature. Low-dose angiotensin II (10 ng/kg per minute, initially sub-pressor) or saline vehicle was infused intravenously for 21 days in rats, and the effects were compared in renal and mesenteric arteries. Mean arterial pressure averaged 12±2 mm Hg higher than in vehicle-infused rats at 21 days. Using electron microscopy, the innervation density of renal arcuate, but not mesenteric arteries of equivalent size, was significantly higher in angiotensin II-infused than in vehicle-infused rats. Functional testing on a pressure myograph revealed that constrictions evoked by nerve stimulation in arcuate arteries were 2.3±0.7-fold greater in vessels from angiotensin II-infused compared with vehicle-infused rats (
P
<0.0001), whereas there was no significant difference in nerve-induced constrictions in mesenteric arteries. Sensitivity to and maximum amplitude of constrictions evoked by phenylephrine were not different in renal or mesenteric arteries between groups, suggesting that the increased neurally evoked constriction in renal arcuate arteries was not caused by postsynaptic changes. Endothelium-dependent vasorelaxation and the vessel wall physical properties were not different between the two groups in either artery. Thus, angiotensin II infusion appeared to evoke renal-specific increases in vessel innervation and increased vasoconstriction to nerve stimulation. These changes appear early and occur before changes in renal endothelial function are apparent. Thus, “slow pressor” angiotensin II hypertension is associated with increased renal innervation, compatible with a pathogenetic role.
Association Between Blood Pressure and Resting Energy Expenditure Independent of Body Size
Authors: Luke Amy , Adeyemo Adebowale , Kramer Holly , Forrester Terrence , Cooper Richard S.
Published: February 2004

Obesity is an important risk factor for hypertension; however, the pathway through which it raises blood pressure (BP) is poorly understood. Body size is also the primary determinant of energy expenditure, and we therefore examined the joint relationship of energy expenditure and body size to blood pressure. Resting energy expenditure (REE) was measured using respiratory gas exchange in population-based samples of 997 Nigerians and 452 African Americans. In a third sample of 118 individuals, nonresting energy expenditure (ie, physical activity) was measured in addition to REE. The univariate correlation between REE and BP ranged from 0.10 to 0.22 in the 3 samples (
P
<0.001). In multivariate models, adiposity, whether defined by body mass, fat mass, or leptin, was no longer associated with BP, while REE remained highly significant (
P
<0.001). The REE–BP association also persisted after adjustment for physical activity measured with doubly labeled water. The odds ratio for hypertension among persons in the highest quartile versus the lowest quartile of REE, after adjustment for body size, was 1.7. This relationship was not the result of hypertension among the obese, because it did not vary across the range of BMI and was the same in lean Nigerians as in obese Americans. These data suggest that metabolic processes represented by REE may mediate the effect of body size on BP. The interrelationship of REE with sympathetic tone, transmembrane ion exchange, or other metabolic processes that determine energy costs at rest could provide physiological explanations for this observation.
Induction of Hyaluronic Acid Synthase 2 (HAS2) in Human Vascular Smooth Muscle Cells by Vasodilatory Prostaglandins
Authors: Sussmann M. , Sarbia M. , Meyer-Kirchrath J. , Nüsing R.M. , Schrör K. , Fischer J.W.
Published: February 2004

Hyaluronic acid (HA) is a prominent constituent of the extracellular matrix of atherosclerotic vascular lesions in humans known to modulate vascular smooth muscle phenotype. The regulation of HA synthesis by vasodilatory prostaglandins was analyzed in human arterial smooth muscle cells (SMCs). The prostacyclin analogue, iloprost (100 nmol/L), markedly increased pericellular formation of HA coats and HA secretion into the cell culture medium in human arterial SMCs (8.7±1.6-fold). Expression of HA synthase 2 (HAS2) was determined by semiquantitative RT-PCR and found to be strongly upregulated at concentrations of iloprost between 1 and 100 nmol/L after 3 hours. Furthermore, endogenous cyclooxygenase-2 (COX2) activity was required for basal expression of HAS2 mRNA in SMCs in vitro. Total HA secretion in response to iloprost was markedly decreased by RNA interference (RNAi), specific for HAS2. In addition, siRNA targeting HAS2 strongly increased the spreading of human SMCs compared with mock-transfected cells. HAS2 mRNA levels were also stimulated by a selective prostacyclin receptor (IP) agonist, cicaprost (10 nmol/L), prostaglandin E
2
(10 nmol/L), and the EP
2
receptor agonist, butaprost (1 μmol/L). Induction of HAS2 mRNA and HA synthesis by prostaglandins was mimicked by stable cAMP analogues and forskolin. In human atherectomy specimens from the internal carotid artery, HA deposits and COX2 expression colocalized frequently. In addition, strong EP
2
receptor expression was detected in SMCs in HA-rich areas. Therefore, upregulation of HAS2 expression via EP
2
and IP receptors might contribute to the accumulation of HA during human atherosclerosis, thereby mediating proatherosclerotic functions of COX2.
Restitution of Action Potential Duration During Sequential Changes in Diastolic Intervals Shows Multimodal Behavior
Authors: Wu Runze , Patwardhan Abhijit
Published: February 2004

Restitution of action potential duration (APD) is thought to be critical in activation instability. Although restitution is used to predict APD during sequential changes in diastolic interval (DI), currently used protocols to determine restitution do not use sequential changes in DI. We explored restitution using a new pacing protocol to change DI sequentially and independently of APD. Transmembrane potentials were recorded from right ventricular endocardial tissue isolated from six dogs. We used three patterns of DIs: oscillatory, to demonstrate differences in APDs depending on previous activation history; random, to minimize effects of previous activation history, each DI preceding an APD had an equal probability of being short or long; and linear, to compare restitution relationship obtained during sequential changes in DI with those obtained using currently used protocols; DIs mimicked those that resulted using currently used protocols, except that they changed in sequence. During oscillatory DIs, restitution showed bimodal trajectory similar to hysteresis. Decrease in APD during decreasing DIs was faster than increase in APD during increasing DIs. When effects of previous activation history were minimized, we observed that for a given DI there were multiple values of APD. Restitution relationship obtained during sequential changes in DI was shallower than those obtained using currently used protocols. Our results show that the new pacing protocol may permit direct evaluation of effects of memory on APD. Sequential and explicit control of DI suggests that use of a unimodal relationship to predict APD when DIs change in sequence may not be appropriate.
Modulation of In Vivo Cardiac Function by Myocyte-Specific Nitric Oxide Synthase-3
Authors: Champion Hunter C. , Georgakopoulos Dimitrios , Takimoto Eiki , Isoda Takayoshi , Wang Yibin , Kass David A.
Published: February 2004

Nitric oxide (NO) functions principally as a diffusible paracrine effector. The exception is in cardiomyocytes where both NO synthases (NOS) and target proteins coexist, allowing NO to work in an autocrine/intracrine fashion. However, the most abundant myocyte isoform (NOS3) is far more expressed in vascular endothelium; thus, the in vivo contribution of myocyte-NOS3 remains less clear. The present study tested this role by transfecting whole hearts of NOS3-null (NOS3
−/−
) mice with adenovirus-expressing NOS3 coupled to a α-MHC promoter (AdV
NOS3
), comparing results to hearts transfected with marker-gene β-galactosidase (AdVβ
gal
). Total myocardial NOS3 protein and activity were restored to near wild-type (WT) levels in NOS3
−/−
+AdV
NOS3
hearts, and NOS3 relocalized normally with caveolin-3. Ejection function by pressure-volume analysis was enhanced in NOS3
−/−
+AdVβ
gal
over WT or NOS3
−/−
+AdV
NOS3
. More prominently, isoproterenol (ISO)-stimulated systolic and diastolic function in WT was amplified in NOS3
−/−
+AdVβ
gal
, whereas NOS3
−/−
+AdV
NOS3
returned the response to control. ISO-activated systolic function was inhibited 85% by concomitant muscarinic stimulation (carbachol) in NOS3
−/−
+AdV
NOS3
but not NOS3
−/−
+AdVβ
gal
hearts. Lastly, NOS3
−/−
+AdVβ
gal
mice displayed enhanced inotropy and lusitropy over WT at slower heart rates but a blunted rate augmentation versus controls. A more positive rate response was restored in NOS3
−/−
+AdV
NOS3
(
P
<0.001). Thus, myocyte autocrine/intracrine NOS3 regulation in vivo can underlie key roles in β-adrenergic, muscarinic, and frequency-dependent cardiac regulation.
Selective Suppression of Endothelial Cell Apoptosis by the High Molecular Weight Form of Adiponectin
Authors: Kobayashi Hideki , Ouchi Noriyuki , Kihara Shinji , Walsh Kenneth , Kumada Masahiro , Abe Yuki , Funahashi Tohru , Matsuzawa Yuji
Published: February 2004

Adiponectin is an adipocyte-derived, antiatherogenic protein that is present in serum as three isoforms. Total adiponectin levels are decreased in obese or diabetic humans or animal models. This study was designed to elucidate the relative isoform distribution of adiponectin in human disease states and identify the active form of adiponectin toward vascular endothelial cells. The percentage of high molecular weight form (HMW) per total adiponectin was significantly lower in patients with coronary artery disease than control subjects, whereas the hexamer form was similar and the trimer form was significantly higher. During weight reduction in obese subjects, the HMW form increased and the trimer and hexamer forms decreased. Recombinant adiponectin dose-dependently suppressed apoptosis and caspase-3 activity in human umbilical vein endothelial cells (HUVECs). Transduction with dominant-negative AMP-activated protein kinase (AMPK) abolished the suppressive effect of adiponectin on HUVECs. Gel filtration chromatography was used to separate the adiponectin isoforms, and the antiapoptotic effect toward HUVECs was only observed with the HMW form. These data suggest that HMW adiponectin specifically confers the vascular-protective activities of this adipocytokine. The full text of this article is available online at
http://circres.ahajournals.org
.
Active and Passive Smoking and the Risk of Subarachnoid Hemorrhage
Authors: Anderson Craig S. , Feigin Valery , Bennett Derrick , Lin Ruey-Bin , Hankey Graeme , Jamrozik Konrad
Published: February 2004

Background and Purpose—

This study was undertaken to better clarify the risks associated with cigarette smoking and subarachnoid hemorrhage (SAH).

Methods—

The study included 432 incident cases of SAH frequency matched to 473 community SAH-free controls to determine dose-dependent associations of active and passive smoking (at home) and smoking cessation with SAH.

Results—

Compared with never smokers not exposed to passive smoking, the adjusted odds ratio for SAH among current smokers was 5.0 (95% confidence interval [CI], 3.1 to 8.1); for past smokers, 1.2 (95% CI, 0.8 to 2.0); and for passive smokers, 0.9 (95% CI, 0.6 to 1.5). Current and lifetime exposures showed a clear dose-dependent effect, and risks appeared more prominent in women and for aneurysmal SAH. Approximately 1 in 3 cases of SAH could be attributed to current smoking, but risks decline quickly after smoking cessation, even among heavy smokers.

Conclusions—

A strong positive association was found between cigarette smoking and SAH, especially for aneurysmal SAH and women, which is virtually eliminated within a few years of smoking cessation. Large opportunities exist for preventing SAH through smoking avoidance and cessation programs.
Deregulated expression in Ph+ human leukemias of AHI-1, a gene activated by insertional mutagenesis in mouse models of leukemia
Authors: Jiang Xiaoyan , Zhao Yun , Chan Wing-Yiu , Vercauteren Suzanne , Pang Emily , Kennedy Sean , Nicolini Frank , Eaves Allen , Eaves Connie
Published: February 2004

Ahi-1/AHI-1 (Abelson helper integration site-1) encodes a family of protein isoforms containing one Src homology 3 (SH3) domain and multiple tryptophan-aspartic acid 40 (WD40)–repeat domains. The function of these proteins is unknown, but involvement in leukemogenesis has been suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias. Here we show that in both mice and humans, Ahi-1/AHI-1 expression is highest in the most primitive hematopoietic cells with specific patterns of down-regulation in different lineages. Cells from patients with chronic myeloid leukemia (CML; n = 28) show elevated AHI-1 transcripts in all disease phases and, in chronic phase, in the leukemic cells at all stages of differentiation, including quiescent (G0) CD34+ cells as well as terminally differentiating cells. In the most primitive lin–CD34+CD38– CML cells, transcripts for the 2 shorter isoforms of AHI-1 are also increased. Although 15 of 16 human lymphoid and myeloid leukemic cell lines showed aberrant control of AHI-1 expression, this was not seen in blasts obtained directly from patients with acute Philadelphia chromosome–negative (Ph–) leukemia (n = 15). Taken together, our results suggest that down-regulation of AHI-1 expression is an important conserved step in primitive normal hematopoietic cell differentiation and that perturbations in AHI-1 expression may contribute to the development of specific types of human leukemia.
Modeled Microgravity Inhibits Osteogenic Differentiation of Human Mesenchymal Stem Cells and Increases Adipogenesis
Authors: Zayzafoon Majd , Gathings William E. , McDonald Jay M.
Published: February 2004

Space flight-induced bone loss has been attributed to a decrease in osteoblast function, without a significant change in bone resorption. To determine the effect of microgravity (MG) on bone, we used the Rotary Cell Culture System [developed by the National Aeronautics and Space Administration (NASA)] to model MG. Cultured mouse calvariae demonstrated a 3-fold decrease in alkaline phosphatase (ALP) activity and failed to mineralize after 7 d of MG. ALP and osteocalcin gene expression were also decreased. To determine the effects of MG on osteoblastogenesis, we cultured human mesenchymal stem cells (hMSC) on plastic microcarriers, and osteogenic differentiation was induced immediately before the initiation of modeled MG. A marked suppression of hMSC differentiation into osteoblasts was observed because the cells failed to express ALP, collagen 1, and osteonectin. The expression of runt-related transcription factor 2 was also inhibited. Interestingly, we found that peroxisome proliferator-activated receptor γ (PPARγ2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4 are highly expressed in response to MG. These changes were not corrected after 35 d of readaptation to normal gravity. In addition, MG decreased ERK- and increased p38-phosphorylation. These pathways are known to regulate the activity of runt-related transcription factor 2 and PPARγ2, respectively. Taken together, our findings indicate that modeled MG inhibits the osteoblastic differentiation of hMSC and induces the development of an adipocytic lineage phenotype. This work will increase understanding and aid in the prevention of bone loss, not only in MG but also potentially in age-and disuse-related osteoporosis.
Insulin-Like Growth Factor-I Augments Chondrocyte Hypertrophy and Reverses Glucocorticoid-Mediated Growth Retardation in Fetal Mice Metatarsal Cultures
Authors: Mushtaq T. , Bijman P. , Ahmed S. F. , Farquharson C.
Published: February 2004

The study aims were to improve our understanding of the mechanisms of glucocorticoid-induced growth retardation at the growth plate and determine whether IGF-I could ameliorate the effects. Fetal mouse metatarsals were cultured for up to 10 d with dexamethasone (Dex; 10–6m) and/or IGF-I and GH (both at 100 ng/ml). Both continuous and alternate-day Dex treatment inhibited bone growth to a similar degree, whereas IGF-I alone or together with Dex caused an increase in bone growth. GH had no effects. These observations may be explained at the cellular level; cell proliferation within the growing bone was decreased by Dex and increased by IGF-I and these effects were more marked in the cells of the perichondrium than those in the growth plate. However, the most prominent observation was noted in the hypertrophic zone where all treatments containing IGF-I significantly increased (3-fold) the length of this zone, whereas Dex alone had no significant effect. In conclusion, Dex impaired longitudinal growth by inhibiting chondrocyte proliferation, whereas IGF-I stimulated chondrocyte hypertrophy and reversed the growth-inhibitory Dex effects. However, the IGF-I-mediated improvement in growth was at the expense of altering the balance between proliferating and hypertrophic chondrocytes within the metatarsal.
Orphan Nuclear Receptor Small Heterodimer Partner, a Novel Corepressor for a Basic Helix-Loop-Helix Transcription Factor BETA2/NeuroD
Authors: Kim Joon-Young , Chu Khoi , Kim Han-Jong , Seong Hyun-A , Park Ki-Cheol , Sanyal Sabyasachi , Takeda Jun , Ha Hyunjung , Shong Minho , Tsai Ming-Jer , Choi Hueng-Sik
Published: February 2004

Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA binding domain (DBD) and represses the transcriptional activity of various nuclear receptors. In this study, we examined the novel cross talk between SHP and BETA2/NeuroD, a basic helix-loop-helix transcription factor. In vitro and in vivo protein interaction studies showed that SHP physically interacts with BETA2/NeuroD, but not its heterodimer partner E47. Moreover, confocal microscopic study and immunostaining results demonstrated that SHP colocalized with BETA2 in islets of mouse pancreas. SHP inhibited BETA2/NeuroD-dependent transactivation of an E-box reporter, whereas SHP was unable to repress the E47-mediated transactivation and the E-box mutant reporter activity. In addition, SHP repressed the BETA2-dependent activity of glucokinase and cyclin-dependent kinase inhibitor p21 gene promoters. Gel shift and in vitro protein competition assays indicated that SHP inhibits neither dimerization nor DNA binding of BETA2 and E47. Rather, SHP directly repressed BETA2 transcriptional activity and p300-enhanced BETA2/NeuroD transcriptional activity by inhibiting interaction between BETA2 and coactivator p300. We also showed that C-terminal repression domain within SHP is also required for BETA2 repression. However, inhibition of BETA2 activity was not observed by naturally occurring human SHP mutants that cannot interact with BETA2/NeuroD. Taken together, these results suggest that SHP acts as a novel corepressor for basic helix-loop-helix transcription factor BETA2/NeuroD by competing with coactivator p300 for binding to BETA2/NeuroD and by its direct transcriptional repression function.
Complementary Medicine Prescription Patterns in Germany
Authors: Schneider Berthold , Hanisch Jürgen , Weiser Michael
Published: February 2004

BACKGROUND

There is growing interest in complementary medicine worldwide and a corresponding need to know how patients and practitioners interact.

OBJECTIVE

To chart the use of complementary medications compared with conventional treatments among practitioners and patients in Germany.

METHODS

Comparative analysis of patients' data from a prospective cohort study of 4178 patients presenting with chronic as well as acute symptoms by 218 practitioners at 218 centers in all German federal states between 2001 and 2002. Practices focused on either conventional medicine, complementary medicine, or both. Data were gathered on prescription patterns, treatment satisfaction and adherence, and the degree of patient involvement in treatment decisions.

RESULTS

Complementary medicine was preferentially prescribed over conventional medicine in patients <18 and >65 years old, in women, and in patients with chronic symptoms. Patients receiving complementary medications had, on average, higher rates of accompanying illnesses, received more preparations, and were more closely involved in the decision process than patients prescribed conventional therapies. The decision to use complementary medicine was based on both the patient's and the practitioner's wishes in 40.8% of the cases compared with 25.8% of the cases of conventional therapies. Tolerability and satisfaction with treatment appeared greater with complementary than with conventional therapies.

CONCLUSION

Complementary medicine is generally well established in Germany, apparently in a dialogue with conventional medicine. Patients receiving complementary medicine appear to be more closely involved in the decision process and more satisfied with treatments than conventionally treated patients.
Troponin I in Patients without Chest Pain
Authors: Ritter Detlef , Lee Paul A , Taylor James F , Hsu Leo , Cohen Jerome D , Chung Hyung D , Virgo Katherine S
Published: February 2004

Background: Testing for troponin has important clinical value for patients who present with typical symptoms of acute coronary syndromes (ACS) such as chest pain (CP). Much less is known about the value of troponin testing for patients who present with other symptoms of ACS (anginal equivalent symptoms).

Methods: The utilization and prognostic value of cardiac troponin I (cTnI) were evaluated at a Veterans Affairs Acute Care Facility. Clinical charts of 1184 predominantly male patients, who submitted specimens for initial cTnI testing by AxSYM, were evaluated for demographic data, cardiovascular risk factors, major diseases, and complaints at the time of testing. The endpoint was defined as all-cause death during a 200-day period after initial testing.

Results: Sixty-one percent of cTnI tests were ordered for patients who did not present with CP. Patients presenting with symptoms other than CP did not have significantly lower plasma cTnI than patients with CP. However, patients with symptoms other than CP were rarely diagnosed with ACS unless cTnI was ≥2 μg/L. The mortality during the follow-up period was severalfold higher among patients presenting with symptoms other than CP (CP, 6%; without CP, 22%; P <0.0001, χ2 test). cTnI ≥0.2 μg/L provided significant additional predictive information for patients who presented with anginal equivalent symptoms such as shortness of breath or general weakness.

Conclusion: Patients with anginal equivalent symptoms of ACS and low-positive cTnI are less often diagnosed with ACS and have a higher mortality than patients with CP.
Diagnosis of Congenital Disorders of Glycosylation by Capillary Zone Electrophoresis of Serum Transferrin
Authors: Carchon Hubert A , Chevigné Roland , Falmagne Jean-Bernard , Jaeken Jaak
Published: February 2004

Background: Congenital disorders of glycosylation (CDG) are usually diagnosed by isoelectric focusing (IEF) of serum transferrin (Tf). The aim of this study was to evaluate capillary zone electrophoresis (CZE) as a diagnostic alternative to IEF.

Methods: We performed 792 CZE analyses of Tf, using the CEofixTM-CDT (carbohydrate-deficient transferrin) assay. Peak identification was based on relative migration times (RMTs) to reduce migration variability.

Results: Tf profiles comprised three main groups (A–C). Groups A and B were characterized by one or two dominant tetrasialo-Tf peaks, whereas group C showed a widely variable Tf isoform composition. Group A was composed of four subgroups: a major group with a typical Tf profile (considered as reference group), two minor groups with decreased or moderately increased trisialo-Tf isoform, and a group showing the presence of unknown compounds with RMTs similar to mono- and disialo-Tf. However, these compounds were absent on IEF. Group C contained all profiles from patients with confirmed as well as putative CDG. From the reference group, 99% confidence intervals were calculated for the RMTs of the Tf isoforms, and percentiles representing the Tf isoform distributions were defined.

Conclusions: All patients with abnormal IEF results and confirmed CDG were identified by CZE; thus, this method can be used as a diagnostic alternative to IEF in a manner suitable for automation. Because whole serum is analyzed, it should be kept in mind that CZE profiles can show substances other than Tf.
Highly Recurrent RET Mutations and Novel Mutations in Genes of the Receptor Tyrosine Kinase and Endothelin Receptor B Pathways in Chinese Patients with Sporadic Hirschsprung Disease
Authors: Garcia-Barceló Mercè , Sham Mai-Har , Lee Wing-Shan , Lui Vincent Chi-Hang , Chen Benedict Ling-Sze , Wong Kenneth Kak-Yuen , Wong Joyce Suet-Wan , Tam Paul Kwong-Hang
Published: February 2004

Background: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of ganglion cells in the nerve plexuses of the lower digestive tract. HSCR has a complex pattern of inheritance and is sometimes associated with mutations in genes of the receptor tyrosine kinase (RET) and endothelin receptor B (EDNRB) signaling pathways, which are crucial for development of the enteric nervous system.

Methods: Using PCR amplification and direct sequencing, we screened for mutations and polymorphisms in the coding regions and intron/exon boundaries of the RET, GDNF, EDNRB, and EDN3 genes of 84 HSCR patients and 96 ethnically matched controls.

Results: We identified 10 novel and 2 previously described mutations in RET, and 4 and 2 novel mutations in EDNRB and in EDN3, respectively. Potential disease-causing mutations were detected in 24% of the patients. The overall mutation rate was 41% in females and 19% in males (P = 0.06). RET mutations occurred in 19% of the patients. R114H in RET was the most prevalent mutation, representing 7% of the patients or 37% of the patients with RET mutations. To date, such a high frequency of a single mutation has never been reported in unrelated HSCR patients. Mutations in EDNRB, EDN3, and GDNF were found in four, two, and none of the patients, respectively. Two patients with mutations in genes of the EDNRB pathway also harbored a mutation in RET. Three novel and three reported polymorphisms were found in EDNRB, EDN3, and GDNF.

Conclusion: This study identifies additional HSCR disease-causing mutations, some peculiar to the Chinese population, and represents the first comprehensive genetic analysis of sporadic HSCR disease in Chinese.
Detection of the Factor V Leiden Mutation by a Modified Photo-Cross-Linking Oligonucleotide Hybridization Assay
Authors: French Cynthia , Li Conan , Strom Charles , Sun Weimin , Van Atta Reuel , Gonzalez Belen , Wood Michael
Published: February 2004

Background: Our previously developed assay for detection of the factor V Leiden mutation (G1691A) based on a nucleic acid photo-cross-linking technology used two allele-specific capture probes and six fluorescein-modified signal-generating reporter probes. We wished to improve the sensitivity and performance of the method.

Methods: We developed new reporter probes with ∼10-fold more fluorescein molecules than the original probes. The single, cross-linker-modified capture probe was replaced by a three-probe system, separating the probe–target cross-linking function and the allelic differentiation function. The capture probe cross-linked to either or both of two flanking probes through stem structures at the capture-probe/flanking-probe junctions. The flanking probes cross-linked to target DNA through two cross-linking sites each. Genomic DNA was extracted from 0.2 mL of whole blood and restriction-enzyme digested to create a defined 677 bp target sequence. Preliminary genotype ranges were determined for the assay by testing of pretyped samples. We then tested 1054 clinical samples, using an automated sample processor.

Results: The new assay had a 10-fold increase in signal-to-background ratio. Genotype results for 1039 of 1054 clinical samples (98.6%) agreed with those of a PCR-based method. Of the 15 remaining samples, 10 produced an indeterminate result outside the defined genotype ranges, 2 yielded insufficient signal to be genotyped, and 3 gave a discordant result. All 15 samples were genotyped correctly after reextraction of genomic DNA and retesting.

Conclusion: The modified photo-cross-linking assay for factor V Leiden detection is a sensitive non-PCR-based assay with potential for use in high-throughput clinical laboratories.
High-Throughput Mutation Detection Method to Scan BRCA1 and BRCA2 Based on Heteroduplex Analysis by Capillary Array Electrophoresis
Authors: Esteban-Cardeñosa Eva , Duran Mercedes , Infante Mar , Velasco Eladio , Miner Cristina
Published: February 2004

Background: Scanning for mutations in BRCA1 and BRCA2 in a large number of samples is hampered by the large sizes of these genes and the scattering of mutations throughout their coding sequences. Automated capillary electrophoresis has been shown to be a powerful system to detect mutations by either single-strand conformation polymorphism or heteroduplex analysis (HA).

Methods: We investigated the adaptation of gel-based HA of BRCA1 and BRCA2 to a fluorescent multicapillary platform to increase the throughput of this technique. We combined multiplex PCR, three different fluorescent labels, and HA in a 16-capillary DNA sequencer and tested 57 DNA sequence variants (11 insertions/deletions and 46 single-nucleotide changes) of BRCA1 and BRCA2.

Results: We detected all 57 DNA changes in a blinded assay, and 2 additional single-nucleotide substitutions (1186 A>G of BRCA1 and 3624 A>G of BRCA2), previously unresolved by conformation-sensitive gel electrophoresis. Furthermore, different DNA changes in the same PCR fragment could be distinguished by their peak patterns.

Conclusions: Capillary-based HA is a fast, efficient, and sensitive method that considerably reduces the amount of “hands-on” time for each sample. By this approach, the entire coding regions of BRCA1 and BRCA2 from two breast cancer patients can be scanned in a single run of 90 min.
Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays
Authors: Poon Leo L M , Chan Kwok Hung , Wong On Kei , Cheung Timothy K W , Ng Iris , Zheng Bojian , Seto Wing Hong , Yuen Kwok Yung , Guan Yi , Peiris Joseph S M
Published: February 2004

Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens.

Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV.

Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar.

Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.
Detection of Human Polyomaviruses in Urine from Bone Marrow Transplant Patients: Comparison of Electron Microscopy with PCR
Authors: Biel Stefan S , Nitsche Andreas , Kurth Andreas , Siegert Wolfgang , Özel Muhsin , Gelderblom Hans R
Published: February 2004

Background: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients.

Methods: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples.

Results: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 106 genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <103 GE/mL and 100% for urine samples containing 109 GE/mL.

Conclusions: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.
Diagnostic Accuracy and Prognostic Relevance of the Measurement of Cardiac Natriuretic Peptides: A Review
Authors: Clerico Aldo , Emdin Michele
Published: February 2004

Background: The pathophysiologic and clinical relevance of cardiac natriuretic hormone (CNH) assays has been investigated in numerous experimental and clinical studies. Authors have sought to evaluate the diagnostic accuracy and prognostic relevance of the measurement of CNHs according to evidence-based laboratory medicine principles.

Methods: In June 2003, we ran a computerized literature search on National Library of Medicine using keywords “ANP” and “BNP” and found more than 12 300 and 1200 articles, respectively. A more refined search with keywords “ANP or BNP assay” extracted ∼7000 and 800 articles, respectively. Only studies specifically designed to evaluate the diagnostic accuracy and prognostic relevance of CNH measurements were selected from this huge mass of articles to be discussed in this review.

Content: Several studies suggested that CNH assays may be clinically useful for the screening and classification of patients with heart failure, as a prognostic marker in cardiovascular disease, in the follow-up of patients with heart failure, and because they may reduce the need for further cardiac investigation. However, it is difficult to compare even the best-designed studies because not only did the authors evaluate different populations, they also used different gold standards.

Conclusions: CNH assays and conventional diagnostic work-ups provide complementary information for evaluation of the presence and severity of cardiac dysfunction and clinical disease. Several aspects of CNH assays are still to be elucidated, and further work is needed to carefully assess their diagnostic accuracy and prognostic value in cardiac disease.
Size Distributions of Maternal and Fetal DNA in Maternal Plasma
Authors: Chan K C Allen , Zhang Jun , Hui Angela B Y , Wong Nathalie , Lau Tze K , Leung Tse N , Lo Kwok-Wai , Huang Dolly W S , Lo Y M Dennis
Published: February 2004

Background: The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive prenatal diagnosis. Despite the rapid expansion in clinical applications, the molecular characteristics of plasma DNA in pregnant women remain unclear.

Methods: We investigated the size distribution of plasma DNA in 34 nonpregnant women and 31 pregnant women, using a panel of quantitative PCR assays with different amplicon sizes targeting the leptin gene. We also determined the size distribution of fetal DNA in maternal plasma by targeting the SRY gene.

Results: The median percentages of plasma DNA with size >201 bp were 57% and 14% for pregnant and nonpregnant women, respectively (P <0.001, Mann–Whitney test). The median percentages of fetal-derived DNA with sizes >193 bp and >313 bp were 20% and 0%, respectively, in maternal plasma.

Conclusion: Plasma DNA molecules are mainly short DNA fragments. The DNA fragments in the plasma of pregnant women are significantly longer than those in the plasma of nonpregnant women, and the maternal-derived DNA molecules are longer than the fetal-derived ones.
Liquid Chromatography–Tandem Mass Spectrometry Assay for Simultaneous Measurement of Estradiol and Estrone in Human Plasma
Authors: Nelson Robert E , Grebe Stefan K , O’Kane Dennis J , Singh Ravinder J
Published: February 2004

Background: Estradiol (E2) and estrone (E1) measurements form an integral part of the assessment of female reproductive function and have expanding roles in other fields. However, many E1 and E2 immunoassays have limited functional sensitivity, suffer from cross-reactivity, and display poor intermethod agreement. To overcome these problems, we developed a sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of E1 and E2.

Methods: After dansyl chloride derivatization, samples were separated by fast gradient chromatography and injected into a tandem mass spectrometer after formation of positive ions with atmospheric pressure chemical ionization. The limits of detection and quantification, recovery, linearity, precision, and reference intervals were determined, and performance was compared with several immunoassays.

Results: Total run time per sample was 5 min. The multiple-reaction monitoring ion pairs were m/z 506/171 for 3-dansyl-estradiol and m/z 504/171 for 3-dansyl-estrone. The limits of detection for E1 and E2 were 12.9 pmol/L (3.5 ng/L) and 10.3 pmol/L (2.8 ng/L), respectively. Interassay imprecision (CV) was 4–20% (n = 20). The limits of quantification (functional sensitivities) for E1 and E2 were 44.1 pmol/L (11.9 ng/L) and 23.2 pmol/L (6.3 ng/L), respectively. The assay was linear to >2200 pmol/L (∼600 ng/L) for each analyte. Recoveries were 93–108% for E1 and 100–110% for E2. No cross-reactivity was observed. Method comparison with several immunoassays revealed that the latter were inaccurate and prone to interferences at low E1 and E2 concentrations.

Conclusions: LC-MS/MS allows rapid, simultaneous, sensitive, and accurate quantification of E1 and E2 in human serum.
Online Measurement of Urea Concentration in Spent Dialysate during Hemodialysis
Authors: Olesberg Jonathon T , Arnold Mark A , Flanigan Michael J
Published: February 2004

Background: We describe online optical measurements of urea in the effluent dialysate line during regular hemodialysis treatment of several patients. Monitoring urea removal can provide valuable information about dialysis efficiency.

Methods: Spectral measurements were performed with a Fourier-transform infrared spectrometer equipped with a flow-through cell. Spectra were recorded across the 5000–4000 cm−1 (2.0–2.5 μm) wavelength range at 1-min intervals. Savitzky–Golay filtering was used to remove baseline variations attributable to the temperature dependence of the water absorption spectrum. Urea concentrations were extracted from the filtered spectra by use of partial least-squares regression and the net analyte signal of urea.

Results: Urea concentrations predicted by partial least-squares regression matched concentrations obtained from standard chemical assays with a root mean square error of 0.30 mmol/L (0.84 mg/dL urea nitrogen) over an observed concentration range of 0–11 mmol/L. The root mean square error obtained with the net analyte signal of urea was 0.43 mmol/L with a calibration based only on a set of pure-component spectra. The error decreased to 0.23 mmol/L when a slope and offset correction were used.

Conclusions: Urea concentrations can be continuously monitored during hemodialysis by near-infrared spectroscopy. Calibrations based on the net analyte signal of urea are particularly appealing because they do not require a training step, as do statistical multivariate calibration procedures such as partial least-squares regression.
Definitive Diagnosis of Mitochondrial Neurogastrointestinal Encephalomyopathy by Biochemical Assays
Authors: Martı́ Ramon , Spinazzola Antonella , Tadesse Saba , Nishino Ichizo , Nishigaki Yutaka , Hirano Michio
Published: February 2004

Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the gene encoding thymidine phosphorylase (TP). The clinical manifestations of MNGIE are recognizable and homogeneous, but in the early stages, the disease is often misdiagnosed. This study assesses the reliability of biochemical assays to diagnose MNGIE.

Methods: We studied 180 patients with clinical features suggestive of MNGIE, 14 asymptomatic TP mutation carriers, and 20 controls. TP enzyme activity in the buffy coat was determined by a fixed-time method, and the plasma nucleosides thymidine (dThd) and deoxyuridine (dUrd) were assessed by a gradient-elution reversed phase HPLC method. TP was sequenced through standard procedures in patients who met the clinical criteria for MNGIE.

Results:Twenty-five of the 180 patients fulfilled the clinical criteria for MNGIE and had homozygous or compound heterozygous TP mutations. All had drastically decreased TP activity [mean (SD), 10 (15) nmol thymine formed · h−1 · (mg protein)−1 vs 634 (217) nmol thymine formed · h−1 · (mg protein)−1 for the controls]. Relative to the control mean, TP activities were reduced to 35% in mutation carriers and 65% in MNGIE-like patients. All 25 MNGIE patients had detectable plasma dThd [8.6 (3.4) μmol/L] and dUrd [14.2 (4.4) μmol/L]. Controls, carriers, and MNGIE-like patients showed no detectable plasma dThd and dUrd.

Conclusions:We propose a diagnostic algorithm based on the determination of plasma dThd and dUrd, TP activity in buffy coat, or both to make a definitive diagnosis of MNGIE. Increased concentrations of dThd (>3 μmol/L) and dUrd (>5 μmol/L) in plasma or a decrease in buffy coat TP activity to ≤8% relative to controls is sufficient to diagnose MNGIE.
Determination of Folate Vitamers in Human Serum by Stable-Isotope-Dilution Tandem Mass Spectrometry and Comparison with Radioassay and Microbiologic Assay
Authors: Pfeiffer Christine M , Fazili Zia , McCoy Les , Zhang Ming , Gunter Elaine W
Published: February 2004

Background: Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-methyltetrahydrofolic acid (5MeTHF), 5-formyltetrahydrofolic acid (5FoTHF), and folic acid (FA) in human serum.

Methods: We quantitatively isolated folates from 275 μL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic acid in organic solvent on a C8 analytical column. 13C-labeled folates were used as internal standards.

Results: Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was <7% for 5MeTHF and <10% for 5FoTHF at concentrations >0.5 nmol/L, and <10% for FA at concentrations >2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were ∼9% lower than results obtained with LC/MS/MS. The microbiologic assay gave ∼15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5–75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3% 5MeTHF, 2.3% FA, and 4.4% 5FoTHF. Ten samples with TFOL >50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF.

Conclusions: This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.