bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and Functional Analysis of Mitochondrial DNA‐Encoded Complex I Genes
Authors: BAI YIDONG , HU PEIQING , PARK JEONG SOON , DENG JIAN‐HONG , SONG XIUFENG , CHOMYN ANNE , YAGI TAKAO , ATTARDI GIUSEPPE
Published: May 2004

Abstract: Mammalian mitochondrial NADH dehydrogenase (complex I) is a multimeric complex consisting of at least 45 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). The function of these subunits is largely unknown. We have established an efficient method to isolate and characterize cells carrying mutations in various mtDNA‐encoded complex I genes. With this method, 15 mouse cell lines with deficiencies in complex I‐dependent respiration were obtained, and two near‐homoplasmic mutations in mouse ND5 and ND6 genes were isolated. Furthermore, by generating a series of cell lines with the same nuclear background but different content of an mtDNA nonsense mutation, we analyzed the genetic and functional thresholds in mouse mitochondria. We found that in wild‐type cells, about 40% of ND5 mRNA is in excess of that required to support a normal rate of ND5 subunit synthesis. However, there is no indication of compensatory upsurge in either transcription or translation with the increase in the proportion of mutant ND5 genes. Interestingly, the highest ND5 protein synthesis rate was just sufficient to support the maximum complex I‐dependent respiration rate, suggesting a tight regulation at the translational level. In another line of research, we showed that the mitochondrial NADH‐quinone oxidoreductase of Saccharomyces cerevisiae (NDI1), although consisting of a single subunit, can completely restore respiratory NADH dehydrogenase activity in mutant human cells that lack the essential mtDNA‐encoded subunit ND4. In particular, in these transfected cells, the yeast enzyme becomes integrated into the human respiratory chain and fully restores the capacity of the cells to grow in galactose medium.
Genome‐Wide Analysis of Signal Transducers and Regulators of Mitochondrial Dysfunction in Saccharomyces cerevisiae
Authors: SINGH KESHAV K. , RASMUSSEN ANNE KARIN , RASMUSSEN LENE JUEL
Published: May 2004

Abstract: Mitochondrial dysfunction is a hallmark of cancer cells. However, genetic response to mitochondrial dysfunction during carcinogenesis is unknown. To elucidate genetic response to mitochondrial dysfunction we used Saccharomyces cerevisiae as a model system. We analyzed genome‐wide expression of nuclear genes involved in signal transduction and transcriptional regulation in a wild‐type yeast and a yeast strain lacking the mitochondrial genome (rho0). Our analysis revealed that the gene encoding cAMP‐dependent protein kinase subunit 3 (PKA3) was upregulated. However, the gene encoding cAMP‐dependent protein kinase subunit 2 (PKA2) and the VTC1, PTK2, TFS1, CMK1, and CMK2 genes, involved in signal transduction, were downregulated. Among the known transcriptional factors, OPI1, MIG2, INO2, and ROX1 belonged to the upregulated genes, whereas MSN4, MBR1, ZMS1, ZAP1, TFC3, GAT1, ADR1, CAT8, and YAP4 including RFA1 were downregulated. RFA1 regulates DNA repair genes at the transcriptional level. RFA is also involved directly in DNA recombination, DNA replication, and DNA base excision repair. Downregulation of RFA1 in rho0 cells is consistent with our finding that mitochondrial dysfunction leads to instability of the nuclear genome. Together, our data suggest that gene(s) involved in mitochondria‐to‐nucleus communication play a role in mutagenesis and may be implicated in carcinogenesis.
Enhanced Detection of Deleterious Mutations by TTGE Analysis of Mother and Child's DNA Side by Side
Authors: KWON HAEYOUNG , TAN DUAN JUN , BAI REN‐KUI , WONG LEE‐JUN C.
Published: May 2004

Abstract: Mitochondrial DNA (mtDNA) disorders represent a group of heterogeneous diseases that are caused by mutations in mtDNA. We examined 45 pairs of mother and the affected child, by screening the entire mitochondrial genome with temporal temperature gradient gel electrophoresis (TTGE), using 32 pairs of overlapping primers. TTGE is an effective method of mutation detection. It detects and distinguishes heteroplasmic mutations from homoplasmic mutations. By running the mother and child's DNA samples side by side and sequencing only the DNA fragments showing different TTGE patterns, excessive sequencing can be avoided, particularly because most sequence variations represent benign polymorphisms. Mutations identified by sequencing were further confirmed by PCR/ASO (allele‐specific oligonucleotide) dot blot analysis or PCR/RFLP (restriction fragment length polymorphism). A total of seven differences in sequence between mother and child pairs were identified: A189G, T5580C, G5821A, C5840T, A8326G, G12207A, and G15995A. All but two mutations were novel. The most significant are the A8326G, G12207A, and G15595A mutations. The A8326G is located at the anticodon region of tRNALys, right next to the first nucleotide of the triplet codon, and it is highly conserved throughout evolution. The G12207A mutation is located at the first base of tRNAser (AGY). The G15995A mutation occurs at a stem region that results in the disruption of the first base pair at the anticodon loop of tRNAPro and is highly conserved throughout evolution from sea urchins to mammals. Running TTGE side by side with DNAs from mother and the affected child is a novel method to detect deleterious mutations.
Quantitative PCR Analysis of Mitochondrial DNA Content in Patients with Mitochondrial Disease
Authors: BAI REN‐KUI , PERNG CHERNG‐LIH , HSU CHANG‐HUNG , WONG LEE‐JUN C.
Published: May 2004

Abstract: Molecular diagnosis of mitochondrial DNA disorder is usually focused on point mutations and large deletions. In the absence of detectable mtDNA mutations, abnormal amounts of mtDNA, either depletion or elevation, can be indicative of mitochondrial dysfunction. The amount of mitochondrial DNA (mtDNA), however, varies among individuals of different ages and among different tissues within the same individual. To establish a range of mtDNA levels, we analyzed 300 muscle and 200 blood specimens from patients suspected of having a mitochondrial disorder by real‐time quantitative polymerase chain reaction (PCR) method. Copy numbers were calculated from the standard curve and threshold cycle number using TaqMan probes; 6FAM 5′TTACCGGGCTCTGCCATCT3′‐TAMRA and VIC‐5′AGCAATAACAGGTCTGTGATG3′‐TAMRA for mtDNA and 18S rRNA gene (nDNA), respectively. The copy number ratio of mtDNA to nDNA was used as a measure of mtDNA content in each specimen. The mtDNA content in muscle increases steadily from birth to about 5 years of age; thereafter, it stays about the same. On the contrary, the mtDNA content in blood decreases with age. The amount of mtDNA in skeletal muscle is about 5‐20 times higher than that in blood. About 7% of patients had mtDNA levels in muscle below 20% of the mean of the age‐matched group, and about 10% of patients had muscle mtDNA levels 2‐ to 16‐fold higher than the mean of the age‐matched group. Patients with abnormal levels of mtDNA, either depletion or proliferation, had significant clinical manifestations characteristic of mitochondrial disease in addition to abnormal respiratory enzymes and mitochondrial cytopathies. Cardiomyopathy, lactic acidosis, abnormal brain MRI findings, hypotonia, developmental delay, seizures, and failure to thrive are general clinical pictures of patients with mtDNA depletion. The average age of patients with mtDNA depletion is 4.1 years, compared to 23.6 years in patients with mtDNA proliferation. Mutations in nuclear genes involved in mtDNA synthesis and deoxynucleotide pools are probably the cause of mtDNA depletion. Our results demonstrate that real time quantitative PCR is a valuable tool for molecular screening of mitochondrial diseases.
Somatic Mitochondrial DNA Mutations in Oral Cancer of Betel Quid Chewers
Authors: TAN DUAN‐JUN , CHANG JULIA , CHEN WOAN‐LING , AGRESS LESLEY J. , YEH KUN‐TU , WANG BAOTYAN , WONG LEE‐JUN C.
Published: May 2004

Abstract: Somatic mitochondrial DNA alteration is a general phenomenon that occurs in cancerous cells. Although numerous mtDNA mutations have been identified in various tumors, the pathogenic significance of these mutations remains unclear. In order to better understand the role of mtDNA mutations in the neoplastic process of oral cancer, the occurrence of mtDNA mutations in oral squamous cell carcinomas was screened by temporal temperature gradient gel electrophoresis (TTGE). The entire mitochondrial genome was amplified with 32 pairs of overlapping primers. The DNA fragments showing different banding patterns between normal and tumor mtDNA were sequenced for the identification of the mutations. Fourteen of 18 (77.8%) tumors had somatic mtDNA mutations with a total of 26 mutations. Among them, 6 were in mRNA coding region. Three were missense mutations (C14F, H186R, T173P) in NADH dehydrogenase subunit 2 (ND2). One frameshift mutation, 9485delC, was in cytochrome c oxidase subunit III. Eight (44%) tumors had insertion or deletion mutations in the np303‐309 poly C region of the D‐loop. Our results demonstrate that somatic mtDNA mutations occur in oral cancer. The missense and frameshift mutations in the evolutionary conserved regions of the mitochondrial genome may have functional significance in the pathogenesis of oral cancer.
Association of the Mitochondrial DNA 16189 T to C Variant with Lacunar Cerebral Infarction: Evidence from a Hospital‐Based Case‐Control Study
Authors: LIOU CHIA‐WEI , LIN TSU‐KUNG , HUANG FENG‐MEI , CHEN TZU‐LING , LEE CHENG‐FENG , CHUANG YAO‐CHUNG , TAN TENG‐YEOW , CHANG KU‐CHOU , WEI YAU‐HUEI
Published: May 2004

Abstract: A transition of T to C at nucleotide position 16189 in the hypervariable D‐loop region of mitochondrial DNA (mtDNA) has attracted research interest for its probable correlation with increasing insulin resistance and development of diabetes mellitus (DM) in adult life. In this article, we present our observations of the positive relationship between this variant and cerebral infarction. Six hundred and one subjects in two groups—one with cerebral infarction (307 cases), the other with no cerebral infarction (294 cases)—were recruited. Their clinical features, fasting blood sugar and insulin levels, and insulin resistance index, were recorded. Patients with cerebral infarction were further categorized into four different subgroups according to the TOAST criteria for stroke classification. The results showed the occurrence of the mtDNA 16189 variant in 34.2% of patients with cerebral infarction and in 26.5% of normal controls. The difference in the occurrence rates between the two groups was statistically significant (P= 0.041). Further studies of the occurrence rate in each stroke subgroup revealed that the variant occurred at the highest frequency in the small vessel subgroup (41.5%). The difference in occurrence rate between this subgroup and the normal controls is highly significant (P= 0.006). These results correlated well with the findings of significantly increased levels of average fasting blood insulin and a higher index of average insulin resistance in the small vessel subgroup of patients harboring this mtDNA variant. Taken together, we suggest that the mtDNA 16189 variant is a predisposing genetic factor for the development of insulin resistance and may be related to various phenotypic expressions in adult life such as development of DM and vascular pathologies involved in stroke and cardiovascular diseases.
Mechanisms of Cell Death Induced by Cadmium and Arsenic
Authors: JIMI SHIRO , UCHIYAMA MASANOBU , TAKAKI AYA , SUZUMIYA JYUNJI , HARA SYUJI
Published: May 2004

Abstract: Cadmium (Cd) and arsenic (As) are known toxic metals in humans. As trioxide (As203) has been recently used as a mitochondria‐targeting drug in acute promyelocytic leukemia. In the present study, we examined the intracellular action of these metals using rat kidney tubular cells and cells tolerant to the metals. The cells were cultured with CdCl2 (1‐10 μM) or As2O3 (1‐2.5 μM). Cells tolerant to Cd and As (Cd‐T and As‐T, respectively) were defined as cells that survived at toxic concentrations of each metal. Both Cd and As induced cell toxicity in a dose‐dependent fashion, which was accompanied by fragmented DNA and decreased mitochondrial membrane potential. Intracellular glutathione (GSH) increased with the increase of Cd and As concentration. In Cd‐T and As‐T cells, GSH levels were twice those observed in normal cells. When each metal‐tolerant culture was exposed to the other different metal, i.e., As or Cd, the protective property was maintained. However, when buthionine sulfoximine (BSO) was added to the metal‐tolerant cultures, apoptosis was restored in both Cd‐T and As‐T. Our results indicate that (1) although GSH is increased in NRK52E by the addition of Cd and As, mitochondria‐mediated apoptosis can be still induced, (2) the protective property against metal‐induced cytotoxicity is identical in Cd‐T and As‐T cultures, and (3) although GSH was higher in the metal‐tolerant cell lines, depression of GSH by BSO induced apoptosis. We conclude that Cd‐ and As‐induced apoptosis is mediated by an identical mechanism involving intracellular GSH reactive oxidation.
Cadmium‐Induced Nephropathy in Rats Is Mediated by Expression of Senescence‐Associated Beta‐Galactosidase and Accumulation of Mitochondrial DNA Deletion
Authors: TAKAKI AYA , JIMI SHIRO , SEGAWA MASARU , IWASAKI HIROSHI
Published: May 2004

Abstract: Long‐term exposure to cadmium (Cd) induces perturbation of kidney proximal tubular epithelial cells. Mitochondrial dysfunction in renal cortical cells may contribute to the pathogenesis of Cd‐induced nephropathy. In this study, we examined the accumulation of mitochondrial DNA (mtDNA) with a large deletion and cellular senescence in the renal cortex. Wistar rats at 8 weeks of age were intraperitoneally injected with 1 mL of 1 mM CdCl2 or saline, 3 times/week for 5, 20, 40, or 80 weeks. Mitochondrial Cd content in the renal cortex was quantified by atomic absorption analysis. Cytochrome c oxidase (CCO) and senescence‐associated beta‐galactosidase (SA‐β‐gal) activity were determined in renal cortex by enzyme‐histochemistry. mtDNA in total DNA extracted from the renal cortex was amplified by PCR, and mtDNA deletions, including 4,834‐bp (nt8118‐nt12937) deletion, were determined and semiquantified. After 40 weeks of Cd injection, Cd levels in the renal cortex reached a saturation level, and 30% of the level of the whole‐cell fraction was found in the mitochondria. CCO activity in the renal cortex, which was predominantly found in proximal tubular cells, decreased after 40 weeks of Cd exposure. Expression of SA‐β‐gal was detected primarily in the proximal tubular cells and significantly increased after 80 weeks of Cd exposure. After 40 weeks of study, accumulation of 4,834‐bp deletion in mtDNA was evident in both groups of rats; however, the amount of the deletion was significantly greater in Cd‐treated rats than in control rats. Our results indicate that long‐term Cd exposure induced a post‐regenerative state of proximal tubular cells, which accelerated accumulation of 4,834‐bp mtDNA deletions in the renal cortex, suggesting that Cd may be a senescence acceleration factor for kidney proximal tubular epithelial cells, which results in Cd‐induced nephropathy.
Investigation of Common Mitochondrial Point Mutations in Korea
Authors: KWON SEON‐JOO , PARK SUNG‐SUP , KIM JONG‐MIN , AHN TAE‐BEOM , KIM SEUNG HYUN , KIM JUHAN , LEE SUNG‐HYUN , HA CHOONG‐KUN , AHN MOO‐YOUNG , JEON BEOM S.
Published: May 2004

Abstract: Between 1997 and 2002, 65 patients with suspected mitochondrial diseases were screened for the mitochondrial point mutations A3243G, T3271C, A8344G, and T8356C. Among these patients, 15 were found to have one of these mutations: 12 with A3243G and 3 with A8344G. The phenotypes of A3243G and A8344G mutations were MELAS and MERRF, respectively. Many asymptomatic family members had the same mutations. In this report, detailed clinical and laboratory findings are presented.
Leber's Hereditary Optic Neuropathy: The Spectrum of Mitochondrial DNA Mutations in Iranian Patients
Authors: HOUSHMAND M , SHARIFPANAH F , TABASI A , SANATI M‐H , VAKILIAN M , LAVASANI SH , JOUGHEHDOUST S
Published: May 2004

Abstract: We studied 14 patients with Leber's hereditary optic neuropathy (LHON) to investigate the mtDNA haplotypes associated with the primary mutation(s). Eleven patients carried the mitochondrial DNA (mtDNA) G11778A mutation, while one had the T14484C mutation; one patient had the G3460A mutation and one the G14459A mutation. The Iranian G11778A LHON mutation was not associated with two mtDNA haplogroups—M (0.0% compared with 3.2% in healthy controls) and J (7.7% compared with 10% in healthy controls). Our results showed a similarity in the pattern of LHON primary point mutations between Iranian families with LHON and those of Russian, European, and North American origin. Our results also do not support an association between mtDNA haplogroups J and M with LHON primary point mutations.
Impact of Growth Hormone (GH) Treatment on Cardiovascular Risk Factors in GH-Deficient Adults: A Metaanalysis of Blinded, Randomized, Placebo-Controlled Trials
Authors: Maison Patrick , Griffin Simon , Nicoue-Beglah Marc , Haddad Nabila , Balkau Beverley , Chanson Philippe
Published: May 2004

Patients with hypopituitarism have an increased risk of cardiovascular mortality. GH treatment could modify the cardiovascular risk in adults with GH deficiency, but most published clinical trials involved few patients and the results are variable.

We conducted a systematic review of blinded, randomized, placebo-controlled trials of GH treatment in adult patients with GH deficiency published up to August 2003. Thirty-seven trials were identified. We combined the results for effects on lean and fat body mass; body mass index; triglyceride and cholesterol [high-density lipoprotein, low-density lipoprotein (LDL), and total] levels; blood pressure; glycemia; and insulinemia. Overall effect size was used to evaluate significance, and weighted differences between GH and placebo were used to appreciate the size of the effect.

GH treatment significantly reduced LDL cholesterol [−0.5 (sd 0.3) mmol/liter], total cholesterol [−0.3 (0.3) mmol/liter], fat mass [−3.1 (3.3) kg], and diastolic blood pressure [−1.8 (3.8) mm Hg] and significantly increased lean body mass [+2.7 (2.6) kg], fasting plasma glucose [+0.2 (0.1) mmol/liter], and insulin [+8.7 (7.0) pmol/liter]. All effect sizes remained significant in trials with low doses and long-duration GH treatment.

Thus, GH treatment has beneficial effects on lean and fat body mass, total and LDL cholesterol levels, and diastolic blood pressure but reduces insulin sensitivity. The global cardiovascular benefit remains to be determined in large trials with appropriate clinical endpoints.
Male Hormonal Contraception: Effects of Injections of Testosterone Undecanoate and Depot Medroxyprogesterone Acetate at Eight-Week Intervals in Chinese Men
Authors: Gu Yi-qun , Tong Jian-sun , Ma Ding-zhi , Wang Xing-hai , Yuan Dong , Tang Wen-hao , Bremner William J.
Published: May 2004

Surveys indicate that one form of acceptable male hormonal contraception would consist of injections given at 2- to 3-month intervals. This report describes a study of depot medroxyprogesterone acetate (DMPA) and testosterone undecanoate (TU) injected at 8-wk intervals for suppression of spermatogenesis in healthy Chinese men. After screening, 30 healthy volunteers were enrolled and randomly assigned to one of three dose groups (n = 10/group): 1000 mg TU (group A); 1000 mg TU plus 150 mg DMPA (group B); 1000 mg TU plus 300 mg DMPA (group C). All doses were given as im injections at 8-wk intervals. The study consisted of an 8-wk control (baseline) period, a 24-wk treatment period, and a 24-wk recovery period. Consistent azoospermia or severe oligozoospermia was achieved and maintained in all volunteers during the treatment period, except for two men in the TU-alone group who experienced a rebound in sperm concentrations. An 8-wk regimen of TU plus DMPA at both tested combination doses effectively suppressed spermatogenesis to azoospermia in Chinese men. All volunteers tolerated the injections; no serious adverse effects were reported. The lower-dose combination is recommended for further testing in an expanded clinical trial or contraceptive efficacy study.
Cytotoxic Activity of Camptothecin and Paclitaxel in Newly Established Continuous Human Medullary Thyroid Carcinoma Cell Lines
Authors: Kaczirek K. , Schindl M. , Weinhäusel A. , Scheuba C. , Passler C. , Prager G. , Raderer M. , Hamilton G. , Mittlböck M. , Siegl V. , Pfragner R. , Niederle B.
Published: May 2004

At the time of diagnosis, more than one quarter of patients with medullary thyroid carcinoma (MTC) has distant metastases. Only few of these patients can be cured by surgery. Standard chemotherapy is characterized by low response rates and short response time. The establishment of eight human MTC cell lines provides a new basis for in vitro investigation of cytotoxic drugs. Camptothecin (CPT) and paclitaxel, which never have been investigated in the treatment of MTC, were tested for their cytotoxic profile in comparison with the clinically ineffective dacarbazine.

Eight MTC cell lines were established from seven patients with MTC. IC50 values were calculated from dose-response relationships using cell counts and a formazan dye assay (WST-1).

IC50 values were 3.5 ± 1.2 nmol/liter for CPT and 8.2 ± 1.9 nmol/liter for paclitaxel. Dacarbazine showed no reduction of cell proliferation at concentrations 10-fold higher than clinically achievable.

Given peak plasma concentrations of 65 ± 20 nmol/liter for CPT and 1 μmol/liter for paclitaxel, these promising in vitro results provide a basis for the performance of clinical trials in patients with advanced MTC.
Obstructive Sleep Apnea Presenting as Pseudopheochromocytoma: A Case Report
Authors: Hoy L. J. , Emery M. , Wedzicha J. A. , Davison A. G. , Chew S. L. , Monson J. P. , Metcalfe K. A.
Published: May 2004

Sudden arousal from sleep causes a transient surge in sympathetic nervous activity. Repeated arousals, as occur in obstructive sleep apnea (OSA), are well documented to cause a more prolonged sympathetic overactivity and consequent elevations in 24-h urinary catecholamine levels. We describe here a series of five patients, each presenting with a clinical and biochemical picture indistinguishable from that of pheochromocytoma. Thorough investigations have failed to find catecholamine-secreting tumor in any of these subjects, but all have been diagnosed with OSA. Primary treatment of OSA with nasal continuous positive airways pressure has led to normalization of systemic blood pressure and urinary catecholamines. Pseudopheochromocytoma is therefore a rare, but treatable, presentation of obstructive sleep apnea.
Spinal Volumetric Bone Mineral Density and Vertebral Fractures in Female Patients with Adrenal Incidentalomas: The Effects of Subclinical Hypercortisolism and Gonadal Status
Authors: Chiodini Iacopo , Guglielmi Giuseppe , Battista Claudia , Carnevale Vincenzo , Torlontano Massimo , Cammisa Mario , Trischitta Vincenzo , Scillitani Alfredo
Published: May 2004

Although adrenal incidentalomas (AI) are not associated with clinically evident syndromes, some patients display biochemical features of subclinical hypercortisolism (SH). Previous studies indicated a negative effect of SH on bone in AI patients, but the prevalence of vertebral fractures and the roles of SH and gonadal status in volumetric bone mineral density are unknown. In 70 female AI patients and 84 controls, the prevalence of vertebral fractures and spinal bone mineral density (by quantitative computed tomography) were evaluated. Subjects were subdivided according to menopausal status into groups Pre (21 patients and 23 controls) and Post (49 patients and 61 controls); there were 14 and 35 patients without SH (SH−) and 7 and 14 patients with SH (SH+) in groups Pre and Post, respectively. The prevalence of fractures was higher in SH+ than in controls and in SH− subjects in both groups Pre [SH+, 42.9%; controls, 0% (P = 0.001); SH−, 7.1% (P = 0.049)] and post [SH+, 78.6%; controls, 37.7% (P = 0.006); SH− 42.9% (P = 0.024)]. In group Post, the mean z-score quantitative computed tomography values were lower in SH+ patients (−0.78 ± 0.29) than in controls (0.06 ± 0.14; P = 0.011) and SH− patients (0.02 ± 0.19; P = 0.034). Evaluation of spinal bone is indicated in female AI patients with SH.
Autosomal-Dominant Pseudohypoaldosteronism Type 1 in a Turkish Family Is Associated with a Novel Nonsense Mutation in the Human Mineralocorticoid Receptor Gene
Authors: Riepe Felix G. , Krone Nils , Morlot Michel , Peter Michael , Sippell Wolfgang G. , Partsch Carl-Joachim
Published: May 2004

Pseudohypoaldosteronism type 1 (PHA1) is a rare congenital disease inherited in either an autosomal-recessive or an autosomal-dominant trait. The autosomal-dominant form manifests with renal salt loss in infancy and a gradual improvement with advancing age. PHA1 presents with potential life-threatening salt wasting and failure to thrive in early infancy. Autosomal-dominant forms of PHA1 are often caused by heterozygous mutations of the MR gene coding for the mineralocorticoid receptor. Whether heterozygous mutations of the MR gene impair biological function as a result of haplo-insufficiency or due to a dominant-negative effect needs further clarification. We report a case of a renal form of PHA1 in a Turkish family. A heterozygous nonsense mutation c3055C>T (R947X) in exon 9 of the MR gene leading to a premature stop codon was identified in the index patient. The truncated receptor is free of aldosterone binding. The segregation analysis revealed the identical mutation in the patient’s father, who never showed any symptoms of PHA. This shows the incomplete penetrance of the phenotype, although a mild salt loss might have been overlooked in the father’s childhood.
Effect of the Combination of Methyltestosterone and Esterified Estrogens Compared with Esterified Estrogens Alone on Apolipoprotein CIII and Other Apolipoproteins in Very Low Density, Low Density, and High Density Lipoproteins in Surgically Postmenopausal Women
Authors: Chiuve Stephanie E. , Martin Lisa A. , Campos Hannia , Sacks Frank M.
Published: May 2004

Androgens are known to lower plasma triglycerides, an independent risk factor for coronary heart disease (CHD). Triglycerides are carried in plasma on very low density (VLDL) and low density (LDL) lipoprotein particles. Apolipoprotein CIII (apoCIII), a strong predictor of CHD, impairs the metabolism of VLDL and LDL, contributing to increased triglycerides. The objective of this study was to assess the effect of oral methyltestosterone (2.5 mg/d), added to esterified estrogens (1.25 mg/d), on concentrations of apolipoproteins and lipoproteins, specifically those containing apoCIII, compared with esterified estrogens alone in surgically postmenopausal women. The women in the methyltestosterone plus esterified estrogen group had significant decreases in total triglycerides, apoCI, apoCII, apoCIII, apoE, and high density lipoprotein (HDL) cholesterol compared with those in the esterified estrogen group. The decreases in apoCIII concentrations occurred in VLDL (62%; P = 0.02), LDL (35%; P = 0.001), and HDL (17%; P < 0.0001). There were also decreases in cholesterol and triglycerides concentrations of apoCIII containing LDL, and apoCI concentration of apoCIII containing VLDL. There was no effect on VLDL and LDL particles that did not contain apoCIII or on apoB concentrations. In conclusion, methyltestosterone, when administered to surgically postmenopausal women taking esterified estrogen, has a selective effect to reduce the apoCIII concentration in VLDL and LDL, a predictor of CHD. Methyltestosterone may lower plasma triglycerides through a reduction in apoCIII.
Plasma Ghrelin Concentrations, Food Intake, and Anorexia in Liver Failure
Authors: Marchesini Giulio , Bianchi Giampaolo , Lucidi Paola , Villanova Nicola , Zoli Marco , De Feo Pierpaolo
Published: May 2004

Ghrelin is related to feeding behavior and nutrition in several physiological and pathological conditions. We tested the hypothesis that the anorexia and the decreased food intake of advanced liver failure might be associated with hyperghrelinemia. Fasting ghrelin was measured in 43 cirrhotic patients, food intake was self-assessed using the Corli score and a 3-d dietary record (n = 25), and anorexia/hunger was tested by a Likert scale. Fifty healthy subjects, matched for age and body mass index, served as controls. Ghrelin levels were not systematically increased in cirrhosis (414 ± 164 vs. 398 ± 142 pmol/liter in controls) but increased with decreasing Corli score (P = 0.014) and along the scale of anorexia/hunger (P = 0.0001), which were both related to the 3-d dietary record (P = 0.009 and P < 0.0001, respectively). Logistical regression confirmed that high ghrelin (>500 pmol/liter) was significantly associated with a low calorie intake [odds ratio (OR), 3.03 for any 100-calorie reduced intake; P = 0.015], a reduced Corli score (OR, 3.09; P = 0.031), and the anorexia score (OR, 3.37; P = 0.009), after adjustment for body mass index. The study confirms the previously observed relationship of fasting ghrelin with food intake in disease-associated malnutrition. In the presence of anorexia, hyperghrelinemia might indicate a compensatory mechanism trying to stimulate food intake, which is nonetheless ineffective in the physiological range.
Differential Effects of Metformin and Exercise on Muscle Adiposity and Metabolic Indices in Human Immunodeficiency Virus-Infected Patients
Authors: Driscoll Susan D. , Meininger Gary E. , Ljungquist Karin , Hadigan Colleen , Torriani Martin , Klibanski Anne , Frontera Walter R. , Grinspoon Steven
Published: May 2004

The HIV-lipodystrophy syndrome is associated with fat redistribution and metabolic abnormalities, including insulin resistance (IR). The mechanisms and treatment strategies for IR in HIV-lipodystrophy are unclear, but data suggest that intramuscular lipids contribute to IR in this population. We previously showed that metformin and exercise improve hyperinsulinemia more than metformin alone in HIV-lipodystrophy. Now we investigate the effects of these treatment strategies on thigh muscle adiposity measured by computed tomography and additional body composition measures. Twenty-five HIV-infected patients on stable antiretroviral therapy with hyperinsulinemia and fat redistribution participated in a prospective, randomized, 3-month study of metformin alone or metformin and resistance training three times a week. Thigh muscle adiposity decreased significantly more as shown by increased muscle attenuation [2.0 (range, 0.5–5.0) vs. −1.0 (–3.5–0), P = 0.04] and sc leg fat tended to decrease more [–3.3 (–7.5–4.3) vs. 0.8 (–2.1–9.5), P = 0.06] in the combined treatment group in comparison with metformin alone. In multivariate analysis, change in thigh muscle adiposity remained a significant predictor of change in insulin (P = 0.04), controlling for changes in other body composition measurements. These data suggest that muscle adiposity, in addition to other fat depots, is an important determinant of hyperinsulinemia and that exercise has complex effects on regional fat depots in HIV-infected patients. Reduction in muscle adiposity may be an important mechanism by which exercise improves hyperinsulinemia in this population.
Familial Concordance of Thyroid and Other Head and Neck Tumors in an Irradiated Cohort: Analysis of Contributing Factors
Authors: Momani Munther S. , Shore-Freedman Eileen , Collins Barbara J. , Lubin Jay , Ron Elaine , Schneider Arthur B.
Published: May 2004

Relatively little is known about variations in susceptibility to the effects of radiation in the general population. We have been studying 4296 individuals exposed as children to head and neck radiation. The present study was designed to evaluate the pattern of thyroid, parathyroid, salivary, and neural tumors in irradiated siblings for evidence of heritable susceptibility factors. We also wanted to determine whether the characteristics of thyroid cancers were influenced by familial factors. The following criteria were met by 251 sibling pairs: both irradiated, both with follow-up (average, 44.3 ± 9.4 yr; range, 9.4–59.5 yr), and both with organ-dose estimates. For each sibling pair we derived a quantitative score, taking into account the length of follow-up and known risk factors, for their concordance and used the sum of these scores to characterize the population. Whether we used thyroid cancer or all thyroid nodules as an end point, the degree of concordance did not exceed what could be explained by the length of follow-up and known risk factors. For thyroid cancer, neither the presenting characteristics nor their rates of recurrence were influenced by their concordance status. In summary, we were unable to identify familial factors that modify the strong effects of radiation exposure. There is no reason to alter the evaluation or treatment of thyroid cancer in an irradiated patient based on whether another member of the family has radiation-related tumors.
Quantitative contributions of CtBP-dependent and -independent repression activities of Knirps
Authors: Struffi Paolo , Corado Maria , Kulkarni Meghana , Arnosti David N.
Published: May 2004

The Drosophila Knirps protein is a short-range transcriptional repressor that locally inhibits activators by recruiting the CtBP co-repressor. Knirps also possesses CtBP-independent repression activity. The functional importance of multiple repression activities is not well understood, but the finding that Knirps does not repress some cis-regulatory elements in the absence of CtBP suggested that the co-factor may supply a unique function essential to repress certain types of activators. We assayed CtBP-dependent and -independent repression domains of Knirps in Drosophila embryos, and found that the CtBP-independent activity,when provided at higher than normal levels, can repress an everegulatory element that normally requires CtBP. Dose response analysis revealed that the activity of Knirps containing both CtBP-dependent and-independent repression activities is higher than that of the CtBP-independent domain alone. The requirement for CtBP at certain enhancers appears to reflect the need for overall higher levels of repression, rather than a requirement for an activity unique to CtBP. Thus, CtBP contributes quantitatively, rather than qualitatively, to overall repression function. The finding that both repression activities are simultaneously deployed suggests that the multiple repression activities do not function as cryptic `backup' systems, but that each contributes quantitatively to total repressor output.
Wingless eliminates ommatidia from the edge of the developing eye through activation of apoptosis
Authors: Lin Hua V. , Rogulja Ana , Cadigan Ken M.
Published: May 2004

The Drosophila compound eye is formed by selective recruitment of undifferentiated cells into clusters called ommatidia during late larval and early pupal development. Ommatidia at the edge of the eye, which often lack the full complement of photoreceptors and support cells, undergo apoptosis during mid-pupation. We have found that this cell death is triggered by the secreted glycoprotein Wingless, which activates its own expression in peripheral ommatidia via a positive feedback loop. Wingless signaling elevates the expression of the pro-apoptotic factors head involution defective,grim and reaper, which are required for ommatidial elimination. We estimate that approximately 6-8% of the total photoreceptor pool in each eye is removed by this mechanism. In addition, we show that the retinal apoptosis previously reported in apc1 mutants occurs at the same time as the peripheral ommatidial cell death and also depends on head involution defective, grim and reaper. We consider the implications of these findings for eye development and function in Drosophila and other organisms.
dMyc is required for larval growth and endoreplication in Drosophila
Authors: Pierce Sarah B. , Yost Cynthia , Britton Jessica S. , Loo Lenora W. M. , Flynn Erin M. , Edgar Bruce A. , Eisenman Robert N.
Published: May 2004

Members of the Myc family of proto-oncogenes have long been implicated in regulating proliferation, apoptosis and oncogenesis. Recently, transcriptional and biological studies have suggested a direct role for Myc in regulating growth. We have used dm4, a new null allele of the Drosophila diminutive (dm) gene, which encodes dMyc on the X chromosome, to investigate a role for dMyc in larval endoreplicating tissues,where cellular growth and DNA replication occur in the absence of cell division. Hemizygous dm4/Y mutants arrest as second instar larvae, and fat body nuclei of dm4/Y mutants fail to attain normal size and normal levels of DNA, resulting from a reduced frequency of S-phase. Thus, dMyc is required for endoreplication and larval growth. In support of this, dMyc, as well as its antagonist dMnt, are expressed in larval tissues in a pattern consistent with their involvement in regulating endoreplication. Overexpression of dMyc in endoreplicating cells results in dramatic increases in nuclear DNA content and cell and nucleolar size, whereas dMnt overexpression has the opposite effect. BrdU incorporation and Cyclin E protein levels continue to oscillate in dMyc-overexpressing cells, indicating that the normal cell cycle control mechanisms are not disrupted. dMyc driven growth and endoreplication are strongly attenuated when the endocycle is blocked with Cyclin E or the cdk inhibitor p21. By contrast,the ability of dMyc to promote growth and endoreplication is only partly reduced when PI3K activity is blocked, suggesting that they influence distinct growth pathways. Our results indicate that larval growth and endoreplication are coupled processes that, although linked to cell cycle control mechanisms,are regulated by dMyc and dMnt.
Notch signaling patternsDrosophilamesodermal segments by regulating the bHLH transcription factortwist
Authors: Tapanes-Castillo Alexis , Baylies Mary K.
Published: May 2004

One of the first steps in embryonic mesodermal differentiation is allocation of cells to particular tissue fates. In Drosophila, this process of mesodermal subdivision requires regulation of the bHLH transcription factor Twist. During subdivision, Twist expression is modulated into stripes of low and high levels within each mesodermal segment. High Twist levels direct cells to the body wall muscle fate, whereas low levels are permissive for gut muscle and fat body fate. We show that Su(H)-mediated Notch signaling represses Twist expression during subdivision and thus plays a critical role in patterning mesodermal segments. Our work demonstrates that Notch acts as a transcriptional switch on mesodermal target genes, and it suggests that Notch/Su(H) directly regulates twist, as well as indirectly regulating twist by activating proteins that repress Twist. We propose that Notch signaling targets two distinct `Repressors of twist' - the proteins encoded by the Enhancer of split complex[E(spl)C] and the HLH gene extra machrochaetae(emc). Hence, the patterning of Drosophila mesodermal segments relies on Notch signaling changing the activities of a network of bHLH transcriptional regulators, which, in turn, control mesodermal cell fate. Since this same cassette of Notch, Su(H) and bHLH regulators is active during vertebrate mesodermal segmentation and/or subdivision, our work suggests a conserved mechanism for Notch in early mesodermal patterning.
The first cell cycle after transfer of somatic cell nuclei in a non-human primate
Authors: Ng Soon-Chye , Chen Naiqing , Yip Wan-Yue , Liow Swee-Lian , Tong Guo-Qing , Martelli Barbara , Tan Lay Geok , Martelli Paolo
Published: May 2004

Production of genetically identical non-human primates through somatic cell nuclear transfer (SCNT) can provide diseased genotypes for research and clarify embryonic stem cell potentials. Understanding the cellular and molecular changes in SCNT is crucial to its success. Thus the changes in the first cell cycle of reconstructed zygotes after nuclear transfer (NT) of somatic cells in the Long-tailed Macaque (Macaca fascicularis) were studied. Embryos were reconstructed by injecting cumulus and fibroblasts from M. fascicularis and M. silenus, into enucleated M. fascicularis oocytes. A spindle of unduplicated premature condensed chromosome (PCC spindle) from the donor somatic cell was formed at 2 hours after NT. Following activation, the chromosomes segregated and moved towards the two PCC spindle poles, then formed two nuclei. Twenty-four hours after activation, the first cell division occurred. A schematic of the first cell cycle changes following injection of a somatic cell into an enucleated oocyte is proposed. Ninety-three reconstructed embryos were transferred into 31 recipients, resulting in 7 pregnancies that were confirmed by ultrasound;unfortunately none progressed beyond 60 days.
Investigating the origins of triploblasty: `mesodermal' gene expression in a diploblastic animal, the sea anemoneNematostella vectensis(phylum, Cnidaria; class, Anthozoa)
Authors: Martindale Mark Q. , Pang Kevin , Finnerty John R.
Published: May 2004

Mesoderm played a crucial role in the radiation of the triploblastic Bilateria, permitting the evolution of larger and more complex body plans than in the diploblastic, non-bilaterian animals. The sea anemone Nematostella is a non-bilaterian animal, a member of the phylum Cnidaria. The phylum Cnidaria (sea anemones, corals, hydras and jellyfish) is the likely sister group of the triploblastic Bilateria. Cnidarians are generally regarded as diploblastic animals, possessing endoderm and ectoderm,but lacking mesoderm. To investigate the origin of triploblasty, we studied the developmental expression of seven genes from Nematostella whose bilaterian homologs are implicated in mesodermal specification and the differentiation of mesodermal cell types (twist, snailA, snailB, forkhead,mef2, a GATA transcription factor and a LIMtranscription factor). Except for mef2, the expression of these genes is largely restricted to the endodermal layer, the gastrodermis. mef2is restricted to the ectoderm. The temporal and spatial expression of these`mesoderm' genes suggests that they may play a role in germ layer specification. Furthermore, the predominantly endodermal expression of these genes reinforces the hypothesis that the mesoderm and endoderm of triploblastic animals could be derived from the endoderm of a diploblastic ancestor. Alternatively, we consider the possibility that the diploblastic condition of cnidarians is a secondary simplification, derived from an ancestral condition of triploblasty.
A regulatory code for neurogenic gene expression in theDrosophilaembryo
Authors: Markstein Michele , Zinzen Robert , Markstein Peter , Yee Ka-Ping , Erives Albert , Stathopoulos Angela , Levine Michael
Published: May 2004

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permitted the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. We discuss the prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression.
Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy
Authors: Nath Anjali K. , Enciso Josephine , Kuniyasu Misako , Hao Xiao-Ying , Madri Joseph A. , Pinter Emese
Published: May 2004

Nitric oxide (NO) has been demonstrated to mediate events during ovulation,pregnancy, blastocyst invasion and preimplantation embryogenesis. However,less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this structure is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development,an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO(L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species(ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor that modulates normal yolk sac vascular development, and decreased NO bioavailability and NO-mediated sequela may underlie high glucose induced vasculopathy.
The role ofMixerin patterning the earlyXenopusembryo
Authors: Kofron Matt , Wylie Chris , Heasman Janet
Published: May 2004

The transcription factor VegT, is required in early Xenopusembryos for the formation of both the mesoderm and endoderm germ layers. Inherited as a maternal mRNA localized only in vegetal cells, VegT activates the transcription of a large number of transcription factors, as well as signaling ligands that induce cells in the vegetal mass to form endoderm, and the marginal zone to form mesoderm. It is important now to understand the extent to which transcription factors downstream of VegT play individual, or overlapping, roles in the specification and patterning of the endoderm and mesoderm. In addition, it is important to understand the mechanism that specifies the boundary between endoderm and mesoderm. One of the downstream targets of VegT, the homeodomain protein Mixer, is expressed at high levels at the mesoderm/endoderm boundary at the late blastula stage. We therefore examined its functions by blocking its translation using morpholino oligos. In Mixer-depleted embryos, the expression of many signaling ligands and transcription factors was affected. In particular, we found that the expression of several genes, including several normally expressed in mesoderm,was upregulated. Functional assays of Mixer-depleted vegetal cells showed that they have increased mesoderm-inducing activity. This demonstrates that Mixer plays an essential role in controlling the amount of mesoderm induction by the vegetal cells.