Analysis of the uncoupling protein-1 (UCP1) gene in obese and lean subjects: identification of four amino acid variants

We report the case of a patient with fatal obstructive lung disease after an HLA-haploidentical sibling cord blood transplant (CBT), with severe acute GVHD. A 2-year-old girl developed expiratory air trapping gradually with acute and chronic GVHD after CBT for the treatment of ALL. Anti-CMV and immunosuppressive therapy were ineffective, and the patient died of progressive respiratory acidosis. Necropsy of the lung revealed severe bronchiolitis obliterans with cytomegalic inclusion cells in the granulation tissues of the bronchiolitis. Thus, immunologic and GVHD problems can occur even in CBT.     

Increased uncoupling protein-2 and -3 mRNA expression during fasting in obese and lean humans

Uncoupling protein-2 and -3 (UCP2 and UCP3) are mitochondrial proteins that show high sequence homology with the brown adipocyte-specific UCP1. UCP1 induces heat production by uncoupling respiration from ATP synthesis. UCP2 is widely expressed in human tissues, whereas UCP3 expression seems restricted to skeletal muscle, an important site of thermogenesis in humans. We have investigated the regulation of UCP2 and UCP3 gene expression in skeletal muscle and adipose tissue from lean and obese humans. UCP2 and -3 mRNA levels were not correlated with body mass index (BMI) in skeletal muscle, but a positive correlation (r = 0.55, P < 0.01, n = 22) was found between UCP2 mRNA level in adipose tissue and BMI. The effect of fasting was investigated in eight lean and six obese subjects maintained on a hypocaloric diet (1,045 kJ/d) for 5 d. Calorie restriction induced a similar 2-2.5-fold increase in UCP2 and -3 mRNA levels in lean and obese subjects. To study the effect of insulin on UCP gene expression, six lean and five obese subjects underwent a 3-h euglycemic hyperinsulinemic clamp. Insulin infusion did not modify UCP2 and -3 mRNA levels. In conclusion, the similar induction of gene expression observed during fasting in lean and obese subjects shows that there is no major alteration of UCP2 and -3 gene regulation in adipose tissue and skeletal muscle of obese subjects. The increase in UCP2 and -3 mRNA levels suggests a role for these proteins in the metabolic adaptation to fasting.     

Uncoupling protein-2 messenger ribonucleic acid expression during very-low-calorie diet in obese premenopausal women

An omental flap is useful in reconstructive surgery, but harvesting such a flap generally requires laparotomy. However, endoscopic surgery facilitates harvesting an omental flap without open laparotomy. We performed endoscopic omental harvest in two patients. We described the procedure of endoscopic omental harvest, which is different from that reported previously. Four access ports were required: two placed lateral to the right rectus margin, one placed lateral to the left rectus margin, and one placed in the infraumbilical area. The stomach was suspended from the peritoneum for the dissection of the gastric rami. The vessels from the gastroepiploic arcade to the greater curvature of the stomach were individually clipped and divided. The omentum then was dissected to the transverse colon and the lower portion of the omentum was dissected along the transverse colon. Finally, the right side of the omentum was dissected. The omentum was transferred using the right gastroepiploic vessels for anastomosis. The advantages of endoscopic harvest are an inconspicuous scar, minimal operative pain, and early recovery. The disadvantages include a long procedure time. At the present time, endoscopic harvest of the left side of the omentum is problematic because of difficulty in identifying the left omental artery and the risk of injury to the spleen. However, these limitations will likely be resolved in the future.     

A direct repeat sequence at the Rasgrf1 locus and imprinted expression

Genomic imprinting is an epigenetic modification that can lead to parental-specific monoallelic expression of specific autosomal genes. While methylation of CpG dinucleotides is thought to be a strong candidate for this epigenetic modification, little is known about the establishment or maintenance of parental origin-specific methylation patterns. We have recently identified a portion of mouse chromosome 9 containing a paternally methylated region associated with a paternally expressed imprinted gene, Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1). This area of chromosome 9 also contains a short, direct tandem repeat in close proximity to a paternally methylated NotI site 30 kb upstream of Rasgrf1. Short, direct tandem repeats have been found associated with other imprinted genes and may act as important regulatory structures. Here we demonstrate that two rodent species (Mus and Rattus) contain a similar direct repeat structure associated with a region of paternal-specific methylation. In both species, the Rasgrf1 gene shows paternal-specific monoallelic expression in neonatal brain. A more divergent rodent species (Peromyscus) appears to lack a similar repeat structure based on Southern Blot analysis. Peromyscus animals show biallelic expression of Rasgrf1 in neonatal brain. These results suggest that direct repeat elements may play an important role in the imprinting process.     

Polymorphic expression of the UDP-glucuronosyltransferase UGT1A gene locus in human gastric epithelium

OBJECTIVES: Adenosine, a potent coronary vasodilator is used as a pharmacologic stress agent for the assessment of coronary artery disease. A paucity of data exists on its effects on filling dynamics. Accordingly, this study was undertaken to evaluate the effects of adenosine on left ventricular filling as assessed by Doppler echocardiography. METHODS AND RESULTS: We studied 69 patients (45 men, 24 women, aged 61+/-11 years) referred for evaluation of coronary artery disease. Two-dimensional echocardiography and pulsed-Doppler recordings at the mitral valve tips and annulus were performed at baseline and at maximal adenosine infusion of 140 microg/kg/min. During adenosine infusion, an increase in heart rate occurred (70+/-14 beats/min to 85+/-16 beats/min), with a mild decrease in blood pressure (130/75+/-26/13 mm Hg vs 119/66+/-25/13 mm Hg); both p < 0.02. Changes in filling dynamics included an increase in peak early inflow velocity, E/A ratio, and normalized peak filling rate. Of the patients investigated, 23 had one-vessel coronary artery disease, 29 had coronary disease in two vessels or more by angiography, and 17 had no significant disease. Patients without coronary artery disease (controls) had mild changes in E/A ratio (mean 7%). Patients with coronary artery disease had a more heterogeneous change in filling dynamics (range 43% to 369%, mean 26%), with a significant overlap with controls. However, changes in E/A ratio during adenosine infusion that exceeded the confidence limits of normal (-20% to +30%) were specific for coronary artery disease, with a positive predictive value of 84%. CONCLUSIONS: Normally, adenosine induces significant increases in early filling as assessed by Doppler. The changes in patients with coronary stenosis are more variable. When these changes fall outside the confidence limits of normal, they are predictive of coronary artery disease.     

Linkage disequilibrium at the human phosphoglucomutase 1 locus

By considering the four common PGM1 alleles as haplotypes, that is, combinations of mutations at two polymorphic intragenic sites (1/2 and A/B), we investigated the levels of linkage disequilibrium in 142 human population samples. These groups showed considerable diversity in their disequilibrium (Drel) and heterogeneity. In all the populations the disequilibrium was found to be due to an excess of 1A and 2B haplotypes, although this direction is the opposite of that expected according to the proposed phylogeny of the system. Natural selection could be one of the major causes for such a disequilibrium direction.     

Human obese gene expression: alternative splicing of mRNA and relation to adipose tissue localization

The aim of this work was to obtain further information about the origin of fluoride profiles in cementum. Fluoride was administered to rats at varying doses (0.50, 100 ppm F in drinking water) and for different durations (4, 13 and 25 weeks). Fluoride distribution across the full thickness of molar cementum in rats was measured by means of an abrasive micro-sampling technique. The average fluoride concentrations in cementum increased significantly with increasing dose and duration of fluoride administration. The relative reduction of the average fluoride concentrations after cessation of fluoride administration was 94.2-36.5% at 50 ppm F and 62.2-49.2% at 100 ppm F in the outer layers (1-60 microns) and 91.5-24.1% at 50 ppm F and 74.1-7.6% at 100 ppm F in the middle (61-120 microns) layers of the cementum, respectively. The reduction rates were more closely related to the time intervals following cessation rather than fluoride concentrations in drinking water or specificity within the cementum. Two factors which may influence this are new cementum formation after withdrawal of fluoride and some fluoride release from cementum surfaces when the fluoride supply stopped. It was concluded that the cessation of fluoride administration reduced the fluoride concentration on the outer layers of cementum differing from bone where reduction occurs across the entire thickness.     

Leishmania major: histone H1 gene expression from the sw3 locus

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.     

Endothelin converting-enzyme-1 mRNA expression in human cardiovascular disease

Endothelin-1 converting-enzyme (ECE-1) cleaves the precursor, big-endothelin-1, to the active peptide endothelin-1. The aim of this study was to investigate whether ECE-1 mRNA expression is modified in human cardiovascular disease. Tissue samples from the left human atrium were analyzed for ECE-1 expression and related to different clinical parameters. A quantitative PCR assay (qPCR) with competitive and non-competitive standards was established. The ECE-1 measurements were normalized by a GAPDH qPCR. Patients who suffered from a myocardial infarction had elevated ECE-1 levels when compared to controls (5.81+/-0.76 vs. 3.20+/-0.51 fg ECE-1, ng GAPDH, p<0.05). Drug treatment with the beta-blocker metoprolol was associated with a decreased ECE-1 expression level (3.90+/-0.58 vs. 5.81+/-0.76 fg ECE-1, ng GAPDH, p<0.1). We conclude that the expression of ECE-1 is altered in the atrial tissue depending on the physiological status of the heart. This suggests a differential role of ECE-1 in cardiovascular diseases.     

Uncoupling PR gene expression from NPR1 and bacterial resistance: characterization of the dominant Arabidopsis cpr6-1 mutant

In Arabidopsis, NPR1 mediates the salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance (SAR). Here, we report the identification of another component, CPR 6, that may function with NPR1 in regulating PR gene expression. The dominant CPR 6-1 mutant expresses the SA/NPR1-regulated PR genes (PR-1, BGL 2, and PR-5) and displays enhanced resistance to Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 in the absence of SAR induction. cpr 6-1-induced PR gene expression is not suppressed in the cpr 6-1 npr1-1 double mutant but is suppressed when SA is removed by salicylate hydroxylase. Thus, constitutive PR gene expression in cpr 6-1 requires SA but not NPR1. In addition, resistance to P. s. maculicola ES4326 is suppressed in the cpr 6-1 npr1-1 double mutant, despite expression of PR-1, BGL 2, and PR-5. Resistance to P. s. maculicola ES4326 must therefore be accomplished through unidentified antibacterial gene products that are regulated through NPR1. These results show that CPR 6 is an important regulator of multiple signal transduction pathways involved in plant defense.     

Allelic diversity at the merozoite surface protein-1 (MSP-1) locus in natural Plasmodium falciparum populations: a brief overview

The merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum codes for a major asexual blood-stage antigen currently proposed as a major malaria vaccine candidate. The protein, however, shows extensive polymorphism, which may compromise its use in sub-unit vaccines. Here we compare the patterns of allelic diversity at the MSP-1 locus in wild isolates from three epidemiologically distinct malaria-endemic areas: the hypoendemic southwestern Brazilian Amazon (n = 54), the mesoendemic southern Vietnam (n = 238) and the holoendemic northern Tanzania (n = 79). Fragments of the variable blocks 2, 4a, 4b and 6 or 10 of this single-copy gene were amplified by the polymerase chain reaction, and 24 MSP-1 gene types were defined as unique combinations of allelic types in each variable block. Ten different MSP-1 types were identified in Brazil, 23 in Vietnam and 13 in Tanzania. The proportion of genetically mixed infections (isolates with parasites carrying more than one MSP-1 version) ranged from 39% in Brazil to 44% in Vietnam and 60% in Tanzania. The vast majority (90%) of the typed parasite populations from Brazil and Tanzania belonged to the same seven most frequent MSP-1 gene types. In contrast, these seven gene types corresponded to only 61% of the typed parasite populations from Vietnam. Non-random associations were found between allelic types in blocks 4a and 6 among Vietnamese isolates, the same pattern being observed in independent studies performed in 1994, 1995 and 1996. These results suggest that MSP-1 is under selective pressure in the local parasite population. Nevertheless, the finding that similar MSP-1 type frequencies were found in 1994 and 1996 argues against the prominence of short-term frequency-dependent immune selection of MSP-1 polymorphisms. Non-random associations between MSP-1 allelic types, however, were not detected among isolates from Brazil and Tanzania. A preliminary analysis of the distribution of MSP-1 gene types per host among isolates from Tanzania, but not among those from Brazil and Vietnam, shows significant deviation from that expected under the null hypothesis of independent distribution of parasites carrying different gene types in the human hosts. Some epidemiological consequences of these findings are discussed.     

The human histone gene cluster at the D6S105 locus

The sequences and organization of the histone genes in the histone gene cluster at the chromosomal marker D6S105 have been determined by analyzing the Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (YAC) 964f1. The insert of the YAC was subcloned in cosmids. In the established contig of the histone-gene-containing cosmids, 16 histone genes and 2 pseudogenes were identified: one H1 gene (H1.5), five H2A genes, four H2B genes and one pseudogene of H2B, three H3 genes, and three H4 genes plus one H4 pseudogene. The cluster extends about 80 kb with a nonordered arrangement of the histone genes. The dinucleotide repeat polymorphic marker D6S105 was localized at the telomeric end of this histone gene cluster. Almost all human histone genes isolated until now have been localized within this histone gene cluster and within the previously described region of histone genes, about 2 Mb telomeric of the newly described cluster or in a small group of histone genes on chromosome 1. We therefore conclude that the data presented here complete the set of human histone genes. This now allows the general organization of the human histone gene complement to be outlined on the basis of a compilation of all known histone gene clusters and solitary histone genes.     

Upregulation of leptin receptor mRNA expression in obese mouse brain

Leptin receptor gene expression in the brains of lean (+/+) and obese (ob/ob) C57Bl/6 mice was examined using a non-radioactive in situ hybridization detection method. Significant increases in leptin receptor mRNA expression were found in the ventromedial and arcuate hypothalamic nuclei, piriform and olfactory cortices and medial habenular nucleus. There were very minor changes in the amount of leptin receptor mRNA expression in hippocampus proper (CA1-3). Results indicated that leptin receptor is upregulated when there is a lack of functional leptin, as in hereditary obese (ob/ob) mice. It is also suggested that leptin receptor may be an autoreceptor.     

Alteration of heart uncoupling protein-2 mRNA regulated by sympathetic nerve and triiodothyronine during postnatal period in rats

To provide tissue-specific and developmental characteristics of gene expression of rat heart uncoupling protein-2 (UCP2), we investigated developmental alterations of UCPs mRNA expression in the heart and brown adipose tissue (BAT), and examined possible up-regulators of heart UCP2 expression using in vitro studies. Heart UCP2 mRNA expression was low during the early postnatal days followed by a rapid and significant increase in the 2nd postnatal week. Heart UCP3 mRNA remained undetectable until the 2nd postnatal week when the expression reached a small but significant peak. BAT UCP1 mRNA was abundantly expressed in the neonate, but the expression rapidly decreased to the adult level. The studies using cultured cardiomyocytes demonstrated that both 10(-8) M triiodothyronine and 10(-7) M isoproterenol, but not phenylephrine, increased UCP2 mRNA expression. These results indicate that the sympathetic nervous system and/or thyroid hormones may be involved in the up-regulation of heart UCP2 gene expression during postnatal development. The increase in postnatal heart UCP2 may provide a key link between the postnatal energy shift and adaptation of rat pups to their novel environment.     

The transcriptional activator HlyU of Vibrio cholerae: nucleotide sequence and role in virulence gene expression

HlyU upregulates expression of the haemolysin, HlyA, of Vibrio cholerae. DNA sequence analysis indicates that HlyU is an 11.9 kDa protein containing a putative helix-turn-helix motif and belonging to a family of small regulatory proteins, including NoIR (Rhizobium meliloti), SmtB (Synechococcus PCC 7942) and ArsR (plasmids R773, Escherichia coli; pI258, Staphylococcus aureus; and pSX267, Staphylococcus xylosus). An hlyU mutant was constructed by insertional inactivation, and found to be deficient in the production of both the haemolysin and a 28 kDa secreted protein. The mutant was assessed for virulence in the infant mouse cholera model, revealing a 100-fold increase in the LD50. This suggests that HlyU promotes expression of virulence determinant(s) in vivo.