Exosomal lncRNA FOXD3-AS1 upregulates ELAVL1 expression and activates PI3K/Akt pathway to enhance lung cancer cell proliferation, invasion, and 5-fluorouracil resistance.

Long non-coding RNA (lncRNA) FOXD3-AS1 expression is upregulated in lung cancer; however, its effect and mechanism on 5-fluorouracil (5-FU) resistance remain unclear. In this study, we determined the effects of FOXD3-AS1-enriched exosomes derived from lung cancer cells on the proliferation, invasion, and 5-FU resistance of lung cancer cells. Online bioinformatics database analysis showed that FOXD3-AS1 was upregulated in lung cancer progression. Real-time quantitative PCR results confirmed that FOXD3-AS1 expression was upregulated in lung cancer tissues and cell lines, and FOXD3-AS1 was greatly enriched in lung cancer cell-derived exosomes. ELAV-like RNA-binding protein 1 (ELAVL1) was identified as an RNA-binding protein of FOXD3-AS1. The lung cancer cell-derived exosomes promoted A549 cell proliferation and invasion and inhibited apoptosis caused by 5-FU, and transfection of si-FOXD3-AS1 or si-ELAVL1 in exosome-incubated A549 cells reversed these effects. Moreover, exosome-incubated A549 cells were co-transfected with si-FOXD3-AS1 and pcDNA-ELAVL1, showing the same cell proliferation, invasion, and 5-FU resistance as those of A549 cells treated with lung cancer cell-derived exosomes alone. Mechanistic studies identified that lung cancer cell-derived exosomes activated the PI3K/Akt pathway, and transfection of si-FOXD3-AS1 or treatment with the PI3K inhibitor LY294002 reversed the activation of the PI3K/Akt axis induced by exosomes. In conclusion, our study revealed that lung cancer cell-derived exosomal FOXD3-AS1 upregulated ELAVL1 expression and activated the PI3K/Akt pathway to promote lung cancer progression. Our findings provide a new strategy for lung cancer treatment.

Mutations of P450c21 (steroid 21-hydroxylase) at Cys428, Val281, and Ser268 result in complete, partial, or no loss of enzymatic activity, respectively.

Steroid 21-hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH), a common genetic disease. To define the relationship between gene mutations and enzyme deficiency, we generated missense mutations of the 21-hydroxylase cDNA at three different sites and characterized the mutant proteins after expressing them in cultured mammalian and yeast cells. Among them, Ser268 and Val281 have been found to be mutated in CAH patients, whereas Cys428 has been implicated as the heme ligand. Our results show mutations at these sites result in complete, partial, or no loss of the enzymatic activity. All the Cys428 mutants had neither enzymatic activity nor P450 absorption, thus supporting the notion that Cys428 is the heme ligand. All the 268-mutants exhibited the same activity as normal 21-hydroxylase, demonstrating that the clinically observed Ser268----Thr change represents a polymorphism rather than the cause of the enzyme deficiency. The 281-mutants had normal Km but greatly reduced Vmax values that also paralleled the reduction in the heme content, in the order Val281 (normal, 100%) greater than Ile281 (50%) greater than Leu281 (20%) greater than Thr281 (10%). Our findings suggest that the methyl group at the beta-carbon of Val281 is required for heme incorporation and consequently enzymatic activity.

Quantitative trait locus mapping of human blood pressure to a genetic region at or near the lipoprotein lipase gene locus on chromosome 8p22.

Resistance to insulin-mediated glucose disposal is a common finding in patients with non-insulin-dependent diabetes mellitus (NIDDM), as well as in nondiabetic individuals with hypertension. In an effort to identify the generic loci responsible for variations in blood pressure in individuals at increased risk of insulin resistance, we studied the distribution of blood pressure in 48 Taiwanese families with NIDDM and conducted quantitative sib-pair linkage analysis with candidate loci for insulin resistance, lipid metabolism, and blood pressure control. We found no evidence for linkage of the angiotensin converting enzyme locus on chromosome 17, nor the angiotensinogen and renin loci on chromosome 1, with either systolic or diastolic blood pressures. In contrast, we obtained significant evidence for linkage or systolic blood pressure, but not diastolic blood pressure, to a genetic region at or near the lipoprotein lipase (LPL) locus on the short arm of chromosome 8 (P = 0.002, n = 125 sib-pairs, for the haplotype generated from two simple sequence repeat markers within the LPL gene). Further strengthening this linkage observation, two flanking marker loci for LPL locus, D8S261 (9 cM telomeric to LPL locus) and D8S282 (3 cM centromeric to LPL locus), also showed evidence for linkage with systolic blood pressure (P = 0.02 and 0.0002 for D8S261 and D8S282, respectively). Two additional centromeric markers (D8S133, 5 cM from LPL locus, and NEFL, 11 cM from LPL locus) yielded significant P values of 0.01 and 0.001, respectively. Allelic variation around the LPL gene locus accounted for as much as 52-73% of the total interindividual variation in systolic blood pressure levels in this data set. Thus, we have identified a genetic locus at or near the LPL gene locus which contributes to the variation of systolic blood pressure levels in nondiabetic family members at high risk for insulin resistance and NIDDM.

Transcription of the human ferredoxin gene through a single promoter which contains the 3',5'-cyclic adenosine monophosphate-responsive sequence and Sp 1-binding site.

We have investigated the functional elements involved in cAMP-stimulated transcription of the human ferredoxin gene. Unlike the bovine gene, the human gene lacked a second upstream RNA initiation site as demonstrated by sequence analysis of the exon boundary, lack of upstream RNA, and analysis of the promoter. The presence of a single promoter was determined by testing the ability of various gene segments to drive the expression of the chloramphenicol acetyltransferase gene after transfection into a mouse adrenal cell line Y1. Full promoter activity was conferred by a DNA fragment spanning -209 to +55, although the -94 to +55 fragment already provided some promoter activity. Transcription from the -94 to +55 segment was stimulated by 2-fold when 8-bromo-cAMP was added to the cell. Footprinting analyses showed two GC boxes at -50 to -70 and -87 to -108 were protected by proteins from both Y1 and HeLa cells. Competition experiments showed that a protein with a recognition sequence indistinguishable from Sp1 bound to these sites. When connected to a heterologous TATA box, the sequence at -76 to -42, which contained the proximal GC box, was able to confer a high level of basal transcription and cAMP stimulation. This sequence does not show sequence homology with the known cAMP-responsive element. Mutations or deletion of the Sp 1-binding site showed diminished basal transcription and defined the cAMP responsive sequence to be from -76 to -62. Therefore the cAMP-responsive sequence of the human ferredoxin gene was located at -76 to -62, which was adjacent to the Sp 1-binding site.

Abnormalities of carbohydrate and lipid metabolism in patients with hypertension.

Plasma glucose, insulin, and lipoprotein concentrations were determined in 20 men with hypertension, and compared with values in 20 normotensive men of comparable age and body mass index. The results demonstrated a significant increase in both the plasma glucose and insulin response to a 75-g oral glucose challenge (two-way analysis of variance). In addition, a significant correlation existed between the plasma blood pressure. Finally, the greater the plasma glucose insulin response to oral glucose and both systolic and diastolic and insulin responses to oral glucose, the lower the plasma high-density lipoprotein concentrations, and the higher the ratio of plasma low-density lipoprotein cholesterol to high-density lipoprotein cholesterol. Thus, abnormalities of plasma glucose, insulin, and lipoprotein metabolism exist in patients with untreated hypertension, and these changes may contribute to the increased risk for coronary artery disease associated with hypertension.

Resistance to insulin-stimulated-glucose uptake in patients with hypertension.

Plasma glucose and insulin responses to a glucose challenge and insulin-stimulated glucose uptake were measured in 24 age-, weight-, and sex-matched Chinese men (8 with normal blood pressure, 8 with untreated hypertension, and 8 patients with hypertension treated with thiazide and beta-adrenergic antagonist drugs). Plasma glucose and insulin responses were determined by measuring plasma glucose and insulin concentrations before and at 30-min intervals for 2 h after a 75-g oral glucose dose. Insulin-stimulated glucose uptake was estimated by measuring the steady state plasma glucose (SSPG) and insulin (SSPI) concentrations achieved during the last 60 min of a 180-min continuous infusion of somatostatin, insulin, and glucose (insulin suppression test). Under these conditions endogenous insulin secretion was suppressed, and similar SSPI concentrations were achieved in all men; thus, the differences in the resultant SSPG concentrations allowed direct comparison of insulin's ability to stimulate disposal of an identical glucose load in different individuals. The results indicated that the men with hypertension, whether treated or untreated, had significantly elevated plasma glucose (P less than 0.001) and insulin (P less than 0.001) responses to the oral glucose dose compared to the normal men. Mean (+/- SE) SSPG concentrations were also higher (P less than 0.001) in the men with either untreated hypertension [219 +/- 9 mg/dL (12.2 +/- 0.5 mmol/L)] or treated hypertension [211 +/- 18 mg/dL (11.7 +/- 1.0 mmol/L)] than in the normal men [134 +/- 13 mg/dL (7.4 +/- 0.7 mmol/L)]. Since the mean SSPI concentrations were similar in the 3 groups [approximately 70 microU/mL (502 pmol/L)], insulin was less effective in promoting glucose disposal in both groups with hypertension. These results document the fact that patients with hypertension, whether treated or untreated, are insulin resistant, hyperglycemic, and hyperinsulinemic compared to a well-matched control group.

Metabolic effects of diuretic and beta-blocker treatment of hypertension in patients with non-insulin-dependent diabetes mellitus.

Patients with non-insulin-dependent diabetes mellitus (NIDDM) and hypertension were studied before and after three months of combined beta-blocker-diuretic treatment. Blood pressure fell significantly (P less than .001) from (mean +/- SEM) 167 +/- 3/99 +/- 1 to 142 +/- 3/88 +/- 1 mm Hg. However, mean (+/- SEM) fasting plasma glucose concentration increased significantly (P less than .001) from 132 +/- 11 to 153 +/- 10 mg/dL. In addition, significant increases (P less than .05) were noted in fasting concentration of plasma total triglyceride, very-low-density lipoprotein (VLDL)-triglyceride and VLDL-cholesterol, whereas fasting plasma high-density lipoprotein (HDL)-cholesterol was significantly lower (P less than .05). Thus, a common treatment program for hypertension exacerbated the abnormalities of carbohydrate and lipid metabolism commonly present in patients with NIDDM. Since the changes noted would increase risk of vascular disease, attention should be focused on selection of treatment programs for lowering blood pressure in patients with NIDDM in order to avoid this outcome.

Regulation of ferredoxin gene in steroidogenic and nonsteroidogenic cells.

Ferredoxin is an electron transport intermediate for all the mitochondrial cytochromes P450. It is especially abundant in steroidogenic organs where it functions in steroid biosynthesis. The regulation of ferredoxin gene expression was studied in both steroidogenic and nonsteroidogenic cell lines. In steroidogenic cell line Y1, the expression of ferredoxin was stimulated by cAMP and repressed slightly by angiotensin II and phorbol ester PMA. These drugs exhibited the same effect on the basal promoter of the ferredoxin gene, which includes one TATA box and an SP1 site. In human adrenocortical cell line H295, the stimulation of the ferredoxin gene by cAMP was blocked by cycloheximide, as observed in bovine adrenocortical cell culture. In nonsteroidogenic cell lines such as HeLa and COS-1, the stimulation of ferredoxin gene expression by cAMP was not observed, although basal expression was strong. Transfection studies showed that the ferredoxin promoter could not be stimulated by cAMP in nonsteroidogenic cells. Therefore the steroidogenic cell-specific regulation and the general expression pattern appears to be a property unique to the ferredoxin gene.

Expression and functional study of wild-type and mutant human cytochrome P450c21 in Saccharomyces cerevisiae.

The most common cause of congenital adrenal hyperplasia is deficiency of cytochrome P450c21 (21-hydroxylase), which catalyzes the synthesis of adrenal steroids. We have cloned the human P450c21 cDNA into yeast expression vectors under the control of either the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) promoter or the aldehyde-dehydrogenase (ADH) promoter. P450c21 RNA, protein, and enzyme activity can be detected, indicating that both promoters drive the synthesis of P450c21. The expressed P450c21 catalyzes the conversion of both of its substrates, with Km and Vmax values of 0.33 microM and 280 nmoles/hr.nmole of P450c21 protein for progesterone, and 0.23 microM and 450 nmoles/hr.nmole for 17-hydroxyprogesterone. These kinetic properties are similar to those of human P450c21 expressed in COS-1 cells. The microsomal fraction containing P450c21 exhibited an absorption peak at 450 nm upon binding to CO, demonstrating its hemoprotein nature. The CO-difference spectra indicated that there were about 0.08 nmole P450c21 hemoprotein/mg microsomal protein. Coupling this expression system with site-directed mutagenesis, the Asn-172 mutant of P450c21 had about 20-100 lower Vmax values; yet it retained normal affinity toward both substrates. This mutant protein also exhibited an altered absorbance with a peak at 420 nm rather than at 450 nm.

Structure, sequence, chromosomal location, and evolution of the human ferredoxin gene family.

Ferredoxin is an iron-sulfur protein that serves as an electron transport intermediate for mitochondrial cytochromes P450 involved in steroid, vitamin D, and bile acid metabolism. We cloned and characterized the human ferredoxin gene family, which includes two expressed genes and two pseudogenes. Sequence analysis of this gene family revealed that it encodes only one protein product. The expressed genes were assigned to chromosome 11 and pseudogenes to chromosomes 20 and 21 by identifying single-copy probes from each gene segment and hybridizing them to DNA from rodent-human hybrid cells. The pseudogenes lacked introns and contained numerous mutations, including insertion, deletion, and substitution which rendered them inactive. They were 96% and 85% homologous to the expressed gene, yet they were only 78% homologous with each other. The intronless nature, higher diversity among themselves, and distinct chromosomal location of the pseudogenes suggests that they arose by independent, retroposon-mediated events.

Insulin secretion and insulin action in Taiwanese with type 2 diabetes.

In contrast to the United State, type 2 diabetes appears to be a common occurrence in non-obese Asians. In order to evaluate the possibility that this epidemiologic difference was indicative of a basic metabolic phenomenon, estimates of insulin secretion and insulin action were generated in 32 Chinese males, 16 with type 2 diabetes and 16 with normal glucose tolerance. Half of the individuals in each diagnostic category were obese (body mass index greater than 28 kg/m2) and half were non-obese (less than 26 kg/m2). Plasma glucose responses to a 75-g oral glucose challenge were significantly higher in patients with type 2 diabetes, but did not vary significantly within either group as a function of obesity. Plasma insulin concentrations were lower than normal when patients with type 2 diabetes were compared to their weight-matched controls. In addition, the absolute insulin values also varied as a function of body weight, with higher plasma insulin concentrations observed in the obese individuals. Insulin action was estimated by determination of the steady-state plasma insulin (SSPI) and glucose (SSPG) concentrations during the last 60 min of a continuous 180-min intravenous infusion of somatostatin, crystalline insulin, and glucose. Under these conditions endogenous insulin secretion is suppressed, SSPI concentrations are similar in all individuals, and SSPG concentrations provide a quantitative estimate of insulin-stimulated glucose disposal. The results of these studies indicated that patients with type 2 diabetes had significantly elevated SSPG concentrations as compared to normals, and this was true whether the diabetic subjects were obese or non-obese.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of lovastatin and gemfibrozil in subjects with high ratios of total cholesterol to high-density lipoprotein cholesterol.

Insulin resistance is associated with hypertriglyceridemia, low serum high-density lipoprotein cholesterol (HDL-C) concentrations and high serum total cholesterol (TC) to HDL-C ratios. Several reports have demonstrated that either lovastatin or gemfibrozil may favorably lower serum lipid concentrations. However, their effects on insulin sensitivity are unknown. The primary aim of this study was to compare the effects of lovastatin and gemfibrozil on insulin sensitivity and serum leptin concentrations in subjects with high TC/HDL-C ratios. We enrolled 25 nondiabetic patients, similar in terms of age and weight with TC/HDL-C ratios greater than 5. Thirteen subjects were treated with lovastatin 20 mg per day, and 12 received gemfibrozil 300 mg twice per day. Plasma lipids, glucose, and leptin were measured, and a 75-g oral glucose tolerance test (OGTT) and a modified insulin suppression test were performed before and after 3 months of treatment. The study showed the mean plasma TC, low-density lipoprotein cholesterol (LDL-C) concentrations, and TC/HDL-C ratio were significantly reduced in the lovastatin-treated group, but no obvious effects on plasma triglyceride (TG) and HDL-C were noted. In the gemfibrozil group, plasma TG and HDL-C were markedly lowered, but no significantly different effects in other plasma lipids were found. Gemfibrozil did not affect steady-state plasma glucose (SSPG) concentrations, whereas lovastatin significantly increased SSPG concentrations. Neither drug affected the serum leptin concentration during the OGTT. We conclude that lovastatin significantly lowers plasma TC and LDL-C ratio, and TC/HDL-C concentrations and adversely affects insulin sensitivity, while gemfibrozil markedly reduces plasma TG concentrations without altering insulin sensitivity in subjects with high TC/HDL-C ratios.

Alstrom syndrome in two siblings.

Alstrom syndrome is a very rare autosomal recessive inherited disorder. Only 50 cases have been reported since the syndrome was first described in 1959. This syndrome is characterized by obesity, impaired glucose tolerance with insulin resistance, retinal degeneration, neurosensory deafness, acanthosis nigricans, hepatic dysfunction, and some endocrine disorders. The index case of this report was a 12-year-old girl who became blind at the age of 6 years as the result of progressively impaired vision. At the age of 12, diabetes mellitus was diagnosed and acanthosis nigricans presented in the neck, axilla, and groin regions. Her 10-year-old brother had similar symptoms. Electroretinography and audiometry disclosed generalized pigmentary epithelial change, decreased to absent cone and rod responses, and moderate sensorineural hearing loss in both siblings. Biochemistry and oral glucose tolerance tests showed diabetes mellitus, dyslipidemia, and hepatic dysfunction in the index case. Elevations of insulin, C-peptide, and leptin concentrations were found in both siblings. Insulin resistance was also demonstrated in both siblings using the modified insulin suppression test with constant infusion of somatostatin and exogenous insulin.

The relationships between insulin resistance and components of metabolic syndrome in Taiwanese Asians.

Metabolic syndrome (MetS) is a complicated clinicopathological entity with clustering of cardiovascular and metabolic risk factors, which includes central obesity, hypertension, dyslipidemia and glucose intolerance. There were many studies investigating a wide variety of clinical and pathophysiological aspects of this syndrome. However, the cutoffs of the components of MetS are not yet being evaluated by measured the insulin resistance (IR) directly. In this study, we enrolled 564 (male/female: 250/314) middle-aged healthy subjects. Each of the male and the female group was further divided into four subgroups (group 1 to group 4). Group 4 had the top 25 percentile of most severe IR determined by insulin suppression test. We then obtain the mean values of each component of the MetS in group 4 and compared them with the definitions of World Health Organization, National Cholesterol Education Program Adult Treatment Panel III, European Study Group of Insulin Resistance and International Diabetes Federation. The means of the blood pressure (BP) (male, 125/81; female, 125/80 mmHg) and the triglyceride (TG) (male, 1.6; female, 1.4 mmol/l) in group 4 were lower, and the fasting plasma glucose (6.2 mmol/l) was higher than the cutoffs of the other four sets of the criteria. The means of the high-density lipoprotein cholesterol (male, 0.9; female, 1.03 mmol/l) and the body mass index (male, 26.9; female 26.1 kg/m(2)) in group 4 were consistent with the cutoffs of other four groups and also the Taiwan Health Department criteria. In conclusion, we suggest to lower the cutoffs of the BP from 140/90 to 125/80 mmHg, TG from 1.7 to 1.6 mmol/l for males and 1.4 mmol/l for females for MetS definition, at least in Taiwan. This may help to early detect subjects under high risk of future coronary heart disease and diabetes. Still, these newly proposed cutoffs need larger-scale epidemiological studies to confirm.

Structure and expression of the CYP21 (P450c21, steroid 21-hydroxylase) gene with respect to its deficiency.

Steroid 21-hydroxylase (P450c21) deficiency is the major cause of a common genetic disease, congenital adrenal hyperplasia, with the symptoms of virilization due to steroid imbalance. We have devised a fast diagnostic method to detect common mutations in the c21B gene by a two-step gene amplification procedure coupled to restriction digestion. This procedure does not require isotopes and is suitable for routine use in a hospital setting. In addition, we have developed a procedure for the production of active P450c21 in E. coli. We tested many different vector and bacterial strain combinations to find out the best condition for P450c21 expression. The bacteria harboring the P450c21 expression plasmid were grown in a rich media supplemented with trace metals, heme biosynthesis precursor delta-levulinic acid, and induced with IPTG at 20 degrees C for 48 h. We found that low growth temperature and long induction time were important for abundant synthesis of P450c21 in E. coli.

Cloning and structure of the human adrenodoxin gene.

Adrenodoxin is an iron-sulfur protein that serves as an electron transport intermediate for all mitochondrial forms of cytochrome P450. To facilitate studying the regulation of adrenodoxin, we have cloned and determined the structure of the human adrenodoxin gene. It spans more than 20 kb, containing four exons and three introns. The first exon encodes the 60-amino-acid signal peptide, directing transport of the protein into the inner mitochondrial matrix. The mature peptide of 124 amino acids is encoded by the other three exons. The third exon encodes the portion of the protein containing the iron-sulfur center and a domain which binds other components of the electron transport chain. The transcriptional start sites were determined by primer extension and S1 nuclease mapping. The 5'-flanking region of this gene contains canonical promoters including a TATA box at nucleotide position -30 and two GC boxes at nucleotide positions -60 and -100. The sequence at nucleotides -234 to -252 is also highly homologous to the glucocorticoid-responsive element and the estrogen-responsive element.

Morphometric description of the wandering behavior in Drosophila larvae: aberrant locomotion in Na+ and K+ channel mutants revealed by computer-assisted motion analysis.

Wandering is a simple behavior in Drosophila larvae prior to metamorphosis. Using the Dynamic Image Analysis System (DIAS) initially developed for analyzing amoeboic movements of single cells, we have analyzed videotaped behaviors of Drosophila larvae at the wandering stage. Previous studies show that mutations in the Na+ channel gene paralytic (para) cause paralysis at 29 degrees C, and mutations in the K+ channel beta subunit gene Hyperkinetic (Hk) lead to leg-shaking under ether anesthesia. The application of DIAS revealed quantifiable abnormalities in the larval locomotion of both ion channel mutants even under "permissive" conditions. Analysis of centroid movement indicates that, compared to wild type, both Hk and para larvae crawled at a slower average speed, but a similar peak instantaneous speed during a contraction cycle. Nevertheless, contraction in the body length was greater in mutants, implying a lower efficiency in conversion of muscular contraction to distance translocation. In addition, each mutant produced a characteristic crawling pattern distinct from the wild-type control. The larval crawling pattern was determined by periods of linear locomotion interposed by non-locomotive, "searching and decision-making" episodes, after which the crawling was resumed in a new direction. Our results demonstrate that mutations in single ion channel subunits resulted in stereotypic modifications in locomotion control and crawling patterns, and that DIAS is a powerful tool in revealing subtle differences in animal behavior and quantifying mutational effects on the interplay of discrete behavioral components.

Assignment of the functional gene for human adrenodoxin to chromosome 11q13----qter and of adrenodoxin pseudogenes to chromosome 20cen----q13.1.

Adrenodoxin is a small iron/sulfur protein serving as an electron-transport intermediate for all mitochondrial forms of cytochrome P450. Southern blots of normal genomic DNA cleaved with six restriction endonucleases probed with full-length human adrenodoxin cDNA revealed complex patterns indicating the presence of multiple adrenodoxin genes. Southern blots of DNA from a panel of mouse/human somatic cell hybrids identified cross-hybridizing adrenodoxin DNA in two loci, chromosome 11q13----qter and chromosome 20cen----q13.1. Examination of adrenodoxin clones from a genomic DNA library in phage lambda revealed some clones bearing gene fragments interrupted by introns and other clones bearing processed pseudogenes. By probing the mouse/human hybrids with unique intronic DNA and by correlating restriction maps of the phage clones with that of uncloned genomic DNA, we show that the authentic transcribed adrenodoxin gene lies on chromosome 11, while pseudogenes lie on chromosome 20.