Correlation between single nucleotide polymorphisms in hypoxia-related genes and susceptibility to acute high-altitude pulmonary edema.

This study aimed to explore the relationship between genetic changes and high-altitude pulmonary edema (HAPE) susceptibility, and to screen for the key single nucleotide polymorphism (SNP) loci in the HAPE-susceptibility gene, by investigating the SNPs occurring in hypoxia-related genes in HAPE-susceptible and control (non-susceptible) populations. This research was conducted on Han recruits, who travelled to the Lhasa plateau (altitude, 3658 m). Ten loci located on ten genes extracted from the HAPE and healthy populations were amplified by polymerase chain reaction, and subsequently sequenced. The investigated genes included those coding for aldosterone synthase 2 (CYP11B2), angiotensin-converting enzyme (ACE), heat-shock protein 70 (HSP70), nuclear factor kappa B (NF-κB), surfactant protein A2 (SP-A2), plasminogen activator inhibitor-1 (PAI-1), nitric oxide synthetase (NOS), vascular endothelial growth factor (VEGF), prolyl hydroxylase (EGLN1), and zinc finger protein A20. The gene distribution of each SNP loci and its correlation with HAPE was analyzed. Statistical analyses of the genotype frequencies of the SNPs revealed significant differences in the ACE (rs4309), EGLN1 (rs480902), SP-A2 (rs1965708), HSP70 (rs1008438), PAI-1 (rs1799889), and NOS (rs199983) expressions between the HAPE and healthy control groups (P < 0.05); therefore, these SNP loci were believed to indicate HAPE susceptibility. HAPE is correlated with multiple- SNP loci. A correlation analysis between genetic polymorphism and HAPE susceptibility revealed that 6 hypoxia-related genes were key sites accounting for HAPE. These findings could help assess the risk of HAPE in populations expressing different genotypes, in order to reduce the occurrence of HAPE.

Ubiquitin-related proteins regulate interaction of vimentin intermediate filaments with the plasma membrane.

Integrin-associated protein (IAP, CD47) is a plasma membrane receptor for thrombospondins and signal regulatory proteins (SIRPs) that has an essential role in host defense through its association with integrins. The IAP gene encodes alternatively spliced carboxyterminal cytoplasmic tails that have no previously described function. IAP cytoplasmic tails can bind two related proteins that mediate interaction between IAP and vimentin-containing intermediate filaments, named proteins linking IAP with cytoskeleton (PLICs). Integrins interact with PLICs indirectly, through IAP. Transfection of PLICs induces redistribution of vimentin and cell spreading in IAP-expressing cells. This novel connection between plasma membrane and cytoskeleton is likely to be significant in many adhesion-dependent cell functions.

ROD1, a novel gene conferring multiple resistance phenotypes in Saccharomyces cerevisiae.

Glutathione-dependent detoxification reactions are catalyzed by the enzyme glutathione S-transferase and are important in drug resistance in organisms ranging from bacteria to humans. The yeast Issatchenkia orientalis expresses a glutathione S-transferase (GST) protein that is induced when the GST substrate o-dinitrobenzene (o-DNB) is added to the culture. In this study, we show that overproduction of the I. orientalis GST in Saccharomyces cerevisiae leads to an increase in o-dinitrobenzene resistance in S. cerevisiae cells. To recover genes that influence o-DNB resistance in S. cerevisiae, a high copy plasmid library was screened for loci that elevate o-DNB tolerance. One gene was recovered and designated ROD1 (resistance to o-dinitrobenzene). This locus was found to encode a novel protein with no significant sequence similarity with proteins of known function in the data base. An epitope-tagged version of Rod1p was produced in S. cerevisiae and shown to function properly. Subcellular fractionation experiments indicated that this factor was found in the particulate fraction by differential centrifugation. Overproduction of Rod1p leads to resistance to not only o-DNB but also zinc and calcium. Strains that lack the ROD1 gene are hypersensitive to these same compounds. Rod1p represents a new type of molecule influencing drug tolerance in eukaryotes.

GSH1, which encodes gamma-glutamylcysteine synthetase, is a target gene for yAP-1 transcriptional regulation.

Changes in gene dosage of the YAP1 gene, encoding the yAP-1 transcriptional regulatory protein, cause profound alterations in cellular drug and metal resistance. Previous studies on yAP-1 action in yeast cells have used the AP-1 response element (ARE) from simian virus 40 as an artificial site for yAP-1-mediated transcriptional activation. No authentic yeast target sites for control of gene expression by yAP-1 are known. Here we show that the GSH1 gene, encoding gamma-glutamylcysteine synthetase, is transcriptionally responsive to the yAP-1 protein. GSH1 encodes the rate-limiting step in yeast glutathione biosynthesis and contains within its promoter region a DNA element that matches the ARE in 11 of 12 positions. The GSH1 yAP-1 response element (YRE) was recognized by yAP-1 protein in vitro. Northern (RNA) blot analysis showed that GSH1 mRNA levels were responsive to YAP1 gene dosage. A site-directed mutation in the YRE that blocked yAP-1 binding in vitro prevented the mutant GSH1 promoter from responding to elevation in YAP1 gene dosage. A delta gsh1 mutant strain was constructed and unable to grow in the absence of exogenous glutathione. A mutant GSH1 gene lacking the YRE was unable to confer normal cadmium tolerance, although other yAP-1-mediated phenotypes remained normal. Thus, GSH1 is one of several genes that are transcriptionally controlled by yAP-1 and influence drug resistance.

Transcriptional activation mediated by the yeast AP-1 protein is required for normal cadmium tolerance.

The yeast YAP1 gene encodes a transcriptional regulatory protein that utilizes a basic region-leucine zipper (bZip) DNA-binding domain to recognize its cognate DNA element. A synthetic reporter gene containing a SV40 AP-1 response element (ARE) cloned upstream of a TRP5 promoter-lacZ gene fusion shows yAP-1-dependent transactivation in vivo. Recent work has shown that changes in the gene dosage of this factor can dramatically alter the ability of a cell to tolerate a host of toxic agents including cadmium, cycloheximide, and sulfometuron methyl. We have focused on the YAP1-dependent cadmium resistance as cells that lack a functional YAP1 gene are hypersensitive to this metal. Deletion mapping experiments define two domains in the carboxyl-terminal region of the yAP-1 protein that are required for normal cadmium tolerance and ARE-TRP5-lacZ expression. Single amino acid substitutions in the bZip domain of yAP-1 indicate that this region is required for normal DNA binding and in vivo function of the protein. Replacement of a non-canonical asparagine with leucine in the yAP-1 leucine zipper leads to production of a defective protein. A substitution mutation in the basic domain converts this mutant protein into a dominant negative factor. The ability of yAP-1 to act as a positive regulator of transcription is required for its biological action.

Mutations in the yeast PDR3, PDR4, PDR7 and PDR9 pleiotropic (multiple) drug resistance loci affect the transcript level of an ATP binding cassette transporter encoding gene, PDR5.

The yeast pleiotropic (multiple drug) resistance gene PDR5 encodes a product with homology to a large number of membrane transport proteins including the mammalian multiple drug resistance family. In this study, we identified four genes on chromosome II that affect the steady-state level of PDR5 transcript in addition to a previously identified positive regulator, PDR1. The genes in question are PDR3, PDR4, PDR7 and PDR9. We also analyzed the interaction between PDR5 and YAP1. YAP1 encodes a positive regulator with a leucine zipper motif that causes pleiotropic drug resistance when overproduced. YAP1-mediated pleiotropic drug resistance is not dependent on the presence of PDR5 and must act through other genes.

An epidemiological study on farmer's lung among hay grinders in Dafeng County.

This article reports on an epidemiological survey of the presence of farmer's lung among 1054 farmers who grind moldy hay. The prevalence was 8.3-11.4%. The main pathogen was Thermoactinomyces vulgaris. The factors which influenced farmer's lung were the moldy state of the hay and the dust concentration. The epidemiological features of farmer's lung are analyzed and discussed and certain preventive measures are suggested.

Variant forms of plasma apolipoprotein B. Hepatic and intestinal biosynthesis and heterogeneous metabolism in the rat.

Recent studies of circulating lipoproteins in man and the rat have shown that apolipoprotein B in plasma exists in two immunologically distinct forms with apparent Mr, in the rat, of approximately 335,000 (apoB335K) and 240,000 (apoB240K). With isolated, perfused organs and single and double isotope labeling methods in vivo (1979) J. Biol. Chem. 254, 7316), we have now found that the rat liver produces both apoB variants in abundance while the small intestine produces apoB240K but only trace amounts of apoB335K. The hepatic apoB335K/apoB240Kleucine incorporation ratio ranged from 0.5-1.7 under different conditions, suggesting physiological regulation of the relative amounts produced. Disappearance of both newly synthesized hepatic apoB variants from plasma followed first order kinetics. The circulating half-life of hepatic apoB335K was 2.4 times that of hepatic apoB240K, indicating independent catabolism of the two variants and consistent with the hepatic production of multiple apoB-containing lipoproteins with different metabolic properties. The half-life of apoB240K from the intestine was even shorter than that of apoB240K from the liver.

Biosynthesis of plasma apolipoproteins by rat small intestine without dietary or biliary fat.

Using two complementary methods developed and applied earlier in rats absorbing triacylglycerols, we have now determined the intestinal contribution of individual plasma apolipoproteins when the rate of intestinal lipid absorption is very low. After all dietary fat was withheld for 16-20 h, the intestine produced approximately 50% of the plasma apolipoprotein A-I (apo-A-I) and apo-A-IV, similar to its contribution during fat absorption. When rats were fed a fat-free diet for 12 days and, in addition, biliary fat was diverted from the intestine for 16 h, the intestine still produced approximately 50% of the apo-A-I and only slightly less of the apo-A-IV. Under both conditions of reduced fat absorption, a larger proportion of the intestinally-derived apo-A-I and apo-A-IV bypassed the mesenteric lymph and was released directly into intestinal venous blood. The intestinal apo-B contribution, 16% of the total in fat-fed rats, was reduced to approximately 5% when dietary fat was withheld. Intestinal apo-B was released entirely into the lymph. Intestine produced only small amounts of apo-C and little or no apo-E under all conditions. The results indicate that production of apo-A-I and apo-A-IV by the small intestine is not regulated by the rate of intestinal triacylglycerol transport.

Reduced intestinal absorption of vitamin E by low dietary levels of retinoic acid in rats.

It was observed that rats fed a low dietary level of retinoic acid had markedly lower plasma concentrations of alpha-tocopherol than did rats fed the same amount of retinol. In this report, the possible mechanisms by which retinoic acid alters vitamin E metabolism has been investigated. Weanling male rats were fed a complete purified diet with either retinol or retinoic acid at 4 mg/kg diet; plasma and tissues were analyzed after 2-5 weeks. The plasma alpha-tocopherol concentration in rats ingesting retinoic acid was one-half that of rats ingesting retinol, and this difference also occurred in the liver and adipose tissue. Similar effects occurred in chicks. This low dietary level of retinoic acid had no effect on plasma triglyceride concentration, as has been reported for higher intakes, and plasma cholesterol and total lipids were also unaffected. Retinoic acid did not affect the rate of decrease in endogenous alpha-tocopherol in normal rats fed a vitamin E-free diet for 3 weeks. In rats with mesenteric lymph cannulas, dietary retinoic acid caused a reduced absorption of 3H-labeled alpha-tocopherol. In chicks fed retinoic acid, plasma and liver radioactivity 2.5 hours after an oral dose of 3H-alpha-tocopherol was one-fifth that of chicks fed retinol. More oxidation of alpha-tocopherol occurred during absorption in rats fed retinoic acid than in those fed retinol, as evidenced by more alpha-tocopherylquinone in the collected lymph. We postulate that dietary retinoic acid reduces the intestinal absorption of alpha-tocopherol and may also promote its oxidation.

Effect of sucrose on intestinal very low-density lipoprotein production.

The effect of 2 to 3 weeks of 73% sucrose feeding and intestinal sucrose infusion upon intestinal lipoprotein formation was studied in mesenteric lymph cannulated female rats. Lymph lipoprotein fractions were analyzed for lipid and apoprotein content and were compared to chow-fed control rats. The sucrose regimen increased mesenteric lymph triglyceride output by 54%, the increase being confined to the very low-density lipoprotein fraction. Sucrose infusion in chow-fed control rats did not increase lymph triglyceride transport when compared to infusion of a glucose polymer. Unesterified cholesterol output in whole lymph was stimulated by sucrose by 76%. Both the d greater than 1.006 and d less than 1.006 g/ml fractions of lymph were found to have an increase in unesterified cholesterol output. Since no difference in the chemical composition of very low-density lipoproteins in the two groups of animals was detected, sucrose appeared to increase the number of very low-density lipoprotein particles secreted by the intestine. Sucrose did not alter the relative proportions of apo A1, apo A4, apo ARP, and total C peptides present in very low-density lipoproteins. However, differences in the proportion of apo C subunits after sucrose were detected by isoelectric focusing with a pronounced increase in the apo CIII0 peak. The present studies demonstrate that the intestine participates in changes in lipoprotein formation and metabolism that accompany experimental sucrose induced hyperlipidemia.

Composition of lymph chylomicrons from proximal or distal rat small intestine.

The composition of lymph chylomicrons secreted by rat proximal and distal small intestine were compared during constant perfusion of a lecithin-stabilized tri (1-14C) oleoyl glycerol emulsion, in pairs of unanesthesized rats with mesenteric lymph fistulas. By the 6th hr of infusion when 14C-triglyceride output was constant, the distal intestine secreted 33% less chylomicron phospholipid. Distal chylomicrons were larger and had higher triglyceride:phospholipid and higher apoprotein:phospholipid ratios than chylomicrons secreted by proximal intestine. The major apoprotein classes--apoB, apoA-I, apoE(= ARP), and C peptides--were present in both groups of chylomicrons, but in different proportions. Distal chylomicrons contained less apoA-I and more C peptides, with an increase in apoC-III3, and a decrease in apoC-III0, compared with proximal chylomicrons. The present study suggests that the distal intestine is defective in the utilization of phospholipid from the intestinal lumen for chylomicron phospholipid synthesis. Whether the observed changes in the size and phospholipid or apoprotein content of distal chylomicrons affect their system metabolism is presently not known.

Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.

Apoproteins of chylomicrons, very low density lipoprotein (VLDL), and a low density + high density fraction secreted by proximal and distal rat small intestine into mesenteric lymph were examined during triglyceride (TG) absorption. Apoprotein output and composition were determined and the turnover rates of labeled non-apoB (soluble) apoproteins in lipoprotein fractions were measured after an intraluminal [(3)H]leucine pulse during stable TG transport into lymph. The output of VLDL apoproteins exceeded that of chylomicrons during the absorption of 45 micro mol of TG per hour. More [(3)H]leucine was incorporated into VLDL than into chylomicrons and the decay of newly synthesized VLDL apoproteins was more rapid than that of chylomicrons, in part due to higher concentrations of apoA-I and apoA-IV with a rapid turnover rate. Chylomicrons from proximal intestine contained more apoA-I and less C peptides than chylomicrons from distal intestine. Ninety percent of [(3)H]leucine incorporated into soluble apoproteins was in apoA-I and apoA-IV, but little apoARP was labeled. The turnover rate of apoA-I and apoA-IV differed significantly in the lymph lipoproteins examined. Although total C peptide labeling was small, evidence for intestinal apoC-II formation and differing patterns of apoC-III subunit labeling was obtained. [(3)H]Leucine incorporation and apoprotein turnover rates in lipoprotein secreted by proximal and distal intestine were similar. The different turnover rates of apoA-I and apoA-IV in individual lipoproteins suggest that these A apoproteins are synthesized independently in the intestine.-Holt, P. R., A-L. Wu, and S. Bennett Clark. Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.

Identification of circulating apolipoproteins synthesized by rat small intestine in vivo.

Apolipoproteins synthesized by intestine were identified in mesenteric lymph and plasma collected from an animal preparation in which exposure to [3H]leucine was restricted to the mucosal epithelial cells of a fat-transporting small intestinal segment in situ. The intestine released labeled chylomicrons and very low density lipoproteins only into lymph but labeled high density lipoproteins into both plasma and lymph. Of the 3H incorporated into individual apolipoproteins, separated by polyacrylamide gel electrophoresis, 25% was recovered in apolipoprotein B (apoB), 50% in apoA-I, 21% in apoA-IV, and 4% in C apolipoproteins, nearly all in apoC-II and apoC-III-0. Only traces of radioactivity were associated with the arginine-rich protein and none was detected in apoA-II. Labeled apoB was found in chylomicrons and very low density lipoproteins and labeled apoA-I and apoA-IV in all lipoproteins. Evidence suggested that substantial amounts of labeled apoA-IV were also present in the lipoprotein-free lymph fraction (d greater than 1.21 g/ml), further indicating that apoA-IV is a major apolipoprotein product in intestine. Thus, the pattern of apolipoprotein synthesis by intestine is notably different from that reported for liver.

Immunological control mechanism against cholera toxin: interference with toxin binding to intestinal receptors.

The immunological control mechanism against cholera toxin (CT) in the small intestine of rats was studied in vivo. CT binding to intestinal receptors was determined by injected radiolabeled CT into the loops of rat small intestine and subsequently separating purified microvillus membranes from mucosal scrapings of those loops. substantial radioactivity (10(5) cpm/mg of microvillus protein) was present in microvillus fractions of small intestine exposed to 125I-labeled CT compared to radioactivity (10(2) cpm/mg) in fractions from intestine exposed to radiolabeled bovine serum albumin (BSA) used as a control. CT binding to intestinal receptors was significantly inhibited (P less than 0.02) in rats immunized with crude toxin by a combined intraperitoneal and oral method compared to CT binding in animals immunized with BSA or controls, suggesting a specific relationship between intestinal antitoxin and inhibition of binding. Furthermore, ligated ileal loops from CT-immunized animals showed a significant decrease in fluid accumulation when exposed to CT compared to loops from control or BSA-immunized animals, suggesting that antitoxins also interfered with the biological action of CT under conditions of immunization. These studies provide direct evidence that intestinal antitoxins protect against CT-induced diarrhea by interfering with the attachment of the toxin to the intestinal microvillus surface.

Resistance of intestinal triglyceride transport capacity in the rat to adaptation to altered luminal environment.

Previous studies have shown that the transmucosal transport capacity for tiolein is slower in distal compared with proximal regions of rat small intestine. The effect of altered luminal conditions on the relative capacities for [14C]triolein transport through different regions of intestinal wall were examined by determining the net accumulation of 14C-lipid in the mucosa during maximal steady [14C]triolein absorption. The high 14C-lipid accumulation in distal mucosa was not reduced after direct distal infusion of oleic acid and monolein for 7 days before challenge (substrate induction). No decrease in distal intestinal lipid accumulation was found 1 month after removal of the proximal 40% of intestine beyond the duodenum. Conversely, alteration of the luminal environment in the proximal intestine by 3 days of starvation or by ileal resection did not increase the tissue 14C-lipid concentration in this region during maximal [14C]triolein challenge. When proximal and distal segments were exchanged without reduction in intestinal length, by jejunoileal transposition, major 14C-lipid accumulation still occurred in originally distal segments during 3 hr of [14C]triolein infusion 1 month later. Mucosal synthesis of triglyceride was equally efficient in jejunum and ileum, suggesting that the intrinsic differences in proximal and distal lipid accumulation during maximal rate transport were related to a relatively limited capacity for chylomicron synthesis or secretion in the distal intestine.

Transmucosal triglyceride transport rates in proximal and distal rat intestine in vivo.

Transmucosal transport rates for triolein in proximal and distal intestine were compared in unanesthetized rats. Emulsified [1-14-C] triolein together with bile and pancreatic juice from donor rats was infused for 6 hr into either the duodenum or the midpoint of the small intestine at such a rate that absorption was essentially complete in both regions of the intestine. Lymph was collected from the thoracic duct during triolein infusion and for an additional 6-hr period. The decrease in the rate of lymphatic output of labeled fat was found to follow a simple exponential function in all animals. This rate of decrease (decay rate) was used to calculate the half-times of lipid turnover through the intestinal wall and the fractional output rates. Distal intestine transported lipid 40% more slowly than proximal intestine, and the difference was associated with a greater accumulation of triglyceride in the distal intestinal wall. Chylomicron synthesis and/or release is the rate-limiting step for distal lymphatic fat transport in vivo, whereas fat uptake from the lumen is rate limiting for proximal intestine.