Phenotypic characterization of lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice. Pathophysiology Of biliary lipid secretion.

The inbred C57L strain but not the AKR strain of mice carry Lith genes that determine cholesterol gallstone susceptibility. When C57L mice are fed a lithogenic diet containing 15% fat, 1% cholesterol, and 0.5% cholic acid, gallbladder bile displays rapid cholesterol supersaturation, mucin gel accumulation, increases in hydrophobic bile salts, and rapid phase separation of solid and liquid crystals, all of which contribute to the high cholesterol gallstone prevalence rates (D. Q-H. Wang, B. Paigen, and M. C. Carey. J. Lipid Res. 1997. 38: 1395;-1411). We have now determined the hepatic secretion rates of biliary lipids in fasting male and female C57L and AKR mice and the intercross (C57L x AKR)F(1) before and at frequent intervals during feeding the lithogenic diet for 56 days. Bile flow and biliary lipid secretion rates were measured in the first hour of an acute bile fistula and circulating bile salt pool sizes were determined by the "washout" technique after cholecystectomy. Compared with AKR mice, we found that i) C57L and F(1) mice on chow displayed significantly higher secretion rates of all biliary lipids, and larger bile salt pool sizes, as well as higher bile salt-dependent and bile salt-independent flow rates; ii) the lithogenic diet further increased biliary cholesterol and lecithin outputs, but bile salt outputs remained constant. Biliary coupling of cholesterol to lecithin increased approximately 30%, setting the biophysical conditions necessary for cholesterol phase separation in the gallbladder; and iii) no gender differences in lipid secretion rates were noted but male mice exhibited significantly more hydrophobic bile salt pools than females.We conclude that in gallstone-susceptible mice, Lith genes determine increased outputs of all biliary lipids but promote cholesterol hypersecretion disproportionately to lecithin and bile salt outputs thereby inducing lithogenic bile formation.     

Mapping genes encoding drug-metabolizing enzymes in recombinant inbred mice.

Probes for cytochrome P450IVA (P450IVA), alpha- and pi-class glutathione S-transferases (GST), and phenol-metabolizing UDP-glucuronyltransferase (UDPGT-K39) detected restriction fragment length variants (RFLVs) between C57BL/6J and DBA/2J mice. These variants were used to map the P450IVA genes (Cyp4 alpha) to chromosome 4, close to Mtv-13 and Pmv-19, midway between brown (b) and Gpd-1; GST alpha genes were mapped to chromosome 9, with a cross-hybridizing sequence mapping to another chromosome; the GST pi genes were mapped to the distal end of chromosome 1 near Pmv-21; one UDPGT-K39 variant to chromosome 1, between Acrg and Emv-17, and another showed linkage to Odc-10 on an unidentified chromosome. No RFLVs were detected with probes for P450IID, P450 reductase, androsterone-metabolizing UDPGT, GST mu, or microsomal GST.     

Quantitative trait loci that determine lipoprotein cholesterol levels in DBA/2J and CAST/Ei inbred mice

To investigate genetic contributions to individual variations of lipoprotein cholesterol concentrations, we performed quantitative trait locus/loci (QTL) analyses of an intercross of CAST/Ei and DBA/2J inbred mouse strains after feeding a high-cholesterol cholic acid diet for 10 weeks. In total, we identified four QTL for HDL cholesterol. Three of these were novel and were named Hdlq10 [20 centimorgans (cM), chromosome 4], Hdlq11 (48 cM, chromosome 6), and Hdlq12 (68 cM, chromosome 6). The fourth QTL, Hdl1 (48 cM, chromosome 2), confirmed a locus discovered previously using a breeding cross that employed different inbred mouse strains. In addition, we identified one novel QTL for total and non-HDL cholesterol (8 cM, chromosome 9) that we named Chol6. Hdlq10, colocalized with a mutagenesis-induced point mutation (Lch), also affecting HDL. We provide molecular evidence for Abca1 as the gene underlying Hdlq10 and Ldlr as the gene underlying Chol6 that, coupled with evidence generated by other researchers using knockout and transgenic models, causes us to postulate that polymorphisms of these genes, different from the mutations leading to Tangier's disease and familial hypercholesterolemia, respectively, are likely primary genetic determinants of quantitative variation of lipoprotein levels in mice and, by orthology, in the human population.     

Genetic analysis of cholesterol accumulation in inbred mice.

Genetic linkage analysis in the laboratory mouse identified chromosomal regions containing genes that contribute to cholesterol accumulation in the liver and plasma. Comparisons between five inbred strains of mice obtained from the Jackson Laboratory (DBA/2, AKR, C57BL/6, SJL, and 129P3) revealed a direct correlation between intestinal cholesterol absorption and susceptibility to diet-induced hypercholesterolemia. This correlation was lost in the F1 generation arising from crosses between high- and low-absorbing strains. Linkage analyses in AKxD recombinant inbred strains and 129xSJL129F1 N2 backcross mice identified four quantitative trait loci (QTL) that influenced Liver cholesterol accumulation (Lcho1-4) and one locus that affected Plasma cholesterol accumulation (Pcho1). These loci map to five chromosomes and, with one exception, are different from the seven QTL identified previously that influence intestinal cholesterol absorption. We conclude that a large number of genes affects the amount of cholesterol absorbed in the small intestine and its accumulation in the liver and plasma of inbred mice.     

Echinococcus multilocularis: susceptibility and responses to infection in inbred mice

Kroeze W. K. and Tanner C. E. 1987. Echinococcus multilocularis: susceptibility and responses to infection in inbred mice. International Journal for Parasitology17: 873–883. Of six strains of mice examined, C57L/J mice were the most susceptible to intraperitoneal infections with Echinococcus multilocularis. Five of the six strains developed splenomegaly during the infection. Changes in leukocyte levels in infected mice were most pronounced in the C57L/J and BALB/cJ strains. Two of the six strains of mice, C57BL/6J and C57BL/6J (bg^J), showed low specific IgG responses to E. multilocularis when measured using an ELISA. Many of the responses observed were directly correlated with the parasite burden in infected animals. It is concluded that susceptibility or resistance to E. multilocularis in mice is probably controlled by non-H-2 gene(s); additionally, hematological and immunological responses to infection, although correlated to parasite burden, varied among strains of mice, suggesting some degree of host genetic control of these responses.     

Genetic analysis of intestinal cholesterol absorption in inbred mice.

A genetic mapping strategy was employed to identify chromosomal regions harboring genes that influence the absorption of intestinal cholesterol in the mouse. Analysis of seven inbred strains of male mice (129P3, AKR, BALB/c, C3H/He, C57BL/6, DBA/2, and SJL, all from Jackson Laboratories) revealed substantial differences in their abilities to absorb a bolus of cholesterol delivered by gavage. Crosses between high (AKR, 129) and low (DBA/2, SJL) absorbing strains revealed evidence for the presence of dominant genes that increase and decrease cholesterol absorption. Backcrosses between F1 offspring and parental strains (DBA/2xAKD2F1 and 129xSJL129F1) followed by linkage analyses revealed four quantitative trait loci that influenced cholesterol absorption. Analyses of recombinant inbred strains identified an additional three loci affecting this phenotype. These seven quantitative trait loci, which map to different chromosomes and are termed Cholesterol absorption 1-7 (Chab1-7) loci, together influence the absorption of intestinal cholesterol in mice and are likely to be involved in different steps of this complex pathway.     

Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibility with in vitro defective macrophage microbicidal activities

Eleven mouse strains were inoculated in footpads with amastigotes of Leishmania tropica and observed for 12 weeks. Liver and spleen impression smears from infected mice were examined for the presence of intracellular parasites. Four strains (BALB/cJ, C57L/J, NZW/N, and P/J) failed to heal the subcutaneous lesion and showed evidence of systemic infection; the remaining seven strains (A/J, C3H/HeJ, C3H/HeN, C3HeB/FeJ, C57BL/6J, C57BL/10J, and C57BL/10ScN) were each resistant to infection and resolved their lesions by Week 10. Macrophages from the four susceptible strains could not be activated to kill L. tropica amastigotes by treatment with soluble lymphocyte products in vitro. In contrast, macrophages from all seven resistant strains responded to lymphokine treatment and eliminated 80-90% of intracellular parasites. These results suggest that in vitro macrophage microbicidal activities predict the course of systemic leishmanial disease.     

Effect of portacaval anastomosis on the activities of hepatic enzymes related to cholesterol and bile acid metabolism in rats.

1. The effect of a portacaval anastomosis on the activities of hepatic enzymes related to cholesterol metabolism was investigated in rats. 2. Portacaval anastomosis led to a fall in body weight and liver weight/body weight ratio, and to a rise in the activities of hydroxymethylglutaryl-CoA reductase and cholesterol 7alpha-hydroxylase per g of liver. The net effect was to maintain a normal activity of both enzymes per 100 g of rat. Diurnal rhythm in the activities of both enzymes was maintained after portacaval anastomosis. 3. The rate of excretion of total bile acids, per 100 g of rat, in bile fistula rats was not significantly decreased by portacaval anastomosis.     

Identification of regulatory domains in ADP-ribosyltransferase-1 that determine transferase and NAD glycohydrolase activities

Mono-ADP-ribosyltransferases (ART1-7) transfer ADP-ribose from NAD+ to proteins (transferase activity) or water (NAD glycohydrolase activity). The mature proteins contain two domains, an alpha-helical amino terminus and a beta-sheet-rich carboxyl terminus. A basic region in the carboxyl termini is encoded in a separate exon in ART1 and ART5. Structural motifs are conserved among ART molecules. Successive amino- or carboxyl-terminal truncations of ART1, an arginine-specific transferase, identified regions that regulated transferase and NAD glycohydrolase activities. In mouse ART1, amino acids 24-38 (ART-specific extension) were needed to inhibit both activities; amino acids 39-45 (common ART coil) were required for both. Successive truncations of the alpha-helical region reduced transferase and NAD glycohydrolase activities; however, truncation to residue 106 enhanced both. Removal of the carboxyl-terminal basic domain decreased transferase, but enhanced NAD glycohydrolase, activity. Thus, amino- and carboxyl-terminal regions of ART1 are required for transferase activity. The enhanced glycohydrolase activity of the shorter mutants indicates that sequences, which are not part of the NAD binding, core catalytic site, exert structural constraints, modulating substrate specificity and catalytic activity. These functional domains, defined by discrete exons or structural motifs, are found in ART1 and other ARTs, consistent with conservation of structure and function across the ART family.     

Trypanosoma congolense: inheritance of susceptibility to infection in inbred strains of mice

Inbred strains of mice have shown marked differences in susceptibility to infection with Trypanosoma congolense, as judged by survival and levels of parasitemia. The underlying genetic basis of the susceptibility was examined with F1 hybrids and backcrosses derived from mouse strains of high and low susceptibility. The influence of H-2 haplotype on susceptibility was studied using H-2 congenic resistant strains of mice. F1 hybrids between the most susceptible strain (A/J) and the least susceptible strain (C57Bl/6) showed similar survival to that of the C57Bl/6 parent. This was reflected in a similar undulating pattern of parasitemia, although the level of parasitemia was consistently higher in the F1 hybrids than in the C57Bl/6. Backcrosses of the F1 hybrids with C57Bl/6 also had a similar pattern of parasitemia although there was a greater scatter in survival times so that a few animals survived longer than either of the parental strains. Backcrosses of F1 hybrids with A/J showed a range of survival times; approximately 25% of these animals died during the period when the A/J mice died, approximately 25% had a similar survival to that of C57Bl/6, while the remaining animals showed an intermediate duration of survival. All these backcrosses had a high initial peak of parasitemia; in about 70% of the mice the early parasitemia showed a distinct undulating pattern. F1 hybrids of A/J and C57Bl/6 with C3H/He mice, which are known to be of intermediate susceptibility, were also examined. The degree of dominance for low susceptibility was much less pronounced in these hybrid combinations than in the A/J × C57Bl/6 hybrids. The H-2 congenic resistant strains, all of which were on a C57Bl/10 genetic background, showed a similar pattern of parasitemia and survival. However, although the majority of all these strains survived for more than 100 days, there was a significant difference in survival between the C57Bl/10 mice and the H-2 congenic resistant strains. It was concluded that susceptibility of mice to T. congolense infection is likely to be under complex genetic control and that, at least in C57Bl/mice, H-2 haplotype has little influence on susceptibility.     

Trichinella spiralis: anaphylactic antibody formation and susceptibility in strains of inbred mice.

The anaphylactic antibody response of various strains of inbred mice of different H-2 specificities was investigated using the passive cutaneous anaphylactic technique (PCA) for the detection of the antibody response. Neither IgC₁ nor reaginic antibody were detected in serum samples obtained at the end of the first week of infection with Trichinella spiralis. Subsequently, all animals had detectable IgG₁ antibodies, although in some strains the titers were very low. Reaginic antibody was detected in relatively high titers in C57L, A, and DBA/1 mice. Two other strains were very poor responders (SJL and AKR). In most strains, reagin and IgG₁ remained detectable for 14 wk or longer. The pattern of response of all strains was very reproducible, indicating genetic control of the anaphylactic antibody production to the infection. In F₁ hybrids obtained from crosses between good and poor anaphylactic antibody responders, intermediate levels of both antibody classes were detected.Adult worm recovery rates were established at various points during the intestinal phase of infection, and no correlation between worm numbers and reaginic antibody titers in the various strains of mice could be demonstrated. There were noticeable differences in larval yields obtained after muscle digestion of mice belonging to the different inbred strains. In fact, we generally observed an inverse relationship between the number of larvae recovered from a given strain and their reaginic antibody titer.The intravenous injection of newborn larvae (NBL), obtained upon in vitro incubation of adult worms, produced detectable antibodies only in mice of the DBA/1 strain. These antibodies were consistently of low titer and became detectable only after the administration of two additional injections of NBL. This contrasted with the results observed after “per os” infection of DBA/1 mice, where high titers of these antibodies were always obtained, in spite of comparable ratios of muscle larval yield.     

Nanolitre-scale assays to determine the activities of enzymes in individual plant cells

There are a variety of methods for characterising gene expression at the level of individual cells and for demonstrating that the cells also contain the encoded proteins. However, measuring the activity of enzymes at the resolution of single cells in complex tissues, such as leaves, is problematic. We have addressed this by using single-cell sampling to extract 10-100 pl droplets of sap from individual plant cells and then measuring enzyme activities in these droplets with nanolitre-scale fluorescence-based assays. We have optimised these assays and used them to measure and characterise the activities of acid phosphatase, cysteine protease and nitrate reductase in sap samples from epidermal and mesophyll cells of barley (Hordeum vulgare L.) and Arabidopsis thaliana leaves exposed to different developmental and environmental conditions. During leaf senescence in barley, we found that the dynamics with which acid phosphatase and protease activities changed were different in each cell type and did not mirror the changes occurring at the whole-leaf level. Increases in nitrate reductase activities after exposure of barley plants to nitrate were large in mesophyll cells but small in epidermal cells. The technique was applied successfully to Arabidopsis and, as in barley, revealed cell-specific differences in the activities of both acid phosphatase and nitrate reductase. The assays add to the spectrum of techniques available for characterising cells within complex plant tissues, thus extending the opportunity to relate gene expression to biochemical activities at the single-cell level.     

Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.

Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6(Scp2(-/-)) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6(Scp2(-/-)) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6(Scp2(-/-)) mice. When C57BL/6(Scp2(-/-)) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution.     

Quantitative trait locus analysis of susceptibility to diet-induced atherosclerosis in recombinant inbred mice

Quantitative trait locus (QTL) analysis is a statistical method that can be applied to identify loci making a significant impact on a phenotype. For the phenotype of susceptibility to diet-induced atherosclerosis in the mouse, we have studied four quantitative traits: area of aortic fatty streaks and serum concentrations of high-density lipoprotein-bound cholesterol (HDL-cholesterol), apolipoprotein A-I, and apolipoprotein A-II (apo A-II). QTL analysis revealed a significant locus on chromosome 1 distal impacting serum apo A-II concentration on a high-fat diet and serum HDL-cholesterol concentration on a chow diet. This locus is presumablyApoa-2, the structural gene for apo A-II. QTL analysis of aortic fatty streaks failed to reveal a significant locus.     

Endotoxin increases hepatic susceptibility to lipid peroxidation: a possible role of iron

The purpose of this study was to investigate the possible mechanism by which endotoxin enhances peroxidative damage to membrane lipids. Male B6C3 mice were treated with endotoxin intraperitoneally 0 or 20 mg/kg body weight for 24 h. Freshly prepared liver homogenate was incubated with either 1-5 mM of reduced glutathione (GSH), glucose, H(2)O(2), ascorbic acid (AA), FeSO(4), FeCl(3), EDTA, FeCl(3) plus AA, AA plus EDTA or EDTA plus FeCl(3) in phosphate-buffered saline (PBS), pH 7.0, or PBS, at 37 degrees C for 60 min. The levels of lipid peroxidation products, thiobarbituric acid reactants (TBAR), were significantly higher in the liver of endotoxin-treated mice, and the values were markedly increased following incubation. Compared to PBS, incubation with H(2)O(2), FeCl(3), FeSO(4), and AA, but not glucose, significantly enhanced TBAR formation. The greatest increase of TBAR was found when AA and FeCl(3) were added together. On the other hand, EDTA and GSH inhibited the formation of TBAR during incubation. When added before AA, EDTA completely inhibited the peroxidative effect of AA or FeSO4, and when added subsequent to AA, EDTA partially prevented the adverse effect of AA. The results obtained suggest that ionic iron plays an important role in initiating endotoxin-induced peroxidative damage to membrane lipids, and that AA may be involved in releasing iron from its protein complex and/or maintaining ionic iron in a reduced or catalytic state.     

Synergism of iron and hexachlorobenzene inhibits hepatic uroporphyrinogen decarboxylase in inbred mice.

The combination of a single subcutaneous dose of iron (12.5 mg/mouse) and subsequent treatment with hexachlorobenzene (0.02% of the diet) caused a progressive inhibition of hepatic uroporphyrinogen decarboxylase in male C57BL/10 mice leading to the accumulation of uroporphyrin in 4-6 weeks. There was only a slight inhibition of the enzyme in the absence of iron and none without hexachlorobenzene. Females were less sensitive than males. In addition, comparisons between the C57BL/10, BALB/c, AKR and DBA/2 strains indicated that the susceptibilities of mice to induction of porphyria did not completely correlate with their classification as Ah-responsive or Ah-non-responsive.     

The susceptibility of inbred mice to infection with Brachylaima cribbi (Digenea: Brachylaimidae)

Susceptibility to infection with Brachylaima cribbi was studied in eight strains of inbred mice (AKR, C3H/HeJ, CBA/CaH, BALB/c, DBA/2J, SJL/J, A/J, C57BL/6J) and Swiss albino outbred mice by quantifying faecal egg excretion over the period of the infection. Preliminary experiments indicated that a combination of filtration/sedimentation/diethyl ether sedimentation was the most sensitive and reliable technique for quantification of eggs in faeces. Mice were infected with 13-15 wild-type B. cribbi metacercariae from naturally infected Cernuella virgata and in a second experiment with human-derived B. cribbi from laboratory-reared Helix aspersa. In both experiments C57BL/6J mice were the most susceptible having the highest egg excretion levels and the longest duration of infection. Worm burdens were assessed at 12 wpi for the wild-type and at 9 wpi for the human-derived infections, when the majority of mice were no longer excreting eggs. The numbers of worms recovered from the small intestine were few and there were no significant differences among the inbred or outbred groups of mice. We have found that C57BL/6J mice were the most susceptible to Brachylaima cribbi infection as assessed by excretion of eggs and provide a suitable model for a laboratory life-cycle.     

Isolation and characterization of Rhizobium (IC3342) genes that determine leaf curl induction in pigeon pea.

Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.