A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by ≈31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

Conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: Implications for c type cytochrome biogenesis and folding

Cytochrome c552 from Hydrogenobacter thermophilus, a thermophilic bacterium, has been converted into a b type cytochrome, after mutagenesis of both heme-binding cysteines to alanine and expression in the cytoplasm of Escherichia coli. The b type variant is less stable, with the guanidine hydrochloride unfolding midpoint occurring at a concentration 2 M lower than for the wild-type protein. The reduction potential is 75 mV lower than that of the recombinant wild-type protein. The heme can be removed from the b type variant, thus generating an apo protein that has, according to circular dichroism spectroscopy, an α-helical content different from that of the holo b type protein. The latter is readily reformed in vitro by addition of heme to the apo protein. This reforming suggests that previously observed assembly of cytochrome c552, which has the typical class I cytochrome c fold, in the E. coli cytoplasm is a consequence of spontaneous thioether bond formation after binding of heme to a prefolded polypeptide. These observations have implications for the general problem of c type cytochrome biogenesis.

Blockade of type β transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat

We eliminated type β transforming growth factor (TGF-β) signaling by adenovirus-mediated local expression of a dominant-negative type II TGF-β receptor (AdCATβ-TR) in the liver of rats treated with dimethylnitrosamine, a model of persistent liver fibrosis. In rats that received a single application of AdCATβ-TR via the portal vein, liver fibrosis as assessed by histology and hydroxyproline content was markedly attenuated. All AdCATβ-TR-treated rats remained alive, and their serum levels of hyaluronic acid and transaminases remained at low levels, whereas all the AdCATβ-TR-untreated rats died of liver dysfunction. The results demonstrate that TGF-β does play a central role in liver fibrogenesis and indicate clearly in a persistent fibrosis model that prevention of fibrosis by anti-TGF-β intervention could be therapeutically useful.

γ-Aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes

γ-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of γ-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K+ (Kir3) conductance. These GABA type B (GABAB)-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABAB agonist baclofen. Recent studies of native GABAB receptors (GABABRs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABABRs assemble heteromeric complexes from the GABABR1 and GABABR2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABAB agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K+ current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABAB antagonists, and prevented by pertussis toxin pretreatment. Both GABABR1 and GABABR2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABABRs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease

Niemann–Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

Decreased GA1 Content Caused by the Overexpression of OSH1 Is Accompanied by Suppression of GA 20-Oxidase Gene Expression

We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA1 was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) of OSH1 on GA metabolism. GA53 and GA20, precursors of GA1, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA20, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA1 and GA20 contents in OSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA19, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression of OSH1 causes a reduction of the level of GA1 by suppressing GA 20-oxidase expression.

Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.