Paul W. Denny

The NOD Idd9 Genetic Interval Influences the Pathogenicity of Insulitis and Contains Molecular Variants of Cd30, Tnfr2, and Cd137

Previous analyses of NOD mice have shown that some genes control the development of both insulitis and diabetes, while other loci influence diabetes without reducing insulitis. Evidence for the existence of a gene only influencing diabetes, Idd9 on mouse chromosome 4, is provided here by the development of a novel congenic mouse strain, NOD.B10 Idd9. NOD.B10 Idd9 mice display profound resistance to diabetes even though nearly all develop insulitis. Subcongenic analysis has demonstrated that alleles of at least three B10 genes, Idd9.1, Idd9.2, and Idd9.3 are required to produce Idd9-mediated diabetes resistance. Candidate genes with amino acid differences between the NOD and B10 strains have been localized to the 5.6 cM Idd9.2 interval (Tnfr2, Cd30) and to the 2.0 cM Idd9.3 interval (Cd137).

The gene mutated in bare patches and striated mice encodes a novel 3β-hydroxysteroid dehydrogenase

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa ) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.

Mapping of the IDDM locus Idd3 to a 0.35-cM interval containing the interleukin-2 gene.

Currently, 16 loci that contribute to the development of IDDM in the NOD mouse have been mapped by linkage analysis. To fine map these loci, we used congenic mapping. Using this approach, we localized the Idd3 locus to a 0.35-cM interval on chromosome 3 containing the Il2 gene. Segregation analysis of the known variations within this interval indicated that only one variant, a serine-to-proline substitution at position 6 of the mature interleukin-2 (IL-2) protein, consistently segregates with IDDM in crosses between NOD and a series of nondiabetic mouse strains. These data, taken together with the immunomodulatory role of IL-2, provide circumstantial evidence in support of the hypothesis that Idd3 is an allelic variation of the Il2 gene, or a variant in strong linkage disequilibrium.

Localization of two insulin-dependent diabetes (Idd) genes to the Idd10 region on mouse Chromosome 3

Multiple genes control the development of autoimmune diabetes both in humans and in the nonobese diabetic (NOD) strain of mouse. Previously, three insulin-dependent diabetes (Idd) genes, Idd3, Idd10, and Idd17, were localized to mouse Chromosome (Chr) 3. The B10-or B6-derived resistance alleles at Idd10 and Idd3 together provide the NOD mouse with nearly complete protection from diabetes. In the present study, the 10.2-cM region encoding Idd10 was defined further with newly developed congenic strains. A locus, located in the centromeric 2.1 cM of the 10.2 cM region, contributed to the Idd10 trait. However, this locus did not account for the full effect of Idd10, suggesting the presence of a second gene in the distal portion of the 10.2-cM region. This second gene is designated as Idd18 and is localized to a 5.1-cM region. The resolution of the originally defined Idd3 locus into at least four separate loci, Idd3, Idd10, Idd17, and Idd18, illustrates the complex poly genic nature of diabetes.

Ether Phospholipids and Glycosylinositolphospholipids Are Not Required for Amastigote Virulence or for Inhibition of Macrophage Activation by Leishmania major

Ether phospholipids are major components of the membranes of humans and Leishmania. In protozoan parasites they occur separately or as part of the glycosylphosphatidylinositol (GPI) anchor of molecules implicated in virulence, such as lipophosphoglycan (LPG), smaller glycosylinositolphospholipids (GIPLs), and GPI-anchored proteins. We generated null mutants of the Leishmania major alkyldihydroxyacetonephosphate synthase (ADS), the first committed step of ether lipid synthesis. Enzymatic analysis and comprehensive mass spectrometric analysis showed that ads1- knock-outs lacked all ether phospholipids, including plasmalogens, LPG, and GIPLs. Leishmania ads1- thus represents the first ether lipid-synthesizing eukaryote for which a completely null mutant could be obtained. Remarkably ads1- grew well and maintained lipid rafts (detergent-resistant membranes). In virulence tests it closely resembled LPG-deficient L. major, including sensitivity to complement and an inability to survive the initial phase of macrophage infection. Likewise it retained the ability to inhibit host cell signaling and to form infectious amastigotes from the few parasites surviving the establishment defect. These findings counter current proposals that GIPLs are required for amastigote survival in the mammalian host or that parasite lyso-alkyl or alkylacyl-GPI anchors are solely responsible for inhibition of macrophage activation.

Thiostrepton binds to malarial plastid rRNA

Binding of the thiazolyl peptide antibiotic thiostrepton to the GTPase domain of 23S rRNA involves a few crucial nucleotides, notably A1067 (E. coli). Small RNA transcripts were prepared corresponding to the GTPase domain of the plastid 23S rRNA and the two forms of cytosolic 28S rRNAs found in the human malaria parasite Plasmodium falciparum, as well as the plastid form of rRNA of the AIDS‐related pathogen Toxoplasma gondii. Binding affinities of the wild type and mutated RNA sequences were as predicted; the malarial plastid sequence had by far the highest affinity, whereas that from toxoplasma did not bind thiostrepton.

GPI-anchored proteins and glycoconjugates segregate into lipid rafts in Kinetoplastida.

The plasma membranes of the divergent eukaryotic parasites, Leishmania and Trypanosoma, are highly specialised, with a thick coat of glycoconjugates and glycoproteins playing a central role in virulence. Unusually, the majority of these surface macro‐molecules are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. In mammalian cells and yeast, many GPI‐anchored molecules associate with sphingolipid and cholesterol‐rich detergent‐resistant membranes, known as lipid rafts. Here we show that GPI‐anchored parasite macro‐molecules (but not the dual acylated Leishmania surface protein (hydrophilic acylated surface protein) or a subset of the GPI‐anchored glycoinositol phospholipid glycolipids) are enriched in a sphingolipid/sterol‐rich fraction resistant to cold detergent extraction. This observation is consistent with the presence of functional lipid rafts in these ancient, highly polarised organisms.

Sphingolipid-free Leishmania are defective in membrane trafficking, differentiation and infectivity.

Sphingolipids are structural components of the eukaryotic plasma membrane that are involved, together with cholesterol, in the formation of lipid microdomains (rafts). Additionally, sphingolipid metabolites have been shown to modulate a wide variety of cellular events, including differentiation and apoptosis. To investigate the role of de novo sphingolipid biosynthesis in Leishmania, we have focused on serine palmitoyltransferase (SPT), which catalyses the first, rate‐limiting step in the synthetic pathway. Genetic ablation of one SPT subunit, LmLCB2, yields viable null parasites that can no longer synthesize ceramide and sphingolipids de novo. Unexpectedly, LmLCB2 expression (and sphingolipid biosynthesis) is stage regulated in Leishmania, being undetectable in intramacrophage parasites. As expected from this observation, the LmLCB2 null mutants maintain infectivity in vivo. However, they are compromised in their ability to form infective extracellular parasites, correlating with a defect in association of the virulence factor, leishmanolysin or GP63, with lipid rafts during exocytosis and an observed relocalization of a second virulence factor, lipophosphogycan, during differentiation. Thus, de novo sphingolipid biosynthesis is critical for membrane trafficking events in extracellular Leishmania but has at best a minor role in intracellular pathogenesis.

Mechanistic basis of infertility of mouse intersubspecific hybrids

Significance Hybrid sterility contributes to speciation by restricting gene flow between related taxa. Although four hybrid sterility genes have been identified in Drosophila and mouse so far, the underlying molecular mechanisms are largely unknown. We describe extensive asynapsis of chromosomes in male and female meiosis of F1 hybrids between two closely related mouse subspecies. Using the intersubspecific chromosome-substitution strains, we demonstrate that the heterospecific pairing of homologous chromosomes is a preexisting condition of asynapsis and may represent a universal mechanism of pachytene arrest in interspecific hybrids. Sex-specific manifestation of asynapsis can explain the mechanism of Haldane’s rule. According to the Dobzhansky–Muller model, hybrid sterility is a consequence of the independent evolution of related taxa resulting in incompatible genomic interactions of their hybrids. The model implies that the incompatibilities evolve randomly, unless a particular gene or nongenic sequence diverges much faster than the rest of the genome. Here we propose that asynapsis of heterospecific chromosomes in meiotic prophase provides a recurrently evolving trigger for the meiotic arrest of interspecific F1 hybrids. We observed extensive asynapsis of chromosomes and disturbance of the sex body in >95% of pachynemas of Mus m. musculus × Mus m. domesticus sterile F1 males. Asynapsis was not preceded by a failure of double-strand break induction, and the rate of meiotic crossing over was not affected in synapsed chromosomes. DNA double-strand break repair was delayed or failed in unsynapsed autosomes, and misexpression of chromosome X and chromosome Y genes was detected in single pachynemas and by genome-wide expression profiling. Oocytes of F1 hybrid females showed the same kind of synaptic problems but with the incidence reduced to half. Most of the oocytes with pachytene asynapsis were eliminated before birth. We propose the heterospecific pairing of homologous chromosomes as a preexisting condition of asynapsis in interspecific hybrids. The asynapsis may represent a universal mechanistic basis of F1 hybrid sterility manifested by pachytene arrest. It is tempting to speculate that a fast-evolving subset of the noncoding genomic sequence important for chromosome pairing and synapsis may be the culprit.

The protozoan inositol phosphorylceramide synthase: a novel drug target that defines a new class of sphingolipid synthase.

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilize phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalyzed by IPC synthase. Because of the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome data bases of these species. By utilizing bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species, strongly indicating that IPC synthase activity is reconstituted. Furthermore, the gene product can be specifically inhibited by an anti-fungal-targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme that defines a new class of protozoan sphingolipid synthase. The identification and characterization of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side affects.