Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Haemophilus influenzae type b strain A2 has multiple sialyltransferases involved in lipooligosaccharide sialylation

The lipooligosaccharide (LOS) of Haemophilus influenzae contains sialylated glycoforms, and a sialyltransferase, Lic3A, has been previously identified. We report evidence for two additional sialyltransferases, SiaA, and LsgB, that affect N-acetyllactosamine containing glycoforms. Mutations in genes we have designated siaA and lsgB affected only the sialylated glycoforms containing N-acetylhexosamine. A mutation in siaA resulted in the loss of glycoforms terminating in sialyl-N-acetylhexosamine and the appearance of higher molecular weight glycoforms, containing the addition of phosphoethanolamine, N-acetylgalactosamine, and N-acetylneuraminic acid. Chromosomal complementation of the siaA mutant resulted in the expression of the original sialylated LOS phenotype. A mutation in lic3A resulted in the loss of sialylation only in glycoforms lacking N-acetylhexosamine and had no effect on sialylation of the terminal N-acetyllactosamine epitope. A double mutant in siaA and lic3A resulted in the complete loss of sialylation of the terminal N-acetyllactosamine epitope and expression of the higher molecular weight sialylated glycoforms seen in the siaA mutant. Mutation of lsgB resulted in persistence of sialylated glycoforms but a reduction in N-acetyllactosamine containing glycoforms. A triple mutant of siaA, lic3A, and lsgB contained no sialylated glycoforms. These results demonstrate that the sialylation of the LOS of H. influenzae is a complex process involving multiple sialyltransferases.     

RNA binding properties of the AU-rich element-binding recombinant Nup475/TIS11/tristetraprolin protein

Regulation of messenger RNA stability by AU-rich elements is an important means of regulating genes induced by growth factors and cytokines. Nup475 (also known as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding Cys(3)His motif proteins required for proper regulation of tumor necrosis factor mRNA stability in macrophages. We developed an Escherichia coli expression system to produce soluble Nup475 protein in quantity to study its RNA binding properties. Nup475 protein bound a tumor necrosis factor AU-rich element over a broad range of pH and salt concentrations by RNA gel shift. This binding was inhibited by excess zinc metal, providing a potential mechanism for previous reports of zinc stabilization of AU-rich element (ARE) containing messenger RNAs. Immobilized Nup475 protein was used to select its optimal binding site by RNA SELEX and revealed a strong preference for the extended sequence UUAUUUAUU, rather than a simple AUUUA motif. These findings were confirmed by site-directed mutagenesis of the tumor necrosis factor ARE and RNA gel shifts on c-fos, interferon-gamma, and interferon-beta ARE fragments. A weaker binding activity toward adenine-rich sites, such as a poly(A) tail RNA fragment, can partially disrupt the Nup475-tumor necrosis factor AU-rich element complex.     

Lessons from genetically engineered animal models XI. Novel mouse models to study pathogenic mechanisms of Crohn's disease

Crohn's Disease (CD) affects more than 500,000 individuals in the United States and represents the second most common chronic inflammatory disorder after rheumatoid arthritis. Although major advances have been made in defining the basic mechanisms underlying chronic intestinal inflammation, the precise etiopathogenesis of CD remains unknown. We have recently characterized two novel mouse models of enteritis that express a CD-like phenotype, namely the TNF DeltaARE model of tumor necrosis factor (TNF) overexpression and the SAMP1/Yit model of spontaneous ileitis. The unique feature of these models is that they closely resemble CD for location and histopathology. These genetically manipulated new models of intestinal inflammation offer a powerful tool to investigate potential causes of human disease and may allow the development of novel disease-modifying therapeutic modalities for the treatment of CD.     

Norepinephrine mediates glucoprivic-induced increase in GABA in the ventromedial hypothalamus of rats

Noradrenergic mechanisms in the hypothalamus may be involved in counterregulatory responses to glucoprivic episodes. After 2-deoxy-D-glucose (2-DG; 1.2 mmol/kg iv), extracellular norepinephrine (NE) concentration in the ventromedial hypothalamus (VMN) increased in a bimodal fashion to 251 +/- 39% (P < 0.001) and 150 +/- 17% (P < 0.001) of baseline during the first 30 min. In the lateral hypothalamus (LHA), NE decreased by 30 min (61 +/- 4%, P < 0.001) and no consistent changes were measured in the paraventricular nucleus (PVN). Because the NE response in the VMN after 2-DG followed the same pattern as GABA, the interaction between NE and GABA was evaluated. In the VMN, GABA had little effect on extracellular NE concentrations but NE increased GABA concentrations 166 +/- 13%, (P < 0.01). In the presence of yohimbine (alpha(2)-adrenoceptor antagonist) the first GABA peak after 2-DG was absent, and the second GABA peak was absent in the presence of timolol (beta-adrenoceptor antagonist). These results support an interaction among noradrenergic and GABAergic systems in the VMN during glucoprivation and that increased NE mediates the increase in extracellular GABA after 2-DG.     

Commensal bacteria exacerbate intestinal inflammation but are not essential for the development of murine ileitis

The pathogenesis of Crohn's disease has been associated with a dysregulated response of the mucosal immune system against intraluminal Ags of bacterial origin. In this study, we have investigated the effects of germfree (GF) conditions in the SAMP1/YitFc murine model of Crohn's disease-like ileitis. We show that the bacterial flora is not essential for ileitis induction, because GF SAMP1/YitFc mice develop chronic ileitis. However, compared with disease in specific pathogen-free (SPF) mice, ileitis in GF mice is significantly attenuated, and is associated with delayed lymphocytic infiltration and defective mucosal expression of Th2 cytokines. In addition, we demonstrate that stimulation with purified fecal Ags from SPF, but not GF mice leads to the generation of IL-4-secreting effector lymphocytes. This result suggests that commensal bacteria drive Th2 responses characteristic of the chronic phase of SAMP1/YitFc ileitis. Finally, adoptive transfer of CD4-positive cells from GF, but not SPF mice induces severe colitis in SCID recipients. These effects were associated with a decreased frequency of CD4(+)CD25(+)Foxp3(+) T cells in the mesenteric lymph nodes of GF mice compared with SPF mice, as well as lower relative gene expression of Foxp3 in CD4(+)CD25(+) T cells in GF mice. It is therefore apparent that, in the absence of live intraluminal bacteria, the regulatory component of the mucosal immune system is compromised. All together, our results indicate that in SAMP1/YitFc mice, bacterial flora exacerbates intestinal inflammation, but is not essential for the generation of the chronic ileitis that is characteristic of these mice.