Peroxisome proliferator-activated receptor alpha agonists as therapy for autoimmune disease

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. PPAR gamma ligands, which include the naturally occurring PG metabolite 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), as well as thiazolidinediones, have been shown to have anti-inflammatory activity. The PPAR alpha agonists, gemfibrozil, ciprofibrate, and fenofibrate, have an excellent track history as oral agents used to treat hypertriglyceridemia. In the present study, we demonstrate that these PPAR alpha agonists can increase the production of the Th2 cytokine, IL-4, and suppress proliferation by TCR transgenic T cells specific for the myelin basic protein Ac1-11, as well as reduce NO production by microglia. Oral administration of gemfibrozil and fenofibrate inhibited clinical signs of experimental autoimmune encephalomyelitis. More importantly, gemfibrozil was shown to shift the cytokine secretion of human T cell lines by inhibiting IFN-gamma and promoting IL-4 secretion. These results suggest that PPAR alpha agonists such as gemfibrozil and fenofibrate, may be attractive candidates for use in human inflammatory conditions such as multiple sclerosis.     

Effect of dietary copper on selenium toxicity in Fischer 344 rats

The purpose of this study was to investigate the ameliorating effects of dietary copper supplementation on selenium toxicity. Nine groups (n = 6) of weanling Fischer 344 female rats were randomly assigned to treatment groups and fed diets containing nontoxic levels of copper as CuCl2 and/or selenium as selenite or selenocystamine. Weight gain, liver and spleen weights, plasma lipid peroxidation, and liver selenium and copper content were analyzed after the 6-wk treatment period. Concentrations of up to 10 times the daily lethal dose of dietary selenium were well tolerated in rats supplemented with dietary copper. As the dietary level of selenium was increased, the ratio of selenium to copper measured in the liver decreased. In the groups of rats in which dietary copper supplementation was absent and dietary selenium was supplemented, copper stores in the liver remained unchanged from control values. Copper's protective effects from dietary selenium toxicity may come from the formation of a copper-selenide complex that renders both selenium and copper metabolically unavailable and nontoxic.     

Telomere length is reflected by plumage coloration and predicts seasonal reproductive success in the barn swallow

Individuals differ in realized fitness but the genetic/phenotypic traits that underpin such variation are often unknown. Telomere dynamics may be a major source of variation in fitness traits because physiological telomere shortening depends on environmental and genetic factors and may impair individual performance. Here, we showed that, in a population of a socially monogamous, biparental passerine bird, the barn swallow (Hirundo rustica), breeding in northern Italy, telomere length (TL) of both adult males and females positively correlated with seasonal reproductive and fledging success, as expected because long telomeres are supposed to boost performance. Telomere length was correlated with sexually dimorphic coloration in both sexes, showing for the first time in any species that coloration reliably reflects TL and may mediate mutual mate choice, leading to the observed positive assortative mating for TL in the barn swallow. Thus, TL appears to be associated with variation in a major fitness trait and may be an ultimate target of mate choice, as individuals of both sexes can use coloration to adaptively choose high‐quality mates that possess long telomeres.     

Resistance to streptolysin O in mammalian cells treated with oxygenated derivatives of cholesterol cholesterol content of resistant cells and recovery of streptolysin O sensitivity

Cultures of L cells and HeLa cells were made resistant to the cytolytic toxin, streptolysin O, by incubating them in the presence of 20α-hydroxycholesterol or 25-hydroxycholesterol. Such cells were also found to be more resistant to the cytotoxic effects of saponin and digitonin, agents known to interact with membrane cholesterol. Sterol synthesis in L cells that had been treated with either of the oxygenated derivatives of cholesterol was reduced by almost 90%, and the free cholesterol content of streptolysin O-resistant HeLa and L cells fell to approx. 50% of control cell levels. Significant recovery of sensitivity to streptolysin O occurred in about 6 h when refractory L cells were incubated in serum or cholesterol. Partial recovery was observed when the cultures were incubated for 24 h in mevalonate or lipid-depleted serum. The results provide further support for the role of membrane cholesterol in the cytotoxic action of streptolysin O on mammalian cells.     

Mutational Insertion of a ROSA26�CEGFP Transgene Leads to Defects in Spermiogenesis and Male Infertility in Mice

Pronuclear injection has been a successful strategy for generating genetically engineered mouse models to better understand the functionality of genes. A characteristic of pronuclear injection is that random integration of the transgene into the genome can disturb a functional gene and result in a phenotype unrelated to the transgene itself. In this study, we have characterized a mouse model containing an insertional mutation that, in the homozygous state, severely affects spermatogenesis as characterized by lack of sperm motility and acrosomal aplasia. Whereas homozygous female mice had normal fertility, male mice homozygous for the insertional mutation were unable to produce pups by natural mating with either homozygous or wild-type female mice. No fertilized embryos were produced by matings to homozygous male mice, and no sperm were present in the reproductive tract of mated female mice. Spermatozoa isolated from homozygous male mice exhibited head and midpiece defects, but no major defects in the principal piece of these sperm. Histologic examination and immunohistochemical staining of the testes revealed vacuolar degeneration of Sertoli cells and loss of structural seminiferous tubule integrity and organization, indicating that spermatogenesis is severely affected in this mouse model. Although the males are always infertile, the severity of the histologic and sperm morphologic defects appeared to be age-related.Abbreviations: EGFP, enhanced green fluorescent protein; GOPC, Golgi-associated proteinThe production of genetically engineered mice has enabled unprecedented advancements in understanding the functionality of genes. Genetically engineered mice can be produced in many ways, but pronuclear injection has been the standard method for many years.^2,4,7,23 Despite its success, pronuclear injection is associated with several problems. One important problem is that random integration of the transgene into the genome can disturb a functional gene and lead to associated problems. However, as we present here, random insertion of the transgene sometimes can result in interesting and important discoveries about gene function.Numerous reports describe problems in spermatogenesis associated with transgene insertion,^10,16,18,24 and these mice have provided powerful research tools with which to study the complexities of male infertility and the production and maturation of spermatozoa. Here we characterize the male infertility phenotype of the FVB/NTac-Tg(Gt(ROSA)26Sor-EGFP)130910Eps/Mmmh strain¹³ The original mouse strain was donated to the University of Missouri Mutant Mouse Regional Resource Center (http://www.mmrrc.org) and assigned designation MMRRC:000366. The founder animal for this strain was generated by random insertion of a transgene construct containing the enhanced green fluorescent protein (EGFP) under control of the mouse ROSA 26 promoter by pronuclear injection of FVB/NTac embryos. The strain has since been maintained for more than 20 generations by backcrossing to FVB/NTac mice. Efforts to cryopreserve the strain revealed that male mice homozygous for the transgene insertion were infertile, thereby leading to the studies presented here.To determine the precise site of integration of the transgene, a chromosome-walking technique³ was used and revealed a single integration site on chromosome 3 within the intronic region of a novel gene (ENSMUSG00000027939). Based on the predicted protein sequence for this gene, it represents a novel nucleoporin with shared similarity to members of the nuclear pore membrane glycoprotein 210 family. Nucleoporins are protein components of the nuclear pore complex and play a key role in nucleocytoplasmic transport. Importantly in the context of the infertility phenotype described for this mouse model, we speculate that the gene disrupted by the mutational insertion may be involved in nucleocytoplasmic trafficking, which is important for male germ cell differentiation.In this report, we describe the infertility phenotype of the FVB/NTac-Tg(Gt(ROSA)26Sor-EGFP)130910Eps/Mmmh strain (ROSA–EGFP) and document age-related sperm defects and Sertoli cell abnormalities that underlie the male infertility seen in animals homozygous for the insertional mutation. These mice exhibited severe defects in spermatogenesis, as manifested by sperm head and midpiece abnormalities and a loss of sperm motility. In addition, we discuss the results of the histologic examinations, which revealed vacuolar degeneration of Sertoli cells, rare multinucleated cells, and reduced numbers of all stages of germinal cells in the seminiferous tubules as well as occasional vacuolation of the epididymal epithelium. The sperm head defects coupled with the Sertoli cell degeneration suggest possible disruption of normal Sertoli cell–spermatid interactions in homozygotes for this transgene insert. Furthermore matings with homozygous male mice failed to produce any pups, whereas heterozygous and homozygous female mice bred with wild-type male mice had normal litter sizes. Functional analysis of the homozygous male mice may provide additional insight into the molecular mechanisms of spermiogenesis, particularly those involving acrosomal biogenesis and the development of functional flagella.     

Endogenous APOBEC3A DNA Cytosine Deaminase Is Cytoplasmic and Nongenotoxic

APOBEC3A (A3A) is a myeloid lineage-specific DNA cytosine deaminase with a role in innate immunity to foreign DNA. Previous studies have shown that heterologously expressed A3A is genotoxic, suggesting that monocytes may have a mechanism to regulate this enzyme. Indeed, we observed no significant cytotoxicity when interferon was used to induce the expression of endogenous A3A in CD14⁺-enriched primary cells or the monocytic cell line THP-1. In contrast, doxycycline-induced A3A in HEK293 cells caused major cytotoxicity at protein levels lower than those observed when CD14⁺ cells were stimulated with interferon. Immunofluorescent microscopy of interferon-stimulated CD14⁺ and THP-1 cells revealed that endogenous A3A is cytoplasmic, in stark contrast to stably or transiently transfected A3A, which has a cell-wide localization. A3A constructs engineered to be cytoplasmic are also nontoxic in HEK293 cells. These data combine to suggest that monocytic cells use a cytoplasmic retention mechanism to control A3A and avert genotoxicity during innate immune responses.     

116 Deamination of both methyl- and normal-cytosine by the foreign DNA restriction enzyme APOBEC3A

Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methyl-cytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC-to-T activity and 10-fold less C-to-U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for non-chromosomal DNA MeC-to-T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.