Molecular, biological, and in vivo characterization of the guinea pig cytomegalovirus (CMV) homologs of the human CMV matrix proteins pp71 (UL82) and pp65 (UL83)

We recently identified the genes encoding the guinea pig cytomegalovirus (GPCMV) homologs of the upper and lower matrix proteins of human CMV, pp71 (UL82) and pp65 (UL83), which we designated GP82 and GP83, respectively. Transient-expression studies with a GP82 plasmid demonstrated that the encoded protein targets the nucleus and that the infectivity and plaquing efficiency of cotransfected GPCMV viral DNA was enhanced by GP82. The transactivation function of GP82 was not limited to GPCMV, but was also observed for a heterologous virus, herpes simplex virus type 1 (HSV-1). This was confirmed by its ability to complement the growth of an HSV-1 VP16 transactivation-defective mutant virus in an HSV viral DNA cotransfection assay. Study of a GP82 "knockout" virus (and its attendant rescuant), generated on a GPCMV bacterial artificial chromosome construct, confirmed the essential nature of the gene. Conventional homologous recombination was used to generate a GP83 mutant to examine the role of GP83 in the viral life cycle. Comparison of the one-step growth kinetics of the GP83 mutant (vAM409) and wild-type GPCMV indicated that GP83 protein is not required for viral replication in tissue culture. The role of GP83 in vivo was examined by comparing the pathogenesis of wild-type GPCMV, vAM409, and a control virus, vAM403, in guinea pigs. The vAM409 mutant was significantly attenuated for dissemination in immunocompromised strain 2 guinea pigs, suggesting that the GP83 protein is essential for full pathogenicity in vivo.     

Identification of blottin: a novel gastric trefoil factor family-2 binding protein

The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.     

Chlamydia heat shock protein 60 induces trophoblast apoptosis through TLR4

Intrauterine infection affects placental development and function, and subsequently may lead to complications such as preterm delivery, intrauterine growth retardation, and preeclampsia; however, the molecular mechanisms are not clearly known. TLRs mediate innate immune responses in placenta, and recently, TLR2-induced trophoblast apoptosis has been suggested to play a role in infection-induced preterm delivery. Chlamydia trachomatis is the etiological agent of the most prevalent sexually transmitted bacterial infection in the United States. In this study, we show that in vitro chlamydial heat shock protein 60 induces apoptosis in primary human trophoblasts, placental fibroblasts, and the JEG3 trophoblast cell line, and that TLR4 mediates this event. We observed a host cell type-dependent apoptotic response. In primary placental fibroblasts, chlamydial heat shock protein 60-induced apoptosis was caspase dependent, whereas in JEG3 trophoblast cell lines it was caspase independent. These data suggest that TLR4 stimulation induces apoptosis in placenta, and this could provide a novel mechanism of pathogenesis for poor fertility and pregnancy outcome in women with persistent chlamydia infection.     

DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.     

RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage

Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or poleta only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm.     

Cupiennius salei and Achaearanea tepidariorum: Spider models for investigating evolution and development

The spiders Cupiennius salei and Achaearanea tepidariorum are firmly established laboratory models that have already contributed greatly to answering evolutionary developmental questions. Here we appraise why these animals are such useful models from phylogeny, natural history and embryogenesis to the tools available for their manipulation. We then review recent studies of axis formation, segmentation, appendage development and neurogenesis in these spiders and how this has contributed to understanding the evolution of these processes. Furthermore, we discuss the potential of comparisons of silk production between Cupiennius and Achaearanea to investigate the origins and diversification of this evolutionary innovation. We suggest that further comparisons between these two spiders and other chelicerates will prove useful for understanding the evolution of development in metazoans.     

CD36 N-terminal cytoplasmic domain is not required for the internalization of oxidized low-density lipoprotein

The uptake of OxLDLs (oxidized low density lipoproteins) by CD36-expressing macrophages in the arterial intima and the subsequent 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. The present study has addressed the function of the CD36 N-terminal cytoplasmic domain in the binding and internalization of OxLDL. A selection of CD36 N-terminal cytoplasmic domain mutants were generated and stably expressed in HEK-293 (human embryonic kidney) cells. The capacity of three mutants [CD36_C3/7-A (CD36-C3A/C7A), CD36_D4/R5-A (CD36-D4A/R5A) and CD36_nCPD(-) (CD36 lacking the N-terminal cytoplasmic domain)] to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluorimetry. Each of the CD36 constructs was expressed at differing levels at the cell surface, as measured by flow cytometry and Western blotting. Following incubation with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-OxLDL, cells bearing the CD36_wt (wild-type CD36), CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD(-) constructs all internalized DiI-OxLDL into endosomal structures, whereas empty-vector-transfected cells failed to do so, indicating that, unlike the C-terminal cytoplasmic domain, the N-terminal cytoplasmic domain is not essential for the endocytosis of OxLDL. In conclusion, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 N-terminal cytoplasmic domain. However, the N-terminal cytoplasmic domain may conceivably be implicated in the maturation of CD36.     

Inhibition of DNA replication fork progression and mutagenic potential of 1, N6-ethenoadenine and 8-oxoguanine in human cell extracts

Comparative mutagenesis of 1,N⁶-ethenoadenine (εA) and 8-oxoguanine (8-oxoG), two endogenous DNA lesions that are also formed by exogenous DNA damaging agents, have been evaluated in HeLa and xeroderma pigmentosum variant (XPV) cell extracts. Two-dimensional gel electrophoresis of the duplex M13mp2SV vector containing these lesions established that there was significant inhibition of replication fork movement past εA, whereas 8-oxoG caused only minor stalling of fork progression. In extracts of HeLa cells, εA was weakly mutagenic inducing all three base substitutions in approximately equal frequency, whereas 8-oxoG was 10-fold more mutagenic inducing primarily G→T transversions. These data suggest that 8-oxoG is a miscoding lesion that presents a minimal, if any, block to DNA replication in human cells. We hypothesized that bypass of εA proceeded principally by an error-free mechanism in which the undamaged strand was used as a template, since this lesion strongly blocked fork progression. To examine this, we determined the sequence of replication products derived from templates in which a G was placed across from the εA. Consistent with our hypothesis, 93% of the progeny were derived from replication of the undamaged strand. When translesion synthesis occurred, εA→T mutations increased 3-fold in products derived from the mismatched εA: G construct compared with those derived from the εA: T construct. More efficient repair of εA in the εA: T construct may have been responsible for lower mutation frequency. Primer extension studies with purified pol η have shown that this polymerase is highly error-prone when bypassing εA. To examine if pol η is the primary mutagenic translesion polymerase in human cells, we determined the lesion bypass characteristics of extracts derived from XPV cells, which lack this polymerase. The εA: T construct induced εA→G and εA→C mutant frequencies that were approximately the same as those observed using the HeLa extracts. However, εA→T events were increased 5-fold relative to HeLa extracts. These data support a model in which pol η-mediated translesion synthesis past this adduct is error-free in the context of semiconservative replication in the presence of fidelity factors such as PCNA.     

Intraguild predation by the generalist predator Dicyphus hesperus on the parasitoid Encarsia formosa

Predation was measured in the laboratory by Dicyphus hesperus females on patches of greenhouse whitefly (Trialeurodes vaporariorum) pupae. The patches contained different proportions of unparasitized pupae and those parastisized as nymphs by Encarsia formosa. Dicyphus hesperus fed readily on parasitized pupae and the number of parasitized pupae consumed increased with the proportion available indicating a capacity for intraguild predation. However, D. hesperus females did not display a preference for feeding on parasitized pupae versus unparasitized pupae. Dicyphus hesperus adults were released at three densities into greenhouse enclosures containing tomato plants harbouring greenhouse whitefly and immature E. formosa. Numbers of killed and parasitized whitefly nymphs and pupae and the total number of healthy whitefly were compared among Dicyphus density treatments. On upper leaves, the amount of predation on whitefly nymphs was higher at the highest predator density than at the lower density or in enclosures where D. hesperus was not released. On lower leaves, the number of parasitized pupae was lowest with the highest predator density indicating a negative effect of intraguild predation by D. hesperus on E. formosa. Despite this, Dicyphus density did not affect the density of whitefly in the presence of E. formosa. The results are discussed as they relate to concurrent use of D. hesperus and E. formosa for regulation of greenhouse whitefly populations.