Characterization of the microheterogeneity of transthyretin in plasma and urine using SELDI-TOF-MS immunoassay

Background  

It has been shown that transthyretin (TTR) exists in different molecular variants. Besides point mutations associated with different diseases such as amyloidosis, other posttranslational modifications occur that might be of diagnostic interest.     

Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation

In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome.     

Pollinator attractiveness increases with distance from flowering orchids

Orchids are extraordinary among plants because many species are pollinated through sexual duplicity by producing flowers that mimic female insects to lure unsuspecting males. Previous work showed that sexual deception by the orchid Chiloglottis trapeziformis can have a negative impact on its wasp pollinator Neozeleboria cryptoides. We report that female wasps may be capable of mitigating the cost of the orchids' deception. Although male wasps quickly habituated to areas planted with unrewarding flower decoys, we found that the effectiveness of the chemical cue used by the wingless females to attract males increases with increasing distance from an orchid patch. The apparent specificity of the males' site-based avoidance strategy means that females emerging in areas occupied by flowering orchids could, potentially, leave the orchid colony by walking to increase their attractiveness.     

Increased luminal pH in the epididymis of infertile c-ros knockout mice and the expression of sodium-hydrogen exchangers and vacuolar proton pump H+-ATPase

Transgenic mice targeted for the c-ros gene, which are fertile when heterozygous (HET), but infertile when homozygous (knockout, KO) and associated with failure in pubertal differentiation of the epididymal initial segment, provide a model for studying the role of the epididymal luminal environment in sperm development. Luminal fluid from the cauda epididymidis was measured by both ion-selective microelectrodes and pH strips to be 0.3 pH units higher in the KO than HET. Of the genes responsible for luminal acidification, expression of mRNA of vacuolar H(+)-ATPase was found in all epididymal regions, but with no difference between KO and HET. Immunohistochemistry showed its presence in epithelial apical cells and clear cells. The Na(+)-hydrogen exchanger NHE2 was expressed at mRNA and protein levels in the caput but only marginally detectable if at all in the distal epididymis. This was compensated for by NHE3 which was expressed strongest in the cauda region, in agreement with immunohistochemical staining. Quantification of Western blot data revealed slight, but significant, decreases of NHE2 in the caput and of NHE3 in the cauda in the KO mice. The increase in luminal fluid pH in the KO mice could also be contributed to by other epithelial regulating factors including the Na(+)-dependent glutamate transporter EAAC1 formerly reported to be down regulated in the KO.     

Transfection of adherent and suspended cells by calcium phosphate

DNA-calcium phosphate coprecipitates have been used for 30 years as an efficient method to introduce genetic material into cells. The method involves simple solutions that can be prepared or purchased by the experimentalist. All the numerous variations of the protocol found in the literature are based on the same principle--a spontaneous precipitation that occurs in supersaturated solutions. When DNA is present during this process, it is readily incorporated into the forming calcium phosphate precipitate. Although a wide range of conditions will lead to precipitates, high transfection efficiencies are only obtained within a narrow range of optimized parameters that assure certain properties of the precipitate. This paper describes several physico-chemical parameters that are critical to adapt the method to a particular cell line and/or cultivation condition. Examples of protocols that were established and tested within the authors' laboratory are presented. The article also emphasizes differences between transfections of adherent and suspended cells.     

Identification of an elite sorghum genotype with high <Emphasis Type="Italic">In vitro</Emphasis> performance capacity

Summary The use of plant genetic engineering to augment plant breeding programs is significantly strengthened if novel trait(s) can be introduced directly into elite germplasm. Implementing this technology to sorghum breeding programs has been hampered by the lack of an efficient and transferable protocol that is suitable with elite genotypes. This study was conducted to identify parameters that maximize in vitro culture performance in sorghum targeting a specific elite genotype, C2-97, which possesses enhanced agronomic characteristics. Three different tissue culture media formulations, MS, N6, and M11 were evaluated. M11 medium contains approximately 16% and 85% more total nitrogen and sevenfold and threefold higher levels of potassium phosphate than MS and N6 formulations, respectively. Culture performance of C2-97 across the three media formulations was compared to sorghum genotypes that were previously reported to be amenable to genetic engineering, namely Tx430, P898012. Bwheatland, and C401. Maximum embryogenesis induction was observed on M11 medium for all genotypes tested, with greater than 70% embryogenic calluses occurring on immature embryos derived from the C2-97 genotype cultured on M11 medium.     

Increased platelet-activating factor-induced periventricular brain microvascular constriction associated with immaturity

Oxidant stress contributes to the pathogenesis of hypoxic-ischemic encephalopathies. Platelet-activating factor (PAF) is generated during oxidant stress. We studied the vasomotor mode of actions of PAF on periventricular (PV) microvessels of fetal ( approximately 75% of term), newborn (1-3 days), and adult pigs. PAF constricted PV microvessels from fetal (29.27 +/- 2.6%) and newborn (22.14 +/- 3.2%) pigs but was ineffective in adults (<2.5%). Specific [(3)H]PAF binding was greater in fetus and newborn than in adults; a concordant developmental PAF-induced inositol phosphate formation was observed. PAF-induced vasoconstriction was abrogated by thromboxane A(2) (TXA(2)) synthase and receptor inhibitors, calcium channel blockers, and by removal of endothelium; vasoconstriction to TXA(2) mimetic U-46619 did not differ with age. Immunoreactive TXA(2) synthase expression and PAF-evoked TXA(2) formation revealed a fetus> newborn>adult profile. Thus the greater PAF-induced PV microvascular constriction in younger subjects seems attributable to greater PAF receptor density and mostly secondary to TXA(2) formation from endothelium. The resulting decrease in blood flow may contribute to the increased vulnerability of the PV brain regions to oxidant stress-induced injury in immature subjects.     

Plasma distribution of apoA-IV in patients with coronary artery disease and healthy controls

Recent studies showed lower apolipoprotein A-IV (apoA-IV) plasma concentrations in patients with coronary artery disease (CAD). The actual distribution of the antiatherogenic apoA-IV in human plasma, however, is discussed controversially and it was never investigated in CAD patients. We therefore developed a gentle technique to separate the various apoA-IV-containing plasma fractions. Using a combination of precipitation of all lipoproteins with 40% phosphotungstic acid and 4 M MgCl2, as well as immunoprecipitation of all apoA-I-containing particles with an anti-apoA-I antibody, we obtained three fractions of apoA-IV: lipid-free apoA-IV (about 4% of total apoA-IV), apoA-IV associated with apoA-I (LpA-I:A-IV, 12%), and apoA-I-unbound but lipoprotein-containing apoA-IV (LpA-IV, 84%). We compared these three apoA-IV fractions between 52 patients with a history of CAD and 52 age- and sex-matched healthy controls. Patients had significantly lower apoA-IV levels when compared to controls (10.28 +/- 3.67 mg/dl vs. 11.85 +/- 2.82 mg/dl, P = 0.029), but no major differences for the three plasma apoA-IV fractions. We conclude that our gentle separation method reveals a different distribution of apoA-IV than in many earlier studies. No major differences exist in the apoA-IV plasma distribution pattern between CAD patients and controls. Therefore, the antiatherogenic effect of apoA-IV has to be explained by other functional properties of apoA-IV (e.g., the antioxidative characteristics).     

Identification and molecular characterization of the RNA polymerase-binding motif of infectious bursal disease virus inner capsid protein VP3

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most important infectious poultry diseases. Major aspects of the molecular biology of IBDV, such as assembly and replication, are as yet poorly understood. We have previously shown that encapsidation of the putative virus-encoded RNA-dependent RNA polymerase VP1 is mediated by its interaction with the inner capsid protein VP3. Here, we report the characterization of the VP1-VP3 interaction. RNase A treatment of VP1- and VP3-containing extracts does not affect the formation of VP1-VP3 complexes, indicating that formation of the complex requires the establishment of protein-protein interactions. The use of a set of VP3 deletion mutants allowed the mapping of the VP1 binding motif of VP3 within a highly charged 16-amino-acid stretch on the C terminus of VP3. This region of VP3 is sufficient to confer VP1 binding activity when fused to an unrelated protein. Furthermore, a peptide corresponding to the VP1 binding region of VP3 specifically inhibits the formation of VP1-VP3 complexes. The presence of Trojan peptides containing the VP1 binding motif in IBDV-infected cells specifically reduces infective virus production, thus showing that formation of VP1-VP3 complexes plays a critical role in IBDV replication.     

Identification and characterization of a nuclear interacting partner of anaplastic lymphoma kinase (NIPA)

Anaplastic large-cell lymphoma is a subtype of non-Hodgkin lymphomas characterized by the expression of CD30. More than half of these lymphomas carry a chromosomal translocation t(2;5) leading to expression of the oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). NPM-ALK is capable of transforming fibroblasts and lymphocytes in vitro and of causing lymphomas in mice. Previously, we and others demonstrated phospholipase C-gamma and phosphatidylinositol 3-kinase as crucial downstream signaling mediators of NPM-ALK-induced oncogenicity. In this study, we used an ALK fusion protein as bait in a yeast two-hybrid screen identifying NIPA (nuclear interacting partner of ALK) as a novel downstream target of NPM-ALK. NIPA encodes a 60-kDa protein that is expressed in a broad range of human tissues and contains a classical nuclear translocation signal in its C terminus, which directs its nuclear localization. NIPA interacts with NPM-ALK and other ALK fusions in a tyrosine kinase-dependent manner and is phosphorylated in NPM-ALK-expressing cells on tyrosine and serine residues with serine 354 as a major phosphorylation site. Overexpression of NIPA in Ba/F3 cells was able to protect from apoptosis induced by IL-3 withdrawal. Mutations of the nuclear translocation signal or the Ser-354 phosphorylation site impaired the antiapoptotic function of NIPA. In NPM-ALK-transformed Ba/F3 cells, apoptosis triggered by wortmannin treatment was enhanced by overexpression of putative dominant-negative NIPA mutants. These results implicate an antiapoptotic role for NIPA in NPM-ALK-mediated signaling events.