Specific delivery of AtBT1 to mitochondria complements the aberrant growth and sterility phenotype of homozygous Atbt1 Arabidopsis mutants

It has been shown that homozygous AtBT1::T-DNA Arabidopsis mutants display an aberrant growth and sterility phenotype, and that AtBT1 is a carrier that is exclusively localized to the inner plastidial envelope and is required for export of newly synthesized adenylates into the cytosol. However, a recent demonstration that AtBT1 is localized to both plastids and mitochondria suggested that plastidic AtBT1 is not necessary for normal growth and fertility of Arabidopsis. To test this hypothesis, we produced and characterized homozygous AtBT1::T-DNA mutants stably expressing either dually localized AtBT1 or AtBT1 specifically localized to the mitochondrial compartment. These analyses revealed that the aberrant growth and sterility phenotype of homozygous AtBT1::T-DNA mutants was complemented when expressing both the dual-targeted AtBT1 and AtBT1 specifically delivered to mitochondria. These data confirm that (i) plastidic AtBT1 is not strictly required for normal growth and fertility of the plant, and (ii) specific delivery of AtBT1 to mitochondria is enough to complement the aberrant growth and sterility phenotype of homozygous AtBT1::T-DNA mutants. Furthermore, data presented here question the idea that the requirement for AtBT1 is due to its involvement in transport of newly synthesized adenylates from the plastid to the cytosol, and suggest that the protein may play as yet unidentified functions in plastids and mitochondria.     

Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia

Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2′-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.     

Anatomy of carbonate mounds from the Middle Anisian of Nakhlak (Central Iran): architecture and age of a subtidal microbial-bioclastic carbonate factory

The Anisian succession of Nakhlak (in Central Iran) is characterized by a siliciclastic succession with minor carbonate units, with massive carbonate mounds up to 50?m thick in its upper part. The mounds, constrained in age to the late Bithynian (Ismidicus Zone) by ammonoids and conodonts, are characterized by a flat top and a lateral pinch-out marked by clinostratified slopes (about 15° in dip). Stratigraphic and microfacies analyses document an inner part of the mound characterized by massive microbial carbonates with open-space structures (stromatactis) filled with fine-grained internal sediments and marine cements. Isolated sponges (up to 5?cm), serpulids and bryozoans are present, which grew on the calcimicrobial limestone. A narrow bioclastic margin (mainly with crinoids and brachiopods) produces most of the slope facies (consisting of bioclastic grainstone and packstone, with intraclasts from the inner part of the mounds) which interfinger basinward with volcaniclastic sandstones. The demise of carbonate productivity is marked on the top of the carbonate mounds by a condensed surface, rich in ammonoids, glaucony grains, and articulated crinoids, documenting a rapid drowning. Paleolatitude data support deposition in a tropical setting, and sedimentological constraints indicate deposition close to the fair-weather wave base, within the photic zone. The late Bithynian Nakhlak carbonate mounds developed before the appearance (documented since the Pelsonian in different parts of the world) of scleractinians which, despite the favorable environmental conditions, are absent at Nakhlak. The Nakhlak mounds thus represent one of the last occurrences of the microbial factories (which developed after the Permo-Triassic extinction event and persisted for most of the Middle Triassic, but with a gradually increasing role played by scleractinians) before the first appearance of the Mesozoic corals.     

A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

Synthesized by glycogen synthase and starch synthases (SS) using ADP-glucose as the sugar donor molecule, glycogen and starch accumulate as predominant storage carbohydrates in most bacteria and plants, respectively. We have recently shown that the so-called “starch-less” Arabidopsis thaliana adg1–1 and aps1 mutants impaired in ADP-glucose pyrophosphorylase do indeed accumulate low starch content in normal growth conditions, and relatively high starch content when plants were cultured in the presence of microbial volatiles. Our results were strongly supported by data obtained using a highly sensitive method for confocal fluorescence microscopic visualization of iodine stained starch granules. Using Arabidopsis leaves from WT plants, aps1 plants, ss3/ss4 plants lacking both class III and class IV SS, gbss plants lacking the granule-bound SS, and sus1/sus2/sus3/sus4 plants lacking four genes that code for proteins with sucrose synthase activity, in this work we precisely describe the method for preparation of plant samples for starch microscopic examination. Furthermore, we show that this method can be used to visualize glycogen in bacteria, and pure starch granules, amylose and amylopectin.     

Occurrence of more than one important source of ADPglucose linked to glycogen biosynthesis in Escherichia coli and Salmonella

To explore the possible occurrence of sources, other than GlgC, of ADPglucose linked to bacterial glycogen biosynthesis we characterized Escherichia coli and Salmonella DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery. These mutants displayed the expected glycogen-less phenotype but accumulated ADPglucose. Importantly, DeltaglgCAP cells expressing the glycogen synthase encoding glgA gene accumulated glycogen. Protein chromatographic separation of crude extracts of DeltaglgCAP mutants and subsequent activity measurement analyses revealed that these cells possess various proteins catalyzing the conversion of glucose-1-phosphate into ADPglucose. Collectively these findings show that enterobacteria possess more than one important source of ADPglucose linked to glycogen biosynthesis.     

Aortic Intima-Media Thickness and Aortic Diameter in Small for Gestational Age and Growth Restricted Fetuses

ObjectiveThe objective of this study is to measure aortic intima-media thickness (aIMT) and aortic diameter (AD) in appropriate for gestational age (AGA) fetuses, small for gestational age (SGA) fetuses, and intrauterine growth restricted (IUGR) fetuses.MethodsCase-control study performed between June 2011 and June 2012. Forty-nine AGA fetuses, 40 SGA fetuses, and 35 IUGR fetuses underwent concomitant measurement of aIMT and AD at a mean gestational age of 34.4 weeks.ResultsMedian aIMT was higher in fetuses with IUGR (0.504 mm [95%CI: 0.477-0.530 mm]), than in SGA fetuses (0.466 mm [95% CI: 0.447–0.485 mm]), and AGA fetuses (0.471 mm [95% CI: 0.454-0.488 mm]) (p = 0.023). Mean AD was significantly lower in fetuses with IUGR (4.451 mm [95% CI: 4.258–4.655 mm]), than in AGA fetuses (4.74 mm [95% CI: 4.63-4.843 mm]) (p = 0.028).ConclusionsGrowth restricted fetuses have a thicker aortic wall than AGA and SGA fetuses, which possibly represents preclinical atherosclerosis and a predisposition to later cardiovascular disease.     

Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus

Although Leishmania metacyclic promastigotes are generally considered resistant to human complement, studies of in vitro-cultured axenic stationary promastigotes using serum concentrations that approximate physiological plasma conditions indicate complement sensitivity. Natural Leishmania infection is caused by sand fly-inoculated promastigotes, whose complement resistance has not been analyzed systematically. We compared Leishmania susceptibility to human complement in L. infantum promastigotes derived from in vitro cultures and from sand flies. Phlebotomus perniciosus sand flies were fed with axenic promastigotes, L. infantum-infected U-937 cells, or spleen cells from L. infantum-infected hamsters. On selected days post-feeding, flies were dissected and promastigotes isolated; in addition, axenic promastigotes were obtained from culture at equivalent days of growth. In near-physiological serum concentration and temperature conditions, measurement of real-time kinetics of propidium iodide uptake showed that approximately 90% of axenic- and sand fly-derived promastigotes were rapidly killed by complement. We found no substantial differences between promastigotes from axenic culture, those isolated from flies on different post-feeding days, or those generated in flies fed with distinct inocula. The results indicate that Leishmania susceptibility to human complement is independent of promastigote developmental stage in the sand fly mid-gut and in axenic culture.     

Ganglioside GM(3) is stably associated to tyrosine-phosphorylated ErbB2/EGFR receptor complexes and EGFR monomers, but not to ErbB2

Gangliosides are known to modulate the activation of receptor tyrosine-kinases (RTKs). Recently, we demonstrated the functional relationship between ErbB2 and ganglioside GM(3) in HC11 epithelial cell line. In the present study we investigated, in the same cells, the ErbB2 activation state and its tendency to form stable molecular complexes with the epidermal growth factor receptor (EGFR) and with ganglioside GM(3) upon EGF stimulation. Results from co-immunoprecipitation experiments and western blot analyses indicate that tyrosine-phosphorylated ErbB2 and EGFR monomers and stable ErbB2/EGFR high molecular complexes (heterodimers) are formed following EGF stimulation, even if the receptors co-immunoprecipitates also in the absence of the ligand; these data suggest the existence of pre-dimerization inactive receptor clusters on the cell surface. High performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of the ganglioside fractions extracted from the immunoprecipitates demonstrate that GM(3), but not other gangliosides, is tightly associated to the tyrosine-phosphorylated receptors. Furthermore, we show that GM(3) is preferentially and in a SDS-resistant manner associated to the activated ErbB2/EGFR complexes and EGFR monomer, but not to ErbB2. Altogether our data support the hypothesis that the modulating effects produced by GM(3) on ErbB2 activation are mediated by EGFR.