Chondrocyte protein with a poly-proline region (CHPPR) is a novel mitochondrial protein and promotes mitochondrial fission

We have recently identified a chondrocyte protein with a poly-proline region, referred to as CHPPR, and showed that this protein is expressed intracellularly in chick embryo chondrocytes. Conventional fluorescence and confocal localization of CHPPR shows that CHPPR is sorted to mitochondria. Furthermore, immunoelectron microscopy of CHPPR transfected cells demonstrates that this protein is mostly associated with the mitochondrial inner membranes. Careful analysis of CHPPR expressing cells reveals, instead of the regular mitochondrial tubular network, the presence of a number of small spheroid mitochondria. Here we show that the domain responsible for network-spheroid transition spans amino acid residues 182-309 including the poly-proline region. Functional analyses of mitochondrial activity rule out the possibility of mitochondrial damage in CHPPR transfected cells. Since cartilage expresses high levels of CHPPR mRNA when compared to other tissues and because CHPPR is associated with late stages of chondrocyte differentiation, we have investigated mitochondrial morphology in hypertrophic chondrocytes by MitoTracker Orange labeling. Confocal microscopy shows that these cells have spheroid mitochondria. Our data demonstrate that CHPPR is able to promote mitochondrial fission with a sequence specific mechanism suggesting that this event may be relevant to late stage of chondrocyte differentiation.     

Plasmalogens in rat liver chromatin: new molecules involved in cell proliferation

A minor component of chromatin, the phospholipid fraction, changes during cell cycle as result of the activation of intranuclear lipid metabolism enzymes including phosphatidylcholine-dependent phospholipase C activity. It is known that this enzyme may be activated by phosphatidylcholine plasmalogen (Plg). Until now, there has been little evidences for the presence of Plgs inside the nucleus. The aim of our study is to ascertain if they are present in the nucleus and are responsible of the activation of phosphatidylcholine-dependent phospholipase C during cell proliferation and apoptosis. Therefore, we have analysed the Plg composition of the whole homogenate, cytosol, nuclei and chromatin of hepatocytes. The phosphatidylcholine-dependent phospholipase C activity was assayed using both phosphatidylcholine and plasmalogenyl-phosphatidylcholine as substrates. Our results show, for the first time, that Plgs are present in chromatin and the plasmalogenyl-phosphatidylcholine stimulates the phosphatidylcholine-dependent phospholipase C activity more than phosphatidylcholine. Finally, in order to verify the possible role of these molecules during cell proliferation and apoptosis, we used liver of rats fed with ciprofibrate which stimulates hepatocytes proliferation during the treatment and, after withdrawal, apoptosis. After 3 days of ciprofibrate treatment, the chromatin plasmalogenyl-phosphatidylcholine increases as well as the phosphatidylcholine-dependent phospholipase C activity. After drug withdrawal, when the hepatocytes undergo to apoptosis, the plasmalogenyl-phosphatidylcholine content together with phosphatidylcholine-dependent phospholipase C activity decreases. Therefore, it can be concluded that plamalogens are present in the chromatin, and probably may have a function both in regulating phosphatidylcholine dependent phospholipase C and cell cycle.     

Vaccine-stimulated, adoptively transferred CD8+ T cells traffic indiscriminately and ubiquitously while mediating specific tumor destruction

It has been suggested that antitumor T cells specifically traffic to the tumor site, where they effect tumor destruction. To test whether tumor-reactive CD8(+) T cells specifically home to tumor, we assessed the trafficking of gp100-specific pmel-1 cells to large, vascularized tumors that express or do not express the target Ag. Activation of tumor-specific CD8(+) pmel-1 T cells with IL-2 and vaccination with an altered peptide ligand caused regression of gp100-positive tumors (B16), but not gp100-negative tumors (methylcholanthrene 205), implanted on opposing flanks of the same mouse. Surprisingly, we found approximately equal and very large numbers of pmel-1 T cells (>25% of all lymphocytes) infiltrating both Ag-positive and Ag-negative tumors. We also found evidence of massive infiltration and proliferation of activated antitumor pmel-1 cells in a variety of peripheral tissues, including lymph nodes, liver, spleen, and lungs, but not peripheral blood. Most importantly, evidence for T cell function, as measured by production of IFN-gamma, release of perforin, and activation of caspase-3 in target cells, was confined to Ag-expressing tumor. We thus conclude that CD8(+) T cell-mediated destruction of tumor is the result of specific T cell triggering at the tumor site. The ability to induce ubiquitous homing and specific tumor destruction may be important in the case of noninflammatory metastatic tumor foci.     

Paenibacillus larvae larvae spores in honey samples from Uruguay: a nationwide survey

American foulbrood is a severe bacterial disease affecting larvae of the honeybee Apis mellifera and it is caused by Paenibacillus larvae larvae. The disease is present worldwide and cases have been reported in almost all the beekeeping regions of the five continents. During 2001 and 2002 we carried out a nationwide study to assess the presence and amount of P. l. larvae spores in honey samples from Uruguay, combining classic bacteriological, and molecular approaches. The distribution of P. l. larvae spores in honey of the whole country showed a clear pattern and may provide useful data for a control and prevention strategy of American foulbrood.     

Storage of human plasma samples leads to alterations in the lipoprotein distribution of apoC-III and apoE

The effect of frozen storage on lipoprotein distribution of apolipoprotein C-III (apoC-III) and apoE was investigated by measuring apoC-III and apoE by ELISA in HDL and apoB-containing lipoproteins of human plasma samples (n = 16) before and after 2 weeks of frozen storage (-20 degrees C). HDLs were separated by heparin-manganese precipitation (HMP) or by fast-protein liquid chromatography (FPLC). Total plasma apoC-III and apoE levels were not affected by frozen storage. HDL-HMP apoC-III and apoE levels were significantly higher in frozen versus fresh samples: 7.7 +/- 0.7 versus 6.7 +/- 0.7 mg/dl (P < 0.05) and 2.0 +/- 0.1 versus 1.2 +/- 0.1 mg/dl (P < 0.001), respectively. HDL-FPLC apoC-III and apoE, but not triglyceride (TG) or cholesterol, levels were also higher in frozen samples: 12.0 +/- 1.2 versus 7.5 +/- 0.6 mg/dl (P < 0.001) and 2.7 +/- 0.2 versus 1.6 +/- 0.2 mg/dl (P < 0.001), respectively. Frozen storage led to a decrease in apoC-III (-17 +/- 9%) and apoE (-19 +/- 9%) in triglyceride-rich lipoprotein. Redistribution of apoC-III and apoE was most evident in samples with high TG levels. HDL apoC-III and apoE levels were also significantly higher when measured in plasma stored at -80 degrees C. Our results demonstrate that lipoprotein distribution of apoC-III and apoE is affected by storage of human plasma, suggesting that analysis of frozen plasma should be avoided in studies relating lipoprotein levels of apoC-III and/or apoE to the incidence of coronary artery disease.     

Characterization and quantification of triple helix formation in chromosomal DNA

DNA-binding molecules that recognize specific sequences offer a high potential for the understanding of chromatin structure and associated biological processes in addition to their therapeutic potential, e.g. as positioning agents for validated anticancer drugs. A prerequisite for the development of DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the human genome. We have further developed a capture assay to assess triplex-forming oligonucleotide (TFO) binding efficiency quantitatively. This assay is based on bifunctional, psoralen and biotin-conjugated, TFOs and real-time PCR analysis. We have applied this novel quantification method to address two issues that are relevant for DNA-binding molecules. First, we have compared directly the extent of TFO-binding in three experimental settings with increasing similarity to the situation in vivo, i.e. naked genomic DNA, isolated cell nuclei, or whole cells. This comparison allows us to characterize factors that influence genomic triplex formation, e.g. chromosomal DNA organization or intracellular milieu. In isolated nuclei, the binding was threefold lower compared to naked DNA, consistent with a decreased target accessibility int he nucleosomal environment. Binding was detected in whole cells, indicating that the TFO enters the nucleus and binds to its target in intact cells in vivo, but the efficiency was decreased (tenfold) compared to nuclei. Secondly, we applied the method to characterize the binding properties of two different TFOs targeting the same sequence. We found that an antiparallel-binding GT-containing TFO bound more efficiently, but with less target sequence selectivity compared to a parallel-binding CU-containing TFO. Collectively, a sensitive method to characterize genomic triplex formation was described. This may be useful for the determination of factors driving TFO binding efficiency and, thus, may improve the usefulness of triplex-mediated gene targeting for studies of chromatin structure as well as for therapeutic antigene strategies.     

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.     

Species composition of assemblages of Ceratomyxa (Myxozoa), parasites of lings Genypterus (Ophidiidae) in the southeastern Pacific Ocean: an ecomorphometric approach

Studies on Myxozoa have emphasized their potential impact on aquatic animal health but less on ecological aspects. In this investigation, we assess the importance of host and latitudinal variations in the morphometry of spores of Ceratomyxa Théohan, 1892 (Myxozoa) from the gall bladder of 3 ling Genypterus (Pisces; Ophidiidae) species. Discriminant analyses indicated that several morphospecies of Ceratomyxa coexist in these hosts, despite the fact that the specific level of taxonomic resolution of each spore was not attempted. At least 4 species, i.e., Ceratomyxa hokarari Meglitsch, 1960; Ceratomyxa inversa Meglitsch, 1960; Ceratomyxa laxa Meglitsch, 1960; and Ceratomyxa elongata Meglitsch, 1960 occur in the study area, and at least 2 species co-occur in each host and geographical location. The most widespread pattern found is that spores are larger in golden ling (Genypterus blacodes) and spore size decreases with increasing latitude; this may be associated with the species composition of these assemblages, as well as with the habitat and diet segregation of the host species.     

Preextinction viral RNA can interfere with infectivity

When the error rate during the copying of genetic material exceeds a threshold value, the genetic information cannot be maintained. This concept is the basis of a new antiviral strategy termed lethal mutagenesis or virus entry into error catastrophe. Critical for its success is preventing survival of residual infectious virus or virus mutants that escape the transition into error catastrophe. Here we document that mutated, preextinction foot-and-mouth disease virus (FMDV) RNA can interfere with and delay viral production up to 30 h when cotransfected in BHK-21 cells with standard RNA. Interference depended on the physical integrity of preextinction RNA and was not observed with unrelated RNAs or with nonmutated, defective FMDV RNA. These results suggest that this type of interference requires large size, preextinction FMDV RNA and is mediated neither by small interfering RNAs nor by RNAs that can compete with infectious RNA for host cell factors. A model based on the aberrant expression of mutated RNA as it is expected to occur in the initial stages of the transition into error catastrophe is proposed. Interference mediated by preextinction RNA indicates an advantage of mutagenesis versus inhibition in preventing the survival of virus escape mutants during antiviral treatments.