Insertional editing of mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes

tRNAs encoded on the mitochondrial DNA of Physarum polycephalum and Didymium nigripes require insertional editing for their maturation. Editing consists of the specific insertion of a single cytidine or uridine relative to the mitochondrial DNA sequence encoding the tRNA. Editing sites are at 14 different locations in nine tRNAs. Cytidine insertion sites can be located in any of the four stems of the tRNA cloverleaf and usually create a G. C base pair. Uridine insertions have been identified in the T loop of tRNALys from Didymium and tRNAGlu from Physarum. In both tRNAs, the insertion creates the GUUC sequence, which is converted to GTPsiC (Psi = pseudouridine) in most tRNAs. This type of tRNA editing is different from other, previously described types of tRNA editing and resembles the mRNA and rRNA editing in Physarum and Didymium. Analogous tRNAs in Physarum and Didymium have editing sites at different locations, indicating that editing sites have been lost, gained, or both since the divergence of Physarum and Didymium. Although cDNAs derived from single tRNAs are generally fully edited, cDNAs derived from unprocessed polycistronic tRNA precursors often lack some of the editing site insertions. This enrichment of partially edited sequences in unprocessed tRNAs may indicate that editing is required for tRNA processing or at least that RNA editing occurs as an early event in tRNA synthesis.     

Assays for investigating RNA editing in plant mitochondria

Dendritic cells (DCs) pulsed with unfractionated tumor cell lysates or defined tumor peptides provide potent vaccines which elicit strong antitumor immunity. In this study, we generated DCs from the 2-h adherent progenitor cells obtained from the peripheral blood of melanoma patients. These DCs were able to capture biotinylated melanoma tumor cell lysates. We examined the efficacy of immunogens composed of DCs loaded either with the melanoma peptide gp100 [amino acids 280-288 (DC/gp100)] or with lysates from melanoma tumor cells (DC/lysates) in inducing cytotoxic T-cells from autologous PBLs of HLA-A2 melanoma patients. After four to five weekly stimulations of bulk PBLs with DC/gp100 or DC/lysates, the cultures were enriched with CD3+ T-cells and exhibited one of three phenotypic and functional patterns: (1) Predominant expression of CD8+ and MHC class I-restricted CTLs which displayed strong lytic activity against melanoma cells and T2 cells loaded with the gp100 peptide, (2) mixed CD4+/CD8+ phenotype and weak lytic activity, or (3) nonlytic and predominantly CD4+ cultures. Interestingly, T-cell cultures from each patient exhibited similar phenotypes and lytic activities whether the stimulant was DC/gp100 or DC/cell lysates. Our study demonstrates that DCs pulsed with soluble melanoma peptides or cell lysates are capable of inducing CD8+ CTLs from autologous PBLs of some, but not all, melanoma patients. The function and phenotype of the generated T-cell cultures are governed by DCs since both antigens (the gp100 peptide and melanoma lysates), when presented by a given DC preparation, induced similar T-cell cultures. In summary, it may be difficult to predict the nature of the cellular responses elicited by DC/tumor antigen vaccines from patient to patient.     

Accurate and efficient insertional RNA editing in isolated Physarum mitochondria

RNA editing is a process whereby nucleotide insertion, deletion, or base substitution results in the production of an RNA whose sequence differs from that of its template. The mitochondrial RNAs of Physarum polycephalum are processed specifically at multiple sites by both mono- and dinucleotide insertions, as well as apparent cytidine (C) to uridine (U) changes. The precise mechanism and timing of these processing events are currently unknown. We describe here the development of an isolated mitochondrial system in which exogenously supplied nucleotides can be incorporated into RNAs under defined conditions. The results of S1 nuclease protection, nearest neighbor and RNase T1 fingerprint analyses indicate that the vast majority of these newly synthesized mitochondrial RNAs have been accurately and efficiently processed by both mono- and dinucleotide insertions. This work provides a direct demonstration of faithful nucleotide insertion in a mitochondrial editing system. In contrast, the newly synthesized RNAs are not processed by C to U changes in the isolated mitochondria, suggesting that the base changes observed in Physarum are unlikely to occur via a deletion/insertion mechanism.     

Cis- and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria

In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22 kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNA(Tyr) is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation.     

Processing differences between simple and choice reactions affect bottleneck localization in overlapping tasks.

Recent models disagree on bottleneck localization in overlapping tasks in which participants make 2 responses to 2 stimuli presented with varying stimulus onset asynchrony (SOA). Key evidence for a peripheral bottleneck is that response selection difficulty and SOA underadditively affect reaction times in Task 2 if response selection difficulty is varied by increasing the number of alternatives from 1 to 2. Three experiments examined an alternative explanation that suggests that differences in response preparation and stimulus anticipation between 1 and 2 alternative conditions are responsible for this effect. A manipulation of preparational state and number of anticipatory reactions directly affected the likelihood of obtaining the underadditive interaction. In the absence of advance response preparation and stimulus anticipation, an additive interaction was found, providing key evidence for a central bottleneck in overlapping-task performance. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Intranuclear mitochondria in human myocardial cells

Dentin phosphoproteins are thought to have a primary role in the deposition of mineral on the collagen of dentin. In this study we determined the type of binding between collagen and phosphoproteins necessary for mineral formation onto collagen fibrils and whether the phosphate esters are required. Bovine dentin phosphophoryn or phosvitin from egg yolk were immobilized on reconstituted skin type I collagen fibrils by adsorption or by covalent cross-linking. In some samples the ester phosphate was removed from the covalently cross-linked phosphoproteins by treatment with acid phosphatase. All samples were incubated at 37 degrees C in metastable solutions that do not spontaneously precipitate. Reconstituted collagen fibrils alone did not induce mineral formation. The phosphoproteins adsorbed to the collagen fibrils desorbed when the mineralization medium was added, and mineral was not induced. The mineral induced by the cross-linked phosphoproteins was apatite, and the crystals were confined to the surface of the collagen fibrils. With decreasing medium saturation the time required for mineral induction increased. The interfacial tensions calculated for apatite formation by either phosphoprotein cross-linked to collagen were about the same as that for phosphatidic acid liposomes and hydroxyapatite. This similarity in values indicates that the nucleation potential of these highly phosphorylated surfaces is about the same. It is concluded that phosphoproteins must be irreversibly bound to collagen fibrils for the mineralization of the collagen network in solutions that do not spontaneously precipitate. The phosphate esters of phosphoproteins are required for mineral induction, and the carboxylate groups are not sufficient.     

Processing and editing of kinetic data of substances for the data and program base system Kindas

On the basis of a Dbase system in the programming languages Fortran and Clipper, the data processing system Kindat has been created. With the aid of this system, approx. 120,000 kinetic data of substances have been compiled, approximated and annotated. These data are available to the metallurgical industry both in tabular form or as approximation formulae. Furthermore, the user can apply the Kindat system directly to solve metallurgical problems, or to process and manage own data.     

ATP-dependent proteolysis in mitochondria. m-AAA protease and PIM1 protease exert overlapping substrate specificities and cooperate with the mtHsp70 system

To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at non-permissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.     

Differential partitioning of tRNAs between micellar and aqueous phases: a convenient gel filtration method for separation of tRNAs

A case of Blue Rubber Bleb Nevus in a 11 year-old boy is reported. The patient exhibited characteristic hemangiomas of the skin and gastrointestinal tract with an iron deficiency anemia. Historical, clinical, pathological, and surgical features of the condition are discussed. Value of fibroscopic examination in delineation of the cause of persistent gastrointestinal bleeding is pointed out.     

In vivo assessment of human skeletal muscle mitochondria respiration in health and disease

One of many problems to be faced when assessing in vivo human muscle mitochondria respiration by phosphorus magnetic resonance spectroscopy (31P-MRS) is the definition of the correct reference population and the values of reference range. To take into account most factors that influence muscle activity as age, sex, physical activity; nutritional state etc., an exceedingly high number of different reference groups are needed. To overcome this problem we developed specific tests to assess separately in vivo the activity and the functionality of muscle mitochondria by 31P-MRS in clinical settings. By activity we refer to muscle whole metabolic activity, i.e. the total oxidative capacity of muscle mitochondria which is influenced by many factors (age, sex, physical activity, nutritional state etc.). By functionality we refer to the qualitative aspects of mitochondrial respiration which depends on the integrity of mitochondrial multienzyme systems and on substrate availability. Our tests have been experienced on some 1200 patients and are currently used to detect deficits of mitochondrial respiration and ion transport in patients with suspected primary or secondary muscle mitochondrial malfunctioning.     

Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy

It has been shown that adoptive immunotherapy can be curative for established malignant tumors. The key to this treatment lies in obtaining sufficient numbers of lymphocytes which are sensitized to recognize tumor antigens and carry out immunological reactions to destroy tumor cells. Reported here are the results of experiments to: 1) sensitize lymphocytes to the antigens of rat glioma cells and expand them ex vivo for use in adoptive immunotherapy, 2) characterize the cells of the expanded population, and 3) evaluate antitumor activity in a cohort of rats with well-established intracranial gliomas. Viable RT-2 glioma cells were injected into the hind foot pads of syngeneic Fischer 344 rats. After 10 days, the tumor draining lymph nodes (DLN) were harvested from the injected limbs and mechanically dissociated. The cells of the DLN were then suspended in culture medium supplemented with low dose interleukin-2 (IL-2) and incubated for 18 hours with Bryostatin-1 and ionomycin (Bryo/Io) to stimulate expansion. The cells were next washed to remove the Bryo/Io and resuspended in culture medium and IL-2. Population expansions of 40- to 100-fold were seen after 8 days. Flow cytometric analysis showed these cells to be a nearly pure population of T lymphocytes of the CD3+CD8+ phenotype. Intravenous injection of the ex vivo expanded DLN cells did not significantly improve survival of rats with a seven-day intracerebral RT-2 glioma, although, compared to untreated controls, the tumors of the treated animals were smaller, showed no necrosis, and appeared to be less infiltrative. Furthermore, the treated animals had a pronounced lymphocytic infiltration of their tumors with greater associated degrees of hemorrhagic change and peritumoral edema. When the ex vivo expanded DLN cells were intravenously injected into three-day intracerebral RT-2 glioma models, tumors were almost always eliminated and the animals survived their tumor challenge. We conclude that successful expansion of glioma-sensitized DLN lymphocytes is possible and that adoptive immunotherapy using these cells is capable of effectively limiting the progression of large gliomas, while totally eradicating small ones.     

Regulation of progesterone biosynthesis in human placental mitochondria by Krebs cycle metabolites

1. 2-Oxoglutarate, succinate, fumarate, malate and citrate, cis-aconitate and isocitrate stimulate conversion of cholesterol to progesterone in human placental mitochondria. 2. The stimulatory effect of dicarboxylic and tricarboxylic acids depends on the activity of malate dehydrogenase (decarboxylating) (NADP+) (EC 1.1.1.40) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42), respectively.     

Processing of adenosine receptor agonists in rat and human whole blood

A stability study of adenosine receptor agonists in rat and human whole blood was performed. The compounds were incubated at 37 degrees in fresh blood, and aliquots of the incubation mixture were hemolyzed at regular time intervals and analyzed with HPLC. N6-cyclopentyladenosine (CPA) and N6-cyclobutyladenosine (CBA) were degraded, whereas N6-cyclohexyladenosine, N6-cycloheptyladenosine and N6-sulfophenyladenosine were not. 2-Chloroadenosine had a half-life very similar to that of CPA. However, the 2'-, 3'-, and 5'-deoxyribose derivatives of CPA remained intact. The nucleoside transport inhibitor nitrobenzylthioinosine attenuated CBA and CPA metabolism in rat blood as did the inhibitor of adenosine deaminase erythro-9-(2-hydroxy-3-nonyl)adenine, albeit at relatively high concentrations. Complete blockade of CBA and CPA degradation was achieved by a preincubation of rat and human blood with the adenosine kinase (AK) inhibitor 5'-amino-5'-deoxyadenosine. We conclude that the two adenosine analogues are metabolized by AK both in rat and in human whole blood.     

Amiodarone affects membrane water permeability properties of human erythrocytes and rat mitochondria

Dose-dependent water exchange times and intracellular water contents were measured by NMR (nuclear magnetic resonance) in erythrocytes and mitochondria interacted with the anti-anginal and anti-arrhytmic agent, amiodarone. Addition of the drug up to 26 microM yielded 80% enhancement of the water exchange rate in erythrocytes at 37 degrees C and 41% enhancement at 22 degrees C with 40% and 9%, respectively, increases in the intracellular water content. Similar enhancements were obtained in mitochondria at 22 degrees C. The data suggests a somewhat higher affinity of amiodarone to mitochondrial than to erythrocyte membranes.     

Proofreading and aminoacylation of tRNAs before export from the nucleus

After synthesis and processing in the nucleus, mature transfer RNAs (tRNAs) are exported to the cytoplasm in a Ran.guanosine triphosphate-dependent manner. Export of defective or immature tRNAs is avoided by monitoring both structure and function of tRNAs in the nucleus, and only tRNAs with mature 5' and 3' ends are exported. All tRNAs examined can be aminoacylated in nuclei of Xenopus oocytes, thereby providing a possible mechanism for functional proofreading of newly made tRNAs. Inhibition of aminoacylation of a specific tRNA retards its appearance in the cytoplasm, indicating that nuclear aminoacylation promotes efficient export.     

The role of exportin-t in selective nuclear export of mature tRNAs

Exportin-t (Xpo-t) is a vertebrate nuclear export receptor for tRNAs that binds tRNA cooperatively with GTP-loaded Ran. Xpo-t antibodies are shown to efficiently block tRNA export from Xenopus oocyte nuclei suggesting that it is responsible for at least the majority of tRNA export in these cells. We examine the mechanism by which Xpo-t-RanGTP specifically exports mature tRNAs rather than other forms of nuclear RNA, including tRNA precursors. Chemical and enzymatic footprinting together with phosphate modification interference reveals an extensive interaction between the backbone of the TPsiC and acceptor arms of tRNAPhe and Xpo-t-RanGTP. Analysis of mutant or precursor tRNA forms demonstrates that, aside from these recognition elements, accurate 5' and 3' end-processing of tRNA affects Xpo-t-RanGTP interaction and nuclear export, while aminoacylation is not essential. Intron-containing, end-processed, pre-tRNAs can be bound by Xpo-t-RanGTP and are rapidly exported from the nucleus if Xpo-t is present in excess. These results suggest that at least two mechanisms are involved in discrimination of pre-tRNAs and mature tRNAs prior to nuclear export.     

Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire

In the bone marrow, diversity in the primary antibody repertoire is created by the combinatorial rearrangement of different gene segments and by the association of different heavy and light chains. During the secondary response in the germinal centres, antibodies are diversified by somatic mutation and possibly by further rearrangements, or "receptor editing". Here, we have analysed the pairings of heavy and light chain variable domains (VH and VL) in 365 human IgG+ B cells from peripheral blood, and established that these pairings are largely random. The repertoire is dominated by a limited number of pairings of segments and folds. Among these pairings we identified two identical mutated heavy chains in combination with two different mutated light chains (one kappa and one lambda). This shows that receptor editing occurs in the human periphery and that the same antibody lineage can be subjected to both receptor editing and somatic hypermutation. This suggests that receptor editing may be used together with somatic mutation for the affinity maturation of antibodies. We also propose that receptor editing has shaped variable gene segment use and the evolution of V gene families.