Influence of Nematode Parasitism,Body Size,Temperature, and Diel Period on the Flight Capacity of <Emphasis Type="Italic">Sirex noctilio</Emphasis> F. (Hymenoptera: Siricidae)

Sirex noctilio F. (Hymenoptera: Siricidae) is a woodwasp of pine trees that has recently invaded and established in North American forests. Although S. noctilio has had a limited impact in North America to date, there is some concern that it could have a significant impact on pine plantations, especially in the southeastern U.S.A. Moreover, there are few data on the flight capacity of male S. noctilio. We found no association between parasitism by D. siricidicola and whether or not S. noctilio initiated flight on the flight mill. Male wasps that were parasitized by nematodes were heavier than non-parasitized males, but there was no significant difference in mass between parasitized and non-parasitized females. We also examined the flight capacity of male and female S. noctilio in relation to nematode parasitism, body mass, temperature (for only males), and diel period. Body mass, temperature, and diel period affected flight in S. noctilio such that wasps were generally observed to fly faster, farther, and more frequently if they were heavier, flying at warmer temperatures, and flying during the photoperiod. The fact that nematode-parasitized male wasps were found to fly farther than the non-parasitized males is consistent with the hypothesis that nematode parasitism does not negatively affect the flight capacity of S. noctilio.     

Aberrant heteroplasmic transmission of mtDNA in cloned pigs arising from double nuclear transfer

Double nuclear transfer begins with the transfer of nuclear DNA from a donor cell into an enucleated recipient oocyte. This reconstructed oocyte is allowed to develop to the pronuclear stage, where the pronuclei are transferred into an enucleated zygote. This reconstructed zygote is then transferred to a surrogate sow. The genetic integrity of cloned offspring can be compromised by the transmission of mitochondrial DNA from the donor cell, the recipient oocyte and the recipient zygote. We have verified through the use of sequence analysis, restriction fragment length polymorphism analysis, allele specific PCR and primer extension polymorphism analysis that following double nuclear transfer the donor cell mtDNA is eliminated. However, it is likely that the recipient oocyte and zygote mitochondrial DNA are transmitted to the offspring, indicating bimaternal mitochondrial DNA transmission. This pattern of mtDNA inheritance is similar to that observed following cytoplasmic transfer and violates the strict unimaternal inheritance of mitochondrial DNA to offspring. This form of transmission raises concerns regarding the genetic integrity of cloned offspring and their uses in studies that require metabolic analysis or a stable genetic environment where only one variable is under analysis, such as in knockout technology.     

Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.     

Effect of electric field vectoriality on electrically mediated gene delivery in mammalian cells

Electropermeabilization is a nonviral method used to transfer genes into living cells. Up to now, the mechanism is still to be elucidated. Since cell permeabilization, a prerequired for gene transfection, is triggerred by electric field, its characteristics should depend on its vectorial properties. The present investigation addresses the effect of pulse polarity and orientation on membrane permeabilization and gene delivery by electric pulses applied to cultured mammalian cells. This has been directly observed at the single-cell level by using digitized fluorescence microscopy. While cell permeabilization is only slightly affected by reversing the polarity of the electric pulses or by changing the orientation of pulses, transfection level increases are observed. These last effects are due to an increase in the cell membrane area where DNA interacts. Fluorescently labelled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable and is not affected by pulses of reversed polarities. Under such conditions, DNA interacts with the two sites of the cell facing the two electrodes. When changing both the pulse polarity and their direction, DNA interacts with the whole membrane cell surface. This is associated with a huge increase in gene expression. This present study demonstrates the relationship between the DNA/membrane surface interaction and the gene transfer efficiency, and it allows to define the experimental conditions to optimize the yield of transfection of mammalian cells.     

Cranial Morphology of a Pantolestid Eutherian Mammal from the Eocene Bridger Formation,Wyoming, USA: Implications for Relationships and Habitat

Pantolestinae is a eutherian subfamily of mammals whose members are known from the middle early Paleocene through at least the beginning of the Oligocene of North America. They are also known from Europe, and possibly Africa. A lack of information on pantolestine skulls has prevented the use of cranial anatomy in evaluation of this group’s enigmatic higher-level phylogenetic relationships. Conversely, postcranial skeletons are well known and locomotor interpretations based on them are robust. The most complete known skull of a pantolestine, Pantolestes longicaudus (YPM 13525), is described here and compared to potential close fossil relatives and extant mammals. Semicircular canal morphology is used to test locomotor hypotheses. YPM 13525 lacks an ossified bulla. It has a mediolaterally broad basioccipital, a large entoglenoid process, and a deeply incised glaserian fissure of the squamosal, caudal and rostral tympanic processes on the petrosal, a foramen for an internal carotid artery (ICA) that entered the tympanic cavity from a posteromedial position, bony tubes enclosing the main stem and transpromontorial branch of the ICA, a large anterior carotid foramen formed within the basisphenoid, evidence of a stapedial artery ramus superior, a groove on the dorsal aspect of the basisphenoid leading to the piriform fenestra possibly for drainage of the cavernous sinus to an extracranial inferior petrosal sinus, a dorsum sellae with well-developed posterior clinoid processes, a foramen rotundum within the alisphenoid, and a sphenorbital fissure between the alisphenoid and orbitosphenoid. Overall, the morphology is not strikingly similar to any potential close relative and the phylogenetic position of Pantolestinae cannot be estimated without cladistic analysis of a character matrix that includes this new morphology and broadly samples extant and extinct eutherian taxa. Semicircular canal morphology differs from that of two likely terrestrial Paleocene mammals, Aphronorus (another pantolestid) and Eoryctes (a palaeoryctid), suggesting a different, possibly semi-aquatic, lifestyle for Pantolestes.     

Translation initiator EIF4G1 mutations in familial Parkinson disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.     

Quantitative trait loci controlling light and hormone response in two accessions of Arabidopsis thaliana

We have mapped quantitative trait loci (QTL) responsible for natural variation in light and hormone response between the Cape Verde Islands (Cvi) and Landsberg erecta (Ler) accessions of Arabidopsis thaliana using recombinant inbred lines (RILs). Hypocotyl length was measured in four light environments: white, blue, red, and far-red light and in the dark. In addition, white light plus gibberellin (GA) and dark plus the brassinosteroid biosynthesis inhibitor brassinazole (BRZ) were used to detect hormone effects. Twelve QTL were identified that map to loci not previously known to affect light response, as well as loci where candidate genes have been identified from known mutations. Some QTL act in all environments while others show genotype-by-environment interaction. A global threshold was established to identify a significant epistatic interaction between two loci that have few main effects of their own. LIGHT1, a major QTL, has been confirmed in a near isogenic line (NIL) and maps to a new locus with effects in all light environments. The erecta mutation can explain the effect of the HYP2 QTL in the blue, BRZ, and dark environments, but not in far-red. LIGHT2, also confirmed in an NIL, has effects in white and red light and shows interaction with GA. The phenotype and map position of LIGHT2 suggest the photoreceptor PHYB as a candidate gene. Natural variation in light and hormone response thus defines both new genes and known genes that control light response in wild accessions.     

Novel pyrrolidine melanin-concentrating hormone receptor 1 antagonists with reduced hERG inhibition

We discovered novel pyrrolidine MCHR1 antagonist 1 possessing moderate potency. Profiling of pyrrolidine 1 demonstrated that it was an inhibitor of the hERG channel. Investigation of the structure-activity relationship of this class of pyrrolidines allowed us to optimize the MCHR1 potency and decrease the hERG inhibition. Increasing the acidity of the amide proton by converting the benzamide in lead 1 to an anilide provided single digit nanomolar MCHR1 antagonists while replacing the dimethoxyphenyl ring of 1 with alkyl groups possessing increased polarity dramatically reduced the hERG inhibition.     

Extent of intracellular pH changes during H+ extrusion by maize root-tip cells

³¹P-Nuclear-magnetic-resonance spectra of maize (Zea mays L.) root tips, that had been induced to extrude large amounts of H⁺ in response to fusicoccin (FC) in the presence of potassium salts, indicate that the cytoplasmic pH does not become higher than that of controls. In fact, the cytoplasmic pH may become slightly (approx. 0.1 pH unit) lower in cells extruding H⁺. Estimations of the buffer capacity of the cells show that without active intracellular pH regulation, H⁺ extrusion caused by FC would cause the intracellular pH to rise by at least 0.6 pH unit h^-1. Our results indicate that intracellular pH is tightly regulated even during extreme rates of acid extrusion, and that a rise in cytoplasmic pH is not the signal linking H⁺ extrusion with enhanced organic-acid synthesis or other intracellular responses to H⁺ pumping.Abbreviations FC fusicoccin - P_i inorganic phosphate - NMR nuclear magnetic resonance - chemical shift - MDP methylene diphosphonic acid     

Plasmid DNA fermentation strain and process‐specific effects on vector yield,quality, and transgene expression

Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication. We also investigated the impact of differences in epigenetic dcm methylase‐directed cytosine methylation on plasmid production, transgene expression, and immunogenicity. While Escherichia coli plasmid production yield and quality are unaffected, dcm− versions of CMV and CMV‐HTLV‐I R promoter plasmids had increased transgene expression in human cells. Surprisingly, despite improved expression, dcm− plasmid is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e., production strain) early in product development and that dcm− strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial. Biotechnol. Bioeng. 2011;108: 354–363. © 2010 Wiley Periodicals, Inc.