ROLE OF SERUM AND ION CHANNEL BLOCK ON GROWTH AND HORMONALLY‐INDUCED DIFFERENTIATION OF Spodoptera frugiperda (Sf21) INSECT CELLS

A neuronal morphological phenotype can be induced in cultured Spodoptera frugiperda insect cells (Sf21) by supplementing serum‐containing media with 20‐hydroxyecdysone (20‐HE) and/or insulin. In this study, the primary objectives were to determine any role of ion channels in mediating the morphological change in cells treated with 20‐HE and insulin, and whether serum was required to observe this effect. Results showed serum‐free media also induced growth of processes in Sf21 cells, but at a lower percentage than that found previously in cells bathed in serum‐containing media. Veratridine, a sodium channel activator, increased cell survival when applied in combination with 20‐HE to Sf21 cells, and the effect was blocked by tetrodotoxin (1 μM) a known sodium channel blocker. Cobalt, a calcium channel blocker, showed significant inhibition of cell process growth when applied in combination with both 20‐HE and 20‐HE plus veratridine. Cobalt also showed significant inhibition of cell process growth when applied in combination with insulin. Thus, some type of sodium channel, as well as a mechanism for transmembrane calcium ion movement, are apparently expressed in Sf21 cells and are involved in the differentiation process. These cell lines may be used in a wide variety of endeavors, including the screening of insecticides, as well as foster basic studies of neurodevelopment and ecdysone action.     

Species‐specific responses to island connectivity cycles: refined models for testing phylogeographic concordance across a Mediterranean Pleistocene Aggregate Island Complex

The contribution of Pleistocene sea level changes to diversification patterns in archipelagos around the world, and specifically whether the repeated cycles of island connectivity and isolation acted as a ‘species pump’ is debated. The debate has been perpetuated in part because of the type of evidence used to evaluate the species‐pump hypothesis. Specifically, existing tests of the ‘Pleistocene Aggregate Island Complex’ (PAIC) model of diversification interpret the lack of concordant divergence times among multiple codistributed taxa as a rejection of the PAIC model. However, the null expectation of concordance disregards taxon‐specific ecological traits and geographic characteristics that may affect population persistence and gene flow among islands. Here, we study the factors affecting population divergence in thirteen flightless darkling beetle species (Coleoptera: Tenebrionidae) across the PAIC system of the Cycladic plateau in the Aegean archipelago. Based on isolation‐by‐resistance analyses, hierarchical amova and the degree of genealogical sorting on individual islands, we identify a major effect of bathymetry and habitat stability on the levels of genetic divergence across the PAIC, with island size and body size playing a secondary role as well. We subsequently use bathymetric maps and habitat association to generate predictions about the set of islands and group of taxa expected to show phylogeographic concordance. We test these predictions using hierarchical approximate Bayesian computation and show how our interpretations regarding the role of PAICs as drivers of divergence change when relying on a null expectation of concordance compared to a refined model that takes geography and ecological traits into account.     

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H₂ into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H₂ oxidation, H₂ production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H₂ oxidation and H₂ production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H₂ oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H₂ oxidation/production.     

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

Replication pauses of the wild-type and mutant mitochondrial DNA polymerase gamma: a simulation study

The activity of polymerase γ is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase:DNA complex. Pausing of pol-γ might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-γ that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-γ variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-γ (exo(-)) in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo(-) polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase.     

Enzyme kinetics of the mitochondrial deoxyribonucleoside salvage pathway are not sufficient to support rapid mtDNA replication

Using a computational model, we simulated mitochondrial deoxynucleotide metabolism and mitochondrial DNA replication. Our results indicate that the output from the mitochondrial salvage enzymes alone is inadequate to support a mitochondrial DNA replication duration of as long as 10 hours. We find that an external source of deoxyribonucleoside diphosphates or triphosphates (dNTPs), in addition to those supplied by mitochondrial salvage, is essential for the replication of mitochondrial DNA to complete in the experimentally observed duration of approximately 1 to 2 hours. For meeting a relatively fast replication target of 2 hours, almost two-thirds of the dNTP requirements had to be externally supplied as either deoxyribonucleoside di- or triphosphates, at about equal rates for all four dNTPs. Added monophosphates did not suffice. However, for a replication target of 10 hours, mitochondrial salvage was able to provide for most, but not all, of the total substrate requirements. Still, additional dGTPs and dATPs had to be supplied. Our analysis of the enzyme kinetics also revealed that the majority of enzymes of this pathway prefer substrates that are not precursors (canonical deoxyribonucleosides and deoxyribonucleotides) for mitochondrial DNA replication, such as phosphorylated ribonucleotides, instead of the corresponding deoxyribonucleotides. The kinetic constants for reactions between mitochondrial salvage enzymes and deoxyribonucleotide substrates are physiologically unreasonable for achieving efficient catalysis with the expected in situ concentrations of deoxyribonucleotides.     

Species diversity of <Emphasis Type="Italic">Trichoderma</Emphasis> in Poland

In the present study, we reinvestigate the diversity of Trichoderma in Poland utilizing a combination of morphological and molecular/phylogenetic methods. A total of 170 isolates were collected from six different substrata at 49 sites in Poland. These were divided among 14 taxa as follows: 110 of 170 Trichoderma isolates were identified to the species level by the analysis of their ITS1, ITS2 rDNA sequences as: T. harzianum (43 isolates), T. aggressivum (35), T. citrinoviride (11), T. hamatum (9), T. virens (6), T. longibrachiatum (4), T. polysporum (1), and T. tomentosum (1); 60 isolates belonging to the Viride clade were identified based on a fragment of the translation-elongation factor 1-alpha (tef1) gene as: T. atroviride (20 isolates), T. gamsii (2), T. koningii (17), T. viridescens (13), T. viride (7), and T. koningiopsis (1). Identifications were made using the BLAST interface in TrichOKEY and TrichoBLAST (). The most diverse substrata were soil (nine species per 22 isolates) and decaying wood (nine species per 75 isolates). The most abundant species (25%) isolated from all substrata was T. harzianum.     

The case for locus-specific databases

Locus-specific databases are the most useful repositories of the sequence information underlying medical genetic conditions and, for this reason, they need our continued support.     

Genetic diversity in tetraploid switchgrass revealed by AFLP marker polymorphisms

Switchgrass (Panicum virgatum) is a perennial warm-season grass native to North America that has been identified as a dedicated cellulosic biofuel crop. We quantified genetic diversity in tetraploid switchgrass germplasm collected at Oklahoma State University and characterized genetic relatedness among the collections from distinct regions. Fifty-six tetraploid accessions, including seven upland and 49 lowland genotypes from throughout the US, were examined. The amplified fragment length polymorphism (AFLP) procedure was utilized to generate DNA profiling patterns that were scored visually. Sixteen selective AFLP primer combinations were used to amplify 452 polymorphic bands. The accessions' genetic similarity coefficients, UPGMA (unweighted pair-group method with arithmetic averaging) cluster analysis and principle coordinate analysis, were performed. The upland and lowland accessions clustered according to ecotypes, with one exception (TN104). Genetic similarity coefficients among the accessions ranged from 0.73 to 0.95. Analysis of molecular variance (AMOVA) was performed, showing significant differences between the upland and lowland genotypes. The trnL marker confirmed that TN104 was a lowland genotype, but the trnL marker identification of upland and lowland genotypes was not consistent with the AFLP analysis in two germplasms (Miami and AR4).