Gibberellic acid production using different solid-state fermentation systems

Gibberellic acid production in liquid fermentation was compared with production of this compound in solid-state fermentation systems using cassava flour, sugar cane bagasse and low density polyurethane. Gibberella fujikuroi produced 23 mg of gibberellin/ml in 120h of liquid fermentation. Solid-state fermentation on bagasse showed excellent growth but presented gibberellin extraction problems. Very low production and growth was observed in solid-state fermentation with low density polyurethane as an inert support. Solid-state fermentation on cassava flour showed high production (250 mg/kg of dry solid medium) in a very short time (36h).     

Molecular phylogenetics at the population/species interface in cave spiders of the southern Appalachians (Araneae:Nesticidae:Nesticus)

This paper focuses on the relationship between population genetic structure and speciation mechanisms in a monophyletic species group of Appalachian cave spiders (Nesticus). Using mtDNA sequence data gathered from 256 individuals, I analyzed patterns of genetic variation within and between populations for three pairs of closely related sister species. Each sister-pair comparison involves taxa with differing distributional and ecological attributes; if these ecological attributes are reflected in basic demographic differences, then speciation might proceed differently across these sister taxa comparisons. Both frequency-based and gene tree analyses reveal that the genetic structure of the Nesticus species studied is characterized by similar and essentially complete population subdivision, regardless of differences in general ecology. These findings contrast with results of prior genetic studies of cave-dwelling arthropods that have typically revealed variation in population structure corresponding to differences in general ecology. Species fragmentation through both extrinsic and intrinsic evolutionary forces has resulted in discrete, perhaps independent, populations within morphologically defined species. Large sequence divergence values observed between populations suggest that this independence may extend well into the past. These patterns of mtDNA genealogical structure and divergence imply that species as morphological lineages are currently more inclusive than basal evolutionary or phylogenetic units, a suggestion that has important implications for the study of speciation mechanisms.      

Conformational lability of vitronectin: induction of an antigenic change by alpha-thrombin-serpin complexes and by proteolytically modified thrombin

We previously showed that the alpha-thrombin-antithrombin III complex causes antigenic change in vitronectin as monitored by the monoclonal anti-vitronectin antibody 8E6 (Tomasini & Mosher, 1988). We have extended these studies to other protease-serpin complexes and to gamma-thrombin, a proteolytic derivative of alpha-thrombin. In the presence of heparin, recognition of vitronectin by 8E6 was increased 64- or 52-fold by interaction with the complex of alpha-thrombin and heparin cofactor II or the Pittsburgh mutant (Met358----Arg) of alpha 1-protease inhibitor, respectively. This was comparable to the value obtained with the alpha-thrombin-antithrombin III complex. Factor Xa-serpin complexes were approximately 4-fold less effective than the corresponding thrombin complexes. alpha-Thrombin-serpin complexes but not Xa-serpin complexes formed disulfide-bonded complexes with vitronectin. Antigenic changes and disulfide-bonded complexes were not detected when trypsin- or chymotrypsin-serpin complexes were incubated with vitronectin. gamma-Thrombin caused 7- and 34-fold increases in recognition of vitronectin by MaVN 8E6 in the absence and presence of heparin, respectively. In contrast, alpha-thrombin by itself had no effect. The antigenic change induced by gamma-thrombin was maximal when gamma-thrombin and vitronectin were equimolar, was not dependent on cleavage of vitronectin, and was abolished by inhibition of gamma-thrombin with Phe-Pro-Arg-chloromethyl ketone but not with diisopropyl fluorophosphate. These data indicate that alpha-thrombin is the component in alpha-thrombin-serpin complexes that induces the antigenic change in vitronectin, probably via a region that is preferentially exposed in gamma-thrombin.     

Structures of the PKA RIα Holoenzyme with the FLHCC Driver J-PKAcα or Wild-Type PKAcα

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Mendelian randomization supports causality between maternal hyperglycemia and epigenetic regulation of leptin gene in newborns

Leptin is an adipokine that acts in the central nervous system and regulates energy balance. Animal models and human observational studies have suggested that leptin surge in the perinatal period has a critical role in programming long-term risk of obesity. In utero exposure to maternal hyperglycemia has been associated with increased risk of obesity later in life. Epigenetic mechanisms are suspected to be involved in fetal programming of long term metabolic diseases. We investigated whether DNA methylation levels near LEP locus mediate the relation between maternal glycemia and neonatal leptin levels using the 2-step epigenetic Mendelian randomization approach. We used data and samples from up to 485 mother-child dyads from Gen3G, a large prospective population-based cohort. First, we built a genetic risk score to capture maternal glycemia based on 10 known glycemic genetic variants (GRS₁₀) and showed it was an adequate instrumental variable (β = 0.046 mmol/L of maternal fasting glucose per additional risk allele; SE = 0.007; P = 7.8 × 10⁻¹¹; N = 467). A higher GRS₁₀ was associated with lower methylation levels at cg12083122 located near LEP (β = −0.072 unit per additional risk allele; SE = 0.04; P = 0.05; N = 166). Direction and effect size of association between the instrumental variable GRS₁₀ and methylation at cg12083122 were consistent with the negative association we observed using measured maternal glycemia. Lower DNA methylation levels at cg12083122 were associated with higher cord blood leptin levels (β = −0.17 log of cord blood leptin per unit; SE = 0.07; P = 0.01; N = 170). Our study supports that maternal glycemia is part of causal pathways influencing offspring leptin epigenetic regulation.     

β-Peptoids: synthesis of a novel dimer having a fully extended conformation

Chiral imines 1a,b, already synthesized in our laboratory, were converted in good yield by reduction into the corresponding N-benzyl-γ-lactams 2a,b. Desilylation followed by oxidation of the hydroxymethyl functionality gave the N-benzyl-β-amino acids 5a,b in good yield and high purity. Starting from compound 6a, the corresponding β-peptoid dimer 8 was prepared, together with its derivatives 9 and 10, these latter displaying conformational restriction about the peptide bond, as evidenced by NMR data.     

Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks

Background  

Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum).     

Early and late molecular events of glucose-induced pexophagy in Pichia pastoris require Vac8

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like "segmented" extensions that almost completely surrounded the adjacent peroxisomes. Vac8-GFP was found at the vacuolar membrane and concentrated at the base of the arm-like protrusions that extend from the vacuole to sequester the peroxisomes. The localization of Vac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8 resulted in decreased protein stability, impaired vacuolar association and reduced degradation of peroxisomal alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a non-functional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.