Yarrowia lipolytica lipase production enhanced by increased air pressure

Aims: To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures. Methods and Results: Batch cultures of the yeast were conducted in a pressurized bioreactor at 4 and 8 bar of air pressure and the cellular behaviour was compared with cultures at atmospheric pressure. No inhibition of cellular growth was observed by the increase of pressure. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization enhanced the extracellular lipase activity from 96·6 U l^?1 at 1 bar to 533·5 U l^?1 at 8 bar. The extracellular protease activity was reduced by the air pressure increase, thereby eliciting further lipase productivity. Cell morphology was slightly affected by pressure, particularly at 8 bar, where cells kept the predominant oval form but decreased in size. Conclusions: OTR improvement by total air pressure rise up to 8 bar in a bioreactor can be applied to the enhancement of lipase production by Y. lipolytica. Significance and Impact of the Study: Hyperbaric bioreactors can be successfully applied for yeast cells cultivation, particularly in high‐density cultures used for enzymes production, preventing oxygen limitation and consequently increasing overall productivity.     

Mutations in methylenetetrahydrofolate reductase and in cysthationine beta synthase: is there a link to homocysteine levels in peripheral arterial disease?

Peripheral arterial disease (PAD) is an atherosclerotic disturbance characterized by a progressive obstruction of lower limb arteries. Many risk factors associated with PAD development have being reported in the literature. The present study aimed to investigate whether mutations in the methylenetetrahydrofolate reductase (MTHFR) or in the cystathionine beta synthase (CBS) genes are associated with higher levels of homocysteine and the risk of PAD in patients from Brazil. This study analyzed 39 patients with PAD and 32 without PAD in whom risk factors and C677T mutations in the MTHFR gene and both 844ins68 and T833C mutations in the CBS gene were investigated. Although higher levels of homocysteine could be observed in patients with PAD compared to controls, no association between the increase of homocysteine and the frequency of C677T, 844ins68, and T833C mutations could be observed. The results suggest that these mutations do not appear to be related to either homocysteine levels or the development of the disease. However, hyperhomocysteinemia and smoking are important factors in PAD development.     

Comparative ability of rat seminiferous tubules, interstitium, and whole testes to utilize cholesterol-1,5-3H as a substrate for testosterone synthesis and the intratesticular distribution of cholesterol side-chain cleavage enzyme.

Homogenates of rat seminiferous tubules, interstitium and intact testis tissues were assessed for their ability to convert cholesterol -1,2-3H to testosterone in vitro. While 3H-testosterone synthesis was observed in incubates of interstitial and whole testis homogenates, no synthesis was detectable in homogenates of seminiferous tubules. To determine whether cholesterol side-chain cleavage enzyme (CSCCE) was deficient or absent in tubules, mitochondria from tubules, interstitium and whole testes were analyzed for CSCCE activity by measuring conversion of cholesterol -26-14C to 14C-isocaproate (+pregnenolone). Interstitial mitochondrial preparations from each of six testes were found to be approximately 200 times more active in CSCCE than the corresponding tubule mitochondria, and 1600-1800 times more active on a specific activity basis. Although caution is required in extrapolation of in vitro data to the in vivo state, these findings suggest rat seminiferous tubules may be incapable of de novo testosterone biosynthesis and that this lack of synthetic ability may be due to a deficiency of CSCCE.     

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides

Cellular and Molecular Neurobiology - Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses....     

Direct seeding of dry forest tree species in abandoned pastures: effects of grass canopy and seed burial on germination

Natural tree regeneration in abandoned pastures can be hampered by various biotic and abiotic filters, including seed removal and predation. We tested the effects of maintenance and removal of grass and seed deposition (buried and unburied) on seed germination of 12 tree species in dry forest pastures. We obtained evidence supporting the hypothesis that seeds attain higher germination under a grass canopy than on bare ground. For most species, grass cover provides safety from seed predators and facilitates germination by providing a suitable microclimate with soil humidity similar to the forest. The hypothesis that buried seeds attain higher germination was not supported by our data. Predation and removal of unburied seeds ranged from 0 to 77 % and, alone or together, were the major causes of non-germination. Direct seeding is a promising technique for revegetation of recently abandoned pastures in areas originally covered by tropical dry forests. The high germination rate of seeds deposited on the ground and under grass reduces costs during initial restoration stages, potentially facilitating the spread and use of this technique.     

Heterogeneity in high performance liquid chromatography of a variant surface glycoprotein of Trypanosoma brucei

High performance liquid chromatography (HPLC) procedures have been used to analyze a preparation of the variant surface glycoprotein AnTat 1.1A of Trypanosoma brucei. The native preparation gives several peaks with a high reproducibility both by reverse-phase (RP-) and gel permeation (GP-) HPLC. Under RP-HPLC conditions, nine fractions are fully resolved. The RP-HPLC fractions migrate with the same molecular weight VSG band on polyacrylamide slab gel electrophoresis and no significant differences are observed in amino acid composition among these fractions. The RP-HPLC resolution is found to be related to the ability of the VSG to polymerize as shown using GP-HPLC. These results suggest the existence of a microheterogeneity of the AnTat 1.1A VSG preparation in relation to post-translational modification of the VSG molecule.     

Two modes of FEN1 binding to PCNA regulated by DNA

The FEN1 nuclease functions during Okazaki fragment maturation in the eukaryotic cell. Like many other proliferating cell nuclear antigen (PCNA)-binding proteins, FEN1 interacts with the interdomain connector loop (IDCL) of PCNA, and PCNA greatly stimulates FEN1 activity. A yeast IDCL mutant pcna-79 (IL126,128AA) failed to interact with FEN-1, but, surprisingly, pcna-79 was still very active in stimulating FEN1 activity. In contrast, a C-terminal mutant pcna-90 (PK252,253AA) showed wild-type binding to FEN1 in solution, but poorly stimulated FEN1 activity. When PCNA was loaded onto a DNA substrate coupled to magnetic beads, it stabilized retention of FEN1 on the DNA. In this DNA-dependent binding assay, pcna-79 also stabilized retention of FEN1, but pcna-90 was inactive. Therefore, in the absence of DNA, FEN1 interacts with PCNA mainly through the IDCL. However, when PCNA encircles the DNA, the C-terminal domain of PCNA rather than its IDCL is important for binding FEN1. An FF-->GA mutation in the PCNA-interaction domain of FEN1 severely decreased both modes of interaction with PCNA and resulted in replication and repair defects in vivo.     

Pseudomonas community structure and antagonistic potential in the rhizosphere: insights gained by combining phylogenetic and functional gene-based analyses

The Pseudomonas community structure and antagonistic potential in the rhizospheres of strawberry and oilseed rape (host plants of the fungal phytopathogen Verticillium dahliae) were assessed. The use of a new PCR-DGGE system, designed to target Pseudomonas-specific gacA gene fragments in environmental DNA, circumvented common biases of 16S rRNA gene-based DGGE analyses and proved to be a reliable tool to unravel the diversity of uncultured Pseudomonas in bulk and rhizosphere soils. Pseudomonas-specific gacA fingerprints of total-community (TC) rhizosphere DNA were surprisingly diverse, plant-specific and differed markedly from those of the corresponding bulk soils. By combining multiple culture-dependent and independent surveys, a group of Pseudomonas isolates antagonistic towards V. dahliae was shown to be genotypically conserved, to carry the phlD biosynthetic locus (involved in the biosynthesis of 2,4-diacetylphloroglucinol - 2,4-DAPG), and to correspond to a dominant and highly frequent Pseudomonas population in the rhizosphere of field-grown strawberries planted at three sites in Germany which have different land use histories. This population belongs to the Pseudomonas fluorescens phylogenetic lineage and showed closest relatedness to P. fluorescens strain F113 (97% gacA gene sequence identity in 492-bp sequences), a biocontrol agent and 2,4-DAPG producer. Partial gacA gene sequences derived from isolates, clones of the strawberry rhizosphere and DGGE bands retrieved in this study represent previously undescribed Pseudomonas gacA gene clusters as revealed by phylogenetic analysis.     

In silico and in vivo analysis of ABI3 and VAL2 genes during somatic embryogenesis of Coffea arabica: competence acquisition and developmental marker genes

Plant Cell, Tissue and Organ Culture (PCTOC) - The employment of biotechnology-based approaches such as somatic embryogenesis has been applied to several plants including Coffea sp. Despite the...