Bacteria-induced static batch fungal fermentation of the diterpenoid cyathin A<Subscript>3</Subscript>, a small-molecule inducer of nerve growth factor

Cyathin A₃, produced by the fungus Cyathus helenae, is a member of the cyathane family of diterpene natural products. While many of the cyathanes display antibacterial/antimicrobial activity or have cytotoxic activity against human cancer cell lines, their most exciting therapeutic potential is derived from their ability to induce nerve growth factor (NGF) release from glial cells, making the cyathanes attractive lead molecules for the development of neuroprotective therapeutics to prevent/treat Alzheimer’s disease. To investigate if cyathin A₃ has NGF-inducing activity, we set out to obtain it using published C. helenae bench-scale fungal fermentations. However, to overcome nonproducing fermentations, we developed an alternative, bacteria-induced static batch fermentation approach to the production of cyathin A₃, as described in this report. HPLC, UV absorption spectra, and mass spectrometry identify cyathin A₃ in fungal fermentations induced by the timely addition of Escherichia coli K12 or Bacillus megabacterium. Pre-filtration of the bacterial culture abolishes cyathin A₃ induction, suggesting that bacteria-associated media changes or physical interaction between the fungus and bacteria underlie the induction mechanism. Through alteration of incubation conditions, including agitation, the timing of induction, and media composition, we optimized the fermentation to yield nearly 1 mg cyathin A₃/ml media, a sixfold increase over previously described yields. Additionally, by comparison of fermentation profiles, we reveal that cyathin A₃ biosynthesis is regulated by carbon catabolite repression. We have used an enzyme-linked immunosorbent assay to illustrate that cyathin A₃ induces NGF release from cultured glial cells, and therefore cyathin A₃ warrants further examination in the development of neuroprotective therapeutics.     

Incorporation and degradation by kidney lysosomes of cystatin alpha injected intravenously.

Cystatin alpha was purified from Escherichia coli transfected with a recombinant cystatin alpha gene and injected into the tail vein of rats. Its fate was then followed using a sensitive enzyme immunoassay for cystatin alpha. Results showed that it was rapidly removed from the blood and taken up by the kidney. Percoll density-gradient analysis showed that it was rapidly incorporated into lysosomes in the kidney. Its level in the kidney was maximal 30 min after its injection then rapidly decreased. The activity of cathepsin H in the kidney lysosomal fraction was markedly decreased 30 min after injection of cystatin alpha but recovered rapidly. Anti-(cystatin alpha) antibody precipitated cathepsin H and anti-(cathepsin H) antibody precipitated cystatin alpha in an extract of the lysosome-rich fraction of the kidney 30 min after injection of cystatin alpha. These results indicate that cystatin alpha was taken up by kidney lysosomes, formed a complex with cathepsin H and initially decreased the activity of cathepsin H, but that later the level of cystatin alpha in the kidney rapidly decreased.     

Isolation and characterization of bacterial strains that have high ability to degrade 1,4-dioxane as a sole carbon and energy source

Four novel metabolic 1,4-dioxane degrading bacteria possessing high ability to degrade 1,4-dioxane (designated strains D1, D6, D11 and D17) were isolated from soil in the drainage area of a chemical factory. Strains D6, D11 and D17 were allocated to Gram-positive actinomycetes, similar to previously reported metabolic 1,4-dioxane degrading bacteria, whereas strain D1 was allocated to Gram-negative Afipia sp. The isolated strains could utilize a variety of carbon sources, including cyclic ethers, especially those with carbons at position 2 that were modified with methyl- or carbonyl-groups. The cell yields on 1,4-dioxane were relatively low (0.179–0.223 mg-protein (mg-1,4-dioxane)^?1), which was likely due to requiring energy for C–O bond fission. The isolated strains showed 2.6–13 times higher specific 1,4-dioxane degradation rates (0.052–0.263 mg-1,4-dioxane (mg-protein)^?1 h^?1) and 2.3–7.8 fold lower half saturation constants (20.6–69.8 mg L^?1) than the most effective 1,4-dioxane degrading bacterium reported to date, Pseudonocardia dioxanivorans CB1190, suggesting high activity and affinity toward 1,4-dioxane degradation. Strains D1 and D6 possessed inducible 1,4-dioxane degrading enzymes, whereas strains D11 and D17 possessed constitutive ones. 1,4-Dioxane degradation (100 mg L^?1) by Afipia sp. D1 was not affected by the co-existence of up to 3,000 mg L^?1 of ethylene glycol. The effects of initial pH, incubation temperature and NaCl concentration on 1,4-dioxane degradation by the four strains revealed that they could degrade 1,4-dioxane under a relatively wide range of conditions, suggesting that they have a certain adaptability and applicability for industrial wastewater treatment.     

Comparison between mechanical stress and bone mineral density in the femur after total hip arthroplasty by using subject-specific finite element analyses

The mechanism underling bone mineral density (BMD) loss that occurs in the femur after total hip arthroplasty (THA) remains unknown. We compared the equivalent stress and strain energy density (SED) to BMD in the femur after THA using subject-specific finite element analyses. Twenty-four patients who had undergone primary cementless THA were analysed. BMD was measured using dual-energy X-ray absorptiometry (DEXA) at 1 week and 3, 6 and 12 months after THA. Seven regions of interest (ROIs) were defined in accordance with Gruen's system (ROIs 1–7). Computed tomography images of the femurs were acquired pre- and postoperatively, and the images were converted into three-dimensional finite element (FE) models. Equivalent stress and SED were analysed and compared with DEXA data. BMD was maintained 1 year after THA in ROIs 3, 4, 5 and 6, whereas BMD decreased in ROIs 1, 2 and 7. FE analysis revealed that equivalent stress in ROIs 3, 4, 5 and 6 was much higher than that in ROIs 1, 2 and 7. A significant correlation was observed between the rate of changes in BMD and equivalent stress. Reduction of equivalent stress may contribute to decrease in BMD in the femur after THA.     

An improved CARV process for bioethanol production from a mixture of sugar beet mash and potato mash

A mixed mash of sugar beet roots and potato tubers with a sugar concentration of 23.7% w/v was used as a feedstock for bioethanol production. Enzymatic digestion successfully reduced the viscosity of the mixture, enabling subsequent heat pretreatment for liquefaction/sterilization. An energy-consuming thick juice preparation from sugar beet for concentration and sterilization was omitted in this new process.     

Leviserpin: A Serine Peptidase Inhibitor (Serpin) from the Sugarcane Weevil <Emphasis Type="Italic">Sphenophorus levis</Emphasis>

Serine peptidase inhibitors (serpins) form a superfamily of proteins covering abroad spectrum of different biological functions. Here we describe the inhibitory characterization of leviserpin, the first serpin from the sugar cane weevil Sphenophorus levis. Leviserpin was able to inhibit bovine trypsin by the formation of the covalent complex serpin-peptidase, demonstrated by SDS–PAGE and mass spectroscopy analysis. We also have determined the cleavage site at the reactive center loop, by the analysis of the polypeptides released from de C-terminus of leviserpin. Moreover we investigated the mRNA expression of leviserpin in different stages of S. levis development. Thus the specificity of leviserpin, in addition with its mRNA coding being transcribed through all lifecycle of the insect, can suggest a possible role in defense mechanism by regulating the action of prophenoloxidase (proPO) activating enzyme.     

Anatomical aspects of the gynoecium of species of Anthurium sect. Urospadix Engl. (Araceae,Alismatales)

Anthurium sect. Urospadix includes approximately 70 species differently distributed in Eastern Brazil, Western South America, and Central America. The present work studied the gynoecia of species of section Urospadix to describe their anatomical features. In all the species studied, the apical portion of the gynoecium differentiates into a stigma covered with short trichomes and an adjacent region formed by isodiametric cells. The stylar canal comprises short, unicellular secretory trichomes extending up to the ovarian aperture. Carpels are apically symplicate and basally synascidiate with a septum that does not reach the apical portion of the locule. The apical portion of the septum differentiates into long, secretory trichomes while the subapical one differentiates into the placenta. Locules are filled with mucilage. Since the epidermis of the region adjacent to the stigma, the length of the stylar portion, and the number of cell layers of the ovarian septum vary among the species studied, they may be useful for further taxonomic and systematic purposes. The present work is the first detailed report of trichome location and distribution on the gynoecia of Anthurium.     

Rapid and Liquid-Based Selection of Genetic Switches Using Nucleoside Kinase Fused with Aminoglycoside Phosphotransferase

The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3’)-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.     

Two Models for Interaction in a 3-way Analysis of Variance With One Observation Per Cell

The paper shows that three factor interaction can be represented by two different models in a 3-way classification without replications. Each model is developed as a product of the three main effects plus the sum of the products of a main effect and a two-factor interaction. A conditional F-test for non-additivity is derived from each model. A pseudo-correlation exists between the models and as this correlation coefficient tends to unity the models are shown to converge. Extension of the procedure to four-factor interaction and higher is indicated.