Effect of dilution rate on eicosapentaenoic acid productivity ofPhaeodactylum tricornutum utex 640 in outdoor chemostat culture

Summary Eicosapentaenoic acid (EPA) volumetric productivity from an outdoor chemostat culture ofPhaeodactylum tricornutum UTEX 640 in a 50-l tubular photobioreactor varies with dilution rate, reaching a maximum of 47.8 mg l^–1 d^–1 at D=0.36 d^–1. Continuous culture at high dilution rates' is proposed as the most adequate operating mode to maximize polyunsaturated fatty acid production.     

Gram-scale purification of eicosapentaenoic acid (EPA, 20:5n-3) from wetPhaeodactylum tricornutum UTEX 640 biomass

Eicosapentaenoic acid (EPA, 20:5n-3) was obtained from the microalgaPhaeodactylum tricornutum following a three-step process: fatty acid extraction by direct saponification of wet biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds and EPA isolation by preparative HPLC. Direct saponification of wet biomass was carried out with KOH-ethanol (96% v:v) (1 h, 60 °C), extracting 91% of the EPA. PUFAs were concentrated by the urea method with an urea/fatty acid ratio of 4:1 at a crystallization temperature of 28 °C using methanol as the urea solvent. An EPA concentration ratio of 1.5 (55.2/36.3) and recovery of 79% were obtained. This PUFA concentrate was used to obtain 95.8% pure EPA by preparative HPLC, using a reverse-phase column (C₁₈, 4.7 cm i.d. × 30 cm) and methanol-water (1% AcH) 80:20 w/w as the mobile phase. Ninety-seven per cent of EPA loaded was recovered and 70% EPA present in theP. tricornutum biomass was recovered in a highly pure form by means of this three-step downstream processing. In each of the HPLC preparative runs, 635 mg PUFA concentrate were loaded, obtaining 326 mg of a highly concentrated EPA fraction (2.46 g d^–1). Finally, a preliminary cost statement has been calculated.     

Modelling of the mechanical and mass transport properties of auxetic molecular sieves: an idealised inorganic (zeolitic) host–guest system

Force field based simulations have been employed to model the structure, and mechanical and mass transport properties of the all-silica zeolite MFI (ZSM5—Si₉₆O₁₉₂). Undeformed and deformed MFI subject to uniaxial loading in each of the three principal directions were investigated. The mechanical properties are predicted to include negative on-axis Poisson's ratios (auxetic behaviour) in the x ₁–x ₃ plane of the undeformed structure, and are strain-dependent. Transformation from positive-to-negative Poisson's ratio behaviour, and vice versa, is predicted for most on-axis Poisson's ratios at critical loading strains. Simulations of the simultaneous sorption of neopentane and benzene guest molecules onto the undeformed host MFI framework indicate a low neopentane-to-benzene loading ratio, consistent with experimental observation. The sorption of these two molecular species onto deformed MFI is Poisson's ratio- and strain-dependent. Uniaxial tensile loading along a direction containing a negative on-axis Poisson's ratio leads to an increase in the loading of the larger neopentane molecules with respect to benzene, strongly correlated with the increase in volume associated with auxetic behaviour.     

Two new male contraceptives exert their effects by depleting germ cells prematurely from the testis

The three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3-7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4-14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.     

FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P

The molecular mechanisms underlying the formation of carriers trafficking from the Golgi complex to the cell surface are still ill-defined; nevertheless, the involvement of a lipid-based machinery is well established. This includes phosphatidylinositol 4-phosphate (PtdIns(4)P), the precursor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). In yeast, PtdIns(4)P exerts a direct role, however, its mechanism of action and its targets in mammalian cells remain uncharacterized. We have identified two effectors of PtdIns(4)P, the four-phosphate-adaptor protein 1 and 2 (FAPP1 and FAPP2). Both proteins localize to the trans-Golgi network (TGN) on nascent carriers, and interact with PtdIns(4)P and the small GTPase ADP-ribosylation factor (ARF) through their plekstrin homology (PH) domain. Displacement or knockdown of FAPPs inhibits cargo transfer to the plasma membrane. Moreover, overexpression of FAPP-PH impairs carrier fission. Therefore, FAPPs are essential components of a PtdIns(4)P- and ARF-regulated machinery that controls generation of constitutive post-Golgi carriers.     

Cultivation of explants of the marine sponge Crambe crambe in closed systems

Explants of the sponge Crambe crambe were cultured in natural seawater, with or without marine microalga (Phaeodactylum tricornutum) in discontinuous flow through systems and in continuous flow-through systems (DFTHS and CFTHS, respectively). Growth was measured as the increase in underwater weight. In the experiment carried out in the CFTHS, the explants average underwater weight increased by up to 1380% of the initial weight in 22-45 days. Growth in DFTHS was much slower producing a gain of up to an average value of 322% of the initial weight in 100-210 days. Growth kinetics varied considerably for different explants. Explants grew fastest in the first 10-days of subculture. The sponges grew better in CFTHS compared with the DFTHS. The high growth rates observed in CFTHS suggest that this technique is a promising method for culturing C. crambe in closed systems.     

Rat testicular myotubularin, a protein tyrosine phosphatase expressed by Sertoli and germ cells, is a potential marker for studying cell-cell interactions in the rat testis

The full-length cDNA encoding the entire open reading frame (ORF) of rat myotubularin (rMTM) was isolated from a rat testis expression library by PCR. Among the three approximately 2.9-kb cDNAs that were sequenced, one clone was different from the other two clones. It contained seven extra amino acids of FVVLNLQ; this short stretch of extra sequence was found between Gln(421) and Phe(422) within the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) interacting domain (SID) of rMTM. The rMTM ORF had 1,713 bp encoding for a 571 amino acid polypeptide and a calculated molecular weight of 65.8 kDa. A comparison between its deduced amino acid sequence and the GenBank database using BLAST revealed a 53.1% identity with human myotubularin protein (hMTM1), which is a member of the protein tyrosine phosphatase (PTP) family associated with X-linked myotubular myopathy. A 22 amino acid peptide NH(2)-TKVNERYELCDTYPALLAVPAN was synthesized based on the deduced amino acid sequence of rMTM and used for antibody production. By using immunoblot analysis, a 66-kDa protein was indeed detected in both Sertoli and germ-cell cytosols. rMTM mRNA was found in various tissues but was predominantly expressed in the testis, ovary, and skeletal muscle. Sertoli cell rMTM expression was stimulated by germ cells and enhanced when inter-Sertoli junctions were being assembled in vitro. A drastic reduction in testicular rMTM steady-state mRNA level correlated with the depletion of germ cells from the testis in vivo following either glycerol or lonidamine treatment. These results indicate that rMTM is a rat homologue of hMTM1 that may be a useful marker in monitoring the events of cell-cell interactions in the testis.