An inhibitor-like binding mode of a carbonic anhydrase activator within the active site of isoform II

The 2,4,6-trimethylpyridinium derivative of histamine is an effective activator of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). However, unlike other CA activators, which bind at the entrance of the active site cavity, an X-ray crystal structure of hCA II in complex with the 1-[2-(1H-imidazol-4-yl)-ethyl]-2,4,6-trimethylpyridinium salt evidenced a binding mode never observed before either for activators or inhibitors of this enzyme, with the 2,4,6-trimethylpyridinium ring pointing towards the metal ion deep within the enzyme cavity, and several strong hydrophobic interactions stabilizing the adduct. Indeed, incubation of the activator with the enzyme for several days leads to potent inhibitory effects. This is the first example of a CA activator which after a longer contact with the enzyme behaves as an inhibitor.     

Biomass production and fatty acid accumulation in Chlorella sp. (strain DEC1B) isolated from a petrol refinery in Huelva (Spain)

A microalgal strain was established from Cepsa's refinery wastewater treatment plant in Huelva (southwest of Spain). Genetic analysis of the chloroplastic rbcL gene encoding for the large subunit of the ribulose bisphosphate carboxylase enzyme (Rubisco) showed the strain had high homology with other known rbcL sequences of the genus Chlorella. The strain grows well autotrophically in minimum mineral medium, with a growth rate of 0.28 ± 0.012 day^?1 and a biomass productivity of 138.9 ± 6.7 mg L^?1 day^?1. N‐starvation and/or over illumination with 650 µmol photons m^?2 s^?1 of PAR light on the cultures induced a significant increase in the intracellular content of lipids in this microalga. Total lipids were extracted from the strain biomass with 2:1 chloroform‐methanol, and they accounted for approximately 50% of the dry biomass. Polyunsaturated fatty acids (PUFAs) represented 60.4% of the total fatty acids found in the strain, thus making this biomass attractive as a high added‐value product source. The strain was able to grow efficiently in the refinery treated wastewater from which it was isolated, providing an attractive advantage for further development of more sustainable algal biomass production processes at reduced costs close to a petrol refinery area.     

Effects of siRNA-dependent knock-down of cardiolipin synthase and tafazzin on mitochondria and proliferation of glioma cells

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function, and cell proliferation. Changes in CL are often paralleled by changes in the lipid environment of mitochondria that may contribute to mitochondrial function and proliferation. This study aimed to separate the effects of CL content and CL composition from cellular free fatty acid distribution on bioenergetics and proliferation in C6 glioma cells. To this end, cardiolipin synthase and the CL remodelling enzyme, tafazzin, were knocked-down by siRNA in C6 cells. After 72?h of cultivation, we analysed CL composition by means of LC/MS/MS, distribution of cellular fatty acids by means of gas chromatography, and determined oxygen consumption and proliferation. Knock-down of cardiolipin synthase affected the cellular CL content in the presence of linoleic acid (LA) in the culture medium. Knock-down of tafazzin had no consequence with respect to the pattern of cellular fatty acids but caused a decrease in cell proliferation. It significantly changed the distribution of molecular CL species, increased CL content, decreased oxygen consumption, and decreased cell proliferation when cultured in the presence of linoleic acid (LA). The addition of linoleic acid to the culture medium caused significant changes in the pattern of cellular fatty acids and the composition of molecular CL species. These data suggest that tafazzin is required for efficient bioenergetics and for proliferation of glioma cells. Supplementation of fatty acids can be a powerful tool to direct specific changes in these parameters.     

Microtubule defects in mesenchymal stromal cells distinguish patients with Progressive Supranuclear Palsy

Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease whose etiopathogenesis remains elusive. The intraneuronal accumulation of hyperphosphorylated Tau, a pivotal protein in regulating microtubules (MT), leads to include PSP into tauopathies. Pathological hallmarks are well known in neural cells but no word yet if PSP‐linked dysfunctions occur also in other cell types. We focused on bone marrow mesenchymal stromal cells (MSCs) that have recently gained attention for therapeutic interventions due to their anti‐inflammatory, antiapoptotic and trophic properties. Here, we aimed to investigate MSCs biology and to disclose if any disease‐linked defect occurs in this non‐neuronal compartment. First, we found that cells obtained from patients showed altered morphology and growth. Next, Western blotting analysis unravelled the imbalance in α‐tubulin post‐translational modifications and in MT stability. Interestingly, MT mass is significantly decreased in patient cells at baseline and differently changes overtime compared to controls, suggesting their inability to efficiently remodel MT cytoskeleton during ageing in culture. Thus, our results provide the first evidence that defects in MT regulation and stability occur and are detectable in a non‐neuronal compartment in patients with PSP. We suggest that MSCs could be a novel model system for unravelling cellular processes implicated in this neurodegenerative disorder.     

Use and exploitation of channel waters by the black-browed albatross

Black-browed albatrosses are the most abundant albatross species of the southern hemisphere, breeding on sub-Antarctic and Antarctic oceanic islands around the globe. Their foraging habitat during the breeding season is reasonably well known along its distributional range, indicating a preferred use of waters <500 m deep. The discovery of a colony inserted within the Admiralty Sound, Tierra del Fuego, poses an interesting challenge to the known precepts on foraging behavior for the species. In this study, we present the first record on the foraging distribution of the only known inner-channel colony of albatrosses in the world, using high-resolution GPS loggers. Black-browed albatrosses breeding at the Albatross Islet used exclusively inner-channel waters, at least during the chick-guard stage. Our results indicate a significant smaller foraging range during chick-guard compared with conspecifics from Diego Ramirez and Falklands/Malvinas Islands. Implications for the conservation of this colony are discussed.     

Extended use of a selective inhibitor of acid lipase for the diagnosis of Wolman disease and cholesteryl ester storage disease

Lysosomal acid lipase (LAL) deficiency produces two well defined inborn disorders, Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD is a severe, early-onset condition involving massive storage of triglycerides and cholesteryl esters in the liver, with death usually occurring before one year of life. CESD is a more attenuated, later-onset disease that leads to a progressive and variable liver dysfunction. Diagnosis of LAL deficiency is mainly based on the enzyme assay of LAL activity in fibroblasts. Recently, a selective acid lipase inhibitor was used for the determination of enzyme activity in dried-blood filter paper (DBFP) samples. To extend and to validate these studies, we tested LAL activity with selective inhibition on DBFP samples, leukocytes and fibroblasts. Our results showed a clear discrimination between patients with LAL deficiency and healthy controls when using DBFP, leukocytes or fibroblasts (p < 0.001). Deficiency of LAL was also demonstrated in individuals referred to our laboratory with suspected clinical diagnosis of WD, CESD, and Niemann–Pick type B. We conclude that the assay of LAL using selective inhibitor is a reliable and useful method for the identification of LAL deficiency, not only in DBFP samples but also in leukocytes and fibroblasts. This is important as enzyme replacement therapy for LAL deficiency is currently being developed, making the correct diagnosis a critical issue.     

Regional Variation in Tissue Composition and Biomechanical Properties of Postmenopausal Ovine and Human Vagina

Objective

There are increasing numbers of reports describing human vaginal tissue composition in women with and without pelvic organ prolapse with conflicting results. The aim of this study was to compare ovine and human posterior vaginal tissue in terms of histological and biochemical tissue composition and to assess passive biomechanical properties of ovine vagina to further characterise this animal model for pelvic organ prolapse research.

Study Design

Vaginal tissue was collected from ovariectomised sheep (n = 6) and from postmenopausal women (n = 7) from the proximal, middle and distal thirds. Tissue histology was analyzed using Masson''s Trichrome staining; total collagen was quantified by hydroxyproline assays, collagen III/I+III ratios by delayed reduction SDS PAGE, glycosaminoglycans by dimethylmethylene blue assay, and elastic tissue associated proteins (ETAP) by amino acid analysis. Young''s modulus, maximum stress/strain, and permanent strain following cyclic loading were determined in ovine vagina.

Results

Both sheep and human vaginal tissue showed comparable tissue composition. Ovine vaginal tissue showed significantly higher total collagen and glycosaminoglycan values (p<0.05) nearest the cervix. No significant differences were found along the length of the human vagina for collagen, GAG or ETAP content. The proximal region was the stiffest (Young''s modulus, p<0.05), strongest (maximum stress, p<0.05) compared to distal region, and most elastic (permanent strain).

Conclusion

Sheep tissue composition and mechanical properties showed regional differences along the postmenopausal vaginal wall not apparent in human vagina, although the absolute content of proteins were similar. Knowledge of this baseline variation in the composition and mechanical properties of the vaginal wall will assist future studies using sheep as a model for vaginal surgery.     

Different temperature profiles of enzyme secretion by two common strains ofTrichoderma reesei

Summary We have investigated the effects of high and low temperature on the synthesis and secretion of cellulases and other enzymes by two common and readily available strains ofTrichoderma reesei. While some effects were similar in both strains QM9414 and RUT-C30 (a reduction in cellulase production but stimulation of xylanase production at high temperature, and alterations in expression of the cellulase complex at low temperature), some specific differences between the strains were determined, most significantly an enhanced specific secretion rate (secretion/growth) at low growth temperature for QM9414.