A systematic study of fundamentals in α-helical coiled coil mimicry by alternating sequences of β- and γ-amino acids

Aimed at understanding the crucially important structural features for the integrity of α-helical mimicry by βγ-sequences, an α-amino acid sequence in a native peptide was substituted by differently arranged βγ-sequences. The self- and hetero-assembly of a series of αβγ-chimeric sequences based on a 33-residue GCN4-derived peptide was investigated by means of molecular dynamics, circular dichroism, and a disulfide exchange assay. Despite the native-like behavior of βγ alternating sequences such as retention of α-helix dipole and the formation of 13-membered α-helix turns, the αβγ-chimeras with different βγ substitution patterns do not equally mimic the structural behavior of the native parent peptide in solution. The preservation of the key residue contacts such as van der Waals interactions and intrahelical H-bonding, which can be met only by particular substitution patterns, thermodynamically favor the adoption of coiled coil folding motif. In this study, we show how successfully the destabilizing structural consequences of α → βγ modification can be harnessed by reducing the solvent-exposed hydrophobic surface area and placing of suitably long and bulky helix-forming side chains at the hydrophobic core. The pairing of αβγ-chimeric sequences with the native wild-type are thermodynamically allowed in the case of ideal arrangement of β- and γ-residues. This indicates a similarity in local side chain packing of β- and γ-amino acids at the helical interface of αβγ-chimeras and the native α-peptide. Consequently, the backbone extended residues are able to participate in classical “knob-into-hole” packing with native α-peptide.     

A novel fluorogenic substrate for the measurement of endothelial lipase activity

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.     

Variation in fibular robusticity reflects variation in mobility patterns

During hominin plantigrade locomotion, the weight-bearing function of the fibula has been considered negligible. Nevertheless, studies conducted on human samples have demonstrated that, even if less than that of the tibia, the load-bearing function of the fibula still represents a considerable portion of the entire load borne by the leg. The present study assesses whether variation in habitual lower limb loading influences fibular morphology in a predictable manner. To achieve this, both fibular and tibial morphology were compared amongst modern human athletes (field hockey players and cross-country runners) and matched sedentary controls. Peripheral quantitative computed tomography was used to capture two-dimensional, cross-sectional bone images. Geometric properties were measured at the midshaft for each bone. Results show a trend of increased fibular rigidity from control to runners through to field hockey players. Moreover, relative fibular robusticity (fibula/tibia) is significantly greater in hockey players compared with runners. These results are likely the consequence of habitual loading patterns performed by these athletes. Specifically, the repeated directional changes associated with field hockey increase the mediolateral loading on the lower leg in a manner that would not necessarily be expected during cross-country running. The present study validates the use of the fibula in association with the tibia as a mean to provide a more complete picture of leg bone functional adaptations. Therefore, the fibula can be added to the list of bones generally used (tibia and femur) to assess the correspondence between mobility patterns and skeletal morphology for past human populations.     

Evidence of calcium- and SNARE-dependent release of CuZn superoxide dismutase from rat pituitary GH3 cells and synaptosomes in response to depolarization

The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH(3) cells that express voltage-dependent calcium channels and are subjected to Ca(2+) oscillations, we found that treatment with high K(+)-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH(3) cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH(3) cells and rat brain synaptosomes showed that K(+)-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulinum toxin A. Subcellular fractionation analysis showed that SOD1 was present in large dense core vesicles. These data clearly show that, in addition to the constitutive SOD1 secretion, depolarization induces an additional rapid calcium-dependent SOD1 release in GH(3) cells and in rat brain synaptosomes. This likely occurs through exocytosis from SOD1-containing vesicles operated by the SNARE complex.     

From mass of body elements to fish biomass: a direct method to quantify food intake of fish eating birds

The winter diet of great crested grebe (Podiceps cristatus) was studied in the major lakes of Insubria region, N Italy, in 2000–2003 by analysing stomach and oesophagus contents of birds found dead. Winter diet of cormorant (Phalacrocorax carbo) was studied using pellet analysis. Diet was expressed in terms of numerical abundance, frequency and biomass of prey. A detailed methodology is described of how to prepare a reference collection of otoliths, pharyngeal bones and chewing pads (key-elements), essential for species-specific identification of fish remains in stomachs and pellets, and to estimate ingested fish biomass. Correlations between mass (mg) of key-elements and mass of corresponding fish were significant, and exponential regression models were used to calculate biomass of each single prey item directly from the mass of its key-elements. Advantages of this method are compared with the more traditional two step approach in which size of elements is used to estimate fish length, followed by calculating fresh mass from fish length. Cyprinids formed the largest part of the diet for both predators. Great crested grebes selected small prey, feeding mainly on bleak (Alburnus alburnus alborella), while diet composition of cormorants varied significantly in relation to prey availability. Handling editor: K. Martens     

Chitosan-mediated changes in cell wall composition, morphology and ultrastructure in two wood-inhabiting fungi

The effect of chitosan on cell wall deposition was investigated in the two wood-inhabiting fungal species Trichoderma harzianum (CBS 597.91) and Sphaeropsis sapinea (NZFS 2725). The study used three independent analytical techniques to quantify chitin in the fungal mycelium. A colorimetric method for the detection of d-glucosamine was compared with two gas chromatography–mass spectroscopy (GC-MS) methods employing alditol acetates analysis and pyrolysis. The latter used a stable-isotope-labelled internal standard, d₃-N-acetyl glucosamine. At least in the case of S. sapinea, the study provided evidence of an increase in the chitin content in the mycelium due to chitosan treatment, indicating that chitosan treatment affected cell wall deposition. Electron microscopy techniques showed alteration in surface morphology and cell wall texture due to chitosan treatment. The implications of these results are discussed with a view to analysing possible mechanisms for growth inhibitory effects of chitosan on fungal hyphae.     

Phenylpiperidine-benzoxazinones as urotensin-II receptor antagonists: synthesis, SAR, and in vivo assessment

Various 4-phenylpiperidine-benzoxazin-3-ones were synthesized and biologically evaluated as urotensin-II (U-II) receptor antagonists. Compound 12i was identified from in vitro evaluation as a low nanomolar antagonist against both rat and human U-II receptors. This compound showed in vivo efficacy in reversing the ear-flush response induced by U-II in rats.