Progress in understanding the role of lipids in membrane protein folding

Detailed investigations of membrane protein folding present a number of serious technical challenges. Most studies addressing this subject have emphasized aspects of protein amino acid sequence and structure. While it is generally accepted that the interplay between proteins and lipids plays an important role in membrane protein folding, the role(s) played by membrane lipids in this process have only recently been explored in any detail. This review is intended to summarize recent studies in which particular lipids or membrane physical properties have been shown to play a role in the folding of intact, functionally competent integral membrane proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Water use and water-use efficiency of coppice and seedling Eucalyptus globulus Labill.: a comparison of stand-scale water balance components

Aims

Growers of Eucalyptus globulus Labill. plantations can establish second and later rotations from coppice or by replanting with seedlings. At most locations where E. globulus is grown commercially, water availability is a major driver for productivity. Thus growers must consider which reestablishment technique will maximize productivity whilst sustaining site resources for subsequent rotations. In this study we aimed to compare the stand-scale water balance components of young coppice and seedling E. globulus.

Methods

A second rotation E. globulus coppice and seedling trial was monitored for two successive seasonal cycles. Coppice and seedling plots were instrumented with sap flow- and meteorological-sensors so that stand-scale water balance components could be estimated on a daily time step.

Results

Stand-scale transpiration rate (E) and rate of interception (E _I) were larger in coppice compared to seedlings, but the rate of soil evaporation (E _S) was lower. At approximately 2?years of age each coppice stump was reduced to a single dominant stem, a standard management practice for E. globulus growers, which reduced stem biomass by approximately 70% and caused E to fall to a value approximating that in seedlings. The cumulative transpiration of coppice was 425?mm greater than seedlings up to 34?months of age. Without the coppice reduction (down to one stem/stump), we estimate that the difference would have been much greater. The water-use efficiency of stem production (WUE_stem) was greater in young coppice compared to seedlings but this benefit is likely to be offset by the loss of biomass (and thus transpired water) during coppice stem reduction.

Conclusion

Under the conditions of this study, which included reducing coppice to a single stem, reestablishment with seedling E. globulus resulted in a higher water-use efficiency of stem biomass production compared to coppice of a similar age.     

Aragonite precipitation by "proto-polyps" in coral cell cultures

The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag)~4), the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM) and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 μm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective) similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.     

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ～90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.     

An Observational Study on the Perceptive and Physiological Variables During a 10,000-m Race Walking Competition

ABSTRACT: Vernillo, G, Agnello, L, Drake, A, Padulo, J, Piacentini, MF, and Torre, AL. An observational study on the perceptive and physiological variables during a 10,000-m race walking competition. J Strength Cond Res 26(10): 2741-2747, 2012-In this study, we observed the variations on physiological and perceptual variables during a self-paced 10,000-m race walking (RW) event with the aim to trace a preliminary performance profile of the distance. In 14 male athletes, the heart rate (HR) was monitored continuously throughout the event. The rating of perceived exertion (RPE) was collected using the Borg's 6-20 RPE scale placed at each 1,000 m of an outdoor tartan track. Pacing data were retrieved from the official race results and presented as percent change compared with the first split time. The athletes spent 95.4% at 90-100% of the HRpeak, whereas the other work (4.6%) was negligible. During the race, a shift toward higher HR values was observed because % HRpeak increased by 3.6% in the last vs. the first 1,000-m sector (p = 0.002, effect size [ES] = 1.55 ± 0.68, large). The mean RPE reported by the athletes in the last 1,000 m was significantly higher than in the first 5 sectors (p < 0.02, ES = 1.93-2.96, large to very large). The mean percent change increased between the first 6 sectors and the last 1,000-m sector (p < 0.01, ES = 1.02-2.1, moderate to very large). The analysis of walking velocity at each 1,000-m sector suggested the adoption of a negative pacing. In conclusion, the RPE may be a valid marker of exercise intensity even in real settings. Match physiological and perceptual data with work rate are required to understand race-related regulatory processes. Pacing should be considered as a conscious behavior decided by the athletes based on the internal feedback during the race.     

Inter-relationship between testicular dysgenesis and Leydig cell function in the masculinization programming window in the rat

The testicular dysgenesis syndrome (TDS) hypothesis proposes that maldevelopment of the testis, irrespective of cause, leads to malfunction of the somatic (Leydig, Sertoli) cells and consequent downstream TDS disorders. Studies in rats exposed in utero to di(n-butyl) phthalate (DBP) have strongly supported the TDS concept, but so far no direct evidence has been produced that links dysgenesis per se to somatic cell dysfunction, in particular to androgen production/action during the ‘masculinization programming window’ (MPW; e15.5–e18.5). Normal reproductive tract development and anogenital distance (AGD) are programmed within the MPW, and TDS disorders arise because of deficiencies in this programming. However, DBP-induced focal testicular dysgenesis (Leydig cell aggregation, ectopic Sertoli cells, malformed seminiferous cords) is not evident until after the MPW. Therefore, we used AGD as a read-out of androgen exposure in the MPW, and investigated if this measure was related to objectively quantified dysgenesis (Leydig cell aggregation) at e21.5 in male fetuses exposed to vehicle, DBP (500 or 750 mg/kg/day) or the synthetic glucocorticoid dexamethasone (Dex; alone or plus DBP-500) from e15.5–e18.5 (MPW), e13.5–e20.5 or e19.5–e20.5 (late window). Dysgenesis was found only in animals exposed to DBP during the MPW, and was negatively correlated (R² = −0.5) with AGD at e21.5 and at postnatal day 8, irrespective of treatment period. Dysgenesis was also negatively correlated (R² = –0.5) with intratesticular testosterone (ITT) at e21.5, but only when treatments in short windows (MPW, late window) were excluded; the same was true for correlation between AGD and ITT. We conclude that AGD, reflecting Leydig cell function solely within the MPW, is strongly related to focal dysgenesis. Our results point to this occurring because of a common early mechanism, targeted by DBP that determines both dysgenesis and early (during the MPW) fetal Leydig cell dysfunction. The findings provide strong validation of the TDS hypothesis.     

The effect of periphyton stoichiometry and light on biological phosphorus immobilization and release in streams

Periphyton stoichiometry can vary substantially as a result of differences in stream nutrient availability. A decrease in the periphyton carbon to phosphorus (C:P) ratio should decrease the demand for new P to be immobilized from stream water, but no studies to our knowledge have explored the relationship between periphyton stoichiometry and net P immobilization and release by periphyton. We sought to model biological P immobilization and release (flux) in streams by measuring periphyton stoichiometry and light availability. We measured P flux to and from intact periphyton on stream cobbles (20–100 mm diameter) in 1 L microcosms incubated with streamwater under variable light conditions. Net P immobilization occurred in 75% of microcosms, net P release occurred in only 5% of microcosms, and 20% of microcosms had neither net immobilization nor net release. When normalized to stream conditions, net P immobilization was highest when light availability was high (<60% canopy attenuation) and the periphyton C:P ratio was also high. In contrast, net P release occurred only when light availability was low (>60% canopy attenuation) and the periphyton C:P ratio was also low. A multiple regression model that included both periphyton stoichiometry and light availability from the growing season only, and the interaction term of these two variables, explained 99% of the variation in daily periphyton P flux observed in the study. These results indicate that in order to predict periphyton P immobilization, periphyton stoichiometry and light availability should be considered together. Furthermore, the results indicate that net P immobilization occurs even in very P-rich periphyton, which can act as a P sink when light availability is high.     

The three faces of riboviral spontaneous mutation: spectrum, mode of genome replication, and mutation rate

Riboviruses (RNA viruses without DNA replication intermediates) are the most abundant pathogens infecting animals and plants. Only a few riboviral infections can be controlled with antiviral drugs, mainly because of the rapid appearance of resistance mutations. Little reliable information is available concerning i) kinds and relative frequencies of mutations (the mutational spectrum), ii) mode of genome replication and mutation accumulation, and iii) rates of spontaneous mutation. To illuminate these issues, we developed a model in vivo system based on phage Qß infecting its natural host, Escherichia coli. The Qß RT gene encoding the Read-Through protein was used as a mutation reporter. To reduce uncertainties in mutation frequencies due to selection, the experimental Qß populations were established after a single cycle of infection and selection against RT ⁻ mutants during phage growth was ameliorated by plasmid-based RT complementation in trans. The dynamics of Qß genome replication were confirmed to reflect the linear process of iterative copying (the stamping-machine mode). A total of 32 RT mutants were detected among 7,517 Qß isolates. Sequencing analysis of 45 RT mutations revealed a spectrum dominated by 39 transitions, plus 4 transversions and 2 indels. A clear template•primer mismatch bias was observed: A•C>C•A>U•G>G•U> transversion mismatches. The average mutation rate per base replication was ≈9.1×10⁻⁶ for base substitutions and ≈2.3×10⁻⁷ for indels. The estimated mutation rate per genome replication, μ_g, was ≈0.04 (or, per phage generation, ≈0.08), although secondary RT mutations arose during the growth of some RT mutants at a rate about 7-fold higher, signaling the possible impact of transitory bouts of hypermutation. These results are contrasted with those previously reported for other riboviruses to depict the current state of the art in riboviral mutagenesis.     

Identification of an iron-hepcidin complex

Following its identification as a liver-expressed antimicrobial peptide, the hepcidin peptide was later shown to be a key player in iron homoeostasis. It is now proposed to be the 'iron hormone' which, by interacting with the iron transporter ferroportin, prevents further iron import into the circulatory system. This conclusion was reached using the corresponding synthetic peptide, emphasizing the functional importance of the mature 25-mer peptide, but omitting the possible functionality of its maturation. From urine-purified native hepcidin, we recently demonstrated that a proportion of the purified hepcidin had formed iron-hepcidin complexes. This interaction was investigated further by computer modelling and, based on the sequence similarity of hepcidin with metallothionein, a three-dimensional model of hepcidin, containing one atom of iron, was constructed. To characterize these complexes further, the interaction with iron was analysed using different spectroscopic methods. Monoferric hepcidin was identified by MS, as were possibly other complexes containing two and three atoms of iron respectively, although these were present only in minor amounts. UV/visible absorbance and CD studies identified the iron-binding events which were facilitated at a physiological pH. EPR spectroscopy identified the ferric state of the bound metal, and indicated that the iron-hepcidin complex shares some similarities with the rubredoxin iron-sulfur complex, suggesting the presence of Fe(3+) in a tetrahedral sulfur co-ordination. The potential roles of iron binding for hepcidin are discussed, and we propose either a regulatory function in the maturation of pro-hepcidin into active hepcidin or as the necessary link in the interaction between hepcidin and ferroportin.