Reliability of egg morphology to detect conspecific brood parasitism in goldeneyes Bucephala clangula examined using protein fingerprinting

Eadie (1989) developed a method based on variation between females in egg length, width and weight to detect conspecific brood parasitism in the field: using these three egg measures, Euclidean distance between all pairs of eggs within a clutch is calculated, and if maximum Euclidean distance (MED) between any two eggs exceeds a threshold value the nest is considered parasitized. The MED method has been tested in Finnish and Scottish common goldeneye Bucephala clangula populations but the results have been contradicting. Here we use protein fingerprinting to assess the validity of the MED method. Data comprised 35 clutches of which we knew, based on protein fingerprinting, how many different females laid the clutch (range 1–5 females). The mean MED of non-parasitized clutches (laid by 1 female only) was 1.470 (95% CI: lower 1.169, upper 1.771; n=21) and that of parasitized clutches (laid by 2 or more females) was 3.654 (95% CL: lower 3.083, upper 4.225; n=14). Using a MED>3.0 as a criterion to identify parasitized clutches 89% of all clutches were classified correctly either parasitized or non-parasitized when compared to the identification based on protein fingerprinting. Clutch size and the number of females (beyond 2 females) did not affect the clutch MED, whereas the status of parasitism did. Repeatability of egg length, width and weight were: 0.63, 0.76 and 0.80, respectively, implying that, variation in these egg measures occurs among rather than within females. Our new results confirm that the MED method is reliable enough to detect parasitism in common goldeneye.     

Characterisation of the role of Vrp1 in cell fusion during the development of visceral muscle of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

In Drosophila muscle cell fusion takes place both during the formation of the somatic mesoderm and the visceral mesoderm, giving rise to the skeletal muscles and the gut musculature respectively. The core process of myoblast fusion is believed to be similar for both organs. The actin cytoskeleton regulator Verprolin acts by binding to WASP, which in turn binds to the Arp2/3 complex and thus activates actin polymerization. While Verprolin has been shown to be important for somatic muscle cell fusion, the function of this protein in visceral muscle fusion has not been determined.     

Translational control of collagen prolyl 4-hydroxylase-alpha(I) gene expression under hypoxia

Hypoxia is a pro-fibrotic stimulus, which is associated with enhanced collagen synthesis, as well as with augmented collagen prolyl 4-hydroxylase (C-P4H) activity. C-P4H activity is controlled mainly by regulated expression of the alpha C-P4H subunit. In this study we demonstrate that the increased synthesis of C-P4H-alpha(I) protein in human HT1080 fibroblasts under long term hypoxia (36 h, 1% oxygen) is controlled at the translational level. This is mediated by an interaction of RNA-binding protein nucleolin (approximately 64 kDa form) at the 5'- and 3'-untranslated regions (UTR) of the mRNA. The 5'/3'-UTR-dependent mechanism elevates the C-P4H-alpha(I) expression rate 2.3-fold, and participates in a 5.3-fold increased protein level under long term hypoxia. The interaction of nucleolin at the 5'-UTR occurs directly and depends on the existence of an AU-rich element. Statistical evaluation of the approximately 64-kDa nucleolin/RNA interaction studies revealed a core binding sequence, corresponding to UAAAUC or AAAUCU. At the 3'-UTR, nucleolin assembles indirectly via protein/protein interaction, with the help of another 3'-UTR-binding protein, presumably annexin A2. The increased protein level of the approximately 64-kDa nucleolin under hypoxia can be attributed to an autocatalytic cleavage of a high molecular weight nucleolin form, without alterations in nucleolin mRNA concentration. Thus, the alteration of translational efficiency by nucleolin, which occurs through a hypoxia inducible factor independent pathway, is an important step in C-P4H-alpha(I) regulation under hypoxia.     

Inoculation of selenium hyperaccumulator Stanleya pinnata and related non‐accumulator Stanleya elata with hyperaccumulator rhizosphere fungi—investigation of effects on Se accumulation and speciation

Little is known about how fungi affect elemental accumulation in hyperaccumulators (HAs). Here, two rhizosphere fungi from selenium (Se) HA Stanleya pinnata, Alternaria seleniiphila (A1) and Aspergillus leporis (AS117), were used to inoculate S. pinnata and related non‐HA Stanleya elata. Growth and Se and sulfur (S) accumulation were analyzed. Furthermore, X‐ray microprobe analysis was used to investigate elemental distribution and speciation. Growth of S. pinnata was not affected by inoculation or by Se. Stanleya elata growth was negatively affected by AS117 and by Se, but combination of both did not reduce growth. Selenium translocation was reduced in inoculated S. pinnata, and inoculation reduced S translocation in both species. Root Se distribution and speciation were not affected by inoculation in either species; both species accumulated mainly (90%) organic Se. Sulfur, in contrast, was present equally in organic and inorganic forms in S. pinnata roots. Thus, these rhizosphere fungi can affect growth and Se and/or S accumulation, depending on host species. They generally enhanced root accumulation and reduced translocation. These effects cannot be attributed to altered plant Se speciation but may involve altered rhizosphere speciation, as these fungi are known to produce elemental Se. Reduced Se translocation may be useful in applications where toxicity to herbivores and movement of Se into the food chain is a concern. The finding that fungal inoculation can enhance root Se accumulation may be useful in Se biofortification or phytoremediation using root crop species.     

Substrate complexation and aggregation influence the cyclodextrin glycosyltransferase (CGTase) catalyzed synthesis of alkyl glycosides

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) was used to convert dodecyl-β-maltoside (DDM) to dodecyl-β-maltooctaoside (DDMO) using α-cyclodextrin (α-CD) or starch as glycosyl donors. At 300 mM α-CD, varied DDM concentration and 60 °C, the reaction obeyed Michaelis-Menten kinetics with a K_m value of 18 mM and a V_max value of 100 U/mg enzyme. However, at 25 mM α-CD the reaction rate decreased with increasing DDM concentration (5-50 mM), and when the α-CD concentration was varied at fixed DDM concentration an S shaped curve was obtained. The deviations from Michaelis-Menten kinetics were interpreted as being caused by formation of inclusion complexes between α-CD and DDM and by micellation of DDM. To achieve a high reaction rate, a high concentration of free α-CD is necessary, since α-CD in the form of a complex has low reactivity. When starch is used as glycosyl donor in the CGTase catalyzed alkyl glycoside elongation reaction, it is thus important to choose reaction conditions under which the cyclization of starch to α-CD is efficient.     

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid‐encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β‐barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion‐conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco‐harboring pathogens.     

Studying the uptake of cell-penetrating peptides

More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.     

Changes of the proteinase binding properties and conformation of bovine alpha 2-macroglobulin on cleavage of the thio ester bonds by methylamine

Cleavage of the thio ester bonds of human alpha2-macroglobulin (alpha 2M) by methylamine leads to an extensive conformational change and to inactivation of the inhibitor. In contrast, cleavage of these bonds in bovine alpha 2M only minimally perturbs the hydrodynamic volume of the protein [Dangott, L. J., & Cunningham, L. W. (1982) Biochem. Biophys. Res. Commun. 107, 1243-1251], as well as its spectroscopic properties, as analyzed by ultraviolet difference spectroscopy, circular dichroism, and fluorescence in this work. A conformational change analogous to that undergone by human alpha 2M thus does not occur in the bovine inhibitor. However, changes of several functional properties of bovine alpha 2M are induced by the amine. The apparent stoichiometry of inhibition of trypsin thus is reduced from about 1.2 to about 0.7 mol of enzyme/mol of inhibitor. In spite of this decrease, the interaction with the proteinase induces similar conformational changes in methylamine-treated alpha 2M as in intact alpha 2M, as revealed by spectroscopic analyses, indicating that the mode of binding of the proteinase to the inhibitor is essentially unperturbed by thio ester bond cleavage. The reaction with methylamine also greatly increases the sensitivity of bovine alpha 2M to proteolysis by trypsin at sites other than the "bait" region. Moreover, the second-order rate constant for the reaction with thrombin is reduced by about 10-fold. These results indicate that the thio ester bonds of bovine alpha 2M, although not required per se for the binding of proteinases, nevertheless are responsible for maintaining certain structural features of the inhibitor that are of importance for full activity.     

Effect of head-group structure and counterion condensation on phase equilibria in anionic phospholipid-water systems studied by 2H, 23Na, and 31P NMR and X-ray diffraction

The phase equilibria, hydration, and sodium counterion association for the systems DOPA-2H2O, DOPS-2H2O, DOPG-2H2O, and DPG-2H2O were investigated with 2H, 23Na, and 31P NMR and X-ray diffraction. The following one-phase regions were found in the DOPA-water system: a reversed hexagonal liquid-crystalline (HII) phase up to about 35 wt % water and a lamellar liquid-crystalline (L alpha) phase between about 55 and 98 wt % water. The area per DOPA molecule was 36-65 A2 in the HII phase (10-40 wt % water) and 69 A2 in the L alpha phase (60 wt % water). DOPS and DOPG with 10-98 wt % water, and DPG with 20-95 wt % water formed an L alpha phase at temperatures between 25 and 55 degrees C. At temperatures above 55 degrees C, DPG with 20 and 30 wt % water formed a mixture of L alpha, HII, and cubic liquid-crystalline phases, the mole percent of lipid forming nonlamellar phases being smaller at 30 wt % water than at 20 wt % water. DPG with 10 wt % water probably formed a mixture of an L alpha phase and at least one nonlamellar liquid-crystalline phase at 25 and 35 degrees C, and a pure HII phase at 45 degrees C and higher temperatures. At water concentrations above about 50 wt % the 23Na quadrupole splitting was constant for all four lipid-water systems studied, implying that the counterion association to the charged lipid aggregates did not change upon dilution. These experimental observations can be described with an ion condensation model but not with a simple equilibrium model. The fraction of counterions located close to the lipid-water interface was calculated to be greater than 95%. The 2H and 23Na NMR quadrupole splittings of 2H2O and sodium counterions, respectively, indicate that the molecular order in the polar head-group region decreases for the L alpha phase in the order DOPA approximately DPG greater than DOPS greater than DOPG.