Fluorescent and Ferritin Labelling of Cuticle Surface Carbohydrates of Caenorhabditis elegans and Panagrellus redivivus

Caenorhabditis elegans and Panagrellus redivivus were investigated for surface carbohydrates using fluorescent-labelled and ferritin-labelled lectins. Rhodamine-labelled Concanavalin A was specifically located in the cephalic region of both species. Rhodamine-labelled wheat germ agglutinin was located over the entire cuticle of P. redivivus but was absent on C. elegans. Rhodamine-labelled peanut agglutinin and Limax flavus agglutinin did not label nematodes of either species. Galactose and sialic acid were not detected on either species, whereas mannose-glucose residues were specifically localized in the head areas of both species. No detectable N-acetylglucosamine occurred on C. elegans, but it was evenly distributed over the cuticle surface of P. redivivus.     

Nuclear Localization Signals Enhance Germline Transmission of a Transgene in Zebrafish

We report that cytoplasmic injection into zebrafish eggs of 104 copies of plasmid DNA complexed to nuclear localization signal (NLS) peptides, as compared to 106 copies of naked DNA, increased nuclear uptake of transgene DNA early during embryo development and enhanced transgene integration frequency into the germline of founders. Monitoring the dynamics of nuclear uptake of DNA-NLS complexes by fluorescence in situ hybridization (FISH) of interphase nuclei indicates that NLS enhances both the proportion of nuclei importing DNA during early embryo development, and the amount of DNA imported by individual nuclei. The use of NLS increases the proportion of germline transgenic founders from 14 to 43% (P < 0.01) as assessed by polymerase chain reaction analysis of F1s. From germline transgenic DNA-NLS-injected founders, 47% transgenic F1s are obtained in wild-type crosses, as opposed to 6% from naked DNA-injec...     

Satiation-dependent, intra-cohort variations in prey size selection of young roach (Rutilus rutilus)

The tolerance to freezing and thawing of Leucodon sciuroides, a moss growing in mountainous areas of the Mediterranean (south-east Spain), was investigated by means of CO₂ gas exchange, modulated chlorophyll (Chl) a fluorescence and pigment analysis by high-performance liquid chromatography. Evidence is presented for freezing-induced decreases in CO₂ fixation that enhance non-radiative dissipation of absorbed light energy, a process which protects the photosynthetic apparatus. The photosynthetic apparatus of L. sciuroides remained fully recuperable after freezing, as indicated by the recovery of photosynthetic CO₂ fixation and Chl fluorescence parameters to pre-freezing values during thawing. The rapid recovery of photosynthesis activity during thawing in L. sciuroides suggests that this moss is capable of tolerating freeze-thaw cycles in a manner similar to that found at higher latitudes or in the Antarctic. The resistance of the photosynthetic apparatus of this moss to freezing might be achieved, at least partially, through the employment of dissipative pathways, such as non-radiative dissipation of absorbed light energy. Received: 4 June 1998 / Accepted: 15 February 1999     

Stomatal conductance is a key parameter to assess limitations to photosynthesis and growth potential in barley genotypes

Fourteen genotypes of barley were compared for response to salinity by monitoring the parameters gas exchange and chlorophyll fluorescence. We present relationships between stomatal conductance (gs) gas exchange chlorophyll fluorescence parameters and aboveground dry matter (AGDM). We found that genetic variability provided a continuum of data for gs across control and saline conditions. We used this continuum of gs values to test the overall relationships between gs and net photosynthesis (A), leaf internal CO2 concentration (Ci), actual quantum yield of PSII electron transport (PhiPSII), relative electron yield over net CO2 assimilation rate (ETR/A), and AGDM. The relationship between gs and A was highly significant (P < 0.0001) for both control and saline treatments, while correlations between gs and Ci, and Ci and A were significant only under control conditions. Unexpectedly, we found positive correlations between gs and PhiPSII (P < 0.0001) for both conditions. A comparison between relationships of gs and A, and gs and PhiPSII seemed to indicate a possible acclimation to salinity at the chloroplastic level. Finally, the relationships between gs and ETR/A were exceptionally strong for both growing conditions (P < 0.0001) indicating that, as gs values were negatively affected in barley by genetics and salinity as main or interactive effects, there was a progressive increase in photorespiration in barley. Overall, we found that stomatal conductance was a key parameter in the study of barley responses to limiting situations for photosynthesis. We also found a strong relationship between AGDM and gs regardless of growing conditions and genotypes. For breeding evaluations to select barley genotypes for salinity tolerance, it may be possible to replace all measurements of gas exchange and chlorophyll fluorescence by the simple use of a porometer.     

Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIAB(Man) subunit of the mannose transporter of Escherichia coli.

The hydrophilic subunit of the mannose transporter (IIAB(Man)) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N-terminal domain (Trp12, Trp33, and Trp69) and one in the C-terminal domain (Trp182). Single and double Trp-Phe mutants of IIABMan and of the IIA domain were produced. Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment. Stabilities of the Trp mutants of dimeric IIA(Man) and IIAB(Man) were estimated from midpoints of the GdmHCl-induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively. W12F exhibited increased stability, but only 6% of the wild-type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active. Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F. These results suggest that flexibility is required for the adjustments of protein-protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIA(Man), IIB(Man), and the incoming mannose bound to the transmembrane IIC(Man)-IID(Man) complex.     

Photosynthetic response to water stress and changes in metabolites in <Emphasis Type="Italic">Jasminum sambac</Emphasis>

Gas exchange, chlorophyll (Chl) fluorescence, and contents of some metabolites in two genotypes of jasmine (Jasminum sambac), single petal (SP) and double petal (DP) one, were analyzed during dehydration and re-hydration. Water stress significantly decreased net photosynthetic rate, stomatal conductance, and maximum photochemical efficiency (F_v/F_m) in both jasmine genotypes, but increased minimum fluorescence (F₀) only in DP-jasmine. Water stress also decreased starch content, while increased contents of total soluble sugars and proline in leaves of both genotypes. SP-jasmine demonstrated higher drought tolerance as evidenced by maintaining higher gas exchange and photochemical efficiency and lower alteration of metabolites than DP-jasmine. Recovery analysis revealed that drought-induced injury in photosynthetic machinery in jasmine plants was reversible. DP-jasmine exhibited a slow recovery of drought-induced impairment in photosynthetic activity and associated metabolites, suggesting that this genotype had lower capacity to adapt to water limited condition. Higher yield stability of SP-than that of DP-jasmine under rain-fed condition finally confirmed higher drought tolerance of SP-jasmine.     

Protochlorophyllide forms in non-greening epicotyls of dark-grown pea (Pisum sativum)

Low-temperature fluorescence emission spectra of 6.5-day-old dark-grown epicotyls of pea ( Pisum sativum ) revealed the presence of protochlorophyll(ide). The upper part of the epicotyl contained 30% of the protochlorophyll(ide) content per fresh weight found in pea leaves, whereas the lower part contained 3%. Three discrete spectral forms of protochlorophyll(ide) were clearly distinguished after Gaussian deconvolution of fluorescence excitation and emission spectra. Adding the satellite bands of the Qy_(0-0) transitions (the emission vibrational (Emv) bands with correlated amplitudes, gave the following delineation: Ex439–Em629–Emv684, Ex447–Em636–Emv700 and Ex456–Em650–Emv728. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunodetection of whole tissue extracts of the epicotyl indicated the presence of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). Electron micrographs showed prolamellar bodies in at most 11 % of the plastid profiles of the epicotyl cells. These prolamellar bodies were smaller, and many of them showed less regular structure than those of the leaves. Taken together, the results indicate that the protochlorophyll(ide) in epicotyls is arranged in a different way than in leaves.     

Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.     

Interference of low‐molecular substances with the thioflavin‐T fluorescence assay of amyloid fibrils

Abnormal fibrillization of amyloidogenic peptides/proteins has been linked to various neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as with type‐II diabetes mellitus. The kinetics of protein fibrillization is commonly studied by using a fluorescent dye Thioflavin T (ThT) that binds to protein fibrils and exerts increased fluorescence intensity in bound state. Recently, it has been demonstrated that several low‐molecular weight compounds like Basic Blue 41, Basic Blue 12, Azure C, and Tannic acid interfere with the fluorescence of ThT bound to Alzheimers' amyloid‐β fibrils and cause false positive results during the screening of fibrillization inhibitors. In the current study, we demonstrated that the same selected substances also decrease the fluorescence signal of ThT bound to insulin fibrils already at submicromolar or micromolar concentrations. Kinetic experiments show that unlike to true inhibitors, these compounds did neither decrease the fibrillization rate nor increase the lag‐period. Absence of soluble insulin in the end of the experiment confirmed that these compounds do not disaggregate the insulin fibrils and, thus, are not fibrillization inhibitors at concentrations studied. Our results show that interference with ThT test is a general phenomenon and more attention has to be paid to interpretation of kinetic results of protein fibrillization obtained by using fluorescent dyes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.