Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure,backbone dynamics and protein association

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of¹H-¹⁵N-correlated 2D HSQC,¹⁵N-edited 3D TOCSY-HMQC, and¹⁵N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T₁ and T₂ relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH Src homology - GST glutathione-S-transferase - IPTG isopropyl--D-galactopyranoside - DTT dithiothreitol - PMSF phenyl-methyl-sulphonyl-fluoride - TBS 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 - MWCO molecular weight cut off - NMR nuclear magnetic resonance - HSQC heteronuclear single-quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Power frequency magnetic field exposures among nurses in a neonatal intensive care unit and a normal newborn nursery

Given the current interest in potential carcinogenic and developmental effects of exposure to extremely-low-frequency electromagnetic fields, there is a need to identify cohorts of exposed female workers for future epidemiologic investigations. This study was designed to test the hypothesis that nurses working in neonatal intensive care units (NICU) may be significantly exposed to power-frequency magnetic fields. An electromagnetic field monitor was used to measure magnetic fields at distances of 5, 15, 30, and 60 cm from the surfaces of each device used in the NICU. Six female nurses assigned to the NICU (the “exposed” group) and six female nurses working in the normal newborn nursery (the “referent” group) wore EMDEX dosimeters for the entire duration of their 12 h shifts. An investigator kept a detailed log of each NICU subject's whereabouts for the first one-third of her shift. Magnetic fields at 5 cm from the front (defined by the nurses' usual work area) of the NICU devices ranged from less than 0.1 to 114 μT and in all cases decreased considerably with increasing distance. The geometric mean of the shift-time-weighted average exposure of the NICU nurses was 0.17 μT compared with 0.11 μT for the normal newborn nurses. The percentage of time when subjects were exposed to magnetic fields of 0.4 μT or greater ranged from 5.8% to 15.6% for the NICU nurses, 0.4% to 2.9% for five of the comparison group nurses, and was 9.4% for one of the normal newborn nurses with unidentified aberrantly high exposures. Log data revealed that the vast majority of observed peaks among NICU nurses occurred while subjects were in close proximity to infant bed units. We conclude that NICU nurses represent one female-intensive job sector with intermittent high exposures to ELF magnetic fields and encourage larger exposure studies of nurses in a variety of medical settings. © 1994 Wiley-Liss, Inc.     

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%–19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.     

PYCNOCOCCUS PROVASOLII GEN. ET SP. NOV., A COCCOID PRASINOXANTHIN-CONTAINING PHYTOPLANKTER FROM THE WESTERN NORTH ATLANTIC AND GULF OF MEXICO1

The new genus Pycnococcus Guillard is based on several clones from the western North Atlantic and Gulf of Mexico. The type and only described species, Pycnococcus provasolii Guillard, sp. nov., is typified by clone Ω48-23 from the North Atlantic. Cells of Pycnococcus provasolii are solitary, spherical, 1.5–4.0 μm in diameter, have a resistant cell wall lacking sporopollenin, and have the ultrastructural characteristics of green algae. With the light microscope they are scarcely distinguishable from cells of other coccoid planktonic organisms. In pigmentation P. provasolii resembles Micromonas pusilla, Mantoniella squamata, and Mamiella gilva in having chl a, much chl b, Mg 2,4-divinylphaeoporphyrin a₅ monomethyl ester (presumably), and prasinoxanthin as a major xanthophyll. The pyrenoid of P. provasolii has a cytoplasmic channel, which is unique among species closely related to it. Flagellates, occurring rarely in culture, are similar to but distinguishable from known Pedinomonas species by size and shape. Pycnococcus provasolii is referred to the new family Pycnococcaceae Guillard, in the order Mamiellales of the class Micromonadophyceae (Chlorophyta). Clones of Pycnococcus provasolii are oceanic in nutritional characteristics, require only vitamin B₁₂ in culture, and are well adapted to growth under blue or blue-violet light of low intensity.     

THE IMPACT OF A FLOWER-COLOR POLYMORPHISM ON MATING PATTERNS IN EXPERIMENTAL POPULATIONS OF WILD RADISH (RAPHANUS RAPHANISTRUM L.)

We conducted field experiments to determine how a naturally occurring petal-color polymorphism influences mating patterns in wild radish (Raphanus raphanistrum). The polymorphism is controlled at a single genetic locus, with white petal color being completely dominant to yellow. In experimental populations with equal numbers of yellow- and white-flowered homozygous individuals, insect visitors strongly discriminated against white flowers. Pieris rapae, the most frequent pollinator, was almost 50% more likely to visit yellow than white flowers. Maternal fecundity did not differ between the morphs and was not significantly influenced by a plant's compatibility with potential donors, suggesting that seed production was not limited by receipt of compatible pollen. In contrast, the yellow-flowered morph sired approximately 75% of all seeds produced during the study. This paternity proportion was consistently greater than that expected on the basis of postpollination compatibility measures and was indistinguishable from that expected on the basis of pollinator-visitation frequency. We conclude that the male-fitness advantage of the yellow morph resulted from enhanced pollen export due to the greater attractiveness of its flowers to insect pollinators. With color morphs evenly distributed in experimental arrays, insects did not move assortatively on the basis of petal color, and we found no evidence for assortative pollen flow due to the floral polymorphism. Once postpollination compatibility relationships within populations were taken into account, paternal success of yellow donors did not differ between yellow- and white-flowered maternal plants.     

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.     

Construction of an ∼2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2

A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artifical chromosome (YAC) physical map is described. An ∼2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.     

Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to transdifferentiate semi-synchronously into tracheary elements (TEs). This system has been used to establish the precise time point at which the TE cell fate becomes determined, and then changes have been looked for in cell-wall composition and architecture that are associated with the establishment of competence, determination, and differentiation with the transition from primary to secondary cell wall formation. At very early stages in this time course, changes in the repertoire of proteins and polysaccharides both in the cell wall and secreted into the culture medium were found. Changes in the secretion of pectic polysaccharides, xyloglucans and arabinogalactan proteins (AGPs) have been detected using the monoclonal antibodies JIM 7, CCRC-M1 and JIM 13, that recognize these three classes of cell-wall molecule, respectively. Twenty-four hours before secondary thickenings are visible, an AGP is present in the primary walls of a subpopulation of cells, and is secreted into the culture medium. This molecule is present in the secondary thickenings of mature TEs but not in their surrounding primary walls. Methyl-esterified pectic polysaccharides are present in all cell walls and are secreted into the culture medium throughout the time course of differentiation, though at an increased rate in inductive medium. However, sugar and linkage analysis of culture media shows that a relatively unbranched rhamnogalacturonan is enriched in inductive medium around the time of determination and increases rapidly in concentration. The amount of fucosylated xyloglucan in cell walls increases during the time course, but appears in inductive medium 24 h earlier than in control medium and may have a subtly different structure. The fucose-containing epitope on the xyloglucan disappears abruptly and entirely from inductive medium 6 h before any secondary thickenings are visible in the cells. The disappearance of the epitope is correlated with secretion of several hydrolytic enzyme activities. In Zinnia leaves, the mesophyll cell walls contain neither the fucosylated xyloglucan nor the AGP, although methylesterified pectin is present. All three epitopes are expressed in the vascular bundles, and the AGP is specifically localized in the xylem cells. Fucosylated xyloglucan is also present in the epidermal tissue, and the AGP is present in guard cells. The dynamic behaviour of these specific cell-wall molecules is tightly correlated with differentiation events in vitro, and can be clearly distinguished from the production of new wall material found in expanding and elongating cells. The precise timing of the appearance and disappearance of these proteins and polysaccharides compared with the point of cell-fate determination provides us with a series of cell-surface markers for cell states at very early times in the transdifferentiation pathway.     

Enhanced production of mouse hybridomas to picomoles of antigen using EL-4 conditioned media with an in vitro immunization protocol

Summary We report here that the use of murine thymoma cell EL-4 conditioned medium enhances hybridoma yield in a low-antigen dose in vitro immunization protocol. This improved protocol allowed the production of a panel of monoclonal antibodies toDrosophila yolk proteins using less than 1 nanomole of antigen. We believe this refinement will be valuable for the application of hybridoma technology to biologically active materials that are hard to isolate and purify due to their low concentration in the biological fluids. This research was supported by the College of Agriculture and Life Sciences, University of Maryland, USDA-University of Maryland Cooperative Editor's Statement This observation should simplify in vitro immunization approaches and shed new light on the factors required for the in vitro immune response. Wallace L. McKeehan