Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.     

Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution

The chemical shift assignments and secondary structure of a murine–human chimera,MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa,have been determined from multidimensional heteronuclear NMR spectra acquired on auniformly 13C,15N-labelled sample. Secondary structure elements were defined on the basisof chemical shifts, NH-CH coupling constants, medium-range NOEs and the location ofslowly exchanging amide protons. The protein contains four -helices, the relativeorientations of which were determined on the basis of long-range, interhelical NOEs. The fourhelices are arranged in an up-up-down-down orientation, as found in other four-helical bundlecytokines. The overall topology of MH35-LIF is similar to that of the X-ray crystallographicstructure for murine LIF [Robinson et al. (1994) Cell, 77, 1101–1116]. Differencesbetween the X-ray structure and the solution structure are evident in the N-terminal tail, wherethe solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystalstructure and the small antiparallel -sheet encompassing residues in the N-terminus andCD loop in the crystal structure is less stable.     

The role of skylight polarization in the orientation of a day-migrating bird species

To assess the role of skylight polarization in the orientation system of a day-migrating bird, Yellow-faced Honeyeaters (Lichenostomus chrysops, Meliphagidae) were tested in funnel cages for their directional preferences. In control tests in the natural local geomagnetic field under the clear natural sky, they preferred their normal migratory course. Manipulations of the e-vector by depolarizing the skylight or rotating the axis of polarization failed to affect the orientation as long as the natural geomagnetic field was present. When deprived of magnetic information, the birds continued in their normal migratory direction as long as they had access to information from the natural sky, or when either the sun or polarized light was available. However, when sun was hidden by clouds, depolarizers caused disorientation. — These findings indicate that polarized skylight can be used for orientation when no other known cues are available. However in the hierarchy of cues of this species, the polarization pattern clearly ranks lower than information from the geomagnetic field.     

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.     

Selection of a proteolytic enzyme to solubilize lean beef tissue

Many proteases are available for the hydrolysis of various protein substrates. The qualitative effect of most experimental variables on reaction progress is known, so it is possible to devise a rational procedure for selecting the best enzyme. Reaction time and enzyme concentration should be chosen in the region where they have little effect on reaction progress. Substrate concentration should be low to avoid possible product inhibition. Each enzyme should be tested at its optimum pH, and at a range of temperatures around (mainly below) the reported temperature optimum. Enzyme cost and other relevant factors should also be considered in the enzyme selection. Using this selection procedure Alcalase was chosen as the most appropriate enzyme for solubilizing lean beef tissue.     

A role for intracellular calcium and calmodulin in the release of triiodothyronine from human thyroid-cell monolayer cultures

Human thyroid cells in monolayer responded to acute stimulation by TSH with an increase in the secretion of T₃. This process appeared to be dependent on a rise in the cytosolic calcium concentration since the antagonist of intraceliular calcium mobilization, TMB-8, was found to inhibit the release of T₃ in response to TSH. The importance of intracellular calcium was further shown using the agent veratridine which increases the free calcium level within cells; veratridine potentiated the stimulation of T₃ secretion by TSH and itself stimulated the release of T₃ to a level higher than that seen in the presence of TSH alone. The calcium ionophore A23197 produced a biphasic effect on T₃ secretion from human thyroid monolayers; at low concentrations, A23187 caused a decrease in both unstimulated and TSH-stimulated T₃ secretion but above a concentration of 1 M, T₃ secretion was increased. The calmodulin antagonist W7 was found to inhibit T₃ release in response to TSH, indicating a role for calmodulin in mediating the effects of intracellular calcium on T₃ secretion.     

Calmodulin activation of adenylate cyclase in the mouse B16 melanoma.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.     

THE PERMISSIVE ROLE OF HYPERTHERMIA IN THE DISAGGREGATION OF BRAIN POLYSOMES BY l-DOPA OR d-AMPHETAMINE1

Abstract— l -DOPA or d -amphetamine administration disaggregates brain polyribosomes in animals maintained in an environment warm enough (26°C) so that the drugs concurrently elevate their body temperatures to above 39°C. The production of equivalent hyperthermia (by keeping control rats at ambient temperatures of 40–44° C) does not cause similar disaggregation of brain polysomes. Hence, the role of hyperthermia in the drug-induced disaggregation is permissive.     

Morphologic and pathologic findings in teeth and jaws from the middle bronze age (Pitten, Lower Austria)

The detailed examination of the masticating apparatus of 18 skulls from the Middle Bronze Age in Pitten (Lower Austria) revealed numerous pathological findings of the teeth. Most remarkable were frequency and extent of dental abrasions. In addition to it, indications on inflammatory processes in the marginal parodontium were obtained, combined with partly immense formations of concrements on the surfaces of the teeth. The incidence of dental caries was relatively low. Abnormal positions of the teeth and pathological processes concerning the development of dentition as well as the eruption of the wisdom teeth could be observed repeatedly. Conclusions on insufficiences of the oral hygiene in this time follow especially from the concrement findings and from the inflammatory reactions of the marginal parodontium.