A rapid simple radiometric assay for phospholipase A2 activity

A rapid, simple radiochemical assay for phospholipase A₂ (PLA₂) activity is described in which incubations of homogenized tissue were directly applied to columns of silica gel absorbent. The metabolites and substrate were eluted from the columns with two different solvent systems so that each could be separated and quantified.     

Nuclear spin-relaxation studies of hydrated elastin.

The nuclear magnetic spin-lattice and transverse relaxation processes for the ¹H and ²D nuclei in purified elastin (ligamentum nuchae), exchanged and hydrated with excess D₂O, have been studied in the temperature range 276°–340°K. The ²D relaxation results clearly show the presence of D²O (1) external to the bulk elastin sample, (2) in spaces within the bulk elastin, and (3) as an integral part of the protein on a molecular level. It is shown from these measurements that the protein on a molecular level. It is shown from these measurements that the water content of the protein itself changes from ～0.8 g D₂O/g dry elastin at ～280°K to ～0.2 g D₂O/g dry elastin at ～335°K, a decrease of 400%. The D₂O content of the interfiber spaces decreases by less than 20% over the same temperature range. This fact throws considerable doubt on the validity of the values of β, the thermal expansion coefficient of elastin, used by other workers in discussion of the elastic mechanism in elastin. The elastin proton transverse relaxation shows the presence of three regions in elastin having different degrees of molecular mobility. These are assigned to protons associated with the crosslinks, a fairly mobile, hydrophobic, and low-water-content region, and a more mobile higher water-content region. The temperature variation of the relative proportions of these three regions is explained in terms of a hypothetical temperature-composition phase diagram in which the two mobile regions are represented as two partially miscible phases with different negative temperature coefficients of ‘solubility’ in water. The implications of these observations for current views of the nature of elastin are assessed. It is concluded that the spin-relaxation results are consistent with a multiphase structural model for elastin. An approximate sorption isotherm for the water/elastin system is reported and shows the relatively weak nature of the water/elastin interaction.     

Aporphine alkaloids and lignans formed in response to injury of sapwood in Liriodendron tulipifera

Two new phenolic aporphine alkaloids, (+)-lirioferine and (+)liriotulipiferine, were isolated from discolored sapwood of L. tulipifera. Injury to the tree stem greatly stimulated biosynthesis of glaucine and phenolic alkaloids related to glaucine including thaliporphine, predicentrine, N-methylaurotetanine and corunine as well as the above two compounds. Injury also stimulated synthesis of oxoaporphine related and other polymeric pigments. Corunine was responsible for at least part of the color of discolored sapwood. None of the above compounds except glaucine was detected in normal sapwood or heartwood of L. tulipifera. Thus, formation of alkaloids and lignans in discolored sapwood differs both quantitatively and qualitatively from that observed during the normal transition of sapwood to heartwood in this tree. The compounds formed in response to injury differed substantially from one zone of injury to another within the same tree.     

Temporoparietal fascial flap for orbital and eyelid reconstruction.

The temporoparietal fascial flap is a recognized technique for the transfer of vascularized tissue in the craniofacial region. The flap has a predictable axial vessel, provides thin vascularized tissue, and can be harvested with minimal donor-site morbidity. The temporoparietal fascial flap is well suited for orbital or eyelid reconstruction because of its proximity to the orbit. The flap is useful for reconstruction of anatomic barriers between the orbit, intracranial cavity, and paranasal sinus spaces. We present four patients in whom the temporoparietal fascial flap was used for orbital reconstruction following extirpative surgery for orbital neoplasm and two patients in whom the flap was used for lower eyelid and malar reconstruction.     

Fungal Elicitor-Mediated Responses in Pine Cell Cultures : III. Purification and Characterization of Phenylalanine Ammonia-Lyase

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is involved in the lignification of pine suspension cultures in response to an elicitor prepared from an ectomycorrhizal fungus. To elucidate the molecular basis of this response, PAL was purified to homogeneity from jack pine (Pinus banksiana) suspension cultures using anion-exchange and chromatofocussing fast protein liquid chromatography. Physical characterization of the enzyme revealed that pine PAL was similar to PAL from other plant sources. Pine PAL had a pH optimum of 8.8, an isoelectric point of 5.75, and a native molecular mass of 340 kilodaltons. The enzyme appears to be a tetramer composed of 77 kilodalton subunits. Chromatographic and western blot analyses were used to identify possible isoenzymic changes in pine PAL in response to elicitation and to determine the nature of the increase in PAL activity associated with inducible lignification in these cultures. Only one species of PAL was detected in P. banksiana cell cultures and increased quantities of this protein were correlated with the enhanced enzyme activity observed in elicited cultures. P. banksiana PAL was not feedback-inhibited by a wide range of phenolic compounds at micromolar concentrations, including the reaction product cinnamic acid. Our data suggest that a different set of metabolic and molecular controls must be in place for the regulation of PAL in pine.     

Tubulin-isotype analysis of two grass species-resistant to dinitroaniline herbicides

Trifluralin-resistant biotypes of Eleusine indica (L.) Gaertn. (goosegrass) and Setaria viridis (L.) Beauv. (green foxtail) exhibit cross-resistance to other dinitroaniline herbicides. Since microtubules are considered the primary target site for dinitroaniline herbicides we investigated whether the differential sensitivity of resistant and susceptible biotypes of these species results from modified tubulin polypeptides. One-dimensional and two-dimensional polyacrylamide gel electrophoresis combined with immunoblotting using well-characterised anti-tubulin monoclonal antibodies were used to display the family of tubulin isotypes in each species. Seedlings of E. indica exhibited four -tubulin isotypes and one -tubulin isotype, whereas those of S. viridis exhibited two -tubulin and two -tubulin isotypes. Comparison of the susceptible and resistant biotypes within each species revealed no differences in electrophoretic properties of the multiple tubulin isotypes. These results provide no evidence that resistance to dinitroaniline herbicides is associated with a modified tubulin polypeptide in these biotypes of E. indica or S. viridis.Abbreviations 1-D, 2-D one-, two-dimensional - ID₅₀ dose causing 50% inhibition - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - S susceptible - R resistant We gratefully acknowledge Professor B.J. Gossett (Clemson University, South Carolina, USA) and Professor I.N. Morrison (University of Manitoba, Winnipeg, Canada) for providing seed stocks of Eleusine indica and Setaria viridis, respectively. Antibodies TAT-1 and KMX-1 were a kind gift from Professor K. Gull (University of Manchester, UK). Technical assistance with seed germination was provided by Ms. K.E. Cronin. We acknowledge financial support from the Science and Engineering Research Council for a studentship to T.R.W.     

The effect of primary and secondary immunization on the lymphoid tissues of the carp, Cyprinus carpio L

Mirror carp immunized with human gamma globulin (HGG) in Freund's complete adjuvant (FCA) show a proliferative response involving cells whose cytoplasm stains deep red with methyl green-pyronin (pyroninophilic cells). This response occurs particularly in the haemopoietic parenchyma of the pronephros and mesonephros. It peaks at week 3, with the formation of clusters of pyroninophilic cells in the pronephros. Immunization with Aeromonas salmonicida elicited a less intense pyroninophilic response but caused a larger increase in pigment-containing cells. After a secondary immunization with HGG in FCA, a distinct response was observed in the spleen: Pyroninophilic cells collected within the ellipsoid sheaths in large numbers and formed nodules. The reticulum of such nodules acquired spherical proportions and resembled the white pulp reticulum of the tetrapod spleen. The roles of such pyroninophilic cells and the possibility that aggregations of them may be functionally analogous to homoiotherm germinal centres are discussed.     

Acute effects of testosterone on serum PGE2 levels in male dogs

Serum prostaglandin levels are influenced by testosterone. To test the hypothesis that the effect of testosterone is mediated through the prostate gland, testosterone was given acutely to intact and to prostatectomized male dogs. Intact dogs responded to testosterone with an abrupt, transient rise in plasma PGE₂ levels; prostatectomized dogs did not respond. We conclude that testosterone has an acute effect on the prostate gland which results in release of PGE₂ into the blood stream.