R. B. Knox

Pollen-Wall Proteins: Localization and Enzymic Activity

Cytochemical methods have been used to examine the distribution of acid phosphatase, ribonuclease, esterase, amylase and protease activity and protein in the walls of pollen grains and spores. Enzyme activity was detected in the walls of 50 angiosperm pollens, in pine, and in the spore wall of a species of Equisetum. Activity was absent from the 2 species of ferns examined. The survey covered all major structural pollen types. In all cases enzyme activity was associated principally with the intine, the inner cellulosic part of the wall. In all aperturate grains activity was concentrated in or around the apertural intine; that is, over or near the potential sites of emergence of the pollen tube. Protein concentrations in the intine at these sites could be demonstrated in several pollen types by staining methods and by absorption at 285 nm. Developmental study showed that the enzymes are incorporated in the intine during the early period of wall growth following the release of the spores from the meiotic tetrads. During this period, stratified nbosomal endoplasmic reticulum lies adjacent to the inner spore wall over the areas of incorporation. In Cosmos bipinnatus, a composite, the material is incorporated as ribbons or leaflets, which interleave with cellulose lamellae. In other species the wall protein may take the form of granules, tubules or vesicles, embedded in the intine cellulose. At maturity the intine is separated from the spore cytoplasm by an intact plasmalemma, so the wall enzymes are to be regarded as being extracellular. In some species enzyme activity was detected in materials on the surface of the pollen exine derived probably from the surrounding tapetal tissue; however, this was usually insignificant compared with that shown by the intine. The intine enzymes are very readily leachable, and their function is probably connected with the early nutrition of the pollen tube and the penetration of the stigma. It is likely that the wall proteins make up a very large proportion of mobile protein of the pollen grain, and they may therefore be important in pollen allergenicity.SUMMARY Recent years have witnessed significant progress towards understanding the molecular mechanism of nucleotide excision repair in living cells. Biochemical studies in Escherichia coli, and genetic and molecular studies in lower and higher eukaryotes have revealed an unexpected complexity suggesting interesting protein–protein and protein–DNA interactions. This review considers selected aspects of nucleotide excision repair in E. coli, Saccharomyces cerevisiae and mammalian cells, with a particular emphasis on new observations and on models that may provide explanations for the complexity evident from genetic and biochemical studies.

Botanical immunocytochemistry: a review with special reference to pollen antigens and allergens

Antigen-antibody recognition is an essential component of the immune system of defence reactions in vertebrates, but is foreign to plant cell biology. Plants exhibit some of the features of immunity: they are able to discriminate self from non-selL though they have largely lost the capacity for phagocytosis, now confined to certain algae (see review by Clarke & Knox, 1979). Plant responses appear to be largely cellular in nature, resembling allograft recognition seen in coelenterates, and there is little evidence as yet for hurnoral responses exemplified by the circulating antibodies of cyclostomes and higher vertebrates (Cooper, 1976). The closest analogy in plants is perhaps the production of inhibitory phytoalexins by cells of some species in

Immunofluorescent localization of two water-soluble glycoproteins including the major allergen from the pollen of ryegrass, Lolium perenne.

SummaryTwo major glycoproteins have been localized in sectioned grains of ryegrass pollen by direct and indirect immunofluorescence methods using Fluorescein isothiocyanate (FITC)-labelled IgC fractions of antisera. These glycoproteins are the major allergen Group 1 allergen, and a principal antigen Antigen A. Four methods of fixation were employed: freeze-drying, methanol, 2.5% glutaraldehyde and 4% paraformaldehyde for 1 h at 4°C. The post-embedding staining technique of immunocytochemistry was used: anthers were embedded directly, or after dehydration, in JB-4 plastic resin and antibody reacted with sectioned pollen.The effects of these fixatives on the antibody combining sites of the antigens were quantified by a solid phase radioimmunoassay using [125I]protein A to measure antibody binding. Glutaraldehyde was the only fixative to significantly depress antibody binding of both Antigen A and Group 1 allergen to their homologous antisera. This radioimmunoassay was modified to reyeal that FITC conjugation to either antibody did not impair antigen binding. In mature pollen, these antigens were located in the cytoplasm and in the complex wall. In developing grains early in the maturation period, specific fluorescence was concentrated at the periphery of the cytoplasm.

Rapid Batch Fractionation of Ryegrass Pollen Allergens

A rapid batch fractionation procedure employing ion-exchange matrices has been used to fractionate ryegrass pollen antigens and allergens. After sequential elution through CM-Sephadex and DEAE-Sephadex, a group of four electrophoretically distinct allergens with a molecular weight of approximately 32,000 was fractionated. These allergens correspond to the group I allergens previously isolated by Johnson and Marsh. This material may be suitable for clinical use where a characterised group I allergen preparation is required.