Grass group I allergens (beta-expansins) are novel, papain-related proteinases.

Expansins are a family of proteins that catalyse long-term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the alpha-expansins and beta-expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post-translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino-acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active-site amino acids of the C1 (papain-like) family of cysteine proteinases. Moreover, the significantly homologous alpha-expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.     

Recent rhinos do not lack character(s): neither do fossil rhinos!

Concepts, methods, and interest of phylogenetic reconstruction are briefly examined. As large data sets are considered and refutable results are proposed, there is no need to use the argument of authority concerning relationships between taxons. Cladistic analysis in vertebrate palaeontology has gained considerable strength in the last decade, based on sets of hundreds of anatomical characters. One example is selected, which concerns the rhino family, i.e. rhinocerotids. Although underrepresented in recent times, these perissodactyl mammals flourished throughout the Cenozoicera (4 recent genera vs. 50 fossil genera). The main results of a recent cladistic analysis of elasmotheriine rhinocerotids, based on 282 anatomical characters, are listed. Such results concern phylogenetics (monophyly of both elasmotheriines and recent rhinos; branching of elasmotheriines among rhinocerotids) and methodology (definition of a "branching group"; location and processing of homoplasy; influence of taxonomic sampling). The implications are both biostratigraphical and palaeobiogeographical (evolution of the diet and spatial distribution; intercontinental dispersals; ghost lineages and heuristic use of the phylogenetic tree). Finally, forthcoming developments of the available data set for rhinocerotids are examined: controversial phylogenetic relationships among recent rhinos will be refined (thanks to close extinct taxa) and an exhaustive phylogeny of fossil and recent rhinocerotids will be reconstructed (54 genera).     

Anther wall and pollen development in Ophrys mammosa L. (Orchidaceae)

The objective of this study was to investigate anther wall and pollen development in Ophrys mammosa. Primary sporogen tissue resembles longitudinal cells with divided archeosporal cells. Thereafter these primary sporogen tissue cells re-divide anticlinally and periclinally forming secondary sporogen tissue. Microsporogenesis was successive type. Microgametogenesis occurred at the distal poles of the microspores. In addition, dense starch accumulation was detected in the pollen. Pollinia and massulae are separated from each other by dead cells filled with callose, according to histochemical preparations. The anther wall was a four-layered “monocotyledon” type. There was ring-like wall thickening in the endothecium. The tapetum is of the glandular type. When these two developmental processes are compared, it is seen that the anther wall has become mature by the sporogen tissue phase and is composed of only epidermis and endothecium at the beginning of microgametogenesis.     

Development of structure-less pollen wall inCeratophyllum demersum L. (Ceratophyllaceae)

Developmental process of structure-less exine is studied in a hydrophilous plant,Ceratophyllum demersum L., with electron microscopy. The plant shows a characteristic feature in tetrad formation. A callose wall is not synthesized and exine initiation does not occur during the tetrad stage. After release of microspores, a trilaminar layer with two electron-dense lines is formed in the surface of each microspore. The trilaminar layer develops to a thin structure-less exine that is considered to consist of only an endexine. The unusual exine would be an adaptive feature for submersed pollination in fresh water.     

Immunochemical studies of Lolium perenne (rye grass) pollen allergens, Lol p I, II, and III

It was reported earlier that human immune responses to three perennial rye grass (Lolium perenne) pollen allergens, Lol p I, II, and III, are associated with histocompatibility leukocyte antigen (HLA)-DR3. Rye-allergic people are often concordantly sensitive to all three of these allergens. Since earlier studies suggested that these antigens are non-cross-reactive, their immunologic relatedness by double antibody radioimmunoassay (DARIA) was studied in order to understand further the immunochemical basis for the concordant recognition of the three allergens. Direct binding DARIA studies were performed with human sera from 189 allergic subjects. Inhibition DARIA studies were carried out with 17 human sera from grass-allergic patients who were on grass immunotherapy, one goat anti-serum, and six rabbit antisera. None of the sera detected any significant degree of two-way cross-reactivity between Lol p I and II, or between Lol p I and III. However, the degree of two-way cross-reactivity between Lol p II and III exhibited by individual human and animal antisera varied between undetectable and 100%. In general, the degree of cross-reactivity between Lol p II and III was higher among human sera than among animal sera. Taken together with earlier findings that antibody responses to Lol p I, II and III are associated with HLA-HDR3, and that most Lol p II and III responders are also Lol p I responders, but not vice versa, our present results suggest the following: the HLA-DR3-encoded Ia molecule recognizes a similar immunodominant Ia recognition site (agretope) shared between Lol p I and Lol p II and/or III; in addition, Lol p I appears to contain unique Ia recognition site(s) not present in Lol p II and III. However, further epitope analyses are required to investigate these possibilities.     

Properties of group I allergens from grass pollen and their relation to cathepsin B, a member of the C1 family of cysteine proteinases.

Expansins are a family of proteins that catalyze pH-dependent long-term extension of isolated plant cell walls. They are divided into two groups, alpha and beta, the latter consisting of the grass group I pollen allergens and their vegetative homologs. Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall. Our group reported papain-like properties of beta-expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin-mediated wall extension. Here, we report additional data showing that beta-expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases. Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N-glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein. We also show that deglycosylated, full-length allergen is not activated efficiently and therefore is relatively stable. Motif and homology search tools detected significant similarity between beta-expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino-acid residues, as well as a conserved location of these features within these molecules. Lastly, we demonstrate by site-directed mutagenesis that the conserved His104 residue is involved in the catalytic activity of beta-expansins. These results indicate a common origin of cathepsin B and beta-expansins, especially if taken together with their previously known biochemical properties.     

Plasmodial tapetum and pollen wall development in Butomus umbellatus (Butomaceae)

This report describes the ultrastructural development of plasmodial tapetum and pollen wall of Butomus umbellatus. The tapetum contains extensive arrays of rough endoplasmic reticulum, vesicles from which are responsible for the formation of sporopollenin-like bodies. The tapetum is also involved in the formation of other forms of sporopollenin precursors. Development of pollen wall continues after microspores are released from their callosic walls; they are then enclosed by plasmodial tapetum. The activity and products of the plasmodial tapetum show substantial correlation with pollen wall development, particularly ektexine formation. In B. umbellatus, the tapetum intrudes into the anther locule at early microspore stage. This timing of plasmodial intrusion occurs at a later stage of pollen development as compared to those in the advanced monocotyledons. We report the rough endoplasmic reticulum origin of sporopollenin-like bodies and their occurrence in the plasmodial tapeta of B. umbellatus.     

Magnetic fields generated by an induction heating (IH) cook top do not cause genotoxicity in vitro

The use of induction heater (IH) cook tops in homes has become widespread, especially in Japan, but there are concerns about the safety of intermediate frequency (IF) electromagnetic fields associated with these cooking appliances. Since the cellular genotoxicity of IF magnetic fields has not been examined in cultured cells, we examined the effects of these fields at a magnetic flux density of 532 +/- 20 microT at 23 kHz, using an exposure unit with a built-in CO2 incubator. Exposure to the IF magnetic field at 532 microT for 2 h did not affect the growth of CHO-K1 cells and caused no mutagenic effects in bacterial mutation assays. Exposure to the IF magnetic field for 2 h induced neither single nor double DNA strand breaks in comet assays, and caused no significant change in the mutation frequency at the HPRT locus compared to sham exposure. The magnetic field used in this study is more than 80 times higher than the level recommended as safe in the International Commission on Non-ionizing Radiation Protection (ICNIRP) guidelines. From these results, we suggest that exposure to an IF magnetic field for 2 h does not cause cellular genotoxicity in bacteria and in Chinese hamster cells. However, the possibility of effects on other cellular functions remains, and further studies on the cellular effects of IF magnetic fields are required.     

Glycosylasparaginase activity requires the alpha-carboxyl group, but not the alpha-amino group, on N(4)-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond in N(4)-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine in the catabolism of N-linked oligosaccharides. A deficiency, or absence, of enzyme activity gives rise to aspartylglycosaminuria, the most common disorder of glycoprotein metabolism. The enzyme catalyzes the hydrolysis of a variety of asparagine and aspartyl compounds containing a free alpha-carboxyl group and a free alpha-amino group; computational studies suggest that the alpha-amino group actively participates in the catalytic mechanism. In order to study the importance of the alpha-carboxyl group and the alpha-amino group on the natural substrate to the reaction catalyzed by the enzyme, 14 analogues of the natural substrate were studied where the structure of the aspartyl group of the substrate was changed. The incremental binding energy (DeltaDeltaGb) for those analogues that were substrates was calculated. The results show that the alpha-amino group may be substituted with a group of comparable size, for the alpha-amino group contributes little, if any, to the transition state binding energy of the natural substrate. The alpha-amino group position acts as an "anchor" in the binding site for the substrate. On the other hand, the alpha-carboxyl group is necessary for enzyme activity; removal of the alpha-carboxyl group or changing it to an alpha-carboxamide group results in no hydrolysis reaction. Also, N-acetyl-D-glucosamine is not sufficient for binding to the active site for efficient hydrolysis by the enzyme. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction for the enzyme. However, the results raise a question as to an important role for the alpha-amino group in the catalytic mechanism as indicated in computational studies.     

Polyplax serrata: cutaneous cytologic reactions in mice that do (CFW strain) and do not (C57BL strain) develop resistance.

The cutaneous inflammatory response of CFW mice at 2 weeks after infestation with lice, Polyplax serrata, was more rapid, produced five times as many polymorphonuclear neutrophils, half as many eosinophils, and in the last 8 weeks twice as many mast cells and fibroblasts as that of the C57BL mice. It was concluded that possibly a factor chemotactic for neutrophils was missing in the sequential response to infestation and prevented the full expression of acquired resistance in the C57BL mice, as compared both to the CFW and to the RML mice of earlier work. Possible explanations for other differences observed are discussed.     

Lipoprotein(a) binds to fibronectin and has serine proteinase activity capable of cleaving it.

The plasma concentration of human lipoprotein(a) [Lp(a)] is correlated with the risk of heart disease. A distinct feature of the Lp(a) particle is the apolipoprotein (a) [apo(a)], which is associated with apoB-100, the main protein component of low-density lipoprotein. We now report that apo(a), which has extensive homology to plasminogen, binds to immobilized fibronectin. The binding of Lp(a) was localized to the C-terminal heparin-binding domain of fibronectin. Incubation of Lp(a) with fibronectin resulted in fragmentation of fibronectin. The cleavage pattern, as visualized by gel electrophoresis and immunoblotting, was reproducibly obtained with Lp(a) purified from five different individuals and was distinct from that obtained upon proteolysis of fibronectin by plasmin or kallikrein. The use of synthetic peptide substrates demonstrated that the amino acid specificity for Lp(a) was arginine rather than lysine. The proteolytic activity of Lp(a) was localized to apo(a) and experiments with inhibitors indicated that the proteolytic activity was of serine proteinase-type.     

Altered specificity of lactococcal proteinase p(i) (lactocepin I) in humectant systems reflecting the water activity and salt content of cheddar cheese

By using various humectant systems, the specificity of hydrolysis of alpha(s1)-, beta-, and kappa-caseins by the cell envelope-associated proteinase (lactocepin; EC 3.4.21.96) with type P(1) specificity (i.e., lactocepin I) from Lactococcus lactis subsp. lactis BN1 was investigated at water activities (a(w)) and salt concentrations reflecting those in cheddar type cheese. In the presence of polyethylene glycol 20000 (PEG 20000)-NaCl (a(w) = 0.95), hydrolysis of beta-casein resulted in production of the peptides comprising residues 1 to 6 and 47 to 52, which are characteristic of type P(III) enzyme activity (lactocepin III) in buffer. The fragment comprising residues 1 through 166, inclusive (fragment 1-166), which is typical of lactocepin I activity in buffer systems, was not produced. Similarly, peptide 152-160 from kappa-casein, which is usually produced in aqueous buffers exclusively by lactocepin III, was a major product of lactocepin I. Most of the specificity differences obtained in the presence of PEG 20000-NaCl were also obtained in the presence of PEG 20000 alone (a(w) = 0.99). In addition, alpha(s1)-casein, which normally is resistant to lactocepin I activity, was rapidly hydrolyzed in the presence of PEG 20000 alone. Hydrolysis of casein in the presence of PEG 300-NaCl or glycerol-NaCl (both having an a(w) of 0.95) was generally as expected for lactocepin I activity except that beta-casein peptide 47-52 and kappa-casein fragment 1-160 were produced; both of these are normally formed by lactocepin III in buffer. The differences in lactocepin specificity obtained in the humectant systems can be attributed to a combination of a(w) and humectant hydrophobicity, both of which are parameters that are potentially relevant to the cheese-ripening environment.     

Doppel and PrP(C) do not share the same membrane microenvironment

Doppel is a paralog of the normal prion protein, PrP^C. It has been suggested that Doppel can compensate for the absence of PrP^C in PrP^0/0 mice. In this work, we tested whether Doppel and PrP^C share the same cell location, thereby sharing the same neighboring cell components, probably required to share the same cell function. Our results show that, at detergent conditions in which membrane rafts were intact, neither PrP^C and Doppel co-immunoprecipitate with the appropriate antibodies, nor was Doppel retained by a Cu²⁺IMAC resin, as PrP^C does. This indicates that, although Doppel is a raft-associated protein as is PrP^C, both proteins are not present in the same membrane microenvironment, and they probably do not perform the same function.     

Self-splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family.

A new family of protein domains consisting of 50-80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group II introns; eubacterial genomes and plasmids; and phages. The name "EX1HH-HX3H" was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EX1HH and HX3H. The "His" motifs often alternate with amino- and carboxy-terminal motifs of a new type of Zn-finger-like structure CX2,4CX29-54[CH]X2,3[CH]. The EX1HH-HX3H domain in eubacterial E2-type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EX1HH-HX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group II introns encoding the EX1HH-HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules.