Abscisic acid,phaseic acid and gibberellin contents associated with dormancy and germination in barley

Analyses of abscisic acid (ABA), ent -kaurenoids and gibberellins (GAs) showed that there were major changes in the contents of these compounds associated with germination of after-ripened barley ( Hordeum vulgare cv. Schooner and cv. Proctor) grain but not in hydrated dormant grain. Embryos from dormant and after-ripened dry grain contained similar amounts of ABA, of ent -kaurenoids and of GAs, determined by gas chromatography-mass spectrometry-selected ion monitoring. In embryos of after-ripened grain, ABA content decreased rapidly after hydration and ABA appeared to be metabolized (inactivated) to phaseic acid (PA) rather than diffusing into the endosperm or the surrounding medium as previously thought. Similar changes in ABA occurred in hydrated dormant grain during germination in darkness. Accumulation of ent -kaurenoids and GAs, including GA_1, the first biologically active GA in the early 13-hydroxylation biosynthetic pathway, occurred to a much greater extent in after-ripened than in dormant grain and these changes occurred mainly after 18 h of hydration when ABA had already decreased and germination was occurring. The block in ent -kaurenoid and GA synthesis in dormant grain appeared to occur prior to ent -kaurene in the biosynthetic pathway. These results are consistent with the view that ABA is the primary effector of dormancy and that after-ripening involves the development of the ability to reduce the amount of ABA quickly following hydration. Accumulation of GAs does not appear to be causally related to loss of dormancy but it does appear to be related to germination.     

Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs

Oxidant stress, in vivo or in vitro, isknown to induce oxidative changes in human red blood cells (RBCs). Ourobjective was to examine the effect of augmenting RBC glutathione(GSH) synthesis on 1) degenerative protein loss and2) RBC chemokine- and free radical-scavenging functions inthe oxidatively stressed human RBCs by using banked RBCs as a model.Packed RBCs were stored up to 84 days at 1-6°C in Adsol or inthe experimental additive solution (Adsol fortified with glutamine,glycine, and N-acetyl-L-cysteine). Supplementingthe conventional additive with GSH precursor amino acids improved RBCGSH synthesis and maintenance. The rise in RBC -glutamylcysteineligase activity was directly proportional to the GSH content andinversely proportional to extracellular homocysteine concentration,methemoglobin formation, and losses of the RBC proteins band 3, band4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffyantigen (P < 0.01). Reduced loss of Duffy antigencorrelated well with a decrease in chemokine RANTES (regulated uponactivation, normal T-cell expressed, and secreted) concentration. Weconclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSHsynthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, inwhich RBCs are subjected to chronic/acute oxidant stresses.

     

Characterisation of a homogeneous plant aminoaldehyde dehydrogenase

According to our knowledge, this is the first purification method developed, enabling isolation of a homogeneous aminoaldehyde dehydrogenase (AMADH) from etiolated pea seedlings. The procedure involved initial purification with precipitants followed by three low pressure chromatographic steps. Partially purified enzyme was further subjected to fast protein liquid chromatography on a Mono Q column and to affinity-interaction chromatography on 5'-AMP Sepharose. Purity of the final enzyme preparation was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and chromatofocusing. Pea AMADH exists as a tetramer of 230 kDa in the native state, a molecular mass of one subunit was determined as 57 kDa. The enzyme was found to be an acidic protein with pI 5.4. AMADH showed a broad substrate specificity utilising various aminoaldehydes (C3-C6) as substrates. The best substrate of pea AMADH was 3-aminopropionaldehyde, the enzyme also efficiently oxidised 4-aminobutyraldehyde and omega-guanidinoanalogues of the aminoaldehydes. Pea AMADH was inhibited by SH reagents, several elementary aldehydes and metal-binding agents. Although AMADH did not oxidise betaine aldehyde at all, the N-terminal amino acid sequence of the enzyme shows a high degree of homology with those of plant betaine aldehyde dehydrogenases (BADHs) of spinach, sugar beet and amaranth. Several conserved amino acids were found in comparison with BADH from cod liver of known crystal structure.     

Calumenin interacts with serum amyloid P component

We recently reported the identification of human calumenin, a novel Ca(2+) binding, transformation-sensitive and secreted protein [Vorum et al. (1998) Biochim. Biophys. Acta 1386, 121-131; Vorum et al. (1999) Exp. Cell Res. 248, 473-481] belonging to the family of multiple EF-hand proteins of the secretory pathway that include reticulocalbin, ERC-55, Cab45 and crocalbin. In order to further investigate the extracellular functions of calumenin we immobilized the recombinant protein to a column. After application of a placental tissue extract we were able to elute one protein that interacts with calumenin in the presence of Ca(2+). Amino acid sequencing identified this protein as serum amyloid P component (SAP). Furthermore, we verified and characterized the calumenin-SAP interaction by the surface plasmon resonance technique. The findings indicate that calumenin may participate in the immunological defense system and could be involved in the pathological process of amyloidosis that leads to formation of amyloid deposits seen in different types of tissues.     

Altered binding of mutated presenilin with cytoskeleton-interacting proteins

The majority of familial Alzheimer's disease (AD) cases are linked to mutations on presenilin 1 and 2 genes (PS1 and PS2). The normal function of the proteins and the mechanisms underlying early-onset AD are currently unknown. To address this, we screened an expression library for proteins that bind differentially to the wild-type PS1 and mutant in the large cytoplasmic loop (PS1L). Thus we isolated the C-terminal tail of the 170 kDa cytoplasmic linker protein (CLIP-170) and Reed-Sternberg cells of Hodgkin's disease-expressed intermediate filament-associated protein (Restin), cytoplasmic proteins linking vesicles to the cytoskeleton. PS1L binding to CLIP-170/restin requires Ca(2+). Treating cells with thapsigargin or ionomycin increased the mutated PS1 in CLIP-170 immunoprecipitates. Further, PS1 and CLIP-170 co-localize in transfected cells and neuronal cultures.     

Diel variation in habitat use by planktivores in field enclosure experiments: the effect of submerged macrophytes and predation

The importance of submerged macrophytes and predation risk for habitat use by 0+ perch ( Perca fluviatilis ) and 0+ roach ( Rutilus rutilus ) was investigated in triplicate 78-m² field enclosures with and without macrophytes in the middle. During three experimental runs, habitat use by fish were monitored every 6 h with Breder traps. Each period included 2 days of fish monitoring before stocking with piscivorous perch, and 2 days after. Predation risk significantly changed habitat use by 0+ perch in the morning, midday and evening, but not at night. By comparing with the unvegetated controls, we found a refuging effect of macrophytes in the morning. Under predation risk there was significant diel variation in habitat use by 0+ perch, suggesting a migration from the open water habitat at night into the macrophytes in the morning. Roach continued to use open water even after predators were stocked, but responded like perch by reducing overall activity.     

Autotetraploids and genetic mapping using common AFLP markers: the R2 allele conferring resistance to Phytophthora infestans mapped on potato chromosome 4

 Due to the complexity of tetrasomic inheritance, mapping studies in potato (Solanum tuberosum L.) are generally conducted at the diploid level. In the present study we tested the feasibility of Bulked Segregant Analysis (BSA) using a tetraploid offspring for the identification of AFLP markers linked to the R2 allele, which confers race-specific resistance to Phytophthora infestans. Eleven bulk-specific AFLP markers, detected in fingerprints of 205 AFLP primer combinations, could be mapped in a linkage group encompassing the R2 locus. The efficiency of BSA at the tetraploid level, determined by the frequency of single-dose restriction fragments (SDRF), was much higher than expected on the basis of overall genetic dissimilarity between the parental clones. The fortuitous detection of AFLPs with linkage to the R2 allele is explained on the basis of specific genetic dissimilarity between cultivated potato and the chromosomal segment introgressed from S. demissum carrying the resistant R2 allele. AFLP markers common to those with linkage to R2 were visually recognized by their electrophoretic mobility in the AFLP fingerprint in a parental clone of a reference mapping population. Using these common AFLP markers we anchored the linkage group comprising the R2 allele to potato chromosome 4. Received: 30 October 1997 / Accepted: 6 November 1997     

Plant regeneration from protoplasts isolated from friable embryogenic callus of cassava

Protoplasts were isolated from friable embryogenic callus (FEC) and from suspensions derived from FEC of cassava genotype TMS60444. Suspensions yielded the highest number of protoplasts (1.5×10⁶ protoplasts/g fresh weight). Protoplasts plated at a density of 10⁵–10⁶/ml in a medium supplemented with 0.5 mg/l α-naphthaleneacetic acid and 1 mg/l zeatin began dividing after 3 days, and after 30 days this resulted in an absolute plating efficiency as high as 2.5%. After 2 months of culture, 60% of the developed calli were highly friable and in appearance identical to the original FEC. The protoplast derived FEC was first purified through two rounds of selection of 3 weeks each before beeing cultured for regeneration of plants. This was done by culturing the protoplast-derived FEC for 11 weeks on maturation medium, yielding a maximum of 184 organized embryos per 10.000 initially cultured protoplasts. Most of the organized embryos were torpedo shaped and matured after they had been isolated from the calli and transferred to fresh medium. Mature embryos were multiplied by secondary somatic embryogenesis at high efficiency (>90%) on a medium supplemented with 8 mg/l 2,4-dichlorophenoxyacetic acid. About 30% of the mature secondary somatic embryos developed into shoots after transfer to a medium supplemented with 1 mg/l N⁶-benzylaminopurine (BAP). Shoots rooted readily on a medium without BAP. Received: 30 August 1996 / Revision received: 9 June 1997 / Accepted: 1 October 1997     

Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts.     

Interspecific crosses inHordeum (Poaceae)

A crossing programme including 30 species and 40 cytotypes within the genusHordeum was undertaken. Viable hybrids were obtained in 302 combinations, 15 of which were intraspecific. Differences in seed set and in germination were observed in crosses between different groups of species. Obtaining crosses between different taxonomic groups was generally more difficult when diploid material was used. Some species, e.g.,H. lechleri, H. jubatum, andH. brachyantherum showed a higher crossability than others. The chromosome numbers of the hybrids were usually those expected from the parental numbers but aneuploid series around the expected numbers were rather frequent. Three cases of unreduced gametes were found. Selective chromosome elimination was restricted to combinations including eitherH. vulgare orH. bulbosum.—Despite a very diverse morphology, all South American diploid species together with the two North American diploidsH. intercedens andH. pusillum appear to be closely related. The hexaploid American speciesH. procerum, H. lechleri, andH. arizonicum are also related. The two North American tetraploid speciesH. jubatum andH. brachyantherum sometimes form semifertile hybrids. The Asiatic speciesH. roshevitzii appears to be related to both North and South American taxa.