Recurrent hybridisation events between Primula vulgaris,P. veris and P. elatior (Primulaceae,Ericales) challenge the species boundaries: using molecular markers to re‐evaluate morphological identifications

Three Primula species, Primula vulgaris, P. veris and P. elatior, have been objects of fascination for gardeners and botanists over several centuries. The species are able to hybridise, and where they co‐occur, hybrids are commonly found. In Denmark, Møns Klint on the island of Møn and Købelev Skov on Lolland are examples of localities where all three species occur and where the hybrids P. × digenea, the hybrid between P. vulgaris and P. elatior, and P. × polyantha, the hybrid between P. veris and P. vulgaris, can also be found. To investigate relationships between the species and their hybrids, we sampled 168 specimens from 10 geographical locations and subjected them to genetic analysis. The samples were also identified based on morphological traits: primarily inflorescense structure, the size, shape, colour and markings of corolla and leaf basis, leaf blade texture and hairiness. After identifying species‐specific SNPs in the Internal Transcribed Spacer sequence, these were used to resolve species and hybrid boundaries and status through a cleaved amplified polymorphic sequence assay. Polymorphisms in the chloroplast trnL sequence were used as a high‐throughput marker and used to determine the maternal parent of hybrids. Ten simple sequence repeat markers were applied to obtain further insight into the genetic makeup of the accessions using structure and Introgress, providing information of genetic variability within and between populations. Our results indicated that backcrossing of P. × digenea hybrids with parental species has occurred, and that many of the P. × digenea sampled were later‐generation hybrids rather than F₁s. Analyses of P. × polyantha specimens show mostly the expected pattern for primary hybrids but indications of P. veris ancestry of a P. vulgaris plant was discovered. Our results further indicate that some of the specimens initially identified as P. elatior include P. vulgaris among their progenitors and thus challenge currently accepted species boundaries.     

Cellular characteristics of peripheral blood lymphocytes and tumour-infiltrating lymphocytes in patients with gynaecological tumours

 Immunotherapy of gynaecological cancer with tumour-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) has become a valid treatment modality with varying degrees of success in obtaining an antitumour response. TIL consist of lymphocytes, mainly T cells and minor populations of natural killer cells or B cells. Conventional cytogenetic studies of tumour cells from patients with breast and ovarian cancer have shown multiple chromosomal abnormalities including chromosomes 7 and 12. This study was designed to analyse the surface further, as well as investigate the intracellular, characteristics of TIL by multicolour flow cytometry and the cytogenetic features by fluorescence in situ hybridization. Tumour cell, peripheral blood and TIL samples from 25 patients (15 ovarian tumours, 8 breast cancers, 1 uterine sarcoma, 1 cervical carcinoma) were analysed for their phenotype, the expression of major cytokines [interleukin-2 (IL-2), IL-4 and interferon γ (IFNγ)], their proliferation rate, their cytotoxic ability and for the presence of numerical aberrations of chromosomes 7 and 12. All the tumour cells showed a high frequency of numerical aberration in chromosomes 7 and 12, especially trisomies or tetrasomies and combined aberrations. Trisomies of both chromosomes also occured at a low percentage in TIL and PBL. Received: 20 June 1996 / Accepted: 4 January 1997     

Similar Organization of the Lipopolysaccharide-Binding Protein (LBP) and Phospholipid Transfer Protein (PLTP) Genes Suggests a Common Gene Family of Lipid-Binding Proteins

The transfer of lipids in aqueous environments such as serum has been attributed to a recently characterized class of proteins. Abnormal regulation of serum lipids by these proteins is thought to be a key event in the pathophysiology of cardiovascular diseases. Lipopolysaccharide (endotoxin) binding protein (LBP) was identified by virtue of its ability to bind bacterial lipid A. We have analyzed the exon–intron organization of the LBP gene and the nucleotide sequence of its approximately 20 kb spanning 5′- and 3′-untranslated regions. When comparing the genomic organization of LBP with that of two other genes coding for lipid transfer proteins, significant homologies were found. The LBP gene includes 15 exons, and the 2-kb promoter contains recognition elements of acute phase-typical reactants and a repetitive 12-mer motif with an as yet unknown protein-binding property. Detailed sequence comparison revealed a closer relatedness of LBP with PLTP than with CETP as demonstrated by an almost identical intron positioning. This high degree of similarity supports functional studies by others suggesting that like LBP, PLTP may also be able to bind and transport bacterial lipopolysaccharide.     

Molecular characterisation of gibberellin 20-oxidases. Structure-function studies on recombinant enzymes and chimaeric proteins

Gibberellin (GA) 20-oxidases are multifunctional enzymes that catalyse reactions at an important branch point in the GA biosynthetic pathway. These enzymes oxidise the C-20 methyl group of a diterpene carboxylic acid precursor (e.g. GA₁₂) to form an alcohol (in our case GA₁₅-open lactone) and an aldehyde (GA₂₄). The aldehyde is either oxidised to a tricarboxylic acid (GA₂₅) or, with loss of carbon-20 and lactonisation, to a C₁₉-GA (GA₉). This branching is interesting to study, because C₁₉-GA derivatives function as plant hormones in different tissues, whereas the C₂₀-GA tricarboxylic acids have no known function. We have constructed chimaeric proteins by combining a GA 20-oxidase from immature seeds of Cucurbita maxima L., which produces mainly C-20 carboxylic acids, with a 20-oxidase from Marah macrocarpus immature seeds, which forms predominantly CC₁₉-GAs. The cDNAs encoding these two very similar 20-oxidases were digested with restriction endonucleases Van 911. Bcl 1, and Bsa WI, and six chimaeric sequences were produced by recombination of the DNA fragments. The pCM1 -construct was obtained by exchanging nt 303–809 of the Cucurbita cDNA with the homologous DNA from the March 20-oxidase. In pCM2, pCM3, pCM4, pCM5 and pCM6, nt 810–992, nt 993–end, nt 303–992, nt 810–end, and nt 311–end were exchanged, respectively. All constructs were cloned in a pUC18 vector and functionally expressed in E. coli NM522 cells. GA 20-oxidase activity was detectable in cell-lysates from the transformed E. coli, but the extent and kind of conversion depended on the construct. Highest conversion of GA₁₂was found with pCM1 and pCM3, one-tenth of this conversion was observed with pCM5 and pCM6, and one-hundredth was obtained with the hybrid proteins from pCM2 and pCM4. With pCM2 and pCM4, neither the C₁₉-end product, GA₉, nor the C₂₀-end product, GA₂₅-was formed. However, after transformation with constructs pCM1, pCM3, pCM5 or pCM6. GA₉accounted for 30, 40, 60 and 90%, respectively, of the end products formed. Thus, the segments originating from M. macrocarpus conferred upon the chimaeric proteins an increasing ability to direct the biosynthetic flow into C₁₉-GAs in this order. Although GA₂₄is the immediate precursor, much less end products were formed by using this substrate.     

Influence of Cadmium and Mercury on Activities of Ligninolytic Enzymes and Degradation of Polycyclic Aromatic Hydrocarbons by Pleurotus ostreatus in Soil

The white-rot fungus Pleurotus ostreatus was able to degrade the polycyclic aromatic hydrocarbons (PAHs) benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, and benzo[ghi]perylene in nonsterile soil both in the presence and in the absence of cadmium and mercury. During 15 weeks of incubation, recovery of individual compounds was 16 to 69% in soil without additional metal. While soil microflora contributed mostly to degradation of pyrene (82%) and benzo[a]anthracene (41%), the fungus enhanced the disappearance of less-soluble polycyclic aromatic compounds containing five or six aromatic rings. Although the heavy metals in the soil affected the activity of ligninolytic enzymes produced by the fungus (laccase and Mn-dependent peroxidase), no decrease in PAH degradation was found in soil containing Cd or Hg at 10 to 100 ppm. In the presence of cadmium at 500 ppm in soil, degradation of PAHs by soil microflora was not affected whereas the contribution of fungus was negligible, probably due to the absence of Mn-dependent peroxidase activity. In the presence of Hg at 50 to 100 ppm or Cd at 100 to 500 ppm, the extent of soil colonization by the fungus was limited.     

The role of spatial scale and the perception of large-scale species-richness patterns

Despite two centuries of exploration, our understanding of factors determining the distribution of life on Earth is in many ways still in its infancy. Much of the disagreement about governing processes of variation in species richness may be the result of differences in our perception of species‐richness patterns. Until recently, most studies of large‐scale species‐richness patterns assumed implicitly that patterns and mechanisms were scale invariant. Illustrated with examples and a quantitative analysis of published data on altitudinal gradients of species richness (n = 204), this review discusses how scale effects (extent and grain size) can influence our perception of patterns and processes. For example, a hump‐shaped altitudinal species‐richness pattern is the most typical (c. 50%), with a monotonic decreasing pattern (c. 25%) also frequently reported, but the relative distribution of patterns changes readily with spatial grain and extent. If we are to attribute relative impact to various factors influencing species richness and distribution and to decide at which point along a spatial and temporal continuum they act, we should not ask only how results vary as a function of scale but also search for consistent patterns in these scale effects. The review concludes with suggestions of potential routes for future analytical exploration of species‐richness patterns.     

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.     

Antibacterial activity of marine-derived fungi

A total of 227 marine isolates of ubiqituous fungi were cultivated on different media and the secondary metabolite content of the extracts (ethyl acetate/chloroform/methanol 3 : 2 : 1) characterized by HPLC. The fungi were secured from animals, plants and sediments of Venezuelan waters (0–10 m) including mangroves and lagoonal areas. The extracts were tested for antibacterial activity. A total of 7 were active towards Vibrio parahaemolyticus and 55 towards Staphylococcus aureus, representing 18 different fungal species from 8 ascomycetous genera. For 61 strains of Penicillium citrinum antibacterial activity correlated well with content of secondary metabolites as measured by HPLC. Thirteen isolates of Penicillium steckii produced very similar profiles of secondary metabolites and 6 of these had activity against either V. parahaemolyticus or S. aureus or both. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S₂ and for positively charged residues in S₁. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S₂ pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.