Interactions of calcium with the pyridine nucleotides

Association constants were determined for the 1:1 interactions of calcium with NAD⁺, NADH, NADP⁺, and NADPH in aqueous systems (pH 7, 25 °C) by use of a calcium-sensitive electrode. The order of binding of calcium to these pyridine nucleotides appears to be NAD⁺ < NADH < NADP⁺ < NADPH with association constants of 0.2 × 10², 0.3 × 10², 0.9 × 10², and 2 × 10², respectively. Calorimetric experiments revealed that all of these interactions are endothermic with enthalpy changes of 1, 2, 2, and 3 kcal/mol, respectively.     

Structural and functional processes during water vapour uptake and desiccation in selected lichens with green algal photobionts

Structural alterations of the photobiont and mycobiont cells of lichens have been related to CO₂-gas exchange during experiments involving water vapour uptake and desiccation of liquid-water-saturated thalli. Increasing water vapour uptake of air dry lichens led to a gradual unfolding of the photobiont cells in Lobaria pulmonaria, Pseudevernia furfuracea, Ramalina maciformis and Teloschistes lacunosus as studied by low-temperature scanning electron microscopy. The data indicated that globular, probably turgid, cells and also slightly infolded or even heavily collapsed cells contributed to positive net photosynthesis, which was reached after water vapour uptake by the four species studied. During desiccation of fully water-saturated thalli of L. pulmonaria, extrathalline water films gradually evaporated before maximum values of CO₂-gas exchange were measured and before photobiont cells started to shrivel. In contrast, in P. furfuracea the CO₂-gas exchange maximum was reached when a considerable percentage of photobiont cells had already collapsed and while other parts of the thalli were still covered with liquid water. Further desiccation led to cavitation of the cortical cells in both species, this occurring at water contents at which net photosynthesis was still positive.Abbreviations EF exoplasmic fracture face - LTSEM low-temperature scanning electron microscopy - NP net photosynthesis - PAR photosynthetic active radiation (400–700 nm) - PF plasmic fracture face We thank D. Pichier, P. Hatvani, H. Müller, Birmensdorf, and J.B. Winkler, Kiel, for technical assistance, and J. Innes, Birmensdorf, for correcting the English text. Stimulating discussion with R. Honegger (Institut für Pflanzenbiologie, Universität Zürich, Switzerland), L. Kappen (Botanisches Institut, Universität Kiel, Germany), T.G.A. Green (Department of Biological Sciences, Hamilton, New Zealand), and O.L. Lange (Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, Germany) are gratefully acknowledged.     

Cell suspension cultures of spruce (Picea abies): inactivation of extracellular enzymes by fungal elicitor-induced transient release of hydrogen peroxide (oxidative burst)

Elicitation of suspension culture cells of spruce [Picea abies (L.) Karst] with a fungal cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii Bubak induced inactivation of extracellular enzymes. Extracellular peroxidase, -glucosidase and acid phosphatase, secreted by the cells during growth, and also -amylase and pectinase from Aspergillus strains, added to an elicited cell culture, were inactivated. Inactivation is caused by an elicitor-mediated transient release of H₂O₂ from the cells (oxidative burst). H₂O₂ released into the medium was determined with ABTS (2,2'-Azino-bis-(3-ethylbenthiazoline-6-sulfonate)) (formation of blue colour) and with phenol red (destruction of pH indicator). The release started only minutes after beginning of elicitation and its inactivating effect existed for more than 1 day. Release of H₂O₂ is a biphasic process with a first smaller maximum at 1 h, followed by a second larger increase, peaking at 5–6 h and returning to approximately the control levels thereafter. Also H₂O₂ is transiently released in small quantities from cell incubations in the absence of elicitor as a stress response of the cells to manipulations of the cultures. Extracellular enzymes secreted into the medium could also be inactivated by direct addition of exogenous H₂O₂. Catalase prevents inactivation of the secreted extracellular enzymes, however, to a limited extent only because, as a result of contact of cells and medium, catalase becomes inactivated. The ionophores A 23187 and cycloheximide induced release of H₂O₂ and, when present together with elicitor, induction was synergistically increased.     

Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations

The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat for its relationship to CFTR mutations and intragenic markers on 200 chromosomes from German patients with cystic fibrosis (CF). Four frequent length variations were strongly associated with the four predominant haplotypes previously defined by intragenic marker dimorphisms. One of these alleles displayed absolute linkage disequilibrium to the major CF mutation F508. Other frequent CFTR mutations were linked to one particular splice site haplotype indicating that differential exon 9 skipping contributes little to the clinical heterogeneity among CF patients with an identical mutation. We also identified a novel missense mutation (V456F) and a novel nonsense mutation (Q414X) within the coding region of exon 9. The missense mutation V456F adjacent to Walker motif A was present in a pancreas-sufficient CF patient. In contrast, the pancreas-insufficient Q414X/F508 compound heterozygote suffered from a severe form of the disease, indicating that alternative splicing of exon 9 does not overcome the deleterious effect of a stop codon within this exon.     

Detection of more than 50 different CFTR mutations in a large group of German cystic fibrosis patients

We have conducted a comprehensive study of the molecular basis of cystic fibrosis (CF) in 350 German CF patients. A screening approach based on single-strand conformation analysis and direct sequencing of genomic polymerase chain reaction products has allowed us to detect the molecular defects on 95.4% of the CF chromosomes within the coding region and splice sites of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The spectrum of sequence changes comprises 54 different mutations, including 17 missense mutations, 14 nonsense mutations, 11 frameshift mutations, 10 splice site variants and two amino acid deletions. Eleven of these mutations have not previously been described. Our results reflect the marked mutational heterogeneity of CF in a large sample of patients from a non-isolated population.     

Altered CYP21 genes in HLA-haplotypes associated with congenital adrenal hyperplasia (CAH): a family study

Disorders of the CYP21 gene, which is located within the major histocompatibility complex on the short arm of chromosome 6, are the leading causes of congenital adrenal hyperplasia (CAH). The coding gene and a highly homologous pseudogene are tandemly arranged with the two genes for the fourth component of complement (C4A and C4B). To analyse the prevalence rates of mutations of the CYP21 genes and the segregation of the CYP21 genes with their corresponding human leucocyte antigen (HLA)-haplotypes, 21 families with one or two children with the severe form of 21-hydroxylase deficiency were studied. Mutations of the CYP21 gene on their corresponding HLA-haplotype were detected by hybridisation of polymerase chain reaction (PCR)-amplified genomic DNA with sequence-specific oligonucleotides and solid phase direct sequencing. Our study has shown the following. (1) A single basepair mutation (AG or CG) within the second intron is the most frequent mutation leading to impaired 21-hydroxylase activity. This mutation is only detected in HLA-haplotypes associated with the salt-wasting form of CAH. (2) A large deletion of part or all of the CYP21 gene is associated with the HLA-haplotype A3, BW47, C6, DR7, DR53, DQ2 but is also observed in other HLA-haplotypes and can be detected by a simple rapid PCR restriction fragment length polymorphism method. (3) Two alleles of the coding CYP21 gene differing in a leucine codon within the first exon, (formerly described as a mutation associated with 21-hydroxylase deficiency) have been found with an equal distribution in patients with 21-hydroxylase deficiency, non-disease HLA-haplotypes and the local healthy controls.     

Degradation of polychlorinated biphenyls by hairy root culture of Solanum nigrum

Polychlorinated biphenyls (PCBs) present in the commercial mixture Delor 103 were transformed by hairy root culture of Solanum nigrum. Plant growth regulators kinetin, 2,4-dichlorophenoxy-acetic acid, benzylaminopurin and/or naph-thaleneacetic acid, influenced the cells' growth and transformation of PCBs in a different manner. The cells were able to transform PCBs even if they ceased growing. Young inoculum (16 days) had a 20% lower PCB conversionthan did older inocula (37, 68 days) while biomass increase was much higher using young inoculum. With increasing size of inoculum, transformation of PCBs was stimulated. After 30 days of incubation the average amount of residual PCBs was 40% of the controls at initial PCB concentration of 100 ppm.     

Intra- and extragenic marker haplotypes of CFTR mutations in cystic fibrosis families

Summary In order to facilitate the screening for the less common mutations in the cystic fibrosis (CF) gene viz., the CF transmembrane conductance regulator gene (CFTR), marker haplotypes were determined for German nonCF (N) and CF chromosomes by polymerase chain reaction analysis of four polymorphisms upstream of the CF gene (XV-2c, KM.19, MP6-D9, J44) and six intragenic polymorphisms (GATT, TUB9, M470V, T854T, TUB18, TUB20) that span the CFTR gene from exon 6 through exon 21. Novel informative sequence variants of CFTR were detected in front of exons 10 (1525-61 A or G), 19 (3601-65 C or A), and 21 (4006-200 A or G). The CF locus exhibits strong long-range marker-marker linkage disequilibrium with breakpoints of recombination between XV-2c and KM.19, and between exons 10 and 19 of CFTR. Marker alleles of GATT-TUB9 and TUB18-TUB20 were found to be in absolute linkage disequilibrium. Four major haplotypes encompass more than 90% of German N and CF chromosomes. Fifteen CFTR mutations detected on 421 out of 500 CF chromosomes were each identified on one of these four predominant 7-marker haplotypes. Whereas all analysed F508 chromosomes carried the same KM.19-D9-J44-GATT-TUB9-M470V-T854T haplotype, another frequent mutation in Germany, R553X, was identified on two different major haplotypes. Hence, a priori haplotyping cannot exclude a particular CF mutation, but in combination with population genetic data, enables mutations to be ranked by decreasing probability.     

A termination mutation (2143delT) in the CFTR gene of German cystic fibrosis patients

German patients with cystic fibrosis (CF) were screened for molecular lesions in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by single strand conformation polymorphism (SSCP) and chemical cleavage of mismatch analyses. Direct sequencing of four samples that displayed the same SSCP pattern and that were susceptible to cleavage of heteroduplexes by osmium tetroxide revealed, in all cases, a deletion of a single T residue at nucleotide position 2143 within codon 671 of the CFTR gene. As a result, leucine codon 671 is changed into a termination codon. In total, the 2143delT mutation was confirmed in 6 out of 271 German non-F508 CF chromosomes by artificial restriction fragment length polymorphism analysis, indicating that this frameshift mutation accounts for about 2% of German non-508 mutations. The 6 pancreas insufficient patients who are compound heterozygous for 2143-delT suffer from the typical features of pulmonary and gastrointestinal CF disease. The 2143delT mutation completes the panel of the more frequent CFTR mutations that reside on the F508 haplotype and that contribute to its overpresentation among German non-F508 alleles that are associated with severe forms of disease.     

l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies)

The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate.In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light.With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.