Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.     

Influence of osmotic stress on the respiration of potato tuber callus

Potato tuber ( Solanum tuberosum L. cv. Bintje) callus shows a decrease in fresh weight and an increase in dry weight upon transfer to nutrient medium supplemented with 0.3 or 0.5 M mannitol. The osmolarity of the intracellular fluid increases simultaneously. Probably mannitol is taken up from the medium till the osmolarity of the tissue is in equilibrium with that of the medium. After osmotic adaptation, on a medium with 0.5 M mannitol, growth is negligible, although the tissue retains its viability.
Respiration increases upon transfer to medium with extra mannitol, especially when expressed on a fresh weight basis. On this basis cytochrome and alternative pathway capacities do not change appreciably. The respiratory increase is exclusively caused by an increased engagement of the alternative pathway. The participation of this pathway in uninhibited respiration increases from about 10 to 90% upon transfer to medium with extra mannitol. The increase in respiration is partly correlated with the decrease in fresh weight upon transfer. Per disc, the capacities of the cytochrome and alternative pathway decline. Yet, total respiration per disc significantly increases due to the increased participation of the alternative pathway. This results in an almost equal ATP-production per disc before and after transfer. We suggest, that the alternative pathway functions as a reserve capacity in potato callus, which is switched on when ATP-production coupled to the cytochrome pathway is impaired.     

cis-Platinum induced distortions in DNA. Conformational analysis of d(GpCpG) and cis-pt(NH3)2[d(GpCpG)], studied by 500-MHz NMR

Proton NMR studies at 500 MHz in aqueous solution were carried out on the G-G chelated deoxytrinucleosidediphosphate platinum complex cis-Pt(NH3)2[d(GpCpG], on the uncoordinated trinucleotide d(GpCpG) and on the constituent monomers cis-Pt(NH3)2[d(Gp)]2, cis-Pt(NH3)2[d(pG)]2, d(Gp), d(pCp) and d(pG). Complete NMR spectral assignments are given and chemical shifts and coupling constants are analysed to obtain an impression of the detailed structure of d(GpCpG) and the distortion of the structure due to chelation with [cis-Pt(NH3)2]2+. Platination of the guanosine monophosphates affects the sugar conformational equilibrium to favour the N conformation of the deoxyribose ring. This feature is also apparent in ribose mononucleotides and is possibly caused by an increased anomeric effect. In cis-Pt(NH3)2[d(pG)]2 the phase angle of pseudorotation of the S-type sugar ring is 20 degrees higher than in 'free' d(pG) which might be an indication for an ionic interaction between the positive platinum and the negatively charged phosphate. It appears that d(GpCpG) reverts from a predominantly random coil to a normal right-handed B-DNA-like single-helical structure at lower temperatures, whereas the conformational features of cis-Pt(NH3)2[d(GpCpG)] are largely temperature-independent. In the latter compound much conformational freedom along the backbone angles is seen. The cytosine protons and deoxyribose protons exhibit almost no shielding effect as should normally be exerted by the guanine bases in stacking positions. This is interpreted in terms of a 'turning away' of the cytosine residue from both chelating guanines. Conformational features of cis-Pt(NH3)2[d(GpCpG)[ are compared with the 'bulge-out' of the ribose-trinucleotide m6(2)ApUpm6(2)A.     

Cultural Aspects of Health and Illness Behavior in Hospitals

Health care attitudes reflect the basic world view and values of a culture, such as how we relate to nature, other people, time, being, society versus community, children versus elders and independence versus dependence. Illness behavior determines who is vulnerable to illness and who agrees to become a patient—since only about one third of the ill will see a physician. Cultural values determine how one will behave as a patient and what it means to be ill and especially to be a hospital patient. They affect decisions about a patient''s treatment and who makes the decisions. Cultural differences create problems in communication, rapport, physical examination and treatment compliance and follow through. The special meaning of medicines and diet requires particular attention. The perception of physical pain and psychologic distress varies from culture to culture and affects the attitudes and effectiveness of care-givers as much as of patients. Religious beliefs and attitudes about death, which have many cultural variations, are especially relevant to hospital-based treatment. Linguistic and cultural interpreters can be essential; they are more available than realized, though there are pitfalls in their use. Finally, one must recognize that individual characteristics may outweigh the ethnic and that a good caring relationship can compensate for many cultural missteps.     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Cortisol and Growth Hormone Secretion in Relation to Linear Growth: Patients with Still's Disease on Different Therapeutic Regimens

Linear growth was studied in 20 children suffering from Still''s disease on various treatment regimens, and their ability to secrete growth hormone and cortisol was investigated. Growth recovered on reducing daily corticosteroid therapy or on changing to an alternative regimen. Retardation of growth was not due to an absolute inability to secrete growth hormone. Basal plasma cortisol levels and the plasma cortisol response to hypoglycaemia were reduced in patients on daily steroid therapy, but patients on alternate-day prednisone did not differ significantly in this respect from those on non-steroid regimens. Those on alternate-day corticotrophin showed preservation of the circadian rhythm but a subnormal response to hypoglycaemia.     

The dieback ofZostera marina in the 1930's in the Waddensea; An eye-witness account by A. van der Werff

In the archives of the State Institute of Fisheries Research a handwritten report of A. van der Werff on the wasting disease ofZostera marina L. in the Waddensea in 1932 and 1933 is kept. It contains an eye-witness account of the decline of the eelgrass beds. In 1932 the first attack of the disease took place in May and caused heavy devastation. Regeneration started soon, but a second attack at the end of August seemed to destroy the beds definitively. The next year was a year of struggle for the few poorly developed surviving plants. In 1938 the last surviving specimens were seen in the sublittoral.     

Quantitative Determination of the Spatial Distribution of Nitrosomonas europaea and Nitrobacter agilis Cells Immobilized in κ-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of Nitrosomonas europaea (ATCC 19718) and Nitrobacter agilis (ATCC 14123) were homogeneously distributed in a κ-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either N. europaea or N. agilis in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for N. europaea and N. agilis in κ-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.