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.     

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.     

A phosphorus NMR study of the reaction products of cis-diamminedichloroplatinum (II) with a double-helical oligonucleotide and with DNA

The structural distortion of oligonucleotides upon cis-PtCl2(NH3)2[d(T-C-T-C-G-G-T-C-T-C)-N7(5), N7(6)] reveals shifting of 4 phosphorus resonances due to platination. 3 Resonances could be assigned by selective 31P-irradiation, showing P(6) (P between the two Gs) to be shifted 1.5 ppm to low field. In the concomitant double strands P(6) is shifted 0.9 ppm to lower field. A similar peak has been observed in platinated salmon sperm DNA (37 degrees C), indicating that Pt-binding to GpG-fragments in DNA is similar to that found for the decanucleotide, so the distortion of DNA might be comparable.     

Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.     

Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.     

Maxillary reconstruction: functional and aesthetic considerations

Maxillary reconstruction is a challenging endeavor in functional and aesthetic restoration. Given its central location in the midface and its contributions to the key midfacial elements--the orbits, the zygomaticomaxillary complex, the nasal unit, and the stomatognathic complex--the maxilla functions as the keystone of the midface and unifies these elements into a functional and aesthetic unit. Maxillary defects are inherently complex because they generally involve more than one midfacial component. In addition, most maxillary defects are composite in nature, and they often require skin coverage, bony support, and mucosal lining for reconstruction. In the reconstruction of maxillary defects secondary to trauma, ablative tumor surgery, or congenital deformities, the following goals must be met: (1) obliteration of the defect; (2) restoration of essential functions of the midface, such as mastication and speech; (3) provision for adequate structural support to each of the midfacial units; and (4) aesthetic reconstruction of the external features. This review will discuss the pertinent anatomic considerations, the historical approaches to maxillary reconstruction, and the merits of the techniques in use today, with an emphasis on state-of-the-art reconstruction and dental rehabilitation of extensive maxillary defects.