Temperature coefficients of peptides dissolved in hexafluoroisopropanol monitor distortions of helices

Temperature coefficients are widely used as an indication of solvent accessibility to amide protons. Low temperature coefficients are related to low accessibility and are often interpreted as evidence for intramolecular hydrogen bonding. Conformational shifts, i.e. the difference between chemical shifts of a particular residue in a structured and in a random-coil conformation, provide information on secondary structure. In particular, negative CH conformational shifts are often used to delineate the extent of helical stretches. NH conformational shifts show large oscillations within a helix that have been interpreted as the result of helix distortions affecting hydrogen bond lengths. In the course of the study of different peptides that adopt a helical structure in the presence of the structure-inducing solvent hexafluoroisopropanol (HFIP), we have found a strong correlation between temperature coefficients and amide conformational shifts. However, contrary to the initial expectations, lower temperature coefficients were associated to amide protons involved in longer, and presumably weaker, hydrogen bonds. The correlation can be explained, however, assuming that, in helical peptides dissolved in HFIP, temperature affects the chemical shift of amide protons mainly by changing the average length of intramolecular hydrogen bonds and changes in solvent accessibility play only a secondary role under these experimental conditions. The pattern of temperature coefficients in helical peptides can therefore be used to identify short or long hydrogen bonds causing bending of the helix axis.     

In Vitro Induction of Apoptosis or Differentiation by Dopamine in an Immortalized Olfactory Neuronal Cell Line

Abstract: Amyloid β-peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that Aβ25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25-35 were specific because Aβ1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1-40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25-35. Damage and death of ECs induced by Aβ25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

Physiology of sulfide tolerance in a thermophilic Oscillatoria

Oscillatoria amphigranulata is a fast-growing (3 doublings/day) cyanobacterium isolated from sulfide hot springs in New Zealand. Photosynthesis, as measured by incorporation of [¹⁴C]-HCO ₃ ^- , was initially inhibited by 0.3–1.5 mM sulfide at pH 7.9–8.1. However, conversion to sulfide-dependent anoxygenic photosynthesis occurred in about 2 h or less under light intensities of 3–14 klx. Under the stimulation of higher light intensity (8–14 klx) a partial recovery of oxygenic photosynthesis also occurred. It was concluded that oxygenic photosynthesis was responsible for 21–42% of the total incorporation at sulfide concentrations of 1.0–0.3 mM, respectively. This contribution was suppressed at 1.5 mM sulfide and not elicited under lower light intensities (3–7 klx). As judged by the inhibitory effect of 10 g/ml chloramphenicol protein synthesis was required for attainment of both anoxygenic photosynthesis and photosystem II recovery. Sulfide could not be replaced by thiosulfate, elemental sulfur or dithionite as electron donors in photosynthesis, but elemental sulfur could serve as the sole assimilatory source of sulfur. Oxygenic photosynthesis was inhibited by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] or DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but sulfide relieved the effect of either inhibitor in adapted cells, indicating that electrons derived from sulfide enter the photosynthetic electron transport chain at a point beyond plastoquinone.Uncommon abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DSPD disalicyclidene propanediamine - DNP-INT 2-4-dinitrophenyl ether of 2-iodo-4-nitrothymol - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - PPO 2,5-diphenyloxazole - POPOP 1,4-bis-2-(5-phenyl oxzolyl) benzene     

Loss of sulfide adaptation ability in a thermophilic Oscillatoria

A spontaneous variant incapable of anoxygenic photosynthesis was derived from a fully competent strain of Oscillatoria amphigramulata which was originally isolated from a high sulfide-containing hot spring of New Zealand. Although the variant (Oa-2) acquired a slight ability to photosynthesize in the presence of 0.3–0.4 mM sulfide, this was only after a 24 h exposure to sulfide and represented oxygenic photosynthesis only. Unlike the parent strain, the incompetent variant never grew in the presence of sulfide >0.05 mM, nor was there any relief of the inhibition by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] of CO₂ photoincorporation when sulfide was present. The variant strain has retained all of these characteristics over a 4 year period with monthyl transfers in non-sulfide medium. The wild type, under identical conditions, has retained all of its competence with respect to sulfide.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Oxygen metabolism by the anaerobic bacteriumVeillonella alcalescens

Veillonella alcalescens contained a membrane-bound lactate oxidase system. Studies on the effect of inhibitors on lactate oxidase showed the participation of non-heme iron, quinone and cytochromesb andd. Superoxide anion radicals ( ) and H₂O₂ were shown to be formed at lactate oxidation and presumably arose from cyanide- and azide-resistant side chains of the respiratory system. The H⁺/O ratio withL-lactate as a hydrogen donor was 2.3. When an anaerobic culture growing on lactate was shifted to a high dissolved oxygen tension (d.o.t.=15 kPa) rapid inhibition of growth and lactate conversion occurred. This could be correlated with a rapid inactivation of lactate dehydrogenase. The effects of high d.o.t.'s on lactate dehydrogenase, lactate conversion and growth were reversible. After a shift to low d.o.t.'s (<2.5 kPa) growth ofV. alcalescens continued for one or two doublings whereafter lysis did occur. Acetate and pyruvate were the main fermentation products. P/O ratio's were calculated from molar growth yields and fermentation balances. A P/O value of 0.66 was found after a shift to a very low oxygen supply at which the d.o.t. presumably was zero. Shifts to higher d. o. t.'s gave much lower growth yields. Presumably, under these conditions uncoupling between growth and energy production occurred. Accumulation of toxic oxygen compounds was given as an explanation for the behaviour ofV. alcalescens at low d.o.t.'s.Abbreviations HQNO 2-n-heptyl-4-hydroxy-quinoline-N-oxide - CCCP carbonyl cyanide m-chlorophenyl-hydrazone - ABTS 2,2-azino-di-3-ethyl-benzthiazoline sulfonate - DCPIP 2,6 dichlorophenolindophenol - PMS phenazine methosulfate - NBT p-nitro blue tetrazolium chloride - d.o.t. dissolved oxygen tension - SOD superoxide dismutase     

Nitrogenase from Rhodospirillum rubrum Relation between ‘switch-off’ effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios

Nitrogenase activity of ‘membrane-free’ extracts, produced from nitrogenstarved Rhodospirillum rubrum to which 4 mM NH⁺₄ had been added is only about 10% of the activity in the control. The activity could be restored to 80% by including the membrane component, earlier found to activate R. rubrum nitrogenase, in the reaction mixture. The relation between this ‘switch-off/switch-on’ effect and the function of the membrane component is discussed.Hydrogen production catalyzed by R. rubrum nitrogenase is also dependent on activation by the membrane component. Hydrogen production is inhibited by acetylene but the degree of inhibition is dependent on the nitrogenase component ratio. The strongest inhibition is achieved at low MoFe protein/Fe protein ratios. The $\text{ATP}\text{2 e}{}^{\mathrm{?}}$ values are 4–5 at the component ratios giving the highest activity and increase at high MoFe protein/Fe protein ratios. CO inhibits acetylene reduction but has no effect on the hydrogen production.     

Regulation of adenosine 5′-phosphosulfate sulfotransferase activity by H2S and cyst(e)ine in primary leaves of Phaseolus vulgaris L.

During chloroplast development in the primary leaves of Phaseolus vulgaris, the extractable activity of adenosine 5-phosphosulfate sulfotransferase increased ten-fold. When chloroplast development took place in air enriched with 3.5 l H₂S·l^-1 there was a decrease in adenosine 5-phosphosulfate sulfotransferase activity. Cyst(e)ine in concentrations up to 1 mM (in the external medium) did not affect the increase in adenosine 5-phosphosulfate sulfotransferase activity in intact plants. In plants with excised roots, 0.75 mM cyst(e)ine inhibited this increase. In green primary leaves, H₂S or cyst(e)ine treatment resulted in a decrease of extractable adenosine 5-phosphosulfate sulfotransferase activity. In intact plants, this effect of cyst(e)ine was observed at a concentration of 1 mM, and in plants with excised roots, 0.25 mM had a comparable effect.In developing plants, the extractable activities of O-acetyl-L-serine sulfhydrylase (EC 4.2.99.9) and ribulosebisphosphate carboxylase (EC 4.1.1.39.) were not affected by H₂S or cyst(e)ine.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5phosphosulfate sulfotransferase - BSA bovine serum albumin - DTE dithioerythritol - EDTA ethylenediaminetetra-acetic acid - OASSase O-acetyl-L-serine sulfhydrylase - PAPS adenosine 3-phosphate 5-phosphosulfate - POPOP 1,4 Di 2-(5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazol - RubP ribulose-bisphosphate - RubPCase ribulosebiphosphate carboxylase This is no. 8 in the series Regulation of Sulfate Assimilation in Plants. The term cysteine is used when it is clear that cystine is not involved; cyst(e)ine is used for an undefined mixture of cysteine and cystine. The concentrations are expressed in all cases relative to cysteine     

Theoretical investigations on circular and chain-like hydrogen bonded structures found in two crystal forms of α-cyclodextrin hexahydrate: Models for hydration and water clusters

Hydration of macromolecules and the structure of water of crystallization are not understood in detail because in these complex systems. H-atoms cannot be located and the hydrogen bonding schemes are not known. X-ray and neutron diffraction studies on a hydrated oligosaccharide, α-cyclodextrin 6H₂O, ((C₆H₁₀O₅)₆·6H₂O), crystals forms A and B, gave insight into the chain-like and circular arrangement of hydrogen bonds. In the circles, homodromic (unidirectional) and antidromic (counter-running) orientation of five to six hydrogen bounds is observed. PCILO calculations showed that homodromic circles and chains are approx. 8% per hydrogen bond more stable than antidromic circles, that the changes in electronic charges on H and O atoms are greater in homo than in antidromic systems and that the dipole moments are only approx. 3 D in the homodromic circles but 6–8 D in chain-like and antidromic arrangement. These results have been interpreted in terms of cooperative effect. Circular systems are considered as structural elements in hydration shells of macromolecules and in the assembly of ‘flickering’ water clusters.