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Biomedical subjects

D J Kyle

Publications and source records attributed to D J Kyle.

67 records · Page 4Linked to original sources
PubMed1984

Membrane protein damage and repair: Selective loss of a quinone-protein function in chloroplast membranes.

A loss of electron transport capacity in chloroplast membranes was induced by high-light intensities (photoinhibition). The primary site of inhibition was at the reducing side of photosystem II (PSII) with little damage to the oxidizing side or to the reaction center core of PSII. Addition of herbicides (atrazine or diuron) partially protected the membrane from photoinhibition; these compounds displace the bound plastoquinone (designated as Q(B)), which functions as the secondary electron acceptor on the reducing side of PSII. Loss of function of the 32-kilodalton Q(B) apoprotein was demonstrated by a loss of binding sites for [(14)C]atrazine. We suggest that quinone anions, which may interact with molecular oxygen to produce an oxygen radical, selectively damage the apoprotein of the secondary acceptor of PSII, thus rendering it inactive and thereby blocking photosynthetic electron flow under conditions of high photon flux densities.

Journal Article↗
PubMed1984

Undernutrition in sheep. The effect of supplementation with protein on protein accretion.

In a comparative-slaughter experiment, individually rationed wether lambs initially of 42 kg were given 235, 362 or 456 kJ metabolizable energy (ME)/kg live weight (LW)0.75 per d as sodium hydroxide-treated barley straw with urea (six lambs per treatment), or NaOH-treated barley straw with urea plus 125 g/d white-fish meal to give 307 or 488 kJ ME/kg LW0.75 per d (seven lambs per treatment) for 92 d. All unsupplemented lambs lost both fat and body protein. The changes in fat were -3.53, -2.75 and -1.40 (SE 0.59) kg (initial value 8.6 kg), and the changes in body protein were -0.47, -0.09 and -0.14 (SE 0.13) kg (initial value 4.9 kg) for the three unsupplemented groups respectively. When supplemented with fish meal, fat was again lost as -1.53 and -0.93 (SE 0.55) kg, but wool-free body protein was increased, and gains were 0.48 and 0.89 (SE 0.12) kg for the two supplemented groups respectively. All animals lost wool-free body energy, total changes being -150, -111, -59 and -49 and -16 MJ respectively. When corrected to an equal ME intake the supplemented lambs, when compared with the unsupplemented lambs, gained (instead of losing) body protein (P less than 0.001) and lost less fat (P less than 0.05). Wool growth did not respond to supplemental protein, but was related to ME intake with an increase of 0.78 g wool fibre for each additional MJ ME. The maintenance requirements of the unsupplemented and supplemented groups respectively were estimated by regression analysis to be 554 and 496 kJ ME/kg LW0.75 per d. The apparent utilization of ME below energy equilibrium (km) was 0.31 (SE 0.08) for the unsupplemented animals, and 0.12 (SE 0.10) for the supplemented animals, well below a km of 0.70 which current UK standards (Agricultural Research Council, 1980) would predict. Most of these differences could be reconciled if basal metabolism was assumed not to be constant. It is concluded that lambs in negative energy balance can continue lean body growth at the expense of body fat, provided sufficient dietary protein is available. It is also concluded that since the animals at the lowest ME intakes required less ME than predicted by current feeding standards, the effect was that it would have been difficult to distinguish between the apparent utilization of ME for maintenance (km) and for fattening (kf).

Animals↗
PubMed1984

Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes.

Incubation of Chlamydomonas reinhardii cells at light levels that are several times more intense than those at which the cells were grown results in a loss of photosystem II function (termed photoinhibition). The loss of activity corresponded to the disappearance from the chloroplast membranes of a lysine-deficient, herbicide-binding protein of 32,000 daltons which is thought to be the apoprotein of the secondary quinone electron acceptor of photosystem II (the QB protein). In vivo recovery from the damage only occurred following de novo synthesis (replacement) of the chloroplast-encoded QB protein. We believe that the turnover of this protein is a normal consequence of its enzymatic function in vivo and is a physiological process that is necessary to maintain the photosynthetic integrity of the thylakoid membrane. Photoinhibition occurs when the rate of inactivation and subsequent removal exceeds the rate of resynthesis of the QB protein.

Chlamydomonas↗
PubMed1984

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye.

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20 degrees C (nonhardened rye, RNH) or 5 degrees C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20 degrees C indicated that RNH and RH thylakoids contained the same proportions of fast (alpha) and slow (beta) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for alpha components increased and the rate constant for beta components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5 degrees C.

Journal Article↗
PubMed1983

Lateral mobility of the light-harvesting complex in chloroplast membranes controls excitation energy distribution in higher plants.

Chloroplast thylakoid protein phosphorylation produces changes in light-harvesting properties and in membrane structure as revealed by freeze-fracture electron microscopy. Protein phosphorylation resulted in an increase in the 77 degrees K fluorescence signal at 735 nm relative to that at 685 nm. In addition, a decrease in connectivity between Photosystem II centers (PS II) and a dynamic quenching of the room temperature variable fluorescence was observed upon phosphorylation. Accompanying these fluorescence changes was a 23% decrease in the amount of stacked membranes. Microscopic analyses indicated that 8.0-nm particles fracturing on the P-face moved from the stacked into the unstacked regions upon phosphorylation. The movement of the 8.0-nm particles was accompanied by the appearance of chlorophyll b and 25 to 29 kD polypeptides in isolated stroma lamellae fractions. We conclude that phosphorylation of a population of the light-harvesting chlorophyll a/b protein complexes (LHC) in grana partitions causes the migration of these pigment proteins from the PS II-rich appressed membranes into the Photosystem I (PS I) enriched unstacked regions. This increases the absorptive cross section of PS I. In addition, we suggest that the mobile population of LHC functions to interconnect PS II centers in grana partitions; removal of this population of LHC upon phosphorylation limits PS II leads to PS II energy transfer and thereby favors spillover of energy from PS II to PS I.

Chloroplasts↗
PubMed1982

Development of photochemical activity in relation to pigment and membrane protein accumulation in chloroplasts of barley and its virescens mutant.

The development of photochemical activity in relation to pigment and membrane protein accumulation in chloroplasts of greening wild-type barley (Hordeum vulgare L. cv. Gateway) and its virescens mutant were studied. The rate of chlorophyll accumulation per plastid was faster in the wild-type than in the mutant seedlings upon illumination after 6 days of etiolation, but was not different after 8 days. Although the protein content per plastid did not vary during greening, there was a change in the sodium dodecyl sulfate-polyacrylamide gel polypeptide profiles. High molecular weight proteins of 96,000 and 66,000 decreased whereas those at 34,000, 27,000 and 22,000 increased in relative quantity as a function of greening. The fully greened mutant seedlings were not deficient in the light-harvesting chlorophyll protein complex (LHC) or the reaction centers of photosystem I and photosystem II. Photosystem I-associated photochemical activities appeared within the first hour of plastid development and photosystem II associated activities and O(2) evolution within the next 6 hours. In all cases, the developmental rates per unit protein were slower in the mutant following 6 days of etiolation, but no differences between the two genotypes could be seen after 8 days due to a decrease in the developmental rate of the wild-type chloroplasts. An increase in photosynthetic unit size associated with plastid morphogenesis was faster in the wild-type seedlings after 6 days, but again the difference was negligible after 8 days. It was concluded that no single measured photochemical parameter is affected by this mutation, but rather, all aspects of chloroplast development are affected similarly by an overall reduction in the rate of chloroplast morphogenesis. This mutant, therefore, undergoes the normal pattern of proplastid to chloroplast development, but at a markedly reduced rate.

Journal Article↗
PubMed1982

Photosystem II Activity, Plastoquinone A Levels, and Fluorescence Characterization of a Virescens Mutant of Barley.

Chloroplasts isolated from seedlings of a virescens mutant of barley (Hordeum vulgare L cv Gateway) grown for 6 days under continuous illumination had lower levels of photosystem II activities on a chlorophyll basis than wild-type seedlings. After 8 days, however, the photosystem II rates of the mutant and wild-type were approximately equal. Lower levels of the photosystem II activities in the mutant were correlated with a smaller functional plastoquinone pool size as determined by room temperature fluorescence induction. Higher levels of extractable plastoquinone A on a chlorophyll basis, however, were obtained from mutant chloroplasts. An increased room temperature fluorescence yield in the mutant was shown to be due to a higher level of initial fluorescence. A decreased sigmoidicity in the room temperature fluorescence induction transient in the presence of diuron and an increased 77 K fluorescence emission at 680 nanometers lead us to believe that a certain population of the light harvesting chlorophyll protein complex in the mutant membranes is unconnected to photo-system II reaction centers. Although photochemical activities of the mutant approach wild-type values as the mutant develops, the population of dissociated light harvesting complexes does not appear to change.

Journal Article↗
PubMed1979

Stabilization of Thylakoid Membranes by Spermine during Stress-induced Senescence of Barley Leaf Discs.

The effect of spermine on photochemical activity and polypeptide composition of chloroplasts from barley leaf discs during senescence in the dark was studied. Chloroplast membranes did not show photosystem II activity after spermine treatment when water was the electron donor, but in the presence of diphenylcarbazide, this activity was observed. The diphenylcarbazide-stimulated photoreduction of dichloroindophenol was 3-fold greater in leaf discs incubated for 72 hours in spermine than in water. Photosystem I activity was reduced by about 90% within the first 24 hours in the spermine-treated samples. This reduction, however, was not due to a decrease in the photosynthetic unit size. A preferential loss of polypeptides other than those associated with photosystem II was observed during senescence of the leaf discs in water, but this loss was reduced by spermine. Spermine treatment also prevented the appearance of several additional chlorophyll proteins found in the controls during senescence. The results have been interpreted on the basis of the interaction of spermine with thylakoid membranes resulting in stabilization of membrane function during senescence.

Journal Article↗
PubMedDate not supplied

Alkylation of DNA with aziridine produced during the hydrolysis of N,N',N''-triethylenethiophosphoramide.

A reaction pathway by which thiotepa (N,N',N''-triethylenethiophosphoramide) and tepa (N,N',N''-triethylenethiophosphoramide), its major metabolite in humans, alkylate and depurinate DNA involves hydrolysis to aziridine (ethylene imine), a highly reactive monofunctional alkylating agent. Hydrolytic cleavage of an N-P bond of thiotepa releases aziridine which reacts with DNA, resulting in depurination and formation of the stable N-7 adduct 7-(2-aminoethyl)guanine and an aminoethyl adduct of adenine. Chromatographically identical alkylated products were observed in the reaction of thiotepa and tepa with individual nucleosides. Adducts with deoxycytidine or thymidine were not detected. Aziridine was measured by HPLC after derivatization with 1,2-naphthoquinone 4-sulfate. On the basis of the identity of the DNA adducts and the rate of formation of aziridine by hydrolysis in vitro, thiotepa is concluded to be a lipophilic, stabilized form of aziridine which serves as a cell-penetrating carrier of aziridine.

Alkylating Agents↗
PubMedDate not supplied

A novel beta-turn mimic useful for mapping the unknown topology of peptide receptors.

Ethers of cis or trans D-4-hydroxyproline (Hype), adjacent to octahydroindole-carboxylic acid (Oic), introduce a beta-turn into the backbone of peptides when positioned respectively at the i+1 and i+2 position of the turn. This is confirmed by NMR experiments performed on a model tetrapeptide in water. Synthetic alteration of the ether group allows simple probing of the steric limits and electrostatic potential of a receptor binding site, a technique applied successfully to the bradykinin receptor.

Amino Acid Sequence↗
PubMedDate not supplied

Novel pseudopeptides with high affinities for the human bradykinin B2 receptor.

We recently proposed a model of bradykinin bound to the rat bradykinin B2 receptor that is constructed on the basis of structural homology modeling to the criomicroscopic structure of the seven transmembrane domains of bacteriorhodopsin, extensive conformational searches and experimental mutagenesis results. On the basis of that model, a novel third-generation pseudopeptide antagonist, NPC 18325 (D-Arg1-Arg2-[aminotridecanoyl]3-Ser4-D-Tic5-Oic6++ +-Arg7) (Ki = 440 nM, guinea pig ileum), was designed and also reported. NPC 18325 has been proposed to adopt a C-terminal beta turn separated from N-terminal positive charges by a linear 12 carbon chain spacer. Experimentally, the four amino acids making up the C-terminus have been shown by NMR to preferentially adopt a beta turn at neutral pH in aqueous solution. We now present a series of peptides, related to and including NPC 18325, that explore the relationship between the length of the carbon chain and the affinity to the human bradykinin B2 receptor. The results show that there is a structure-activity relationship (SAR) associated with the chain length and that these pseudopeptides have better affinity to the human bradykinin receptor than they have to the guinea pig ileal-derived B2 receptor. Specifically, peptide I (a 12-methylene linker) had a measured Ki of 31 nM and peptide V (a 4-methylene linker) had a Ki of 471 nM. Implications regarding conformation and hydrophobicity are also described.

Amino Acid Sequence↗