Theoretical assessment of alternative mechanisms for non-photochemical quenching of PS II fluorescence in barley leaves

The components of non-photochemical chlorophyll fluorescence quenching (qN) in barley leaves have been quantified by a combination of relaxation kinetics analysis and 77 K fluorescence measurements (Walters RG and Horton P 1991). Analysis of the behaviour of chlorophyll fluorescence parameters and oxygen evolution at low light (when only state transitions — measured as qN_t — are present) and at high light (when only photoinhibition — measured as qN_i — is increasing) showed that the parameter qN_t represents quenching processes located in the antenna and that qN_i measures quenching processes located in the reaction centre but which operate significantly only when those centres are closed. The theoretical predictions of a variety of models describing possible mechanisms for high-energy-state quenching, measured as the residual quenching, qN_e, were then tested against the experimental data for both fluorescence quenching and quantum yield of oxygen evolution. Only one model was found to agree with these data, one in which antennae exist in two states, efficient in either energy transfer or energy dissipation, and in which those photosynthetic units in a dissipative state are unable to exchange energy with non-dissipative units.Abbreviations: F_o, F_m room-temperature chlorophyll fluorescence yield with all centres open, closed - F_v variable fluorescence yield - LHC II light-harvesting chlorophyll-protein complex of PS II - PS I, PS II Photosystem I, II - P700, P680 primary donor in Photosystem I, II - Q_A primary electron acceptor of PS II - P_max maximum quantum yield of oxygen evolution - qN coefficient of non-photochemical quenching of variable fluorescence - qN_e, qN_t, qN_i coefficient of non-photochemical quenching due to high-energy-state, state transition, photoinhibition - qO coefficient of quenching of dark level fluorescence - qP coefficient of photochemical quenching of variable fluorescence - _P intrinsic quantum yield of open PS II reaction centres = _s/qP - _{PS 2} quantum yield of PS = qP × F_v/F_m - _S quantum yield of oxygen evolution = rate of oxygen evolution/light intensity     

Myeloperoxidase from Neutrophil Peroxisomes

Peroxisomal myeloperoxidase plays a key role in synthesis of oxidants by neutrophilic leukocytes. This heme protein consists of two subunits connected by a disulfide bond. The enzyme uses ₂₂ and l^– for synthesizing HOCl, the major oxidant produced by neutrophils. In addition to the chlorination reaction, myeloperoxidase exhibits some other properties depending on its oxidation state. The enzyme significantly affects synthesis of oxidants in the cells depending on the competing substrate concentrations and other factors. ^¯ ₂ is also a physiological substrate of myeloperoxidase. Its reaction with the enzyme determines how the cells utilize ^¯ ₂ for pathogen elimination. ^¯ ₂ affects the chlorinating and peroxidase activities of myeloperoxidase. In addition, ^¯ ₂ reacts with the enzyme yielding the catalytically active compound III that hydroxylates phenols.     

Spatiotemporal expression patterns of sialoglycoconjugates during nephron morphogenesis and their regional and cell type-specific distribution in adult rat kidney

The expression of 2,6- and 2,3-linked sialic acids on N-glycans was studied in embryonic, postnatal, and adult rat kidney. Histochemistry and blotting using Polyporus squamosus and Sambucus nigra lectins for 2,6-linked sialic acids and the Maackia amurensis lectin for 2,3-linked sialic acids were performed and sialyltransferase activity was assayed. N-glycans with 2,6- and 2,3-linked sialic acid were differently expressed in the two embryonic anlagen and early stages of nephron. Metanephrogenic mesenchyme was positive for 2,3-linked sialic acid but not for the 2,6-linked one, which became detectable initially in the proximal part of S-shaped bodies. Collecting ducts were positive for 2,6-linked sialic acid, whereas 2,3-linked sialic acid was restricted to their ampullae. Although positive in embryonic kidney, S1 and S2 of proximal tubules became unreactive for 2,3-linked sialic acid in postnatal and adult kidneys. In adult kidney, intercalated but not principal cells of collecting ducts were reactive for 2,3-linked sialic acid. In contrast, 2,6-linked sialic acids were detected in all cells of adult kidney nephron. Blot analysis revealed a different but steady pattern of bands reactive for 2,6- and 2,3-linked sialic acid in embryonic, postnatal, and adult kidney. Activity of 2,6 and 2,3 sialyltransferases was highest in embryonic kidney and decreased over postnatal to adult kidney with the activity of 2,6 sialyltransferase always being three to fourfold that of 2,3 sialyltransferase. Thus, 2,6- and 2,3-linked sialic acids are differently expressed in embryonic anlagen and mesenchyme-derived early stages of nephron and show regional and cell type-specific differences in adult kidney.     

Constant-time NOESY: An aid in the analysis of protein NMR spectra

Summary A constant-time version of the homonuclear NOESY experiment (CT-NOESY) is described. The experiment yields simplified protein spectra, in which cross peaks arising from protons with zero or small couplings are differentiated from other cross peaks, thus partially overcoming the problem of signal overlap. In addition, the CT-NOESY spectrum provides information on the magnitude of³J_NH- and³J coupling' constants, and is thus useful to determine torsion angle constraints and to perform stereospecific assignments of CHH protons in the case of³J constants.     

Hydrogen isotope discrimination in higher plants: Correlations with photosynthetic pathway and environment

Summary The ratio of deuterium to hydrogen (expressed as D) in hydrogen released as water during the combustion of dried plant material was examined. The D value (metabolic hydrogen) determined on plant materials grown under controlled conditions is correlated with pathways of photosynthetic carbon metabolism. C₃ plants show mean D values of-132 for shoots and -117 for roots; C₄ plants show mean D values of -91 for shoots and-77 for roots and CAM plants a D value of-75 for roots and shoots. The difference between the D value of shoot material from C₃ and C₄ plants was confirmed in species growing under a range of glasshouse conditions. This difference in D value between C₃ and C₄ species does not appear to be due to differences in the D value (tissue water) in the plants as a result of physical fractionation of hydrogen isotopes during transpiration. In C₃ and C₄ plants the hydrogen isotope discrimination is in the same direction as the carbon isotope discrimination and factors contributing to the difference in D values are discussed. In CAM plants grown in the laboratory or collected from the field D values range from-75 to +50 and are correlated with ¹³C values. When deprived of water, the D value (metabolic hydrogen) in both soluble and insoluble material in leaves of Kalanchoe daigremontiana Hamet et Perr., becomes less negative. These changes may reflect the deuterium enrichment of tissue water during transpiration, or in field conditions, may reflect the different D value of available water in areas of increasing aridity. Whatever the origin of the variable D value in CAM plants, this parameter may be a useful index of the water relations of these plants under natural conditions.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Mechanisms for controlling balance between light input and utilisation in the salt tolerant alga Dunaliella C9AA

The yield of photosynthetic O₂ evolution was measured in cultures of Dunaliella C9AA over a range of light intensities, and a range of low temperatures at constant light intensity. Changes in the rate of charge separation at Photosystem I (PS I) and Photosystem II (PS II) were estimated by the parameters _{PS I} and _{PS II} . _{PS I} is calculated on the basis of the proportion of centres in the correct redox state for charge separation to occur, as measured spectrophotometrically. _{PS II} is calculated using chlorophyll fluorescence to estimate the proportion of centres in the correct redox state, and also to estimate limitations in excitation delivery to reaction centres. With both increasing light intensity and decreasing temperature it was found that O₂ evolution decreased more than predicted by either _{PS I} or _{PS II}. The results are interpreted as evidence of non-assimilatory electron flow; either linear whole chain, or cyclic around each photosystem.Abbreviations F₀ dark level of chlorophyll fluorescence yield (PS II centres open) - F_m maximum level of chlorophyll fluorescence yield (PS II centres closed) - F_v variable fluorescence (F_m-F₀) - PS I Photosystem I - PS II Photosystem II - P₇₀₀ reaction centre chlorophyll(s) of PS I - q_N coefficient of non-photochemical quenching of chlorophyll fluorescence - q_P coefficient of photochemical quenching of fluorescence yield - q_E high-energy-state quenching coefficient - _{PS I} yield of PS I - _{PS II} yield of PS II - _S yield of photosynthetic O₂ evolution - _P intrinsic yield of open PS II centres     

Photosynthetic electron transport and carbon-reduction-cycle enzyme activities under long-term drought stress in Casuarina equisetifolia Forst. & Forst.

The inhibition of photosynthetic electron transport and the activity of photosynthetic carbon reduction cycle (PCR) enzymes under long-term water stress after slow dehydration was studied in non-nodulated Casuarina equisetifolia Forst. & Forst. plants. Initially, drought increased the fraction of closed Photosystem II (PS II) reaction centres (lowered qP) and decreased the quantum yield of PS II electron transport (_PSII) with no enhancement of non-radiative dissipation of light energy (qN) because it increased the efficiency of electron capture by open PS II centres (F_v/F_m). As drought progressed, F_v/F_m fell and the decrease in _PSII was associated with an increased qN. The kinetics of dark relaxation of fluorescence quenching pointed to an increase in a slowly-relaxing component under drought, in association with increased contents of zeaxanthin and antheraxanthin. Total NADP-dependent malate dehydrogenase activity increased and total stromal fructose-1,6-bisphosphatase activity decreased under drought, while the activation state of these enzymes remained unchanged. Water stress did not alter the activity and the activation state of ribulose bisphosphate carboxylase oxygenase.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Genomic organization of human complement protein C8α and further examination of its linkage to C8β

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8, C8, C8) encoded by separate genes. C8 and C8 are linked on chromosome 1p32, whereas C8 is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 gene. The gene contains at least 11 exons spanning 70kb of DNA. When compared to C6, C8 and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 and C8 linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5kb) and in a 5- 3 orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.     

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F₀F₁) is driven by an electrochemical potential of H⁺ (H⁺). F₀F₁ is composed of an ion-conducting portion (F₀) and a catalytic portion (F₁). The subunit composition of F₁ is ₃₃. The active ₃₃ oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F₁ (TF₁). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F₀. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of ₃₃. The torque of the rotation is estimated to be 420 pN·å from the H⁺ and H⁺-current through F₀F₁. The angular velocity () of is the rate-limiting step, because H⁺ increased theV _max of H⁺ current through F₀, but not theK _m (ATP). The rotational unit of F₀ (=ab₂c₁₀) is /5, while that in ₃₃ is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN₃-ATP), and tight Mg-ATP binding in ₃₃ was shown by ESR. The rotational relaxation of TF₁ is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of ₃₃ measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF₁) and F₀-d to hold free rotation of ₃₃.     

Amylase activity and growth in internodes of deepwater rice

Isoelectrofocusing, product analysis, thermal denaturation studies and affinity chromatography on cycloheptaamylose-Sephadex were used to identify the amylolytic enzymes in internodes of deepwater rice (Oryza sativa L.). Amylolytic activity in internodes of deepwater rice consists of -amylase (sometimes separated into two isoforms) and of -amylase. During submergence of whole plants, -amylase activity increases in young, growing internodes, but -amylase activity declines. Although non-growing, mature internodes contain higher levels of -amylase than do the elongating younger internodes, the effect of submergence on amylase activities in both tissues follows the same trend. Submergence, gibberellic acid (GA₃) and ethylene all promote -amylase activity in growing and non-growing internodes of excised deepwater-rice stem sections. Inhibitor studies showed that submergence and ethylene promote -amylase activity in the absence of endogenous gibberellin (GA), and GA₃ enhances -amylase activity when ethylene action is inhibited. Therefore, ethylene and GA appear to increase -amylase activity independently of each other. Enhanced -amylase activities are probably responsible for the mobilization of carbohydrates which are needed to support internode elongation during submergence of deepwater rice.Abbreviations CHA cycloheptaamylose - GA₃ gibberellic acid - NBD 2,5-norbornadiene - TCY tetcyclacis     

Water stress-induced oxidative damage and antioxidant responses in micropropagated banana plantlets

Oxidative injury and antioxidant responses were investigated in two banana genotypes (Musa AAA Berangan and Musa AA Mas) subjected to 40 % PEG-induced water stress. PEG treatment resulted in oxidative injury, as expressed in increased lipid peroxidation and reduced membrane stability index, in both cultivars; however, greater oxidative injury was detected in Mas. Under PEG treatment, catalase activity and glutathione reductase activity were enhanced in both cultivars, but were higher in Mas. Ascorbate peroxidase activity was enhanced in Berangan under water stress, but was unaffected in Mas. Meanwhile, superoxide dismutase activity was inhibited in both cultivars under water stress, but higher activity was detected in Berangan. Higher ascorbate peroxidase and superoxide dismutase activities were associated with greater protection against water stress-induced oxidative injury.     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Demonstration of ‘cardiac-specific’ myosin heavy chain in masticatory muscles of human and rabbit

Summary Human and rabbit masticatory muscles were analyzed immuno-and enzyme-histochemically using antibodies specific to cardiac , slow and fast myosin heavy chain isoforms. In human masseter, temporalis, and lateral pterygoid muscle cardiac myosin heavy chain is found in fibres that contain either fast, or fast and slow myosin heavy chain. In rabbit masseter, temporalis and digastric muscles, fibres are present that express cardiac myosin heavy chain either exclusively, or concomitantly with slow myosin heavy chain or fast myosin heavy chain. Our results demonstrate a much broader distribution of cardiac myosin heavy chain than hitherto recognized and these might explain in part the specific characteristics of masticatory muscles. The cardiac myosin heavy chain is only found in skeletal muscles originating from the cranial part of the embryo (including the heart muscle) suggesting that its expression might be determined by the developmental history of these muscles.