Effect of phytohormones on chlorophyll degradation during aging of chloroplastsin vivo andin vitro

Summary Phytohormones like IAA and kinetin inhibit chlorophyll loss during aging of wheat chloroplasts duringin vivo andin vitro. GA, on the other hand, stimulates the pigment degradation during aging of attached leaves in contrast to its senescence inhibiting action in detached leaves and isolated chloroplasts. A shift in optimum concentration of hormone in inhibiting chlorophyll degradation suggests a differential pool size of endogenous hormone regulating aging of chloroplastsin vivo andin vitro. The retardation of chlorophyll loss by kinetin, IAA and GA during aging of chloroplastsin vitro would indicate that the effect of hormones in preventing yellowing of senescing leaves may be mediated through their direct action on chloroplasts.     

Charge-transfer complexes of some linear conjugated polyenes.

On adsorption of some electron-acceptor molecules on the solid films of all-trans-beta-carotene, beta-apo-8'-carotenal, astacene and methylbixin a new absorption band appears on the longer-wavelength side of the spectrum in addition to the original bands. The position of this new band is dependent on the electron affinity (EA) of the acceptor molecules, and the intensity of this band increases with the amount of adsorbed acceptor molecules. A linear relationship between the vmax. of the new band and EA was observed. The value of the ionization potential of the polyenes estimated from such linear relationship agrees satisfactorily with the value obtained by other methods. It has been concluded that the polyenes behave as electron donor and first form molecular charge-transfer complexes (of type [polyene . I2] with iodine) with electron acceptors, these finally dissociating to yield ionic complexes (of type [polyene . I+] with iodine).     

Studies on the chemical modification of potato (Solanum tuberosum) lectin and its effect on haemagglutinating activity

1. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the haemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. 2. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. 3. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1m-NN′N″-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. 4. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulphonic acid modified 0.9 and 2.6 residues per molecule of lectin respectively with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulphonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of haemagglutination activity was prevented under these conditions. 5. Reaction of carboxy groups, activated with carbodi-imide, with α-aminobutyric acid methyl ester led to the incorporation of 5.3 residues of the ester per molecule of lectin. Presence of inhibitor in this case, although protecting activity, did not prevent modification of carboxy groups; in fact an increase in the number of modified residues was seen. This effect could be imitated by performing the reaction in 8m-urea. In both cases the number of carboxy groups modified was close to the total number of free carboxy groups as determined by the method of Hoare & Koshland [(1967) J. Biol. Chem. 242, 2447–2453]. Guanidination of lysine residues after carboxy-group modification gave less homoarginine than did the unmodified lectin under the same conditions, suggesting the formation of intramolecular cross-links during carbodi-imide activation. 6. It is suggested from the results presented that amino, arginyl, methionyl, histidyl and carboxyl groups are not involved in the activity of the lectin and that tyrosyl and tryptophyl groups are very closely involved. These findings are similar to those reported for other proteins that bind N-acetylglucosamine oligomers and also fit the general trend in other lectins.     

Superoxide dismutase: a photochemical augmentation assay.

Cell envelope vesicles containing bacteriorhodopsin, prepared from Halobacterium halobium, have previously been shown to accumulate glutamate to high concentration gradients when illuminated. This active transport is energized by a sodium gradient (Na_out⁺ ? Na_in⁺), which arises from Na⁺-efflux coupled to the light-induced H⁺-gradient. The oxidation of dimethyl phenylenediamine (DPD) by the vesicles also can drive uphill glutamate transport, and such transport is inhibited by KCN, azide, ionophores, or uncouplers. K_T for glutamate is 1.4 × 10^?7m under these conditions, as compared to 1.3 × 10^?7m for light-induced transport. The respiration-induced transport of glutamate is dependent on high Na⁺ concentrations on the vesicle exterior and requires low Na⁺ concentrations in the interior. When Na⁺ of increasing concentrations is included in the vesicles, transport proceeds with increasing lags, similarly to the case of light-driven transport. In vesicles to which DPD is added first, and then KCN at increasing time intervals (5 to 15 min), glutamate transport occurs after the addition of KCN, with increasing rates, even though respiration is inhibited. This indicates that the energy generated by DPD-oxidation is conserved over several minutes. These results suggest that in the case of respiration-dependent glutamate transport the translocation is also driven by a Na⁺-gradient; thus, there is a single glutamate transport system independent of the source of energy. The generation of such an Na⁺-gradient during DPD-oxidation implies that the respiration component involved, cytochrome oxidase, is functionally equivalent to bacteriorhodopsin, which acts as a proton pump.     

Studies on electrolyte-nonelectrolyte interactions: Viscosity behavior of alkali halides aqueous sucrose solutions

The effect of various added electrolytes (sodium and potassium halides of progressively increasing molecular volume) on the viscosity behavior of aqueous sucrose solution (292mM) has been investigated. Data are presented for the concentration range 0.125–3M of the electrolytes over the temperature range of 25 to 40°. The data have been found to satisfy Moulik's equation,(η/η₀)²M +K¹C², for concentrated solutions of electrolytes beyond the Einstein region. The “effective” rigid, molar volume (V_e) and apparent. Jones-Dole B-coefficient (in sucrose solution) have been computed by employing the Breslau-Miller treatment. The change in the viscosity behavior is attributed to structuring effects in solution.     

Loss of heterozygosity for alleles on chromosome II in cervical carcinoma.

The HeLa cell (a cervical carcinoma cell line) tumor-suppressor gene has been localized to the long arm of chromosome 11 by molecular genetic studies of nontumorigenic and tumorigenic hybrids derived from normal chromosome 11 x HeLa cell fusions. In the present study, 33 primary cervical carcinoma samples were analyzed using chromosome 11-specific polymorphic DNA markers. The RFLP analysis indicated a somatic loss of chromosome 11 heterozygosity in 10 (30%) of the primary tumors. Preferential loss of the long arm of the chromosome was observed in two of the primary tumors. In addition, at least eight-fold amplification of sequences in the q13 region, including those coding for the fibroblast growth factor-related gene (int-2), was observed in one of the primary tumors. These results suggest a possible role for gene(s) localized to chromosome 11, possibly that localized to the long arm in the development and/or progression of cervical carcinomas.     

Effect of organic anions on the crystallization of the Ca2(+)-ATPase of muscle sarcoplasmic reticulum

The effect of varying the solute species on the crystallization of the Ca2(+)-ATPase from rabbit muscle reticulum (SR) is reported. We have found that substitution of KCl with salts of organic acids in the crystallization protocol reported by Pikula et al. has a profound effect on the size of two-dimensional crystalline arrays. Crystalline arrays of up to 3 microns diameter have been obtained by incubating purified calcium ATPase in standard crystallization medium but with 0.8 M sodium propionate substituted for KCl. These two-dimensional (2-D) arrays display a reduced tendency to stack in addition to having larger planar dimensions. Increasing the KCl concentration does not have the same effect on stacking or crystal growth that sodium propionate has. The production of 2-D sheets has some dependence on the hydrocarbon chain length of the salt because crystals formed in propionate were larger and less stacked than those formed in acetate or formate. There seems to be no dependence on cation. These observations suggest that in addition to reducing the forces that lead to stacking of the sheets, propionate may facilitate incorporation of the detergent-solubilized protein into the 2-D sheet.     

Regulation of urea synthesis in sublobular regions of the liver lobule by oxygen

Periportal and pericentral regions of the liver lobule were isolated from perfused rat liver using a micropunch and incubated in Krebs-Henseleit buffer (pH 7.6) containing 2% poly(ethylene glycol) in Eagle's basal medium, PMSF (50 micrograms/ml) and leupeptin (20 micrograms/ml) for 2 h at 25 degrees C under and O2/CO2 (95:5%) gas phase. Maximal rates of urea production from ammonium chloride were 96.4 +/- 8.7 and 32.8 +/- 5.4 mumol/g per h at 800 and 200 microM O2. Thus, urea synthesis was 2-3-times greater at high than low O2 tension in plugs from periportal and pericentral regions of the liver lobule.     

Effect of aflatoxin B1 on chromatin-bound ribonucleic acid polymerase and nucleic acid and protein synthesis in germinating maize seeds.

The treatment of germinating maize seeds (cv. Ganga 2) with aflatoxin B1 resulted in suppression of ribonucleic acid (RNA), protein, and deoxyribonucleic acid (DNA) synthesis at 3, 4, and 5 h, respectively. At or below the concentrations inhibitory for these in vivo syntheses, the toxin inhibited chromatin-bound DNA-dependent RNA polymerase activity. The synthesis of both polyadenylated and non-polyadenylated RNA was inhibited, but the effect on the former was more pronounced. Equilibrium dialysis and difference spectral and viscometric analyses showed a binding of aflatoxin B1 to DNA isolated from the seeds. It is proposed that the inhibition of RNA synthesis in maize seeds by the toxin is due to the interference with the RNA polymerase activity, which seems, at least partially, due to the impairment of DNA template functions.     

Avian Retrovirus pp32 DNA-Binding Protein I. Recognition of Specific Sequences on Retrovirus DNA Terminal Repeats

The avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA.