Effects of divalent cations on the ultrasonic absorption coefficient of negatively charged liposomes (LUV) near their phase transition temperature

The ultrasonic absorption coefficient per wavelength (alpha lambda), as a function of temperature and frequency, was determined for large unilamellar vesicles (LUV) in the vicinity of their phospholipid phase transition temperature, using a double crystal acoustic interferometer. (The vesicles were composed of a 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). It has been found that alpha lambda reaches a maximum (alpha lambda)max at the phase transition temperature (tm) of the phospholipids in the bilayer, at an ultrasonic relaxation frequency of 2.1 MHz. Divalent cations (Ca2+ and Mg2+), added to LUV suspensions, shifted (alpha lambda)max to higher temperatures, dependent upon the concentration of divalent cation. It was also found that the shape of the alpha lambda versus t curve was significantly changed, representing changes in the Van't Hoff enthalpy of the transition, and therefore, the cooperative unit of the transition. This suggests that divalent cations interact individually with the negatively charged phospholipid headgroups of DPPG and with DPPC headgroups, thus decreasing the cooperative unit of the transition. The observed upward shift in tm suggests an interaction that increases the activation energy and, therefore, the temperature of the phase transition. However, alpha lambda as a function of frequency did not change with the addition of divalent cations and, thus, the relaxation time of the event responsible for the absorption of ultrasound is not changed by the addition of divalent cations.     

Exopolysaccharides Produced by Phytopathogenic Pseudomonas syringae Pathovars in Infected Leaves of Susceptible Hosts

Bacterial exopolysaccharide (EPS) was extracted from infected leaves of several host plants inoculated with phytopathogenic strains of Pseudomonas syringae pathovars. Extraction was by a facilitated diffusion procedure or by collection of intercellular fluid using a centrifugation method. The extracted EPS was purified and characterized. All bacterial pathogens which induced watersoaked lesions on their host leaves, a characteristic of most members of this bacterial group, were found to produce alginic acid (a polymer consisting of varying ratios of mannuronic and guluronic acids). Only trace amounts of bacterial EPS could be isolated from leaves inoculated with a pathovar (pv. syringae) which does not induce the formation of lesions with a watersoaked appearance. Guluronic acid was either present in very low amounts or absent in the alginic acid preparations. All bacterial alginates were acetylated (7-11%). Levan (a fructan) was apparently not produced as an EPS in vivo by any of the pathogens tested.     

Hormonal Regulation, and Intracellular Localization of a 33-kD Cationic Peroxidase in Excised Cucumber Cotyledons

Ethylene enhanced chlorosis and levels of 33-kilodalton cationic peroxidase (33-CPO) in excised cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. Compared to other hormones, such as kinetin, indoleacetic acid, gibberellic acid, and abscisic acid, ethylene was the only effective promoter of 33-CPO synthesis. The hypothesis that peroxidase plays a role in chlorophyll degradation was tested by comparing levels of 33-CPO in cotyledons treated with compounds thought to either retard (kinetin, indoleacetic acid and gibberellic acid), or promote (abscisic acid and methyl jasmonate [MJ]) senescence. It was concluded that 33-CPO did not play a role in senescence since no direct correlation between chlorophyll content and 33-CPO was observed. MJ was as effective as ethylene in inducing senescence. However, ethylene did not appear to be involved in the action of MJ. Using immunocytochemistry, 33-CPO was found to be located primarily around starch grains and near the plasmalemma. High levels of 33-CPO were also found in cells destined to be vascular tissue.     

Oxidation of glycated proteins: age-dependent accumulation of N epsilon-(carboxymethyl)lysine in lens proteins

N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of fructoselysine (FL) in glycated (nonenzymatically glycosylated) proteins in vitro and has also been detected in human tissues and urine [Ahmed et al. (1986) J. Biol. Chem. 261, 4889-4894]. In this study, we compare the amounts of CML and FL in normal human lens proteins, aged 0-79 years, using specific and sensitive assays based on selected ion monitoring gas chromatography-mass spectrometry. Our results indicate that the lens content of FL increases significantly between infancy and about age 5 but that there is only a slight, statistically insignificant increase in FL between age 5 and 80 (mean +/- SD = 1.4 +/- 0.4 mmol of FL/mol of Lys). In contrast, the lens content of the oxidation product, CML, increased linearly with age, ranging from trace levels at infancy up to 8 mmol of CML/mol of lysine at age 79. The ratio of CML to FL also increased linearly from 0.5 to 5 mol of CML/mol of FL between age 1 and 79, respectively. These results indicate that CML, rather than FL, is the major product of glycation detectable in adult human lens protein. The age-dependent accumulation of CML in lens protein indicates that products of both glycation and oxidation accumulate in the lens with age, while the constant rate of accumulation of CML in lens with age argues against an age-dependent decline in free radical defense mechanisms in this tissue.     

Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus

An aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, contained a polypeptide neurotoxin (Sh I) selectively toxic to crustaceans. The polypeptide was purified by G-50 Sephadex, phosphocellulose, and sulfopropyl-Sephadex chromatography and shown to have a molecular size of 5200 daltons and a pI of 8.3. The amino acid sequence determined by automatic Edman degradations of whole RCM Sh I and of its clostripain, staphylococcal protease, and cyanogen bromide digest peptides is A1ACKC5DDEGP10DIRTA15PLTGT20VDLGS25CNAGW30EKCAS35YYTII40ADCCR45KKK . Only 33% of this sequence is identical with the sequence of Anemonia sulcata toxin II, a sea anemone toxin isolated from the taxonomic family Actiniidae. The six half-cystines are located in equivalent positions to those of the actiniid toxins and account for nearly half of the residues common to all of the toxins. However, 69% of the Sh I sequence is identical with that of toxin II from Heteractis paumotensis, another sea anemone belonging to the family Stichodactylidae. Stichodactylid toxins lack the initial N-terminal residue of actiniid toxins and possess three consecutive acidic residues at positions 6-8, a single tryptophan at position 30, and four consecutive basic residues at positions 45-48 (C-terminus). A rabbit IgG prepared by Sh I immunization bound Sh I with a K0.5 of 4.7 nM but failed to bind homologous actiniid (Anemonia sulcata II, Condylactis gigantea III) or bolocerid (Bolocera tuedae II) polypeptide neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection by beta-carotene and related compounds against oxygen-mediated cytotoxicity and genotoxicity: implications for carcinogenesis and anticarcinogenesis.

beta-Carotene protects against photooxidative dermatitis in porphyric humans and mice by quenching of photoactivated species. Other actions of beta-carotene in vivo are explained on the basis of its ability to scavenge free radicals in vitro. For example, in guinea pigs treated with CCl4, beta-carotene decreases pentane and ethane production. Epidemiological studies link low serum beta-carotene levels to elevated risk of lung and other cancers, and in intervention trials, beta-carotene diminishes preneoplastic lesions. However, the dose/response relationships are not well established, and antineoplastic mechanisms await clarification. Given a radical quenching mechanism, beta-carotene should block tumor promotion, but more typically the site of action is progression and an even later role in invasion has not been ruled out. Some antineoplastic actions of carotenoids (such as increased rejection of fibrosarcomas in mice) are attributed to immunoenhancement; others may reflect conversion to retinoids and subsequent gene regulation. Carotenoids other than beta-carotene may act at an earlier stage of carcinogenesis or be more effective as anticarcinogens at certain target sites. As scavengers of hydroxyl radicals, canthaxanthin and astaxanthin are more effective than beta-carotene. Canthaxanthin is sometimes more effective than beta-carotene in chemoprevention, but it is sometimes completely ineffective. Lycopene quenches singlet oxygen more than twice as effectively as beta-carotene. However, the antineoplastic actions of lycopene or astaxanthin remain untested. Explorations of the interactions of carotenoids with other nutrients are just beginning. Dietary fat increases absorption of carotene but decreases antineoplastic effectiveness. Research is hampered by technical problems, including the unavailability of rigorous controls, the instability of carotenoids, and the heterogeneous phase structure induced by hydrophobic compounds in aqueous media. Areas of current controversy and promising approaches for future research are identified.     

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.     

N-terminal amino acid sequence analysis of the subunits of pea photosystem I

Six 'core' subunits of pea photosystem I have been isolated and their N-terminal amino acid sequences determined by gas-phase or solid-phase sequencing. On average more than thirty residues were determined from the N-terminus of each polypeptide. This sequence analysis has revealed three polypeptides with charged N-terminal regions (21, 17 and 11 kDa subunits), one polypeptide with a predominantly hydrophobic N-terminal region (9 kDa subunit), one polypeptide which is cysteine-rich (8 kDa subunit) and one which is alanine-rich (13 kDa subunit).