Production and Characterization of a Pressure-induced Gel from Freeze-concentrated Milk

A process was developed to produce a characteristic milk gel. Raw and market milk samples were freeze-concentrated using bacterial ice nuclei. The concentrates were kept at 5°C and compressed at 300–600 MPa for 5 min. The combination of the freeze concentration and the pressurization gave a milk gel without adding any gelling agents. The addition of sugar at 10% to the concentrated milk improved its gel strength and viscoelasticity. The gel was characterized by a phase transition at about 62–75°C.     

Pyrenocine C,A phytotoxin-related metabolite produced by onion pink root fungus,Pyrenochaeta terrestris

The structure of pyrenocine C, a new metabolite isolated from onion pink root fungus, Pyrenochaeta terrestris (Hansen) has been elucidated as (±)-(2′E)-5-(1′-hydroxybut-2′-enyl)-4-methoxy-6-methyl-2-pyrone by spectroscopic methods and chemical correlation with pyrenocine A.     

Thiolytic cleavage of the anti-tumour compound 4′-(9-acridinylamino)-methanesulphon-m-anisidine (m-AMSA,NSC 156 303) in blood

4′-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA), a compound with a broad spectrum of experimental anti-tumour activity, was found to have a short biological half-life in mice bearing L1210 leukaemia. The fate of m-AMSA [³H]-labelled in the acridine nucleus, was determined following injection into mice. There was rapid formation of covalent adducts with plasma proteins. Adducts were also formed in freshly isolated blood samples following incubation at 37°C, and were found to be highly fluorescent. The formation of adducts was accompanied by a decrease in the free thiol concentration in plasma, and the concomitant addition of radioactivity from [³H]acridine nuclei. Acid or alkaline hydrolysis of the plasma protein adduct liberated acridone, while digestion with a protease produced unstable fluorescent compounds. A comparison of the rates of acid hydrolysis of the adducts and of model compounds suggested that the adducts were produced as a result of nucleophilic attack at the C-9 position of m-AMSA by protein thiol groups. The side chain of m-AMSA was liberated as 4-amino-3-methoxymethanesulphonanilide. Several congeners of m-AMSA were shown to form similar or identical adducts both in vivo and in vitro, and at rates which correlated with their reactivity towards simple organic thiols.     

Evidence that Fe2+ complexes of 3-aminopicolinate and 3-mercaptopicolinate activate and inhibit phosphoenolpyruvate carboxykinase

3-Mercaptopicolinic acid is known to be an inhibitor of phosphoenolpyruvate carboxykinase and 3-aminopicolinic acid permits Fe²⁺ to activate the enzyme. The potency of mercaptopicolinate is increased by incubating the enzyme with Fe²⁺ prior to assaying for activity. In the present work, the average combining ratio of either pyridine carboxylate with Fe²⁺ at pH 7.5 was determined to be 2:1 when measured by the method of continuous variation of Job or by elemental analysis of the isolated pyridine carboxylate-Fe²⁺ complexes. The ratio of 3-mercaptopicolinate or 3-aminopicolinate to Fe²⁺ that caused the greatest inhibition or activation of purified phosphoenolpyruvate carboxykinase was 2:1. In the absence of Fe²⁺, neither pyridine carboxylate altered the activity of the enzyme. These results indicate that the two pyridine carboxylates can interact with phosphoenolpyruvate carboxykinase as Fe²⁺ coordination complexes.     

Bound nucleotides and phosphorylation in Rhodospirillum rubrum.

Chromatophores from $\text{Rhodospirillum rubrum}$ contain 12 × 10^?3 mol ATP and 8.3 × 10^?3 mol ADP per mol chlorophyll, tightly bound to the coupling ATPase. Under energised conditions, these exchange slowly with added nucleotide. Using single turnover light flashes, it is demonstrated that the release of bound ATP is too slow to be on the direct pathway of photophosphorylation.     

Daphneticin,a coumarinolignoid from Daphne tangutica

A new coumarinolignoid, daphneticin, has been isolated from the roots of Daphne tangutica and its structure elucidated on the basis of chemical and NMR spectroscopic evidence.     

In vitro alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl-adducts of guanine and thymine and carboxyethyl-adducts of adenine and cytosine

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.     

Characterization of the low temperature thermoluminescence band Zv in leaf an explanation for its variable nature

Out of the six thermoluminescence bands reported for a mature leaf, one band (Z_v) appearing at the lowest temperatures is dependent on the temperature of illumination. The characteristics of this band in fresh leaf are compared with those in a leaf heated to 60°C for 5 min. It is concluded here that this band, following illumination at temperatures lower than 173 K, is part of Arnold and Azzi's Z band (Arnold, W. and Azzi, J.R. (1971) Photochem. Photobiol. 14, 233–240). However, it is a part of peak I when observed subsequent to illumination beyond 173 K. An explanation for the appearance of this band at different temperatures is proposed.     

Synthesis and characterization of a new fluorescent phospholipid

A novel fluorescent phospholipid, whose structure was tentatively assigned as 1-(2′-thio-1′-hydroxyethyl)-2-(ethylphosphatidyl)isoindole), was synthesized by reacting O-phthalaldehyde and β-mercaptoethanol with phosphatidylethanolamine. The fluorescent lipid product was purified by silicic acid chromatography. The purity was demonstrated by thin-layer chromatography. This fluorescent phospholipid could not form stable lipid vesicles. However, a mixture of phosphatidylcholine and this fluorescent phospholipid did form stable vesicles after sonication, as demonstrated by Sepharose 4B column chromatography and electron microscopy. The absorption and fluorescence properties of this lipid, both as aqueous micelles or incorporated into vesicles, have been determined. The potential usage of this new fluorescent phospholipid in membrane studies is discussed.     

A new method for oligo(ADP-ribose) fractionation according to chain length

A new method was developed to separate mono- and oligo-(ADP-ribose) with chain lengths below 11 ADP-ribose units by size difference of one ADP-ribose residue. The separation was performed on a DEAE-cellulose column by elution with a NaCl gradient (0–0.3 $\text{M}$) in the presence of 7 $\text{M}$ urea at pH 7.6. Using this method, the chain length distribution of oligo(ADP-ribose) molecules attached to histones by incubation of isolated nuclei with radioactive NAD was determined. The average chain length estimated from this distribution coincided exactly with the value obtained by the phosphodiesterase digestion method, suggesting that the oligomers were synthesized directly on histones and not elongated from pre-existing ADP-ribose.     

Macrantaline and macrantoridine,new alkaloids from a turkish sample of Papaver pseudo-orientale

The major alkaloids of the aerial parts of a Turkish sample of Papaver pseudo-orientale are salutaridine and a new alkaloid, macrantaline, UV, IR, PMR, MS and CD have been used to establish the structure of macrantaline as 1-(2′-hydroxymethylene-3′,4′-dimethoxybenzyl)-2-methyl-6,7-methylenedioxy-8-methoxy-1,2,3,4-tetrahydroisoquinoline. The corresponding 2′-methyl substituted analogue prepared from (?)-α-narcotine and also from macrantaline proved to have identical properties, including CD spectra, thus confirming the structure and establishing the absolute configuration of macrantaline. A new minor alkaloid, macrantoridine, yielded macrantaline on lithium aluminium hydride reduction and differs from the latter in that the 2′-substituent is a carboxyl instead of hydroxymethylene. UV, IR, PMR, MS and CD data are reported for macrantoridine.     

Fluorescent probes in model membranes. II. monolayer studies of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole, d-3-aminodesoxyequilenin and n-octadecylnaphthyl-2-amino-6-sulfonic acid with host-lipid tetradecanoic acid

Film studies at the air-water interface have been carried out for pure films of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole (VPBO), d-3-aminodesoxy-equlenin (EQ) and N-octadecylnapthyl-2-amino-6-sulfonic acid (ONS), and for mixed films with tetradecanoic acid for the first two fluorescent probes. Pure film isotherms indicate highly rigid non-monomolecular films for both VPBO and EQ, revealing the presence of strong intermolecular forces. In mixed films with tetradecanoic acid VPBO rapidly segregates with resultant film loss over a wide concentration range. EQ, however, can be stabilized by the host-lipid at low concentrations. This, coupled with an ability to only slightly affect the host-lipid liquid-condensed/liquid-expanded phase change, suggests that EQ can be regarded as “non-perturbing” and should be retained in condensed lipid phases.ONS, because of its unusual polar headgroup, resembled hexadecanoic acid more than octadecanoic acid. While difficulties in spreading ONS precluded the study of mixed films, the indications are that it would be a satisfactory expanded lipid state probe if mixing can be brought about.     

Comparative 5-doxylstearoyllecithin and 3-doxylcholestane EPR spin labeling study of phospholipid bilayer perturbation by different oxidized lecithin species

A 3-doxylcholestane spin label was employed in addition to 5-doxylstearoyl lecithin for a more detailed study of the different effects exerted by variously oxidized lecithins on fatty acid alignment in phospholipid planar bilayers. Either spin label was enclosed in oriented PLPC planar samples also containing in turn a variety of conjugated-dienes lecithins and cleaved chain lecithins, in order to monitor EPR spectral angular dependence loss. Data obtained by use of arachidonoyl-hydroxystearoyl-PC and palmitoyl-hydroxylinoleoyl-PC confirm that the 5-DSPC nitroxide ring almost completely retains its orientation in CD-PCs-containing planar membranes, in contrast with angular dependence loss observed in the presence of the CC-PC molecular species palmitoyl-oxononanoyl-PC and palmitoyl-oxovaleroyl-PC, already seen with cleaved-chain palmitoyl-glutaroyl-PC and palmitoyl-azelaoyl-PC. However, the 3-DC nitroxide ring also loses its orientation with CD-PCs, in addition to being disoriented by cleaved chain-lecithins, similarly to 5DSPC. Joint information from the two spin labels will help to clarify whether OXPC-related disordering involved the whole bilayer structure or only the hydrophobic core. In addition, the propensity of different OXPCs to form bilayer vesicles in water suspension was also determined by Sepharose 4B gel-chromatography. The results suggest that CD-PCs might yield SPB bilayer structures with a disordered hydrophobic core, while pure CC-PC more probably forms non-bilayer disordered structures, possibly micelles or mixed micelle/bilayers.     

The Interaction of Flavonoids with Membranes: Potential Determinant of Flavonoid Antioxidant Effects

Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2′-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2′-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid–water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.     

Mutagenicity of BCNU and related chloroethylnitrosoureas in Drosophila.

BCNU and 10 related chloroethylnitrosoureas were tested for their ability to induce sex-linked recessive lethals in Drosophila spermatozoa. All chloroethylnitrosoureas tested were potent mutagens. Among the substances with one chloroethylnitrosourea group, chlorozotocin, BCNU and methanesulfonyloxyethyl chloroethylnitrosourea exhibited the strongest mutagenic effects. Two hydroxyalkyl chloroethylnitrosoureas behaved as potent mutagens too, although the mutation frequencies obtained were one order of magnitude lower relative to the other substances. Among the compounds with two chloroethylnitrosourea groups, bisCNU-ethane and bisCNU-diphenylmethane were most active. When the interconnecting polymethylene chain was elongated from 2 methylene groups (bisCNU-ethane) to 6 methylene groups (bisCNU-hexane), the mutagenic activity decreased by a factor of 2. The mutagenic activity of polymethylene bischloroethylnitrosoureas with connecting chains of intermediate length was not different from bisCNU-hexane. Differences in mutagenic activity were supposed to reflect different concentrations reaching the target cells, possibly in part as a result of differences in transportability of the substances.