Age-related changes in oxidized proteins

We have previously described the oxidative inactivation of several key metabolic enzymes by a variety of mixed function oxidation systems. Because many of the enzymes which are inactivated have been shown by others to accumulate as inactive or less active forms during cellular aging, we have examined the levels of oxidatively modified proteins in two model systems used for studies on aging. The results show that levels of oxidatively modified proteins increase with age in circulating erythrocytes, and this change is correlated with the loss of marker enzyme activity. Our studies also show that in cultured fibroblasts from normal donors the levels of oxidatively modified proteins increase only after the age of 60. However, the levels of oxidatively modified proteins in fibroblasts from individuals with progeria or Werner's syndrome are significantly higher than age-matched controls. Moreover, treatment of glucose-6-phosphate dehydrogenase with a mixed function oxidation system leads to oxidative modification and increased heat lability of the enzyme. Taken together these results suggest that loss of functional enzyme activity and increased heat lability of enzymes during aging may be due in part to oxidative modification by mixed function oxidation systems.     

Synthesis and biochemical studies of analogs of platelet-activating factor bearing a methyl group at C2 of the glycerol backbone

Two platelet-activating factor (PAF) analogs containing a methyl group at C2 of the glycerol moiety were synthesized, and some of their biochemical properties were investigated. 1-O-Hexadecyl-2-C,O-dimethyl-rac-glycero-3-phosphocholine (2-methyl-2-methoxy PAF) was prepared in a synthetic scheme beginning with the etherification of 2-methylpropen-1-ol. A reaction sequence involving hydroxylation, tritylation, alkylation, and detritylation afforded 1-O-hexadecyl-2-C,O-dimethyl-rac-glycerol, which was converted into the phosphocholine. A 2-lyso derivative of this PAF analog (2-methyl-lyso PAF) was synthesized from 1-O-hexadecyl-2-C-methyl-3-O-trityl-rac-glycerol. Benzylation followed by detritylation gave 1-O-hexadecyl-2-C-methyl-2-O-benzyl-rac-glycerol, which was converted into the phosphocholine compound. Hydrogenolysis afforded 1-O-hexadecyl-2-C-methyl-rac-glycero-3-phospholine (2-methyl-lyso PAF). The 2-methyl-lyso PAF analog served as a substrate for the acetyl-CoA-dependent acetyltransferase that acetylates 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine. However, 2-methyl-lyso PAF did not have a significant effect on the activities of a CoA-independent transacylase or of the acetylhydrolase that inactivates PAF, and thus does not appear to be a substrate or an inhibitor, respectively, for these enzymes. In addition, this analog exhibited only one-half of the antitumor activity of rac-1-O-alkyl-2-methoxy-rac-glycero-3-phosphocholine in human leukemic (HL-60) cells, and elicited no hypotensive response in rats and no platelet-activating activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Structure and thermotropic properties of hydrated 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine bilayer membranes

The ether-linked phosphatidylcholines 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine (EDPC) and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine (DEPC) have been investigated by differential scanning calorimetry (DSC) and X-ray diffraction. DSC of hydrated EDPC shows a single endothermic transition at 34.8 degrees C (delta H = 11.2 kcal/mol) after storage at -4 degrees C while DEPC shows three endothermic transitions at 7.7 and approximately 9.0 degrees C (combined delta H approximately 0.4 kcal/mol) and at 25.2 degrees C (delta H = 4.7 kcal/mol). Both the single transition of EDPC and the two higher temperature transitions of DEPC are reversible, while the approximately 7.7 degrees C transition of DEPC increases in enthalpy on low-temperature incubation. At 23 degrees C, X-ray diffraction of hydrated EDPC shows a sharp reflection at 4.2 A together with lamellar reflections corresponding to a bilayer periodicity, d = 56.2 A. Electron density profiles derived from swelling experiments show a phosphate-phosphate intrabilayer distance, dp-p, of 36 A at all hydrations. This, together with calculated lipid thickness and molecular area considerations, suggests an interdigitated, three chains per head group, bilayer gel phase, L beta*, with no hydrocarbon chain tilt. This is structurally analogous to the bilayer gel phase of hydrated 18:0/10:0 ester PC [McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038]. In contrast, DEPC at -4 degrees C shows an L beta' bilayer gel phase with tilted hydrocarbon chains (d = 61.1 A). However, this transforms above 9 degrees C to an interdigitated, triple-chain, L beta* bilayer gel phase (identical with that of EDPC) with d = 56.6 A and a phosphate-phosphate distance of 36 A. Above their respective chain melting transitions, Tm, EDPC and DEPC exhibit liquid-crystalline L alpha bilayer phases with d = 64.5 and 65.0 A at 55 and 45 degrees C, respectively. The ability of both EDPC and DEPC to form triple-chain interdigitated gel-state bilayers suggests that the conformational inequivalence at the sn-1 and sn-2 positions is less pronounced in the ether-linked PCs compared to the ester-linked PCs, where only one of the positional isomers, e.g., 18:0/10:0 PC but not 10:0/18:0 PC, forms the triple-chain structure (J. Mattai, unpublished results). Thus, a different conformation around the glycerol is predicted for ether-linked PC compared to ester-linked PC.     

Ability ofVibrio vulnificus to obtain iron from transferrin and other iron-binding proteins

The ability of virulent and avirulent strains ofVibrio vulnificus to overcome iron limitations by using iron bound to iron-binding proteins was examined. While no strains were able to obtain iron from lactoferrin or ferritin when these proteins were not fully saturated with iron, growth was enhanced by the iron-saturated form of these proteins. None of the strains was able to scavange iron from 30% saturated transferrin, but there were strain differences in the ability to obtain iron from the saturated form. The virulent strains were able to compete more efficiently with transferrin when it was fully saturated with iron than were the avirulent strains.     

SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.

We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea-treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea-treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.     

Terpenoid precursors of strigol as seed germination stimulants of broomrape (Orobanche ramosa) and witchweed (Striga asiatica)

Strigol and some of its synthetic precursors and analogs are known to be germination stimulants for broomrape (Orobanche ramosa) and witchweed (Striga asiatica). Fifteen synthetic terpenoids, similar in structure to one of the four rings of the strigol molecule, were evaluated in two bioassays as seed germination stimulants with broomrape, and nine were found to be active. Five of the more active compounds contained ester groups. Whereas the study was intended primarily to evaluate forced germination of broomrape by aqueous solutions, the results are almost qualitatively identical for broomrape and witchweed. Monocyclic compounds with chemical structures similar to two of the rings of strigol have now been shown to possess significant bioactivity as germination stimulants.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Distribution of the Glucose-1,6-Bisphosphate System in Brain and Retina

The distribution of glucose-1,6-bisphosphate (G16P2) synthase was measured in more than 70 regions of mouse brain, and nine layers of monkey retina. Activities in gray areas varied as much as 10-fold, in a hierarchical manner, from highest in telencephalon, especially the limbic system, to lowest in cerebellum, medulla, and spinal cord. The synthase levels were significantly correlated among different regions with G16P2 itself, as well as with previously published levels of a brain specific IMP-dependent G16P2 phosphatase. In contrast, neither G16P2 nor either its synthase or phosphatase correlated positively with phosphoglucomutase, and in all regions the G16P2 levels greatly exceeded requirements for activation of this mutase. This strengthens the view that G16P2 has some function besides serving as coenzyme for phosphoglucomutase. However, attempts to correlate the "G16P2 system," as defined by the three coordinately related elements, synthase, phosphatase, and G16P2, with other enzymes of carbohydrate metabolism, or with regional data of Sokoloff et al. [J. Neurochem. 28, 897-916 (1977)] for glucose consumption, were unsuccessful. This leaves open the possibility that brain G16P2 might serve as a phosphate donor for specific nonmetabolic effector proteins.