Linear dichroism and orientational studies of carotenoid Langmuir-Blodgett films

The linear dichroism of single monolayers of lutein, zeaxanthin and a mixture of lutein and synthetic phosphatidylcholine has been measured. The angle of orientation of the carotenoid molecules was found to lie between 45° and 51° relative to the plane of the solid support. Although the adsorbed monolayers were mostly in a monomeric state, microscopic observations, as well as the II-A isotherms, indicated the existence of crystalline islets. The results have been interpreted in connection with Haidinger's polarization brushes.     

Expression of human salivary protein genes

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamulaet al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to study the molecular and cellular biology of PRP production. Cell-free translation and mRNA characterization with EO27 indicate that the numerous PRPs seen in saliva are produced from relatively few, large precursors, probably by posttranslational cleavage. This supports an hypothesis originally proposed by Friedman and Karn in 1977 (Am. J. Hum. Genet. 29:44A;Biochem. Genet. 15:549) and later supported by biochemical studies (Karnet al., Biochem Genet. 17:1061, 1979) and molecular studies (Mamulaet al., Fed. Proc. 43:1522, 1984; Maedaet al., J. Biol. Chem. 260:1123, 1985). EO27 was also used in this study to localize PRP mRNA production to the acinar cells of the parotid gland byin situ hybridization.     

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and β-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. (i) Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. (ii) Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H₂O₂ system. But (iii) they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

Involvement of plastoquinone and lipids in electron transport reactions mediated by the cytochrome b6-f complex isolated from spinach

The isolation of a cytochrome b₆-f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol-plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol-plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.     

ATP-induced ΔpH formation in chloroplast ATP synthase proteoliposomes

Summary A procedure to reconstitute CF₀CF₁ proteoliposomes by gel filtration through a Sephadex-column pre-equilibrated with valinomycin and potassium is described. Proteoliposomes reconstituted by this procedure catalyze an ATP-induced pH of 2.5 to 3.5 units. pH was measured with either 9-aminoacridine or with the pH indicator pyranine trapped inside the proteoliposomes. CF₀CF₁ proteoliposomes prepared by conventional techniques catalyzed an ATP-induced formation, but were unable to catalyze an ATP-induced pH even in the presence of valinomycin.The ATP-induced pH was sensitive to uncouplers and energy transfer inhibitors and was increased at low temperatures. It is suggested that ATP-induced pH was observed in these proteoliposomes due to the efficient removal of intravesicular ammonium introduced with the CF₀CF₁ preparation. The ammonium acted as an internal buffer, and thus prevented an observable pH formation.     

Foliage sesquiterpenes of Dacrydium cupressinum: identification,variation and biosynthesis

The major sesquiterpenes in the foliage of Dacrydium cupressinum are α-longipinene, longifolene, longibornyl acetate, caryophyllene, caryophyllene oxide, humulene, α- and β-selinene, β- and δ-elemene, aromadendrene and the rare 9βH-caryophyllene. Sesquiterpene levels vary greatly from tree to tree. As this variation is largely independent of environmental factors, genetic control is proposed. Longifolene and α-longipinene levels are closely correlated, as are those of caryophyllene and humulene. The biosynthetic implications of these correlations are discussed.     

Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Electrostatic effect of trypsin binding on the hydrogen exchange rate of bovine pancreatic trypsin inhibitor beta-sheet NH's

The changes of H-D exchange rates upon protein-protein interactions are generally interpreted as a result of the changes of the dynamic properties of the proteins. The effect of trypsin binding on the H-D exchange kinetics of some trypsin inhibitor amide H's was reported (Simon et al., 1984). In this paper the electrostatic potential originating from the trypsin molecule is calculated at the positions of the studied amide H's in the trypsin-trypsin inhibitor complex. We conclude that the observed decrease of the exchange rates is mainly due to the electrostatic field of the trypsin molecule.     

Reaction of some antiinflammatory 17 beta-(2-aminooxazol-4-yl) steroids with hydrogen peroxide. Synthesis of steroid-17-spiro-5'-oxazolidine-2',4'-diones

The heterocyclic moiety of 17 beta-(2-aminooxazol-4-yl) steroids is sensitive to the oxidizing action of hydrogen peroxide and yields products mainly from the opening of the amino-oxazole ring. Unlike simple 2-aminooxazoles, it does not rearrange to 2-imidazolone and the expected steroidal hydroperoxyimidazolidinones were not detected. Among the substances we isolated, N-(aminocarbonyl)-17 alpha-hydroxy-17-carboxamides (2a) and (3a) undergo spontaneous cyclization, in the reaction conditions, giving steroid-17-spirooxazolidinediones (2d) and (3d). Spirane (2d) was synthesized in high yields from (2a) in strongly alkaline medium.