C(2)H(4) metabolism in morning glory flowers

Flowers of Ipomoea tricolor Cav. (cv. Heavenly Blue) were cut at various stages of development and evaluated for their ability to metabolize ethylene. Freshly cut buds or flowers were treated in glass containers for 8 hours with 6 μl/liter of highly purified ¹⁴C₂H₄. Following removal of dissolved ¹⁴C₂H₄, radioactivity was determined for the different flower tissues and trappd CO₂. ¹⁴C₂H₄ oxidation to ¹⁴CO₂ and tissue incorporation occurred at very low to nondetectable levels 2 to 3 days prior to flower opening. About 1 day prior to full bloom, just at the time when mature buds become responsive to ethylene (Kende and Hanson, Plant Physiol 1976, 57: 523-527), there was a dramatic increase in the capacity of the buds to oxidize ¹⁴C₂H₄ to ¹⁴CO₂. This activity continued to increase until the flower was fully opened reaching a peak activity of 2,500 dpm per three flowers per 8 hours. It then declined as the flower closed and rapidly senesced. A similar but smaller peak occurred in tissue incorporation and it was followed by a second peak during late flower senescence. This first peak in tissue incorporation and the dramatic peak in ethylene oxidation slightly preceded a large peak of natural ethylene production which accompanied flower senescence. The ethylene metabolism observed was clearly dependent on cellular metabolism and did not involve microorganisms since heat killing destroyed this activity and badly contaminated heat-killed flowers were unable to metabolize ethylene.     

Species-specific aggregation factor in sponges. XIII. Entire and core structure of the large circular proteid particle from Geodia cydonium

High-molecular weight particles have been isolated from the sponge Geodica cydonium. In the "native" from these particles consist of a spherical center and have 25-30 filaments attached to it. The core structure of the particles is assembled of a central circle and 25 radially-arranged filaments. The core structure is obtained from the entire structure by incubation in a medium, containing a non-ionic detergent and EDTA. The molecular weight of the enitre structure was in the range of 1.4 X 10(9) daltons or more and of the core structure 6.1 x 10(8) daltons. Two functional proteins are released from the "native" particles: the aggregation factor and the sialytransferase.     

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.     

Studies on facial temperature rise and involvement of serotonin in the respiratory stimulation by CRH.

Following an intravenous injection of 100 micrograms hCRH a facial flushing can frequently be observed along with respiratory stimulation. Both effects can be mediated by a common transmitter. Serotonin is well known to produce facial flush as well as to modulate respiration. In order to clarify is serotonin is a common mediator for facial flush and respiratory stimulation after i.v. application of hCRH, we studied the time course of facial skin temperatures and respiratory stimulation after intravenous injection of 100 micrograms hCRH in 10 healthy subjects. Furthermore, we measured respiratory stimulation after i.v. administration of 100 micrograms hCRH in 10 healthy subjects pretreated with the serotonin antagonist cyproheptadine. Facial skin temperatures reached maximum levels 9 min after CRH administration and remained raised for more than 60 min. Respiratory stimulation occurred within the first minute after CRH administration and reached a maximum during the second minute, but could no longer be observed after 10 min. Serum serotonin levels did not change after CRH stimulation in doses up to 3 micrograms/kg body weight), and cyproheptadine did not abolish the respiratory stimulation effect of hCRH in a dosage sufficient to suppress CRH.-induced cortisol secretion.     

Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product.

The gene encoding the multifunctional protein (MFP) of peroxisomal beta-oxidation in Saccharomyces cerevisiae was isolated from a genomic library via functional complementation of a fox2 mutant strain. The open reading frame consists of 2700 base pairs encoding a protein of 900 amino acids. The predicted molecular weight (98,759) is in close agreement with that of the isolated polypeptide (96,000). Analysis of the deduced amino acid sequence revealed similarity to the MFPs of two other fungi but not to that of rat peroxisomes or the multifunctional subunit of the Escherichia coli beta-oxidation complex. The FOX2 gene was overexpressed from a multicopy vector (YEp352) in S. cerevisiae and the gene product purified to apparent homogeneity. A truncated version of MFP lacking 271 carboxyl-terminal amino acids was also overexpressed and purified. Experiments to study the enzymatic properties of the wild-type MFP demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA). The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity. These results clearly demonstrate that the beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate.     

The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles.

The 2.2 A resolution crystal structure of recombinant human manganese superoxide dismutase, a homotetrameric enzyme that protects mitochondria against oxygen-mediated free radical damage, has been determined. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical 4-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form novel tetrameric interfaces that stabilize the active sites. Structurally altered polymorphic variants with reduced activity, such as tetrameric interface mutant Ile-58 to Thr, may produce not only an early selective advantage, through enhanced cytotoxicity of tumor necrosis factor for virus-infected cells, but also detrimental effects from increased mitochondrial oxidative damage, contributing to degenerative conditions, including diabetes, aging, and Parkinson's and Alzheimer's diseases.     

Chin marking behavior, sexual receptivity, and pheromone emission in steroid-treated, ovariectomized rabbits

The effect of daily injections of estradiol benzoate (1 or 10 micrograms) and of progesterone (10 mg) on chin marking activity, sexual receptivity, and emission of nipple-search pheromone in ovariectomized rabbits was investigated. Both estradiol treatments resulted in a significant increase in all three measures over baseline and control group levels within 1-3 days, and withdrawal in a return to pretreatment levels within 2 weeks (Experiment I). In contrast, the administration of progesterone to such estradiol-primed does resulted in an almost immediate suppression of chin marking and lordosis, but in marked enhancement of pheromone emission and aggressive behavior (Experiment II). However, progesterone given alone to nonprimed does had no effect on any of these measure (Experiment III). The response profiles resulting from these treatments correspond well to patterns reported for intact does during estrus (= estradiol alone), pregnancy (= estradiol plus progesterone), and at parturition (= progesterone withdrawal).     

A deoxyribonucleic acid-replication intermediate in the growing mosquito.

In previous experiments on growth and aging in the yellow-fever mosquito, Aedes aegypti, a low mol. wt. (500000) DNA species was found in the supernatant fraction after ultracentrifugation of homogenates of rapidly-growing larvae. This DNA species, "sDNA", constituted 30-40% of total DNA in 2-4-day-old larvae, but was less than 5% in older larvae, pupae and adults. We have now isolated and characterized sDNA and initiated experiments to determine its metabolic role. Isolated sDNA has the same physical and chemical characteristics as bulk DNA, "pDNA", and differs only in size. In CsCl isopycnic centrifugation the buoyant densities of sDNA and pDNA were 1.700 and 1.697 g/cm3 respectively. The "melting" temperature of both DNA species was 84 degrees C. Base compositions calculated from these data and other methods were 38.9 mol% of guanine-plus-cytosine for sDNA, and 38.5 for pDNA. Also, the size of newly-synthesized DNA was investigated by pulse-labelling and pulse-chase experiments. In neutral sucrose gradients the labelled DNA component after a 2h pulse had a sedimentation coefficient of about 8S, but after a 4h pulse sedimented in a broad band from 10-19S. In alkaline sucrose gradients a single peak around 7S was observed for pulse times up to 4h. After a 6h pulse and a 1 day "chase", labelled DNA species had sedimentation coefficients ranging from 10-15S in alkaline sucrose, and after a 2-day chase the values were 17-31S, similar to those of pDNA under alkaline conditions. These results suggest that sDNA represents an intermediate form in the replication of DNA in mosquito larvae.