Oxysterols: biological activities and physicochemical studies.

To improve the understanding of the various biological activities of oxysterols (oxygenated derivatives of cholesterol), studies of their physicochemical properties have been undertaken. Oxysterols modify membrane dynamic properties which consequently trigger several biological effects. Despite the presence of at least one oxygenated group in addition to the C3 beta-hydroxyl, oxysterols insert perfectly into the lipidic bilayer of the membrane inducing a condensing effect similar to, but less potent than, that of cholesterol. In biological membranes oxysterols probably interact with membrane components as they are not easily exchanged after their incorporation into the cell membrane. These lipid-protein interactions are probably crucial for the expression of the biological activities of the oxysterols.     

Studies of the oxysterol inhibition of tumor cell growth

The oxysterols 3 beta-hydroxy-5 alpha-cholest-8-en-11-one, 3 beta-hydroxy-5 alpha-cholest-8-en-7-one, 3 beta-hydroxy-5 alpha-cholest-8(14)-en-7-one, 3 beta-hydroxy-4,4'-dimethylcholest-5-ene-7 one, 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol, lanost-8-ene-3 beta, 25-diol, 25-hydroxylanost-8-en-3-one, 9 alpha, 11 alpha-epoxy-5 alpha-cholest-7-en-3 beta-ol, 3 beta-hydroxycholest-5 alpha-en-22-one, and 3 beta-hydroxycholest-5-en-22-one oxime were evaluated with respect to their ability to inhibit cell growth. All of the sterols were found to possess cytotoxicity when incubated with hepatoma (HTC) and lymphoma (RDM-4) cells in culture at 10-30 microM concentrations.     

The structure and evolution of the spider monkey delta-globin gene

We have isolated the delta-globin gene of the New-World spider monkey, Ateles geoffroyi, and compared its nucleotide sequence with those of other primate delta- and beta-globin genes. Among primate delta-globin genes, the rate of nonsynonymous substitutions is much less than the rate of synonymous substitutions. This suggests that primate delta- globin genes may remain under evolutionary conservation, perhaps because hemoglobin A2 has an as yet unknown physiological importance.      

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

A specific immunological probe for the major myelin proteolipid. Confirmation of a deletion in DM-20

Major myelin proteolipid (MMPL, also called PLP) and DM-20 are the two major intrinsic membrane proteins of CNS myelin. A specific immunological probe was obtained for MMPL by raising antibodies against the synthetic tridecapeptide 117-129 of MMPL. Antibodies against this peptide reacted with the MMPL but did not cross react with DM-20, while both proteolipids had been shown previously to be recognized by antibodies directed against the C-terminal hexapeptide of MMPL. This is in accordance with previous findings showing that DM-20 differs only from MMPL by a deletion of residues 100-140 (+/- few units). Furthermore, this site-specific immunological probe also recognizes MMPL in its native form in oligodendrocytes in primary glial cell cultures.     

Biochemical pathways in prokaryotes can be traced backward through evolutionary time

For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA. This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured. This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.      

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

USE OF SENSORY EVALUATION FOR MEASURING DEODORIZING EFFECTS OF AN AIR CONDITIONER

The application of sensory methodology for measuring deodorizing effect of an air conditioner equipped with electric plasma was introduced. Deodorizing effect was measured using chemical and sensory methods at different time (0, 30 and 60 min) and mode (control, blowing and cooling) of an air conditioner. Smoke from a roll of cigarette in a closed room was used as a source of odor and the concentrations of acetic acid and ammonia were measured as odorous chemical components. As one of the sensory methods triangle test was used and as a first step to obtain deodorizing effects by triangle test, the threshold of each panelist was obtained as the log dilution ratio of odor concentration at which the difference from odorless air was detected. The odor concentration at each time and mode was calculated using the threshold of the panel and the deodorizing effect was obtained on the basis of the odor concentration. In addition to a triangle test, scaling methods such as category scaling or magnitude estimation were used to measure deodorizing effect of an air conditioner. Deodorizing effects by scaling methods were calculated based on odor intensity with time at each mode. The regression analysis was done between the efficacy of deodorizing effect by sensory test and those by acetic acid and ammonia, the R² values of the regression equations for triangle test, category scale, and magnitude estimation were 0.84, 0.72 and 0.69, respectively. Deodorizing effect by triangle test explained the decrease of acetic acid and ammonia better than those by category scaling or magnitude estimation while high cost and time consuming labor involved in triangle tests reduced the merit. The results of this study demonstrated that various sensory methods could be used to measure deodorizing effect of air conditioners and further researches on fast and reliable methods are needed to establish the official procedures.     

Monitoring trichloroethylene mineralization by Pseudomonas cepacia G4 PR1

To analyze the extent of mineralization of trichloroethylene (TCE) without disturbing an actively growing biofilm, a minimal growth medium was formulated that reduces the concentration of chloride ions to the extent that the chloride ions generated from TCE mineralization may be detected with a chloride-ion-specific electrode. By substituting chloride salts with phosphates and nitrates, a chloride-free minimal medium was produced that yields a specific growth rate for Pseudomonas cepacia G4 PR1 which was 93% of that in chloride-ion-containing minimal medium. Furthermore, TCE degradation by resting cell suspensions was similar in both media (85% of 75 M TCE degraded in 6 h), and complete mineralization of TCE was slightly superior in the chloride-free minimal medium (77% compared to 60% of 75 M TCE mineralized in 6 h). In addition, indole-containing, minimal-medium agar plates were developed to indicate the presence of the TCE-degrading enzyme toluene ortho-monooxygenase (fire-engine-red colonies) as well as to distinguish this enzyme from other TCE-degrading enzymes (toluene dioxygenase and toluene para-monooxygenase).