Plasma protein profiles in early asthmatic responses to inhalation allergen challenge.

Although mediators, such as lipids, cytokines, and chemokines, are related to the appearance of an IPR, there has been no reliable indicator to predict conditions for the appearance of an IPR. In this study, we adopted a proteomic approach to investigate the pathogenesis at the level of the plasma proteins and to develop plasma markers to predict the appearance of an IPR following an inhalation challenge with Dermatophagoides pteronyssinus (D.p.). Sixteen mild asthmatics were recruited. Plasma was obtained before challenge and when a decline in forced expiratory volume in 1 s (FEV(1)) values greater than 20% from the phosphate-buffered saline value was achieved during D.p. allergen challenge (positive responders), or at 60 min after the highest concentration of D.p. allergen was inhaled (negative responders). After comparing normalized volumes of the spots in the two groups, differentially expressed spots were identified using intra-gel digestion and mass spectrometric analysis. Before D.p. antigen challenge, four spots of gamma fibrinogen and its isoforms were significantly decreased and two spots of complement C3 fragments were significantly increased in the positive responders compared to the negative responders. After D.p. antigen challenge, complement C3 fragment was persistently higher, while gamma fibrinogen was lower in the positive responders than in the negative responders. A validation study using Western blotting showed that gamma fibrinogen expression in the IPR-positive asthmatics was significantly decreased compared to the average of the IPR-negative asthmatic control group. These results indicate that alterations in the complement cascade and fibrinogen may predispose patients to the appearance of an immediate response to D.p. allergen challenge and may provide plasma markers to predict the appearance of an IPR.

Giant polytene chromosomes from the ovaries of a Drosophila mutant.

The chromosomes of the ovarian nurse cells of Drosophila melanogaster fall apart during their cycles of endoreduplication. However, chromosomal synapsis occurs in the pseudonurse cells produced in certain mutant females. The resulting polytene chromosomes undergo developmental changes that are strikingly different from those recorded for the giant chromosomes of the larval salivary gland cells.

Characterization of an upstream regulatory sequence and its binding protein in the mouse apolipoprotein E gene.

The mouse apolipoprotein (apo) E gene from strain C57BL/6 was isolated from a genomic DNA library and its complete nucleotide sequence, together with 1.3 kilobase of 5' flanking DNA and 300 base pairs of the 3' flanking DNA, was determined. Regulatory sequences in the proximal 5' flanking region of the gene were identified. Using a chloramphenicol acetyltransferase transient assay system, positive and negative cis-acting sequences were mapped within 380 base pairs of the 5' flanking region of the mouse apoE gene. Two nuclear protein binding sites were identified within this region by DNase I footprinting. We have characterized one of these regions, termed mouse apoE regulatory sequence (MARS-2), which spans nucleotides -151 to -133. Gel mobility shift assays using oligonucleotides of the MARS-2 sequence having specific deletions or substitutions as probes or competitors showed that the essential sequence of MARS-2 required for nuclear protein binding consists of 16 nucleotides encompassing -151 to -136. When nuclear extracts from different cells were examined, L cells and mouse liver nuclear protein contained the highest levels of binding protein for the MARS-2 probe. This protein, termed MARS-2 binding protein, was purified from mouse liver nuclear extracts to homogeneity using gel filtration and MARS-2 oligonucleotide-specific column chromatographic procedures. The Mr = 66,000 binding protein showed a gel mobility shift band that was identical to that of crude nuclear extracts.

Cholesterol biosynthesis from lanosterol: regulation and purification of rat hepatic sterol 14-reductase.

We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of delta 8,14-diene or delta 7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis in mammals (Paik et al. (1984) J. Biol. Chem. 259, 13413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5% cholestyramine plus 0.1% lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5% cholesterol or 0.01% AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01% AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 microM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a M(r) = 70,000 protein that is composed of two equally-sized subunits having a M(r) = 38,000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.

Biochemical basis for a cholesterol-lowering activity of 2-[2"-(1",3"-dioxolane)]-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6- nitro-2H-1-benzopyran (SKP-450), a novel antihypertensive agent.

Administration (p.o.) of SKP-450, 2-[2"-(1",3"-dioxolane)]-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6-nitro-2H- 1-benzopyran, a novel antihypertensive agent, to hypercholesterolemic Syrian hamsters led to a significant reduction in plasma lipids in a dose-dependent manner, i.e., a 10.8% to 29% reduction in low-density lipoprotein cholesterol at doses of 0.3 to 10 mg/kg of SKP-450. SKP-450 was found to specifically inhibit the hepatic microsomal lanosterol 14alpha-methyl demethylase (14alpha-DM) in a competitive manner (Ki:2.65 microM). Furthermore, a dose-dependent decrease in the 14alpha-DM activity by SKP-450 parallelled the cholesterol synthetic rate in vitro in both the rat hepatic S10 fractions (supernatants at 10,000 g; IC50:20 microM) and Chinese hamster ovary cells (IC50:23 microM). However, this phenomenon was not seen in AR45 cells, which are deficient in 14alpha-DM, suggesting that 14alpha-DM is the major target for the inhibitory action of SKP-450 in regard to cholesterol biosynthesis.

Cholesterol biosynthesis from lanosterol: regulation and purification of rat hepatic sterol 8-isomerase.

The membrane bound sterol-8-isomerase (isomerase) catalyzes the anaerobic conversion of sterol-8-ene to the sterol-7-ene isomer in eucaryotes. To examine the regulatory mechanism as well as molecular characteristics of the isomerase we investigated the consequences of alteration of the enzymic activity under various diet conditions. Feeding 5% cholesterol or 0.1% AY-9944 for a minimum of 2 days caused more than a 70% decrease in microsomal isomerase activity. Feeding 5% cholestyramine plus 0.1% lovastatin (CL-diet) for 7 days led to approximately 4.0-fold induction of the isomerase activity. In addition, diurnal variation in the enzymic activity was observed with this diet. Induction of the isomerase activity by the CL-diet was quantitatively reflected in an increase in the cholesterol synthetic rate in isolated rat hepatocytes. The isomerase was highly purified from liver of rats fed the CL-diet, and its molecular mass was determined to be 21,000 Da by denaturing sodium dodecylsulfate gel electrophoresis.

Characterization of an upstream regulatory element of the human apolipoprotein E gene, and purification of its binding protein from the human placenta.

We have previously demonstrated that expression of the human apolipoprotein (apo) E gene is controlled by multiple regulatory elements in the promoter [Paik et al. (1988) J. Biol. Chem. 263, 13340-13349; Chang et al. (1990) J. Biol. Chem. 265, 9496-9504]. To extend these studies, we have characterized an element in the apoE gene promoter that spans nucleotides -101 to -89, upstream regulatory element 3 (URE3). Transcription of promoter/marker gene constructs in vitro showed that URE3 modulates gene expression. Gel mobility shift assays of URE3 using human placental nuclear extracts detected a specific binding protein whose activity can be modulated by micromolar amounts of divalent copper and zinc. Competitive binding and gel shift assays with mutant oligonucleotides revealed critical nucleotides within URE3 required for its specific nuclear protein-binding activity. Gel filtration and oligonucleotide affinity chromatography were employed to isolate a URE3-binding protein (URE3BP) from human placental nuclear extracts. Purified URE3BP appears to be a M(r) = 300,000 protein that is composed of four equally-sized basic subunits of M(r) = 67,000. These studies indicate that URE3 is an active regulatory component of the apoE gene.

Cholesterol biosynthesis from lanosterol: differential inhibition of sterol delta 8-isomerase and other lanosterol-converting enzymes by tamoxifen.

The fact that administration of tamoxifen (Tam) to humans and laboratory animals (e.g., rats and monkeys) results in both a drastic reduction in cholesterol and a marked accumulation of certain sterol intermediates in their serum led us to undertake more direct biochemical studies on the mechanism of Tam's inhibitory action on the cholesterogenic enzymes. Of the five rat hepatic lanosterol-converting enzymes examined, the enzyme most sensitive to inhibition by Tam was sterol delta 8-isomerase (delta 8-SI) (a 208-fold inhibition relative to lanosterol 14 alpha-methyl demethylase), followed by sterol delta 24-reductase (13-fold) and sterol delta 14-reductase (5.2-fold). The inhibition patterns of all four affected enzymes were found to be noncompetitive, despite widely different inhibition constants (Ki) of 0.21 to 23.5 microM. The inhibitory activity of Tam on delta 8-SI was not affected by detergent-mediated solubilization of the microsomes. In Chinese hamster ovary cells, inhibition of delta 8-SI activity (IC50 = 0.15 microM) was paralleled by a decreased rate of [14C]-mevalonate incorporation into cholesterol (IC50 = 0.70 microM). Our results should provide more insight into an underlying mechanism of Tam's cardioprotective role by interfering the operation of the pathway of cholesterol biosynthesis from lanosterol in mammals.

Overexpression and characterization of the cDNA encoding the coat protein of cucumber mosaic virus (Strain ABI) isolated in Korea.

We constructed a recombinant CMVCP expression vector termed pMALCMV in which cDNA fragment encoding CMVCP is ligated into pMAL-c2, an E. coli expression vector. Overexpression of pMALCMV containing the entire open reading frame of CMV cDNA sequence and the maltose binding protein (MBP) leader gene was facilitated in E. coli TB1 cells, which resulted in the production of a fusion protein of MBP-CMVCP (Mr 67.7 kDa) that was immunoprecipitable with rabbit polyclonal antiserum specific for MBP. The CMVCP (Mr 24.5 kDa) was isolated through a preparative SDS polyacrylamide gel following digestion of the affinity ligand purified fusion protein with Factor Xa. The partial amino acid sequences of the cleaved proteins were confirmed at the amino terminus by peptide sequencing. The CMVCP antiserum was also prepared by intraperitoneal injection of this purified CP into a BALB/c mouse. Immunoblot analysis showed that the purified CMVCP from the Factor Xa cleavage reaction was an authentic overexpression product of the cloned CMVCP. Using an RNA mobility shift assay, it was demonstrated that CMVCP can bind to its own RNA transcript in a concentration dependent manner. However, the complex formed between CMVCP and its RNA was abolished by the addition of a polyclonal antibody that had been raised against CMVCP, confirming that the overexpressed CMVCP specifically interacts with its own RNA. Thus, our results can provide a basis for the development of a hybridoma cell line expressing the monoclonal antibody for CMVCP and molecular cloning of their genes, which may lead to the creation of CMV-resistant transgenic plants.

The synthesis and in vitro activity of some delta 7,9(11)-lanostadienes.

The synthesis of delta 7,9(11)-lanostadiene derivatives functionalized at C(32) starting from 3 beta-acetoxy-7 alpha,32-epoxylanostan-11-one has been presented. The delta 7,9(11) moiety was efficiently introduced in three steps in 71% yield by the regioselective abstraction of allylic 8 beta hydrogen. The formyl group of the key intermediate, 3 beta-benzoyloxylanosta-7,9(11)-dien-32-al, has been stereoselectively alkylated into (32S) derivative, whereas its oxidation unexpectedly afforded 3 beta-benzoyloxy-7-oxolanost-8-ene-32,11 alpha-lactone and not the corresponding acid. delta 7,9(11)-lanostadienes possessing HC(32)=O, C(32) [symbol: see text] N, HC(32S)CH3OH, H2C(32)OH, as well as some 11-keto lanostenes, were tested in vitro against several purified cholesterogenic enzymes showing moderate activity, with most the active aldehyde 16 having IC50 = 86 microM.

A quantitative study of environmental asbestos exposure in Honolulu.

The increased use of asbestos in various industries in past decades has led to increases in environmental asbestos pollution. Incidental exposure to asbestos is inevitable, and has generated public concern. We performed the following study aimed at determining the level of environmental asbestos exposure in Honolulu, and our results indicate that the levels of environmental asbestos in Honolulu are the lowest in the nation.

Cholesterol biosynthesis from lanosterol: development of a novel assay method and characterization of rat liver microsomal lanosterol delta 24-reductase.

The membrane-bound sterol delta 24-reductase (24-reductase) catalyses anaerobic reduction of the 24(25)-enes of lanosterol and other obligatory intermediates of cholesterol biosynthesis from lanosterol. A novel assay method and properties of the 24-reductase are described. More than a 120-fold induction of the 24-reductase activity was achieved by feeding rats a diet containing 5% cholestyramine plus 0.1% lovastatin in chow and by modulating diurnal variation. With this enzyme induction condition, lanosterol was converted efficiently into dihydrolanosterol in both intact hepatic microsomes and freshly isolated hepatocytes only when either miconazole or CO was added to inhibit 14 alpha-demethylation of lanosterol. AR45 cells, which are deficient in 14 alpha-methyl demethylase (14 alpha-DM), exhibit lanosterol 24-reductase activity without addition of either CO or miconazole. Conversely, inhibition of the 24-reductase was not required for the expression of 14 alpha-DM activity. Studies on the substrate specificities for the 24-reductase using different 24(25)-enes showed that the most reactive substrate was 5 alpha-cholesta-7,24-dien-3 beta-ol, which exhibited a maximal 18-fold higher kcat than that of lanosterol without the aid of the 14 alpha-DM inhibitor. In addition, both the kinetic behaviour of lanosterol substrate in relation to the 24-reductase and a non-competitive inhibition mode of U18666A (Ki 0. 157 microM) as well as Triparanol (Ki 0.523 microM), two well-known 24-reductase inhibitors, were determined. On the basis of our new findings on the preferred substrate and on the negative effect of 14 alpha-DM on the 24-reductase, we suggest that C-24 reduction of sterols takes place straight after sterol delta 8-->7 isomerization of zymosterol, which occurs several steps after C-32 demethylation of lanosterol in the 19-step pathway of cholesterol biosynthesis from lanosterol.

Electrophoretic variability in island populations of Drosophila simulans and Drosophila immigrans.

Genetic structure and variability were investigated in several Hawaiian populations of D. simulans and D. immigrans. Genetic variability is lower in Hawaiian populations of D. simulans than in Texas populations, and allelic differences exist as well. For D. immigrans, Hawaiian and Korean populations are similar in variability, allelic content, and gene frequencies. Several hypothesis are advanced to account for the patterns in gene variation observed between island and continental populations of these two colonizing species.

Continuing studies on chromosomal polymorphism in a Korean natural population of Drosophila melanogaster.

Investigations on the chromosomal inversion polymorphism were conducted on a Korean (Taenung) natural population of D. melanogaster during the period 1978 to 1992. A total of 66 different endemic and cosmopolitan inversions were found on both major chromosome pairs II and III. Some of them proved to be rare cosmopolitan types (2LKA, 2LNS, 2LF, 2RCy, 3LM, 3RKI, and 3RK), while others were endemics. The distribution of breakpoints for endemic and rare cosmopolitan inversions are not random along the two autosome arms. With respect to frequency changes, the 15-year survey revealed that five of the cosmopolitan types (2Lt, 2RNS, 3LP, 3RC, and 3RMo) exhibit cyclical frequency changes, whereas gene arrangement 3RP shows relatively stable frequencies. Tests for correlations between gene arrangement frequencies and several climatic variables gave no clear evidence for such relationships. Only one correlation coefficient out of 64 was statistically significant.

Cholesterol biosynthesis from lanosterol. A concerted role for Sp1 and NF-Y-binding sites for sterol-mediated regulation of rat 7-dehydrocholesterol reductase gene expression.

The 7-dehydrocholesterol reductase (Dhcr7) is the terminal enzyme in the pathway of cholesterol biosynthesis. We have previously reported that sterol depletion in vivo caused a significant induction of both liver mRNA and enzyme activity of Dhcr7 (Bae, S.-H., Lee, J. N., Fitzky, B. U., Seong, J., and Paik, Y.-K. (1999) J. Biol. Chem. 274, 14624-14631). In this paper, we also observed liver cell-specific sterol-mediated Dhcr7 gene induction in vitro by sterol depletion in rat hepatoma cells, suggesting the presence of sterol-mediated regulatory elements in the Dhcr7 gene. To understand the mechanisms responsible for regulating Dhcr7 expression, we have isolated the 5'-flanking region of the gene encoding rat Dhcr7 and have characterized the potential regulatory elements of the gene that are responsible for sterol-mediated regulation. The Dhcr7 promoter contains binding sites for Sp1 (at -177, -172, -125, and -20), NF-Y (at -88 and -51), and SREBP-1 or ADD1 (at -33). Deletion analysis of the Dhcr7 gene promoter (-1053/+31), employing a nested series of Dhcr7-luciferase constructs, demonstrated that the -179 upstream region of the gene is necessary and sufficient for optimal efficient sterol-regulated transcription. DNase I footprinting and electrophoretic mobility shift assay showed that the SRE1/E box (-33/-22) involved in sterol response of many sterol-related enzyme genes was protected specifically by the overexpressed recombinant ADD1. Mutational analysis for the functional relationship between the identified cis-elements in this region indicate that one of the binding sites for Sp1 (GC box at -125) and NF-Y (CCAAT box at -88) plays a cooperative role in the sterol-mediated activation, in which the latter site also acts as a co-regulator for SREBP-activated Dhcr7 promoter activity. We believe that Dhcr7 is the first enzyme characterized with a sterol-regulatory function in the post-lanosterol pathway. This may be important for understanding the coordinated control of cholesterol biosynthesis as well as the molecular mechanism of Smith-Lemli-Opitz syndrome-related protein in mammals.