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The past several years have seen substantial progress in the development of antisense oligonucleotides as pharmacological tools and as therapeutic agents. With properly designed and executed experiments, it has been possible to demonstrate selective inhibition of gene expression, owing to an antisense mechanisms of action both in cell culture-based experiments and in vivo. As the field has matured, it has also realized that oligonucleotides can produce a variety of effects in cell culture and in vivo that cannot be ascribed to an antisense mechanism of action. Nevertheless, with proper controls it has been possible to demonstrate that the pharmacological activity of an oligonucleotide is consistent with an antisense mechanism of action. The pharmacokinetic properties of first-generation phosphorothioate oligodeoxynucleotides and their toxicological limitations have been characterized in rodents, primates, and humans. Finally, it has been demonstrated that medicinal chemistry can improve the properties of oligonucleotides, as several modifications have been identified that have increased potency, altered pharmacokinetic properties and potentially decreased toxicological liabilities.

Antisense oligonucleotides represent a new paradigm for drug discovery that holds great promise to deliver potent and specific drugs with fewer undesired side effects. The antisense paradigm offers the opportunity to identify rapidly lead compounds based on knowledge of the biology of a disease process, and a relevant target gene sequence. With this information, the practitioner of antisense drug discovery can rapidly design, synthesize, and test a series of compounds in cell culture and determine if the target gene is specifically inhibited. A compound thus identified can then be tested in an animal model, either to determine whether targeted gene expression can be inhibited in various animal tissues or to determine if there is activity in an animal model of a human disease. The length of time and the resources required to identify a lead compound by the antisense paradigm is much less than by any other drug discovery method.

Significant islet allograft prolongation was achieved by ICAM-1/LFA-1 blockade. ICAM-1 antisense oligonucleotide IP-3082 was effective in improving islet function as well as prolonging graft survival.

We performed a systematic study of 55 solvent-producing clostridial strains, the majority of which are currently classified as Clostridium acetobutylicum strains, by using a combination of biotyping and DNA fingerprint analysis. The biotyping procedures used included rifampin susceptibility testing, bacteriocin typing, and bacteriophage typing. The 55 strains examined exhibited a good correlation between their biotypes and DNA fingerprints, which allowed us to divide them into nine groups. The DNA fingerprints of the nine groups differed markedly, but within each group the DNA fingerprints exhibited a high level of similarity. To determine the phylogenetic relationships of the nine groups, we performed a 16S rRNA gene sequence analysis. The results of a comparative analysis of the partial sequence corresponding to positions 830 to 1383 (Escherichia coli numbering) of the 16S rRNA gene indicated that the nine biotype groups could be assembled into four taxonomic groups. The complete 16S rRNA sequences of strains representing these groups were determined. Our phylogenetic analysis revealed that the amylolytic type strain C. acetobutylicum ATCC 824 (taxonomic group I) was only distantly related to the saccharolytic strains belonging to taxonomic groups II, III, and IV (levels of sequence similarity, 90 to 90.5%). The strains belonging to taxonomic groups II, III, and IV, represented by C. acetobutylicum NCP 262, "Clostridium saccharoperbutylacetonicum" N1-4, and C. acetobutylicum NCIMB 8052T (T = type strain), respectively, were closely related (levels of sequence similarity, 98.2 to 98.9%). C. acetobutylicum NCIMB 8052T exhibited a level of similarity of 100% with the type strain of Clostridium beijerinckii. Reclassification of the saccharolytic solvent-producing strains is necessary, and possible names for the four taxonomic groups are discussed.

Intercellular adhesion molecule-1 (ICAM-1) binds circulating leukocytes through interactions with beta 2 integrins, LFA-1, and macrophage Ag-1. The phosphorothioate antisense oligodeoxynucleotide, IP-3082, specific for ICAM-1 mRNA inhibited ICAM-1, but not vascular cell adhesion molecule-1, mRNA induction and expression of ICAM-1 molecules by mouse endothelioma cells. Scrambled control oligonucleotides were ineffective. Untreated C3H (H-2k) mice rejected C57BL/10 (H-2b) heart allografts with a mean survival time of 7.7 +/- 1.4 days. Administration i.v. of IP-3082 by a 7-day osmotic pump prolonged the survival of heart allografts in a dose-dependent fashion: 1.25 mg/kg, to 11 +/- 0 days; 2.5 mg/kg, to 12 +/- 2.7 days; 5 mg/kg, to 14.1 +/- 2.7 days; and 10 mg/kg, to 15.3 +/- 5.8 days (all p < 0.01). Control IP-1082 (10 mg/kg) was ineffective (7 +/- 0.8 days). Although 7-day anti-LFA-1 mAb (50 micrograms/day; i.p.) prolonged allograft survival to 14.1 +/- 2.7 days, the addition of IP-3082 (5.0 mg/kg x 7 days) induced donor-specific transplantation tolerance (> 150 days). Furthermore, IP-3082 (5 mg/kg x 7 days) acted synergistically with antilymphocyte serum, rapamycin, and brequinar, but not cyclosporin A: a single antilymphocyte serum (0.2 ml) i.p. injection alone prolonged graft survival to 10 +/- 0.5 days (p < 0.01) and in combination with IP-3082 (5 mg/kg), to 32.2 +/- 8.3 days (p < 0.001); rapamycin (0.1 mg/kg x 7 days; i.v.) alone prolonged survival to 13 +/- 7.5 days (p < 0.01), and with IP-3082, to 32.4 +/- 8.9 days (p < 0.03); brequinar (0.5 mg/kg x 7 days; oral gavage) alone to 12 +/- 2.4 days (p < 0.05), and with IP-3082 (5 mg/kg), to 38.8 +/- 30.2 days (p < 0.01); in contrast, cyclosporin A (5 mg/kg x 7 days; i.v.) alone produced graft survival of 9.8 +/- 1.3 days (p < 0.1 and in combination with IP-3082 (5 mg/kg), produced survival of 7.8 +/- 3.5 days (NS). Thus, antisense oligonucleotides may proffer a selective gene-targeted immunosuppressive therapy for organ transplantation.

Keratinocyte intercellular adhesion molecule-1 (ICAM-1) is important in mediating retention of T cells within the epidermal compartment. To determine if antisense oligonucleotides designed to hybridize to various ICAM-1 mRNA regions could selectively influence cultured keratinocyte ICAM-1 expression following gamma interferon (IFN-gamma), cells were exposed to several antisense compounds, in the absence and presence of cationic lipid (lipofectin). Keratinocytes rapidly internalized sense and antisense compounds (within 30-60 min), even in the absence of lipofectin with approximately 30% of the cell possessing positive nuclei. Such nuclear accumulation was not observed in the absence of lipofectin in cultured fibroblasts, smooth muscle cells, or endothelial cells, even though total cellular uptake within the cytoplasm was significantly increased in all these cell types. Using flow cytometry, IFN-gamma-inducible ICAM-1 expression was reduced 50% by antisense compounds with lipofectin, and by 30% without lipofectin. This inhibition was specific as no change was observed for HLA-DR or tumor necrosis factor-alpha receptor expression. Northern blot hybridization studies confirmed that ICAM-1 antisense oligonucleotides selectively and significantly inhibited ICAM-1 expression. These results suggest that such antisense compounds interact with keratinocytes differently than other cell types, and provide the in vitro basis for clinical trials in which reduction (or elimination) of ICAM-1 expression by epidermal keratinocytes could be selectively accomplished without necessarily influencing dermal cell types such as fibroblasts, endothelial cells, or smooth muscle cells.

Treating human malignant melanoma cells with tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) causes a dose- and time-dependent increase in surface expression of ICAM-1. Increased ICAM-1 expression corresponds to greater binding of human leukocyte functional antigen-1 (CD11a/CD18)-expressing peripheral blood mononuclear cells (PBMC) to C8161 monolayers, suggesting that cytokine-treated melanoma cells would be more metastatic due to PBMC-tumor cell emboli. The purpose of this study was: (1) to test whether TNF-alpha-treated human melanoma cells are indeed more metastatic than untreated C8161 and (2) to determine whether ICAM-1 plays a role in metastasis of C8161. When surface ICAM-1 expression is upregulated, formation of lung metastases in nude mice increases 1.5- to 4-fold (P < 0.05) for human melanoma cell lines C8161, MeWo, and A375. Treatment of C8161 with ICAM-1 phosphorothioate antisense oligonucleotides using cationic lipids results in > 90% inhibition of ICAM-1 surface expression as determined by ELISA and flow cytometry. Antisense ICAM-1-treated cells form 41-64% fewer metastases than sham-treated cells. Metastasis does not increase when antisense-treated melanoma cells are exposed to TNF-alpha. However, treatment of C8161 with antisense 5-lipoxygenase (5-LO) oligonucleotides inhibits metastases 39% in Lipofectin-treated cells, but does not inhibit TNF-alpha-induced upregulation of experimental metastases. Similar experiments were performed to measure PBMC adhesion to antisense oligonucleotide-treated C8161 cells; however, TNF-alpha-inducible increase in adhesion was unaffected by ICAM-1 or 5-LO antisense oligonucleotides. These results demonstrate that ICAM-1 is involved in melanoma metastasis, but probably not at the step of PBMC adhesion to C8161 cells.

Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner.

We have identified 20-mer phosphorothioate oligodeoxynucleotides which potently (IC50 values of 100-200 nM) and specifically inhibit protein kinase C (PKC)-alpha mRNA and protein expression in human lung carcinoma (A549) cells. These oligonucleotides target multiple, diverse sites on PKC-alpha mRNA including the AUG translation codon and 3'-untranslated sequences. 2'-O-Methyl phosphorothioate analogs of these oligonucleotides were without effect on PKC-alpha mRNA levels, suggesting that the reduction in targeted PKC-alpha mRNA is through RNase H-mediated cleavage. One oligonucleotide, however, was effective at inhibiting PKC-alpha protein levels as a 2'-O-methyl phosphorothioate at concentrations 2-3-fold greater than its phosphorothioate/deoxy homolog. These results suggest that the ability to serve as an RNase H substrate, although not required for all oligonucleotides, certainly increases their potency. These oligonucleotides have been used to examine the role played by PKC-alpha in mediating the phorbol ester-induced changes in mRNA levels of the cell adhesion molecule ICAM-1. In A549 cells, ICAM-1 mRNA is increased 10-20-fold by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate. When PKC-alpha protein levels are depleted by oligonucleotide treatment of A549 cells, the increase in ICAM-1 expression in response to phorbol 12-myristate 13-acetate is greatly reduced, demonstrating that PKC-alpha plays a major role in this process.

In response to inflammatory stimuli, expression of a group of proteins that bind circulating leukocytes (endothelial-leukocyte adhesion molecules) are induced on the luminal surface of vascular endothelium. A series of phosphorothioate oligonucleotides 18 to 21 bases in length were designed and synthesized to hybridize selectively to the mRNA, which encodes three such endothelial-leukocyte adhesion molecules; human intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were identified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expression in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selective reduction in the respective mRNA levels. In contrast, oligonucleotides that hybridized to 5'-untranslated sequences did not significantly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell surface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of oligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM-1 expression. However, at an oligonucleotide concentration of 50 nM or greater, phosphorothioate oligonucleotides not predicted to hybridize to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independent inhibition of VCAM-1 expression by phosphorothioate oligonucleotides could be the result of a perturbation in the transcriptional regulation of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense oligonucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. These data demonstrate that phosphorothioate oligonucleotides are capable of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC.

Antisense oligonucleotides have the ability to inhibit gene expression in viral infections, malignancy, and other diseases. Even though much work has been accomplished with oligonucleotides demonstrating in vitro therapeutic effects, little work has been done to address how these molecules gain access to the cell. One of the plausible means of entrance could be through passive diffusion of the oligonucleotides through the cellular lipid bilayer. To enhance membrane permeability of oligonucleotides lipophilic moieties at the 2' position of the ribose ring have been added. To evaluate the effect of this modification, a liposome system was used. The oligonucleotides evaluated were a series composed of poly A 10mers phosphorothioates labeled at the 5' end with fluorescein and modified at the 2' position of the ribose ring with lipophilic alkyl chains ranging from methyl to nonyl. Efflux studies were accomplished by monitoring the appearance of the oligonucleotide in the incubation medium. There were modest but significant differences between the efflux half-life times of the 2'-modified compounds and the control compound. The values ranged from approximately 6 days for the control, unmodified compound to 4.6 days for the propyl modification. The nonyl derivative had a longer efflux half-life time (8.3 days) compared with the control, unmodified phosphorothioate oligonucleotide.

The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody (MAbp120) in most human malignant tumors (Freeman et al., Cancer Research, 48, 1244-1251, 1988). Stable transfection of the sense p120 cDNA caused malignant transformation of NIH/3T3 cells in vitro, and the antisense p120 constructs markedly delayed the growth of these transformed cells (Perlaky et al., Cancer Research, 52, 428-436, 1992). Several p120 antisense phosphorothioate oligonucleotides designed to hybridize with different regions of the p120 sequence were screened on human tumor cell lines in vitro. Marked growth inhibition of HeLa, LOX and HRCC cell lines was found, particularly with antisense p120 oligonucleotide ISIS 3466 in combination with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); oligonucleotide ISIS 3466 is complementary to a non-translated region at the 3' end of the molecule. Preliminary in vivo studies on human LOX ascites tumor in nude mice showed marked inhibitory effects on tumor growth by the antisense oligonucleotide ISIS 3466 in the presence of DOTMA when treated on alternate days.

5-Lipoxygenase performs the first two enzymic reactions in the biosynthetic pathway for the leukotrienes. We have utilized HL-60 cells to study the mechanisms regulating expression of 5-lipoxygenase and the recently described 18 kDa membrane protein, 5-lipoxygenase-activating protein (FLAP). Differentiation of HL-60 cells into granulocyte-like cells with dimethyl sulphoxide (Me2SO), retinoic acid or dibutyryl cyclic AMP (Bt2-cAMP) resulted in a 2-3-fold increase in 5-lipoxygenase enzyme activity and a 4-fold increase in leukotriene B4 synthesis. Differentiation of HL-60 cells into monocyte-like cells with 1,25-dihydroxyvitamin D3 induced 5-lipoxygenase activity 5-fold, but leukotriene B4 production was only increased 2-fold. Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (PMA) into macrophage-like cells failed to induce 5-lipoxygenase or leukotriene B4 production. Examination of the levels of the transcript encoding 5-lipoxygenase and FLAP demonstrated that differentiation of HL-60 cells into granulocytes resulted in a co-ordinate induction of 5-lipoxygenase and FLAP mRNA. In contrast, differentiation of HL-60 cells into monocytes resulted in discordant regulation of 5-lipoxygenase and FLAP mRNA. Treatment with 1,25-dihydroxyvitamin D3 resulted in a 6-fold increase in 5-lipoxygenase mRNA and a 1.3-fold increase in FLAP mRNA, while treatment with phorbol ester did not induce 5-lipoxygenase mRNA but did increase FLAP mRNA 2-fold. The transcriptional rate of the 5-lipoxygenase and FLAP genes did not change upon Me2SO or 1,25-dihydroxyvitamin D3 treatment, suggesting that the increase of the mRNA coding for these proteins was not due to transcriptional activation of their respective genes. The mRNA half-life for 5-lipoxygenase did not change significantly upon treatment with Me2SO or 1,25-dihydroxyvitamin D3. The FLAP mRNA half-life increased from approx. 3.5 h to 4.5 h in cells treated with either Me2SO or 1,25-dihydroxyvitamin D3. These data suggest that expression of 5-lipoxygenase and FLAP is controlled by a post-transcriptional event other than stabilization of the mRNA.

We have investigated the use of a cationic lipid preparation to enhance antisense oligonucleotide activity in human umbilical vein endothelial cells. A liposomal preparation containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was found to increase by at least 1000-fold the potency of an antisense oligonucleotide (ISIS 1570) that hybridizes to the AUG translation initiation codon of human intercellular adhesion molecule-1. In the presence of 8 microM DOTMA, 6-15-fold more 35S-ISIS 1570 associated with cells, at oligonucleotide concentrations from 0.01 to 5 microM, than did in the absence of DOTMA. Both 35S-ISIS 1570 association with cells and antisense activity were increased as a function of DOTMA concentration and with increasing time of incubation with the cationic lipid. Fluorescein-labeled ISIS 1570 was used to assess the intracellular distribution of the oligonucleotide in the presence and absence of DOTMA. In the absence of DOTMA, the oligonucleotide localized to discrete structures in the cytoplasm of the cell, resulting in a punctate fluorescence pattern. In the presence of DOTMA, cellular fluorescence markedly increased and the oligonucleotide localized within the nucleus, as well as to discrete structures in the cytoplasm. Accumulation of the oligonucleotide in the nucleus in the presence of DOTMA was time and temperature dependent. Nuclear accumulation was inhibited by preincubation of the cells with monensin but not chloroquine, NH4Cl, nocodazole, colcemid, or brefeldin A. These data demonstrate that cationic lipids increase antisense activity by increasing the amount of oligonucleotide associated with cells and altering intracellular distribution of the oligonucleotide.

The use of antisense oligonucleotide as pharmacologic agents is a derivative of the central dogma of molecular biology and knowledge of the physical and chemical properties that govern the structure of nucleic acids. Oligonucleotides have been reported to inhibit the growth of a large number of viruses in cell culture, as well as the expression of numerous oncogenes, a variety of normal genes and transfected reporter genes controlled by several regulatory elements. The therapeutic activity of antisense compounds in animal disease models have also been reported. This review provides some general conclusions and trends regarding the pharmacologic action of antisense oligonucleotides, that can be formulated from studies previously reported in the literature. In addition, data is highlighted for two specific examples in which antisense oligonucleotides have demonstrated activity against herpes viruses and intracellular adhesion molecule RNA targets.

Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed on the surface of both hemopoietic and nonhemopoietic cells that mediates, in part, the emigration of leukocytes out of the vasculature. Expression of ICAM-1 on the surface of human umbilical vein endothelial cells and a human lung carcinoma cell line (A549) was increased by interleukin-1 beta, tumor necrosis factor alpha, and interferon gamma in a concentration-dependent manner. Phosphorothioate antisense oligonucleotides designed to hybridize to 10 target sites on the human ICAM-1 mRNA were tested for inhibition of ICAM-1 expression in both cell lines by an ICAM-1 enzyme-linked immunosorbent assay. Based upon potency and unique mRNA target sites, two oligonucleotides were studied in greater detail: ISIS 1570, which targeted the AUG translation initiation codon, and ISIS 1939, which targeted specific sequences in the 3'-untranslated region of the mRNA. Both oligonucleotides specifically inhibit expression of ICAM-1 as analyzed by immunoprecipitation of 35S-labeled proteins. Treatment of cells with ISIS 1939 promoted a reduction in ICAM-1 mRNA, whereas ISIS 1570 did not change the level of ICAM-1 mRNA, suggesting that the two oligonucleotides may be inhibiting ICAM-1 expression by two different mechanisms. The activity of both oligonucleotides was blocked by hybridization of the oligonucleotide to its complementary sense strand prior to addition to the cells. Neither ISIS 1570 nor ISIS 1939 changed the transcriptional rate of the ICAM-1 gene, demonstrating that both oligonucleotides were working through a post-transcriptional mechanism. 2'-O-Methyl phosphorothioate analogs, which do not support RNase H-mediated cleavage of target mRNA, were used to determine if the active antisense oligonucleotides inhibited ICAM-1 expression by an RNase H-dependent mechanism. The 2'-O-methyl phosphorothioate analog of ISIS 1939 did not significantly reduce interleukin-1 beta-induced ICAM-1 expression, whereas the 2'-O-methyl phosphorothioate analog of ISIS 1570 did inhibit ICAM-1 expression, suggesting that the reduction of ICAM-1 mRNA following treatment with ISIS 1939 was due, in part, to RNase H-mediated hydrolysis. Adherence of HL-60 cells to human umbilical vein cell monolayers was inhibited by ISIS 1570 and ISIS 1939, demonstrating that the reduced levels of ICAM-1 impact on ICAM-1-associated function.

Coenzyme A-independent transacylase (CoA-IT) mediates the transfer of polyunsaturated fatty acids from the sn-2 position of a donor phospholipid to the sn-2 position of an acceptor lyso-phospholipid. We have characterized this activity in U937 cells, a human monocytic cell line. The microsomes of these cells contained CoA-IT activity which demonstrated a fatty acid preference for transferring arachidonic acid into exogenously added 1-alkyl-2-lyso-GPC. This enzymatic activity was optimum between pH 6.5 and 9, was heat labile and displayed an apparent Km for 1-alkyl-2-lyso-GPC of 0.4 microM. This activity was not dependent on Ca2+, Mg2+, CoA or ATP, was not inhibited by 2-mercaptoethanol nor by addition of product, 1-alkyl-2-acyl-GPC. The activity of this enzyme was not altered by differentiation of U937 cells towards the macrophage with Me2SO. Treatment of U937 cells with dexamethasone had no effect on transacylase activity. The activity of this enzyme was decreased by the serine esterase inhibitors phenylmethyl-sulfonyl fluoride and N-tosyl-L-phenylalanine chloromethyl ketone and by the histidine modifier diethyl pyrocarbonate, suggesting that CoA-IT may belong to a family of acyltransferase enzymes typified by LCAT. CoA-IT activity was not affected by compounds that affect PLA2 activity, such as quinacrine, aristolochic acid and arachidonic acid, suggesting a mechanism of action for CoA-IT different from classical, low molecular weight PLA2 enzymes. In conclusion, U937 cells contain CoA-IT activity and this study extends our previous knowledge of this enzyme by demonstrating the differences between CoA-IT and PLA2 enzymes and suggesting similarities between CoA-IT and LCAT.

To better understand the mechanisms by which leukotriene tone is regulated, we have characterized the mechanisms of genetic regulation of 5-lipoxygenase in HL60 cells induced to differentiate with dimethyl sulfoxide (DMSO) and compared a number of rat and human 5-lipoxygenase introns. We demonstrate that differentiation of HL60 cells with DMSO results in coordinate induction of 5-LO and 5-LO activating protein (FLAP). The production of LTB4, 5-LO protein, 5-LO mRNa and FLAP RNA increased coordinately. However, two approaches demonstrated no increase in the initiation of transcription of 5-LO and FLAP pre-mRNA and no changes in the mRNA half lives. Moreover, cycloheximide inhibits the induction of the mRNAs and proteins. Thus, we suggest that 5-LO and FLAP are coordinately regulated in HL60 cells via mechanisms involving changes in RNA processing. To better understand potential mechanisms involved, we have cloned and sequenced several human 5-LO introns and compared them to analogous rat 5-LO introns. A number of regions of potential regulatory significances are conserved and may be important in controlling the rate of pre-mRNA processing.

Guinea pig lung cytosolic phospholipase A2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16,700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16,700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A2 hydrolyzed phosphatidylcholine and phosphatidylethanolamine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A2 under conditions in which Crotalus adamanteus phospholipase A2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A2 in a concentration-dependent manner (IC50 = 2 microM). Antibodies prepared against porcine pancreatic phospholipase A2 specifically immunoprecipitated guinea pig lung phospholipase A2 suggesting that the major phospholipase A2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A2 in agreement with the biochemical properties of the enzyme.

The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans has been sequenced and its transcript mapped and orientated. A single ORF can encode a protein of 719 amino acids. A 52 amino acid region including a putative 'zinc finger' strongly resembles putative DNA binding regions of the major regulatory protein of erythroid cells. The derived protein sequence also contains a highly acidic region possibly involved in gene activation and 22 copies of the motif S(T)PXX, abundant in DNA binding proteins. Analysis of chromosomal rearrangements and transformation with deletion clones identified 342 N-terminal and 124 C-terminal residues as inessential and localized a C-terminal region required for nitrogen metabolite repressibility. A -1 frameshift eliminating the inessential 122 C-terminal amino acids is a surprising loss-of-function mutation. Extraordinary basicity of the replacement C terminus might explain its phenotype. Mutant sequencing also identified a polypeptide chain termination and several missense mutations, but most interesting are sequence changes associated with specificity mutations. A mutation elevating expression of some structural genes under areA control whilst reducing or not affecting expression of others is a leucine to valine change in the zinc finger loop. It reverts to a partly reciprocal phenotype by replacing the mutant valine by methionine.

The deacylated forms of the phosphoinositides were used to determine whether the guinea pig uterus phosphoinositide-specific phospholipase C (PI-PLC I, Mr 60,000) required fatty acids at the sn-1 and sn-2 positions for the hydrolysis of the sn-3 phosphodiester bond. L-alpha-Glycerophospho-D-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-P), L-alpha-glycerophospho-D-myo-inositol (Gro-PI), or L-alpha-glycerophospho-D-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 microM [3H]phosphatidylinositol ([3H]PI), 10 microM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 microM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP] revealed an IC50 = 380 microM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 microM and 300 microM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 microM. Ca2+ concentrations over the range 1.1 microM to 1 mM did not effect inhibition, suggesting that Gro-PIP inhibition of [3H]PI hydrolysis was calcium-independent. To determine whether Gro-PIP was a substrate, 20 microM and 500 microM [3H]Gro-PIP were incubated with PI-PLC I. Anion-exchange HPLC analysis revealed no [3H]IP2 product formation, indicating that [3H]Gro-PIP was not hydrolyzed. Assays performed with [3H]PI and [3H]PIP substrates in the presence of 500 microM [3H]Gro-PIP revealed approx. 75% less [3H]inositol 1-phosphate ([3H]IP1) and [3H]inositol 1,4-bisphosphate ([3H]IP2) product formation, respectively, indicating that [3H]Gro-PIP inhibited the hydrolysis of the substrates by PI-PLC I. These data suggest that Gro-PIP does not serve as a substrate, and that it inhibits PI-PLC I by competitive inhibition in a Ca2(+)-independent fashion.

Manoalide is a novel sesterterpenoid which has previously been shown to be a potent inhibitor of venom phospholipases A2. To determine whether manoalide inhibited other phospholipases, the sensitivity of phosphoinsitide-specific phospholipase C (PI-PLC) to inactivation by manoalide was examined using crude cytosolic PI-PLC and a PI-PLC purified to homogeneity from guinea pig uterus cytosol (PI-PLC I). Manoalide inhibited both cytosolic and purified PI-PLC I in a concentration-dependent fashion, exhibiting an IC50 of 3-6 microM. Inactivation of PI-PLC I was calcium- and pH-dependent, with greater inactivation occurring at alkaline pH. Manoalide inhibited hydrolysis of all three phosphoinositides by purified PI-PLC I. The substrate kinetics of PI-PLC I suggest that manoalide does not inhibit purified PI-PLC I by simple competitive or noncompetitive inhibition. Enzyme activity was not recovered after dialysis of manoalide-treated PI-PLC I, indicating that inactivation of PI-PLC I was irreversible. To determine whether manoalide inhibited PI-PLC in cells, the effects of manoalide on norepinephrine (NE)-stimulated phosphoinositide hydrolysis and calcium mobilization were investigated in a smooth muscle-like cell line, DDT1MF-2. Manoalide inhibited NE-induced inositol 1,4,5-trisphosphate and inositol 1-phosphate formation in a concentration-dependent manner. The IC50 for inhibition of inositol 1-phosphate formation was 1.5 microM. Manoalide also inhibited NE-induced calcium transients in DDT1MF-2 cells, exhibiting an IC50 of 2 microM. These data suggest that inhibition of PI-PLC may account, in part, for the anti-inflammatory actions of manoalide.

A DNA-binding nonhistone protein, protein BA, was previously demonstrated to co-localize with U-snRNPs within discrete nuclear domains (Bennett, F. C., and L. C. Yeoman, 1985, Exp. Cell Res., 157:379-386). To further define the association of protein BA and U-snRNPs within these discrete nuclear domains, cells were fractionated in situ and the localization of the antigens determined by double-labeled immunofluorescence. Protein BA was extracted from the nucleus with the 2.0 M NaCl soluble chromatin fraction, while U-snRNPs were only partially extracted from the 2.0 M NaCl-resistant nuclear structures. U-snRNPs were extracted from the residual nuclear material by combined DNase I/RNase A digestions. Using an indirect immunoperoxidase technique and electron microscopy, protein BA was localized to interchromatinic regions of the cell nucleus. Protein BA was noted to share a number of chemical and physical properties with a family of cytoplasmic enzymes, the glutathione S-transferases. Comparison of the published amino acid composition of protein BA and glutathione S-transferases showed marked similarities. Nonhistone protein BA isolated from saline-EDTA nuclear extracts exhibited glutathione S-transferase activity with a variety of substrates. Substrate specificity and subunit analysis by SDS polyacrylamide gel electrophoresis revealed that it was a mixture of several glutathione S-transferase isoenzymes. Protein BA isolated from rat liver chromatin was shown by immunoblotting and peptide mapping techniques to be two glutathione S-transferase isoenzymes composed of the Yb and Yb' subunits. Glutathione S-transferase Yb subunits were demonstrated to be both nuclear and cytoplasmic proteins by indirect immunolocalization on rat liver cryosections. The identification of protein BA as glutathione S-transferase suggests that this family of multifunctional enzymes may play an important role in those nuclear domains containing U-snRNPs.

We report the molecular cloning and sequence of a phosphoinositide-specific phospholipase C (PI-PLC), an enzyme that is of particular interest because of its central role in cell signal transduction. The signals in question are those delivered by hormones to their cell-surface receptors that activate PI-PLC by means of a guanine nucleotide binding protein. Activation of the enzyme leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the second of which ultimately mobilizes internal pools of calcium. There are at least five PI-PLC isoenzymes, whose differences in structure and function are unknown. We have focused on isoenzyme I, which we have recently purified and characterized from guinea pig uterus. We have now determined the sequence of a full length complementary DNA of this isoenzyme from the rat. Although the sequence has little similarity with the only other sequenced PI-PLC isoenzyme, it has a surprising degree of similarity to thioredoxins, protein co-factors in thiol-dependent redox reactions.

Manoalide, a novel nonsteroidal sesterterpenoid, is a potent inhibitor of phospholipase A2 isolated from bee and cobra venoms. This report compares the inhibition by manoalide of phospholipase A2 in crude cytosol fractions from four mammalian tissues with that of four purified extracellular phospholipase A2's. Phospholipase A2 isolated from bee venom (Apis mellifera) was the most sensitive to inactivation by manoalide (IC50 approximately equal to 0.12 microM). Extracellular phospholipase A2 from rattlesnake and cobra venom was intermediate in sensitivity to manoalide (IC50 values of 0.7 and 1.9 microM respectively). Porcine pancreatic phospholipase A2 was relatively resistant to inactivation by manoalide (IC50 approximately equal to 30 microM). The phospholipase A2 assayed in crude cytosol fractions from four mammalian tissues exhibited IC50 values of 30 microM or greater. Cytosolic proteins as well as bovine serum albumin and poly-L-lysine (Mr = 57,000) protected purified bee venom phospholipase A2 from inactivation by manoalide. In contrast, amino acids such as lysine and alanine failed to protect the purified enzyme from inactivation. Proteins and certain amino acids, such as lysine, formed a chromogenic product when incubated with manoalide. These data suggest that lysine is capable of reacting with manoalide, but only when it is present in macromolecules is it capable of protecting phospholipase A2 from inactivation by manoalide. Because cellular proteins protect PLA2 from inactivation by manoalide, high concentrations of manoalide must be applied topically to produce statistically significant inactivation of intracellular phospholipase A2. Finally, a chemical model is presented which explains the formation of a chromogenic product when manoalide is incubated with proteins and amino acids.

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.

Incubation of the bovine endothelial cell line, CPAE, with leukotriene D4, leukotriene C4, bradykinin, or the calcium ionophore A23187 results in the release of arachidonic acid metabolites including 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Pretreatment of these cells with the pertussis toxin islet-activating protein (IAP) results in a dose-dependent inhibition of the release of arachidonic acid metabolites and prostacyclin in response to leukotriene D4 and leukotriene C4. In contrast, similar responses evoked by bradykinin or ionophore were not significantly altered by the IAP pretreatment of the cells. IAP in the presence of [32P]NAD specifically [32P]ADP-ribosylates a 41-kDa protein in membranes prepared from CPAE cells. Pretreatment of the intact cells with IAP resulted in a dose-dependent inhibition of subsequent 32P labeling of the toxin substrate in the membranes and correlates with the uncoupling of the leukotriene responses. These results suggest that the 41-kDa IAP substrate, presumably a guanine nucleotide regulatory protein, mediates the response of CPAE cells to leukotriene D4 and leukotriene C4, but not to bradykinin or the calcium ionophore.

Cytosolic calcium is a key determinant of the contractile state of airway smooth muscle (ASM). To investigate the mechanisms by which histamine affects cytosolic calcium, we measured changes in inositol 1,4,5-trisphosphate (IP3) following the addition of histamine to cultured canine ASM cells. The effect of phorbol 12-myristate 13-acetate (PMA) on IP3 formation was investigated under conditions previously shown to abolish histamine-induced calcium release. In both intact cells and ASM membranes, histamine produced a significant increase in IP3 formation, which was inhibited by PMA. The site of this blockade was investigated by examining the effect of PMA on guanine nucleotide-stimulated IP3 formation and on phosphoinositide-specific phospholipase C (PI-PLC) activity in ASM membranes. Guanine nucleotide-stimulated IP3 formation was inhibited by PMA pretreatment. Membrane-associated PI-PLC activity was also decreased, an effect that was not due simply to a shift in the calcium sensitivity of the enzyme. We conclude that in cultured canine ASM cells, PMA blocks histamine-induced IP3 formation and that this inhibition is caused, in part, by a postreceptor site of action of protein kinase C, possibly via a direct effect on PI-PLC.

Vasopressin V1 receptors were solubilized from rat liver plasma membranes with the detergent lysophosphatidylcholine. [[3H]Arginine]vasopressin (AVP) binding to the solubilized preparations was specific and saturable, with a dissociation constant of 0.6 nM. Cross-linking of [125I]vasopressin to the solubilized fraction, studied by SDS/polyacrylamide-gel-electrophoretic analysis, demonstrated the presence of a 65 kDa band which was specifically labelled with [125I]vasopressin. Specific binding of [3H]AVP to these solubilized receptors was decreased by guanine nucleotides, but not by adenosine 5'-[beta gamma-imido]triphosphate. Addition of vasopressin increased specific binding of 35S-labelled guanosine 5'-[gamma-thio]triphosphate (GTP[35S]) to the solubilized fractions, indicating co-solubilization of GTP-binding protein(s) [G-protein(s)] and vasopressin receptors. The solubilized fraction was insensitive to both cholera- and pertussistoxin treatment. Immunoblotting of the solubilized fraction with antibodies specific for a phosphoinositide-specific phospholipase C (PI-PLC I) demonstrated the presence of a 60 kDa protein. Anti-PI-PLC I antiserum immunoprecipitated solubilized vasopressin-binding sites from rat liver (V1), but not solubilized vasopressin-binding sites from hog kidney (V2). Similar results were obtained with an anti-PI-PLC I IgG affinity column. The solubilized (V1) receptors were enriched by ion-exchange and high-performance gel-filtration liquid chromatography. Vasopressin-binding activity was co-eluted with PI-PLC I and GTP[S]-binding activity on a DEAE-Sepharose column. The major vasopressin- and GTP[35S]-binding activities were co-eluted with PI-PLC I activity at approx. 240 kDa suggesting that vasopressin receptors from rat liver membranes can be solubilized as a complex of receptor-coupler-effector by using the detergent lysophosphatidycholine.

We have previously shown that a 30 kDa DNA-binding protein isolated from rat cell nuclei exhibits the chemical and immunological properties of glutathione S-transferase Yb subunits [Bennett, Spector & Yeoman (1986) J. Cell Biol. 102, 600-609]. It was of interest, therefore, to determine whether Yb subunits isolated from rat liver nuclei would return to nuclear fractions upon reintroduction to cell cytoplasms via red-blood-cell-mediated fusion. Labelled Yb subunits were associated with nuclear fractions 60 min after cell fusion. The microinjected protein remained associated with the nuclei for 18 h and was not extractable with low-salt washes. In addition, injected Yb subunits were found to equally distribute between extractable (56%) and residual (44%) nuclear fractions. These experiments demonstrate that glutathione S-transferase Yb subunits isolated from nuclei rapidly translocate to nuclei upon reintroduction into cell cytoplasms.

Procaryotic and eucaryotic cells have evolved multiple pathways for communication with their external environment. The inositol 1,4,5-trisphosphate/diacylglycerol second messenger system is an example of such a signal transduction pathway which is present in multicellular eucaryotic organisms. Binding of an agonist to a specific cell surface receptor promotes rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. The pivotal enzyme for this second messenger system is phosphoinositide-specific phospholipase C which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Recently, much progress has been made in the purification, characterization and cDNA cloning of multiple PI-PLC isoenzymes. The results of the recent studies on phosphoinositide-specific phospholipase C are reviewed.

During the past several years, substantial progress in understanding the receptors and signal transduction processes for peptidyl leukotrienes has been reported. Receptors have been identified and characterized, the major steps in the signal transduction pathway have been described, and the genetic and epigenetic regulatory processes have been characterized. Very recent studies have defined the mechanisms by which LTE4 acts as a partial agonist at the LTD4 receptor. The cloning of the genes for the proteins involved in the major steps of the signalling process has also been initiated. Stanley Crooke and co-authors summarize this recent progress and present their current notions about the LTD4 receptor signalling process.

The most frequently overlooked aspect of conservation of natural resources and economic development is human cultural diversity. However, conservation and development of natural resources are basically human-oriented endeavors and all conservation and developmental efforts ought to start with a clear understanding of the varied needs of the people. In addition, cultural diversity is a natural resource that ought to be protected along with all the more commonly recognized resources of the ecosystems of which humans form an integral part. Cultural diversity in a large measure is an ecological phenomenon because such diversity includes variations in the ways different people perceive and utilize the environments in which they live. Thus, cultural diversity, in large measure, equates with ecological diversity. It has been well established that a high degree of ecological diversity (including taxonomic, niche, biogeochemical and other measures of diversity) is a necessary attribute of humid tropical ecosystems if such ecosystems are to remain viable over long periods of time. The current land-use trends in Central America and Panama are leading toward ever larger areas being devoted to monocultural use with sharply reduced ecological diversity that poses great dangers for the near and long terms. In addition, the resultant removal of people from rural areas results in growing social, economic and political problems, that are not being successfully met by developing nations. Not only is there a growing wastage of human resources, a weakening of the social structures, and an increasing and dangerous dependence upon monocultures oriented toward export markets, but the ecological diversity of the previous existing land-use systems are being lost. This kind of diversity is probably no less valuable to the ecological health of a nation's agriculture, forestry, and general resource utilization than is the genetic diversity of "primitive" crop plant varieties which biologists now recognize and increasingly seek to preserve as "modern" crop plant varieties become ever more simple genetically and hence ever more vulnerable to disease and other perturbations.