L-454,560, a potent and selective PDE4 inhibitor with in vivo efficacy in animal models of asthma and cognition.

Type 4 phosphodiesterases (PDE4) inhibitors are emerging therapeutics in the treatment of a number of chronic disorders including asthma, chronic obstructive pulmonary disease (COPD) and cognitive disorders. This study delineates the preclinical profile of L-454,560, which is a potent, competitive and preferential inhibitor of PDE4A, 4B, and 4D with IC50 values of 1.6, 0.5 and 1.2 nM, respectively. In contrast to the exclusive binding of cilomilast and the preferential binding of roflumilast to the PDE4 holoenzyme state (Mg2+-bound form), L-454,560 binds to both the apo-(Mg2+-free) and holoenzyme states of PDE4. The intrinsic enzyme potency for PDE4 inhibition by L-454,560 also results in an effective blockade of LPS-induced TNFalpha formation in whole blood (IC50 = 161 nM) and is comparable to the human whole blood potency of roflumilast. The cytokine profile of inhibition of L-454,560 is mainly a Th1 profile with significant inhibition of IFNgamma and no detectable inhibition of IL-13 formation up to 1 microM. L-454,560 was also found to be efficacious in two models of airway hyper-reactivity, the ovalbumin (OVA) sensitized and challenged guinea pig and the ascaris sensitized sheep model. Furthermore, L-454560 was also effective in improving performance in the delayed matching to position (DMTP) version of the Morris watermaze, at a dose removed from that associated with potential emesis. Therefore, L-454,560 is a novel PDE4 inhibitor with an overall in vivo efficacy profile at least comparable to roflumilast and clearly superior to cilomilast.

Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer.

Prostaglandin G/H synthase (PGHS), a key enzyme leading to the formation of prostaglandins, is the target of nonsteroidal antiinflammatory drugs. Two forms of the enzyme have been identified, PGHS-1 and PGHS-2. Epidemiological evidence has suggested that aspirin and other nonsteroidal antiinflammatory drugs may reduce the risk of colorectal cancer. We examined by immunoblot analyses the expression of human PGHS-1 and PGHS-2 protein in 25 matched colon cancer and nontumor tissues, 4 premalignant polyps, 5 control colon tissues from noncancer patients, and 3 matched normal and cancerous breast tissue samples. PGHS-1 was detected in all normal and tumor tissue. In contrast, PGHS-2 was not detected in 23 of 25 normal colon tissues but was detected in 19 of 25 colon tumors. PGHS-2 protein was not observed in four human premalignant polyp samples, control colon from noncancer patients, or matched normal or cancerous breast tissues. These results suggest that the beneficial effects of nonsteroidal antiinflammatory drugs in colon cancer may be mediated by inhibition of PGHS-2.

Mutation of serine-516 in human prostaglandin G/H synthase-2 to methionine or aspirin acetylation of this residue stimulates 15-R-HETE synthesis.

Prostaglandin G/H synthase (PGHS) is a key enzyme in cellular prostaglandin (PG) synthesis and is the target of non-steroidal anti-inflammatory agents. PGHS occurs in two isoforms, termed PGHS-1 and PGHS-2. These isoforms differ in several respects, including their enzymatic activity following acetylation by aspirin. While PG synthesis by both isoforms is inhibited by aspirin, 15-R-hydroxyeicosatetraenoic acid (15-R-HETE) synthesis by PGHS-2, but not PGHS-1, is stimulated by preincubation with aspirin. We have mutated the putative aspirin acetylation site of hPGHS-2, and expressed the mutants in COS-7 cells using recombinant vaccinia virus. Enzyme activity and inhibitor sensitivity studies provide evidence that Ser516 is the aspirin acetylation site of human PGHS-2 and that substitution of a methionine residue at this position can mimic the effects of aspirin acetylation on enzyme activity.

5-lipoxygenase-activating protein is an arachidonate binding protein.

5-Lipoxygenase-activating protein (FLAP) is an 18-kDa integral membrane protein which is essential for cellular leukotriene (LT) synthesis, and is the target of LT biosynthesis inhibitors. However, the mechanism by which FLAP activates 5-LO has not been determined. We have expressed high levels of human FLAP in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus, and used this system to demonstrate that FLAP specifically binds [125I]L-739,059, a novel photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK-886, an LT biosynthesis inhibitor which specifically interacts with FLAP. These studies suggest that FLAP may activate 5-LO by specifically binding arachidonic acid and transferring this substrate to the enzyme.

Cloning and expression of rhesus monkey cathepsin K.

Cathepsin K is a cysteine protease involved in degradation of human type I collagen and plays a primary role in bone resorption. We have cloned rhesus monkey cathepsin K by reverse transcriptase-polymerase chain reaction (RT-PCR) from rhesus ovary poly A+ RNA. The sequence for the rhesus enzyme is 98% identical to that of the human with 100% identity within the mature active form of cathepsin K. Rhesus monkey cathepsin K was transiently expressed in Chinese hamster ovary (CHO) cells and found to be secreted as the proenzyme in the culture media and 50% activated to the mature form intracellularly. The substrate specificity preference of aminomethylcoumarin and rhodamine peptide substrates was Leu > Phe > Pro in the P2 position when tested with constant arginine at P1. The enzyme activity expressed in CHO cell extracts was sensitive to inhibition by E-64 and cystatin with IC50s of 3.5 nmol/L and 13 ng/mL, respectively. The apparent second order rate constants of inactivation by E-64 were 66,000 M(-1) s(-1) and 130,000 M(-1) s(-1) for the recombinantly expressed rhesus monkey and human cathepsin K, respectively. The high similarity between the sequences and the kinetic properties of rhesus monkey and human cathepsin K establishes this monkey species as a suitable animal model for development of novel cathepsin K inhibitors as antiresorptive agents.

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor.

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furan one) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX-2 (IC50 = 41 +/- 14 nM) over COX-1 (IC50 > 50 microM). Indomethacin was a potent inhibitor of both COX-1 (IC50 = 18 +/- 3 nM) and COX-2 (IC50 = 26 +/- 6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50 > 50 microM and 4.1 +/- 1.7 nM, respectively). 3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a Ki, value of 140 +/- 68 microM for the initial reversible binding to enzyme and a kappa 2 value of 0.11 +/- 0.06 s-1 for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62 +/- 26 microM and 0.06 +/- 0.01 s-1, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1:1 stoichiometry and to dissociate only very slowly (t1/2 = 1-3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50 = 63 +/- 5 microM at 0.1 microM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5. DFU inhibited lipopolysaccharide (LPS)-induced PGE2 production (COX-2) in a human whole blood assay with a potency (IC50 = 0.28 +/- 0.04 microM) similar to indomethacin (IC50 = 0.68 +/- 0.17 microM). In contrast, DFU was at least 500 times less potent (IC50 > 97 microM) than indomethacin at inhibiting coagulation-induced TXB2 production (COX-1) (IC50 = 0.19 +/- 0.02 microM). 6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 microM), DFU inhibited COX-1 with an IC50 value of 13 +/- 2 microM as compared to 20 +/- 1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac > indomethacin approximately naproxen > nimesulide approximately meloxicam approximately piroxicam > NS-398 approximately SC-57666 > SC-58125 > CGP 28238 approximately etodolac > L-745,337 > DFU. 7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED50 of 1.1 mg kg-1 vs 2.0 mg kg-1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg-1 vs 1.5 mg kg-1 for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED50 = 0.76 mg kg-1 vs 1.1 mg kg-1 for indomethacin). 8. In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg-1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg-1), meloxicam (3 mg kg-1) or etodolac (10-30 mg kg-1). A 5 day administration of DFU in squirrel monkeys (100 mg kg-1) did not affect chromium leakage in contrast to diclofenac (1 mg kg-1) or naproxen (5 mg kg-1). 9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.

Expression, maturation, and rhodamine-based fluorescence assay of human cathepsin K expressed in CHO cells.

Cathepsin K is a cysteine protease that degrades type I human collagen during bone resorption. We have expressed the recombinant human cathepsin K in Chinese hamster ovary (CHO) cells as a pre-proenzyme and demonstrated that it is processed intracellularly to an active enzyme form and that only the proenzyme form is secreted. Immunofluorescence detection of cathepsin K in CHO cells resulted in discrete punctate distribution consistent with a lysosomal localization of the enzyme. With both extract and cell preparations of CHO cells expressing cathepsin K, [Z-Leu-Arg](2)-rhodamine was the best substrate for analyzing cathepsin K activity over background proteases. We have established a cellular-based assay to analyze cell-permeable inhibitors of cathepsin K and validated the assay with detection of intracellular versus extracellular activity, fluorescence-assisted cell sorter (FACS) analysis, and a selective cathepsin K inhibitor. The intracellular activity of cathepsin K was monitored by FACS analysis using the rhodamine substrate, which demonstrated an increased fluorescence over mock-transfected cells that was also inhibitable by (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E64d). A selective cathepsin K inhibitor, 1, 3-bis(CBZ-Leu-NH)-2-propanone, had an IC(50) of 134 nM in the CHO/Cat K cells, which is the same potency as that measured against a purified enzyme preparation of cathepsin K. Therefore, we have established a system to evaluate intracellular cathepsin K activity and inhibition by cell-permeable inhibitors of this thiol protease.

Leisure satisfaction and psychologic well-being in old age: effects of health and income.

From a study (probability sampling methods) of the response of 74 older adults residing in a high-rise pbulic housing complex, it was concluded that the association between leisure satisfaction and psychologic well-being is substantial and seems relatively unaffected by self-rated levels of health and income.

Situational influences on leisure satisfaction and morale in old age.

The relationship between leisure satisfaction and morale was examined within the context of leisure behavior, health, age, and socioeconomic status. Interviews were conducted with 104 noninstitutional adults aged 65 or older living in an urbanized area. Leisure satisfaction and morale were substantially related, and remained so when statistically tested with various third factors. Multivariate analysis indicated that the most important predictors of morale were leisure satisfaction and socioeconomic status. Self-rated health, the illness index, and age were much less significant as predictors.

The binding of leukotriene biosynthesis inhibitors to site-directed mutants of human 5-lipoxygenase-activating protein.

Site-directed mutagenesis was used to develop deletion and point mutants of human 5-lipoxygenase-activating protein (FLAP), which were then expressed in COS-7 cells. Membrane preparations from these cells were analyzed in a radioligand binding assay. Binding of leukotriene biosynthesis inhibitors to FLAP mutants containing deletions of 2 to 6 amino acids within the region from residue 48-61 was undetectable. This finding is consistent with previous studies which suggest that residues amino-terminal to the proposed second transmembrane of FLAP are critical for inhibitor binding. The present study also defines residues of FLAP a) amino-terminal to residue 48, b) between the proposed second and third transmembrane regions and c) in the C-terminal region of the protein which are not involved in inhibitor binding.

Peripheral phosphodiesterase 4 inhibition produced by 4-[2-(3,4-Bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl]-3-methylpyridine-1-oxide (L-826,141) prevents experimental autoimmune encephalomyelitis.

Administration of phosphodiesterase 4 (PDE4) inhibitors suppresses the pathogenesis associated with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In the present study, we compared the effects of rolipram and 4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl]-3-methylpyridine-1-oxide (L-826,141), a novel nonbrain penetrant PDE4 inhibitor, on the onset and severity of clinical signs in a chronic, nonrelapsing/remitting model of EAE. Both rolipram (10 mg/kg p.o.) and L-826,141 (3 mg/kg p.o.) reduced the severity of EAE relative to controls, whereas L-826,141 (3 mg/kg p.o.) also delayed disease onset. To assess whether L-826,141 prevented EAE progression after the first signs of clinical onset, rolipram (10 mg/kg p.o.) or L-826,141 (3 or 30 mg/kg p.o.) were administered 24 h after the first signs of EAE were observed. Only L-826,141 at a dose of 30 mg/kg p.o. significantly decreased the clinical severity of EAE compared with vehicle controls. Immunohistochemical detection of the neuronal activity marker Fos confirmed that L-826,141 did not reach concentrations in the central nervous system sufficient to activate central neurons. Lipopolysaccharide-induced tumor necrosis factor-alpha in whole blood and plasma concentrations of L-826,141 revealed that only the 30-mg/kg dose resulted in levels sufficient to produce a near complete inhibition of PDE4 activity in immune cells. Taken together, these results demonstrate that peripheral PDE4 inhibition, produced by L-826,141, prevents the progression of EAE after the first onset of clinical signs, and suggest that similar compounds may have clinical efficacy in the treatment of MS.

Effects of health and income on control orientation and life satisfaction among aged public housing residents.

Researchers have recently begun to focus to a greater degree on personality factors in old age. One such factor is locus of control. This investigation examines the relationship between life satisfaction and locus of control within the context of health and income, two variables that pervade numerous aspects of the older person's life. The data are derived from a randomly selected sample of people sixty-five years of age and older residing in high-rise public housing complexes (N = 74). The findings of the investigation are consistent with most of the previous research, which had shown internal locus of control and higher life satisfaction to be significantly associated. This association remained stable and significant when controlling on the effects of self-rated health and income, suggesting that internal control is related to life satisfaction independent of health level or monetary resources.

Cloning and characterization of the human leukotriene A4 hydrolase gene.

The human gene encoding the bifunctional aminopeptidase and epoxide hydrolase enzyme, leukotriene A4 hydrolase (LTA4 hydrolase) has been cloned from a placental lambda phage genomic library. The gene is greater than 35 kbp and contains 19 exons ranging in size over 24-312 bp. The introns range in size over 0.26-5.7 kbp. The essential zinc-binding histidine residues and glutamate residue, which delineate the zinc-binding domain required for both enzyme activities of LTA4 hydrolase, are divided between exons 10 and 11. The LTA4 hydrolase gene was localized to chromosome 12q22 utilizing fluorescence in situ hybridization. Based on the chromosome localization and genomic DNA analysis, LTA4 hydrolase was determined to be a single-copy gene. Primer-extension analysis demonstrated that the transcription initiation site of LTA4 hydrolase mRNA is 151 nucleotides upstream of the initiator ATG. Approximately 4 kbp of 5'-flanking region of the LTA4 hydrolase gene has been obtained and sequencing of 1.4 kb of this 5'-flanking region demonstrated several transcription-factor consensus sequences, including a phorbol-ester-response element (AP2) and two xenobiotic-response elements. The cloning and characterization of the human gene for LTA4 hydrolase provides a basis for further insight into transcriptional regulation of this bifunctional enzyme and its role in various inflammatory processes.

Social provisions in adulthood: concept and measurement in close relationships.

Social gerontologists are increasingly concerned about examining the nature of close relationships among the elderly. Theoretically grounded and empirically validated instruments are needed to advance research in this area. We analyzed the psychometric properties of the Social Provisions Scale using data from a probability sample of 494 community residents aged 65 or older. The theoretical foundation of this scale is Weiss's delineation of social support functions of close relationships. Confirmatory factor analysis revealed a pattern in the data that corresponds to the theoretical definition of relational provisions: Factor 1 was Intimacy; Factor 2 was Social Integration; Factor 3 was Reassurance of Worth; and Factor 4 was Opportunity for Nurturance. Alphas for the four scales ranged from .83 to .94. Convergent validity was supported by significant correlations between the Social Provisions Scale (SPS) and respondent's morale, frequency of contact with friends, feelings of closeness with an adult child, relationship control, and relationship conflict. Discriminant validity was supported by nonsignificant correlations between the SPS and the Eysenck Lie Scale.

Dimensions of health and their importance for morale in old age: a multivariate examination.

Numerous researchers have pointed to the centrality of health in personal adaptation in the later years. This investigation examines morale in regard to 16 health indicators. Probability techniques were used to draw a sample of 104 noninstitutionalized people, 65 years of age and older. Product-moment correlations indicated a substantial relatedness among the the health indicators. A principal components factor analysis generated five health dimensions: Assessing and Promoting Own Health, Health Behavior and Facilities, Disease and Its Control, Sensory Abilities, and Source of Care. Morale was bivariately related most to the individual measures of fatigue, comparative health level, visual acuity, and general self-rated health. A multiple regression analysis indicated that morale was reported to be higher among those who felt more rested upon wakening in the morning, who had better visual abilities, and who saw their health level as being at least as good as in the past.

Coupling of recombinant 5-lipoxygenase and leukotriene A4 hydrolase activities and transcellular metabolism of leukotriene A4 in Sf9 insect cells.

High-level expression of human leukotriene (LT) A4 hydrolase has been established in Sf9 insect cells using the recombinant baculovirus system. LTA4 hydrolase activity in this system is at least 50-fold higher than previously achieved in a bacterial cell system. Recombinant viral human LTA4 hydrolase (rvHLTA4h) was used for coinfection studies with recombinant viral 5-lipoxygenase (rvH5LO). When Sf9 cells expressing 5-lipoxygenase are incubated in the presence of A23187 and arachidonic acid, (5S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid 5-H(P)ETE and LTA4 are synthesized in a ratio of 5:1 for 5-H(P)ETE/LT. Coexpression of 5-lipoxygenase and LTA4 hydrolase in these insect cells results in the synthesis of 5-H(P)ETE, LTA4 and in addition LTB4, and the ratio shifts to 2:1 for 5-H(P)ETE/LT. The production of enzymically formed LTB4 after addition of arachidonic acid to the Sf9 cells coinfected with LTA4 hydrolase and 5-lipoxygenase is the first demonstration of channeling of arachidonic acid to LTB4 in an engineered intact cell system. This delineates a novel biological system to synthesize significant amounts of the potent chemotactic agent, LTB4. Studies in which Sf9 cells infected with rvH5LO were incubated with Sf9 cells infected with rvHLTA4h resulted in export of LTA4 from the rvH5LO cells and transcellular metabolism of LTA4 to LTB4 in the rvHLTA4h Sf9cells.

5-Lipoxygenase activity in the human pancreas.

Low levels of 5-lipoxygenase (5-LO), the first committed enzyme in the synthesis of leukotrienes (LTs), have been reported in the porcine pancreas. We have quantitated 5-LO activity in subcellular fractions of pancreas samples from three human donors. 5-LO activity was detectable in all samples, although enzyme activity was lower than in human leukocytes. 5-LO in human pancreas samples displayed highest specific activity in membrane fractions, and did not require arachidonic acid (AA) addition for activity. These unusual characteristics of pancreatic 5-LO appear to be due, at least in part, to the presence of unesterified AA in the pancreas samples. Western blot analysis demonstrated that the human pancreas contains low levels of 5-lipoxygenase-activating protein (FLAP) in addition to 5-LO.

Cellular oxygenation of 12-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid by 5-lipoxygenase is stimulated by 5-lipoxygenase-activating protein.

It has been proposed that 5-lipoxygenase (5-LO)-activating protein (FLAP) is an arachidonate transfer protein for leukotriene biosynthesis. Using the Spodoptera frugiperda (Sf9) insect cells, we demonstrate that FLAP causes a large stimulation (190-fold) of the conversion of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) to 5, 12-diHETE when co-expressed with 5-lipoxygenase. We also demonstrate that FLAP can stimulate (2-2.5-fold) the oxygenation of 15(S)-HETE by 5-LO to 5,15-diHETE. The stimulation of both 12(S)-HETE and 15(S)-HETE oxygenation by 5-LO is completely inhibitable by the FLAP inhibitor, MK-886. In order to determine which residues of FLAP are important for 12(S)-HETE and arachidonic acid utilization by 5-LO, various mutants of FLAP were co-expressed with 5-LO in Sf9 cells. The FLAP deletion mutants del 37-53, del 52-58, del 106-108, and del 148-161 and the point mutant D62N were analyzed. The D62N mutation, which reduces the binding of indole inhibitors to FLAP, had no effect on the stimulation of substrate utilization by 5-LO. In contrast to wild type FLAP, the mutant proteins del 37-53, del 106-108, and del 148-161 failed to stimulate 12(S)-HETE and arachidonic acid utilization by 5-LO. Only one of the latter three mutations (del 37-53) has been shown to abolish the binding of indole inhibitors to FLAP. These results suggest that the lipid binding site of FLAP overlaps the inhibitor binding site and occupies several regions of the protein not essential for inhibitor binding. Because FLAP can stimulate the utilization of 12(S)-HETE, 15(S)-HETE, and arachidonic acid by 5-LO, FLAP may also function as a more general lipid carrier protein for the biosynthesis of multiple oxygenation products of arachidonic acid in addition to its role in leukotriene biosynthesis.

Cloning and expression of the Photobacterium phosphoreum luminescence system demonstrates a unique lux gene organization.

The organization of the lux structural genes (A-E) in Photobacterium phosphoreum has been determined and a new gene designated as luxF discovered. The P. phosphoreum luminescence system was cloned into Escherichia coli using a pBR322 vector and identified by cross-hybridization with Vibrio fischeri lux DNA. The lux genes were located by specific expression of P. phosphoreum DNA fragments in the T7-phage polymerase/promoter system in E. coli and identification of the labeled polypeptide products. The luxA and luxB gene products (luciferase subunits) were shown to catalyze light emission in the presence of FMNH2, O2, and aldehyde. The luxC, luxD, and luxE gene products (fatty acid reductase subunits) responsible for aldehyde biosynthesis could be specifically acylated with 3H-labeled fatty acids. The order of the lux genes in P. phosphoreum was found to be luxCDABFE with luxF coding for a new polypeptide of 26 kDa. The presence of a new gene in the P. phosphoreum luminescence system between luxB and luxE as compared to the organization of the lux structural gene in V. fischeri and Vibrio harveyi (luxCDABE) demonstrates that the luminescent systems in the marine bacteria have significantly diverged. The discovery of the luxF gene provides the basis for elucidating the role of its gene product in the expression of luminescence in different marine bacteria.

A new lux gene in bioluminescent bacteria codes for a protein homologous to the bacterial luciferase subunits.

The nucleotide sequence of a new gene, luxF, located between the luxB and E genes in the bioluminescent system of Photobacterium phosphoreum has been determined. The luxF gene codes for a polypeptide of 231 amino acids which is homologous to the alpha and beta subunits of luciferase coded by the luxA and luxB genes, respectively. The degree of homology of the luxF protein is very high with the beta subunit of luciferase (approximately 30% identity) with greatest similarity to the Vibrio luxB proteins. the luxF gene appears to have evolved by duplication of the luxB gene followed by deletion of approximately 100 codons just penultimate to the 5'-terminal. The close homology with the luciferase beta subunit implicates the luxF protein in a function related to the light-emitting reaction.