Biomedical subjects

J F Evans

Publications and source records attributed to J F Evans.

At least 37 records · Page 2Linked to original sources

PubMed1999

Effect of hypophysectomy on the proliferation and differentiation of rat bone marrow stromal cells.

Conditions such as estrogen deficiency, skeletal unloading, and aging have all been demonstrated to have various effects on the proliferation and differentiation of bone marrow stroma-derived osteoprogenitor cells. Here we have sought to examine the effects of pituitary hormone deficiency on the proliferation and the differentiation of these osteoprogenitor cells using the hypophysectomized (HX) rat as a model. In the present study, we use an in vitro culture system to examine the effects of HX on the osteogenic potential of rat bone marrow stroma. With the intact animal as a control, we used [3H]thymidine incorporation and cell number as indexes of proliferation. We also measured alkaline phosphatase enzyme activity, relative levels of osteocalcin expression with RT-PCR, and osteopontin and bone sialoprotein steady-state levels by Northern blot to delineate the effect on differentiation. Our results indicate that osteoprogenitor cells exposed to a pituitary hormone-deficient environment in vivo demonstrate an enhanced proliferative capacity and also exhibit an augmented expression of differentiation markers when exposed to an optimal environment in vitro.

Animals↗

PubMed1999

Effects of dorsomedial hypothalamic nuclei lesions on intake of an imbalanced amino acid diet.

Within 3 h of ingesting an imbalanced amino acid diet (Imb), rats show attenuated intake, which can be ameliorated by prior administration of the serotonin receptor antagonist tropisetron (Trop). Earlier work in which the dorsomedial hypothalamic nucleus (DMN) was electrolytically lesioned (DMNL) determined that this structure plays a role in the early detection of and subsequent adaptation to Imb. However, that study did not address whether cell bodies in the DMN, fibers of passage, or both were involved in the DMNL response to Imb. In the present investigation in experiment 1, rats were given electrolytic DMNL or a sham operation (Sham). The rats were injected with saline (Sal) or Trop just before introduction of Imb. By 3 h Sal-DMNL rats consumed more Imb than did the Sal-Sham rats; intake was normal by 12 h. Trop enhanced Imb intake, with Trop and DMNL being additive. By day 4 the DMNL rats were eating and gaining weight less than were Sham rats. In experiment 2, DMN cell bodies were destroyed by ibotenic acid (Ibo). Sal-injected Ibo-lesioned and Sham rats showed similar food intake depression on Imb; Trop similarly increased Imb intake in both groups. By day 4 both Ibo-L rats were eating and gaining weight less than were Sham rats. In experiment 3, groups of rats were given knife cuts posterior, lateral, ventral, dorsal, or anterior to the DMN. During the first 3 h of consuming Imb, all cuts except posterior enhanced the intake of Imb. Over the next 24 h the anterior cut group continued to eat more Imb than did the Sham rats. In experiment 4 DMNL rats were given novel diets; the DMNL rats did not display a neophilic response. The data suggest that fiber tracts that pass through the DMN may be involved in the early detection of Imb. DMN cell bodies, or fibers of passage, are not involved in the Trop effect. Finally, DMN cell bodies are necessary for proper long-term adaptation to Imb.

Amino Acids, Essential↗

PubMed1998

Structural characterization of the covalent attachment of leukotriene A3 to leukotriene A4 hydrolase.

Leukotriene A4 (LTA4) hydrolase catalyzes the conversion of the unstable epoxide LTA4 [5(S)-trans-5,6-oxido-11,14-cis-eicosatetraenoic acid] into proinflammatory LTB4. During the process of catalyzing this reaction, the enzyme is suicide inactivated by its substrate. In addition, LTA3, and analogue of LTA4 that lacks the C14-C15 double bond, is a potent suicide inhibitor of LTA4 hydrolase. We have synthesized [3H]LTA3 and used this ligand to demonstrate that LTA3 can covalently label LTA4 hydrolase and that this labeling is specifically competed for by bestatin and LTA4. Incubation of recombinant human LTA4 hydrolase with LTA3 followed by proteolysis (endoproteinase Lys-C) resulted in a peptide map with a single modified peptide defining the location of the LTA3 covalent attachment region. This modified 21-amino-acid peptide had a UV absorption spectrum corresponding to a conjugated triene chromophore which established conservation of this structural unit after covalent interaction of LTA3 with LTA4 hydrolase. MALDI-TOF mass spectrometric analysis of the 21-amino-acid peptide adduct revealed an abundant MH+ at m/z 2658, consistent with the predicted nominal mass of the sequenced peptide with the addition of a single LTA3 moiety. Proteolysis of LTA4 hydrolase modified with LTA3 was performed sequentially with endo-Asp-N and endo-Lys-C. The resulting peptide isolated by reverse-phase high-performance liquid chromatography was analyzed by mass spectroscopy revealing two related peptides, D371-K385 (m/z 2018.0) and D375-K385 (m/z 1577.8), both of which retained the elements of LTA3. Postsource decay of m/z 1577.8 resulted in an abundant ion at m/z 536 and an ion of lesser abundance at m/z 856 consistent with cleavage between V381 and P382 that supported assignment of the modified tyrosine residue at Y383. These results suggest nucleophilic attack of a tyrosine residue (Y383) at the conjugated triene epoxide of LTA3 resulting in a triene ether carbinol covalent adduct.

Amino Acid Sequence↗

PubMed1998

Dorsomedial hypothalamic lesions alter intake of an imbalanced amino acid diet in rats.

Within 3 h of ingesting an imbalanced amino acid diet (IAAD), rats show attenuated intake. The associated conditioned taste aversion can be ameliorated by giving the serotonin3 receptor blocker, tropisetron (TROP). A recent c-fos study indicated that the dorsomedial hypothalamic nucleus (DMN) may be activated 2-3 h after ingestion of IAAD. In Experiment 1, DMN-lesioned rats (DMNL) or sham-operated (SHAM) rats were injected with saline (SAL) or TROP just before introduction of IAAD. By 3 h, SAL-DMNL rats consumed more (P < 0.01) of the IAAD than did the SAL-SHAM rats. Thereafter, over the next 21 h, the intake of the SAL-DMNL group returned to control levels. TROP treatment enhanced the intake of the treated groups; the TROP and the lesion effect were additive (P < 0.01). By d 4 of receiving the IAAD, the DMNL groups were eating less than SHAM rats (P < 0.05). The data suggest that the DMN may be involved in the early detection of the amino acid deficiency induced by IAAD, is not involved in the TROP effect and is necessary for proper long-term adaptation to an IAAD.

Amino Acids↗

PubMed1998

Changing the lens. A position paper on the value of qualitative research methodology as a mode of inquiry in the education of the deaf.

A case study of a young deaf child's conversation development is used to explore the relationship between mode of inquiry and findings resulting from the use of qualitative research methodology. Data were collected through participant observation, videotaping, and interviews in the participants' natural settings. The breadth of findings from thematic analysis of descriptive data and discourse analysis of language samples led to the discovery of the child's language competencies in a field where research usually focuses on language deficits. Additionally, data revealed the contextual features that contributed to the child's conversation development. Scope of findings and implications for educators and researchers provide evidence of the value of qualitative methodology as a mode of inquiry in the field of education of the Deaf.

Child↗

PubMed1998

Radiation absorbed doses to the walls of hollow organs.

UNLABELLED: Many radiopharmaceuticals are excreted from the body through the gastrointestinal (GI) tract. The doses to the walls of the organs involved often are very significant. As significant fractions of the administered activity pass through them, these organs may receive the highest doses in the body for many radiopharmaceuticals. The absorbed dose to these walled organs, from activity in their contents, is typically calculated as 50% of the average absorbed dose to the contents, for nonpenetrating emissions. The internal surface of the GI tract, and to a certain extent the urinary bladder, is lined with a variable thickness of mucus. In addition, the radiosensitive cell populations (crypt or stem cells) are located at some depth into the mucosa. These two factors suggest that the surface dose, often used to characterize the clinically relevant absorbed doses for walled organs, may represent an overestimate in some cases. METHODS: In this study, the radiation transport code MCNP was used to simulate the deposition of energy from nonpenetrating emissions of several radionuclides of interest: 90Y, 99mTc,123I and 131I. Absorbed doses as a function of distance from the wall-contents interface were calculated for three geometric shapes representing different organs along the routes of excretion. RESULTS: The absorbed dose from nonpenetrating emissions to the sensitive cell populations was consistently lower than estimated by the standard model assumption. The simulated absorbed doses to radiosensitive cells in the GI tract for 99mTc and 123I are tenfold lower; those for 131I are fivefold lower and those for 90Y are 20% lower. CONCLUSION: This study demonstrates that the normally reported dose to the walls of hollow organs probably should be modified to account for the attenuation of these nonpenetrating emissions in the linings of the walls. This study also demonstrates that Monte Carlo codes continue to be useful in the evaluation of the dose to sensitive cells in walled organs.

Colon↗

PubMed1996

Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2).

Two cyclooxygenase isozymes catalyze conversion of arachidonic acid to prostaglandin H2: constitutive COX-1 and inducible COX-2. To assess the role of COX-2 in colorectal tumorigenisis, we determined the effects of COX-2 gene (Ptgs2) knockouts and a novel COX-2 inhibitor on Apc delta716 knockout mice, a model of human familial adenomatous polyposis. A Ptgs2 null mutation reduced the number and size of the intestinal polyps dramatically. Furthermore, treating Apc delta716 mice with a novel COX-2 inhibitor reduced the polyp number more significantly than with sulindac, which inhibits both isoenzymes. These results provide direct genetic evidence that COX-2 plays a key role in tumorigenesis and indicate that COX-2-selective inhibitors can be a novel class of therapeutic agents for colorectal polyposis and cancer.

Adenomatous Polyposis Coli↗

PubMed1996

Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells.

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.

Animals↗

PubMed1996

Shielding design of a treatment room for an accelerator-based epithermal neutron irradiation facility for BNCT.

Protecting the facility personnel and the general public from radiation exposure is a primary safety concern of an accelerator-based epithermal neutron irradiation facility. This work makes an attempt at answering the questions "How much?" and "What kind?" of shielding will meet the occupational limits of such a facility. Shielding effectiveness is compared for ordinary and barytes concretes in combination with and without borated polyethylene. A calculational model was developed of a treatment room , patient "scatterer," and the epithermal neutron beam. The Monte Carlo code, MCNP, was used to compute the total effective dose equivalent rates at specific points of interest outside of the treatment room. A conservative occupational effective dose rate limit of 0.01 mSv h-1 was the guideline for this study. Conservative Monte Carlo calculations show that constructing the treatment room walls with 1.5 m of ordinary concrete, 1.2 m of barytes concrete, 1.0 m of ordinary concrete preceded by 10 cm of 5% boron-polyethylene, or 0.8 m of barytes concrete preceded by 10 cm of 5% boron-polyethylene will adequately protect facility personnel.

Boron Neutron Capture Therapy↗

PubMed1995

Thiopyranol[2,3,4-c,d]indoles as inhibitors of 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C4 synthase.

The attachment of an arylacetic or benzoic acid moiety to the thiopyrano[2,3,4-c,d]indole nucleus results in compounds which are highly potent and selective 5-lipoxygenase (5-LO) inhibitors. These compounds are structurally simpler than previous compounds of similar potency in that they contain a single chiral center. From the data presented, 2-[[1-(3-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]- 4, 5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]methoxy]-phenylacetic acid, 14b, was shown to inhibit 5-hydroperoxyeicosatetraenoic acid (5-HPETE) production by human 5-LO (IC50 of 18 nM). The acid 14b is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or human leukocyte leukotriene A4 (LTA4) hydrolase (IC50 > 20 microM). In addition, 14b was inactive in a 5-lipoxygenase-activating protein (FLAP) binding assay at 10 microM. In vivo studies showed that 14b is bioavailable in rat and functionally active in the hyperreactive rat model of antigen-induced dyspnea (74% inhibition at 0.5 mk/kg po; 2 h pretreatment). In the conscious squirrel monkey model of asthma, 14b showed excellent functional activity at 0.1 mg/kg against antigen-induced bronchoconstriction (94% inhibition of the increase in RL and 100% inhibition in the decrease in Cdyn; n = 4). Resolution of this compound gave (-)-14b, the most potent enantiomer (IC50 = 10 nM in the human 5-LO assay), which was shown to possess the S configuration at the chiral center by X-ray crystallographic analysis of an intermediate. Subsequent studies on the aryl thiopyrano[2,3,4-c,d]indole series of inhibitors led to the discovery of potent dual inhibitors of both FLAP and 5-LO, the most potent of which is 2-[[1-(4-chlorobenzyl)-4-methyl-6-(quinolin-2-ylmethoxy)-4, 5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]methoxy]phenylacetic acid, 19. Acid 19 has an IC50 of 100 nM for the inhibition of 5-HPETE production by human 5-LO and is active in a FLAP binding assay with an IC50 of 32 nM. Furthermore, thiopyrano[2,3,4-c,d]indoles such as 1 and 14b are capable of inhibiting the LTC4 synthase reaction in a dose dependent manner (IC50s of 11 and 16 microM, respectively, compared to that of LTC2 at 1.2 microM) in contrast to other, structurally distinct 5-LO inhibitors. It has also been observed that the thiopyrano[2,3,4-c,d]indole class of compounds strongly promotes the translocation of 5-LO from the cytosol to a membrane fraction in the presence or absence of the ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)

5-Lipoxygenase-Activating Proteins↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

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.

Adenomatous Polyps↗

PubMed1995

Specific absorbed fractions of energy from internal photon sources in brain tumor and cerebrospinal fluid.

Transferrin, when injected intracranially into glioblastoma multiforme lesions, acts as a cytotoxic substance. Transferrin, radiolabeled with In-111, can be coinjected and subsequent scintigraphic imaging can demonstrate the biokinetics of the cytotoxic transferrin. The administration of 111In transferrin into a brain tumor results in distribution of radioactivity in the brain, brain tumor, and the cerebrospinal fluid (CSF). Information about absorbed radiation doses to these regions, as well as other nearby tissues and organs, is important for evaluating radiation-related risks from this procedure. The radiation dose is usually estimated for a mathematical representation of the human body. We have included source/target regions for the eye, lens of the eye, spinal column, spinal CSF, cranial CSF, and a 100-g tumor within the brain of an adult male phantom developed by Cristy and Eckerman. The mathematical models of the spinal column, spinal CSF, and the eyes were developed previously, however, these source/targets have not been routinely included in photon transport simulations. Specific absorbed fractions (SAFs) as a function of photon energy were calculated using the ALGAMP computer code, which utilizes Monte Carlo techniques for simulating photon transport. The ALGAMP code was run three times, with the source activity distributed uniformly within the tumor, cranial CSF, and the spinal CSF volumes. These SAFs, which were generated for 12 discrete photon energies ranging from 0.01 to 4.0 MeV, were used with decay scheme data to calculate S-values needed for estimating absorbed doses. S-values for 111In are given for three source regions (brain tumor, cranial CSF, and spinal CSF) and all standard target regions/organs, the eye and lens, as well as to tissues within these source regions. S-values for the skeletal regions containing active marrow are estimated. These results are useful in evaluating the radiation doses from intracranial administration of 111In transferrin. The SAFs are also generally useful for calculation of absorbed dose from any radionuclide in these source regions.

Adult↗

PubMed1995

Inflammatory effector mechanisms in asthma.

Each of these effector systems has the capacity to initiate airway obstruction or alter airway responsiveness in asthma. It is likely that they act in concert in certain asthmatic settings. Further basic and applied research will define their relative roles in asthma.

Arachidonate 5-Lipoxygenase↗

PubMed1995

Conversation at home: a case study of a young deaf child's communication experiences in a family in which all others can hear.

In the past, much research on deaf children's language development has been conducted in schools and clinical settings to examine children's achievement of standard English or to study the effectiveness of mode of communication. The purpose of this study was to examine a deaf child's pragmatic development, her abilities as a language user in the naturally occurring situations of her everyday life at home. Observations, videotaping, and interviews were used to collect descriptive data of the child's conversation experiences with hearing family members. Findings reveal that deafness has had an impact on the family's beliefs and communication practices. Data show the child to be a competent communicator as she uses language appropriately for a variety of purposes, demonstrates her knowledge of the structural features of conversation, and employs an array of communication strategies to achieve mutual understanding with family members.

Adolescent↗

PubMed1994

Structural requirements for the binding of fatty acids to 5-lipoxygenase-activating protein.

5-Lipoxygenase-activating protein is required for cellular leukotriene synthesis and is the target of the leukotriene biosynthesis inhibitors MK-886 (3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H- indol-2-yl]-2,2-dimethylpropanoic acid) and MK-591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)-indol-2-yl] - 2,2-dimethylpropanoic acid). Recent studies demonstrate that 5-lipoxygenase-activating protein binds arachidonic acid and stimulates the utilization of this substrate by 5-lipoxygenase. The present study utilizes a radioligand binding assay to assess the affinity of 5-lipoxygenase-activating protein for arachidonic acid and the specificity of the fatty acid binding site on 5-lipoxygenase-activating protein. Our findings demonstrate that the presence of a free carboxyl group on fatty acids or leukotriene biosynthesis inhibitors which interact with 5-lipoxygenase-activating protein is not required for specific binding to the protein. However, the degree of saturation significantly affects the affinity of fatty acids for 5-lipoxygenase-activating protein.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1994

Protein kinase C-dependent regulation of sulfidopeptide leukotriene biosynthesis and leukotriene C4 synthase in neutrophilic HL-60 cells.

In response to calcium ionophore (A23187) stimulation, human granulocyte/macrophage colony-stimulating factor-primed, dimethylsulfoxide-differentiated HL-60 cells (which resemble mature granulocytes) synthesized leukotrienes (LTs) LTA4, LTB4, LTC4, and LTD4. The synthesis of the sulfidopeptide LTs, LTC4 and LTD4, was specifically inhibited in cells incubated in the presence of both A23187 and phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC). In contrast, neither the synthesis of LTB4, a product of the nonpeptide branch of the LT pathway, nor the formation of LTA4, the precursor for both branches of the LT biosynthetic pathway, was significantly affected by the presence of PMA during A23187 stimulation. The inhibition by PMA of LTC4 production in A23187-stimulated HL-60 cells was dose dependent, with an IC50 value of approximately 3.5 nM. The PKC inhibitor staurosporine completely reversed the inhibition by PMA of LTC4 production in A23187-stimulated cells, in a dose-dependent fashion, with an IC50 value of approximately 30 nM. Bisindolylmaleimide, another PKC inhibitor, was also able to prevent PMA-mediated inhibition of LTC4 formation, whereas inhibitors of protein kinase A, tyrosine kinases, or the respiratory-burst oxidase were not. Measurement of LTC4 synthase enzymatic activity in cells challenged with A23187 and PMA in the presence or absence of staurosporine demonstrated that the activity of the LTC4 synthase enzyme was inhibited in cells costimulated with A23187 and PMA and that inhibition could also be completely prevented by the presence of staurosporine. Because PMA is known to activate PKC, and staurosporine and bisindolylmaleimide are inhibitors of PKC, these results suggest that LTC4 synthase in HL-60 cells may be phosphoregulated.

Alkaloids↗

PubMed1993

5-lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes.

The intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion of arachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells. In addition, subcellular fractionation techniques showed > 83% of immunoblot-detectable FLAP protein and approximately 64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction. In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment. These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope.

5-Lipoxygenase-Activating Proteins↗