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PubMed1989

Influence of acute pulmonary interstitial inflammation on kinetics of phagocytosis by alveolar macrophages.

The pulmonary alveolar macrophage (PAM) is central to lung cellular defenses and is a potential participant in lung injury, but little is known about the influence of the nature and anatomic pattern of acute lung injury on PAM function. To assess the relationship between ongoing pulmonary inflammation and PAM function, we evaluated PAM phagocytic kinetics in a model system of experimental interstitial adjuvant pneumonitis (EIAP) in calves. PAMs were obtained from lung one and seven days postinduction (dpi) of EIAP. Lesions were typical of EIAP, characterized by acute multifocal to coalescing exudative interstitial pneumonitis at 1 dpi, which progressed to granulomatous interstitial pneumonitis by 7 dpi. The total recoverable lung cells and percentage of neutrophils (PMNs) were elevated (P less than 0.01) from animals with EIAP at both 1 and 7 dpi, and there was a four-fold increase (P less than 0.01) in the PAM yield by 7 dpi. Linear regression equations revealed that a larger proportion of control PAMs were phagocytic than were PAMs from animals with EIAP. The mean initial phagocytic rates of PAM following acute lung injury were significantly elevated (P less than 0.05) over controls; this difference was concentration dependent and required a phagocytic bead stimulus concentration in excess of 12.5 x 10(6) beads/ml. PAMs from animals with EIAP had a greater maximum rate of phagocytosis (Vmax) and Km than control PAMs. PAMs from animals with EIAP had a slightly higher proportion of cells which phagocytosed multiple beads. Levels of beta-glucuronidase were elevated (P less than 0.02) in PAM from animals with EIAP at 7 dpi. The results document enhanced PAM phagocytic function in EIAP and differ from our previous experiments in which depressed PAM phagocytic indices were obtained in a model of virus-induced acute bronchiolitis and alveolitis. The functional activities of the PAMs thus appear to be modified by injury-specific events in the lung microenvironment which may, in part, reflect the nature and anatomic pattern of developing pulmonary inflammatory reactions.

Acid Phosphatase↗

PubMed1995

Addition of an endoplasmic reticulum retention/retrieval signal does not block maturation of enzymatically active peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal alpha-amidation of neural and endocrine peptides via a two-step reaction carried out in sequence by the monooxygenase and lyase domains contained in this bifunctional protein. Peptide alpha-amidation is thought to take place primarily in the secretory granules in which mature bioactive peptides are stored, and it is not known where in the secretory compartment newly synthesized PAM protein becomes enzymatically active. To address this question, PAM-3, a soluble bifunctional protein, was modified by addition of the KDEL endoplasmic reticulum (ER) retention/retrieval signal to its COOH terminus. PAM-3-KDEL protein stably expressed in hEK-293 cells or in AtT-20 cells was efficiently retained in the ER based on immunocytochemistry, pulse-chase experiments, and maintained endoglycosidase H sensitivity. The effect of the KDEL sequence was specific since PAM-3 with an inactive ER retention/retrieval signal (PAM-3-KDEV) moved through the secretory pathway like wild type PAM-3. In AtT-20 cells, PAM-3-KDEL was not subjected to the COOH-terminal endoproteolytic cleavage that generates a 75-kDa PAM protein from PAM-3 and PAM-3-KDEV. PAM-3-KDEL protein exhibited both monooxygenase and lyase activities with specific activities similar to those of the wild type PAM-3 and PAM-3-KDEV proteins. Thus, although PAM catalyzes a reaction that occurs primarily in the secretory granules, newly synthesized PAM protein becomes enzymatically competent in the ER.

Amino Acid Sequence↗

PubMed1994

Differential trafficking of soluble and integral membrane secretory granule-associated proteins.

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.

Amino Acid Sequence↗

PubMed1991

Kinetic analyses of peptidylglycine alpha-amidating monooxygenase from pancreatic islets.

Peptidylglycine alpha-amidating monooxygenase (PAM) plays an important role in the post-translational processing of bioactive neuropeptides by participating in C-terminal amidation. We have examined PAM activity in the pancreatic islets of the anglerfish (AF), Lophius americanus. It was previously demonstrated that the cofactor requirements and pH optimum for the fish PAM are essentially identical to PAM obtained from other tissues and species. The present study was performed to examine the enzymatic characteristics of the fish islet PAM in more detail. One of the questions addressed was the suitability of the AF islet neuropeptide Y-like peptide, aPY-Gly, as a substrate for the islet PAM. Partially purified PAM from AF islet secretory granules was incubated with [125I] aPY-Gly and the resulting products were analyzed by HPLC. The islet PAM readily mediated the formation of aPY-amide from aPY-Gly. PAM purified from bovine adrenal chromaffin granules also catalyzed the amidation of [125I] aPY-Gly. The kinetic parameters of the islet PAM were examined using trinitrophenylated-D-Tyr-Val-Gly (TNP-D-YVG) and 4-nitrohippuric acid (4-NHA). The Km of the islet PAM was 25 +/- 5 microM for TNP-D-YVG and 3.4 +/- 1 mM for 4-NHA. The competitive inhibitor of mammalian PAM activity, 4-methoxybenzoxyacetic acid, proved to be a potent inhibitor of the islet PAM as well, with an apparent KI of 0.06 mM. These results demonstrate that the AF islet PAM exhibits substrate compatibility, kinetic parameters, and inhibitor susceptibility quite similar to the characteristics of PAM from other tissues and species.

Adrenal Medulla↗

PubMed2026

Fourteen-Day Amoxicillin- or Tetracycline-Containing Bismuth Quadruple Therapy versus 14-Day Metronidazole-Based Triple Therapy as the Treatment for Clarithromycin-Resistant Helicobacter pylori Infection: A Multicenter Randomized Controlled Trial.

BACKGROUND/AIMS: Combination therapy comprising a proton pump inhibitor (PPI), amoxicillin, and metronidazole (PAM) is used to treat clarithromycin-resistant Helicobacter pylori in the Republic of Korea, but eradication rates are decreasing due to an increasing incidence of clarithromycin resistance. We compared PAM, bismuth compounds plus PAM (PAM-B), and the combination of PPI, bismuth compounds, metronidazole, and tetracycline (PBMT) to determine whether PAM-B can achieve an eradication rate higher than that of PAM and comparable to that of PBMT. METHODS: This prospective multicenter study enrolled patients with clarithromycin-resistant H. pylori infections in the Busan and Gyeongsangnam-do of the Republic of Korea between December 2022 and February 2024. RESULTS: In the intention-to-treat (ITT) analysis, the eradication rate was significantly lower in the PAM group (68.2%) than in the PAM-B group (84.8%, p=0.024), whereas the difference from the PBMT group (81.8%) was not significant (p=0.070). The rate remained lowest in the PAM group (75.4%) in the per-protocol (PP) analysis (PAM-B, 96.5%, p=0.001; PBMT, 94.6%, p=0.004). Eradication rates were comparable between the PAM-B and PBMT groups in both the ITT (p=0.640) and PP analyses (p=0.633). Nausea and vomiting were significantly less frequent in the PAM-B group than in the PBMT group (6.8% vs 25.0%; p=0.007). Severe adverse events were rare, and all symptoms resolved after treatment discontinuation. CONCLUSIONS: PAM-B yielded eradication rates higher than those of PAM and comparable to those of PBMT, with no significant increase in the incidence of adverse events, suggesting the potential of PAM-B as a first-line treatment for clarithromycin-resistant H. pylori infection. Further large-scale studies are needed to validate these results. Registered retrospectively with the Clinical Research Information Service (CRIS; KCT0012265).

Humans↗

PubMed1993

Topological switching of the COOH-terminal domain of peptidylglycine alpha-amidating monooxygenase by alternative RNA splicing.

Proteins encompassing the two catalytic domains (monooxygenase and lyase) and the COOH-terminal domain of rat peptidylglycine alpha-amidating monooxygenase (rPAM)3 were purified from recombinant Escherichia coli overexpressing each domain and used to raise domain-specific polyclonal antibodies. Four alternatively spliced forms of PAM RNA (PAM-1, -2, -3, and -4) were transcribed in vitro and used to synthesize PAM proteins in a cell-free translation system. The orientation of the proteins in microsomal membrane vesicles was analyzed using trypsin protection assays and immunoprecipitation with the domain-specific antibodies. Only one of the two potential N-glycosylation sites (Asn765-Phe-Ser) in PAM-1 was efficiently utilized by microsomal membranes. PAM-1 and PAM-2 were shown to be type Ia membrane proteins with their two catalytic domains residing within microsomal vesicles and their COOH-terminal domains exposed to the cytosol. In contrast, PAM-3 and PAM-4 were shown to be soluble proteins contained entirely within vesicles. Thus, the COOH-terminal domain underwent topological switching between the cytosolic (PAM-1 and -2) and luminal (PAM-3) compartments as a function of alternative splicing of exons Ba/Bb. Computer analyses of the PAM protein sequence correlated the exons encoding PAM-1 with a model for the structural and functional domains of the PAM protein. The dual topologies of the PAM proteins confer an important means of functional regulation to this secretory granule associated neuropeptide processing enzyme.

Amino Acid Sequence↗

PubMed1996

Identification of routing determinants in the cytosolic domain of a secretory granule-associated integral membrane protein.

We have investigated the trafficking of integral membrane peptidylglycine alpha-amidating monooxygenase (PAM) in the neuroendocrine AtT-20 cell line. This bifunctional enzyme has two domains which together catalyze the COOH-terminal alpha-amidation of peptidylglycine substrates yielding amidated products stored in secretory granules. As soluble proteins, both catalytic domains were independently targeted to secretory granules. In contrast, membrane PAM was largely localized to the trans-Golgi network (TGN). Upon truncation of its cytoplasmic COOH-terminal domain, membrane PAM was less efficiently cleaved by secretory granule enzymes and accumulated on the plasma membrane. When transferred to the lumenal domain of the interleukin 2 receptor alpha-chain (Tac protein), the cytoplasmic domain of PAM caused rerouting of Tac from the surface to the TGN and supported internalization of Tac antibody from the plasma membrane. To define sequences in the cytoplasmic domain of integral membrane PAM involved in its trafficking, we expressed PAM proteins containing truncations, deletions, or point mutations in the COOH-terminal cytoplasmic domain. PAM proteins were not retained in the TGN when half of the cytoplasmic domain was deleted; such proteins accumulated on the plasma membrane, were not efficiently internalized, and were cleaved to generate a bifunctional PAM protein that was not stored in secretory granules. A tyrosine-based internalization motif was identified, which was not required for efficient cleavage of full-length integral membrane PAM by secretory granule enzymes. Deletion of an 18-amino acid domain surrounding this Tyr residue both diminished cleavage of membrane PAM by secretory granule enzymes and eliminated internalization of PAM from the plasma membrane. The cytoplasmic domain is responsible for retaining membrane PAM in the TGN and for retrieving membrane PAM from the cell surface, while the lumenal catalytic domains of PAM appear to be responsible for targeting the protein to secretory granules.

Amino Acid Sequence↗

PubMed1989

Tissue-specific regulation of peptidyl-glycine alpha-amidating monooxygenase expression.

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is present in a variety of tissues, where it plays a vital role in the processing of numerous peptide substrates, often conferring bioactivity. PAM is present in high amounts in heart atrial myocytes and the pituitary gland, where activity is present in both soluble and membrane forms. We used AtT-20 cells, a mouse corticotrope tumor cell line, and primary heart atrial cultures to establish the occurrence of tissue-specific regulation of PAM expression. In the AtT-20 cells, PAM expression is regulated in parallel with the source of its peptide substrate, pro-ACTH/endorphin. PAM mRNA levels are increased to 132 +/- 5% of control values by treatment with (Bu)2-cAMP and decreased to 55 +/- 7% of control values by treatment with dexamethasone. Treatment with (Bu)2cAMP decreases PAM specific activity in the AtT-20 cells to 68 +/- 4% of the control value, presumably due to secretion of enzyme from the cells; dexamethasone treatment decreases PAM specific activity to 57 +/- 1% of the control value. In contrast, in heart atrial cultures, dexamethasone stimulates PAM expression. In atrial cultures exposed to dexamethasone for 48 h, PAM mRNA and PAM specific activity are elevated to 230 +/- 50% and 220 +/- 20% of control values, respectively; secretion of PAM activity is increased to 230% of the control value. As for AtT-20 cells, treatment of atrial cultures with (Bu)2cAMP increases PAM mRNA levels. Thus, PAM expression is regulated in a tissue-specific manner by dexamethasone in the two tissues examined. In AtT-20 cells, time-course studies and studies with cycloheximide indicate that dexamethasone exerts its effects on PAM mRNA levels by an indirect mechanism involving protein synthesis.

Animals↗

PubMed1997

Estrogen regulation of peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid levels by a nuclear posttranscriptional event.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a bifunctional protein containing two enzymes that act sequentially to catalyze the conversion of glycine-extended peptides into COOH-terminal amidated peptides. We have previously shown that PAM messenger RNA (mRNA) levels in the anterior pituitary of intact cycling adult female rats showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17beta-estradiol was accompanied by a 4.5 +/- 0.5-fold decrease in total PAM mRNA and a 2-fold decrease in PAM activity in the anterior pituitary gland. To investigate the cellular site at which 17beta-estradiol acts to affect the PAM mRNA, we made parallel measurements of the relative levels of PAM mRNA and nuclear precursor RNA and the relative rate of gene transcription after treatments designed to alter the estrogen status. The transcription rate experiments indicated that these 17beta-estradiol effects were not due to reduced PAM gene activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. Primary rat pituitary cell cultures from OVX and OVX-17beta-estradiol-treated rats in the presence of actinomycin D showed that 17beta-estradiol treatment decreased the half-life of PAM mRNA from 15-16 h to 8-9 h. There was no effect of 17beta-estradiol on PAM mRNA poly(A) tail length or site of polyadenylation. However, in this study the down-regulation of PAM was identified as a nuclear event. Analysis of nuclear RNA with probes specific for PAM intron sequences shows that decreased PAM expression after 17beta-estradiol treatment was largely due to intranuclear destabilization of the primary transcript. The levels of nuclear precursor RNA were decreased roughly 5- to 6-fold in OVX + 17beta-estradiol compared with OVX rats. The decrease in PAM mRNA is blocked by cycloheximide, indicating that its requires new protein synthesis. Mechanisms that would generate such an effect include altered stability of unprocessed message in the nucleus. The proportional changes observed in the nuclear precursor and mRNA levels suggest that the site of control is at the level of stability of the nuclear precursor RNA for PAM mRNA.

Animals↗

PubMed1986

Enhanced proliferation of pulmonary alveolar macrophages after carbon instillation in mice depleted of blood monocytes by strontium-89.

The purpose of this study was to evaluate the potential of pulmonary alveolar macrophage (PAM) and interstitial cell division as local sources of PAM in the absence of migratory monocytes. Male B6C3F1 mice were treated with Strontium-89 to deplete the levels of circulating monocytes. Normal and irradiated mice were then instilled intratracheally with carbon particles to increase the demand for PAM. Dividing cells were labeled with tritiated thymidine. The proportion of tritiated thymidine-labeled cells was then determined in lavaged PAM and in IC of lung tissue as an indicator of the proliferative response associated with the demand for increased number of PAM. It was hypothesized that, in the absence of circulating monocytes as a source of PAM, cell division of the local source of PAM would be greater in the monocyte-depleted mice than in the normal mice. It was found that proliferation of lavaged PAM was 112% greater in the monocyte-depleted mice, whereas proliferation of IC in the monocyte-depleted mice was 45% less than that in the normal mice. It was concluded that: cell proliferation of PAM is a significant local source of PAM, and division of cells in the interstitium could not be evaluated as a source of PAM due to the heterogenous nature of the cell types which comprise this population of cells. This study established that cell division of "free" PAM can be a significant local source of PAM and raises questions concerning the nature of the interstitial source of PAM.

Animals↗

PubMed1985

Cytotoxic activity of human pulmonary alveolar macrophages.

The functions of human pulmonary alveolar macrophages (PAMs) have been relatively little studied compared with those of their circulating counterparts, blood monocytes. This study examined the ability of human PAMs to kill primary human tumor cell cultures and control normal fibroblasts in vitro. PAMs were derived by bronchial lavage from patients with lung cancer of various histological types and stages, patients with acute or chronic noncancerous pulmonary disorders, and subjects with a presumed illness who proved to be normal. After extensive washing, the PAMs were cocultured with [3H]proline-labeled tumor cells, principally lung cancers and melanomas, at various effector:target ratios for 60 hr. Cytotoxicity was measured by comparing radioactivity associated with the remaining adherent tumor cells cultured in the presence or absence of PAMs. Twenty-eight of 42 preparations of PAMs from 42 individuals were cytotoxic to one or more short-term primary tumor cultures. All 28 specimens from patients with lung cancer or chronic pulmonary disease were cytotoxic; all of the 14 PAM preparations lacking cytotoxicity were from individuals with acute pulmonary disorders or who were proved free of pulmonary disease. PAMs were cytotoxic even at effector:target ratios of 2.5:1 or 1.25:1. Fibroblasts were unaffected at any ratio. Sarcoidosis patients in remission had noncytotoxic PAMs, whereas the disease in relapse was characterized by cytotoxic PAMs. Serial study of 2 patients confirmed a loss of reactivity during remission. Smoking did not correlate with the presence or absence of spontaneous cytotoxicity and did not influence the degree of cytotoxicity in "reactors." Partially purified alpha-interferon enhanced the killing of cytotoxic PAMs in 10 of 21 instances but did not induce cytotoxicity in 9 tests on nonreactive PAMs. We conclude that human PAMs from patients with lung cancer or chronic pulmonary diseases, including active sarcoidosis, were cytotoxic to several recently explanted tumor cell cultures. PAMs from acute pulmonary dysfunctions and those from patients with inactive sarcoidosis were not spontaneously cytotoxic.

Adenocarcinoma↗

PubMed1986

The significance of local resident pulmonary alveolar macrophage proliferation to population renewal.

The local proliferative capacity of resident pulmonary alveolar macrophages (PAM) was determined in normal CD-1 (ICR) mice and mice made severely monocytopenic with strontium-89 (89Sr). Intravenous injection of 2 muCi/gm BW 89Sr rendered mice monocytopenic for a 1-month period. During this time, in addition to there being no change in the size of the PAM population, flash 3HTdR labeling studies showed that at any one instant about 8.8 X 10(3) of PAM in 88Sr-treated controls and 9.2 X 10(3) PAM in 89Sr-treated mice were in S phase of the cell cycle. Metaphase arrest experiments using vincristine showed that in 88Sr-treated mice PAM entered M phase at a rate (rM) of 0.51%/hr with a duration of M (Tm) of 0.37 hr, whereas rM = 0.23%/hr with Tm = 0.14 hr for PAM in 89Sr-treated mice. These data suggest PAM population half-times of 131 hr in the presence of monocytes and 301 hr in the absence of monocytes. The data show that PAM proliferate locally; moreover, for intervals longer than the estimated half-time, PAM population size was unchanged. By following the accumulation of pulse 3HTdR-labeled mitotic figures it was found that in both 88Sr- and 89Sr-treated mice G2 = 3 hr and Ts = 11 hr. From these data a model of PAM population growth was used to show that PAM renewal may be accounted for by local proliferation alone. Taken together the data show that resident PAM populations may be sustained by local division of cells and that the influx of peripheral blood monocytes was not needed to sustain PAM.

Animals↗

PubMed1997

Embryonic and uterine expression patterns of peptidylglycine alpha-amidating monooxygenase transcripts suggest a widespread role for amidated peptides in development.

Posttranslational processing of peptide precursors frequently includes COOH-terminal amidation by the bifunctional enzyme peptidylglycine alpha-amidating monooxygenase (PAM). We examined the ontogeny of PAM gene expression using in situ hybridization and detected expression in the cardiogenic region beginning at embryonic day 9 (e9) and in decidualizing uterine endometrium and myometrial smooth muscle at even earlier postimplantation stages. PAM expression in the CNS at e10 was highest in the dorsal spinal cord and floor plate and exhibited complex patterning in several CNS regions, including the ventricular zone, over the next several days with PAM expression first detected in neurons at e13. High levels of PAM expression characterized several nonneural cell populations as well, including limb mesoderm and the mesenchyme immediately adjacent to nasal, maxillary, palatal, and dental epithelia during tissue fusion and remodeling. Since alternative splicing generates PAM transcripts encoding proproteins that are differentially localized and processed, we used probes that distinguish major subsets of PAM transcripts to determine that transcripts encoding integral membrane PAM isoforms predominate in most, if not all, PAM-expressing cell types throughout development. Further, transcripts that encode soluble and cleavable PAM isoforms are essentially absent from two CNS areas that are rich in transcripts encoding integral membrane, bifunctional PAM: the ependymal region of the spinal cord and the ventricular zone of the hippocampus. These results provide evidence for widespread expression and cell-type-specific alternative splicing of PAM during development and raise the possibility that region-specific amidation of PAM substrates contributes significantly to several developmental processes.

Animals↗

PubMed1997

Genotoxic changes in the pulmonary alveolar macrophages of mice, rats and hamsters treated with tobacco smoke.

To determine whether tobacco smoke (TS) is genotoxic for lung tissue macrophages (pulmonary alveolar macrophages, PAM) as a general result of its inhalatory action BD6 rats, Syrian golden hamsters and BDF1 (C57BlxDBA2) mice were subjected to wholebody exposure for 90 or 60 min daily (600 cm3 mainstream smoke in 16-1 glass chamber, 9 or 6 exposures of 15 min each, respectively), for different periods ranging up to 30 days. A significant enhancement of the frequency of polynucleated macrophages (BiN PAM) was observed in all animal species after more than 10-days of repeated exposure to TS. The increased level of BiN PAM (the number of bi- (+) poly-nucleated PAM) correlates with the duration of exposure to TS: on day 20 after the start of inhalation, more than 25/1000 of mice PAM were polynucleated, while on day 30 this applied to approximately 50/1000. Furthermore, a highly significant increase in the level of micronucleated PAM (MN PAM) was also established after 10 days TS treatment of mice and persisted to the end of these examinations. TS was effective in enhancing the micronucleated and polynucleated PAM levels in hamsters irrespective of their sex, as it was in male BD6 rats aged 2 or 11 months. It appears that TS induces a more pronounced elevation of polynucleated PAM frequency in rats than in hamsters and mice. These data suggest that inhaled TS is genotoxic in alveolar macrophages in all exposed species of laboratory animals. An attempt was made to trace the possible clastogenic effect of a single i.p. administration of cyclophosphamide (CP, 15 mg/kg) in mice simultaneously in bone marrow and in PAM. A definite clastogenic effect in bone marrow 24 h and 48 h after CP injection and a total absence of changes in PAM from the lungs during the 15-day period after clastogen exposure were established. These data may support the hypothesis of local production of PAM in the lung from their proliferative precursor. The results provide evidence that PAM in laboratory animals are a sensitive and useful target for assessing harmful effects associated with environmental chemical factors that can be inhaled, including TS.

Animals↗

PubMed2003

Melphalan-induced up-regulation of B7-1 surface expression on normal splenic B cells.

We have previously shown that exposure of MOPC-315 or P815 tumor cells to the widely used anticancer drug melphalan ( L-PAM, L-phenylalanine mustard) leads to rapid up-regulation of B7-1 surface expression. Since B7-1-expressing tumor cells depend on B7-expressing host antigen presenting cells (APC) for the generation of CD8(+) T-cell-mediated antitumor immunity, and since L-PAM promotes the acquisition of tumor-eradicating immunity by CD8(+) T-cells from MOPC-315 tumor bearers, the current studies were undertaken to determine if L-PAM also up-regulates B7-1 expression on host APC. Here we show that exposure of normal spleen cells to L-PAM leads within 24 h to up-regulated B7-1 expression on B220(+) cells (B cells). Studies into the mechanism through which L-PAM leads to up-regulated B7-1 expression revealed that within 2 h after exposure of normal spleen cells to L-PAM, accumulation of B7-1 mRNA is evident and this accumulation requires de novo RNA synthesis, indicating that the regulation is at the transcriptional level. The L-PAM-induced accumulation of B7-1 mRNA was prevented with the antioxidant N-acetyl- L-cysteine (NAC), indicating that reactive oxygen species are important for the transcriptional regulation. Although AP-1 and NF-kappa B are considered redox-sensitive transcription factors, L-PAM led only to activation of NF-kappa B that bound specifically to a probe containing the corresponding binding site in the B7-1 gene. Moreover, selective inhibition of NF-kappa B activation prevented the L-PAM-induced B7-1 mRNA accumulation, indicating that NF-kappa B activation is essential for L-PAM-induced B7-1 gene expression in normal spleen cells. Finally, in vivo administration of an immunopotentiating dose of L-PAM to normal mice was found to up-regulate B7-1 mRNA expression in their spleens. Thus, the ability of L-PAM to up-regulate B7-1 expression not only on tumor cells but also on host cells may contribute to the potentiating activity of L-PAM for the acquisition of CD8(+) T-cell-mediated tumor-eradicating immunity in tumor bearers.

Animals↗

PubMed2002

A simple method for the specific detection of Ren-1 renin.

BACKGROUND: Ren-1 and Ren-2 renin are expressed in the kidneys of all mice and in the submandibular gland of several mouse strains. The present study determined the usefulness of modified periodic acid silver-methenamine (PAM) staining for the specific detection of Ren-1 renin. METHODS: Conventional paraffin sections were prepared from kidneys of ICR, BALB/cA, C57BL/6Cr, C3H/HeN, DBA/2Cr, angiotensin II type 1a receptor gene knockout (AT1aKO) mice, Wistar rats and a human, and submandibular glands of C57BL/6Cr and DBA/2Cr mice. Sections were analyzed for the presence of renin using PAM and immunohistochemistry. PAM reactions were terminated at generally or weakly intense (weak PAM staining; W-PAM). In addition, kidneys of DBA/2Cr mice were fixed using various fixatives (formalin, PFA, PLP, Zamboni's, Bouin's, or Carnoy's) and treated using identical procedures. RESULTS: Although PAM-positive reactions were observed in juxtaglomerular (JG) cells, W-PAM reactions were particularly specific for these cells. These findings were observed in all mouse strains. Immunohistochemistry using mirror sections suggested that a W-PAM-positive reaction detected renin. This hypothesis was confirmed by the results from AT1aKO mice. Briefly, W-PAM detected an expansion of renin-positive areas in AT1aKO mice. Rat and human kidneys and mouse submandibular glands were negative for W-PAM. Levels of JG cell detection by W-PAM were similar in samples fixed in formalin, PFA, PLP, or Zamboni's. CONCLUSIONS: The present findings show that W-PAM can identify Ren-1 renin, but not Ren-2, rat or human renin. The W-PAM method is useful for the specific detection of Ren-1 renin.

Animals↗

PubMed1995

Phosphorylation of the cytosolic domain of peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the COOH-terminal alpha-amidation of neural and endocrine peptides through a two-step reaction carried out sequentially by its monooxygenase and lyase domains. PAM occurs in soluble and integral membrane forms. Metabolic labeling of stably transfected hEK-293 and AtT-20 cells showed that [32P]PO4(3-) was efficiently incorporated into Ser and Thr residues of membrane PAM but not into soluble PAM. Truncation of integral membrane PAM proteins (which terminate with Ser976) at Tyr936 eliminated their phosphorylation, suggesting that the COOH-terminal region of the protein was the site of phosphorylation. Recombinant PAM COOH-terminal domain was phosphorylated on Ser932 and Ser937 by protein kinase C (PKC). PAM-1 protein recovered from different subcellular fractions of stably transfected AtT-20 cells was differentially susceptible to calcium-dependent, staurosporine-inhibitable phosphorylation catalyzed by endogenous cytosolic protein kinase(s). Although phorbol ester treatment of hEK-293 cells expressing PAM-1 stimulated the cleavage/release of a bifunctional 105-kDa PAM protein, the effect was an indirect one since it was also observed in hEK-293 cells expressing a truncated PAM-1 protein that was not phosphorylated. AtT-20 cells expressing PAM-1 lacking one of the PKC sites (PAM-1/Ser937-->Ala) exhibited an altered pattern of PAM.PAM antibody internalization, with the mutant protein targeted to lysosomes upon internalization. Thus, phosphorylation of Ser937 in the COOH-terminal cytosolic domain of membrane PAM plays a role in a specific step in the targeting of this protein.

Alanine↗

PubMed1999

Phosphorylation of cytosolic domain Ser(937) affects both biosynthetic and endocytic trafficking of peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM), a bifunctional enzyme, catalyzes the COOH-terminal amidation of bioactive peptides. In test tube assays, PAM is phosphorylated by protein kinase C at Ser(937). The roles of phosphorylation and dephosphorylation of Ser(937) in the biosynthetic and endocytic trafficking of integral membrane PAM were examined using an antiserum specific for the phosphorylation of Ser(937) and using AtT-20 cells expressing membrane PAM in which Ser(937) was mutated to Ala or Asp. Although phosphorylation at Ser(937) can occur while PAM is in the endoplasmic reticulum, early steps in the biosynthetic trafficking of membrane PAM were not affected by Ser(937) phosphorylation. The inability to phosphorylate PAM/S937A increased its intracellular degradation and decreased secretion of the soluble monooxygenase portion of PAM. In contrast, the biosynthetic trafficking of PAM/S937D was indistinguishable from wild-type PAM. Despite the fact that Ser(937) is adjacent to the only Tyr-based internalization motif in PAM, internalization and trafficking through early endosomes were unaffected by phosphorylation. However, PAM antibody internalized by wild-type PAM acquired a perinuclear localization, while antibody internalized by PAM/S937A was routed to lysosomes, and antibody bound to PAM/S937D maintained a dispersed, punctate pattern. In cells stimulated with phorbol ester, phosphorylation of Ser(937) increased and phosphorylated PAM accumulated in large vesicular structures. Therefore, phosphorylation of PAM-1 at Ser(937) directs newly synthesized and internalized protein away from lysosomes, while dephosphorylation is needed for a different step in the late endocytic pathway.

Animals↗