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PubMed1986

Attenuation of cytogenetic damage by 2-mercaptoethanesulfonate in cultured human lymphocytes exposed to cyclophosphamide and its reactive metabolites.

Cyclophosphamide (CP) is metabolized to the reactive intermediates, phosphoramide mustard (PAM) and acrolein (AC), which have generally different molecular binding targets. Sodium 2-mercaptoethanesulfonic acid (MESNA) has been used clinically to alleviate hemorrhagic cystitis caused by CP chemotherapy, has exhibited anticarcinogenic effects in rats exposed to CP during a long-term bioassay, and acts in the urogenital tract by reacting with 4'-OH-CP and AC. The purpose of this study was to: (a) compare the relative abilities of PAM and AC to induce cytogenetic damage and cytotoxicity in cultured human lymphocytes; (b) assess the efficacy of MESNA to attenuate the cytogenetic damage and cytotoxicity induced by CP, AC, PAM, and diethyl-4'-hydroperoxycyclophosphamide (DEHP-CP), an activated AC-generating compound; and (c) determine if concanavalin A-stimulated T-lymphocytes, which differentiate into suppressor cells upon lectin activation, exhibit any heightened cytogenetic sensitivity compared to a variety of cultured mammalian cells during exposure to PAM or AC as reported by other investigators. Purified mononuclear leukocytes were stimulated with concanavalin A and exposed to CP (0.5-2.0 mM) without an exogenous activation system, AC (0.001-40.0 microM), PAM (0.0014-27.1 microM), or DEHP-CP (0.1-100.0 microM) in the presence or absence of MESNA (1, 5, or 10 mM). All four compounds induced significant concentration-related increases in the SCE frequency, but only PAM was clastogenic. On an induced SCE/microM basis, PAM was about 130 and 193 times more potent than were DEHP-CP and AC, respectively. MESNA protected against the cytogenetic damage and cytotoxicity induced by the four compounds, but it was particularly effective against AC and DEHP-CP by abolishing SCE induction completely. SCEs and chromosome aberrations differed considerably in their induction kinetics in lymphocytes exposed to PAM, and these disparities suggested an uncoupling of the two phenomena. Although SCE induction was not consistently associated with cytotoxicity with the four agents, chromosome aberration induction coincided with an inhibition of cell cycle kinetics in PAM-treated cells. The exceptionally high SCE frequency of up to 21 times baseline in cells exposed to PAM indicates that T-suppressor lymphocytes stimulated with concanavalin A may be particularly sensitive to the DNA-damaging effects of PAM. Finally, these data suggest that the anticarcinogenicity of MESNA correlates with its ability to attenuate cytogenetic damage and cytotoxicity induced by reactive CP metabolites.

Acrolein↗

PubMed1988

Membrane-associated peptidylglycine alpha-amidating monooxygenase in the heart.

Recent investigations have shown that the heart atrium is an endocrine tissue. In the present studies, high levels of peptidylglycine alpha-amidating monooxygenase (PAM), which catalyzes the formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in particulate fractions from bovine and rat heart atrium; only low levels of PAM activity were present in soluble fractions. Corresponding fractions from the ventricles contained 20-fold less activity. Immunocytochemical studies demonstrated that PAM was localized primarily to atrial cardiocytes, with a distribution resembling that of atriopeptin. Following differential centrifugation of rat atrial homogenates, most of the PAM activity was associated with crude granule fractions, with lesser amounts of activity associated with crude microsomal fractions. Upon further subcellular fractionation, PAM activity in the rat atrium was found primarily with immunoactive atriopeptin in fractions enriched in secretory granules. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, antisera to purified bovine pituitary PAM identified a 113,000-dalton protein in bovine atrial microsomes and secretory granules; the protein predicted from the sequence of the cDNA encoding bovine pituitary PAM is of similar size (Eipper, B. A., Park, L. P., Dickerson, I. M., Keutmann, H. T., Thiele, E. A., Rodriguez, H., Schofield, P. R., and Mains, R. E. (1987) Mol. Endocrinol. 1, 777-790). Northern blot analysis using cDNA probes encoding bovine pituitary PAM demonstrated higher levels of PAM mRNA in heart atrium than in anterior pituitary. Rat heart contains PAM mRNA species of 3.6 and 3.8 kilobases, the smaller mRNA species corresponding in size to the PAM mRNA expressed in rat anterior pituitary.

Animals↗

PubMed1997

No evidence for direct incorporation of esterified palmitic acid from plasma into brain lipids of awake adult rat.

Awake adult rats were given a solution of [9,10-3H]palmitate ([3H]PAM) by gavage. The appearance of radiolabel in plasma lipid fractions was monitored by thin-layer chromatography at fixed intervals thereafter. At 2 h, the rats were killed by microwave irradiation and radioactivity in whole brain and individual brain phospholipids was determined. In plasma, esterified [3H]PAM was mainly associated with triglyceride, phospholipid, and cholesterol ester. Radioactivity appeared to a larger extent in triglyceride than in unesterified fatty acid, suggesting that unesterified [3H]PAM in plasma was largely due to release from esterified [3H]PAM by lipoprotein lipase hydrolysis. Brain radioactivity could be accounted for entirely by incorporation of unesterified plasma [3H]PAM. Esterified [3H]PAM in chylomicrons or lipoproteins was calculated to make no measurable contribution using a published value for the incorporation coefficient of [3H]PAM into brain in the evaluation. These results suggest that ingested palmitic acid (PAM) in adult rats enters blood as esterified triglyceride within chylomicrons and lipoproteins and, in part, eventually is converted to circulating unesterified PAM. It is the circulating unesterified PAM that is incorporated into brain from blood, whereas esterified PAM within plasma chlomicrons and lipoproteins makes no measurable direct contribution.

Administration, Oral↗

PubMed1994

The use of biodegradable amikacin microspheres to prevent vascular graft infection.

The following study was performed to determine if an antibiotic impregnated in a biodegradable polymer can prevent infection and eradicate inoculum bacteria from contaminated polytetrafluoroethylene vascular grafts. Poly(glycolide-co-dl-lactide) amikacin microspheres (PAM) measuring 50-100 microns were designed to deliver 100 mg (PAM 100) or 300 mg (PAM 300) amikacin per unit dose. Twenty mongrel dogs had a short segment of infrarenal aorta replaced with a graft that had been bathed in a 2 cc solution of Escherichia coli and Staphylococcus aureus (3 x 10(8) CFU/ml). Dogs were divided into three groups: Controls had contaminated grafts placed and received no therapy; PAM 100 and PAM 300 were used, respectively, to cover the grafts in the other two groups. Animals were sacrificed 14 days postoperatively at which time grafts were examined and cultured. Among controls, 7/8 had clinical graft infections and all had positive cultures for S. aureus (8/8) or E. coli (5/8). None of the treated animals had clinical graft infections (P < 0.001). Positive cultures were obtained for S. aureus in 2/8 (P < or = 0.007) and E. coli in 0/8 (P < or = 0.03) PAM 100 dogs and for S. aureus in 0/8 (P < or = 0.0002) and E. coli in 0/8 (P < or = 0.03) PAM 300 dogs. Two PAM 100 and four PAM 300 dogs had rare growth of contaminant bacteria (NS). In conclusion, PAM can prevent clinical graft infection and completely eradicate a standardized bacterial inoculum.

Amikacin↗

PubMed1998

Porcine reproductive and respiratory syndrome virus infection of alveolar macrophages can be blocked by monoclonal antibodies against cell surface antigens.

PRRSV has a restricted macrophage tropism. To explore if the difference in susceptibility of porcine alveolar macrophages (PAM) and peripheral blood mononuclear cells (PBMC) to PRRSV is correlated with certain cellular surface antigens which may serve as a virus receptor, polyclonal antibodies against PAM and PBMC were prepared. Anti-PAM but not anti-PBMC antibodies protected PAM from PRRSV infection suggesting that specific receptor(s) may exist on PAM. Furthermore, monoclonal antibodies (MAbs) against putative receptor(s) were produced. Balb/c mice were firstly immune-tolerized with freshly isolated PBMC after which they were immunized with PAM. Two MAbs (41D3 and 41D5) which blocked PRRSV infection of PAM were obtained. MAb 41D3 and 41D5 prevented the attachment of purified PRRSV to PAM. Both MAbs bound to the cellular membrane of PAM but not to that of porcine peritoneal macrophages, PBMC and three porcine cell lines (SK, ST and PK-15) as revealed by flow cytometry. This membrane reactivity correlates well with the susceptibility of these cells to a PRRSV infection. Taken together, these data suggest that MAb 41D3 and 41D5 recognize a potential cellular receptor for PRRSV on PAM.

Animals↗

PubMed1990

Monocyte-derived macrophage and alveolar macrophage fibronectin production and cathepsin D activity.

Alveolar macrophages are thought to play an important role in ongoing tissue breakdown and repair processes in the normal lung. The secretion and regulation of cathepsin D (important for the final breakdown of collagen) and fibronectin (involved in the healing process) in human peripheral blood monocytes (PBM) and pulmonary alveolar macrophages (PAM) were investigated. Cathepsin D enzyme activity was measured by quantitating the TCA-soluble fragments of [3H]hemoglobin. Freshly isolated PBM contained less cell-associated cathepsin D activity than did freshly isolated PAM (314 +/- 35 micrograms/10(6) cells vs 381 +/- 35 micrograms/10(6) cells, respectively). After 7-10 days in culture, cell-associated enzyme levels in both PBM and PAM were significantly increased (P less than 0.001 for PBM; P less than 0.0001 for PAM). In addition, freshly isolated PAM secreted more cathepsin D than did freshly isolated PBM (5.8 +/- 3.2 micrograms/10(6) cells vs 0.83 +/- 0.83 micrograms/10(6) cells, P less than 0.02). In the presence of LPS (10 micrograms/ml), cell-associated cathepsin D was inhibited in both PBM and PAM. With the addition of gamma-IFN (500 U/ml), both cell-associated and secreted enzyme were increased in freshly isolated and 10-day-cultured PBM and PAM. In parallel studies, fibronectin secretion (by ELISA assay) in both PBM and PAM increased over time in culture. LPS had no effect on PBM or PAM secretion of human fibronectin while gamma-IFN increased PBM and PAM fibronectin levels. Thus, both macrophage cathepsin D activity and fibronectin secretion are increased by gamma-interferon while macrophage cathepsin D activity, but not fibronectin secretion, is decreased by LPS. These studies demonstrate that human macrophage cathepsin D activity is actively modulated by inflammatory mediators and that macrophage mediators of tissue breakdown and repair are not modulated synchronously.

Adult↗

PubMed2005

Purification, cloning, and functional expression of phenylalanine aminomutase: the first committed step in Taxol side-chain biosynthesis.

The conversion of alpha-phenylalanine to beta-phenylalanine is the first committed step in the biosynthesis of the C-13 side chain of Taxol. Thus, the novel enzyme responsible for this step, phenylalanine aminomutase (PAM), is of considerable interest for studies of Taxol biosynthesis and represents a potential target for genetic engineering. A method is described for purifying PAM from Taxus chinensis cell cultures. The purified enzyme has a K(m) of 1.1mM, a V(max) of 110.1 microm/min/mg protein, a pH optimum of 7.5-8.0, and a denatured molecular weight of about 80 kDa. Peptide sequences derived from the purified protein were used to design and synthesize degenerate primers enabling the PCR synthesis of the PAM cDNA. The PAM cDNA encodes a protein of 687 amino acid residues with a deduced molecular weight of 75.3 kDa. The PAM cDNA was cloned and expressed in Escherichia coli, and PAM activity was demonstrated. As a gene symbol for the PAM enzyme, pam is proposed. Protein sequence alignments of PAM, phenylalanine ammonia-lyase (PAL), and histidine ammonia-lyase (HAL) sequences exhibit significant similarity providing insight into potential active site residues of PAM.

Amino Acid Sequence↗

PubMed2005

High resolution computed tomographic features of pulmonary alveolar microlithiasis.

BACKGROUND: Pulmonary alveolar microlithiasis (PAM) is a rare, chronic lung disease with unknown etiology and with a nonuniform clinical course. Nonuniformity of clinical course might be related to the degree of pulmonary parenchymal alterations, which can be revealed with high resolution computed tomography (HRCT). However, HRCT findings of PAM were not fully described in the current literature. AIM: The aim of this study was to interpret and to contribute to describe HRCT findings of PAM and to investigate a correlation between profusion of micro nodules (MN) and pulmonary parenchymal alterations in patients with PAM. MATERIAL AND METHODS: Ten male patients with PAM (mean age: 22+/-3.2) were included into the study. HRCT images were assessed for patterns, distribution, and profusion of pulmonary abnormalities. Dividing the lungs into three zones, profusion of abnormalities was assessed. A profusion score (1-4) was given and the scores of each zone were then summed to obtain a global profusion score for HRCT ranging from 0 to 12. Also a parenchymal alteration score (PAS) was defined with respect to profusion of abnormalities. Chest X-rays were also scored. RESULTS: All of ten patients with PAM had findings of interstitial lung disease in varying degrees on their HRCTs. HRCT findings of patients with PAM were as following: MN, parenchymal bands (PB), ground glass opacity (GGO) and, sub pleural interstitial thickening (SPIT) in 10 patients; interlobular septal thickening (ILST), in 9 patients; paraseptal emphysema (PSA) in 8 patients; centrilobular emphysema (CLA) in 7 patients; bronchiectasis (BE), confluent micro nodules (CMN) in 6 patients; peri bronchovascular interstitial thickening (PBIT) in 5 patients; panacinar emphysema (PANAA) in 3 patients; pleural calcification (PC) in 2 patients. A significant correlation between MN scores and PAS (r=0.68, p=0.031, MN scores and GGO scores (r=0.69, p=0.027) and, MN scores and CLA scores (r=0.67, p=0.034) was detected. We also found significant correlations between HRCT scores and results of pulmonary function tests (PFTs), HRCT scores and chest X-ray score (CXRS) and, CXRS and results of PFTs. CONCLUSION: We conclude that patients with PAM may have all findings of interstitial lung disease in varying degrees as well as MNs on their HRCTs. More importantly, this study suggests a proportional relationship between profusion of MNs and parenchymal alterations in patients with PAM. This study also suggests that the degree of parenchymal alterations closely related with the degree of pulmonary function loss in patients with PAM.

Adult↗

PubMed2004

Adsorption of bilayers and multilayers of cationic and anionic co-polymers of acrylamide on silicon oxide.

Silica surfaces were consecutively treated with copolymers of cationic and anionic polyacrylamides (C-PAM and A-PAM, respectively) and the layer-by-layer build-up was continuously monitored with the aid of stagnation point adsorption reflectometry (SPAR). Four different charge densities of the cationic polymer and one charge density of the anionic polymer were studied. The solid substrate used in the investigation was an oxidized Si wafer, the charge of which was varied by performing the measurements at different pH. Adsorption measurements were performed both in deionized water and with a background electrolyte concentration of 0.01 M NaCl. The results show that the adsorption of C-PAM at pH 6 was dominated by electrostatic interactions. However, a significant nonionic contribution to the adsorption of C-PAM on SiO(2) was detected-when the results of adsorption measurements conducted in deionized water and in 0.01 M NaCl were compared. At pH 9, the adsorption of C-PAM onto SiO(2) was found to be geometrically restricted since the adsorption stoichiometry between the polymer charges and the charges on the surface was less than 1 irrespective of the charge of the C-PAM. Adsorption of the A-PAM onto the C-PAM covered surface increases as a function of the adsorbed charges in the first layer. Experiments showed that it was possible to form multilayers of polyelectrolytes on the SiO(2) surface provided the charge of the C-PAM was high enough. The critical charge of the polyelectrolyte for the formation of multilayers was also dependent on the charge of the substrate; that is, the lower the surface charge the higher the critical charge of the C-PAM. The substrate affected the amount of polyelectrolyte adsorbed up to the fifth layer. For further layers there was almost a stoichiometric relationship between the charges of the polyelectrolytes in consecutive layers. Results from studies of the formed multilayers with a quartz crystal microbalance (QCM-D) indicated that there was a close correlation between energy dissipation into the multilayers and a decrease in the adsorption as detected with SPAR. This in turn indicates that a decrease in the reflectometer signal does not necessarily indicate a decrease in adsorption.

Journal Article↗

PubMed2002

In vitro cytokine release from rat type II pneumocytes and alveolar macrophages following exposure to JP-8 jet fuel in co-culture.

Alveolar type II epithelial cells (AIIE) and pulmonary alveolar macrophages (PAM) are involved in pulmonary toxicity of JP-8 jet fuel exposure. To further elucidate their inflammatory mechanisms, the effect(s) of JP-8 jet fuel on cytokine secretion were examined in a transformed rat AIIE cell line (RLE-6TN) culture alone, primary PAM (from Fischer 344 rats) culture alone, and the co-culture of AIIE and primary PAM. A series of JP-8 jet fuel concentrations (0-0.8 microg/ml), which may actually be encountered in alveolar space of lungs exposed in vivo, were placed in cell culture for 24 h. Cultured AIIE alone secreted spontaneously interleukin (IL)-1beta and -6 [below detectable limits for IL-10 and tumor necrosis factor-alpha (TNF-alpha)], whereas cultured PAM alone secreted IL-1beta, -10, and TNF-alpha, in a concentration-dependent manner. These data suggest that the release of cytokines, not only from PAM but also from AIIE cells, may contribute to JP-8 jet fuel-induced inflammatory response in the alveolar space. However, the co-cultures of AIIE and PAM showed no significant changes in IL-1beta, -6, and TNF-alpha at any JP-8 jet fuel concentration compared to control values. These cytokine levels in co-cultures of AIIE and PAM were inversely related to these of cultured AIIE or PAM alone. Interestingly, IL-10 levels in the co-culture system were concentration-dependently increased up to 1058% at JP-8 concentrations of 0.8 microg/ml, although under detectable limits in cultured AIIE alone and no significant concentration change in cultured PAM alone. It appears that PAM may possibly act via paracrine and/or autocrine pathways to signal AIIE cells to regulate cytokine release.

Animals↗

PubMed1990

Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) catalyzes the formation of alpha-amidated peptides from their glycine-extended precursors, thus playing a key role in the processing of peptide neurohormones. We now report that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-dealkylation. Thus, (4-nitrobenzyl)thioacetic acid is converted to the analogous sulfoxide, N-(4-nitrobenzyl)glycine is converted to 4-nitrobenzylamine and glyoxylate, and [(4-nitrobenzyl)oxy]acetic acid is converted to 4-nitrobenzyl alcohol and glyoxylate. All these new activities display the characteristics expected for the normal PAM-catalyzed reductive oxygenation pathway and produce an equimolar amount of glyoxylate together with the heteroatom-containing dealkylation products. The ester [(4-methoxybenzoyl)oxy]acetic acid is not a PAM substrate, but is instead a good competitive inhibitor (KI = 0.48 mM). In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition. Monoethyl fumarate is also a time-dependent inactivator of PAM. Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid. These compounds are the smallest amide substrates yet reported for PAM, and it is thus apparent that only the minimal structure of an acylglycine is required for PAM-catalyzed oxygenative amidation.

Alkenes↗

PubMed2001

Spectrophotometric determination of polyacrylamide in waters containing dissolved organic matter.

Using polyacrylamide (PAM) to reduce soil erosion in irrigated land has increased rapidly in recent years. A simple and reliable method to measure the PAM concentration in waters containing dissolved organic matter (DOM) is of great importance in assessing the fate and efficiency of PAM application. In this research, an analytical method to determine the PAM concentration of waters with correction for DOM interference was developed and tested. The method is based on a combination of determining the total concentration of amide groups by the N-bromination method (NBM) and determining the DOM content spectrophotometrically. The total concentration of amide groups of both PAM and DOM was determined by NBM at 570 nm. The DOM moiety, which is proportional to DOM concentration, was determined by spectrophotometry using a UV 254-nm wavelength. The actual PAM concentration of a water sample (soil extract containing PAM in this study) was obtained from NBM readings subtracted by the interferential DOM contribution using a correction curve. Analysis of PAM in two soil-water samples showed that the recoveries ranged from 94 to 100.3% for the 2 mg/L PAM sample and from 98.4 to 101.4% for the 10 mg/L PAM sample with various DOM concentrations. The coefficients of variation were <6% in all cases.

Acrylic Resins↗

PubMed1994

NN-dimethyl-1,4-phenylenediamine as an alternative reductant for peptidylglycine alpha-amidating mono-oxygenase catalysis.

C-terminal alpha-amidation is a structural feature essential to the biological activity of many peptide hormones. Peptidylglycine alpha-amidating mono-oxygenase (PAM; EC 1.14.17.3) catalyses conversion of glycine-extended peptide hormone precursors into their corresponding alpha-hydroxyglycine derivatives. This reaction is the first step in the C-terminal amidation process. We report here that in the presence of molecular O2, copper and PAM substrate, NN-dimethyl-1,4-phenylenediamine (DMPD) serves as the requisite electron donor for the mono-oxygenase, being oxidized in the process to a stable and highly chromophoric cation radical. By monitoring the rate of increase in absorbance at 515 nm, PAM activity can be easily followed. This provides a spectrophotometric assay for PAM, which represents the first continuous assay reported for this enzyme. DMPD-supported PAM-catalysed mono-oxygenation exhibits normal Michaelis-Menten kinetic behaviour. Steady-state kinetic studies established that both the ascorbate-supported and DMPD-supported PAM reactions exhibit apparent 'Ping Pong' kinetics. In addition, both electron donors give rise to similar pH profiles and identical inhibition patterns towards known competitive inhibitors of PAM. The stoichiometry between formation of the DMPD cation radical and the alpha-hydroxyglycine PAM product was determined to be 2:1, the value expected for a monooxygenase-catalysed reaction. The optimum pH for the DMPD-supported continuous PAM assay was found to be about 5.5. The major advantage of this assay over all previously reported methods is that it is continuous; thus accurate initial rates are easily obtained. Moreover, unlike previous assay methods, 125I-labelled or chromophorically modified substrates are not required. Kinetic parameters for a broad range of PAM substrates and inhibitors have been successfully obtained using this assay.

Amino Acid Sequence↗

PubMed1998

Evidence of high expression of peptidylglycine alpha-amidating monooxygenase in the rat uterus: estrogen regulation.

In the present study, high levels of peptidylglycine alpha-amidating monooxygenase (PAM), which catalyzes the two-step formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in the uterus. Expression of PAM was evaluated in the uterus of intact cycling adult female rats and after experimental manipulation of the estrogen status of the rats. During the estrous cycle, PAM mRNA levels exhibited striking changes inversely related to the physiological variations of plasma estrogen levels. The levels of PAM transcripts changed markedly during the estrous cycle, reaching the highest levels at metestrus. There was a 15-fold increase in the abundance of PAM mRNA between metestrus and proestrus. Chronic treatment of ovariectomized rats with 17beta-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. Progesterone was without effect on PAM mRNA levels, indicating that the effect was specific for estradiol. In situ hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM. High levels of PAM mRNA were localized in the endometrium at the level of luminal and glandular cells. A weak signal was observed in stromal cells, and the myometrium cells were negative. 17beta-Estradiol treatment induced an overall decrease of the hybridization signal, as compared with ovariectomized rats. These results demonstrate the presence of high levels of PAM in the uterus and indicate that estrogens are involved in regulating the expression of the enzyme in this tissue. However, the present study provides no information regarding whether this regulation takes place at the level of transcription or influences mRNA stability.

Animals↗

PubMed1980

Kinetics of interaction and fate of Pasteurella hemolytica in bovine alveolar macrophages.

To study the role of pulmonary alveolar macrophages (PAMs) in phagocytizing Pasteurella hemolytica, we developed an in vitro cultivation method for preparing them. This procedure provided an adherent monolayer of PAMs which were nonspecific esterase-positive and phagocytized latex beads. The phagocytosis and fate of P. hemolytica (biotype A, serotype 1) by PAMs in suspension were studied. The kinetics of phagocytosis were determined by quantitatively measuring the uptake of 24-h [(3)H]thymidine-labeled bacteria by the PAMs in the presence of opsonins. Results showed that the uptake of P. hemolytica was enhanced in the presence of normal serum or antiserum. A total of 90% of the bacteria were phagocytized in the presence of normal adult bovine serum, and up to 95% were phagocytized in the presence of an antiserum. These studies also showed that normal serum, but not fetal calf serum, contained heat-stable natural antibodies which readily initiated the opsonization of P. hemolytica. The heat-labile complement system was also involved in the opsonization. The fate of P. hemolytica inside the PAMs was investigated by transmission electron microscopy and by the viable plate count method. Approximately 90% of the normal serum- or antiserum-opsonized P. hemolytica were phagocytized by PAMs at a bacteria/PAM ratio of 20:1 and were completely degraded after 60 min of exposure. Prolonged incubation of this mixture of bacteria and PAMs resulted in cytotoxic changes and destruction of PAMs. At a low bacteria/PAM ratio (10:1 or less), there was phagocytosis and killing of bacteria but no cytotoxic changes on the PAMs. The exact mechanism which initiated this phenomenon was not demonstrated. Perhaps toxic substance(s) released by the excess unphagocytized bacteria caused the cytotoxic changes to the PAMs.

Animals↗

PubMed1979

Drug uptake into everted intestinal sacs. II. Inhibition of secretion by hypertonicity.

Mucosal hypertonicity, metabolic inhibitors, or absence of glucose and oxygen enhance mucosal-to-serosal influx of the cationic drug, pralidoxime (PAM), into sacs of everted rat jejunum in vitro. Conversely, efflux of PAM, which is twice the influx rate, is inhibited by mucosal hypertonicity or cyanide and iodoacetate. When sacs containing PAM, 0.87 mM, and glucose, 10 mM, were placed in identical drug- and sugar-containing mediums, the inside (serosal) concentration of PAM fell by over half in 120 min, whereas that of glucose more than doubled. Mucosal hypertonicity depressed PAM efflux and glucose influx regardless of serosal osmolarity. Although azide and mucosal hypertonicity each depressed glucose uptake and oxygen consumption while accelerating net PAM influx, azide more effectively depressed glucose and oxygen uptake, whereas hypertonicity caused greater acceleration of PAM uptake. Hypertonicity did not affect PAM binding to intestinal tissue. Varying mucosal pH did not change PAM or glucose uptake. Thus, mucosal hypertonicity apparently enhances net mucosal-to-serosal transfer of PAM by blocking its active secretion from serosa to mucosa.

Animals↗

PubMed1990

Immunocytochemical and in situ hybridization studies of peptidylglycine alpha-amidating monooxygenase in pituitary gland.

The pituitary gland contains high levels of peptidylglycine alpha-amidating monooxygenase (PAM) activity and mRNA. Using affinity-purified rabbit polyclonal antisera generated to synthetic PAM peptides and PAM RNA probes, immunocytochemical and in situ hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM in the adult male rat pituitary gland. PAM immunoreactivity was present at varying levels in nearly all of the anterior pituitary cells; one cell population stained intensely, while others stained moderately or weakly. These results correlated well with in situ hybridization studies that demonstrated high levels of PAM mRNA in a subpopulation comprising approximately 10-15% of the anterior pituitary cells. Based upon immunocytochemistry, intermediate pituitary lobe cells were divided into an intensely stained and a moderately stained group. PAM staining was also present in neural lobe fibers. Immunocytochemical staining of serial pituitary tissue sections for PAM and other pituitary hormones demonstrated that the anterior pituitary cells intensely stained for PAM represented a subpopulation of the gonadotropes. PAM was also identified at moderate levels in corticotropes and at lower levels in sommatotropes and lactotropes. These results suggest that many anterior pituitary cells are capable of producing amidated peptides along with their major peptide hormone.

Adrenocorticotropic Hormone↗

PubMed2000

Breeding stock-specific variation in peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid splicing in rat pituitary.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.

Alternative Splicing↗