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PubMed1978

Appraisal of the PAM cell effect as a diagnostic test for multiple sclerosis.

The selective reduction of PAM cell yield reported by Carp and associates in the presence of tissue from patients with multiple sclerosis (MS) has been attributed to replication in culture of a viral agent associated with MS (MSAA). We investigated the diagnostic potential of the PAM cell effect in MS and in optic neuritis (ON). Six serum and 7 CSF samples from 7 patients with MS, 3 serum and 3 CSF samples from 4 patients with idiopathic ON, and 4 sera from 4 patients with ON induced by ethambutal toxicity were tested. Blind counting showed no reduction in PAM cell yield in the MS group nor any significant difference between the two ON groups. Disturbing inconsistencies in PAM cell growth rates over time and between control flasks were demonstrated.

Cells, Cultured↗

PubMed1994

Purification and characterization of PAM-1, an integral membrane protein involved in peptide processing.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the two-step alpha-amidation of peptidylglycine intermediates. PAM-1, a Type I integral membrane protein, was solubilized from the membranes of stably transfected hEK-293 cells and purified to homogeneity by antibody affinity chromatography. Purified PAM-1 exhibits an acidic pH optimum and a lower maximal velocity than soluble bifunctional PAM. Limited tryptic digestion of this integral membrane protein releases monofunctional peptidylglycine alpha-hydroxylating monooxygenase, increasing its specific activity almost fourfold and shifting its pH optimum to coincide with the pH optimum of peptidyl-alpha-hydroxyglycine alpha-amidating lyase.

Animals↗

PubMed1993

Localization of the gene encoding peptidylglycine alpha-amidating monooxygenase (PAM) to human chromosome 5q14-5q21.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Southern blot analysis of human placental DNA demonstrated that PAM is encoded by a single gene. The chromosomal localization of the PAM gene was established using in situ hybridization. A 2.2-kb human PAM cDNA hybridized to human metaphase chromosomes revealed a significant clustering of silver grains over chromosome 5 bands q14-q21. The gene encoding another enzyme important in the post-translational processing of neuroendocrine precursors, prohormone convertase 1 (PC1), is localized in the same region (5q15-q21).

Blotting, Southern↗

PubMed1994

The promiscuous plasmids pIP501 and pAM beta 1 from gram-positive bacteria encode complementary resolution functions.

pIP501 and pAM beta 1 are promiscuous plasmids from gram-positive bacteria which carry structurally and genetically related replication functions (L. Jannière, A. Gruss and S.D. Ehrlich, 1993, in "Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics (A.L. Sonenshein, J.A. Hoch, and R. Losick, Eds.), pp. 625-644). We report here the sequence of a approximately 1 kb region located downstream of the pIP501 minimal replicon. This region is 82% homologous to the corresponding region of pAM beta 1 and encodes a resolution system which complements that of pAM beta 1. Additionally, we present evidence that the structural similarity between the two plasmids extends for further approximately 2 kb, through a region which may encode a topoisomerase protein in pAM beta 1.

Amino Acid Sequence↗

PubMed1996

Phosphotriesterase, pralidoxime-2-chloride (2-PAM) and eptastigmine treatments and their combinations in DFP intoxication.

The protective action of i.v. administered eptastigmine, an organophosphate hydrolase (phosphotriesterase), or pralidoxime-2-chloride (2-PAM) and their combination in acute diisopropylfluorophosphate (DFP) intoxication were evaluated in mice. The mice received the physostigmine derivative, eptastigmine (0.9 mg/kg body wt, i.v.), 10 min prior to the i.p. injection of DFP (1.8 mg/kg body wt). Phosphotriesterase (66 micromol/min x ml/g and 6 microg/g body wt) or 2-PAM (30 mg/kg body wt) were given i.v. 30 min after DFP. The animals also received atropine sc (37.5 mg/kg body wt) immediately after DFP. The cholinesterase (ChE) activities were not protected or reactivated by 2-PAM alone. The ChE activities in brain and plasma were protected by phosphotriesterase. Eptastigmine alone assisted the recovery of the brain ChE activities. Also the combination of eptastigmine-phosphotriesterase protected the brain enzymes. It did not, however, provide any additional protection compared with phosphotriesterase-treatment on its own. In brain, the combination of eptastigmine with 2-PAM resulted in partly restored enzyme activities 24 hr after DFP exposure. In plasma, eptastigmine did not prevent the inhibition of ChE by DFP. However, when it was combined with phosphotriesterase, it significantly promoted the recovery of plasma ChE activity. In lung and in erythrocytes, the various combinations of antidotes caused only minor changes in the ChE activities.

Animals↗

PubMed1983

Isolation of a DNA sequence related to several plasmids from Bacillus thuringiensis after a mating involving the Streptococcus faecalis plasmid pAM beta 1.

The transmissible plasmid pAM beta 1, which codes for resistance to erythromycin and lincomycin, was transferred from Streptococcus faecalis to several strains of Bacillus thuringiensis by a filter-mating process. Introduction of pAM beta 1 into the Emr transconjugant strains of B. thuringiensis was confirmed by Southern hybridisation using the 32P-labelled pAM beta 1 as a probe. In the B. thuringiensis transconjugant strains, used as donors, the beta plasmid conserved its ability to be transferred during intraspecific mating, with a frequency of 10(-4) per recipient cell. In addition, the transconjugant clones acted as donors of the erythromycin resistance marker and permitted the transfer of cryptic plasmids present in the B. thuringiensis (beta) strains used as donors. From a transconjugant clone of B. thuringiensis a hybrid plasmid resulting from an in vivo insertion into pAM beta 1 of a 3 Md DNA sequence was isolated. This 3 Md DNA molecule originated from a 54 Md plasmid of a kurstaki strain and is related to several plasmids found in different serotypes of B. thuringiensis.

Bacillus thuringiensis↗

PubMed1990

Enhancement of melphalan (L-PAM) toxicity by reductive metabolites of 1-methyl-2-nitroimidazole, a model nitroimidazole chemosensitizing agent.

Chemosensitization of bifunctional alkylators by misonidazole (MISO) and related nitroimidazoles in vitro has been shown to require hypoxic exposures. Presumably, reductive metabolism of the nitroimidazole under hypoxic conditions results in generation of a chemosensitizing intermediate(s) in a manner analogous to that described for the hypoxic toxicity of these compounds. In an attempt to identify these intermediates, we examined the ability of reductive metabolites of a model 2-nitroimidazole compound, 1-methyl-2-nitroimidazole (INO2), to enhance the toxicity of melphalan (t-PAM) in HT-29 human colon cancer cells. INO2 was a modest chemosensitizing agent, enhancing L-PAM only under hypoxic conditions. The 2-electron reduction product, 1-methyl-2-nitrosoimidazole (INO), was a potent chemosensitizer, enhancing L-PAM toxicity at micromolar concentrations under either aerobic or hypoxic conditions. In contrast, the 4- and 6-electron reduction products, 1-methyl-2-[hydroxylamino]imidazole and 1-methyl-2-aminoimidazole, respectively, failed to modify cell kill by L-PAM even at millimolar concentration. These results suggest that nitrosoimidazoles may be the active chemosensitizing species generated upon the reductive metabolism of nitroimidazoles.

Buthionine Sulfoximine↗

PubMed1987

Comparison of serum concentrations of the acetylcholinesterase oxime reactivators HI-6, obidoxime, and PAM to efficacy against sarin (isopropyl methylphosphonofluoridate) poisoning in rats.

A comparison of serum concentrations of the oximes HI-6 [1-(((4-aminocarbonyl)-pyridino)methoxy)methyl)-2(hydroxy imino)methyl- pyridinium dichloride], PAM [2-[hydroxyimino)methyl-1-methylpyridinium chloride], and obidoxime [1,1'-(oxybis(methylene]bis(4-((hydroxyimino) methyl)-pyridinium dichloride] to the efficacy against sarin (350 micrograms/kg; sc) lethality was evaluated in rats. The oximes were administered prophylactically by means of Alzet osmotic minipumps. Atropine (17.4 mg/kg; im) was administered immediately following sarin (350 micrograms/kg; sc) administration. At serum concentrations of 3.6, 3.6, and 3.3 micrograms/ml for HI-6, obidoxime, and PAM, respectively, the 24-hr mortality following sarin poisoning was 0, 90, and 20%. The serum oxime concentrations (ED50 values) for HI-6, obidoxime, and PAM against a 3 LD50 dose of sarin were 0.72, 9.05, and 2.56 micrograms/ml, respectively. HI-6 was determined to be the most efficacious oxime when combined with atropine against sarin poisoning followed in order by PAM and obidoxime.

Acetylcholinesterase↗

PubMed1982

Effects of PAM, proPAM, and DFP on behavior, thermoregulation, and brain AChE in rats.

The effects of pyridine-2 aldoxime methyl iodide (PAM), N-methyl-1,6-dihydro-pyridine-2-carbaldoxime hydrochloride (proPAM), and diisopropyl phosphorofluoridate (DFP) on performance of a conditioned avoidance response (CAR), body temperature, and in vivo acetylcholinesterase (AChE) activity in five brain regions in the rat were examined. Sublethal doses of DFP (1.5 to 2.5 mg/kg, IP) markedly degraded CAR performance. This effect was antagonized by 5 mg/kg, subcutaneously injected (SC) atropine. A 50 mg/kg, SC dose of PAM had no effect on the CAR, but an equal dose of proPAM caused a transient deterioration of performance. Given 10 min or 2 hr after DFP, 50 mg/kg proPAM initially exacerbated the behaviorally toxic effects of DFP. Neither PAM nor proPAM antagonized DFP-induced hypothermia. PAM did not reactivate DFP-inhibited brain AChE, and proPAM reactivated it by only 6 to 12% of control activity.

Acetylcholinesterase↗

PubMed2006

Plasma peptidylglycine alpha-amidating monooxygenase (PAM) and ceruloplasmin are affected by age and copper status in rats and mice.

In an attempt to identify a sensitive and improved marker of mammalian copper status during neonatal development experiments compared two plasma cuproenzymes, peptidylglycine alpha-amidating monooxygenase (PAM ), an enzyme involved in peptide posttranslational activation, to ceruloplasmin (Cp), a ferroxidase involved in iron mobilization. Dietary Cu deficiency (Cu-) was studied in dams and offspring at postnatal age 3 (P3), P12, and P28. Rodent Cp activity rose during lactation whereas PAM activity fell. Reduction in Cp activity was more severe than reduction in PAM activity in Cu- offspring and dams. Cp activity was greater in rats than mice whereas PAM activity was similar in adults but greater in mouse than rat pups. Both cuproenzymes changed during neonatal development and when dietary copper was limiting. With proper controls, each enzyme can be used to assess copper status.

Age Factors↗

PubMed2005

The PAM-RL ambulatory device for detection of periodic leg movements: a validation study.

BACKGROUND AND PURPOSE: Restless legs syndrome (RLS) is usually associated with periodic leg movements (PLM) occurring during wakefulness and sleep. The PLM index obtained by the polysomnographic method reflects the degree of motor symptoms and their consequences on sleep structure. Automated analysis of PLM using actigraphy can assess this condition and can therefore be used to assess therapeutic effects in clinical trials. In the current study we assessed the reliability of the PAM-RL, an ambulatory device measuring limb movements and PLM with a high-time resolution. PATIENTS AND METHODS: Forty-three patients consecutively referred to the sleep laboratory for insomnia and/or excessive daytime sleepiness underwent one or two nights of polysomnography (PSG) with simultaneous bilateral recording of limb activity by the PAM-RL device. The PSG recordings were blinded and manually analyzed for PLM, while limb actimetry was scored automatically based on the manufacturer's algorithm. RESULTS: There was a significant correlation between PLM derived from PSG and actimetry (r=0.87, P<0.0001) with good agreement across a wide range of values. The sensitivity and specificity of the PAM-RL device in detecting patients having a polysomnographic PLM index >10 were, respectively, 0.88 and 0.76 with a receiving operating curve having an area under the curve (AUC) of 0.86 for the entire group of patients. All patients with clinically definitive RLS and primary PLM disorder (PLMD) had a PLM index >10 on PSG, but among patients with sleep-related breathing disorders (SRBD) 60% reached this cut-off value. Conversely, only 50% of those patients with an actigraphically assessed PLM index >10 had clinically definitive RLS or PLMD, and 40% had SRBD. CONCLUSIONS: We demonstrate that automatic detection of PLM derived from the PAM-RL device is highly reliable when compared to the 'gold standard' of polysomnography in patients with RLS and PLMD. Therefore, limb actigraphy can be used routinely to assess motor restlessness in patients with RLS and PLMD. The higher discrepancy in patients with SRBD and insomnia may preclude the use of the device in these patients.

Adult↗

PubMed2001

Adaptation of a PAM-fluorometer for remote sensing of chlorophyll fluorescence.

The Laser-PAM described in this paper is an adaptation of the PAM 101 fluorometer (Heinz Walz, Effeltrich, Germany) designed for remote sensing and non-invasive laboratory measurements of chlorophyll fluorescence. It is based on a 5 mW laser diode, emitting at 638 nm, and a Fresnel lens coupled to the ED-101 detection unit. Due to these modifications, measurements can be performed at a distance ranging from 0.3 to 2 m. The ED-101 detection unit has been modified to perform simultaneous measurements of both modulated fluorescence and light reflected by the leaf. Reflected light showed a good estimation of the photosynthetically active radiation measured exactly at the same area as the fluorescence. A particular advantage of the Laser-PAM fluorometer is its suitability for remote measurements under field conditions. Simultaneous fluorescence and gas-exchange measurements, performed on grapevine leaves, are reported as an example of an application for the Laser-PAM.

Journal Article↗

PubMed2000

An internal histidine residue from the bacterial surface protein, PAM, mediates its binding to the kringle-2 domain of human plasminogen.

The determinants of binding of a peptide lacking C-termini-exposed lysine residues to a kringle domain were investigated using an up-regulated lysine binding kringle (K2Pg[C4G/E56D/K72Y]) of plasminogen and a peptide (a1-PAM) with a sequence derived from a surface-exposed M-like streptococcal protein. Significant kringle-induced chemical shifts in a His side-chain of a1-PAM were revealed by two-dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His12 in the peptide/ protein complex. In an effort to screen a1-PAM-derived truncation peptides, a combinatorial mixture, a1deltaa2-PAM[H12X] (where X=Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2Pg[C4G/ E56D/K72Y]-containing chip was that containing His12, corresponding to the wild-type sequence. Minor peaks, representing binding, were obtained for Lys12-, Arg12- and Trp12-containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI-based screen.

Bacterial Proteins↗

PubMed1984

Effect of PAM-2 Cl, HI-6, and HGG-12 in poisoning by tabun and its thiocholine-like analog in the rat.

It has been shown that HI-6 was the most potent oxime so far known in poisoning by sarin, VX , and soman, but its protective effect in tabun poisoning, allegedly due to poor reactivation of inhibited ChE, was much less pronounced. We have found that the thiocholine-like analog of tabun , O-ethyl, N-N- dimethyamino -S-(2-diethylaminoethyl)- thiophosphatemethylsul fomethylate (Ta-S-N+), was very useful in resolving this problem and established the relationship between reactivating and protective effects of PAM-2 Cl, HI-6, and HGG-12 in rats. PAM-2 Cl (protective ratio (PR) = 22.1) and HI-6 (PR = 24.8), combined with atropine, were very effective against Ta-S-N+ poisoning and reactivating inhibited RBC AChE in vitro and rat blood ChE in vivo. The inefficiency of PAM-2 Cl (PR = 1.6) and HI-6 (PR = 2) in tabun poisoning was due to their inadequacy to reactive tabun -inhibited ChEs . The protective effects of HGG-12 in tabun (PR = 2.8) and Ta-S-N+ poisoning (PR = 2.6) were low, and in the absence of any reactivation of inhibited ChEs , have been attributed to its direct pharmacological effects, which were much more potent in the comparison with PAM-2 Cl or HI-6. It is concluded that the reactivation of inhibited ChE is of decisive importance in the efficient protection in poisoning by tabun and other known chemical warfare nerve agents, whereas their direct pharmacological effects are of limited value, allowing survival of animals only against a few LD50s .

Animals↗

PubMed1996

Transfer of conjugative plasmid pAM beta 1 from Lactococcus lactis to mouse intestinal bacteria.

Conjugal transfer of plasmid pAM beta 1 from Lactococcus lactis to intestinal bacteria of BALB/c mice was studied. Plasmid transfer was observed to Enterococcus faecalis in vitro by a filter mating method with transfer frequencies of 2.3 x 10-3 and with lower frequencies to other species. In vivo, using gastric intubation with the pAM beta 1-bearing Lactococcus lactis as donor and Ent. faecalis as recipient, a few transconjugants were detected from faecal Ent. faecalis. However, when these mice were given erythromycin through drinking water, a large number of conjugated Ent. faecalis were detected in faeces. Plasmid transfer to Ent. faecalis occurred at high frequency, 1.2 x 10-3, in mice whose anus was artificially closed after gastric intubation with pAM beta 1-bearing Lactococcus lactis. These results demonstrate clearly that pAM beta 1 transfer occurs between Gram-positive bacteria in the gut of mice harbouring many species of bacteria.

Animals↗

PubMed1991

Introduction of Tn916 and pAM beta 1 into Streptococcus bovis JB1 by conjugation.

The transposon Tn916 and self-mobilizing plasmid pAM beta 1 were conjugated from Enterococcus faecalis to the ruminal bacterium Streptococcus bovis JB1. Transconjugants were identified by resistance to tetracycline (Tn916) or erythromycin (pAM beta 1) and by Southern hybridization analyses. Transfer frequencies were 7.0 x 10(-6) and 1.0 x 10(-6) per recipient cell for Tn916 and pAM beta 1, respectively. The transconjugants JB1/Tn916 and JB1/pAM beta 1 were used as donors for matings with E. faecalis, Bacillus subtilis, and the ruminal bacterium Butyrivibrio fibrisolvens. While pAM beta 1 was successfully transferred to all three organisms, Tn916 was transferred only into B. subtilis and B. fibrisolvens at very low frequencies. This is the first report of conjugal DNA transfers between two ruminal organisms.

Conjugation, Genetic↗

PubMed1990

High-frequency, site-specific recombination between lactococcal and pAM beta 1 plasmid DNAs.

In vivo recombination events involving the 75-kilobase lactose proteinase plasmid pCI301 of Lactococcus lactis subsp. lactis UC317 and the conjugative enterococcal plasmid pAM beta 1 were analyzed. A fragment, identified as containing the pCI301 recombination site, mediated greatly elevated levels of mobilization and recombination with pAM beta 1 when cloned in a nonmobilizable L. lactis-Escherichia coli shuttle vector. This latter recombination event was site and orientation specific on both plasmids. Recombination on pAM beta 1 was within the region associated with plasmid replication, but no effect on pAM beta 1 replication functions was detected. Resolution of recombinant plasmids generated derivatives indistinguishable from the parental plasmids.

Conjugation, Genetic↗

PubMed2001

Aggressive atropinisation and continuous pralidoxime (2-PAM) infusion in patients with severe organophosphate poisoning: experience of a northwest Indian hospital.

OBJECTIVE: The aim of the study was to find whether continuous pralidoxime (2-PAM) infusion along with aggressive atropinisation improves the outcome in patients with severe organophosphate poisoning who require assisted ventilation. METHODS: Sixteen patients admitted to the respiratory intensive care unit (RICU) with severe organophosphate poisoning and requiring assisted ventilation were included in the study. The compounds involved were phorate (six), dichlorvos (four), oxydimeton methyl (one), monocrotophos (one), methyl parathion (one) and in three it was unknown. After decontamination, they were given intravenous (iv) bolus atropine 5 mg at onset and then 2.5 mg every 5-10 min till atropinisation was achieved, and then maintained either by intermittent bolus doses or by continuous infusion if the required dose was large. They were also given continuous iv infusion of 2-PAM in dose of 7.5 mg/ kg body weight/h (maximum 500 mg/h) after an initial bolus dose of 2 g. RESULTS: The mean (+/-S.D.) dose of atropine was 735.02 +/- 742.98 mg (range 85-3000 mg) with maximum dose on day 1. The mean (+/-S.D) duration of 2-PAM infusion was 96.4+/-49.4 h (range 10-216 h). The mean (+/-S.D) duration of mechanical ventilation (MV) was 131.5 +/- 95.65 h (range 4-336 h). Fourteen patients could be successfully extubated and two died of bronchopneumonia and sepsis (mortality 12.5%). CONCLUSION: Continuous 2-PAM infusion along with aggressive atropinisation after initial decontamination improved the outcome but not the duration of MV in severely intoxicated patients with organophosphate compounds who required assisted ventilation in this case series.

Adolescent↗