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PubMed2005

Adsorption of cationic polyacrylamide onto sepiolite.

The adsorption of PAM onto sepiolite from aqueous solutions has been investigated systematically as a function of some parameters such as calcination temperature of sepiolite, pH, ionic strength and temperature. The adsorption of cationic polyacrylamide (PAM) increases with pH from 5.50 to 11.00, temperature from 25 to 55 degrees C and ionic strength from 0 to 0.1molL(-1). The sepiolite sample calcined at 200 degrees C has a higher adsorption capacity than the other calcined samples. Adsorption isotherms of PAM onto sepiolite have been determined and correlated with common isotherm equations such as Langmuir and Freundlich isotherm models. The Langmuir isotherm model appeared to fit the isotherm data better than the Freundlich isotherm model. The physical properties of this adsorbent are consistent with the parameters obtained from the isotherm equations. The zeta potentials of sepiolite suspensions have been measured in aqueous solutions of NaCl and different PAM concentrations and pH. From the experimental results: (i) pH strongly alters the zeta potential of sepiolite, (ii) sepiolite has an isoelectric point at about pH 6.6 in water and about pH 8 in 250mgL(-1) PAM concentration, (iii) PAM changes the interface charge from negative to positive for sepiolite. Effect of temperature on adsorption has been quantified by calculating various thermodynamic parameters such as Gibbs free energy, enthalpy and entropy. The dimensionless separation factor (R(L)) has shown that sepiolite can be used for adsorption of PAM from aqueous solutions.

Acrylic Resins↗

PubMed1988

Cryotherapy for conjunctival primary acquired melanosis and malignant melanoma. Experience with 62 cases.

Sixty-two patients were treated by some combination of cryotherapy and surgery with an average follow-up of 3.3 years for one of the following diseases: focal or diffuse flat conjunctival primary acquired melanosis (PAM) with atypia but without a nodule of melanoma (10 cases); unifocal malignant melanoma with or without focal or diffuse PAM (30 cases); and multinodular/multicentric melanoma with and without PAM (22 cases). Of the ten patients who had PAM with atypia, invasive nodules of malignant melanoma did not develop. A second treatment was required to control the disease in four of the ten patients with extensive or diffuse lesions, and one has mild persistent disease. Of the 30 patients with unifocal nodules of malignant melanoma, 27 remained free of recurrence after one treatment, and 2 are asymptomatic after two treatments. One patient with a thick nodule at presentation required a parotidectomy and radical neck dissection for cervical metastases after recurrence in the conjunctival sac. In the group of 22 patients with multinodular malignant melanoma, only two did not have recurrent disease after one treatment. Of those who received multiple therapies, seven remained free of recurrence for at least 2 years after the last treatment; regional or distant metastases developed in nine; four required exenteration; and eight died. Conjunctival adjunctive cryotherapy avoids exenteration in extensive lesions of pure PAM and in unifocal melanoma, but even after multiple therapies, multinodular malignant melanoma had a 45% rate of metastasis. Metastasis was related to the presence of PAM sine pigmento in four patients (microscopically but not clinically detectable PAM); to the location of the nodules (9 of 10 patients who experienced metastases had forniceal, palpebral, and/or caruncular nodules); to the thickness or depth of invasion of the nodules (greater than 2 mm); and to the development of intralymphatic spread ("in-transit" local metastasis) within the conjunctival sac in six patients. No metastases were encountered among patients with strictly limbal nodules and among five patients with invasive nodules composed of spindle cells in part or in toto. Therapeutic success in this spectrum of melanocytic proliferations is closely correlated with the clinical extent of the disease when initiating definitive therapy.

Adult↗

PubMed1995

Potential vision tests in patients with cataracts.

PURPOSE: To determine the limits of usefulness of the potential acuity meter (PAM) and the laser interferometer (LI) in determining potential visual acuity in various severities and types of cataracts. METHODS: The study included 48 patients (67 eyes) who were undergoing cataract surgery and who had PAM and LI tests. The 67 eyes were grouped into (1) eyes with cataracts only (45 eyes) and (2) eyes with cataracts and other ocular diseases (22 eyes). Group 1 eyes were subdivided into two groups according to their Lens Opacities Classification System II (LOCS II) grading: 1a eyes (moderate cataracts) had an LOCS II grade of nuclear opalescence (NO) < 4 and cortical opacity (C) < 5 and posterior sub-capsularcataract (P) < 4, and group 1b eyes (severe cataracts) had an LOCS II grade of NO = 4 or C = 5 or P = 4. The accuracy of the PAM and LI was estimated separately for each group. RESULTS: For moderate cataracts, the accuracy of the PAM was 100% and of the LI was 92%. For severe cataracts, the accuracy of the PAM was 52% and of the LI was 79%. Both instruments tended to underestimate visual outcome more in eyes with posterior subcapsular cataracts. For eyes with other ocular diseases, the LI tends to overestimate the probable visual outcome more than the PAM, especially in patients with poor retinal function. CONCLUSIONS: Several factors such as preoperative visual acuity of 20/200 or worse, the type and severity of the cataract, and coexisting retinal disorders may affect the accuracy of the PAM and LI tests, and these should be considered when interpreting predicted vision, especially in preoperative counseling of patients.

Adult↗

PubMed1998

Conjunctival amelanotic malignant melanoma arising in primary acquired melanosis sine pigmento.

BACKGROUND: The authors describe an amelanotic malignant melanoma of the conjunctiva in association with primary acquired melanosis (PAM) sine pigmento, and highlight the clinical and pathologic features of this rare entity. METHODS: Histopathologic and immunohistochemical studies were performed on a conjunctival tumor in a 54-year-old white woman. STUDY DESIGN: Case report. RESULTS: Histopathologic examination revealed an invasive amelanotic melanoma of the conjunctiva, with anterior orbital extension arising from intraepithelial dysplastic melanocytes that lacked melanin pigment (PAM sine pigmento). Both the malignant melanoma cells and the intraepithelial dysplastic melanocytes in the areas of PAM exhibited S-100 and HMB-45 positivity. The patient underwent an orbital exenteration that disclosed tumor within the anterior orbit inferiorly. CONCLUSIONS: Amelanotic invasive malignant melanoma can arise in association with PAM sine pigmento, as seen in our patient who had orbital invasion necessitating exenteration. This aggressive form of conjunctival melanoma is often associated with a poor prognosis and risk of metastatic disease. Absence of conjunctival pigmentation in PAM sine pigmento prevents early clinical detection of this variant of PAM. This lack of pigmentation also makes clinical diagnosis virtually impossible, and diagnosis can only be established histopathologically. Awareness of this nonpigmented variety of PAM is crucial for early recognition and appropriate management of the associated melanoma.

Antigens, Neoplasm↗

PubMed2003

Characterization of monoaminergic systems in brain regions of prematurely ageing mice.

We have previously shown that differences in life span among members of Swiss mouse populations appear to be related to their exploration of a T-maze, with a slow exploration ("slow mice") being linked to increased levels of emotionality/anxiety, an impaired immune function and a shorter life span. Thus, we proposed the slow mice as prematurely ageing mice (PAM). We have now compared the monoaminergic systems of the PAM and of the non-prematurely ageing mice (NPAM), in discrete brain regions. PAM had decreased noradrenaline (NA) levels in all the brain regions analysed, whereas the 3-methoxy-4-hydroxyphenyl glycol (MHPG)/NA ratios were not significantly modified. PAM also showed decreased serotonine (5-HT) levels in hypothalamus, striatum and midbrain, as well as increased 5-hydroxyindol-3-acetic acid (5-HIAA)/5-HT ratios in hypothalamus and hippocampus. The dopamine (DA) content was lower in PAM in most regions, whereas the 3,4-dihydroxyphenylacetic acid (DOPAC)/DA and homovanillic acid (HVA)/DA ratios were either increased or unchanged depending on the region analysed. In most cases, the differences between PAM and NPAM involved both sexes. One exception was the hypothalamus where the differences only affected the male mice. The neurochemical alterations found in PAM resemble some changes reported for aged animals and are related with their behavioural features.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2000

Influence of polyacrylamide application to soil on movement of microorganisms in runoff water.

Polyacrylamide (PAM) use in irrigation for erosion control has increased water infiltration and reduced soil erosion. This has improved runoff water quality via lower concentrations of nitrogen, phosphorous, and pesticides, and decreased biological oxygen demand. Since non-toxic high molecular weight anionic PAMs removed clay size sediment particles in flowing water, we hypothesized that PAM would effectively remove or immobilize microorganisms in flowing water. In an agricultural field, we determined the efficacy of PAM-treatment of furrow irrigation water to remove several categories of microorganisms in the inflow and runoff. Treatments were: (1) PAM application and a control; (2) three flow rates; (3) two distances from the inflow point; and (4) three times during each irrigation. After water traveled 1 m at 7.5 and 15.5 l min(-1), PAM-treatment reduced total bacterial and microbial biomass and total fungal biomass relative to the control treatment. After water traveled 40 m at 7.5, 15.5, and 22.5 l min(-1), PAM-treatment reduced algae, the numbers active and total bacteria, active and total fungal length, and total bacterial biomass, total fungal and microbial biomass relative to the control treatment. Although specific organisms were not identified or monitored in this study, the results clearly have implications for controlling the spread of soil-borne plant pathogens and other classes of harmful organisms within and among fields via irrigation water and in re-utilized return flows. Beyond furrow-irrigated agriculture, new methods to manage overland transmission of harmful microorganisms could potentially help control transport of pathogens from animal waste in runoff and groundwater.

Journal Article↗

PubMed2003

Direct voltammetry and electrocatalytic properties of catalase incorporated in polyacrylamide hydrogel films.

The direct voltammetry and electrocatalytic properties of catalase (Cat) in polyacrylamide (PAM) hydrogel films cast on pyrolytic graphite (PG) electrodes were investigated. Cat-PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetry peaks for Cat Fe(III)/Fe(II) redox couples at approximately -0.46 V vs. SCE in pH 7.0 buffers. The electron transfer between catalase and PG electrodes was greatly facilitated in the microenvironment of PAM films. The apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') were estimated by fitting square wave voltammograms with non-linear regression analysis. The formal potential of Cat Fe(III)/Fe(II) couples in PAM films had a linear relationship with pH between pH 4.0 and 9.0 with a slope of -56 mV pH(-1), suggesting that one proton is coupled with single-electron transfer for each heme group of catalase in the electrode reaction. UV-Vis absorption spectroscopy demonstrated that catalase retained a near native conformation in PAM films at medium pH. The embedded catalase in PAM films showed the electrocatalytic activity toward dioxygen and hydrogen peroxide. Possible mechanism of catalytic reduction of H(2)O(2) at Cat-PAM film electrodes was proposed.

Acrylic Resins↗

PubMed1998

Potential acuity meter versus scanning laser ophthalmoscope to predict visual acuity in cataract patients.

PURPOSE: To compare the relative abilities of the Potential Acuity Meter (PAM) and the Scanning Laser Ophthalmoscope (SLO) to predict the postoperative visual acuity in patients with mild to dense cataract. SETTING: Gimbel Eye Centre, Calgary, Alberta, Canada. METHODS: In this single-masked prospective study of 31 consecutive eyes with mild to dense cataract and a best corrected visual acuity (BCVA) of 20/40 or less, patients had predictive visual acuity (PVA) testing using the PAM and SLO. Preoperative and postoperative BCVA was recorded using Snellen charts. Differences between PVA and postoperative BCVA results were assessed by a two-tailed t-test and correlation analysis. RESULTS: The SLO was correct within two lines of outcome in 30 of 31 eyes and the PAM, in 19 of 31 eyes. A two-tailed dependent t-test showed a significant difference between predicted and final visual acuity for the PAM (P = .0001) and a nonsignificant difference for the predicted and final visual acuity for the SLO. A correlation analysis showed a mild correlation for the PAM (r = .49) and a good correlation for the SLO (r = .81). For a prediction of better or worse than 20/40, the SLO showed a sensitivity of 0.964 and specificity of 1.0 and the PAM, of 0.67 and 1.0, respectively. CONCLUSION: The SLO was more accurate than the PAM in predicting visual acuity after cataract surgery. The relative cost of the SLO is potentially restrictive to its widespread clinical application. However, the cost/benefit in terms of patient satisfaction with predictable surgical results should be considered.

Aged↗

PubMed2003

Expression and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase.

We report the purification and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase (the bifunctional PAM) expressed in Chinese hamster ovary cells. PAM is in charge of the formation of the C-terminal amides of biologically active peptides. The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5). By introducing a stop codon at 835 Glu, we were able to eliminate the membrane-spanning domain in the C-terminal region and succeeded in purifying a soluble form of bifunctional PAM that was secreted into the medium. Through a three-step purification procedure, we obtained 0.3mg of the purified PAM, which showed a single band at 91 kDa on SDS-PAGE, from 1L of monolayer culture medium. Metals contained in the purified PAM were analyzed and chemical modifications were performed to gain insight into the mechanism of the PAL reaction. Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM. Further, the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+). In a series of chemical modifications, phenylglyoxal almost completely eliminated the PAL activity and diethyl pyrocarbonate suppressed activity by more than 70%. These findings implied that Arg and His residues might play crucial roles during catalysis.

Amidine-Lyases↗

PubMedDate not supplied

Synthesis of thiol-reactive lipopeptide adjuvants. Incorporation into liposomes and study of their mitogenic effect on mouse splenocytes.

Synthetic analogues of triacylated and diacylated lipopeptides derived from the N-terminal domain of respectively bacterial and mycoplasmal lipoproteins are highly potent immunoadjuvants when administered either in combination with protein antigens or covalently linked to small peptide epitopes. Because of their amphipathic properties, lipopeptides, such as S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-glycine (Pam(3)CAG), can be conveniently incorporated into liposomes and serve as anchors for antigens that are linked to them. To design vaccination constructs based on synthetic peptides and liposomes as vectors. we have accordingly synthesized a series of lipopeptides that differ by the number (Pam(3)C vs Pam(2)C) and nature of the acyl chains (palmitoyl vs oleoyl) and by the presence at their C-terminus of thiol-reactive functions, such as maleimide or bromoacetyl. When incorporated into liposomes, these latter functionalized lipopeptides allow, in aqueous media, a well controlled chemoselective conjugation of HS-peptides to the surface of the vesicles. Using a BALB/c mice splenocyte proliferation assay ([(3)H]thymidine incorporation), we have measured the lymphocyte activation potency of the different lipopeptides. We found that, compared to their free (emulsified) forms, the liposomal lipopeptides were endowed with enhanced mitogenic activities; i.e., up to 2 orders of magnitude for Pam(3)CAG which was more potent than Pam(2)CAG. The impact of functionalization on the cellular activity of Pam(3)CAG was dependent on the thiol-reactive group introduced: whereas the bromoacetyl derivative retained its full activity, the presence of a maleimide group virtually abolished the lymphocyte activation of the lipopeptide. Finally, the substitution of saturated palmitoyl chains by unsaturated oleoyl chains was inhibitory. Thus, thiol-reactive Ol(3)CAG derivatives were the least active mitogens in our assay. Taken together, our findings are of importance for the further optimization of antigen-specific liposomal-based synthetic vaccines; the bromoacetyl derivative of Pam(3)CAG should be a promising lipopeptide derivative serving as an anchor for peptide epitopes while retaining its lymphocyte activation activity.

Adjuvants, Immunologic↗

PubMed1991

Functional and structural characterization of peptidylamidoglycolate lyase, the enzyme catalyzing the second step in peptide amidation.

Carboxy-terminal amidation is a prevalent posttranslational modification necessary for the bioactivity of many neurohormonal peptides. We recently reported that in addition to peptidylglycine alpha-monooxygenase (PAM), a second enzyme, which we now call peptidylamidoglycolate lyase (PGL), functions in the enzymatic formation of amides [Katopodis et al. (1990) Biochemistry 29, 4551]. The monooxygenase first catalyzes formation of the alpha-hydroxyglycine derivative of the glycine-extended precursor, and the lyase subsequently catalyzes breakdown of the PAM product to the amidated peptide and glyoxylate. We report here the first primary sequence data for PGL, which establish that it is part of the putative protein precursor which also contains PAM. We also show that PAM and PGL activities are colocalized in the secretory granular fraction of neurointermediate pituitary as would be expected for enzymes sharing the same precursor. Time course studies of the amidation reaction using purified soluble pituitary PAM and PGL indicate that both enzymes are essential for enzymatic amidation. Finally, PGL has no effect on the substrate or inhibition kinetics of PAM, and purified pituitary PAM has an acidic pH optimum consistent with its known localization in secretory granules.

Amidine-Lyases↗

PubMed1990

Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-A resolution and neutral pH.

The T----R transition of the cooperative enzyme aspartate carbamoyltransferase occurs at pH 7 in single crystals without visibly cracking many of the crystals and leaving those uncracked suitable for single-crystal X-ray analysis. To promote the T----R transition, we employ the competitive inhibitors of carbamoyl phosphate and aspartate, which are phosphonoacetamide (PAM) and malonate, respectively. In response to PAM binding to the T-state crystals, residues Thr 53-Thr 55 and Pro 266-Pro 268 move to their R-state positions to bind to the phosphonate and amino group of PAM. These changes induce a conformation that can bind tightly the aspartate analogue malonate, which thereby effects the allosteric transition. We prove this by showing that PAM-ligated T-state crystals (Tpam), space group P321 (a = 122.2 A, c = 142.2 A), when transferred to a solution containing 20 mM PAM and 8 mM malonate at pH 7, isomerize to R-state crystals (Rpam,mal,soak), space group also P321 (a = 122.2 A, c = 156.4 A). The R-state structure in which the T----R transition occurs within the crystal at pH 7 compares very well (rms = 0.19 A for all atoms) with an R-state structure determined at pH 7 in which the crystals were initially grown in a solution of PAM and malonate at pH 5.9 and subsequently transferred to a buffer containing the ligands at pH 7 (Rpam,mal,crys). In fact, both of the PAM and malonate ligated R-state structures are very similar to both the carbamoyl phosphate and succinate or the N-(phosphonoacetyl)-L-aspartate ligated structures, even though the R-state structures reported here were determined at pH 7. Crystallographic residuals refined to 0.16-0.18 at 2.8-A resolution for the three structures.

Amino Acid Sequence↗

PubMed1990

A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine alpha-amidating monooxygenase in peptide amidation.

We report here the isolation of a novel enzyme from bovine neurointermediate pituitary which catalyzes the conversion of alpha-hydroxybenzoylglycine to benzamide. This enzyme, termed HGAD (alpha-hydroxyglycine amidating dealkylase), is a soluble protein with an apparent molecular mass of 45 kDa and no apparent cofactor requirement. Addition of HGAD to purified neurointermediate pituitary PAM (peptidylglycine alpha-amidating monooxygenase, EC 1.14.17.3) increases the rate of formation of amide products by an order of magnitude. Sequential additions of PAM and HGAD gave results consistent with PAM first catalyzing the formation of an intermediate that is subsequently, in a separate reaction, converted by HGAD to the final amide product. Experiments with olefinic inactivators demonstrate that HGAD is not required for turnover-dependent inactivation of PAM and, correspondingly, that HGAD activity is not affected by inactivators of PAM. As expected, HGAD has no effect on the rate of PAM-catalyzed sulfoxidation, where a reaction analogous to that occurring during amidation of glycine-extended substrates is not possible. On the basis of these results, we propose that peptide C-terminal amidation in neurointermediate pituitary is a two-step process, with PAM first catalyzing the conversion of a glycine-extended peptide to the alpha-hydroxyglycine derivative, which is in turn converted to the final amide product by HGAD.

Alkylation↗

PubMed2000

Growth regulated oncogene-alpha expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration and angiogenesis by a host CXC receptor-2 dependent mechanism.

Growth Regulated Oncogene-alpha (GRO-alpha) is an autocrine growth factor in melanoma and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia through binding to CXC Receptor 2. We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo constitutively express increased levels of murine GRO-alpha, designated mGRO-alpha, or KC. We have examined the possible role of mGRO-alpha expression in malignant progression of squamous cell carcinoma PAM 212 in homologous BALB/c and BALB CXC Receptor-2 deficient mice. Transfection of the PAM 212 cell line which exhibits low expression of GRO-alpha and malignant potential with a pActin-KC vector encoding mGRO-alpha enabled isolation of PAM-KC expressing cell lines. These PAM-KC transfectants displayed an increased rate of growth and metastasis in BALB/c mice, similar to the highly malignant phenotype observed in spontaneously occurring metastatic variants. Furthermore, the PAM-KC tumors showed an increase in infiltration of host leukocytes and CD31+ blood vessels, consistent with increased CXC chemokine activity. The increased growth of PAM-KC cells was attenuated in CXCR-2 deficient mice, indicating that the increased growth was dependent in part upon host cells responsive to the CXC chemokine. Together, these results show that a CXC chemokine such as GRO-alpha can promote malignant growth of murine squamous cell carcinoma by a host CXCR-2 dependent pathway. Oncogene (2000) 19, 3477 - 3486

Animals↗

PubMed2001

Differential immunoreactivity of melanocytic lesions of the conjunctiva.

AIMS: To evaluate the expression of S100NKI/C3 and HMB45 antigens in melanocytic lesions of the conjunctiva and the ability of HMB45 to aid assessment of neoplasia. METHODS AND RESULTS: Stored formalin-fixed specimens of conjunctival melanomas and primary acquired melanosis were considered as participants and conjunctival naevi and racial melanosis as controls. Ninety-seven conjunctival melanocytic lesions were analysed using formalin-fixed paraffin-embedded material. These included 20 melanomas arising in the context of primary acquired melanosis (PAM), 22 melanomas arising without evidence of pre-existing PAM, seven cases of PAM with atypia, nine cases of PAM with no atypia, 35 conjunctival naevi and four cases of racial melanosis. S100 and NKI/C3 were similarly expressed in all lesions, with at least one of these markers positive in 100% of the lesions examined. HMB45 was expressed in 72.7% of primary melanomas and 85% of melanomas in the context of PAM; 42.8% of PAM with atypia expressed HMB45 while it was expressed in 11.1% of PAM without atypia and 8.5% of naevi. Racial melanosis cases did not express HMB45. S100 and NKI/C3 were expressed to a similar extent in all groups. CONCLUSIONS: S100 and NKI/C3 are useful markers to assess the extent of melanocytic lesions in the conjunctiva. HMB45 immunoreactivity can act as a useful aid to histopathology for the distinction of benign from malignant conjunctival lesions, particularly in the context of primary acquired melanosis.

Antigens, Neoplasm↗

PubMed1989

Alternative mRNA splicing generates multiple forms of peptidyl-glycine alpha-amidating monooxygenase in rat atrium.

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the conversion of a variety of glycine-extended peptides into biologically active alpha-amidated product peptides in a reaction dependent on copper, ascorbate, and molecular oxygen. We have isolated and sequenced cDNAs representing the two major classes of PAM mRNA in the adult rat heart atrium. The two types of cDNA, rPAM-1 and rPAM-2, are identical except for the deletion of a 315-base-pair segment within the protein coding region in rPAM-2, suggesting that rPAM-1 and rPAM-2 arise by alternative splicing. Northern analysis using a cDNA probe derived from within the 315-base-pair region deleted in rPAM-2 visualized the larger of the PAM mRNAs in adult rat atrium and not the smaller, indicating that the presence or absence of this 315-nucleotide segment is a major feature distinguishing the two size forms of PAM mRNA. The 105 amino acid segment that distinguishes the two forms of atrial PAM contains a consensus N-glycosylation site and a paired basic amino acid site of potential importance in endoproteolytic processing. Comparison of the nucleotide sequences of rat, frog, and bovine PAM cDNAs reveals an extremely well conserved segment in the 3' untranslated region. The high degree of conservation in amino acid sequence throughout the catalytic, intragranular, and cytoplasmic domains of rat atrium, bovine pituitary, and frog skin PAM suggests that both the catalytic and noncatalytic domains of the protein subserve important functions.

Amino Acid Sequence↗

PubMed1998

Identification of a large Myc-binding protein that contains RCC1-like repeats.

The protooncogene MYC plays an important role in the regulation of cellular proliferation, differentiation, and apoptosis and has been implicated in a variety of human tumors. MYC and the closely related MYCN encode highly conserved nuclear phosphoproteins (Myc and NMyc) that apparently function as transcription factors in the cell. We have identified a large and highly conserved nuclear protein that interacts directly with the transcriptional activating domain of Myc (designated "protein associated with Myc" or Pam). Pam contains an extended amino acid sequence with similarities to a protein known as regulator of chromosome condensation (RCC1), which may play a role in the function of chromatin. The gene encoding Pam (PAM) is expressed in all of the human tissue examined, but expression is exceptionally abundant in brain and thymus. Pam binds specifically to Myc, but not NMyc. The region in Myc required for binding to Pam includes a domain that is essential for the function of Myc and that is frequently mutated in Burkitt's lymphomas. PAM is located within a 300-kb region on chromosome 13q22.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Novel proteins that interact with the COOH-terminal cytosolic routing determinants of an integral membrane peptide-processing enzyme.

The steady state distribution of membrane forms of peptidylglycine alpha-amidating monooxygenase (PAM) in the secretory pathway of neurons and endocrine cells depends on signals in its cytosolic COOH-terminal domain (CD). Mutagenesis studies yielded catalytically active PAM proteins that are not properly localized or internalized. Employing the yeast two-hybrid system, we isolated two distinct cDNAs whose protein products showed a strong interaction with the CD of PAM. The interaction of these novel PAM COOH-terminal interactor proteins (P-CIPs) did not occur with a misrouted CD mutant as bait in the yeast system. Both proteins, P-CIP2 and P-CIP10, were expressed as fusion proteins that interacted in vitro with solubilized integral membrane PAM. P-CIP2 was homologous to several serine/threonine and dual specificity protein kinases, while P-CIP10 contained spectrin-like repeats. Endogenous P-CIP2 was localized to the Golgi region of AtT-20 corticotrope tumor cells, and expression of integral membrane PAM disrupted the distribution of endogenous P-CIP2. Both P-CIP2 and P-CIP10 mRNAs were found to be expressed in rat brain neurons also expressing PAM proteins. P-CIP2 and P-CIP10 may be members of a family of cytosolic proteins involved in the routing of membrane proteins that function in the regulated secretory pathway.

Amino Acid Sequence↗