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Combination of the new minor groove alkylator tallimustine and melphalan.

The benzoyl nitrogen mustard derivative of distamycin A, tallimustine, belongs to a new class of alkylating agents, known as DNA minor groove alkylating agents. It alkylates adenine N3 with high sequence specificity, causing no alkylation of guanine N7, the main site of alkylation of clinically used nitrogen mustards such as L-PAM. The present study investigated the in vivo antitumour activity of a combination of tallimustine and melphalan (L-PAM). Two murine tumours were used: i.p. (intraperitoneally) transplanted L1210 leukaemia and i.m. (intramuscularly) transplanted M5076 ovarian reticulum cell sarcoma (M5). In L1210, which is only marginally sensitive to tallimustine, the combination of tallimustine 3 mg/kg i.p. with L-PAM 10 mg/kg i.p. was as effective as 20 mg/kg L-PAM, which is the maximum tolerated dose. In M5, which is sensitive to both drugs, the combination was superior to either drug alone. The results suggest that the combination of tallimustine and L-PAM--or possibly in general, minor groove alkylators and major groove alkylators--may be therapeutically advantageous and therefore should be investigated clinically.

Animals↗

Organotypic slice cultures from rat brain tissue: a new approach for Naegleria fowleri CNS infection in vitro.

The free-living amoeba Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM), a disease leading to death in the vast majority of cases. In patients suffering from PAM, and in corresponding animal models, the brain undergoes a massive inflammatory response, followed by haemorrhage and severe tissue necrosis. Both, in vivo and in vitro models are currently being used to study PAM infection. However, animal models may pose ethical issues, are dependent upon availability of specific infrastructural facilities, and are time-consuming and costly. Conversely, cell cultures lack the complex organ-specific morphology found in vivo, and thus, findings obtained in vitro do not necessarily reflect the situation in vivo. The present study reports infection of organotypic slice cultures from rat brain with N. fowleri and compares the findings in this culture system with in vivo infection in a rat model of PAM, that proved complementary to that of mice. We found that brain morphology, as present in vivo, is well retained in organotypic slice cultures, and that infection time-course including tissue damage parallels the observations in vivo in the rat. Therefore, organotypic slice cultures from rat brain offer a new in vitro approach to study N. fowleri infection in the context of PAM.

Amebiasis↗

Antagonism of free-radical-induced damage of adlay seed and its antiproliferative effect in human histolytic lymphoma U937 monocytic cells.

The goal of our current research was to investigate the antioxidative effects of methanolic extracts from different parts of adlay seed and their antiproliferative activity in malignant human cells. The methanolic extracts from different parts of adlay seeds were from the hull (AHM), testa (ATM), bran (ABM), and polished adlay (PAM). AHM exhibited greater capacity to scavenge superoxide anion radicals in the PMS-NADH system than ATM, ABM, or PAM. The scavenging capacities of AHM and ATM on hydrogen peroxides were about 20% at a dose of 250 microg/mL. Using the method of deoxyribose degradation to assess damage caused by hydroxyl radicals, AHM was found to inhibit damage in deoxyribose at a higher concentration. However, ATM, ABM, and PAM exhibited prooxidative activity at the same concentration. The inhibitory effect on enzymatic oxidation of xanthine to uric acid was found to follow the order AHM > ATM =. ABM. However, PAM was inactive. All test samples were positive for inhibition of TPA-induced free radical formation on neutrophil-like leukocytes and were found to follow the order AHM > ATM > ABM > PAM. When human histolytic lymphoma U937 monocytic cells were exposed to tert-butyl hydroperoxide, AHM protected the cells against the cytotoxicity caused by tert-butyl hydroperoxide. In addition, AHM exhibited antiproliferative activity against human histolytic lymphoma U937 monocytic cells in a dose-dependent manner. The antiproliferative properties of AHM appear to be attributable to its induction of apoptotic cell death as determined by flow cytometry. These results show that AHM displays multiple antioxidant effects and induces apoptosis of malignant human cells.

Antineoplastic Agents↗

Improved delivery through biological membranes. 2. Distribution, excretion, and metabolism of N-methyl-1,6-dihydropyridine-2-carbaldoxime hydrochloride, a pro-drug of N-methylpyridinium-2-carbaldoxime chloride.

N-Methyl-1,6-dihydropyridine-2-carbaldoxime hydrochloride, the pro-drug of 2-PAM, was found to be converted in vivo to 2-PAM, rapidly and quantitatively. The significantly changed properties of the pro-2-PAM resulted in a longer biological half-life and a favorable distribution of 2-PAM formed upon its oxidation. No new metabolite was found when pro-2-PAM was administered intravenously; however, a new metabolic product was formed when the pro-drug was given by oral route.

Administration, Oral↗

The Personal Acquaintance Measure: a tool for appraising one's acquaintance with any person.

The authors developed and evaluated the psychometric properties of the 18-item Personal Acquaintance Measure (PAM) and investigated how the PAM relates to self- other agreement in personality ratings. Results support that 6 factors represent the PAM (Duration, Frequency of Interaction, Knowledge of Goals, Physical Intimacy, Self-Disclosure, Social Network Familiarity), which showed evidence of internal consistency, test-retest reliability over 3 weeks, sensitivity to known group differences, discriminant validity from socially desirable responding, and convergent validity with other relationship inventories. Results also show that the PAM positively predicted self-other agreement. The authors discuss the implications of these findings for the PAM and research in person perception, although this measure may also be used in other research areas.

Adult↗

Hyperplasia of alveolar neuroendocrine cells in rat lung carcinogenesis by silica with selective expression of proadrenomedullin-derived peptides and amidating enzymes.

Pulmonary neuroendocrine (NE) cells are found as clusters called neuroepithelial bodies (NEBs) or as single cells scattered in the respiratory epithelium. They express a variety of bioactive peptides, and they are thought to be the origin of NE lung tumors. Proadrenomedullin N-terminal 20 peptide (PAMP) is a peptide derived from the same precursor as adrenomedullin (AM). AM and PAMP are C-terminally amidated during their processing by a well-characterized amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). We explored AM, PAMP, and PAM expression as markers for NE hyperplasia in three rodent species (Fischer 344 rats, Syrian golden hamsters, and A/J mice) after a single intratracheal instillation of crystalline silica (quartz), which was previously found to induce different reactions in the three species. Rats developed a marked silicosis, with alveolar and bronchiolar hyperplasia and formation of peripheral lung epithelial tumors. Mice developed a moderate degree of silicosis, but not epithelial hyperplasia or tumors. Hamsters showed dust-storage lesions, but not silicosis or tumors. NE cells were immunolabeled for calcitonin gene-related peptide (CGRP), AM, PAMP, and PAM in serial sections of each lung. The numbers of positive NEBs per lung area and positive cells per NEB were quantified. A marked hyperplastic reaction in the NEBs of silica treated rats occurred only in alveolar NEBs, but not in bronchiolar NEBs. From Month 11 onwards, there were marked differences in the number of alveolar NEBs per section and in the number of cells per alveolar NEB immunoreactive for CGRP. No hyperplastic NE cell reaction was observed in silica-treated mice and hamsters. Significant PAMP and PAM expression was seen only in rat hyperplastic alveolar and in bronchiolar NEBs from Month 11 onwards. In E18, rat fetal lung NEBs were found to be strongly positive for PAMP and PAM.

Adrenomedullin↗

An optical sectioning programmable array microscope implemented with a digital micromirror device.

The defining feature of a programmable array microscope (PAM) is the presence of a spatial light modulator in the image plane. A spatial light modulator used singly or as a matched pair for both illumination and detection can be used to generate an optical section. Under most conditions, the basic optical properties of an optically sectioning PAM are similar to those of rotating Nipkow discs. The method of pattern generation, however, is fundamentally different and allows arbitrary illumination patterns to be generated under programmable control, and sectioning strategies to be changed rapidly in response to specific experimental conditions. We report the features of a PAM incorporating a digital micromirror device, including the axial sectioning response to fluorescent thin films and the imaging of biological specimens. Three axial sectioning strategies were compared: line scans, dot lattice scans and pseudo-random sequence scans. The three strategies varied widely in light throughput, sectioning strength and robustness when used on real biological samples. The axial response to thin fluorescent films demonstrated a consistent decrease in the full width at half maximum (FWHM), accompanied by an increase in offset, as the unit cells defining the patterns grew smaller. Experimental axial response curves represent the sum of the response from a given point of illumination and cross-talk from neighbouring points. Cross-talk is minimized in the plane of best focus and when measured together with the single point response produces a decrease in FWHM. In patterns having constant throughput, there appears to be tradeoff between the FWHM and the size of the offset. The PAM was compared to a confocal laser scanning microscope using biological samples. The PAM demonstrated higher signal levels and dynamic range despite a shorter acquisition time. It also revealed more structures in x-z sections and less intensity drop-off with scanning depth.

Adenocarcinoma↗

A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids.

Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e. mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy. Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane. This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol. As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast [Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F. & Daum, G. (1995) Biochim. Biophys. Acta 1234, 214-220], although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several-fold the activity in MAM and also in the bulk endoplasmic reticulum. In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and steroldelta24-methyltransferase (Erg6p), are highly enriched in PAM. A possible role of PAM in the supply of lipids to the plasma membrane is discussed.

Blotting, Western↗

Kalirin, a cytosolic protein with spectrin-like and GDP/GTP exchange factor-like domains that interacts with peptidylglycine alpha-amidating monooxygenase, an integral membrane peptide-processing enzyme.

Although the integral membrane proteins that catalyze steps in the biosynthesis of neuroendocrine peptides are known to contain routing information in their cytosolic domains, the proteins recognizing this routing information are not known. Using the yeast two-hybrid system, we previously identified P-CIP10 as a protein interacting with the cytosolic routing determinants of peptidylglycine alpha-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrin-like repeats and adjacent Dbl homology and pleckstrin homology domains typical of GDP/GTP exchange factors. In the adult rat, expression of P-CIP10 is most prevalent in the brain. Corticotrope tumor cells stably expressing P-CIP10 and PAM produce longer and more highly branched neuritic processes than nontransfected cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to selected members of the Rho subfamily of small GTP binding proteins (Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl family of proteins, may serve as part of a signal transduction system linking the catalytic domains of PAM in the lumen of the secretory pathway to cytosolic factors regulating the cytoskeleton and signal transduction pathways.

Amino Acid Sequence↗

The Caenorhabditis elegans orthologue of mammalian puromycin-sensitive aminopeptidase has roles in embryogenesis and reproduction.

Mammals possess membrane-associated and cytosolic forms of the puromycin-sensitive aminopeptidase (PSA; EC 3.4.11.14). Increasing evidence suggests the membrane PSA is involved in neuromodulation within the central nervous system and in reproductive biology. The functional roles of the cytosolic PSA are less clear. The genome of the nematode Caenorhabditis elegans encodes an aminopeptidase, F49E8.3 (PAM-1), that is orthologous to PSA, and sequence analysis predicts it to be cytosolic. We have determined the spatio/temporal gene expression pattern of pam-1 by using the promoter region of F49E8.3 to control expression in the nematode of a second exon translational fusion of the aminopeptidase to green fluorescent protein. Cytosolic fluorescence was observed throughout development in the intestine and nerve cells of the head. Neuronal expression was also observed in the tail of adult males. Recombinant PAM-1, expressed and purified from Escherichia coli, hydrolyzed the N-terminal amino acid from peptide substrates. Favored substrates had positively charged or small neutral amino acids in the N-terminal position. Peptide hydrolysis was inhibited by the metal-chelating agent 1,10-phenanthroline and by the aminopeptidase inhibitors actinonin, amastatin, and leuhistin. However, the enzyme was approximately 100-fold less sensitive toward puromycin (IC50, 135 mum) than other PSA homologues. Following inactivation of the enzyme, aminopeptidase activity was recovered with Zn2+, Co2+, and Ni2+. Silencing expression of pam-1 by RNA interference resulted in 30% embryonic lethality. Surviving adult hermaphrodites deposited large numbers of oocytes throughout the self-fertile period. The overall brood size was, however, unaffected. We conclude that pam-1 encodes an aminopeptidase that clusters phylogenetically with the PSAs, despite attenuated sensitivity toward puromycin, and that it functions in embryo development and reproduction of the nematode.

Amino Acid Sequence↗

Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators.

The recent discovery of positive allosteric modulators (PAMs) for G-protein-coupled receptors open new possibilities to control a number of physiological and pathological processes. Understanding the mechanism of action of such compounds will provide new information on the activation process of these important receptors. Within the last 10 years, a number of studies indicate that G-protein-coupled receptors can form dimers, but the functional significance of this phenomenon remains elusive. Here we used the metabotropic glutamate receptors as a model, because these receptors, for which PAMs have been identified, are constitutive dimers. We used the quality control system of the GABA(B) receptor to generate metabotropic glutamate receptor dimers in which a single subunit binds a PAM. We show that one PAM/dimer is sufficient to enhance receptor activity. Such a potentiation can still be observed if the subunit unable to bind the PAM is also made unable to activate G-proteins. However, the PAM acts as a non-competitive antagonist when it binds in the subunit that cannot activate G-proteins. These data are consistent with a single heptahelical domain reaching the active state per dimer during receptor activation.

Actins↗

Effects of inhaled ozone on pulmonary immune cells critical to antibacterial responses in situ.

The goal of this study was to examine effects from repeated exposure to ozone (O3) on immune cells involved in cell-mediated antibacterial responses in the lungs. Rats exposed to 0.1 or 0.3 ppm O3 for 4 h/day, 5 days/wk, for 1 or 3 wk were analyzed for the ability to clear an intrapulmonary challenge with Listeria monocytogenes or had their lungs processed to obtain pulmonary alveolar macrophages (PAM) and lung-associated lymphocytes for analyses of select cell functions and surface marker expression. The results indicate that repeated inhalation exposure to O3 affected local cell-mediated immunity (CMI) responses as evidenced by effects on clearance of Listeria. However, this modulation was not consistently dependent on exposure concentration or duration. Short-term repeat exposures had more effect on host resistance than did the more prolonged regimen, with rats exposed to 0.1 ppm O3 most adversely impacted. Clearance patterns suggest modifications in innate resistance following 1 wk of exposure to 0.1 ppm O3, but no similar effect following a 3-wk regimen. Exposure to 0.3 ppm O3 appeared to affect both innate and acquired resistance after a 1-wk regimen, but mainly the former after an additional 2 wk of exposure. We conclude that these two mechanisms of resistance are differentially affected by O3 and that distinct time- and O3 concentration-dependent adaptation phenomena evolve for each; that is, in situ adaptation to higher levels of O3 may occur more readily with acquired than with innate/PAM-dependent resistance. A similar pattern of inconsistent effect on PAM and lung-associated lymphocytes was also evident. For example, while 3-wk exposures had a greater effect on PAM reactive oxygen intermediate ROI production, evidence for a significant effect on antibacterial activity was only notable among PAM from rats exposed for 1 wk. Among lung lymphocytes, while 3-wk exposure to 0.1 ppm O3 led to a significant increase in CD25 expression, there was no corresponding increase in responsivity to concanavalin A (ConA); only among cells from 1-wk-exposed rats did lymphoproliferative responses increase. Though investigations of altered immune cell cytokine receptor expression/binding activity are ongoing, results herein provide further evidence to support our longstanding hypothesis that some well-documented effects of O3 exposure on human health are quite likely linked to changes in local immune cell (i.e., PAM and lung-associated lymphocytes) functions, with the latter being related to changes in the capacities of these cells to interact with immunoregulatory cytokines.

Administration, Inhalation↗

Characterization of 1 alpha-hydroxylation of vitamin D3 sterols by cultured alveolar macrophages from patients with sarcoidosis.

We investigated the 1 alpha-hydroxylation of vitamin D3 sterols by cultured pulmonary alveolar macrophages (PAM) from patients with sarcoidosis with or without clinically abnormal calcium homeostasis. Like the naturally occurring renal 1 alpha-hydroxylase, the PAM 1 alpha-hydroxylation reaction exhibited a high affinity for 25-hydroxyvitamin D3 (25-OH-D3) and a preference for substrates containing a 25-hydroxyl group in the side chain of the sterol. Unlike the renal enzyme, the PAM 1 alpha-hydroxylating mechanism was not accompanied by 24-hydroxylating activity, even after preincubation with 75 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2-D3] or exposure to high concentrations of substrate (500 nM 25-OH-D3). The PAM 25-OH-D3-1 alpha-hydroxylation reaction was stimulated by gamma interferon and inhibited by exposure to the glucocorticoid dexamethasone. The characteristics of the PAM hydroxylation process in vitro appear to reflect the efficiency of the extrarenal production of 1,25-(OH)2-D3 and the therapeutic efficacy of glucocorticoids in patients with sarcoidosis and disordered calcium metabolism.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

Inhibition of the immunosuppressive activity of resident pulmonary alveolar macrophages by granulocyte/macrophage colony-stimulating factor.

Resident pulmonary alveolar macrophages (PAM) play an important role in the maintenance of immunological homeostasis in the lung via downmodulation of local T cell responses in the steady state. The present study demonstrates that this pathway for T cell suppression is reversible via granulocyte/macrophage colony-stimulating factor (GM-CSF). Thus, freshly isolated PAM strongly inhibit mitogen-induced T cell proliferation, and pretreatment of the PAM with cytokine-rich lung-conditioned medium (LCM) generated by exposure of lung to bacterial lipopolysaccharide (LPS) abrogated this suppressive activity. LCM from lungs of normal and athymic nude mice exhibited identical activity. Moreover, the PAM-modulating activity of LCM was inhibited by blocking antibody specific for GM-CSF, and the activity of LCM could be reproduced by recombinant GM-CSF. This suggests that secretion of GM-CSF by mesenchymal cells and/or macrophages under stimulation from agents such as LPS provides a potential mechanism for upregulation of local T cell responsiveness during acute inflammation. In addition, experiments with a range of cytokines indicated that interleukin 4, transforming growth factor beta 1 and tumor necrosis factor alpha (TNF-alpha) exhibited weaker (but significant) modulatory effects on PAM, and (in the case of TNF-alpha) amplified the effects of GM-CSF.

Animals↗

Mutations in SLC34A2 cause pulmonary alveolar microlithiasis and are possibly associated with testicular microlithiasis.

Pulmonary alveolar microlithiasis (PAM) is a rare disease characterized by the deposition of calcium phosphate microliths throughout the lungs. We first identified a PAM locus by homozygosity mapping to 4p15, then identified, by a candidate-gene approach, the gene responsible for the disease as SLC34A2 (the type IIb sodium-phosphate cotransporter gene), which is involved in phosphate homeostasis in several organs. We identified six homozygous exonic mutations in the seven unrelated patients with PAM we studied. Three of the mutations were frameshifts, one was a chain termination, one was an amino acid substitution, and one was a deletion spanning the minimal promoter and the first exon. Absence of functional protein product of the gene is compatible with calcium phosphate deposition in alveolar airspaces. We show that impaired activity of the phosphate transporter is presumably responsible for the microliths and that PAM is a recessive monogenic disease with full penetrance. Testicular microlithiasis (TM) is a disease that is more common than PAM. It is often associated with cancer and infertility. Since the gene we identified is also expressed in testis, we searched for mutations in subjects with TM. In 2 of the 15 subjects with TM we studied, we identified two rare variants, one synonymous and the other noncoding, that are possibly associated with the condition.

Chromosome Mapping↗

Classification of microarrays to nearest centroids.

MOTIVATION: Classification of biological samples by microarrays is a topic of much interest. A number of methods have been proposed and successfully applied to this problem. It has recently been shown that classification by nearest centroids provides an accurate predictor that may outperform much more complicated methods. The 'Prediction Analysis of Microarrays' (PAM) approach is one such example, which the authors strongly motivate by its simplicity and interpretability. In this spirit, I seek to assess the performance of classifiers simpler than even PAM. RESULTS: I surprisingly show that the modified t-statistics and shrunken centroids employed by PAM tend to increase misclassification error when compared with their simpler counterparts. Based on these observations, I propose a classification method called 'Classification to Nearest Centroids' (ClaNC). ClaNC ranks genes by standard t-statistics, does not shrink centroids and uses a class-specific gene-selection procedure. Because of these modifications, ClaNC is arguably simpler and easier to interpret than PAM, and it can be viewed as a traditional nearest centroid classifier that uses specially selected genes. I demonstrate that ClaNC error rates tend to be significantly less than those for PAM, for a given number of active genes. AVAILABILITY: Point-and-click software is freely available at http://students.washington.edu/adabney/clanc.

Algorithms↗

Effects of recombinant soluble CD4 (rCD4) on HIV-1 infection of monocyte/macrophages.

Recombinant soluble CD4 (rCD4) was tested for its ability to block acute human immunodeficiency virus (HIV) infection in the U937 monocytic cell line and in human pulmonary alveolar macrophages (PAM) and for its ability to prevent transfer of virus from chronically infected PAM to target peripheral blood mononuclear leukocytes (PMNL). With an initial virus inoculum of 10(3)-10(4) TCID50/ml, rCD4 completely prevented acute HIV infection of U937 cells at concentrations greater than or equal to 1 microgram/ml and provided substantial but incomplete protection at 0.1 microgram/ml. With an initial virus inoculum of 10(2) TCID50/ml, rCD4 completely prevented acute infection of PAM at concentrations greater than or equal to 0.1 microgram/ml. The transmission of HIV-1 infection to PMNL cocultured with chronically infected PAM was completely inhibited at concentrations greater than or equal to 1 microgram/ml if cell-to-cell contact was prevented. With direct PAM-PMNL contact, substantial inhibition was obtained at an rCD4 concentration of 10 micrograms/ml, and higher concentrations (200 micrograms/ml) could completely block transfer. These results demonstrated that rCD4 can be effective in preventing de novo infection of cells of the monocyte/macrophage lineage, but microenvironments where cell-to-cell contact predominates are likely to pose a formidable challenge to this therapeutic strategy.

CD4 Antigens↗

The immunotherapeutic potential of activated canine alveolar macrophages and antitumor monoclonal antibodies in metastatic canine melanoma.

A variety of immune cell activators can enhance the cytotoxic effects of monocytes/macrophages including interferon-gamma (IFN-gamma) and muramyl peptides, which are under investigation for cancer therapy in humans and dogs. Pulmonary alveolar macrophages (PAMs) in particular, are strategically located within the lung and provide a potential defense against cancer cells metastatic to the lung. For this reason, we examined the in vitro cytotoxic potential of fresh and IFN-gamma-activated PAMs from normal dogs targeted to canine malignant melanoma cells with antiganglioside monoclonal antibodies (mAbs). Antiganglioside mAbs 14.G2a (anti-GD2) and R24 (anti-GD3), both in clinical trials for human neuroectodermal tumors including melanoma, significantly enhanced the cytotoxicity of canine melanoma mediated by canine PAMs. Further, the cytotoxicity mediated by recombinant canine IFN-gamma-activated canine PAMs, in combination with anti-GD2 ganglioside mAb 14.G2a, enhanced melanoma cytotoxicity above that seen with mAb 14.G2a alone. This documentation of antibody-dependent cellular cytotoxicity mediated by activated PAMs suggests that activation and targeting of resident pulmonary immune cells be pursued as a means to control pulmonary metastases.

Adjuvants, Immunologic↗