Biomedical subjects

A Treston

Publications and source records attributed to A Treston.

7 recordsLinked to original sources

PubMed2000

2-Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate.

2-Methoxyestradiol, once considered an inacitve end-metabolite of estradiol, has recently emerged as a very promising agent for cancer treatment. It is orally active in a wide range of tumor models, and inhibits tumor growth at doses showing no clinical signs of toxicity. 2ME2 targets both the tumor cell and endothelial cell compartments by inducing apoptosis in rapidly proliferating cells and inhibiting blood vessel formation at several stages in the angiogenic cascade. Moreover, the ability of 2ME2 to inhibit metastatic spread in several models adds to its therapeutic value for cancer treatment at various stages of the disease. Though the mechanism of action is still undefined, several potential molecular targets and pathways of activation have been suggested.

2-Methoxyestradiol↗

PubMed2000

The metabolism of BW2258U89, a GRP receptor antagonist.

BW2258U89 is a gastrin releasing peptide (GRP) receptor antagonist which inhibits the proliferation of the neuroendocrine tumor small cell lung cancer (SCLC). Here the biological activity of BW2258U89 and its metabolite were investigated. Using mass spectroscopy (LC-ESI/MS) techniques, three major peaks for BW2258U89 were observed with mass/charge (m/z) ratios of 1081.6, 541.4 and 361.4. After metabolism by mouse plasma enzymes, the major product had a m/z ratio of 1082.5, 541.9 and 361.8 suggesting that BW2258U89 was deamidated. Deamidated (Da) BW2258U89 was synthesized and it inhibited ((125)I-Tyr(4)) BB binding to NCI-H345 SCLC cells with an IC(50)value of 450 nM; BW2258U89 had an IC(50)value of 17 nM. BW2258U89 (1 microM) antagonized the ability of 50 nM BB to elevate cytosolic Ca(2+)in NCI-H345 cells, whereas 1 microM (Da) BW2258U89 did not. One micromolar BW2258U89 antagonized the increase in NCI-H345 c-fos mRNA caused by 10 nM BB, whereas 1 microM (Da) BW2258U89 had little effect. One microM BW2258U89 inhibited NCI-H345 clonal growth significantly whereas 1 microM (Da) BW2258U89 did not. These data suggest that an amidated C-terminal is important for antagonism of SCLC GRP receptors by BW2258U89.

Animals↗

PubMed1996

Distribution of peptidyl-glycine alpha-amidating mono-oxygenase (PAM) enzymes in normal human lung and in lung epithelial tumors.

C-terminal alpha-amidation is a post-translational modification necessary for the biological activity of many regulatory peptides produced in the respiratory tract. This modification is a two-step process catalyzed by two separate enzyme activities, both derived from the peptidyl-glycine alpha-amidating mono-oxygenase (PAM) precursor. The distribution of these two enzymes, peptidyl-glycine alpha-hydroxylating monoxygenase (PHM) and peptidyl-alpha-hydroxyglycine a amidating lyase (PAL), was studied in the normal lung and in lung tumors using immunocytochemical methods and in situ hybridization. In normal lung the enzymes were located in some cells of the airway epithelium and glands, the endothelium of blood vessels, some chondrocytes of the bronchial cartilage, the alveolar macrophages, smooth muscle cells, neurons of the intrinsic ganglia, and in myelinated nerves. A total of 24 lung tumors of seven different histological types were studied. All cases contained PAM-immunoreactive cells with various patterns of distribution. All immunoreactive cells were positive for the PHM antiserum but only some of them for the PAL antiserum. The distribution of PAM co-localizes with some other previously described amidated peptides, suggesting that amidation is an important physiological process taking place in the normal and malignant human lung tissue.

Aged↗

PubMed1993

Localization of amidating enzymes (PAM) in rat gastrointestinal tract.

We studied the distribution of the two enzymes involved in post-translational C-terminal alpha-amidation of regulatory peptides in rat digestive tract, using immunocytochemical methods and in situ hybridization techniques. The enzymes were located in most of the fibers and neurons of the myenteric and submucous plexus throughout the entire digestive tract and in endocrine cells of the stomach and colon. Staining of reverse-face serial sections demonstrated that the enzymes in endocrine cells of the stomach co-localized with gastrin in the bottom of the gastric glands. Some gastrin-immunoreactive cells near the neck of the gland were negative for PAM, suggesting that amidation takes place only in the more mature cells. In the colon all cells immunoreactive for glucagon and GLP1 were also positive for peptidylglycine alpha-hydroxylating monooxygenase (PHM) but not for peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The absence of immunoreactivity for the amidating enzymes in endocrine cells of the small intestine, known to produce C-terminally amidated peptides, suggests the existence of other amidating enzymes.

Animals↗

PubMed1993

Immunocytochemical localization of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in human endocrine pancreas.

We studied the distribution of the enzymes that are involved in the post-translational alpha-amidation of regulatory peptides in human endocrine pancreas, using immunocytochemical methods for light and electron microscopy. Immunoreactivity for the two enzymes involved, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), was located in the periphery of the islets of Langerhans and in ductal endocrine cells. Staining of reverse-face serial sections demonstrated that these immunoreactivities co-localize with glucagon but not with pancreatic polypeptide (PP), insulin, or somatostatin. Double immunogold staining for electron microscopy confirmed the previous results and revealed a different localization for each enzyme inside the secretory granule: PHM is present in the central core of the glucagon-containing granules, whereas PAL is predominantly located near the granule membrane. The existence of an amidated peptide, GLP1, in the A-cells explains the presence of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in these cells. The absence of the enzymes in the PR-cells raises the possibility that a different form of amidating enzyme may be involved in the post-translational processing of this peptide.

Amidine-Lyases↗

PubMed1988

Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation.

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.

Amino Acid Sequence↗

PubMedDate not supplied

Rapid mass spectrometric identification of proteins from two-dimensional polyacrylamide gels after in gel proteolytic digestion.

We report a rapid method for identifying proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). In-gel digestion was performed in a way such that the volume ratio of trypsin solution to gel plug was quantitatively controlled to promote reproducible digestion and to maximize the digestion yield. To make the digestion samples more compatible with MALDI-MS, the volatile salt ammonium bicarbonate in the digestion buffer was largely removed prior to peptide extraction. Samples of mixed tryptic peptides from in-gel digestion were used without purification to obtain molecular weights by MALDI-MS with alpha-cyano, 4-hydroxy-cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detection sensitivity by one half to one order of magnitude. The peptide mass peaks in MALDI-MS spectra were distinguished from those of impurities by using several types of controls, and masses were corrected by using trypsin autodigestion fragments as internal calibration standards. Two different peptide-matching computer programs were used to interrogate sequence databases and identify proteins. Identification was enhanced by generation of orthogonal data sets (by using different proteases) and by including experimental values of isoelectric point (pI) and molecular weight to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine protein spots from rat liver 2-D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low-abundance spot was not identified by MALDI-MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2-D PAGE protein spots of medium abundance.

Acrylic Resins↗