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Biomedical subjects

R F Murphy

Publications and source records attributed to R F Murphy.

At least 37 records · Page 2Linked to original sources
PubMed2000

Influence on antiproliferative activity of structural modification and conjugation of gonadotropin-releasing hormone (GnRH) analogues.

The effect of various GnRH analogues, and their conjugates on proliferation, clonogenicity and cell cycle phase distribution of MCF-7 and Ishikawa human cancer cell lines was studied. GnRH-III, a sea lamprey GnRH analogue reduced cell proliferation by 35% and clonogenicity by 55%. Structural modifications either decreased, or did not alter biological activity. Conjugation of GnRH analogues including MI-1544, MI-1892, and GnRH-III with poly(N-vinylpyrrolidone-co-maleic acid) (P) through a tetrapeptide spacer GFLG(X) substantially increased the inhibitory effect of the GnRH analogues. The conjugate P-X-GnRH-III induced significant accumulation of cells in the G2/M phase; from 8% to 15.6% at 24 h and 9.8% to 15% at 48 h. It was concluded that conjugation of various GnRH analogues substantially enhanced their antiproliferative activity, strongly reduced cell clonogenicity and retarded cell progression through the cell division cycle at the G2/M phase.

Animals↗
PubMed2000

Towards a systematics for protein subcelluar location: quantitative description of protein localization patterns and automated analysis of fluorescence microscope images.

Determination of the functions of all expressed proteins represents one of the major upcoming challenges in computational molecular biology. Since subcellular location plays a crucial role in protein function, the availability of systems that can predict location from sequence or high-throughput systems that determine location experimentally will be essential to the full characterization of expressed proteins. The development of prediction systems is currently hindered by an absence of training data that adequately captures the complexity of protein localization patterns. What is needed is a systematics for the subcellular locations of proteins. This paper describes an approach to the quantitative description of protein localization patterns using numerical features and the use of these features to develop classifiers that can recognize all major subcellular structures in fluorescence microscope images. Such classifiers provide a valuable tool for experiments aimed at determining the subcellular distributions of all expressed proteins. The features also have application in automated interpretation of imaging experiments, such as the selection of representative images or the rigorous statistical comparison of protein distributions under different experimental conditions. A key conclusion is that, at least in certain cases, these automated approaches are better able to distinguish similar protein localization patterns than human observers.

Animals↗
PubMed1999

Importance of the aromatic residue at position 6 of [Nle(10)]neurokinin A(4-10) for binding to the NK-2 receptor and receptor activation.

Steric and electrostatic requirements at position 6 of [Nle(10)]NKA(4-10), a full agonist of NK-2 receptors, for molecular recognition by the receptor were studied. Two series of peptide analogues, (a) p-substituted analogues, [p-X-Phe(6), Nle(10)]NKA(4-10), where X = F, Cl, Br, I, NH(2), NO(2), and (b) [D-Phe(6),Nle(10)]NKA(4-10), [Trp(6),Nle(10)]NKA(4-10), and [Chex-Ala(6),Nle(10)]NKA(4-10), were synthesized, and their biological activity was examined. Competition binding experiments with [(3)H]NKA were performed using cloned human NK-2 receptors expressed in CHO cells. Antagonistic and agonistic properties of the analogues were studied using an in vitro functional assay with hamster tracheal rings. The rank order of potency of agonists was [Nle(10)]NKA(4-10) approximately [p-F-Phe(6),Nle(10)]NKA(4-10) > [p-NH(2)-Phe(6),Nle(10)]NKA(4-10) > [p-Cl-Phe(6),Nle(10)]NKA(4-10) > [p-NO(2)-Phe(6),Nle(10)]NKA(4-10) > [Trp(6),Nle(10)]NKA(4-10). Size and planarity of the aromatic side chain were crucially important for the biological activity, whereas electron-donating and electron-withdrawing properties of the para-substituent were less important. The results favor the hypothesis that weakly polar pi-pi interactions exist between the aromatic group and the receptor.

Animals↗
PubMed1999

Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity.

Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(N-vinylpyrrolidone-co-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G2/M phase of the cell cycle after 24 h at lower doses and in the G1 and G2 phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.

Animals↗
PubMed1999

Toward objective selection of representative microscope images.

Scientists wishing to communicate the essential characteristics of a pattern (such as an immunofluorescence distribution) currently must make a subjective choice of one or two images to publish. We therefore developed methods for objectively choosing a typical image from a set, with emphasis on images from cell biology. The methods involve calculation of numerical features to describe each image, calculation of similarity between images as a distance in feature space, and ranking of images by distance from the center of the feature distribution. Two types of features were explored, image texture measures and Zernike polynomial moments, and various distance measures were utilized. Criteria for evaluating methods for assigning typicality were proposed and applied to sets of images containing more than one pattern. The results indicate the importance of using distance measures that are insensitive to the presence of outliers. For collections of images of the distributions of a lysosomal protein, a Golgi protein, and nuclear DNA, the images chosen as most typical were in good agreement with the conventional understanding of organelle morphologies. The methods described here have been implemented in a web server (http://murphylab.web.cmu.edu/services/TyplC).

Animals↗
PubMed1999

Coupling difficulty following replacement of Tyr with HOTic during synthesis of an analog of an EGF B-loop fragment.

During Fmoc synthesis of an analog, [Abu HOTic]hEGF(20-31), of a fragment, Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys, of the B-loop of human EGF, conductivity measurements showed that increased time was necessary for coupling and complete deprotection of the residues Met and Abu which followed the HOTic. Use of different active esterforming reagents, including HOBt and BOP, did not increase the yield. Use of symmetrical anhydride with extended coupling time increased the yield but did not complete the coupling. It appears that inclusion of HOTic in place of Tyr to introduce conformational constrain to peptide analogs can cause or augment a tendency towards conformations with increasing occlusion of N-terminal amino groups and result in the need for altered coupling strategies for completion of analog synthesis.

Amino Acids↗
PubMed1999

Secondary structures and intramolecular interactions in fragments of the B-loops of naturally occurring analogs of epidermal growth factor.

Structures of naturally occurring analogs of the B-loop fragment of human epidermal growth factor-like (hEGF-like) polypeptides were examined by molecular dynamics simulation in order to predict their secondary structures, to find structural similarity and to detect any weakly polar aromatic-aromatic (pi-pi) or amide-aromatic (N-pi) interactions which stabilize the structures. NPT molecular dynamics simulations (1 ns) were performed by the GRO-MACS package with SPC/E water using a weak temperature and pressure coupling method. During the sampling time, the structures of all peptides showed a characteristic secondary structure with a turn and bend at residues 4-7, and a beta-sheet, beta-bridge and random coil at the N- and C-terminal regions. Though the peptide chains were flexible, the stabilization effect of the N-pi interactions was indicated in some cases by the angles and distances between the centroids of aromatic planes of the side-chains and the H-atom of peptide bonds and the planes of the aromatic side-chains, respectively. Pi-pi interactions occurred less frequently because of the flexibility of the short peptide chain.

Amino Acid Sequence↗
PubMed1998

Pharmacokinetics and pharmacodynamics of oral methotrexate and mercaptopurine in children with lower risk acute lymphoblastic leukemia: a joint children's cancer group and pediatric oncology branch study.

We prospectively assessed the pharmacokinetics of methotrexate, mercaptopurine, and erythrocyte thioguanine nucleotide levels in a homogenous population of children with lower risk acute lymphoblastic leukemia and correlated pharmacokinetic parameters with disease outcome. The maintenance therapy regimen included daily oral mercaptopurine (75 mg/m2) and weekly oral methotrexate (20 mg/m2). One hundred ninety-one methotrexate doses and 190 mercaptopurine doses were monitored in 89 patients. Plasma drug concentrations of both agents were highly variable. The area under the plasma concentration-time curve (AUC) of methotrexate ranged from 0.63 to 12 micromol*h/L, and the AUC of mercaptopurine ranged from 0.11 to 8 micromol*h/L. Drug dose, patient age, and duration of therapy did not account for the variability. Methotrexate AUC was significantly higher in girls than boys (P =.007). There was considerable intrapatient variability for both agents. Erythrocyte thioguanine nucleotide levels were also highly variable (range, 0 to 10 pmol/g Hgb) and did not correlate with mercaptopurine dose or AUC. A Cox regression analysis showed that mercaptopurine AUC was a marginally significant (P =.043) predictor of outcome, but a direct comparison of mercaptopurine AUC in the remission and relapsed patient groups failed to show a significant difference. Methotrexate and mercaptopurine plasma concentrations and erythrocyte thioguanine nucleotide levels were highly variable, but measurement of these pharmacokinetic parameters at the start of maintenance will not distinguish patients who are more likely to relapse.

Administration, Oral↗
PubMed1998

Molecular dynamics simulations of epidermal growth factor and transforming growth factor-alpha structures in water.

AMBER v. 4.1 force field in 1.5 ns NPT molecular dynamics simulations of murine epidermal growth factor (mEGF), human epidermal growth factor (hEGF), and human transforming growth factor-alpha (hTGF-alpha) structures with explicit TIP3P solvation were used to investigate differences in backbone stability, changes in secondary structure, interdomain flexibility, and weakly polar interactions. Backbone root mean square deviations of sections of each peptide show that the most stable regions in mEGF and hEGF are the A-, B-, and C-loops, whereas the most stable regions in hTGF-alpha are the A- and B-loops. The secondary structure in the B-loops of mEGF and hEGF differ significantly from the nuclear magnetic resonance (NMR) structures of mEGF and hEGF. The position and type of turns in the B-loop of mEGF and hEGF increase the interstrand distance of the antiparallel beta-sheets thereby disrupting their structure. The interdomain flexibility of simulated hTGF-alpha structure is greater than in either mEGF or hEGF. The phi, psi dihedrals of hTGF-alpha occupy two distinct populations of phase space corresponding to either a Ceq7 or an alpha-helical conformation. This change in dihedral angle is stabilized by Phe15 with Arg42 and Phe17 with Arg42 N-pi weakly polar interactions that are present only in hTGF-alpha but not in mEGF or hEGF.

Amino Acid Sequence↗
PubMed1998

Automated recognition of patterns characteristic of subcellular structures in fluorescence microscopy images.

Methods for numerical description and subsequent classification of cellular protein localization patterns are described. Images representing the localization patterns of 4 proteins and DNA were obtained using fluorescence microscopy and divided into distinct training and test sets. The images were processed to remove out-of-focus and background fluorescence and 2 sets of numeric features were generated: Zernike moments and Haralick texture features. These feature sets were used as inputs to either a classification tree or a neural network. Classifier performance (the average percent of each type of image correctly classified) on previously unseen images ranged from 63% for a classification tree using Zernike moments to 88% for a backpropagation neural network using a combination of features from the 2 feature sets. These results demonstrate the feasibility of applying pattern recognition methods to subcellular localization patterns, enabling sets of previously unseen images from a single class to be classified with an expected accuracy greater than 99%. This will provide not only a new automated way to describe proteins, based on localization rather than sequence, but also has potential application in the automation of microscope functions and in the field of gene discovery.

Image Enhancement↗
PubMed1998

Peptides bind to eosinophils in the rat stomach.

BACKGROUND: An immunological role for eosinophils has been well established. However, roles for eosinophils in the physiological functions of the organs they populate are little explored. METHODS: Fixed, frozen, then vibratomed sections of rat stomach were exposed to biotinylated 1-17 gastrin (bG17), biotinylated gastrin-releasing peptide (bGRP), biotinylated neuromedin C (bNC), biotinylated vasoactive intestinal peptide (bVIP), and biotinylated substance P (bSP). Binding sites were identified using an avidin-biotin-glucose oxidase complex and tetranitroblue tetrazolium staining. RESULTS: bG17, bGRP, and bNC all bound to cells in the lamina propria and to a lesser extent in the submucosa. Neither bVIP nor bSP bound to cells in these sections. Stained cells were identified as eosinophils in the light microscope on the basis of their distribution and staining properties using the Luna stain for eosinophils and in the transmission electron microscope (TEM) on the basis of a light/TEM matching process. Plastic sections viewed in the light microscope showed that stain was localized to a granular component in the cytoplasm of the eosinophils. No other cell type, specifically neither mast cells nor plasma cells, stained. G17 competed for the bG17 binding site better than did NC. A competition study in which polyglutamic acid failed to compete with bG17 for the binding site, and the observation that bG17, bGRP, and bNC did not bind to other positively charged sites (e.g., collagen, red blood corpuscles), demonstrated that binding was not due to nonspecific electrostatic interactions alone. Binding of bG17 to a CCK(B)/gastrin-type receptor was ruled out when specific receptor antagonists failed to block binding. CONCLUSIONS: The particulate nature of the binding site suggests a secretory substance. If so, eosinophils might use that substance to destroy, neutralize, or control the activity of peptide hormones bound to it in the extracellular space.

Animals↗
PubMed1998

Primary cell cultures from murine kidney and heart differ in endosomal pH.

Endosomal and lysosomal pH values have been determined for many established cultured cell lines of different origins. These cell lines may be grouped into two classes based on observed differences in pH of early (recycling) endosomes. Members of the first class typically have an early endosomal pH of 6.2, whereas members of the second class typically have an early endosomal pH of 5.4. Because established cell lines may have developed artificial differences in endosomal pH due to extended culture, it remains to be determined if endosomal pH differences exist in vivo and whether they are functionally significant. To address this question, we generated adherent primary explants from mouse kidney (primarily epithelial cells) and heart (primarily fibroblasts and cardiac muscle cells). Interestingly, enhanced acidification was observed in heart cell endosomes (pH = 5.5) compared with kidney cell endosomes (pH = 6.0). These results indicate that differences in endosomal pH do not solely arise from extended cell culture and imply that such differences may be important for the proper functioning of different cell types.

Animals↗
PubMed1998

Direct anticancer activity of gonadotropin-releasing hormone-III.

In previous studies GnRH-III, a variant of the hypothalamic neurohormone GnRH, was isolated from the brain of the sea lamprey and structurally characterized. GnRH-III is a hypothalamic neurohormone in both female and male sea lampreys. In the present work biological activities of GnRH-III in mammalian systems were examined. In superfused rat pituitary cells, GnRH-III at 1 nM to 100 nM neither induced LH-secretion nor inhibited the LH-secretion elicited by native GnRH and elicited LH release only at 1 microM. At high dose (500 microg/day) in vivo, GnRH-III behaved as a GnRH agonist, though, it was 1000-fold less active than ovurelin. The in vitro and in vivo results were in good agreement in showing that GnRH-III is only a weak agonist of the endocrine activity of GnRH. GnRH-III specifically bound to receptors on cancer cells and recognized not only the high-, but also the low-affinity binding sites. GnRH-III significantly suppressed growth of human cancer cells which have GnRH receptors. The inhibitory effect of GnRH-III on growth of cancer cells was specific and direct since the peptide did not have endocrine activity in the concentration range found to be effective in anticancer assays. GnRH-III inhibited equally the growth of ER-positive and -negative breast and TeR-positive and negative prostate cells.

Animals↗
PubMed1998

Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection.

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713-3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.

Anti-Bacterial Agents↗
PubMed1998

Phase I/II trial of all-trans retinoic acid and tamoxifen in patients with advanced breast cancer.

Because tamoxifen and all-trans-retinoic acid (ATRA) have additive antitumor effects in preclinical systems, we performed a Phase I/II clinical trial of this combination in patients with advanced breast cancer. Patients with potentially hormone-responsive advanced breast cancer were enrolled. All received 20 mg of tamoxifen by mouth daily. Consecutive cohorts of 3-6 patients were treated on odd-numbered weeks with ATRA at doses of 70, 110, 150, 190, or 230 mg/m2/day. Twenty-six patients were entered in this trial; 25 were evaluable. A dose of 230 mg/m2 ATRA produced unacceptable headache and dermatological toxicity, but doses < or = 190 mg/m2 were tolerable. Two of 7 patients with measurable disease responded. Seven of 18 patients with evaluable, nonmeasurable disease achieved disease stability for more than 6 months. Plasma AUCs on day 1 of successive weeks of treatment were stable over time. A nonsignificant decrease in serum insulin-like growth factor I levels was noted during treatment, but this trend was similar to that observed in three "control" patients treated with tamoxifen alone. When given with daily tamoxifen, the maximum tolerated dose of ATRA that could be given on alternate weeks was 190 mg/m2/day. This schedule of ATRA resulted in repeated periods of exposure to potentially therapeutic concentrations of ATRA. Declines in the serum insulin-like growth factor I concentrations observed in patients treated with tamoxifen and ATRA were similar to those observed in patients treated with tamoxifen alone. Objective responses were observed, some in patients who had previously progressed while receiving tamoxifen, suggesting that further studies would be of interest.

Adult↗
PubMed1997

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

Antineoplastic Agents↗