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Biomedical subjects

R M Phillips

Publications and source records attributed to R M Phillips.

At least 19 recordsLinked to original sources
PubMed1995

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.

Animals↗
PubMed1994

Increased activity and expression of NAD(P)H:quinone acceptor oxidoreductase in confluent cell cultures and within multicellular spheroids.

NAD(P)H:quinone acceptor oxidoreductase (NQO1, EC 1.6.99.2) is an enzyme that is believed to play a central role in the bioreductive activation of several compounds, particularly quinones. The results of this study demonstrate that the activity of NQO1 is significantly elevated (2.5-fold) in HT-29 human colon cells that are in the plateau phase of the growth curve as opposed to cells in the exponential phase. Analysis of gene expression using semiquantitative reverse transcription-polymerase chain reaction and Northern blot analysis demonstrates that the increased enzyme activity is associated with increased NQO1 mRNA levels. Sequential trypsinization of layers of cells from HT-29 multicellular spheroids and analysis of gene expression by reverse transcription-polymerase chain reaction demonstrate that NQO1 expression is elevated in cells close to the necrotic center. Maximum expression occurs at a depth of 90-110 microns, with reduced expression as the distance toward both the surface and the necrotic center decreases. HT-29 spheroids were significantly more responsive than monolayers (concentration producing 50% inhibition, 124.6 and 364 nM, respectively) to the experimental drug, 2,5-dimethyl-3,6 diaziridinyl-1,4 benzoquinone. While the environmental stimulus responsible for causing elevated NQO1 expression has not been identified, the fact that NQO1 expression is influenced by microenvironmental conditions will have important implications for those drugs that are activated by NQO1.

Aziridines↗
PubMed1994

Probing the metal binding sites of Escherichia coli isoleucyl-tRNA synthetase.

The metal binding properties of isoleucyl-tRNA synthetase (IleRS) from Escherichia coli were studied by in vivo substitution of the enzyme-bound metals. Purified E. coli IleRS was shown to have two tightly bound zinc atoms per active site. Cobalt- and cadmium-substituted IleRS were also found to contain two tightly bound Co2+ and Cd2+ atoms per polypeptide chain, respectively. The d-d transitions in the low energy absorption spectrum of Co(2+)-substituted IleRS were characteristic of that expected for two tetrahedrally coordinated Co2+ metals. Apo-IleRS was found to be inactive in both the aminoacylation of tRNA(Ile) and in the isoleucine-dependent ATP-pyrophosphate exchange reactions. Both Co(2+)- and Cd(2+)-substituted IleRS were found to have kcat/Km values in the isoleucine-dependent ATP-pyrophosphate exchange assay approximately 5-fold lower than the native Zn2+ enzyme. A single enzyme-bound Zn2+ or Co2+ atom per polypeptide chain could be removed by dialysis of Zn(2+)- or Co(2+)-substituted IleRS against 1,10-phenanthroline. Removal of one of the two enzyme-bound Zn2+ atoms per polypeptide chain with 1,10-phenanthroline was found to decrease (kcat/Km)Ile by approximately 130-fold. The dependence of the kinetic parameters on the identity and number of enzyme-bound metals in the isoleucine-dependent ATP-pyrophosphate exchange reaction suggests that at least one enzyme-bound metal is indirectly involved in aminoacyladenylate formation. Metal substitution or removal of one of the two enzyme-bound metals in IleRS was found to have little effect on the Km value for tRNA(Ile) or the kcat value for aminoacylation of tRNA(Ile).(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1994

Evidence against norepinephrine-stimulated efflux of mitochondrial Mg2+ from intact cardiac myocytes.

We investigated the hypotheses that norepinephrine stimulates Mg2+ efflux from intact isolated adult rat ventricular cardiomyocytes and that adenosine 3',5'-cyclic monophosphate stimulates Mg2+ efflux from permeabilized myocytes and isolated mitochondria. Norepinephrine stimulation of Mg2+ release from cardiac myocytes was observed only when cells at approximately 20 mg protein/ml in Mg(2+)-containing buffer were diluted 50- to 60-fold into an Mg(2+)-free medium. Under these conditions, > 30% of total cellular lactic acid dehydrogenase activity was also released, indicating that a significant portion of the cells had died. In other protocols, where Mg2+ efflux from myocytes was not observed, extracellular Mg2+ removal and administration of 10 microM norepinephrine increased 45Ca2+ accumulation by cells in suspension. In single myocytes, Mg2+ removal and norepinephrine administration increased intracellular free [Ca2+] as measured by fura-2 fluorescence microscopy, and this was accompanied by vigorous spontaneous contractile activity followed by Ca2+ overload hypercontracture. With permeabilized myocytes and isolated mitochondria from a variety of sources, adenosine 3',5'-cyclic monophosphate did not stimulate Mg2+ efflux. These results suggest that recent evidence for direct hormonal regulation of myocardial Mg2+ homeostasis may need to be reevaluated.

Animals↗
PubMed1994

Bioactivation of quinones by DT-diaphorase, molecular, biochemical, and chemical studies.

Because of the elevated DT-diaphorase (DTD) activity in certain tumors such as human nonsmall cell lung cancer (NCSLC), DTD is a potential target on which to base the development of new antitumor compounds. Mitomycin C is the most effective single agent used for the therapy of NSCLC and is metabolized and bioactivated by DTD. Mitomycin C is a poor substrate for DTD, however, and its metabolism is pH-dependent. We have therefore focused on identifying more efficient substrates for DTD. We have developed a metabolic and cytotoxicity screen that identifies compounds which are efficiently bioactivated by DTD. This screen utilizes both aerobic and hypoxic conditions and cell lines with both elevated and deficient DTD activity as an index of selectivity. Using the screen described above, we have identified [3-hydroxy-5-aziridinyl-1-methyl-2-(1H-indole-4,7-indione)-prop-be ta-en- alpha-ol] (E09), 2,5-diaziridinyl-1,4-benzoquinone (MeDZQ), and streptonigrin as compounds that are most efficiently bioactivated by DTD and exert selective cytotoxicity. Although certain tumors such as NSCLC have elevated DTD activity, we have characterized a point mutation at position 609 in the DTD cDNA, which codes for a proline to serine change in the protein and leads to a loss of enzyme activity. We have characterized this mutation in both BE human colon carcinoma cells and H596 human NSCLC cells. This mutation and resulting lack of DTD activity complicates the use of agents designed to target DTD in tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Mitomycin C.

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Animals↗
PubMed1993

Pre-clinical evaluation of a novel chloroethylating agent, Clomesone.

The in vitro activity of the novel chloroethylating agent, Clomesone, was investigated in a panel of established murine and human tumour cell lines. In vivo anti-tumour activity was examined against three transplantable adenocarcinomas of the mouse colon and in vivo bone marrow toxicity was assessed using a spleen colony forming unit assay. The pharmacokinetic behaviour of the drug in vivo and drug stability in vitro was analysed by gas chromatography with electron capture detection. Clomesone exhibited no activity in vitro against the majority of cell lines derived from solid human colorectal carcinomas. Anti-tumour activity against the murine tumours in vivo was not impressive and was accompanied by myelosuppression. Pharmacokinetic data suggested that the lack of in vivo activity was due to the failure to achieve effective anti-neoplastic drug concentrations at the tumour site. It was concluded that this study found no evidence to suggest that Clomesone was toxicologically more selective than the chloroethylnitrosoureas.

Animals↗
PubMed1993

Use of monoclonal antibodies specific for the a determinant of K88 pili for detection of enterotoxigenic Escherichia coli in pigs.

Monoclonal antibodies directed against the a determinant of K88 pili from porcine enterotoxigenic Escherichia coli which react with all three K88 variants have been produced. These antibodies have been used for diagnosis of porcine enterotoxigenic E. coli in a direct enzyme-linked immunosorbent assay with sensitivity to 50 ng of pilus protein per ml.

Animals↗
PubMed1992

Evaluation of the anti-tumour action and acute toxicity of kosins from Hagenia abyssinica.

The kosins are phloroglucinol derivatives isolated from female flowers of Hagenia abyssinica (Rosaceae) and were tested for possible cytotoxic activity in vitro and in vivo against a panel of three transplantable murine adenocarcinomas of the colon of varying growth characteristics and morphology (MAC system). Significant reductions in colony formation were observed in vitro in MAC 15A tumour following 1, 3, 6 and 24 h exposure to all kosins (alpha-kosin, kosotoxin and protokosin). The kosins (kosotoxin and protokosin) were also found to be cytotoxic against MAC tumour cells in vivo in some cases. Kosotoxin was subjected to preliminary toxicity studies in mice. It showed no observable toxicity up to 200 mg kg-1 orally and was found to be toxic at doses in excess of 50 mg kg-1 (i.p.). A single dose of 100 mg kg-1 (i.p.) was lethal for 100% of the animals.

Administration, Oral↗
PubMed1992

In vitro activity of the novel indoloquinone EO-9 and the influence of pH on cytotoxicity.

The novel indoloquinone compound EO-9 is shortly to undergo phase I clinical evaluation as a potential bioreductive drug. Preclinical studies have shown that EO-9 has greater activity against cells derived from human solid tumours than leukaemias in vitro. The results of this study extend the preclinical data available on EO-9 by demonstrating that EO-9 induces a broad spectrum of activity (IC50 values range from 8 to 590 ng ml-1) against a panel of human and murine tumour cell lines. Some evidence exists of selectivity towards leukaemia and human colon cell lines as opposed to murine colon cells. The response of cells to Mitomycin C were not comparable to EO-9 suggesting that the mechanism of action of these compounds is different. The cytotoxic properties of EO-9 under aerobic conditions are significantly influenced by extracellular pH. Reduction of pH from 7.4 to 5.8 increases cell kill from 40% to 95% in DLD-1 cells. In addition, EO-9 is unstable at acidic pH (T1/2 = 37 min at pH 5.5) compared to neutral pH T1/2 = 6.3 h). The major breakdown product in vitro was identified as EO-5A which proved relatively inactive compared to EO-9 (IC50 = 50 and 0.6 ug ml-1 respectively). These studies suggest that if EO-9 can be delivered to regions of low pH within solid tumours, a therapeutic advantage may be obtained.

Animals↗
PubMed1992

Synthesis and cytotoxicity of shikimate analogues. Structure:activity studies based on 1-crotonyloxymethyl-3R,4R,5R-trihydroxycyclohex-2-enone.

Syntheses are described for and structure:activity studies undertaken of the anti-tumour activity of (2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-+ ++enone) (1) and its analogues 1-crotonyloxymethyl-(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-en e (3), 1-crotonyloxymethyl-(3R,4S,5S)-3,4,5-trihydroxycyclohexene (4) and 2-crotonyloxymethyl-2-cyclohexenone (5), which differ from 1 in the presence/absence of the cyclic keto group and/or the stereochemistry at one of the -OH bearing carbon atoms. None of the above compounds, including 1, directly inhibited glyoxalase I, isolated for the first time to homogeneity from rat Yoshida sarcomas and for which a purification protocol was developed. The apparent inhibition of glyoxalase I by 1 and 5 (but not detected for 4 or 3) could be explained by reaction of 1 and 5 with the glutathione present in the assay buffer and the consequent depletion of substrate. 1 and 5 were found to react readily with glutathione whereas 4 and 3 did not react. In vitro chemosensitivity studies against a panel of tumour cell lines of both mouse and human origin showed that in parallel with their thiol reactivity, 1 and 5 exhibited significant in vitro cytotoxicity whereas 4 and 3 did not. Concentrations of drug required to cause 50% cell kill (ID50 values) were in the range 0.5-19 microM (0.1-2.8 micrograms/ml) for 5, and 3-44 microM (0.7-10 micrograms/ml) for 1. The structural features causing the differences in antitumour effects were localized on this basis to the alpha,beta-unsaturated ketone linkage as opposed to the stereochemistry of the (trihydroxy) alcoholic sites.

Animals↗
PubMed1991

Porcine alveolar and pulmonary intravascular macrophages: comparison of immune functions.

Although a substantial amount of information is available on pulmonary alveolar macrophages (PAMs), little is known about pulmonary intravascular macrophages (PIMs), a separate population of lung macrophages found apposed to the endothelium of pulmonary capillaries. We compared these two populations of lung immunocytes to determine their relative immunological activity. Our results suggest that PAMs are more phagocytic than PIMs; however, PIMs may be more efficient at lysing ingested bacteria than PAMs. Although similar in antibody-dependent cellular cytotoxicity, PIMs are more spontaneously cytolytic than PAMs. Depending upon the effector:target cell ratio studied, the tumoricidal activity of PIMs was less than or equal to that of PAMs. Both cell populations produced the cytokines interleukin-1 and tumor necrosis factor-alpha at similar concentrations. These data suggest that PIMs are immunologically active, although the degree of activity may differ between PIMs and PAMs.

Animals↗
PubMed1991

The relationship between the in vitro chemosensitivity of tumor cells and tumor response in vivo in an experimental tumor model.

Cell lines derived from a panel of five histologically distinct murine adenocarcinomas of the colon (MAC) were used to assess whether or not a colony-forming assay could have retrospectively predicted the wide range of in vivo responses to chlorambucil (CHL). The predictive value of the clonogenic assay was significantly improved when fractions (one-tenth) of the plasma drug AUC (from the area under the drug clearance curves); were used to determine clonogenic cell kill in vitro, instead of one-tenth peak plasma drug concentration and total plasma drug AUC exposures. Despite the good correlation between in vitro and in vivo responses observed, the clonogenic assay could not forecast the site-dependent response of MAC 15A to CHL. These site-dependent responses cannot be explained in terms of the inherent sensitivity of tumor cells themselves, suggesting that caution must be applied in the interpretation of in vitro chemosensitivity assays.

Animals↗
PubMed1991

Anti-tumour activity of flavone acetic acid (NSC 347512) in mice--influence of immune status.

Flavone acetic acid (FAA) is a synthetic flavonoid with dramatic pre-clinical anti-tumour activity involving a vascular component in its mechanism but no clinical effects have been seen to date. As FAA also has immunomodulatory activity, immunological factors might explain differences in activity between mouse and man. This study examines the influence of host immune status on the anti-tumour activity of FAA. Two human colon tumour xenografts (COBA, HT-29) fail to respond to FAA in nude mice. The lack of activity of FAA against HT-29 xenografts cannot be explained on the basis of limited drug bioavailability as achievable plasma, and tumour levels of FAA are similar to those seen in sensitive murine colon tumours. The immune status of the host also influences the activity of FAA against two transplantable tumours of the mouse colon. Both these tumours are highly responsive to FAA in their normal NMRI hosts, but neither tumours exhibited significant growth delay in thymectomised NMRI or nude hosts. Histological examination of treated tumours revealed significant areas of haemorrhagic necrosis in all three hosts. These data suggest a clear immunological component in the mechanism of action of FAA which is separate from the previously described haemorrhagic necrosis.

Adenocarcinoma↗
PubMed1991

The relationship between tissue levels of flavone acetic acid (NSC 347512) and site dependent anti-tumour activity in murine colon tumours.

Flavone acetic acid (FAA) is extremely active against subcutaneous transplantable tumours in mice, but disappointingly there has been no demonstrable clinical activity. Previous studies have shown that lung tumour deposits are less responsive than the same cells implanted subcutaneously. The aim of this study is to examine the tissue disposition of FAA in an attempt to explain this site-dependent activity. The data show clearly that FAA clearance curves are influenced by the presence of MAC 15A tumours growing either subcutaneously or systemically. The decreased clearance of FAA from MAC 15A tumour bearing animals does not however explain the resistance of lung deposits. Neither can this be explained by differences in metabolism in these different sites. Cytotoxic metabolites have not been detected either in vitro or in vivo and their role in the mechanism of action of FAA is questionable.

Adenocarcinoma↗
PubMed1991

The effect of neurotoxin on rabies virus binding to mouse neuroblastoma cells.

Mouse neuroblastoma cells were exposed to alpha bungarotoxin, a neurotoxin known to inhibit rabies virus binding to the nicotinic acetylcholine receptor located at the neuromuscular junction in muscle tissue. The total amount of 3H-CVS virus that bound to neurotoxin treated cells was separated into specific and non-specific binding using a cold competition assay. Comparison of untreated and neurotoxin treated cells demonstrated that the majority of cell-associated virus in untreated cells was of a specific nature whereas the majority of the cell-associated virus in neurotoxin treated cells was due to non-specific binding.

Animals↗