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Biomedical subjects

T J Holmes

Publications and source records attributed to T J Holmes.

At least 19 recordsLinked to original sources
PubMed2001

Differential effects of non-genotoxic carcinogens and proliferating agents on cell growth, survival and apoptosis in hepatic cells in vitro.

Many nongenotoxic carcinogen's (ngc) produce hyperplastic lesions from which neoplastic foci may arise. Modulation of the rate of apoptosis by some ngc's within these lesions may be critical to their mechanism of tumour promotion but some may be cytotoxic. To establish if these compounds are apoptotic or necrotic in vitro, three ngc's (12-0-tetradecanoyl phorbol-13-acetate (TPA); nickel, and di(2-ethylhexyl-phthalate (DEHP), two noncarcinogenic hepatoproliferating agents (1,4-dichlorobenzene (DCB; HGF) and an in vitro genotoxic reference compound (7-hydroxy-2-acetylaminofluorene (70H2AAF) were used to induce mitogenic or growth responses in two liver cell-lines HepG2 and JTC-15. MTT and 3H-thymidine incorporation assays were used to measure cell growth and DNA replicative activity respectively. Rates of apoptosis were assayed using FITC-annexin V with propidium iodide staining and flow cytometry. Responses in HepG2 cells were HGF (proliferation at > or = 3 ng/ml), TPA (cell growth at > or = 8 ng/ml), DEHP (proliferation at > or = 0.05 microg/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.001 microg/ml and 100 ng/ml respectively. An equivocal result was obtained for DCB. Responses in JTC-15 cells were HGF (proliferation, 3 ng/ml), TPA (DNA replication, 10 ng/ml), and DEHP (cell mass, 2.5 microl/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.01 microg/ml and 110 mg/ml respectively. Equivocal results were obtained for DCB. In flow cytometry assays apoptotic and necrotic populations were not clearly separable. Approximate rates of apoptosis in HepG2 were: control 8.7%; DEHP, 10.19%. NiCl2, 12.67%; 70H2AAF, 16.56%; TPA, 19.72%; HGF, 23.73%; DCB, 24.59%; positive apoptotic control (taxol) 26.94%. These data show apoptosis was increased in chemically activated populations of HepG2. The ngc, DEHP, unexpectedtly produced proliferation in HepG2 and almost totally suppressed apoptosis in vitro in HepG2 relative to the non-carcinogenic hepatoproliferators. The rate of apoptosis induced by the ngc TPA was not considered to be sufficiently different to the rates of apoptosis induced by the noncarcinogenic hepatoproliferators. The results emphasize the importance of considering necrotic reactions from effects on apoptosis in detecting non-genotoxic carcinogens.

Apoptosis↗
PubMed2000

Case of enantiomer impurity identification by normal-phase chiral high-performance liquid chromatography with optical rotation and mass spectrometric detection.

An impurity produced in the synthesis of compound I is separated and identified as its enantiomer II using normal-phase chiral high-performance liquid chromatography (HPLC) with UV absorbance, optical rotation (OR) and mass spectrometric (MS) detection. The results show that the impurity II and compound I have equal and opposite specific rotations, identical MS spectra and the same MS-MS fragmentation pattern, as required for enantiomers. The procedures presented demonstrate a novel combination of methods for enantiomer identification and characterization that do not require the preparation of individual enantiomer markers or even the racemic mixture, thus reducing the need for additional synthetic work.

Chromatography, High Pressure Liquid↗
PubMed2000

Blind deconvolution of 3D transmitted light brightfield micrographs.

The blind deconvolution algorithm for 3D transmitted light brightfield (TLB) microscopy, published previously [Holmes et al. Handbook of Biological Confocal Microscopy (1995)], is summarized with example images. The main emphasis of this paper is to discuss more thoroughly the importance and usefulness of this method and to provide more detailed evidence, some being quantitative, of its necessity. Samples of horseradish peroxidase (HRP)-stained pyramidal neurones were prepared and evaluated for the ability to see fine structures clearly, including the dendrites and spines. It is demonstrated that the appearance of fine spine structure, and means of identifying spine categories, is made possible by using blind deconvolution. A comparison of images of the same sample from reflected light confocal microscopy, which is the conventional light microscopic way of viewing the 3D structure of these HRP-stained samples, shows that the blind deconvolution method is far superior for clearly showing the structure with less distortion and better resolution of the spines. The main significance of this research is that it is now possible to obtain clear images of 3D structure by light microscopy of absorbing stains. This is important because the TLB microscope is probably the most widely used modality in the life-science laboratory, yet, until now, there has been no reliable means for it to provide visualization of 3D structure clearly. The main importance of the blind deconvolution approach is that it obviates the need to measure the point spread function of the optical system, so that it now becomes realistic to provide a 3D light microscopic deconvolution method that can be pervasively used by microscopists.

Algorithms↗
PubMed1998

Accuracy of nuclear classification in cervical smear images. Quantitative impact of computational deconvolution and 3-D feature computation.

OBJECTIVE: To investigate the accuracy with which the nuclei of cells in overlapped and thick clusters in cervical/ vaginal smears can be classified independent of the segmentation algorithm used and to determine the influence of three-dimensional (3-D) processing as compared to two-dimensional (2-D) methods on classification of the nuclei. STUDY DESIGN: Cell clusters were imaged from 31 ThinPrep smears composed of 808 nuclei, of which 420 were determined to be abnormal by a cytotechnologist. Sets of 2-D and 3-D volumetric features of the detected nuclei were formulated, and classifiers were constructed. The effect of computational deconvolution on classification was assessed using nearest-neighbor and Wiener filter in 2-D and 3-D before calculating features. A "best focus plane" was calculated for each nucleus from the 3-D data set, and the 2-D features in this plane were also analyzed.

Algorithms↗
PubMed1996

Articular cartilage volume in the knee: semiautomated determination from three-dimensional reformations of MR images.

PURPOSE: To determine the accuracy of semiautomated quantification of articular cartilage volume from three-dimensional (3D) reformations of magnetic resonance (MR) images. MATERIALS AND METHODS: Sagittal, fat-suppressed, 3D, spoiled gradient-recalled-echo MR imaging of two bovine and two human cadaver knees was performed. Articular cartilage volume was calculated from 3D reformations of the MR images by using a semiautomated program written at the authors' institution. Calculated volumes were compared with directly measured volumes of the surgically removed articular cartilage. RESULTS: The percentage of error of the MR imaging-determined volumes was 6.53% +/- 4.75 (mean +/- standard deviation). A strong correlation between the two sets of observations was shown (r=.997). Linear regression showed the calculated volumes to be highly accurate (slope=1.002, P>.25). Repeated reformations yielded volumes that were reproducible (mean absolute error, 0.013 mL +/- 0.019) and not significantly different from the measured volume (P>.10). CONCLUSION: Semiautomated quantification of knee articular cartilage from MR images yields highly accurate cartilage volumes.

Animals↗
PubMed1994

Three-dimensional imaging and image analysis of hippocampal neurons: confocal and digitally enhanced wide field microscopy.

The microscopy of biological specimens has traditionally been a two-dimensional imaging method for analyzing what are in reality three-dimensional (3-D) objects. This has been a major limitation of the application of one of science's most widely used tools. Nowhere has this limitation been more acute than in neurobiology, which is dominated by the necessity of understanding both large- and small-scale 3-D anatomy. Fortunately, recent advances in optical instrumentation and computational methods have provided the means for retrieving the third dimension, making full 3-D microscopic imaging possible. Optical designs have concentrated on the confocal imaging mode while computational methods have made 3-D imaging possible with wide field microscopes using deconvolution methods. This work presents a brief review of these methods, especially as applied to neurobiology, and data using both approaches. Specimens several hundred micrometers thick can be sampled allowing essentially intact neurons to be imaged. These neurons or selected components can be contrasted with either fluorescent, absorption, or reflection stains. Image analysis in 3-D is as important as visualization in 3-D. Automated methods of cell counting and analysis by nuclear detection as well as tracing of individual neurons are presented.

Animals↗
PubMed1993

Developments in three-dimensional stereo brightfield microscopy.

We present recent developments of a widefield computer/microscope system and image reconstruction algorithm for producing three-dimensional (3D) increased depth of field images in the form of brightfield stereo pairs of thick specimens. The theoretical principle of this image reconstruction technique is based on Weiner-type inverse filtering. A number of extensions and refinements to our previous work have included further testing of the system with a broader class of specimens and the implementation of several pragmatic refinements important for future 3D microscopy systems. These refinements include histogram modification routines for improving visualization, a preprocessing routine to eliminate edge artifacts due to circular convolution and other effects, stereo viewing angle optimization, a rule of thumb estimate for the axial sampling rate, and incorporation of a variation of the Fast Fourier Transform and filtering operations that significantly reduce computational time. Images of spyrogyra, neonatal rat hippocampal neurons, and cervical/vaginal cell smears are presented to show the utility of these methods for 3D visualization. The primary advantages of these methods are that they operate with an ordinary transmitted light microscope and are inexpensively implemented on a personal computer with reasonable computation time.

Algorithms↗
PubMed1993

Novel 6-alkoxypurine 2',3'-dideoxynucleosides as inhibitors of the cytopathic effect of the human immunodeficiency virus.

Twenty-one 6-alkoxypurine 2',3'-dideoxynucleosides were enzymatically synthesized with nucleoside phosphorylases purified from E. coli. Eighteen analogs exhibited anti-HIV-1 activity in MT4 cells. Two analogs, 6-(hexyloxy)-(17) and 6-(heptyloxy)-(18) purine 2',3'-dideoxynucleoside, were as potent as 2',3'-dideoxyinosine (ddI, didanosine, Videx). Although the antiviral activities of 17 and 18 were equivalent, 18 was more cytotoxic. Analogs containing less than four carbons in the 6-alkoxypurine substituent exhibited weak anti-HIV-1 activity. Analogs containing more than seven carbons in the 6-alkoxypurine substituent were too cytotoxic to be effectively evaluated for antiviral activity. Several 6-alkoxypurine 2',3'-dideoxynucleosides were evaluated for substrate activity with calf intestinal adenosine deaminase (ADA). Increasing the carbon chain length of the 6-alkoxypurine substituent decreased the rate of dealkoxylation. The best substrate in this series was 6-methoxypurine 2',3'-dideoxynucleaside (1); however, the rate of dealkoxylation of 100 microM 1 was 0.17% of the rate of deamination of 100 microM 2',3'-dideoxyadenosine. Compound 17, the most potent anti-HIV-1 analog, was not a substrate for ADA. EHNA (erthro-9-(2-hydroxy-3-nonyl)adenine), a potent inhibitor of ADA, had little effect on the antiviral activities of 17 and ddI. In contrast, coformycin, a potent inhibitor of both ADA and AMP deaminase, dramatically decreased the antiviral activity of 17, but not the antiviral activity of ddI. Thus, AMP deaminase appeared to be involved in the anabolism of 17. The pharmacokinetic profile of 17, the most promising analog in this series, was determined in the rat. At least seventeen metabolites of 17, including ddI, were detected in plasma samples. This analog also had poor oral bioavailability.

Adenosine Deaminase↗
PubMed1993

Iterative, constrained 3-D image reconstruction of transmitted light bright-field micrographs based on maximum likelihood estimation.

We present several image reconstruction algorithms for generating three-dimensional (3-D) renderings of bright-field micrographs that are founded on maximum likelihood estimation (MLE) theory. The basic principle of the algorithms is in estimating the values of the optical densities of the specimen. A computer simulation and initial experimental testing of a steepest ascent version of the algorithm is presented. The computer simulation demonstrates that the MLE algorithm has an advantage over previously used inverse filtering techniques in that it partially restores the zeroed Fourier components in the well-known missing-cone region. We present 3-D reconstructions from real biological data to show the potential of the algorithm in practical applications.

Algorithms↗
PubMed1993

Automated three-dimensional image analysis of thick and overlapped clusters in cytologic preparations. Application to cytologic smears.

Methods are presented for automated analysis of thick and heavily overlapped regions of cytologic preparations, such as cervical/vaginal smears. Current systems are unable to process these regions although they contain diagnostically valuable information. We argue that analysis of such regions is inherently a three-dimensional (3-D) problem that cannot be solved reliably with conventional two-dimensional methods. Furthermore, this issue cannot be side-stepped by special thin preparation methods. Even with 3-D imaging, analysis of these regions is complicated by the high variability in the image gray level and textural features resulting from the uncontrollable cell overlaps and folds and large computational requirements. A novel approach based on 3-D imaging and adaptive 3-D analysis algorithms based on the principles of localization, adaptive data reduction and clustering theory is presented. It was successful in detecting and separating deeply embedded and overlapping nuclei, cytoplasmic folds and creases in thick and overlapped regions of conventional smears and special thin preparations.

Cytological Techniques↗
PubMed1992

Stereo pairs of bright-field micrographs via Wiener-type inverse filtering.

In a three-dimensional (3-D) image data set obtained through optical sectioning, each two-dimensional (2-D) segment is blurred by out-of-focus information from neighbouring focal planes superimposed on the in-focus segments from that plane. Instead of attempting to remove this redundant information over the full 3-D data set, we have developed a technique for restoring stereoscopic views. In this paper we describe the implementation of a Wiener-type inverse filtering method for generating stereo pairs of bright-field micrographs. A theoretical optical transfer function valid under certain simplifying approximations has been used in implementing this filtering technique. In developing this method the slice theorem of computed tomography is used. In this way the image reconstruction problem is reduced to one of processing 2-D arrays rather than 3-D arrays and the problem of restoring missing Fourier components within the missing-cone region is circumvented. Limited experimentation with real micrographs shows that the approach provides images that display an effective increased depth of field and 3-D attributes of the specimen, even though some of the underlying assumptions on which this method is based are difficult to verify explicitly. The method can be implemented with a relatively fast execution time on 386-SX computers.

Fourier Analysis↗
PubMed1992

Blind deconvolution of quantum-limited incoherent imagery: maximum-likelihood approach.

Previous research presented by the author and others into maximum-likelihood image restoration for incoherent imagery is extended to consider problems of blind deconvolution in which the impulse response of the system is assumed to be unknown. Potential applications that motivate this study are wide-field and confocal fluorescence microscopy, although applications in astronomy and infrared imaging are foreseen as well. The methodology incorporates the iterative expectation-maximization algorithm. Although the precise impulse response is assumed to be unknown, some prior knowledge about characteristics of the impulse response is used. In preliminary simulation studies that are presented, the circular symmetry and the band-limited nature of the impulse response are used as such. These simulations demonstrate the potential utility and present limitations of these methods.

Algorithms↗
PubMed1987

Prostaglandin-H synthase inhibition by malonamides. Ring-opened analogues of phenylbutazone.

Recent reports of serious concern regarding the safe clinical use of phenylbutazone and its hydroxylated metabolite (oxyphenbutazone) as antiinflammatory agents have prompted the further investigation of ring-opened (malonamide) derivatives as potentially preferable therapeutic derivatives. Earlier reports have claimed reduced toxicity among similar derivatives. These studies reveal the relative degree of prostaglandin-H (PGH) synthase inhibitory activity among a series of malonamide derivatives. Contrary to observations in the pyrazolidinedione series, incorporation of a nonpolar butyl side chain in these malonamides was not beneficial but, rather, detrimental to enzyme-inhibitory activity. Although none of the reported nonbutylated malonamides was as potent an inhibitor of this enzyme as phenylbutazone, they all showed some inhibitory activity. PGH synthase inhibitory activity was especially pronounced in the bis(p-hydroxy anilide) derivatives, even extending to succinamide and adipamide derivatives. Of some interest is the observation that all of these p-hydroxy anilide derivatives were more potent inhibitors of this enzyme than acetaminophen.

Amides↗
PubMed1987

Preparation and evaluation of electrophilic derivatives of phenylbutazone as inhibitors of prostaglandin-H synthase.

The chemical syntheses and biological evaluation of several potential irreversible inhibitors for prostaglandin (PGH) synthase are described. These inhibitors were modeled after the nonsteroidal antiinflammatory (NSAI) drug phenylbutazone (4-n-butyl-1,2-diphenyl-3,5-pyrazolidinedione). Electrophilic functionalities such as an alpha-bromoacetamide, an alpha-chloroacetamide, a phenylurethane, a propargyl chloride, and several alpha,beta-unsaturated Michael acceptors were incorporated at the 4-position of the pyrazolidinedione ring structure. None of the derivatives showed evidence of irreversible inhibition of PGH synthase, although several were nearly as potent inhibitors of this enzyme as phenylbutazone. The nitrile obtained from 1,4-conjugate addition of cyanide to one of the unsaturated derivatives was considerably more potent as an inhibitor of PGH synthase than was phenylbutazone.

Animals↗
PubMed1986

Inhibition of platelet aggregation by novel triphenylethylene analogs.

The present study has evaluated the effect of some newly synthesized triphenylethylene (TPE) analogs on platelet arachidonic acid metabolism and function. All compounds tested inhibited arachidonic acid induced platelet aggregation and several were superior to aspirin in their relative potency. Introduction of a carboxyl function into the alpha-ring, which should enhance binding according to proposed structural models for cyclooxygenase inhibitors, was not found to be beneficial. Increased structural rigidity, which resulted from covalent linkage of two aromatic rings in this series, did not eliminate anti-aggregatory properties.

Adult↗
PubMed1985

Inhibition of cyclooxygenase mediated by electrochemical oxidation of gentisic acid.

Prostaglandin synthase (EC 1.14.99.1, 8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase), commonly referred to as cyclooxygenase, was inhibited irreversibly upon application of a fixed oxidative potential (+0.4 V versus saturated calomel electrode) in the presence of the aspirin metabolite gentisic acid (2,5-dihydroxybenzoic acid). Electrolyses were carried out at 0 degrees C in a phosphate buffer solution (pH 7.2, 0.1 M) using a carbon felt electrode. This electroinactivation process was time-dependent and pseudo first-order with respect to gentisic acid at concentrations up to 300 microM. These concentrations of gentisic acid are below those normally reported to be inhibitory. The enzyme was stable to this applied potential in the absence of gentisic acid. Similar treatment of apoenzyme (heme-removed) revealed no loss in catalytic activity after reconstitution to the holoenzyme. Oxyphenbutazone, a nonoxidizable competitive inhibitor of cyclolooxygenase, was observed to protect the enzyme from electrolytic inactivation mediated by gentisic acid. Radiolabeling studies indicated the covalent attachment of approximately 1 eq of gentisic acid/subunit of enzyme. These studies support the possible role of quinonoid intermediates in the observed anti-inflammatory action of salicylate derivatives.

Animals↗
PubMed1984

Electrolysis-mediated irreversible inactivation of lipoxygenase directed toward electroaffinity labelling.

Irreversible inhibition of soybean lipoxygenase-1 (SL-1) was accomplished via a controlled potential oxidative electrolysis of 1,5-dihydroxynaphthalene (1,5-DHN) at +0.8 V vs SCE. The inactivation of SL-1 with this known inhibitor was greatly enhanced under these electrolytic conditions to which the enzyme itself was stable. Electrolyses were run at 0 degree C in a 0.05 M phosphate buffer, pH 7.0, using graphite cloth electrodes. The rate of inactivation was observed to be limited by and dependent on the anodic oxidation of 1,5-DHN. The non-oxidizable (at this potential) inhibitor indomethacin was shown to protect the enzyme from irreversible inactivation, however, an external nucleophile (2-mercaptoethanol) had little effect. These initial studies support the capability of such electrochemical methods for the site-specific covalent modification (affinity labelling) of lipoxygenase, and perhaps other enzymes.

Electrochemistry↗