Biomedical subjects

J P O'Connell

Publications and source records attributed to J P O'Connell.

At least 19 recordsLinked to original sources

PubMed2001

Electric manipulation of bioparticles and macromolecules on microfabricated electrodes.

Bioparticle separation, bioparticle enrichment, and electric field-mediated immune detection were carried out on microfabricated semiconductor chips utilizing ac and dc electric fields. Microscale separation on a chip surface having an active area of approximately 16 mm2 was demonstrated for a mixture of Bacillus globigii spores and Escherichia coli bacteria. Dielectrophoretic enrichment was performed by collecting target bioparticles from a flow stream in flow cells of 47.5 microL, achieving a 20-fold increase in the concentration of E. coli bacteria from a diluted sample, a 28-fold enrichment for peripheral blood mononuclear cells from red blood cells, and a 30-fold increase in white blood cells from diluted whole blood. The ability to manipulate and collect bioparticles and macromolecules at microfabricated electrodes with ac and dc fields was further illustrated in electric field-mediated immunoassays for analyzing the biological identities of E. coli bacteria and B. globigii spores. According to these results, the electric methods for manipulating bioparticles present themselves as viable techniques for novel biomedical applications in sample preparations and biochemical assays on microelectrode arrays.

Bacillus↗

PubMed2001

Protein unfolding during reversed-phase chromatography: II. Role of salt type and ionic strength.

While reversed-phase chromatography (RPC) may be a powerful method for purification of proteins at the analytical scale, both preparative and analytical applications have been hindered by the complex chromatographic behavior of proteins compared to small molecules. Further, preparative applications have been limited because of poor yields caused by the denaturing conditions involved. One means for modulating both the stability and chromatographic behavior of proteins is through the use of added salt. In this investigation, we show how salt type and ionic strength affect protein conformation on RPC surfaces. Exposure of amide groups of adsorbed BPTI was monitored using nuclear magnetic resonance (NMR) spectroscopy and hydrogen-deuterium isotope exchange. Sodium chloride, sodium acetate, and ammonium sulfate were studied at ionic strengths up to I = 0.375, with adsorption hold times being 5 min and 2 h. We found that increasing ionic strength decreased exposure of the exchange reporter groups in essentially all cases. However, even at the same ionic strength the level and distribution of residue protection varied with salt type and hold time. NaCl does not protect certain reporter groups at all, while those that it does protect to some degree at short hold times can exchange slightly more at longer times. The pattern and level of protection for NaAc at short times is similar to that for NaCl, but at longer times more uniform protection is seen as the reporter groups completely exposed at short times become more protected. For (NH(4))(2)SO(4) the pattern of protection at short hold time is similar to those of the other salts, although it protects all groups much more. This would be expected from the Hofmeister series. However, at longer times the level of protection with (NH(4))(2)SO(4) decreases below that of the other salts, while it uniquely protects all groups to nearly the same level. Such subtle variations in the protein structure would not have been detected without the measurements and analysis used here. Chromatographic retention times and peak shapes were obtained for the above systems. Variations of behavior were seen that could not be correlated with any of the above protection patterns and levels or even with heuristics such as the Hofmeister series. This suggests further conformational changes upon elution may be critical to the retention process. However, an excellent correlation was found between peak width at half-height and the average degree of unfolding, as indicated by the average level of isotopic exchange. Thus, while further studies are needed to definitively determine the connection between protein unfolding and retention, use of this correlation may improve designing and screening for chromatographic conditions that minimize protein unfolding.

Adsorption↗

PubMed2001

Antimicrobial resistance and bacterial identification utilizing a microelectronic chip array.

Species-specific bacterial identification of clinical specimens is often limited to a few species due to the difficulty of performing multiplex reactions. In addition, discrimination of amplicons is time-consuming and laborious, consisting of gel electrophoresis, probe hybridization, or sequencing technology. In order to simplify the process of bacterial identification, we combined anchored in situ amplification on a microelectronic chip array with discrimination and detection on the same platform. Here, we describe the simultaneous amplification and discrimination of six gene sequences which are representative of different bacterial identification assays: Escherichia coli gyrA, Salmonella gyrA, Campylobacter gyrA, E. coli parC, Staphylococcus mecA, and Chlamydia cryptic plasmid. The assay can detect both plasmid and transposon genes and can also discriminate strains carrying antibiotic resistance single-nucleotide polymorphism mutations. Finally, the assay is similarly capable of discriminating between bacterial species through reporter-specific discrimination and allele-specific amplification. Anchored strand displacement amplification allows multiplex amplification and complex genotype discrimination on the same platform. This assay simplifies the bacterial identification process greatly, allowing molecular biology techniques to be performed with minimal processing of samples and practical experience.

Anti-Bacterial Agents↗

PubMed1998

Isolation of cultured cervical carcinoma cells mixed with peripheral blood cells on a bioelectronic chip.

The separation and subsequent isolation of the metastatic human cervical carcinoma cell line (HeLa cells) from normal human peripheral blood cells has been achieved by exploiting their differential dielectric properties. The isolation process is carried out on a silicon chip containing a five-by-five array of microlocations. These microlocations contain underlying circular platinum electrodes with 80-micron diameters and center-to-center spacing of 200 microns. The surfaces of the electrodes and nonmetallized areas have been coated with a permeation layer to prevent the direct contact of cells with the electrode and also to minimize the nonspecific adhesion of the cells to the chip surface. An inhomogenous ac field is applied to the electrodes to create the conditions for dielectrophoretic separation of cells. Cell separation using dielectrophoresis as well as electronic lysis on a silicon chip would provide essential sample-processing steps which may be combined with a later multiplex electronic hybridization step in an integrated assay system.

Blood Cells↗

PubMed1998

Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips.

Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip. An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes. The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns. After the isolation, the bacteria were lysed by a series of high-voltage pulses. The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA. The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips. Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.

Biosensing Techniques↗

PubMed1998

Matrix metalloproteinases and metastatic cancer.

The rationale for matrix metalloproteinase (MMP) inhibition as a means to treat disease progression in breast cancer stems from the apparent involvement of MMPs in the hydrolysis of basement membranes during tumour cell invasion and subsequent metastasis. MMP-mediated matrix remodelling also appears to promote the growth of tumour cells, possibly by facilitating the proliferation and migration of endothelial cells and the neovascularization of tumour tissue. We found that transfection of the C127 breast cancer cell line by MMP-2 (gelatinase A), but not by MMP-1 or MMP-3 (collagenase and stromelysin respectively), gave rise to an invasive and metastatic phenotype. We were surprised to find that this phenotype depended not only on the catalytic properties of MMP-2 but also on properties associated with the MMP-2 non-catalytic C-terminal domain. Experiments with a synthetic gelatinase inhibitor revealed that a single dose could prevent the lungs of nude mice being colonized by the MMP-2 transfectants, and that the inhibitor had to be administered during or shortly after injection of the cells, indicating that an early event, such as the extravasation of the cells into the lung, is gelatinase-dependent in this system. In other studies employing long-term treatment with CT1746, an orally active gelatinase inhibitor, we have previously demonstrated a reduction in primary tumour growth rates, localized spread, and spontaneous metastasis, even when the treatment was commenced several days after tumour implantation. Furthermore, additive effects were recorded when gelatinase inhibitor therapy was combined with cytotoxic drug treatment. Since the gelatinase inhibitors can also inhibit bone resorption in vitro, these observations point to their potential for delaying disease recurrence and reducing rates of bone loss following conventional therapeutic strategies, in metastatic breast cancer.

Animals↗

PubMed1997

Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control.

We have demonstrated that controlled electric fields can be used to regulate transport, concentration, hybridization, and denaturation of single- and double-stranded oligonucleotides. Discrimination among oligonucleotide hybrids with widely varying binding strengths may be attained by simple adjustment of the electric field strength. When this approach is used, electric field denaturation control allows single base pair mismatch discrimination to be carried out rapidly (<15 sec) and with high resolution. Electric field denaturation takes place at temperatures well below the melting point of the hybrids, and it may constitute a novel mechanism of DNA denaturation.

Bacterial Proteins↗

PubMed1996

Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite.

Peroxynitrite (ONOO-) has recently been implicated in connective tissue destruction in vivo. We have studied the effect of ONOO- on the activity of tissue inhibitor of metalloproteinase-1 (TIMP-1) in vitro. The inactivation of TIMP-1 by ONOO- was dose dependent with 50 microM ONOO- reducing the inhibitory activity of TIMP-1 towards gelatinase-A by 50%. High concentrations of ONOO- (500 microM-5 mM) caused protein fragmentation whilst lower concentrations (<250 microM) inactivated TIMP-1 without altering the molecular weight. Inactivation could be blocked by ONOO- scavengers but not by hydroxyl radical scavengers. Our results show that ONOO- is capable of inactivating TIMP-1, a process which could potentiate metalloproteinase-mediated tissue breakdown.

Amino Acid Sequence↗

PubMed1995

Inhibition of bone resorption in vitro by selective inhibitors of gelatinase and collagenase.

Two low-molecular-mass inhibitors of matrix metalloproteinases (MMPs), CT1166, a concentration-dependent selective inhibitor of gelatinases A and B, and Ro 31-7467, a concentration-dependent selective inhibitor of collagenase, were examined for their effects on bone resorption and type-I collagenolysis. The test systems consisted of measuring (1) the release of [3H]proline from prelabelled mouse calvarial explants; (2) the release of 14C from prelabelled type-I collagen films by mouse calvarial osteoblasts; and (3) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. In 24 h cultures, CT1166 and Ro 31-7467 inhibited both interleukin-1 alpha- (IL-1 alpha; 10(-10) M) and 1,25-dihydroxyvitamin D3 (10(-8) M)-stimulated bone resorption in cultured neonatal mouse calvariae at concentration selective for the inhibition of gelatinase (10(-9) M for CT1166) and collagenase (10(-8) M for Ro 31-7467) respectively. For each compound the inhibition was dose-dependent, reversible, and complete at a 10(-7) M concentration. However, CT1166 (10(-9) M) and Ro 31-7467 (10(-8) M) in combination were required to completely abolish IL-1 alpha-stimulated bone resorption in mouse calvariae throughout a 96 h culture period. Neither of the inhibitors affected protein synthesis, DNA synthesis nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme, beta-glucuronidase. Both CT1166 and Ro 31-7467 partially inhibited IL-1 alpha-stimulated lacunar resorption by isolated osteoclasts, but were without effect on unstimulated lacunar resorption. Rodent osteoclasts produced collagenase and gelatinases-A and -B activity. In contrast the substrate used to assess osteoclast lacunar resorption contained no detectable collagenase or gelatinase activity. Both compounds dose-dependently inhibited 1,25-dihydroxyvitamin D3 (10(-8) M)-stimulated degradation of type-I collagen by mouse calvarial osteoblasts; however, complete inhibition of collagenolysis was only achieved at concentrations at which CT1166 and Ro 31-7467 act as general MMP inhibitors. This study demonstrates that collagenase and gelatinases A and/or B participate in bone resorption. While these MMPs may be primarily involved in osteoid removal, we conclude that they may also be released by osteoclasts, where they participate in bone collagen degradation within the resorption lacunae.

Amides↗

PubMed1994

Reciprocated matrix metalloproteinase activation: a process performed by interstitial collagenase and progelatinase A.

Gelatinase A, a member of the matrix metalloproteinase (MMP) family, is secreted possessing an 80 amino acid N-terminal propeptide that must be removed in order to generate the active enzyme. Purified progelatinase A was activated to 38% of maximum by a 6 h incubation at 37 degrees C with equimolar concentrations of trypsin-activated interstitial collagenase (another MMP). The increase in activity was accompanied by cleavage of the M(r) 72,000 progelatinase A to the M(r) 66,000 active enzyme that has Y81 as its N-terminus. At low concentrations, progelatinase A was processed via an inactive intermediate, suggesting that its activation is a biphasic process. This was confirmed by the action of collagenase on proE375-->A (a mutant of progelatinase A that cannot become active) because, in this instance, only an M(r) 68,000 species with L38 as the N-terminus was produced. The remaining propeptide amino acids to Y81 could be readily removed by added active gelatinase A, indicating that collagenase works by generating an intermediate that is susceptible to autolytic activation. Although relatively slow, the rate of activation could be increased approximately 10-fold by the addition of 100 micrograms/mL heparin. This binds to the C-terminal domain of collagenase and progelatinase A and presumably acts as a template that positions the reactants close to one another. Collagenase activated by trypsin retains 8 or 14 amino acids of its propeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Mutation of the active site glutamic acid of human gelatinase A: effects on latency, catalysis, and the binding of tissue inhibitor of metalloproteinases-1.

Human gelatinase A, a member of the matrix metalloproteinase family, is secreted from cells as the M(r) 72,000 latent precursor, progelatinase A. The autolytic removal of an N-terminal propeptide generates the M(r) 66,000 active form. Mutants of recombinant progelatinase A, altered such that the proposed active site glutamic acid residue (E375) was replaced by either an aspartic acid (proE375-->D), an alanine (proE375-->A) or a glutamine (proE375-->Q), were purified from medium conditioned by transfected NS0 mouse myeloma cells. Like wild-type progelatinase A, the mutant proenzymes were inactive and could bind tissue inhibitor of metalloproteinases (TIMP)-2 but not TIMP-1 to their C-terminal domains. Their rates of autolytic processing induced by the organomercurial (4-aminophenyl) mercuric acetate, however, were markedly slower and, of the three M(r) 66,000 forms so produced, only E375-->D displayed any proteolytic activity against either a synthetic substrate (kcat/Km = 10% that of the wild-type enzyme) or denatured type I collagen (specific activity = 0.9% that of the wild-type enzyme). ProE375-->A and proE375-->Q could be more rapidly processed to their M(r) 66,000 forms by incubation with a deletion mutant of gelatinase A that has full catalytic activity but lacks the C-terminal domain [delta (418-631) gelatinase A]. These two M(r) 66,000 forms displayed low activity on a gelatin zymogram (approximately 0.01% that of the wild-type enzyme) but, like E375-->D were able to bind TIMP-1 with an affinity equal to that of the activated wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Analysis of the role of the COOH-terminal domain in the activation, proteolytic activity, and tissue inhibitor of metalloproteinase interactions of gelatinase B.

Recombinant human progelatinase B and a COOH terminally truncated version, pro-delta426-688 gelatinase B have been prepared from a myeloma cell expression system. Both proenzymes could be processed to active forms by stromelysin-1 to give an NH2 terminus of Phe88, or by treatment with 4-aminophenylmercuric acetate resulting in an NH2-terminal Met75. The kinetics of activation using either treatment was not affected by removal of the enzyme COOH-terminal domain. The specific activities of both gelatinase B and delta426-688 gelatinase B, activated using either method, were found to be similar using either a quenched fluorescent peptide or gelatin as the substrate. Fibroblast monolayers were shown to mediate processing of both progelatinases at similar rates in the presence of either plasminogen or prostromelysin-1. Active wild-type gelatinase B was inhibited by tissue inhibitor of metalloproteinase (TIMP) -1 at a much faster rate than TIMP-2. COOH-terminal truncation of either enzyme or inhibitor gave a marked reduction in the rate constant for TIMP-1 inhibition but had no effect on the rate of TIMP-2 binding. It can be concluded that the COOH-terminal domain of progelatinase B is not involved in autolytic or cellular activation and does not affect the catalytic activity of the enzyme. However, COOH-terminal domain interactions between active gelatinase B and TIMP-1 significantly enhance the rate of complex formation.

Amino Acid Sequence↗

PubMed1993

On the role of the C-terminus of alpha-calcitonin-gene-related peptide (alpha CGRP). The structure of des-phenylalaninamide37-alpha CGRP and its interaction with the CGRP receptor.

alpha-Calcitonin-gene-related peptide (alpha CGRP) lacking its C-terminal phenylalaninamide residue was found not to bind to its receptor as did full-length (amidated) alpha CGRP. Investigation of the structure of these peptides by c.d. and n.m.r. revealed no significant difference, so it seemed that the effect of deleting the C-terminal phenylalaninamide on the biological activity of alpha CGRP was not by disruption of the peptide's structure. Thus the C-terminal phenylalaninamide is an important factor in this ligand-receptor interaction, and the group itself may interact directly with the receptor.

Adenylyl Cyclases↗

PubMed1992

The role of the C-terminal domain in collagenase and stromelysin specificity.

Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.

Amino Acid Sequence↗

PubMed1991

Purification of a hybrid plasminogen activator protein.

Recombinant DNA technology has been employed to produce a hybrid gene in which the kringle and serine protease domains of tissue plasminogen activator are linked to the heavy-chain Fd region of a fibrin-specific antibody. The hybrid gene is co-expressed with antibody light chains. This communication describes a purification procedure for the hybrid protein, involving affinity and ion-exchange chromatography. The purified hybrid protein has been used in vivo and in vitro clot lysis experiments and has been shown to be effective at clot dissolution.

Animals↗

PubMed1988

Phase I trial and clinical pharmacological evaluation of 10-ethyl-10-deazaaminopterin in adult patients with advanced cancer.

10-Ethyl-10-deazaaminopterin (10-EDAM) is an analogue of methotrexate with improved preclinical anticancer activity, more selective entry, and greater conversion to polyglutamate forms in neoplastic cells. In this Phase I trial, we have treated 62 adults with advanced solid tumors, giving 10-EDAM i.v. on either a weekly x 3 schedule (35 patients) or a weekly schedule (27 patients). The dosage levels ranged from 5 to 120 mg/m2. The toxicity observed with 10-EDAM was qualitatively similar to that of methotrexate. Oral mucositis was the dose-limiting toxicity; diarrhea, skin rash, leukopenia, thrombocytopenia, and mild elevations of serum glutamic-oxaloacetic transaminase, prothrombin, and partial thromboplastin times were also observed, but were not dose limiting. A weekly dosage of 80 mg/m2 with escalation or attenuation in accordance with patient tolerance, or 100 mg/m2 weekly for 3 weeks, followed by a 2-week "rest period" are recommended for Phase II assessment. 10-EDAM produced partial remissions in three patients with non-small cell lung cancer and one patient with breast cancer lasting 6, 40+, 26+, and 15 months, respectively. Pharmacokinetic studies carried out at the 5, 30, and 100 mg/m2 dosage levels demonstrated the drug to have a triphasic disappearance from plasma. Elimination was independent of dose over the range tested, with mean plasma half-lives of: alpha = 12.9 min, beta = 1.5 h, and gamma = 11.9 h. Cumulative urinary excretion of the drug ranged from 13 to 55% of the administered dose (mean = 33%); 88% of the urinary drug appeared within the first 4 h following drug administration. The pharmacokinetic behavior of the first and second weekly dosages were consistent within a given patient. The metabolites 7-hydroxy-10-EDAM, and 10-ethyl-10-deaza-2,4-diamino-pteroic acid were demonstrated in the plasma and urine of treated patients. In studies of tissue homogenates from two patients with skin metastases, more extensive retention of the drug and of its polyglutamates was observed in the breast cancer metastases than in the metastases from a kidney cancer or in normal skin.

Adult↗

PubMed1986

Frequency and prognostic importance of pretreatment clinical characteristics in patients with advanced non-small-cell lung cancer treated with combination chemotherapy.

To determine the frequency and prognostic importance of pretreatment clinical characteristics in patients currently undergoing treatment for stage III non-small-cell lung cancer (NSCLC), data were collected on 378 patients receiving high-dose (120 mg/m2) cisplatin plus vinca alkaloid combination chemotherapy regimens since 1978. Variables analyzed included age, sex, weight loss, performance status, histologic subtype, presence of extrathoracic metastases, number of metastatic organ sites, presence of liver, bone, or brain involvement, prior radiation or surgery, and serum lactate dehydrogenase (LDH). The effect of a major response to chemotherapy on survival was also investigated. Using multivariable analyses, the following were found to be associated with outcome: initial performance status, with patients having a performance status of 80% to 100% having an increased major objective response rate and survival; bone metastases, which were adversely predictive of response rate and survival; elevated serum LDH and male sex, both of which were associated with shortened survival and remission duration; and the presence of two or more extrathoracic metastatic organ sites, which was associated with shorter survival. When major objective response with chemotherapy was included in a conditional multivariable analysis, it was strongly associated with longer median survival. Information from this analysis may be useful when comparing the response data of completed studies in similar patients, in designing future trials, and in the selection of cisplatin plus vinca alkaloid therapy for individual patients with advanced NSCLC.

Adult↗

PubMed1986

Phase I trial of taxol given as a 3-hour infusion every 21 days.

Taxol is a unique plant product that promotes in vitro assembly of microtubules. In a phase I trial, adults with advanced solid tumors were given taxol (formulated with cremophor EL and dehydrated alcohol) as a 3-hour iv infusion every 21 days. The total dose administered ranged from 15 to 230 mg/m2 in nine escalation steps. Leukopenia, thrombocytopenia, nausea and vomiting, alopecia, stomatitis, transient rashes, increases in serum triglyceride levels, and hypersensitivity reactions were observed. Hypersensitivity reactions characterized by acute dyspnea, generalized erythema, and hypotension immediately following the initiation of the taxol infusion occurred in three of five patients receiving greater than or equal to 190 mg/m2 (18% of patients overall). No antitumor activity was observed. Hypersensitivity reactions constituted a treatment-limiting toxicity for this preparation of taxol given on this schedule over the dosage range examined. With the severity and unpredictability of the hypersensitivity reactions, further usage of taxol is not indicated with this drug formulation on this administration schedule. Further studies are warranted to uncover ways to permit the safe administration of taxol.

Adult↗