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C R Crawford

Publications and source records attributed to C R Crawford.

At least 19 recordsLinked to original sources
PubMed2000

Cloning, genomic organization and chromosomal localization of the gene encoding the murine sodium-dependent, purine-selective, concentrative nucleoside transporter (CNT2).

A PCR-based strategy was used to isolate a 2653 bp cDNA encoding the mouse sodium-dependent, purine nucleoside selective, concentrative nucleoside transporter (designated mCNT2). The deduced protein sequence exhibits 93 and 80% identity to the previously cloned rat and human sodium-dependent, purine nucleoside selective, nucleoside transporters, respectively. Characterization of 3H-nucleoside uptake by COS-1 cells transiently transfected with the cDNA demonstrated that it encoded a functional nucleoside transport activity with selectivity for purine nucleosides. The cDNA was used to screen a murine (strain 129SvJ/6) genomic library in pBeloBAC11 to identify a clone containing the mCNT2 gene. A PCR strategy was used to identify and sequence the intron-exon boundaries and to determine the approximate sizes of the introns. The mCNT2 gene spans approximately 13.7 kb and is encoded by 15 exons. The gene was mapped to mouse chromosome 2e3 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1998

Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines.

Gemcitabine (2',2'-difluorodeoxycytidine) is a novel pyrimidine nucleoside drug with clinical efficacy in several common epithelial cancers. We have proposed that gemcitabine requires nucleoside transporter (NT) proteins to permeate the plasma membrane and to exhibit pharmacological activity. In humans, there are seven reported distinct NT activities varying in substrate specificity, sodium dependence, and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and dipyridamole. To determine which NTs are required for gemcitabine-dependent growth inhibition, cultures from a panel of 12 cell lines with defined plasma membrane NT activities were incubated with different concentrations of gemcitabine. Cell proliferation was assessed by the sulforhodamine B assay and cell enumeration to identify the concentrations of gemcitabine that inhibited cell replication by 50% (IC50s). NT activity was a prerequisite for growth inhibition in vitro because: (a) the nucleoside transport-deficient cells were highly resistant to gemcitabine; and (b) treatment of cells that exhibited only equilibrative NT activity with NBMPR or dipyridamole increased resistance to gemcitabine by 39- to 1800-fold. These data suggested that the type of NT activities possessed by a cell may be an important determinant of its sensitivity to gemcitabine and that NT deficiency may confer significant gemcitabine resistance. We analyzed the uptake kinetics of [3H]gemcitabine by each of five human NT activities in cell lines that exhibited a single NT activity in isolation; transient transfection of the cDNAs encoding the human concentrative NT proteins (hCNT1 and hCNT2) was used to study the cit and cif activities, respectively. The efficiency of gemcitabine uptake varied markedly among the cell lines with single NTs: es approximately = cit > ei > cib >>> cif. The transportability of [3H]gemcitabine was demonstrated by reconstitution of the human es NT in proteoliposomes, confirming that gemcitabine permeation is a protein-mediated process.

Animals↗
PubMed1998

Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line.

Mammalian cells obtain nucleic acid precursors through the de novo synthesis of nucleotides and the salvage of exogenous nucleobases and nucleosides. The first step in the salvage pathway is transport across the plasma membrane. Several transport activities, including equilibrative and concentrative mechanisms, have been identified by their functional properties. We report here the functional cloning of a 2.6-kilobase pair human cDNA encoding the nitrobenzylmercaptopurine riboside (NBMPR)-insensitive, equilibrative nucleoside transporter ei by functional complementation of the transport deficiency in a subline of CEM human leukemia cells. Expression of this cDNA conferred an NBMPR-insensitive, sodium-independent nucleoside transport activity to the cells that exhibited substrate specificity and inhibitor sensitivity characteristic of the ei transporter. The cDNA contained a single open reading frame that encoded a 456-residue protein with 11 potential membrane-spanning regions and two consensus sites for N-glycosylation in the first predicted extracellular loop. The predicted protein was 50% identical to the recently cloned human NBMPR-sensitive, equilibrative nucleoside transporter ENT1 and thus was designated ENT2. Surprisingly, the carboxyl-terminal portion of the ENT2 protein was nearly identical to a smaller protein in the GenBankTM data base (human HNP36, 326 residues) that has been identified as a growth factor-induced delayed early response gene of unknown function. Comparison of the ENT2 and HNP36 nucleotide sequences suggested that HNP36 was translated from a second start codon within the ENT2 open reading frame. Transient expression studies with the full-length ENT2 and a 5'-truncated construct that lacks the first start codon (predicted protein 99% identical to HNP36) demonstrated that only the full-length construct conferred uridine transport activity to the cells. These data suggest that the delayed early response gene HNP36 is a truncated form of ENT2 and that the full-length open reading frame of ENT2 is required for production of a functional plasma membrane ei transporter.

Affinity Labels↗
PubMed1998

Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line.

Previous studies of nucleoside transport in mammalian cells have identified two types of activities: the equilibrative nucleoside transporters and concentrative, Na+-nucleoside cotransporters. Characterization of the concentrative nucleoside transporters has been hampered by the presence in most cells and tissues of multiple transporters with overlapping permeant specificities. With the recent cloning of cDNAs encoding rat and human members of the concentrative nucleoside transporter (CNT) family, it is now possible to study the concentrative transporters in isolation by use of functional expression systems. We report here the isolation of a nucleoside transport-deficient subline of L1210 mouse leukemia (L1210/DNC3) that is a suitable recipient for stable expression of cloned nucleoside transporter cDNAs. We have used L1210/DNC3 as the recipient in gene transfer studies to develop a stable cell line (L1210/DU5) that produces the recombinant concentrative nucleoside transporter with selectivity for pyrimidine nucleosides (CNT1) that was initially identified in rat intestine (Q.Q. Huang, S.Y. Yao, M.W. Ritzel, A.R.P. Paterson, C.E. Cass, and J.D. Young. 1994. J. Biol. Chem. 269: 17,757-17,760). L1210/DU5 was used to examine the permeant selectivity of recombinant rat CNT1 by comparing a series of nucleoside analogs with respect to (i) inhibition of inward fluxes of [3H]thymidine, (ii) initial rates of transport of 3H-analog, and (iii) cytotoxicity to L1210/DU5 versus the parental transport-deficient cell line. By all three criteria, recombinant CNT1 transported 5-fluoro-2'-deoxyuridine and 5-fluorouridine well and cytosine arabinoside poorly. Although some purine nucleosides (2'-deoxyadenosinedeoxyadeno-2'-deoxyadenosine, 7-deazaadenosine) were potent inhibitors of CNT1, they were poor permeants when uptake was measured directly by analysis of isotopic fluxes or indirectly by comparison of cytotoxicity ratios. We conclude that comparison of analog cytotoxicity to L1210/DU5 versus L1210/DNC3 is a reliable indirect predictor of transportability, suggesting that cytotoxicity assays with a panel of such cell lines, each with a different recombinant nucleoside transporter, would be a valuable tool in the development of antiviral and antitumor nucleoside analogs.

Animals↗
PubMed1995

Optimized gradient waveforms for spiral scanning.

Spiral scanning gradient waveforms can be optimized with respect to blurring from off-resonance effects by minimizing the readout time. This is achieved by maximizing the gradient amplitude during the scan so that the edge of k-space is reached as quickly as possible. Gradient hardware constraints are incorporated by considering a circuit model for the gradient coil and amplifier. The optimized gradient waveforms are determined by a set of coupled differential equations. The resulting solutions have shorter readout time than solutions that do not consider the circuit model.

Acceleration↗
PubMed1994

Oversampled filters for quantitative volumetric PET reconstruction.

Three-dimensional filtered backprojection uses filters generally specified in the Fourier domain. Implementing these filters by direct sampling in the Fourier domain produces an artifact in the reconstructed images consisting primarily of a DC shift. This artifact is caused by aliasing of the reconstruction filter. We have developed a filter construction technique using Fourier domain oversampling, which reduces the artifact. A method to construct the filter efficiently without the need to create and store the entire oversampled filter array is also presented. Quantitative accuracy in filtered backprojection is of particular importance in multiple-pass algorithms used to reconstruct data from cylindrical PET scanners. We are able to implement such algorithms without fitting the reprojected views to the scanner data.

Algorithms↗
PubMed1994

Predictive respiratory gating: a new method to reduce motion artifacts on CT scans.

PURPOSE: To evaluate a gating system, called predictive respiratory gating (PRG), that reduces motion-induced artifacts on computed tomographic (CT) scans of patients who cannot suspend respiration. MATERIALS AND METHODS: PRG uses a respiration monitor and a new algorithm to predict when a motionless period is about to occur. It automatically starts scanning so the scan is temporally centered around the motionless period at end inspiration or end expiration. To demonstrate PRG, CT was performed on a motion phantom and a quietly breathing volunteer with and without gating. RESULTS: Scans of the phantom obtained with PRG contained less motion-induced streaking and blurring than did scans acquired without PRG. Scans of the volunteer gated at end expiration contained significantly less artifact than nongated scans (P < .03). CONCLUSION: PRG reduced motion artifact on scans of a spontaneously breathing volunteer. PRG may be able to reduce motion artifacts on scans of patients unable to suspend respiration.

Adult↗
PubMed1993

Nucleoside transport in normal and neoplastic cells.

The permeation of nucleosides across the plasma membrane of mammalian cells is complex and mediated by at least five distinct transporters that differ in their sensitivity to inhibitors and in their specificity for nucleosides. The basic properties and permeant specificity of these transporters are summarized in Table 3. It appears that there may be differences in the distribution of these transporters in tumors and normal tissues that might be exploited for chemotherapeutic purposes. The human tumor cell lines examined express predominantly the NBMPR-sensitive equilibrative transporter es which can be blocked by low concentrations of NBMPR and dipyridamole. It is reasonable to expect that tumors with transport properties similar to the CCRF-CEM and Rh28 cell lines (Table 1) that have no detectable NBMPR-insensitive transport activity will be highly susceptible to the therapeutic approach of combining a transport inhibitor such as dipyridamole or NBMPR with an inhibitor of de novo pyrimidine biosynthesis. On the other hand, this approach to therapy is unlikely to succeed against tumors with transport phenotypes similar to the WI-L2 cell line that may permit the salvage nucleosides in the presence of these inhibitors. The majority of tumor cells examined, however, fall between these extremes, and it is not yet known what level of NBMPR-insensitive transport activity can be tolerated without seriously compromising this therapeutic approach. With respect to normal tissues, the mature absorptive cells of the intestine have predominantly Na(+)-dependent nucleoside transporters that are insensitive to NBMPR and dipyridamole. The proliferating crypt cells also appear to have Na(+)-dependent nucleoside transport, although they may also have an NBMPR-sensitive component of transport (Belt, unpublished data). Bone marrow granulocyte-macrophage progenitor cells also appear to have one or more concentrative nucleoside transporters. Thus these tissues, which are most vulnerable to the toxicity of antimetabolites, may be able to salvage nucleosides in the presence of inhibitors of equilibrative transport and be protected from the toxicity of de novo synthesis inhibitors. It is likely, however, that a successful application of this therapeutic approach will require the analysis of the nucleoside transport phenotype of individual tumors in order to identify those patients that may benefit from such therapy. Since the development of antibodies and cDNA probes for the various nucleoside transporters is currently underway in several laboratories, it is likely that analysis of the nucleoside transport phenotype of tumors from biopsy material will be feasible in the future.

Animals↗
PubMed1992

Minimum scan speeds for suppression of motion artifacts in CT.

Cardiac and ventilatory motions cause artifacts at chest computed tomography (CT). To determine how short the scan times on third-generation units must be to avoid such artifacts, motion was measured with fast and ultrafast CT scans. Minimum detectable motion was then determined. The longest scan time that avoided a barely perceptible artifact was calculated by dividing the minimum detectable motion by the peak physiologic velocity. The posterior left ventricular wall moved at a maximum velocity of 52.5 mm/sec, necessitating a scan time of 19.1 msec or less to avoid artifact. Lung vessels near the heart moved at 40.5 mm/sec for a scan time of 24.7 msec or less. During quiet breathing, pulmonary vessels moved at 10.7 mm/sec for a scan time of 93.5 msec or less. The authors conclude that the shortest scan time on third-generation units (0.6 second) cannot prevent all artifacts arising from motion in the chest. Even ultrafast scan times (50 msec) are not short enough to eliminate artifacts on these units. Thus, reduction of motion artifacts will require techniques other than fast scanning.

Artifacts↗
PubMed1991

Sodium-dependent, concentrative nucleoside transport in Walker 256 rat carcinosarcoma cells.

Nucleoside transport in Walker 256 cells was reexamined using formycin B, a nonmetabolized analog of inosine. In the presence of dipyridamole to inhibit the equilibrative (facilitated diffusion) transporter previously described in these cells, the initial rate of uptake of 1 microM formycin B was 10-fold greater in Na(+)-containing medium than in Na(+)-free medium. In the presence of Na+ and dipyridamole the intracellular concentration of formycin B exceeded that in the medium within one min and was 6-fold greater than that of the medium by 5 min. Na(+)-dependent transport of formycin B was inhibited by low concentrations of inosine, but not thymidine. Furthermore, Na(+)-dependent transport of uridine, but not thymidine, was apparent in the presence of dipyridamole. These data indicate that Walker 256 cells have, in addition to the previously described equilibrative transporter, a concentrative nucleoside transporter. The specificity of this transporter appears to correspond to one of the two Na(+)-dependent transporters previously described in mouse intestinal epithelial cells.

Animals↗
PubMed1991

Survival of an upper lip aesthetic complex using arterial reanastomosis only.

Venous congestion after microvascular replantation remains a formidable problem even under ideal circumstances. The patient reported is a young woman with a traumatically avulsed upper lip aesthetic complex. Blood flow was reestablished using microanastomosis of the labial artery, however, no veins of adequate size could be recovered. With systemic administration of heparin, allowing the flap to bleed freely in addition to the use of medicinal leeches, we were able to salvage an avulsed upper lip aesthetic complex with the use of a single arterial microanastomosis. This patient report exemplifies alternative methods used in dealing with venous inadequacy in a composite replant.

Adult↗
PubMed1990

Isolation and characterization of an L1210 cell line retaining the sodium-dependent carrier cif as its sole nucleoside transport activity.

Nucleoside permeation across mammalian cell membranes is complex with at least four distinct transporters known. Two of these (es and ei) are equilibrative (facilitated diffusion) carriers that have been studied is considerable detail. The other two (cif and cit) are concentrative, Na(+)-dependent carriers. A major obstacle to the characterization of the latter two mechanisms has been the lack of suitable model systems expressing only a single nucleoside transport activity. The present study describes the isolation of a cell line that has cif as its sole nucleoside transporter. L1210/MC5-1 cells, which have es and cif transport activity, were mutagenized and plated in soft agar containing two cytotoxic nucleosides (tubercidin (7-deazaadenosine) and cytosine arabinoside) that are substrates for es but not cif. A clonal line (L1210/MA-27.1) was isolated which retained the capacity for Na(+)-dependent [3H]formycin B transport but was unable to transport [3H]thymidine, a substrate for es but not cif. Failure of the mutant to transport thymidine was also demonstrated by the inability of thymidine (with adenine as a purine source) to rescue these cells from methotrexate toxicity. Furthermore, the mutant lacked nitrobenzylthioinosine (NBMPR) binding activity (an integral part of the es transporter) as demonstrated by reversible NBMPR binding and photoaffinity labeling with [3H]NBMPR. Loss of es transport activity was also demonstrated by the failure of NBMPR to affect the toxicity of 2-chlorodeoxyadenosine (IC50 approximately 30 nM) in L1210/MA27.1 cells. In contrast, NBMPR decreased the IC50 for 2-chlorodeoxyadenosine from 100 to 30 nM in the parental L1210/MC5-1 cell line. These results are consistent with the mechanism of NBMPR potentiation of 2-chlorodeoxyadenosine toxicity in L1210 cells being a blockade of efflux via es while the nucleoside is pumped into the cells by the concentrative cif carrier.

2-Chloroadenosine↗
PubMed1990

Nucleoside transport in L1210 murine leukemia cells. Evidence for three transporters.

L1210 murine leukemia cells have two nucleoside transport activities that differ in their sensitivity to nitrobenzylmercaptopurine riboside (NBMPR). This study re-examines NBMPR-insensitive nucleoside transport in these cells and finds that it is mediated by two components, one Na(+)-dependent and the other Na(+)-independent. A mutant selected previously for loss of NBMPR-insensitive transport lacks only the Na(+)-independent activity. When NBMPR is used to block efflux via the NBMPR-sensitive transporter, uptake of formycin B (a nonmetabolized analog of inosine) is concentrative in both the parental and mutant cells, but the intracellular concentration of the nucleoside is 5-fold lower in the parental cells. Decreased accumulation of formycin B in the parental cells is due to efflux of the nucleoside via the NBMPR-insensitive, Na(+)-independent transporter that the mutant lacks. The Na(+)-dependent transporter appears to accept most purine, but not pyrimidine, nucleosides as substrates. Two exceptions are uridine, a good substrate, and 7-deazaadenosine, a poor substrate. In contrast, all of the nucleosides tested are substrates for the Na(+)-independent transporter. We conclude that L1210 cells have three distinct nucleoside transporters and that the specificity of the Na(+)-dependent transporter is similar to that of one of the two Na(+)-dependent nucleoside transporters seen in mouse intestinal epithelial cells.

Affinity Labels↗
PubMed1990

Identification and reconstitution of the nucleoside transporter of CEM human leukemia cells.

The major nucleoside transporter of the human T leukemia cell line CEM has been identified by photoaffinity labeling with the transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR). The photolabeled protein migrates on SDS-PAGE gels as a broad band with a mean apparent molecular weight (75,000 +/- 3000) significantly higher than that reported for the nucleoside transporter in human erythrocytes (55,000) (Young et al. (1983) J. Biol. Chem. 258, 2202-2208). However, after treatment with endoglycosidase F to remove carbohydrate, the NBMPR-binding protein in CEM cells migrates as a sharp peak with an apparent molecular weight (47,000 +/- 3000) identical to that reported for the deglycosylated protein in human erythrocytes (Kwong et al. (1986) Biochem. J. 240, 349-356). It therefore appears that the difference in the apparent molecular weight of the NBMPR-sensitive nucleoside transporter between the CEM cell line and human erythrocytes is a result of differences in glycosylation. The NBMPR-binding protein from CEM cells has been solubilized with 1% octyl glucoside and reconstituted into phospholipid vesicles by a freeze-thaw sonication technique. Optimal reconstitution of uridine transport activity was achieved using a sonication interval of 5 to 10 s and lipid to protein ratios of 60:1 or greater. Under these conditions transport activity in the reconstituted vesicles was proportional to the protein concentration and was inhibited by NBMPR. Omission of lipid or protein, or substitution of a protein extract prepared from a nucleoside transport deficient mutant of the CEM cell line resulted in vesicles with no uridine transport activity. The initial rate of uridine transport, in the vesicles prepared with CEM protein, was saturable with a Km of 103 +/- 11 microM and was inhibited by adenosine, thymidine and cytidine. The Km for uridine and the potency of the other nucleosides as inhibitors of uridine transport (adenosine greater than thymidine greater than cytidine) were similar to intact cells. Thus, although the nucleoside transporter of CEM cells has a higher molecular weight than the human erythrocyte transporter, it exhibits typical NBMPR-sensitive nucleoside transport activity both in the intact cell and when reconstituted into phospholipid vesicles.

Affinity Labels↗
PubMed1990

The role of intraarterial vasodilators in the treatment of inadvertent intraarterial injection injuries.

Inadvertent arterial injections, both in cases of drug addiction and in iatrogenic situations, often result in significant loss of tissue and, eventually, loss of function distal to the injection site. Previous experimental studies regarding the effectiveness of agents to reverse vasospasm have been inconclusive. A recent clinical report, however, suggested that intraarterial injection of reserpine was affective in limiting tissue necrosis. This experiment was designed to study the affect of intraarterially injected reserpine and tolazoline hydrochloride in a rabbit ear model of necrosis following intraarterial injection of pentothal. Six groups of 5 rabbits each were studied. Animals in Group 1 received intraarterial saline injections. Animals in Group 2 received intraarterial pentothal. Animals in Group 3 received intraarterially injected pentothal plus intraarterial reserpine 30 minutes later. Animals in Group 4 received intraarterially injected pentothal plus intraarterial tolazoline 30 minutes later. Animals in Group 5 received intraarterially injected pentothal plus intraarterially injected reserpine and intraarterially injected tolazoline both administered immediately. Animals in Group 6 received intraarterially injected pentothal plus intraarterially injected reserpine and intraarterially injected tolazoline, both administered 30 minutes later. Results showed no significant difference in the amount of tissue necrosis between treated and untreated animals. We conclude that the use of intraarterial spasmolytic agents has no effect on the course of tissue necrosis after inadvertent intraarterial injections.

Animals↗
PubMed1985

Antigen stripping from the nematode epicuticle using the cationic detergent cetyltrimethylammonium bromide (CTAB).

The cuticular antigens of adult Nematospiroides dubius were selectively removed using the cationic detergent cetyltrimethylammonium bromide (CTAB). Nonionic, zwitterionic or anionic detergents were ineffective in comparison. The biochemical profile of the antigens removed by detergent was identical to that of surface antigens removed by homogenization, with the added advantage that detergent-stripped antigens lacked many of the background antigens (excretory/secretory--ES and somatic) seen in homogenates. In addition, the detergent was shown to act in a non-invasive manner as electron micrographs failed to reveal any gross damage to the nematode outer cuticle. The observed selective release of significant quantities of relatively clean nematode surface antigen by CTAB in a non-invasive or destructive manner provides the impetus for definitive studies on the relevance of surface antigens (in the absence of ES or somatic antigens) to the overall immunogenicity of this and other parasites.

Animals↗
PubMed1984

Use of zwitterionic detergent for the preparation of an influenza virus vaccine. 1: Preparation and characterization of disrupted virions.

The zwitterionic, Empigen BB an alkylbetaine based on a C12-C14 alcohol was shown to disrupt influenza A and B viruses in such a way as to retain the biological activity of the surface haemagglutinin (HA) and neuraminidase (NA) antigens. The optimal conditions required to obtain the maximum recovery of HA and NA activity from purified influenza X47 (H3N2) virus concentrate after treatment with Empigen, and the nature and the morphological appearance of the Empigen-treated preparations both before and following a sucrose density gradient purification step, are described. Matrix (M) protein was readily removed during purification, but nucleoprotein (NP) could not be separated from the surface protein activity.

Centrifugation, Density Gradient↗