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S Grimes

Publications and source records attributed to S Grimes.

At least 19 recordsLinked to original sources
PubMed2004

Terminal protein-induced stretching of bacteriophage phi29 DNA.

Stretching of DNA molecules helps to resolve detail during the fluorescence microscopy of both single DNA molecules and single DNA-protein complexes. To make stretching occur, intricate procedures of specimen preparation and manipulation have been developed in previous studies. By contrast, the present study demonstrates that conventional procedures of specimen preparation cause DNA stretching to occur, if the specimen is the double-stranded DNA genome of bacteriophage phi29. Necessary for this stretching is a protein covalently bound at both 5' termini of phi29 DNA molecules. Some DNA molecules are attached to a cover glass only at the two ends. Others are attached at one end only with the other end free in solution. The extent of stretching varies from approximately 50% overstretched to approximately 50% understretched. The understretched DNA molecules are internally mobile to a variable extent. In addition to stretching, some phi29 DNA molecules also undergo assembly to form both linear and branched concatemers observed by single-molecule fluorescence microscopy. The assembly also requires the terminal protein. The stretched DNA molecules are potentially useful for observing DNA biochemistry at the single molecule level.

Bacillus Phages↗
PubMed2003

First measurement of transferred polarization in the exclusive ep-->e'K+Lambda--> reaction.

The first measurements of the transferred polarization for the exclusive e-->p-->e(')K+Lambda--> reaction have been performed at Jefferson Laboratory using the CLAS spectrometer. A 2.567 GeV beam was used to measure the hyperon polarization over Q2 from 0.3 to 1.5 (GeV/c)(2), W from 1.6 to 2.15 GeV, and over the full K+ center-of-mass angular range. Comparison with predictions of hadrodynamic models indicates strong sensitivity to the underlying resonance contributions. A nonrelativistic quark-model interpretation of our data suggests that the ssmacr; quark pair is produced with spins predominantly antialigned. Implications for the validity of the most widely used quark-pair creation operator are discussed.

Journal Article↗
PubMed2001

The bacteriophage straight phi29 portal motor can package DNA against a large internal force.

As part of the viral infection cycle, viruses must package their newly replicated genomes for delivery to other host cells. Bacteriophage straight phi29 packages its 6.6-microm long, double-stranded DNA into a 42 x 54 nm capsid by means of a portal complex that hydrolyses ATP. This process is remarkable because entropic, electrostatic and bending energies of the DNA must be overcome to package the DNA to near-crystalline density. Here we use optical tweezers to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force-generating motor. This motor can work against loads of up to 57 pN on average, making it one of the strongest molecular motors reported to date. Movements of over 5 microm are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces. We establish the force-velocity relationship of the motor and find that the rate-limiting step of the motor's cycle is force dependent even at low loads. Notably, the packaging rate decreases as the prohead is filled, indicating that an internal force builds up to approximately 50 pN owing to DNA confinement. Our data suggest that this force may be available for initiating the ejection of the DNA from the capsid during infection.

Bacillus Phages↗
PubMed2001

Antibodies raised against the extracellular tail of the CCKB/gastrin receptor inhibit gastrin-stimulated signalling.

INTRODUCTION: Gastrin acts to stimulate gastric acid secretion and is an acknowledged growth factor for human gastrointestinal (GI) cancer. The identity of the exact receptor type mediating the growth promoting effects of gastrin in tumours is uncertain. However, the best-characterised gastrin receptor is the CCK receptor type B (CCKB)/gastrin receptor. The anti-GRE1 antibody is a polyclonal, affinity-purified antibody raised against GRE1, a synthetic 21 amino acid peptide homologous to part of the extracellular, N-terminal tail of the CCKB receptor. We have recently proven that GRE1 antiserum specifically localises CCKB receptors on CCKB receptor transfected NIH3T3 cells and human gastrointestinal tumour cells by Western blotting and immunocytochemistry. GRE1 antiserum also inhibits liver invasion in the C170HM2 colorectal liver-metastasis model. AIM: To relate the ability of GRE1 antiserum to displace G17 from CCKB receptors with its impact on cellular transduction effects. METHODS: Radioligand binding studies were performed with 125IG17 and Calcium mobilisation studies by use of the fluorescent dye Fura 2-am. RESULTS: GRE1 antiserum competitively displaced 50% radiolabelled gastrin-17 from whole cell NIH3T3 CCKB transfectants at a protein concentration of 250 microg x ml(-1). GRE1 antiserum did not stimulate calcium ion influx in the transfectant NIH3T3 cells when used at a range of protein concentrations. Pre-incubation with GRE1 antiserum was required to inhibit gastrin-stimulated calcium ion influx. This was found to be concentration-dependent, with inhibition shown at 30 and 5 microg x ml(-1) but not at 500 ng x ml(-1) or below. CONCLUSION: The GRE1 antiserum is specific for the CCKB receptor and may act to inhibit gastrin-stimulated signalling in tumour cells.

3T3 Cells↗
PubMed2001

Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage phi29.

A method has been developed for three-dimensional image reconstruction of symmetry-mismatched components in tailed phages. Although the method described here addresses the specific case where differing symmetry axes are coincident, the method is more generally applicable, for instance, to the reconstruction of images of viral particles that deviate from icosahedral symmetry. Particles are initially oriented according to their dominant symmetry, thus reducing the search space for determining the orientation of the less dominant, symmetry-mismatched component. This procedure produced an improved reconstruction of the sixfold-symmetric tail assembly that is attached to the fivefold-symmetric prolate head of phi29, demonstrating that this method is capable of detecting and reconstructing an object that included a symmetry mismatch. A reconstruction of phi29 prohead particles using the methods described here establishes that the pRNA molecule has fivefold symmetry when attached to the prohead, consistent with its proposed role as a component of the stator in the phi29 DNA packaging motor.

Bacillus Phages↗
PubMed2001

Effect of gastrin and anti-gastrin antibodies on proliferation of hepatocyte cell lines.

Gastrin (G-17) and its precursor glycine-extended gastrin (G-17-gly) have been shown to be trophic to some gastrointestinal tumors. This in vitro study assessed the effect of G-17, G-17-gly, anti-gastrin antibodies (anti-G-17), and the CCK-B receptor antagonist PD135,158 on three hepatoma cell lines (PLC/PRF/5, HepG2 and MCA-RH7777) and an embryonic liver cell line (WRL68). The pancreatic adenocarcinoma cell line AR42J was used as a positive control. G-17 and G-17-gly caused significant proliferation of AR42J and WRL68 cell lines. G-17-gly but not G-17 induced significant proliferation of the PLC/PRF/5 cell line. Anti-G-17 and PD135,158 significantly inhibited unstimulated AR42J and WRL68 cell lines. Anti-G-17 also inhibited the proliferative effects of G-17 and G-17-gly on AR42J, WRL68, and PLC/PRF/5 cell lines, whereas PD135,158 inhibited the proliferative effect of G-17 only. G-17 and G-17-gly as well as anti-G-17 and PD135,158 had no effect on HepG2 and MCA-RH77777 cell lines. It is concluded that G-17-stimulated proliferation is mediated via the CCK-B receptor and G-17-gly via a separate, as yet uncharacterized, receptor. There may therefore be a role for gastrin in embryonic hepatocellular proliferation and perhaps also in the proliferation of some hepatocellular tumors.

Animals↗
PubMed2001

The cardiac anesthesia risk evaluation score: a clinically useful predictor of mortality and morbidity after cardiac surgery.

BACKGROUND: The Cardiac Anesthesia Risk Evaluation (CARE) score is a simple risk classification for cardiac surgical patients. It is based on clinical judgment and three clinical variables: comorbid conditions categorized as controlled or uncontrolled, surgical complexity, and urgency of the procedure. This study compared the CARE score with the Parsonnet, Tuman, and Tu multifactorial risk indexes for prediction of mortality and morbidity after cardiac surgery. METHODS: In this prospective study, 3,548 cardiac surgical patients from one institution were risk stratified by two investigators using the CARE score and the three tested multifactorial risk indexes. All patients were also given a CARE score by their attending cardiac anesthesiologist. The first 2,000 patients served as a reference group to determine discrimination of each classification with receiver operating characteristic curves. The following 1,548 patients were used to evaluate calibration using the Pearson chi-square goodness-of-fit test. RESULTS: The areas under the receiver operating characteristic curves for mortality and morbidity were 0.801 and 0.721, respectively, with the CARE score rating by the investigators; 0.786 and 0.710, respectively, with the CARE score rating by the attending anesthesiologists (n = 8); 0.808 and 0.726, respectively, with the Parsonnet index; 0.782 and 0.697, respectively, with the Tuman index; 0.770 and 0.724 with the Tu index, respectively. All risk models had acceptable calibration in predicting mortality and morbidity, except for the Parsonnet classification, which failed calibration for morbidity (P = 0.026). CONCLUSIONS: The CARE score performs as well as multifactorial risk indexes for outcome prediction in cardiac surgery. Cardiac anesthesiologists can integrate this score in their practice and predict patient outcome with acceptable accuracy.

Adult↗
PubMed2000

Structure of the bacteriophage phi29 DNA packaging motor.

Motors generating mechanical force, powered by the hydrolysis of ATP, translocate double-stranded DNA into preformed capsids (proheads) of bacterial viruses and certain animal viruses. Here we describe the motor that packages the double-stranded DNA of the Bacillus subtilis bacteriophage phi29 into a precursor capsid. We determined the structure of the head-tail connector--the central component of the phi29 DNA packaging motor--to 3.2 A resolution by means of X-ray crystallography. We then fitted the connector into the electron densities of the prohead and of the partially packaged prohead as determined using cryo-electron microscopy and image reconstruction analysis. Our results suggest that the prohead plus dodecameric connector, prohead RNA, viral ATPase and DNA comprise a rotary motor with the head-prohead RNA-ATPase complex acting as a stator, the DNA acting as a spindle, and the connector as a ball-race. The helical nature of the DNA converts the rotary action of the connector into translation of the DNA.

Adenosine Triphosphatases↗
PubMed1999

Demonstration of new sites of expression of the CCK-B/gastrin receptor in pancreatic acinar AR42J cells using immunoelectron microscopy.

The CCK-B/gastrin receptor has been characterised in both normal and tumour tissues. Endocytosis of the CCK-B/gastrin receptor has recently been demonstrated and this has similarly been described for other peptide receptors. In addition, ligand and ligand-receptor translocation to the nucleus has been demonstrated for other peptides. The aim of this study was to identify the sites of expression of the CCK-B/gastrin receptor in the known CCK-B/gastrin receptor bearing pancreatic acinar AR42J cells. The specificity of the CCK-B/gastrin receptor antibody (alpha-CCKBR-Ser antibody) was demonstrated by inhibition ELISA studies, radioligand inhibition studies and immunofluorescence binding studies on AR42J cells. Western blotting and immunogold electron microscopy techniques were used to identify the receptor in AR42J cell preparations. The affinity purified alpha-CCKBR-Ser antibody was shown to be specific for the CCK-B/gastrin receptor. The receptor was expressed on the cell membrane, in the cytoplasm and within the nucleus. Isoforms of the receptor protein identified in extra-nuclear and nuclear extracts ranged in molecular weight from 58 to 66 kDa. We conclude that the CCK-B/gastrin receptor is not only expressed on the cell membrane, but also in the cytoplasm and nucleus of AR42J pancreatic acinar cells.

Animals↗
PubMed1999

A comparison of an anti-gastrin antibody and cytotoxic drugs in the therapy of human gastric ascites in SCID mice.

The therapeutic effect of antibodies raised by the immunogen Gastrimmune was compared with both a CCKB/gastrin receptor antagonist, CI-988, and 5-Fluorouracil/leucovorin in a gastric cancer model. The human gastric ascites cell line, MGLVA1asc, produced and secreted progastrin and glycine-extended gastrin as determined by radioimmunoassay and immunocytochemistry. Cells were also stained with an antiserum directed against the human CCKB/gastrin receptor. MGLVAI asc cells were injected i.p. into SCID mice. Antibodies raised by Gastrimmune immunization of rabbits (affinity for G17 of 0.15 nM and GlyG17 of 0.47 nM) were passively infused i.p. and significantly enhanced survival by up to 5 days (p=0.0024 from vehicle controls). The enhancement in survival was not significantly different from that achieved by treatment with 5-Fluorouracil and leucovorin. A CCKB/gastrin receptor antagonist, CI-988, did not affect survival with cells injected at 7.5 x 10(5) cells/mouse but significantly increased the survival of mice injected with a lower cell innoculum of 5 x 10(5) cells/mouse from 30 to 35 days (p=0.0186). At this lower innoculum antibodies raised by Gastrimmune induced complete survival in 2 animals with the remaining dead by day 36 (p=0.0022). Thus, both endocrine and autocrine pathways mediated by precursor and mature gastrin molecules may be jointly operational in the gastric cancer scenario and may be important targets for therapeutic agents.

Animals↗
PubMed1999

Expression and processing of gastrin in hepatocellular carcinoma, fibrolamellar carcinoma and cholangiocarcinoma.

BACKGROUND/AIMS: Gastrin is a trophic factor within the normal gastrointestinal tract and is also a mitogen for a number of gastrointestinal and non-gastrointestinal tumours. Precursor forms of gastrin including progastrin (proG) and glycine-extended gastrin (G-gly) as well as the fully processed amidated gastrin (G-NH2) are expressed by tumours. There has been little study of the role of gastrin in either normal liver or liver tumours. The aim of this study was to identify the expression of CCK-B/gastrin receptor (CCK-BR), proG, G-gly and G-NH2 in normal liver and liver tumours. METHODS: Tissue sections from patients with hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma as well as normal liver biopsies were assessed for expression of CCK-BR and gastrin isoforms. RESULTS: Most liver tumours express CCK-BR and are able to process gastrin as far as proG and G-gly, although not as far as the amidated form. There appears to be little expression of the receptor and no expression of precursor forms of gastrin in normal liver. CONCLUSIONS: Liver tumours express the CCK-BR and precursor forms of gastrin. This expression may be associated with tumour proliferation.

Bile Duct Neoplasms↗
PubMed1998

Expression of CCKB/gastrin receptor isoforms in gastro-intestinal tumour cells.

Anti-serum raised against the human cholecystokinin B (CCKB)/gastrin receptor was used in Western blotting to differentiate the cellular locations of receptor isoforms expressed by human gastro-intestinal (GI) tumour cell lines. Using anti-serum directed against the amino-terminal extracellular tail of the CCKB/gastrin receptor, 8/9 cell lines were shown to express immunoreactive proteins of either m.w. 70 or 40 kDa, or both. Both isoforms were found to be associated with intracellular, non-nuclear membranes, whereas only the 70 kDa protein was expressed in the plasma membrane. Receptor expression was related to gastrin production and secretion. Both progastrin and glycine-extended gastrin-17 were produced and secreted by the tumour cell lines; however, carboxy amidated gastrin was not detected by radioimmunoassay. A CCKB/gastrin receptor transfectant NIH3T3 cell line did not produce detectable gastrin and showed exclusive expression of the 70 kDa receptor on the plasma membrane. One cell line had <50 pg/ml cell-associated progastrin and no detectable receptor form. Cell lines expressing 50-150 pg/ml had both 40 and 70 kDa receptor forms. Those expressing >150 pg/ml progastrin had only the 40 kDa isoform, which was shown to be exclusively expressed on intracellular, non-nuclear membranes, in one of the cell lines. Of the 4 cell lines exclusively expressing the lower m.w. receptor, 3 had gastrin present within the cell, which was not secreted. Thus, if cell-associated gastrin induces a proliferative effect, it may be by an intracrine pathway. Our study has identified the presence of CCKB/gastrin receptor isoforms in different cellular locations and may help toward understanding the complex autocrine and intracrine pathways mediated by gastrin peptides.

3T3 Cells↗
PubMed1998

Pre-clinical evaluation of the Gastrimmune immunogen alone and in combination with 5-fluorouracil/leucovorin in a rat colorectal cancer model.

Mature and post-translational precursor gastrin forms are growth factors for colorectal tumours. The immunogen Gastrimmune is composed of the amino terminus of gastrin-17 linked to diphtheria toxoid and raises antibodies in situ which neutralise amidated and glycine-extended gastrin-17. The aim of the study was to determine the effect of treatment with 5-fluorouracil(5-FU)/leucovorin on the antibody titres induced by Gastrimmune and the effect of combination therapy on the growth of the rat colon tumour DHDK12. Gastrimmune was administered to rats s.c. at 3 weekly intervals. The rat colon tumour line DHDK12 was injected into the abdominal wall of BDIX rats. Combinations of 5-FU/leucovorin were injected i.v. on days 1, 3 and 5, with the cycle repeated every 4 weeks. Antibody titres were measured by an ELISA technique. Antibody titres were followed for 40 weeks after Gastrimmune (500 microg.ml(-1)) immunization, with titres peaking between 10 and 20 weeks after a single immunisation and falling by week 30. At termination, no effect was observed on either the histological appearance of the gastro-intestinal tract or the proliferation of the colonic mucosa. Pre- and post-treatment with 5-FU/leucovorin (30 mg.kg(-1)) had no effect on the kinetics and level of antibody response to Gastrimmune. Gastrimmune (200 microg.ml(-1)) and 5-FU/leucovorin combinations (12.5 and 20 mg.kg(-1)) increased the therapeutic effects on the in vivo growth of DHDK12 tumors when compared to the agents given singly. Gastrimmune immunisation may be a therapeutic option for the treatment of colorectal cancer in combination with 5-FU/leucovorin.

Animals↗
PubMed1997

The bacteriophage phi29 packaging proteins supercoil the DNA ends.

Bacteriophage phi29 DNA with covalently bound terminal protein (DNA-gp3) and its left and right-end restriction fragments (L and R-DNA-gp3) sedimented faster in sucrose density gradients than their proteinase K-treated counterparts, and the faster sedimentation was both gp3 and Mg2+-dependent. Addition of gp16, the phi29 DNA packaging ATPase, further increased the sedimentation rates of both intact DNA-gp3 and L and R-DNA-gp3 fragments. Thus, DNAs with gp3 were more compact than gp3-free DNA, and gp16 further condensed the DNA-gp3 forms. [35S]gp16 cosedimented with the fast-sedimenting DNA-gp3 fragments, and the putative L-DNA-gp3-gp16 complexes were packaged preferentially into proheads in the defined in vitro system. Lariats of DNA-gp3 and L and R-DNA-gp3 observed by electron microscopy rationalized the sedimentation results, and lariats with multiple loops or coils increased tenfold in a preparation of L-DNA-gp3-gp16 complexes. The rapid sedimentation and the structure of the DNA-gp3-gp16 complexes were consistent with supercoiling of lariat loops, and treatment with topoisomerase I shifted fast-sedimenting complexes toward the uncoiled lariat position in sucrose density gradients. DNA-gp3 has a maturation pathway in which the packaging proteins gp3 and gp16 supercoil the DNA ends, probably as a prerequisite for efficient interaction with the prohead.

Adenosine Triphosphatases↗
PubMed1996

Gastrimmune raises antibodies that neutralize amidated and glycine-extended gastrin-17 and inhibit the growth of colon cancer.

The effect of gastrin neutralization was evaluated on the in vivo growth of the rat colon line, DHDK12, which expressed cholecystokinin B/gastrin receptors and secreted glycine-extended gastrin-17 (G17). Gastrin neutralization was achieved by administration of the immunogen, Gastrimmune, which is composed of the amino terminal portion of G17 linked to a diphtheria toxoid. A rat-specific version of Gastrimmune was used to preimmunize rats, with control animals receiving diphtheria toxoid only. The antibodies raised neutralized both carboxy-amidated and glycine-extended G17. The tumor was implanted into the muscle layer of the abdominal wall, and rats immunized with Gastrimmune had significantly reduced median cross-sectional tumor areas (70.2% reduction; P = 0.005) and weights (56.5% reduction; P = 0.0078)) when compared to control rats. Histological analysis revealed that the tumors had an enhanced degree of necrosis, with the area of viable tumor in the Gastrimmune-immunized rat reduced to 40.3% compared to 58.6% in the control rats (P = 0.003). Immunization with Gastrimmune raised antibodies that inhibited the growth of a rat colon tumor. This could have been mediated by neutralization of both serum G17 and cell-associated precursor gastrin molecules.

Amino Acid Sequence↗