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Biomedical subjects

S B Simpson

Publications and source records attributed to S B Simpson.

At least 19 recordsLinked to original sources
PubMed2002

Upper gastrointestinal endoscopic ultrasound and its impact on patient management: 1990-2000.

BACKGROUND: Endoscopic ultrasound (EUS) is a relatively new method used in the investigation and staging of upper gastrointestinal tract (UGIT) disease. AIMS: To review practice and outcomes of EUS in an Australian university teaching hospital. METHODS: The first part of the study was a retrospective review of indications, safety, referral patterns and technical difficulties of all EUS procedures performed at Concord Hospital, New South Wales, Australia, over a 10-year period from 1990 to 2000. The second part of the study examined the impact of EUS on the management of 225 consecutive cases, as determined by a questionnaire completed by each of the referring doctors. RESULTS: A total of 537 EUS examinations was performed over the 10-year period. Indications for EUS included: (i) assessment of oesophageal lesions (241), (ii) assessment of gastric lesions (184) and (iii) assessment of pancreaticobiliary (112) disease. Cancer staging was performed in 46.7% of oesophageal and 31.4% of gastric cases. Sedation was achieved using intravenous midazolam (5.3+/-1.3 mg; mean +/- SD) and 52% of cases required additional intravenous pethidine (48.5+/-10.0 mg; mean +/- SD). Technical difficulties were encountered in 11% of cases and these were mainly related to nontraversable luminal stricturing. Of the 537 referrals, 48.2% were from within Central Sydney Area Health Services, and the remainder were from other Sydney hospitals, New South Wales regional centres and interstate. Of 225 questionnaires sent to referring doctors, 146 questionnaires were completed and returned for analysis. EUS aided staging of malignant disease, and confirmed or established a diagnosis in 86% of cases. The diagnostic accuracy of EUS was 76% when confirmed histologically. EUS avoided further diagnostic investigations in the majority of cases and in 25% of cases surgery was avoided. Fine-needle aspiration biopsy (FNAB) during EUS would have been useful in 30% of cases. Overall, clinical decision-making and management were changed in one-third of cases. CONCLUSIONS: Endoscopic ultrasound is an accurate, safe and useful imaging method in UGIT disease. The increasing demand for EUS and EUS-guided FNAB suggests an expanding future for EUS in Australia.

Adolescent↗
PubMed1998

The upstream, direct repeat sequence of Prague A Rous sarcoma virus is deficient in mediating efficient Gag assembly and particle release.

Rous sarcoma virus (RSV) contains two approximately 135-nt imperfect direct repeats composed of smaller repeats, dr1 (approximately 100 nt) and dr2 (approximately 36 nt), that are between the env and src genes and downstream of src in the 3' untranslated region, respectively. It has previously been shown that a Prague A RSV mutant in which both dr1 sequences are deleted is defective at several points in the virus life cycle, including unspliced RNA and env mRNA stability, unspliced RNA transport, and virus particle assembly. A defect in unspliced RNA transport occurs because a cytoplasmic transport element is present within the dr1. We have suggested that the defect of particle production may arise from the failure of the unspliced RNA to be targeted to sites in the cytoplasm where its translation is favorable for Gag protein assembly. In this report, we have further investigated the function of the direct repeats by comparing virus mutants containing either a single upstream or downstream dr1 sequence. Both mutants were delayed in replication compared to the wild-type; the mutant with a single upstream dr1 (delta DDR) is significantly more defective than the mutant with a single downstream dr1 (delta UDR). While both mutants appear capable of efficiently transporting unspliced RNA to the cytoplasm, the delta DDR mutant with only the upstream dr1 is defective in its ability to support Gag assembly and particle release. The replication defect cannot be repaired by placing the upstream dr1 at the location of the downstream dr1 in the 3' untranslated region. A single point mutation in the upstream dr1 (U to C) restored replication and particle production to near normal levels. The results suggest that unspliced RNA transport and Gag assembly functions may be mediated by different elements within the dr1 and that the Prague A upstream dr1 is defective in the latter but not the former function.

Animals↗
PubMed1997

Rous sarcoma virus direct repeat cis elements exert effects at several points in the virus life cycle.

Two approximately 135-nucleotide (nt) direct repeats flank the Rous sarcoma virus (RSV) oncogene src and are composed of two smaller repeats, dr1 (approximately 100 nt) and dr2 (approximately 36 nt). These sequences have been reported to contain cis-acting signals necessary for RNA packaging and elements that allow cytoplasmic accumulation of unspliced RNA (cytoplasmic transport elements). In this report, we show that avian fibroblasts infected with the Prague A strain of RSV with precise deletions of both dr1 elements express src and are transformed by this mutant virus but production of virus particles is very low and virus spread throughout the culture requires several weeks. We show that the replication defect is due to complex effects on viral RNA transport, viral RNA half-life, and virus particle assembly. The dr1 elements may contain binding sites for a permissive cell-specific factor(s) that facilitates efficient nuclear-cytoplasmic transport, RNA stability, and cytoplasmic utilization of unspliced viral RNA. The implications of these results for understanding the defects of nonpermissive virus infections in mammalian cells are discussed.

Animals↗
PubMed1996

Selection and characterization of replication-competent revertants of a Rous sarcoma virus src gene oversplicing mutant.

All retroviruses require both unspliced and spliced RNA for a productive infection. One mechanism by which Rous sarcoma virus achieves incomplete splicing involves suboptimal env and src 3' splice sites. We have previously shown that mutagenesis of the nonconsensus src polypyrimidine tract to a 14-nucleotide uninterrupted polypyrimidine tract results in an oversplicing phenotype and a concomitant defective replication in permissive chicken embryo fibroblasts. In this report, we show that splicing at the src 3' splice site (3'ss) is further negatively regulated by the suppressor of src splicing cis element which is located approximately 100 nucleotides upstream of the src 3'ss. The increase in splicing at the src 3'ss results in a corresponding increase in splicing at a cryptic 5'ss within the env gene. Two classes of replication-competent revertants of the src oversplicing mutant (pSAP1) were produced after infection, and these mutants were characterized by molecular cloning and sequence analysis. Class I revertants are transformation-defective revertants in which the src 3'ss and the src gene are deleted by homologous recombination at several different sites within the imperfect direct repeat sequences that flank the src gene. Cells infected with these transformation-defective revertants produce lower levels of virus particles than cells infected with the wild-type virus. Class II revertants bear small deletions in the region containing the branchpoint sequence or polypyrimidine tract of the src 3'ss. Insertion of these mutated sequences into pSAP1 restored inefficient splicing at the src 3'ss and efficient replication in chicken embryo fibroblasts. All of these mutations caused reduced splicing at the src 3'ss when they were tested in an in vitro splicing system. These results indicate that maintenance of a weak src 3'ss is necessary for efficient Rous sarcoma virus replication.

Animals↗
PubMed1995

Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a negative cis element and a chicken embryo fibroblast nuclear factor.

In permissive Rous sarcoma virus-infected chicken embryo fibroblasts (CEF), approximately equimolar amounts of env and src mRNAs are present. In nonpermissive mammalian cells, the src mRNA level is elevated and env mRNA level is reduced. A cis element in the region between the env gene and the src 3' splice site, which we have termed the suppressor of src splicing (SSS), acts specifically in CEF but not in human cells to reduce src mRNA levels. The splicing inhibition in CEF is not caused by a base-paired structure which is predicted to form between the SSS and the src 3' splice site. To further investigate the mechanism of the inhibition, we have used human HeLa cell nuclear extracts to compare in vitro the rates of splicing of RNA substrates containing the Rous sarcoma virus major 5' splice site and either the env or src 3' splice sites. We show that the src 3' splice site is used approximately fivefold more efficiently than the env 3' splice site. The efficiency of in vitro splicing at the src 3' splice site is specifically reduced by addition of CEF nuclear extract. The inhibition is dependent on the presence of the SSS element and can be abrogated by addition of competitor RNA. We propose that the SSS region represents a binding site for a negative-acting CEF splicing factor(s).

Animals↗
PubMed1994

Frameshift mutations in the v-src gene of avian sarcoma virus act in cis to specifically reduce v-src mRNA levels.

A portion of the avian sarcoma virus (ASV) primary RNA transcripts is alternatively spliced in chicken embryo fibroblast cells to two different messages, the src and env mRNAs. Frameshift mutations of the viral genome causing premature translation termination within the src gene result in a decreased steady-state level of the src mRNA. In marked contrast, frameshift mutations at various positions of the env gene do not decrease the level of the env mRNA. We show that the src gene product is not required in trans for splicing and accumulation of src mRNA. Conversely, the truncated Src proteins do not act negatively in trans to decrease specifically the levels of src mRNA. Taken together, these results indicate that the frameshift mutations act in cis to reduce src mRNA levels. A double mutant with a lesion in the src initiator AUG and a frameshift within the src gene demonstrated wild-type RNA levels, indicating that the src mRNA must be recognized as a translatable mRNA for the effect on src mRNA levels to occur. Our results indicate that the reduced levels do not result from decreased cytoplasmic stability of the mature src mRNA. We also show that the src gene frameshift mutations affect src mRNA levels when expressed from intronless src cDNA clones. We conclude that the reduction of src mRNA levels triggered by the presence of frameshift mutations within the src gene occurs while it is associated with the nucleus. Our data also strongly suggest that this occurs at a step of RNA processing or transport independent of RNA splicing.

Animals↗
PubMed1993

3H-GABA administration during tail regeneration of lizards and autoradiographical localization.

Thirteen lizards (Anolis carolinensis and Scincella lateralis) were injected with 3H-GABA (tritiated gamma-aminobutyric acid) in order to study its localization inside the regenerating tail spinal cord (SC) by means of light and electron microscopic autoradiography. One and three hours after intraperitoneal injection, the radioactivity was essentially localized in regenerating nerves and pale cells, mostly contacting the central canal of the SC as typical Cerebrospinal-Fluid-Contacting-Neurons (CSFCNs). A smaller number of pale cells did not take up 3H-GABA and some of them appeared to be degenerating. Weaker radioactivity in regenerating SC was observed 6 hours post injection. Among non neural tissues, cells of the regenerating blastema, of the meninges and capillaries surrounding the regenerating SC, showed lower 3H-GABA uptake. This indicates that a certain amount of injected 3H-GABA has been converted to other metabolites. No 3H-GABA uptake was detected 1 or 6 hours post injection in normal SC and brain both in grey and white matter. This research indicates that CSFCNs of the regenerating tail in lizards uptake the aminoacid GABA or derived aminoacids, suggesting these cells subserve a high metabolic activity and may be GABA-ergic

Animals↗
PubMed1992

Axonal sprouting and frank regeneration in the lizard tail spinal cord: correlation between changes in synaptic circuitry and axonal growth.

In our previous studies, we found that the number of supraspinal neurons projecting to the level of tail spinal cord increases by 74% during tail regeneration and that the number of local spinal neurons with descending projections increases 233%. However, only a small fraction of the supraspinal axons (less than 4%) and half of the local spinal axons actually enter the regenerated spinal cord. We suggested that this may be the result of "synaptic capture" in which regrowing axons make synapses on denervated targets rostral to the transection, aborting further regeneration. To examine this hypothesis, morphometric analysis of electron microscope (EM) photomontages was used to test for changes in synaptic distribution on ventral horn neurons rostral to regenerating tail spinal cord. In addition, 3H-thymidine and retrograde markers were used to determine whether the regenerate axons arose from cut axons, neurogenesis, or sprouting from uninjured neurons. 3H-thymidine injections during regeneration, combined with retrograde HRP pathway tracing, did not reveal the production of new neurons in the tail spinal cord. To test whether cut axons regenerate, fluorescein isothiocyanate conjugated latex beads were applied to the exposed end of the tail spinal cord. After tail regeneration, HRP was applied to the new spinal cord in the regenerated tail. Examination of local spinal neurons (the primary source of axons that enter the regenerated tail spinal cord) revealed that 28% of the neurons contained both labels. This indicated that cut axons successfully regrew into the new tail spinal cord. The regenerated axons that fail to enter the new tail spinal cord can be found in the normal spinal cord immediately rostral to the regenerated tail. To determine whether these axons were making synaptic contacts, lamina IX ventral horn neurons were examined. EM photomontages of the spinal cord rostral to the regenerate tail revealed the following properties: (1) neurons rostral to regenerated tails are larger in area compare to non-regenerates (mean increase = 112%); (2) axosomatic contacts cover a greater percentage of the neuronal soma following regeneration compared to normal (mean increase = 23%); and (3) this increased innervation is the result of an increase in the number of synaptic boutons rather than larger boutons. The number of synaptic contacts in regenerated lizards returned to normal following lumbar transection, indicating that supraspinal and/or long descending propriospinal afferents were the major source of the increased synaptic contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

A monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples.

Spleen cells from mice hyperimmunized with a keyhole limpet hemocyanin-tetrodotoxin-formaldehyde conjugate were fused with murine P3X63Ag8.653 myeloma cells. A single hybridoma clone was identified that secretes an IgG1,k monoclonal antibody (MAb), designated T20G10, against tetrodotoxin (TTX), with an estimated affinity of 1.2 x 10(8) L/M. Competitive inhibition enzyme immunoassays (CIEIAs) for detecting TTX were developed using this MAb. A direct CIEIA using alkaline phosphatase-labeled MAb detected TTX with sensitivities at IC50 and IC20 of 6-7 ng/ml and 2-3 ng/ml, respectively. The accuracy of the direct CIEIA was comparable with the high-performance liquid chromatography (HPLC) and the mouse bioassay systems, but the direct CIEIA exhibited greater sensitivity. The direct CIEIA was also more cost effective, as it required less sample preparation, a shorter assay time, and reduced investment in equipment than either of the other assay systems.

Animals↗
PubMed1992

An enzyme immunoassay for determining plasma concentrations of didemnin B.

Didemnin A was conjugated at the amino terminus of the N-methylleucine residue, via the linkers N-succinimidyl-3-(2-pyridyldithio)-propionate and trans-1,4-maleimidomethyl-cyclohexane carboxylic acid, to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). The didemnin-KLH conjugates were used to hyperimmunize rabbits. The resulting high titer antisera were employed with didemnin-BSA conjugate-coated microtiter plate wells to develop an indirect competitive inhibition enzyme immunoassay (CIEIA) that was fully cross reactive with didemnin B. A CIEIA is described that is capable of detecting the drug in plasma from didemnin B-treated patients at concentrations down to 1-3 ng/ml. This simple, sensitive CIEIA has been employed to demonstrate plasma drug clearance profiles with samples from didemnin B-treated patients.

Antineoplastic Agents↗
PubMed1992

Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples.

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection.

Acrylamides↗
PubMed1990

Time course of salamander spinal cord regeneration and recovery of swimming: HRP retrograde pathway tracing and kinematic analysis.

The time course of regeneration of supraspinal and descending brachial intraspinal axons was studied using HRP retrograde tracing and kinematic analysis. Five groups of salamanders (10 salamanders/group) received complete thoracic transection 1.0 cm rostral to the hind limbs abolishing swimming. Groups 1-4 recovered for 2, 4, 6, and 8 weeks, respectively, before being filmed to record the animal's ability to swim. After filming, a second transection was made 1.0 cm caudal to the first (at the level of the lumbar enlargement) and HRP was used to label descending axons which had grown past the first lesion. The fifth group was filmed every 2 weeks for 12 weeks before the second transection was made for HRP application. The films were used to perform frame by frame computer analysis of the amplitude and timing of cyclic lateral flexion waves which make up swimming behavior. The earliest return of coordinated swimming behavior was seen 4 weeks after transection (1 of 20 animals). At 6 weeks post-transection, 5 of 10 animals exhibited coordinated swimming. However, the behavior in these animals was subnormal. In the group surviving 8 weeks post-transection, 5 of 10 animals recovered coordinated swimming behavior. In the group that was filmed every 2 weeks, 5 of the 10 salamanders which did recover, exhibited coordinated swimming behavior by the eighth week post-transection. Kinematic analysis of salamanders that exhibited a return of coordinated swimming revealed quantitative differences compared to normal salamanders. While continuous head to tail undulatory waves were present, the propagation time and period were faster than those in normal salamanders. Retransection of the spinal cord abolished coordinated swimming. The numbers and distribution of HRP-labeled supraspinal neurons varied greatly among the animals that displayed recovery of locomotor abilities. In the salamanders examined 6 weeks post-transection the majority of labeled cells were found in medullary nuclei. In recovered salamanders examined 8 and 12 weeks post-transection, HRP-labeled neurons were found in the red nucleus, in the interstitial nucleus of the fasciculus longitudinalis medialis, and in the mesencephalic as well as the medullary reticular neurons. Recovery of coordinated swimming was only observed in salamanders in which descending supraspinal and intraspinal axons were present at the level of the lumbar enlargement (as demonstrated by HRP retrograde labeling). These results indicate that recovery of locomotion is dependent on the reestablishment of descending input and is not a result of changes in spinal reflexes or propagation of electrical activity through the body wall.

Animals↗
PubMed1989

Bulbospinal and intraspinal connections in normal and regenerated salamander spinal cord.

The salamander is the only limbed adult vertebrate which can regenerate portions of cervical, thoracic, or lumbar spinal cord. While the salamander has been a popular model for regeneration of the spinal cord, it is still not known what portions of the nervous system participate in the regeneration process. In the experiments reported here we examine the bulbospinal and intraspinal projections to the lumbar spinal cord in normal and regenerated salamanders (Notophthalmus viridescens). HRP application to the lumbar enlargement of normal salamanders labeled cells in the ventral thalamus, the rostral tegmentum in the proposed homolog of the red nucleus, the reticular neurons of the rhombencephalon, and the midline regions of the rhombencephalon which are possibly equivalent to raphe nuclei of other vertebrates. In the brachial spinal cord HRP-labeled cells were located in dorsal, intermediate, and ventral regions of the spinal gray matter and tended to be located at the periphery of the gray matter. To examine the spinal circuitry of regenerated salamanders, animals received complete spinal transections at the junction of the thoracic and lumbar spinal cord, abolishing all spontaneous coordinated hindlimb and tail movements. Animals exhibited walking and swimming within 60 days at which time a pledget of HRP was inserted into a gap in the spinal cord made by a transection 10.0 mm (six animals) or 5.0 mm (one animal) caudal to the first lesion. On average, the number of HRP labeled brain stem neurons in regenerated animals was 40% of that found in normal animals. The number of labeled cells in the brachial spinal cord was within the range of normal animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Amputation, Surgical↗
PubMed1988

In vivo test system for tumor production by cell lines derived from lower vertebrates.

The immune suppressed lizard, Anolis carolinensis, can be used to test for in vivo tumor production by cell lines derived from a variety of ectothermic vertebrates. Cell lines tested for tumor production were also assessed for loss of attachment-dependent proliferation and contact inhibition of cell overlap. The results demonstrate that the criteria standardly used to assess transformation and neoplastic change in cultured mammalian cells apply equally well to cultured cells from ectotherms.

Animals↗
PubMed1983

Changes in cell surface antigens during in vitro lizard myogenesis.

Indirect immunofluorescence has been used to examine surface antigens of lizard myogenic cells during in vitro differentiation. At least two developmental stage-specific surface alterations have been identified. One of these is a compositional change and involves the appearance of a cell-surface antigen(s) as the cells differentiate. This antigen(s) (Ag1422) is muscle specific and is characteristic of some rounded-up G0 myosin-positive myocytes, all stretched-back, G0 myosin-positive myocytes, and all identifiable myotubes. The antigen is not found on proliferating myoblasts, extended G1 (myosin-negative) cell-cycle-competent myoblasts or newly differentiated rounded-up, G0 myosin-positive myocytes. Pretreatment of cells with neuraminidase, trypsin, or proteinase K indicates the antigen is not present in "masked" form on normally nonreactive cells. Proteinase K is effective in the removal or destruction of the antigen, indicating it is at least partially protein in nature. The antigen is expressed in a similar developmental stage-specific fashion on early-passage myogenic cells taken from both adult lizard tail regenerates and embryonic muscle. The antibodies identifying Ag1422 can be removed by adsorption with homogenates of mature skeletal muscle. Therefore, Ag1422 is not an artifact due to in vitro conditions or the expression of a transformation antigen unique to the continuous culture line. The second alteration is an apparent restriction in the mobility of surface components (antigens and lectin receptors). Upon treatment with multivalent ligands, undifferentiated myosin-negative myoblasts exhibit rapid patching and capping of cell surface components while well-differentiated myocytes and myotubes do not. This mobility restriction is evident after the appearance of Ag1422. Treatment with cytochalasin B (15 micrograms/ml) and/or colchicine (100 microM) does not alter the restricted mobility of surface components seen on differentiated cells. Therefore, neither microfilaments nor microtubules seem to be involved in the mobility restriction. These observations are discussed in relation to current views of myogenesis.

Adsorption↗