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Biomedical subjects

R Duncan

Publications and source records attributed to R Duncan.

At least 91 records · Page 5Linked to original sources
PubMed1998

Early phase tumor accumulation of macromolecules: a great difference in clearance rate between tumor and normal tissues.

The objective of this study was to investigate the molecular weight (MW) and time-dependence of the phenomenon termed "the enhanced permeability and retention" (EPR) effect in solid tumor, in particular to determine and define the early phase accumulation of macromolecules in tumor and normal tissues and the relationship between blood concentration and tissue clearance. As a model, radioiodinated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers of MW ranging from 4.5 K to 800 K were administered i.v. to mice bearing sarcoma 180 tumor. Within 10 min all HPMA copolymers accumulated effectively in the tumor regardless of MW (1.0-1.5% of injected dose per g of tumor). However, higher MW copolymers (> 50 K) showed significantly increased tumor accumulation after 6 h, while the lower MW copolymers (< 40 K) were cleared rapidly from tumor tissue due to rapid diffusion back into the bloodstream. Blood clearance was also MW-dependent; the lower MW copolymers displayed rapid clearance, with kidney radioactivity of the copolymers of MW < 20 K representing 24% of injected dose per g kidney at 1 min after i.v. administration. Within 10 min these copolymers passed through the kidney and were excreted in the urine. Higher MW copolymers consistently showed kidney levels of 3-5% dose per g kidney in the early phase with no time-dependent accumulation in kidney. There was also no progressive accumulation in muscle or liver, regardless of polymer MW. These results suggest the "EPR effect" in solid tumor primarily arises from in the difference in clearance rate between the solid tumor and the normal tissues after initial penetration of the polymers into these tissues.

Animals↗
PubMed1998

Correlation between interictal regional cerebral blood flow and depth-recorded interictal spiking in temporal lobe epilepsy.

PURPOSE: Single photon emission computed tomography (SPECT) is used as an adjunctive method in preoperative localization of epileptic foci. In temporal lobe epilepsy (TLE), interictal hypoperfusion is observed in 60-70% of cases. Correlation with ictal EEG changes is observed in approximately 50-60% of cases. Relationships with interictal EEG have been studied less. We compared interictal SPECT data obtained in 20 patients with their interictal intracerebral electrical activity recorded by depth electrodes to evaluate a potential relationship. METHODS: We studied 20 sequential patients whose clinical, surface, and depth EEG data indicated seizure originating in the temporal lobe and who had interictal [99mTc]hexamethylpropylene amine oxime (HMPAO)-SPECT stereo-EEG (SEEG). Intracerebral electrodes were placed according to the patient's profile. The interictal extent of epileptiform activity allowed delineation of the irritative zone. Interictal spike frequency was also analyzed semiquantitatively. Visual and numerical SPECT analysis was performed blind to SEEG data. RESULTS: Interictal hypoperfusion was observed in 16 patients, involving the epileptogenic temporal lobe in 14. Except for 1 patient who manifested lateral temporal hypoperfusion corresponding to a mass lesion, two distinctive patterns of hypoperfusion were noted: (a) mesial hypoperfusion (5 patients), and (b) global temporal hypoperfusion (8 patients). In 8 patients, hypoperfusion had also extended into the adjacent cortex. Temporal mesial hypoperfusion was associated with spiking limited to the mesial structures, whereas global temporal hypoperfusion or hypoperfusion extending beyond the temporal lobe was associated with a similar topographic pattern of spikes. CONCLUSIONS: Comparison between SPECT and SEEG data collected in the interictal phase indicated that the extent of the hypoperfused area correlated topographically with that of the underlying irritative zone.

Adolescent↗
PubMed1998

Preclinical toxicology of a novel polymeric antitumour agent: HPMA copolymer-doxorubicin (PK1).

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (PK1) is a novel polymeric anticancer agent containing doxorubicin (approximately 8 wt%) bound to the polymer backbone via a Gly-Phe-Leu-Gly peptidyl linker. The approximate LD50 of PK1 in MF1 mice after a single i.v. injection was 63 mg/kg (doxorubicin-equivalent). Single doses of PK1 were administered to MF1 mice at 22.5 or 45 mg/kg and blood samples taken on days 3, 7 and 14 for haematological examination and clinical chemistry. At day 14 all animals were sacrificed for necropsy. In a multiple dose study, PK1 was administered i.v. to MF1 mice or Wistar rats (20 animals per group) weekly for five consecutive weeks at doses of 12.0 or 22.5 mg/kg (mice) or 3 and 5 mg/kg (rats). After 31 days 10 animals from each group were sacrificed for necropsy and the remainder were sacrificed after 59 days. Blood samples were taken 3 days after administration of each dose and at the end of the experiment, and urine samples were collected on the day prior to sacrifice. Mortality in the single dose mouse and multiple dose rat studies was low. In the multiple dose mouse study 4/10 animals were killed in extremis before the scheduled day 31 and all animals died before day 37. PK1 induced a reduction in WBC and platelets in rats and mice shortly after treatment and RBC at later times, and in the single dose study alanine and aspartate aminotransferase levels were elevated at higher doses. Liver damage was seen only in rat tissue during histological examination. Other histological changes induced by PK1 include thymic and testicular atrophy, bone marrow depletion gastrointestinal tract changes and in the multiple dose study an increase in nuclear size in the proximal tubules of the kidney (although no changes in urine were seen). Recovery from these effects was seen in rats at 59 days. A PK1 dose of 20 mg/m2 (doxorubicin equivalent) was recommended as a safe dose for the start of Phase I clinical trials.

Animals↗
PubMed1997

Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA.

A cellular protein, previously described as p35/38, binds to the complementary (-)-strand of the leader RNA and intergenic (IG) sequence of mouse hepatitis virus (MHV) RNA. The extent of the binding of this protein to IG sites correlates with the efficiency of the subgenomic mRNA transcription from that IG site, suggesting that it is a requisite transcription factor. We have purified this protein and determined by partial peptide sequencing that it is heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an abundant, primarily nuclear protein. hnRNP A1 shuttles between the nucleus and cytoplasm and plays a role in the regulation of alternative RNA splicing. The MHV(-)-strand leader and IG sequences conform to the consensus binding motifs of hnRNP A1. Recombinant hnRNP A1 bound to these two RNA regions in vitro in a sequence-specific manner. During MHV infection, hnRNP A1 relocalizes from the nucleus to the cytoplasm, where viral replication occurs. These data suggest that hnRNP A1 is a cellular factor that regulates the RNA-dependent RNA transcription of the virus.

Animals↗
PubMed1997

Phospholipid peroxidation induces cytosolic phospholipase A2 activity: membrane effects versus enzyme phosphorylation.

Cytosolic phospholipase A2 (cPLA2) is a signal-responsive enzyme that is highly selective to the nature of phospholipid substrates. A mechanism for cPLA2 activity regulation through a signal transduction pathway has been proposed and this signaling appears to be influenced by oxidants. Oxidant-mediated signaling of PLA2 may serve as an alternative mechanism for enzyme regulation; however, the manner of regulation has yet to be delineated. In this report we demonstrate that there is a direct effect of membrane oxidation on cPLA2 phosphorylation and activity. A simple in vitro system consisting of purified cPLA2 and phospholipid vesicles was used to facilitate protein kinase C (PKC) activity and provide substrates for cPLA2. Using these vesicles we found that the activity of cPLA2 was enhanced twofold when the vesicles contained as little as 5 mol% phosphatidylcholine hydroperoxides (PLPCOOH). The order of hydrolytic preference for fatty acyl species was 20:4 > 18:2 > 18:1 > 16:0, and the presence of PLPCOOH stimulated hydrolysis largely of phosphatidylcholine containing 20:4. The Ca2+ concentrations required for stimulated hydrolytic activity were also twofold lower for oxidized compared to unoxidized vesicles. Using phospholipid micelles as substrates, PKC-mediated phosphorylation of cPLA2 increased hydrolytic activity 71% compared to preparations lacking PKC. Using phospholipid vesicles as substrates, PKC-mediated phosphorylation resulted in an 85% increase in cPLA2 activity compared to preparations without PKC. PKC-mediated phosphorylation of cPLA2, therefore, stimulates catalytic activity toward membrane phospholipids and the extent of activation is enhanced directly by peroxidation of membrane phospholipids and involves a peroxide-induced stimulation of cPLA2 phosphorylation.

Animals↗
PubMed1997

Ictal regional cerebral blood flow in frontal lobe seizures.

99mTC (single photon emission computed tomography) (HMPAO SPECT) was carried out during 16 frontal-lobe seizures in 15 patients. Focal changes in regional cerebral blood flow were seen during all seizures. In 9 of 16 seizures SPECT showed hyperperfusion localized to one frontal lobe. In 1 of 16 seizures ictal hypoperfusion was seen in one frontal lobe. In 2 of 16 seizures there was hyperperfusion in both frontal lobes, and in 4 of 16 seizures hyperperfusion involved the frontal lobe or lobes plus other lobes of the brain. These changes were accompanied by hyperperfusion of subcortical structures in 13 seizures. SPECT thus localized to one frontal lobe in 10 of 16 seizures, and localized to the frontal lobes without lateralizing in two further seizures. No seizure showed a pattern of perfusion similar to that reported in mesial-temporal-lobe seizures. We conclude that ictal SPECT may provide useful localizing information in frontal-lobe seizures.

Adolescent↗
PubMed1997

Construction of a homodimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme.

A gene encoding a bifunctional homodimeric dihydrofolate reductase-thymidylate synthase (DHFR-TS) was constructed by destroying the stop codon of Escherichia coli dihydrofolate reductase (DHFR) and joining the coding sequences of the monofunctional enzymes by a five amino acid linker. The protein was designed to mimic features of active site proximity and electrostatics in the protozoan DHFR-TSs which are believed to be important in channeling of the DHFR substrate, H2folate, to TS. The genetically engineered catalytically active homodimeric bifunctional DHFR-TS was expressed, purified and characterized. The component activities of the purified bifunctional enzyme had kinetic properties similar to those of the monofunctional TS and DHFR, but unlike the authentic bifunctional enzymes from protozoa this enzyme did not kinetically channel dihydrofolate from DHFR to TS.

Amino Acid Sequence↗
PubMed1997

Ictal single photon emission computed tomography in occipital lobe seizures.

PURPOSE: Ictal single photon emission computed tomography (SPECT) has been evaluated as an adjunctive localizing technique in temporal lobe epilepsies and, to a lesser degree, in some extratemporal epilepsies. The purpose of this study was to determine whether occipital lobe seizures are associated with distinctive ictal cerebral blood perfusion (rCP) patterns. METHODS: SPECT was used with the tracer 99mTc HMPAO to image ictal rCP in 6 patients in whom clinical, EEG, and imaging data indicated occipital lobe seizures. RESULTS: Two patterns of rCP were seen. Four patients had hyperperfusion that was restricted to the occipital lobe, and two patients had hyperperfusion of the occipital lobe and the ipsilateral mesial temporal lobe, with hypoperfusion of the lateral temporal lobe. The latter 2 patients had clinical and surface EEG evidence of temporal lobe involvement in the seizure discharge. CONCLUSIONS: Ictal rCP patterns in occipital lobe seizures are distinct from those in temporal lobe seizures and may vary according to whether or not ipsilateral temporal lobe structures are involved in the ictal discharge.

Adolescent↗
PubMed1997

Stroke without dissection from a neck holding manoeuvre in martial arts.

Carotid artery trauma is a known cause of stroke in young people. The vessel may occlude, dissect or shower thrombotic emboli into intracranial vessels. This paper reports the use of single photon emission computed tomography (SPECT) imaging in a 29 year old man who developed an embolic stroke after neck holding manoeuvres at a martial arts class. Awareness of the potential consequences of these procedures is matched by the need for rapid and accurate diagnosis of stroke now that thrombolytic and neuroprotective treatments are emerging, which are effective only within a short time window.

Adult↗
PubMed1996

Avian reovirus-induced syncytium formation is independent of infectious progeny virus production and enhances the rate, but is not essential, for virus-induced cytopathology and virus egress.

The nonenveloped avian reoviruses represent a distinct antigenic subgroup of orthoreoviruses. Unlike their mammalian counterparts, the avian reoviruses exhibit the unusual property of inducing rapid and extensive syncytium formation in cell cultures, a cytopathic effect more commonly associated with enveloped virus replication. While the syncytium-inducing capability of avian reovirus has been known for quite some time, the relationship between cell fusion and the virus replication cycle has not been determined. The conservation of the syncytial phenotype among all field isolates of avian reovirus suggests that avian reovirus-induced syncytium formation either reflects an essential step in the virus replication cycle involving intracellular membrane interactions or that cell fusion contributes to enhanced virus replication in infected animals. In order to distinguish between these possibilities, we have examined several aspects of virus replication in the presence of inhibitors of syncytium formation. Inhibitors of intracellular vesicle transport and O-linked glycosylation eliminated or markedly reduced syncytium formation with little effect on the rate or extent of virus macromolecular synthesis and infectious progeny virus production. Moreover, syncytium formation was not required for virus-induced cytopathology or virus egress but did significantly enhance the rate of both of these processes. The data indicate that, unlike the syncytium-inducing enveloped viruses, the membrane interactions and protein trafficking required for avian reovirus-induced syncytium formation do not reflect the sequelae of an essential step in the virus replication cycle. These results suggest that the conservation of the avian reovirus syncytial phenotype may reflect a fortuitous aspect of virus replication which confers advantages associated with the rapid spread of the virus within an infected host.

Animals↗
PubMed1996

The low pH-dependent entry of avian reovirus is accompanied by two specific cleavages of the major outer capsid protein mu 2C.

Avian reoviruses are capable of inducing rapid and extensive syncytium formation, a process that occurs preferentially under conditions of neutral or alkaline pH. In order to ascertain whether the membrane fusion-inducing capability of avian reovirus confers a pH-independent entry mechanism on the virus, virus entry was investigated using internalization assays and several lysomotropic agents that inhibit endosomal acidification. The ability of avian reovirus to infect cells was severely restricted under all conditions that prevented endosomal acidification. The decreased infection efficiency in the presence of the lysomotropic agents correlated with an inhibition in the proteolytic processing of the major outer capsid protein mu 2C. The importance, with respect to virus infection, of the low pH-dependent cleavage of the avian reovirus mu 2C protein was confirmed by demonstrating that infectious subviral particles, generated by proteolytic processing in vitro, were capable of efficiently infecting cells in the presence of the lysomotropic agents. These results indicated that avian reovirus entry-specific membrane interactions are largely dependent on an endosome-mediated proteolytic processing of the virus particle, suggesting that the syncytium-inducing properly of the sigma 3 protein is not sufficient to promote virus uptake. Furthermore, avian reovirus internalization was associated with two distinct cleavages of the major outer capsid protein mu 2C, unlike the entry-specific processing of the analagous mammalian reovirus major outer capsid protein mu 1C. The mu 2C cleavages occured sequentially and appeared to involve distinct cleavage specificities. Moreover, the second cleavage event was observed to be both virus strain- and cell type-independent, suggesting that the cleavage is both specific and biologically significant.

Animals↗
PubMed1996

The Low pH-Dependent Entry of Avian Reovirus Is Accompanied by Two Specific Cleavages of the Major Outer Capsid Protein &mu;2C

Avian reoviruses are capable of inducing rapid and extensive syncytium formation, a process that occurs preferentially under conditions of neutral or alkaline pH. In order to ascertain whether the membrane fusion-inducing capability of avian reovirus confers a pH-independent entry mechanism on the virus, virus entry was investigated using internalization assays and several lysomotropic agents that inhibit endosomal acidification. The ability of avian reovirus to infect cells was severely restricted under all conditions that prevented endosomal acidification. The decreased infection efficiency in the presence of the lysomotropic agents correlated with an inhibition in the proteolytic processing of the major outer capsid protein &mu;2C. The importance, with respect to virus infection, of the low pH-dependent cleavage of the avian reovirus &mu;2C protein was confirmed by demonstrating that infectious subviral particles, generated by proteolytic processing in vitro, were capable of efficiently infecting cells in the presence of the lysomotropic agents. These results indicated that avian reovirus entry-specific membrane interactions are largely dependent on an endosome-mediated proteolytic processing of the virus particle, suggesting that the syncytium-inducing property of the sigma3 protein is not sufficient to promote virus uptake. Furthermore, avian reovirus internalization was associated with two distinct cleavages of the major outer capsid protein &mu;2C, unlike the entry-specific processing of the analagous mammalian reovirus major outer capsid protein &mu;1C. The &mu;2C cleavages occured sequentially and appeared to involve distinct cleavage specificities. Moreover, the second cleavage event was observed to be both virus strain- and cell type-independent, suggesting that the cleavage is both specific and biologically significant.

Journal Article↗
PubMed1996

C-terminal trimerization, but not N-terminal trimerization, of the reovirus cell attachment protein Is a posttranslational and Hsp70/ATP-dependent process.

The C-terminal globular head of the lollipop-shaped final sigma1 protein of reovirus is responsible for interaction with the host cell receptor. Like the N-terminal fibrous tail, it has its own trimerization domain. Whereas N-terminal trimerization (formation of a triple alpha-helical coiled coil) occurs at the level of polysomes (i.e. cotranslationally) and is ATP-independent, C-terminal trimerization is a posttranslational event that requires ATP. Coprecipitation experiments using anti-Hsp70 antibodies and truncated final sigma1 proteins synthesized in vitro revealed that only regions downstream of the N-terminal alpha-helical coiled coil were associated with Hsp70. Hsp70 was also found to be associated with nascent final sigma1 chains on polysomes as well as with immature postribosomal final sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini). These latter structures were true intermediates in the final sigma1 biogenetic pathway since they could be chased into mature final sigma1 trimers with the release of Hsp70. Thus, unlike N-terminal trimerization, C-terminal trimerization is Hsp70- and ATP-dependent. The involvement of two mechanistically distinct oligomerization events for the same molecule, one cotranslational and one posttranslational, may represent a common approach to the generation of oligomeric proteins in the cytosol.

Adenosine Triphosphate↗
PubMed1996

Interictal temporal hypoperfusion is related to early-onset temporal lobe epilepsy.

Previous studies of interictal regional cerebral blood flow (rCBF) in temporal lobe epilepsy have shown variable correlations with clinical measures. We used high spatial resolution hexamethyl propyleneamine oxime single photon emission computed tomography (HMPAO SPECT) in 80 consecutive patients with complex partial seizures (CPS), comparing results with those from a large series of normal subjects. Visual image analysis detected abnormalities of rCBF in 41 of 80 (51%; numeric analysis detected abnormalities in 38 of 80). Age at epilepsy onset was significantly younger in patients with temporal hypoperfusion (p = 0.002), and the frequency distribution of hypoperfusion versus age at epilepsy onset was reverse exponential. The results of numerical image analysis showed that degree of hypoperfusion did not vary with age at epilepsy onset. These data suggest a single insult operating early in life as a cause of temporal hypoperfusion, as has been shown for mesial temporal sclerosis (MTS). We could not demonstrate relationships with other clinical variables, including time since last seizure.

Adolescent↗
PubMed1996

Autoantigens interact with cis-acting elements of rubella virus RNA.

Rubella virus (RV) infections in adult women can be associated with acute and chronic arthritic symptoms. In many autoimmune individuals, antibodies are found targeting endogenous proteins, called autoantigens, contained in ribonucleoprotein complexes (RNPs). In order to understand the molecular mechanisms involved in the RV-associated pathology, we investigated the nature of cellular factors binding RV RNA and whether such RNPs were recognized by antibodies in infected individuals. Previously, we noted that cellular proteins associated with the RV 5'(+) stem-loop (SL) RNA are recognized by serum with Ro reactivity. To better understand the nature of the autoantigens binding RV cis-acting elements, serum samples from individuals with various autoimmune diseases were tested for their ability to immunoprecipitate RNPs containing labeled RV RNAs. A subset of serum samples recognizing autoantigen La, or Ro and La, immunoprecipitated both the RV 5'(+)SL and 3'(+)SL RNA-protein complexes. Autoantigens binding the RV 5'(+)SL and 3'(+)SL RNAs differed in molecular mass, specificities for respective RNA binding substrates, and sensitivity to alkaline phosphatase treatment. The La autoantigen was found to interact with the RV 5'(+)SL RNA as determined by immunological techniques and binding reactions with mixtures containing recombinant La protein. To test whether there is a correlation between La binding to an RV RNA element and the appearance of an anti-La response, we measured anti-La titers in RV-infected individuals. Significant anti-La activity was detected in approximately one-third of RV-infected individuals 2 years postinfection.

Adult↗