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Biomedical subjects

J Sanderson

Publications and source records attributed to J Sanderson.

At least 19 recordsLinked to original sources
PubMed2000

A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy.

Single-chain Fv antibody fragments from the CD20-specific murine monoclonal antibody B9E9 were genetically engineered as streptavidin fusions [single-chain Fv-streptavidin (scFvSA) fusion protein] for use in pretargeted radioimmunotherapy. The scFvSA constructs were expressed as soluble, tetrameric species in the periplasm of Escherichia coli. Expression levels were affected by the order of the variable regions and the length and composition of the single-chain Fv linker. The best expressor was obtained with the variable regions in the heavy chain-light chain configuration separated by a 25-mer Gly4Ser linker. This construct produced 250-300 mg of soluble, tetrameric fusion protein per liter of fermentor culture. The fusion protein (Mr 173,600) was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of about 50% and was analyzed for functionality both in vitro and in vivo. Immunoreactivity of the scFvSA fusion protein and its nanomolar affinity to CD20-positive Ramos cells were comparable with the B9E9 monoclonal antibody. The fusion protein had a biotin dissociation rate identical to recombinant streptavidin and bound an average of 3.6 biotins/molecule of a possible 4 biotins/molecule. Labeled fusion protein cleared from the blood of BALB/c mice with a P half-life of about 16 h. In nude mice bearing Ramos xenografts, the fusion protein demonstrated sufficient tumor localization of functional streptavidin to enable efficient, tumor-specific targeting of a subsequently administered radionuclide-chelate/biotin molecule. These results suggest that large quantities of functional scFvSA can be produced for clinical testing as a therapy for non-Hodgkin's lymphoma.

Amino Acid Sequence↗
PubMed2000

Analysis of single-nucleotide polymorphisms in the interleukin-4 receptor gene for association with inflammatory bowel disease.

Genetic linkage analysis in families with multiple cases of inflammatory bowel disease (IBD) has mapped a gene which confers susceptibility to IBD to the pericentromeric region of chromosome 16 (IBD1). The linked region includes the interleukin(IL)-4 receptor gene (IL4R). Since IL-4 regulation and expression are abnormal in IBD, the IL4R gene is thus both a positional and functional candidate for IBD1. We screened the gene for single-nucleotide polymorphisms (SNPs) by fluorescent chemical cleavage analysis, and tested a subset of known and novel SNPs for allelic association with IBD in 355 families, which included 435 cases of Crohn's disease and 329 cases of ulcerative colitis. No association was observed between a haplotype of four SNPs (val50ile, gln576arg, A3044G, G3289A) and either the Crohn's disease or ulcerative colitis phenotypes using the transmission disequilibrium test. There was also no evidence for association when the four markers were analyzed individually. The results indicate that these variants are not significant genetic determinants of IBD, and that the IL4R gene is unlikely to be IBD1. Linkage disequilibrium analyses showed that the val50ile and gln576arg variants are in complete equilibrium with each other, although they are separated by only about 21 kilobases of genomic DNA. This suggests that a very dense SNP map may be required to exclude or detect disease associations with some candidate genes.

Alleles↗
PubMed2000

A human lens model of cortical cataract: Ca2+-induced protein loss, vimentin cleavage and opacification.

PURPOSE: Cortical cataract in humans is associated with Ca2+ overload and protein loss, and although animal models of cataract have implicated Ca2+-activated proteases in this process, it remains to be determined whether the human lens responds in this manner to conditions of Ca2+ overload. The purpose of these experiments was to investigate Ca2+-induced opacification and proteolysis in the organ-cultured human lens. METHODS: Donor human lenses were cultured in Eagle's minimum essential medium (EMEM) for up to 14 days. The Ca2+ ionophore ionomycin was used to induce a Ca2+ overload. Lenses were loaded with [3H]-amino acids for 48 hours. After a 24-hour control efflux period, lenses were cultured in control EMEM (Ca2+ 1.8 mM), EMEM + 5 microM ionomycin, or EMEM + 5 microM ionomycin + 5 mM EGTA (Ca2+ < 1 microM). Efflux of proteins and transparency were monitored daily. Protein distribution and cytoskeletal proteolysis were analyzed at the end of the experiment. Cytoskeletal proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were probed with anti-vimentin antibody (clone V9) and detected by enhanced chemiluminescence. RESULTS: Lenses cultured under control conditions remained transparent for 14 days in EMEM with no added supplements or serum. The lenses synthesized proteins and had a low rate of protein efflux throughout the experimental period. Ionomycin treatment resulted in cortical opacification, which was inhibited when external Ca2+ was chelated with EGTA. Exposure to ionomycin also led to an efflux of [3H]-labeled protein, amounting to 41% of the labeled protein over the 7-day experimental period, compared with 12% in ionomycin + EGTA-treated lenses. Efflux was accounted for by loss from the lens soluble protein (crystallin) fraction. Western blot analysis of the cytoskeletal protein vimentin (56 kDa) revealed a distinct breakdown product of 48 kDa in ionomycin-treated lenses that was not present when Ca2+ was chelated with EGTA. In addition, high-molecular-weight proteins (approximately 115 kDa and 235 kDa) that cross-reacted with the vimentin antibody were observed in ionomycin-treated lenses. The Ca2+-induced changes were not age dependent. CONCLUSIONS: Human lenses can be successfully maintained in vitro, remaining transparent for extended periods. Increased intracellular Ca2+ induces cortical opacification in the human lens. Ca2+-dependent cleavage and cross-linking of vimentin supports possible roles for calpain and transglutaminase in the opacification process. This human lens calcium-induced opacification (HLCO) model enables investigation of the molecular mechanisms of opacification, and the data help to explain the loss of protein observed in human cortical cataractous lenses in vivo.

Adolescent↗
PubMed1999

Spatial and temporal trends of contaminants in Canadian Arctic freshwater and terrestrial ecosystems: a review.

The state of knowledge of contaminants in Canadian Arctic biota of the freshwater and terrestrial ecosystems has advanced enormously since the publication of the first major reviews by Lockhart et al. and Thomas et al. in The Science of the Total Environment in 1992. The most significant gains are new knowledge of spatial trends of organochlorines and heavy metal contaminants in terrestrial animals, such as caribou and mink, and in waterfowl, where no information was previously available. Spatial trends in freshwater fish have been broadened, especially in the Yukon, where contaminant measurements of, for example, organochlorines were previously non-existent. A review of contaminants data for fish from the Northwest Territories, Yukon and northern Quebec showed mercury as the one contaminant which consistently exceeds guideline limits for subsistence consumption or commercial sale. Lake trout and northern pike in the Canadian Shield lakes of the Northwest Territories and northern Quebec generally had the most elevated levels. Levels of other heavy metals were generally not elevated in fish. Toxaphene was the major organochlorine contaminant in all fish analyzed. The concentrations of organochlorine contaminants in fish appear to be a function not only of trophic level but of other aspects of the lake ecosystem. Among Arctic terrestrial mammals, PCBs and cadmium were the most prominent contaminants in the species analyzed. Relatively high levels (10-60 micrograms g-1) of cadmium were observed in kidney and liver of caribou from the Yukon, the Northwest Territories and northern Quebec, with concentrations in western herds being higher than in those from the east. For the organochlorine contaminants, a west to east increase in zigma PCBs, HCB and zigma HCH was found in caribou, probably as a result of the predominant west to east/north-east atmospheric circulation pattern which delivers these contaminants from industrialized regions of central and eastern North America to the Arctic via long-range atmospheric transport. Radiocesium contamination of lichens and caribou has continued to decrease. Significant contamination by PCBs and lead of soils and vascular plants was observed in the immediate vicinity and within a 20-km radius of DEW line sites in the Canadian Arctic. There was also evidence for transfer of PCBs from plants to lemmings. There was no evidence, however, that large mammals such as caribou living in the general area of the DEW line sites had elevated levels of PCBs. There is very limited temporal trend information for most contaminants in biota of Arctic terrestrial and freshwater environments.

Animals↗
PubMed1999

Barriers to the use of a diagnostic oral microbiology laboratory by general dental practitioners.

OBJECTIVE: To identify barriers on the use of diagnostic microbiology facilities in general dental practice. DESIGN: A cross-sectional survey using a postal questionnaire. SETTING: Primary/secondary care interface between the diagnostic oral microbiology laboratory, University of Glasgow Dental Hospital and School, Glasgow and dental practitioners within the surrounding health boards, 1998. SUBJECTS: All GDPs (797) within Argyll and Clyde, Ayrshire and Arran, Lanarkshire and Greater Glasgow Health Boards. MAIN OUTCOME MEASURES: The responses were expressed as both absolute and relative frequencies. RESULTS: Responses were received from 430 (55%). The most frequent reason for failure to use the service was lack of information, with more than half of the respondents claiming to be unaware of the facility. Lack of request forms and sampling equipment were also viewed as barriers to using the service. CONCLUSIONS: The laboratory is failing to successfully communicate its role in addressing the growing burden of antibiotic resistance in the community and must be more proactive in encouraging appropriate use and increasing accessibility of the service to GDPs.

Attitude of Health Personnel↗
PubMed1999

Thapsigargin inhibits a potassium conductance and stimulates calcium influx in the intact rat lens.

1. An increase in lens cell calcium has long been associated with cortical cataract. Recently, it has been shown that thapsigargin induces a rise in lens cell calcium by release from endoplasmic reticulum stores. The effects of this rise on the optical and membrane characteristics of the lens were studied in the isolated rat lens. 2. The electrical characteristics of the isolated, perifused rat lens were measured using a two-internal microelectrode technique that permits measurement of plasma membrane conductance (Gm), membrane potential (Vm) and junctional conductance in the intact lens. 3. Thapsigargin (1 microM) induced a rapid overall depolarization of Vm that was accompanied by first a decrease and then an increase in Gm. 4. Replacing external Na+ with tetraethylammonium (TEA) abolished the decrease in Gm. However, a transient increase phase was still observed. 5. The changes in conductance were further characterized by measuring 22Na+ and 45Ca2+ influxes into the isolated lens. Thapsigargin (1 microM) induced a transient increase in 45Ca2+, but did not affect Na+ influx. 6. The Ca2+ channel blocker La3+ (10 microM) totally inhibited the thapsigargin-induced Ca2+ influx. It also blocked the increase in Gm observed in control and in Na+-free-TEA medium. In the absence of external calcium, thapsigargin induced a small depolarization in Vm. 7. These data indicate that thapsigargin induces both a decrease in K+ conductance and an increase in Ca2+ conductance. These probably result from release of stored Ca2+ and subsequent activation of store-operated Ca2+ channels (capacitative Ca2+ entry). 8. Thapsigargin application over the time course of these experiments (24 h) had no effect on junctional conductance or on the transparency of the lens.

Animals↗
PubMed1999

Influence of negative childhood experiences on psychological functioning, social support, and parenting for mothers recovering from addiction.

OBJECTIVE: This study was conducted with mothers recovering from drug and alcohol addiction and had three aims: first, to understand the range of negative childhood events these mothers experienced; second, to understand their current level of distress and their parenting experiences; and third, to examine the relationships between negative childhood events and parenting experiences. METHOD: Forty-six mothers participated in a cross-sectional exploratory study and completed a range of self-report measures, including the Child Abuse & Trauma Scale, Social Support Inventory, CES-D, Parenting Stress Index, and the Parenting Scale. RESULTS: When compared to norming samples these mothers reported significantly higher levels of aversive childhood experiences, psychological distress, parenting stress and use of problematic parenting behaviors along with lower levels of social support. Higher levels of neglect and growing up in a negative home environment were significantly correlated with lower levels of social support from the family, higher levels of distress and parenting stress, and greater use of problematic parenting behaviors. CONCLUSION: For this sample there is a greater incidence of aversive childhood experiences and greater problems with maternal functioning. Mothers recovering from addiction have an additional need for clinical attention towards issues of recovery from childhood abuse and responding to parenting difficulties with their own children.

Adult↗
PubMed1999

Quercetin inhibits hydrogen peroxide-induced oxidation of the rat lens.

Cataract results from oxidative damage to the lens. The mechanism involves disruption of the redox system, membrane damage, proteolysis, protein aggregation and a loss of lens transparency. Diet has a significant impact on cataract development, but the individual dietary components responsible for this effect are not known. We show that low micromolar concentrations of the naturally-occurring flavonoid, quercetin, inhibit cataractogenesis in a rat lens organ cultured model exposed to the endogenous oxidant hydrogen peroxide. Other phenolic antioxidants, (+)epicatechin and chlorogenic acid, are much less effective. Quercetin was active both when incubated in the culture medium together with hydrogen peroxide, and was also active when the lenses were pre-treated with quercetin prior to oxidative insult. Quercetin protected the lens from calcium and sodium influx, which are early events leading to lens opacity, and this implies that the non-selective cation channel is protected by this phenolic. It did not, however, protect against formation of oxidized glutathione resulting from H2O2 treatment. The results demonstrate that quercetin helps to maintain lens transparency after an oxidative insult. The lens organ culture/hydrogen peroxide (LOCH) model is also suitable for examining the effect of other dietary antioxidants.

Animals↗
PubMed1998

Vector alkaline phophatase substrate Blue III: one substrate for brightfield histochemistry and high-resolution fluorescence imaging by confocal laser scanning microscopy.

A number of histochemical chromogenic substrates for alkaline phosphatase are commercially available and give reaction products with a range of colours for brightfield examination. Some of these reaction products are also fluorescent, exhibiting a wide excitation range and a broad emission peak. We report here that one of these substrates, Vector Blue III, yields a stable, strongly fluorescent reaction product with an excitation peak around 500 nm and a large Stokes shift to an emission peak at 680 nm. The reaction product can be excited using a mercury lamp with a fluorescein excitation filter or an argon ion laser at 488 nm or 568 nm, and the emission detected using a long-pass filter designed for Cy-5. Thus, a single substrate is suitable for brightfield imaging of tissue sections and high-resolution analysis of subcellular detail, using a confocal laser scanning microscope, in the same specimen.

Adrenocorticotropic Hormone↗
PubMed1998

Acetylcholine-induced membrane potential oscillations in the intact lens.

PURPOSE: Factors that interfere with the acetylcholine signaling system have long been implicated in cataract. The authors sought to investigate the nature of the electrical response of the intact rabbit lens to acetylcholine. METHODS: Membrane potential (Vm) and electrical conductance (Gm) were monitored in the isolated, perifused lens by a technique utilizing two internal microelectrodes. RESULTS: Acetylcholine (100 nM to 1 mM) induced a decrease in membrane conductance and a depolarization of membrane potential in the intact lens. The responses were reversed by application of the muscarinic antagonist, atropine (1 microM to 100 microM). In the presence of 1 microM thapsigargin and 30 microM cyclopiazonic acid, acetylcholine still induced an electrical response. Long exposures to acetylcholine induced sustained oscillations of Vm in 10 of 29 lenses (34%). Oscillations were blocked by atropine and the L-type Ca2+ channel blocker nifedipine (10 microM) but were potentiated by thapsigargin. CONCLUSIONS: The rabbit lens expressed muscarinic receptors that when activated modulate ionic conductances and cause membrane potential oscillations throughout the tissue. Ca2+ influx rather than primary release from intracellular stores appeared to play a major role in the oscillatory response to acetylcholine.

Acetylcholine↗
PubMed1997

Anti-biotin antibodies offer superior organelle-specific labeling of mitochondria over avidin or streptavidin.

The mitochondrial matrix contains endogenously biotinylated proteins. These proteins can cause unexpected background signal when biotin-avidin- or biotin-streptavidin-based detection systems are used in immunocytochemistry. Here we show that this reactivity can be deliberately exploited, using a simple anti-biotin reagent, to obtain strong and highly specific labeling of mitochondria by both light and electron microscopy. The signal is substantially stronger than when either avidin or streptavidin is used to detect the endogenous biotin. These results confirm the accessibility of protein-bound endogenous biotin to exogenous probes, and localize the biotinylated enzymes to the mitochondrial matrix.

Animals↗
PubMed1997

Prevalence of sleep-disordered breathing in diastolic heart failure.

OBJECTIVE: Sleep-disordered breathing (SDB) is common in congestive heart failure. While isolated diastolic heart failure (DHF) accounts for up to a third of all cases of congestive heart failure, the prevalence of SDB in DHF is unknown. We aim to determine the prevalence and characteristics of SDB in a group of patients with symptomatic DHF. METHODS: Twenty subjects with symptomatic DHF (New York Heart Association class II or III) and isolated diastolic dysfunction on echocardiography were assessed with lung function tests, modified sleep and health questionnaire, and overnight polysomnography. Significant SDB was defined as an apnea/hypopnea index (AHI) > 10. RESULTS: Thirteen female and seven male subjects (mean age, 65+/-6.0 years; mean body mass index (BMI), 28+/-3.2) were evaluated, of whom 17 (85%) had a diagnosis of hypertension. Overall sleep quality was poor, with fragmentation and frequent arousals associated with respiratory events. Fifty-five percent of the patients had significant SDB, mainly obstructive apneas. BMI and the prevalence of hypertension were similar in patients with and without SDB. The deceleration time, an index of diastolic dysfunction, was more prolonged in the group with SDB (236+/-40 ms vs 282+/-31 ms; p<0.05). As a group, a lower minimum percentage arterial oxygen saturation during sleep, but not the AHI was associated with more severe degree of diastolic dysfunction on echocardiogram, including a lower ratio between the early peak transmittal flow velocity and the late peak atrial systolic velocity (rho=0.57; p<0.05) and a prolonged isovolumic relaxation time (rho=-0.54; p<0.05). CONCLUSIONS: SDB is common in patients with DHF. Patients with DHF and SDB may be associated with worse diastolic dysfunction than those without SDB, although a causal relationship remains to be established.

Aged↗
PubMed1996

Role of gamma delta T cells in pathogenesis and diagnosis of Behcet's disease.

BACKGROUND: Behcet's disease (BD) is a multisystem disorder of unknown pathogenesis. The diagnosis is based on a set of international clinical criteria. Previous investigations have suggested that immunological cross-reactivity between peptides within streptococcal heat-shock proteins and human peptides might be involved in the pathogenesis of BD. We tested four peptides from mycobacterial heat-shock proteins to see if they specifically stimulated gamma delta T cells from BD patients. We then investigated this response to see whether it could be used as a laboratory test to diagnose BD. METHODS: We used a T-cell proliferative test to assay responses to four mycobacterial 65 kDa heat-shock-protein peptides and to four homologous peptides derived from the sequence of the human 60 kDa heat-shock protein. FINDINGS: We elicited significant gamma delta T-cell responses to the mycobacterial peptides in 25 (76%) of 33 patients with BD, compared with 2 (3.6%) of 55 controls with recurrent oral ulcers, systemic disease, or no disorders. The proportion of BD patients who had false-negative results decreased if the test was done during clinical manifestation of disease activity. There was a correlation between disease activity and T-cell responses. Four homologous peptides from human 60 kDa heat-shock protein also specifically stimulated T cells from patients with BD but with lower stimulation indices. INTERPRETATION: Activation of peripheral-blood mononuclear cells with the four heat-shock-protein peptides elicited significant T-cell proliferative responses by the gamma delta subset of T cells, which may regulate alpha beta T cells. Because these peptides have a high specificity for BD, this assay can be used as a laboratory diagnostic test for BD.

Adult↗