Biomedical subjects

G C Fletcher

Publications and source records attributed to G C Fletcher.

At least 19 recordsLinked to original sources

PubMed2006

CD70 (TNFSF7) is expressed at high prevalence in renal cell carcinomas and is rapidly internalised on antibody binding.

In order to identify potential markers of renal cancer, the plasma membrane protein content of renal cell carcinoma (RCC)-derived cell lines was annotated using a proteomics process. One unusual protein identified at high levels in A498 and 786-O cells was CD70 (TNFSF7), a type II transmembrane receptor normally expressed on a subset of B, T and NK cells, where it plays a costimulatory role in immune cell activation. Immunohistochemical analysis of CD70 expression in multiple carcinoma types demonstrated strong CD70 staining in RCC tissues. Metastatic tissues from eight of 11 patients with clear cell RCC were positive for CD70 expression. Immunocytochemical analysis demonstrated that binding of an anti-CD70 antibody to CD70 endogenously expressed on the surface of A498 and 786-O cell lines resulted in the rapid internalisation of the antibody-receptor complex. Coincubation of the internalising anti-CD70 antibody with a saporin-conjugated secondary antibody before addition to A498 cells resulted in 50% cell killing. These data indicate that CD70 represents a potential target antigen for toxin-conjugated therapeutic antibody treatment of RCC.

Antibodies↗

PubMed2003

hAG-2 and hAG-3, human homologues of genes involved in differentiation, are associated with oestrogen receptor-positive breast tumours and interact with metastasis gene C4.4a and dystroglycan.

hAG-2 and hAG-3 are recently discovered human homologues of the secreted Xenopus laevis proteins XAG-1/2 (AGR-1/2) that are expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development. Although the roles of hAG-2 and hAG-3 in mammalian cells are unknown, both proteins share a high degree of protein sequence homology and lie adjacent to one another on chromosome 7p21. hAG-2 mRNA expression has previously been demonstrated in oestrogen receptor (ER)-positive cell lines. In this study, we have used real-time quantitative RT - PCR analysis and immunohistochemistry on tissue microarrays to demonstrate concordant expression of hAG-2 and hAG-3 mRNA and protein in breast tumour tissues. Tumour expression of both genes correlated with OR (hAG2, P=0.0002; hAG-3, P=0.0012), and inversely correlated with epidermal growth factor receptor (EGFR) (P=0.003). Yeast two-hybrid cloning identified metastasis-associated GPI-anchored C4.4a protein and extracellular alpha-dystroglycan (DAG-1) as binding partners for both hAG-2 and hAG-3, which if replicated in clinical oncology would demonstrate a potential role in tumour metastasis through the regulation of receptor adhesion and functioning. hAG-2 and hAG-3 may therefore serve as useful molecular markers and/or potential therapeutic targets for hormone-responsive breast tumours.

Amino Acid Sequence↗

PubMed2003

Proteomic analysis of the cell-surface membrane in chronic lymphocytic leukemia: identification of two novel proteins, BCNP1 and MIG2B.

B-cell-specific plasma-membrane proteins are potential targets for either small molecule or antibody-based therapies. We have sought to annotate proteins expressed at the cell surface membrane in patients with chronic lymphocytic leukemia (CLL) using plasma-membrane-based proteomic analysis to identify previously uncharacterized and potentially B-cell-specific proteins. Proteins from plasma-membrane fractions were separated on one-dimensional gels and trypsinized fractions subjected to high-throughput MALDI-TOF mass spectrometry. Using this method, many known B-cell surface antigens were detected, but also known proteins not previously described in this disease or in this cellular compartment, including cell surface receptors, membrane-associated enzymes and secreted proteins, and completely unknown proteins. To validate the method, we show that BLK, a B-cell-specific kinase, is located in the CLL-plasma-membrane fraction. We also describe two novel proteins (MIG2B and B-cell novel protein #1, BCNP1), which are expressed preferentially in B cells. MIG2B is in a highly conserved and defined gene family containing two plasma-membrane-binding ezrin/radixin/moesin domains and a pleckstrin homology domain; the Caenorhabditis elegans homolog (UNC-112) is a membrane-associated protein that colocalizes with integrin at cell-matrix adhesion complexes. BCNP1 is a completely unknown protein with three predicted transmembrane domains, with three alternatively spliced final exons. Proteomic analysis may thus define new potential therapeutic targets.

Apoptosis Regulatory Proteins↗

PubMed2002

Haemoglobin level in Scottish intensive care patients--a national one day survey.

UNLABELLED: Objectives were to determine haemoglobin (Hb) levels present in patients and blood ordering habits of clinicians within Scottish Intensive Care Units (ICUs) on one typical day. A questionnaire survey (February 29 2000) was sent to all adult Scottish ICUs. All patients present in the responding adult ICUs in Scotland on the above date were included. MEASUREMENTS AND MAIN RESULTS: Nineteen (73%) of the 26 Scottish Adult Intensive Care Units (ICUs) responded to the questionnaire. Data were received from 78 patients, 8 (10%) received blood. Mean initial Hb was 102 g/l (range 63-138). Modal transfusion trigger haemoglobin was 80 g/l in 38% of subjects at first trigger, 100 g/l in 24% of cases. No intensive care unit allowed haemoglobin to fall below 70 g/l and no patients were transfused when measured Hb was greater than 100 g/l. The presence of ischaemic heart disease was the second most important trigger to transfuse after haemoglobin level. Modal transfusion was 2 units (n = 7). Only one patient received a single unit transfusion. CONCLUSIONS: Scottish ICUs maintain Hb between 70 and 100 g/l but clinicians are currently not consistent when ordering blood. More investigation is required to determine the optimal haemoglobin in our ICU population.

APACHE↗

PubMed2001

Mitochondria are selectively eliminated from eukaryotic cells after blockade of caspases during apoptosis.

Pan caspase inhibitors are potentially powerful cell-protective agents that block apoptosis in response to a wide variety of insults that cause tissue degeneration. In many conditions, however, the blockade of apoptosis by caspase inhibitors does not permit long-term cell survival, but the reasons are not entirely clear. Here we show that the blockade of apoptosis by Boc.Aspartyl(O-methyl)CH2F can result in the highly selective elimination of the entire cohort of mitochondria, including mitochondrial DNA, from both neurons and HeLa cells, irrespective of the stimulus used to trigger apoptosis. In cells that lose their mitochondria, the nuclear DNA, Golgi apparatus, endoplasmic reticulum, centrioles, and plasma membrane remain undamaged. The capacity to remove mitochondria is both specific and regulated since mitochondrial loss in neurons is completely prevented by the expression of the antiapoptotic protein Bcl-2 and partially suppressed by the autolysosomal inhibitor bafilomycin. Cells without mitochondria are more tolerant to an anaerobic environment but are essentially irreversibly committed to death. Prevention of mitochondrial loss may be crucial for the long-term regeneration of tissues emerging from an apoptotic episode in which death was prevented by caspase blockade.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Death commitment point is advanced by axotomy in sympathetic neurons.

Axotomized neurons have several characteristics that are different from intact neurons. Here we show that, unlike established cultures, the axotomized sympathetic neurons deprived of NGF become committed to die before caspase activation, since the same proportion of NGF-deprived neurons are rescued by NGF regardless of whether caspases are inhibited by the pan-caspase inhibitor Boc-Asp(O-methyl)-CH(2)F (BAF). Despite prolonged Akt and ERK signaling induced by NGF after BAF treatment has prevented death, the neurons fail to increase protein synthesis, recover ATP levels, or grow. Within 3 d, all the mitochondria disappear without apparent removal of any other organelles or loss of membrane integrity. Although NGF does rescue intact BAF-treated 6-d cultures after NGF deprivation, rescue by NGF fails when these neurons are axotomized before NGF deprivation and BAF treatment. Moreover, cytosolic cytochrome c rapidly kills axotomized neurons. We propose that axotomy induces signals that make sympathetic neurons competent to die prematurely. NGF cannot repair these NGF-deprived, BAF-treated neurons because receptor signaling (which is normal) is uncoupled from protein renewal, and the mitochondria (which are damaged) go on to be eliminated. Hence, the order of steps underlying neuronal death commitment is mutable and open to regulation.

Adenosine Triphosphate↗

PubMed2000

The effect of 100% CO2 on the growth of nonproteolytic Clostridium botulinum at chill temperatures.

The growth of a cocktail of spores from six nonproteolytic Clostridium botulinum type B and E isolates at 5 and 10 degrees C was used to assess the combined effect of NaCl (0.5-4.5% w/v), pH (5.5-6.5) and atmosphere (10% H2:90% N2, 5% CO2:10% H2:85% N2, or 100% CO2) in buffered peptone, yeast, glucose, starch broth with an Eh of approximately -350 mV. Under all atmospheres growth tended to be slower as the concentration of NaCl increased and with NaCl combined with pH levels below 6.0. Of the atmospheres tested, growth occurred at a slower rate and over a narrower range of conditions when C. botulinum was exposed to 100% CO2. This effect was enhanced when the incubation temperature was 5 degrees C. The results indicate that while CO2 decreased C. botulinum growth at chill temperatures, prevention of growth also depended on the NaCl concentration and the pH of the medium.

Carbon Dioxide↗

PubMed1999

Autophagy is activated by apoptotic signalling in sympathetic neurons: an alternative mechanism of death execution.

Autophagy is a mechanism whereby cells digest themselves from within and so may be used in lieu of apoptosis to execute cell death. Little is known about its role in neurons. In newly isolated sympathetic neurons, two independent apoptotic stimuli, NGF-deprivation or cytosine arabinoside added in the presence of NGF, caused a 30-fold increase in autophagic particle numbers, many autophagosomes appearing before any signs of DNA-fragmentation. The anti-autophagic drug 3-methyladenine also delayed apoptosis, its neuroprotection correlating with inhibition of cytochrome c release from mitochondria and prevention of caspase activation. In contrast, autophagic activity remained elevated in neurons treated with the pan-caspase inhibitor Boc-Asp(OMe)fmk, which inhibited morphological apoptosis but did not inhibit cytochrome c release nor prevent cell death. We propose that the same apoptotic signals that cause mitochondrial dysfunction also activate autophagy. Once activated, autophagy may mediate caspase-independent neuronal death.

Adenine↗

PubMed1999

A simple in vitro method for the evaluation of an ultrasonic nebulizer for drug delivery to intubated, ventilated patients and the effect of nebulizer and ventilator settings on the uptake of fluid from the nebulizer chamber.

Little is known about the performance of ultrasonic nebulizers during different ventilation patterns when these nebulizers are used to deliver drugs to intubated, ventilated patients. A method that enables the performance of an ultrasonic nebulizer to be evaluated is described. We used an in vitro model to examine the performance of the DeVilbiss Ultra-Neb 2000 ultrasonic nebulizer under positive pressure ventilation. Performance was measured at different rates of nebulization and under changing conditions of positive end-expiratory pressure (PEEP), inspiratory flow rate, inspiratory time and minute volume. The volume of saline nebulized was unchanged by variations in positive end expiratory pressure from 0 to 5 cm to 10 cm H2O, in minute ventilation and in inspiratory flow rate. An increase in the inspiratory time resulted in an increase in the volume of saline nebulized and this volume was greater as the power setting of the nebulizer was increased. We conclude that ultrasonic nebulizers may be affected by different patterns of ventilation and that this simple in vitro assessment of nebulizer function in an intensive care setting may be of value prior to nebulizer use.

Aerosols↗

PubMed1998

Thermal death times of Hafnia alvei cells in a model suspension and in artificially contaminated hot-smoked kahawai (Arripis trutta).

In New Zealand the product most frequently implicated in cases of scombroid poisoning is hot-smoked kahawai (Arripis trutta). Using a Hafnia alvei strain, previously isolated from a portion of hot-smoked kahawai with a histamine level of 1,659.4 mg/kg, thermal death trials were carried out in a model suspension (0.1% peptone) at 54, 55, 56, 57, and 58 degrees C. From the linear regression line (R2 = 0.98) fitted to observed D values plotted against temperature, calculated D values for 54, 55, 56, 57, and 58 degrees C were estimated to be 0.63, 0.36, 0.20, 0.11, and 0.06 min, respectively, giving a z value of 4.14 degrees C. Thermal death trials were also carried out for H. alvei associated with hot-smoked kahawai at 54, 55, 55.5, 56, and 57 degrees C. From the linear regression line (R2 = 0.93) fitted to the data, calculated D values for 54, 55, 56, and 57 degrees C were estimated to be 1.42, 0.74, 0.38, and 0.20 min, respectively, giving a z value of 3.57 degrees C. Results indicate that hot smoking has the potential to eliminate H. alvei from seafood products.

Enterobacteriaceae↗

PubMed1998

Levels of histamine and histamine-producing bacteria in smoked fish from New Zealand markets.

Smoked fish has been the most commonly implicated product in presumptive cases of scombroid poisoning in New Zealand. One hundred seven samples of smoked fish were purchased from Auckland retail markets between July 1995 and March 1996, and their histamine and bacterial levels were determined. Eight samples, obtained from five of the nine retail outlets sampled, had histamine levels which exceeded 50 mg/kg, the level set by the FDA as an indicator of decomposition. Histamine levels in only 2 samples (346.4 and 681.8 mg/kg) exceeded a hazard level of 200 mg/kg. Thirty-three of the smoked fish were held at 20 degrees C for 2 days, and 8 of these developed histamine levels above 50 mg/kg with 4 exceeding 200 mg/kg (maximum 1,659.4 mg/kg). The stored samples that exceeded 200 mg/kg were all obtained from two outlets. Within or between fish species there were no consistent relationships between levels of histamine in the samples and either the total aerobic plate counts or the numbers of histamine-producing bacteria. To the contrary, there was evidence that histamine had been formed prior to smoking and that histamine-producing bacteria were eliminated during smoking.

Bacteria↗

PubMed1996

Pupil changes during cardiopulmonary bypass.

Pupil diameter is used during anaesthesia to assess depth of anaesthesia and indicate cerebral hypoxia. This is especially so during cardiac bypass when other autonomic signs cannot be monitored. We have used a pupillometer to determine the effect of cardiopulmonary bypass on the pupil. We have also investigated if any effect was caused by washout of opioid from the central nervous system by allocating patients to one of two groups: in one the bypass pump was preloaded with fentanyl, in the other with 0.9% saline. Cardiopulmonary bypass caused pupil dilatation of between 17% and 53%, which was unaffected by preloading the bypass pump with fentanyl. This effect lasted for the duration of the study, which ended 30 min after the start of cardiopulmonary bypass. Sympathetic nervous system reflexes and hypothermia may account for this observation, but further research is necessary to exclude other contributory factors.

Aged↗

PubMed1996

The effect of temperature upon pain during injection of propofol.

Propofol has been shown to cause pain on injection. This study investigated the effect of warming propofol to 37 degrees C on the pain of intravenous injection. One hundred and one women on outpatient gynaecology lists were allocated to receive propofol either at room temperature or at 37 degrees C. Warming propofol decreased the incidence of pain on injection by 37% (p < 0.001), and also decreased the severity of pain reported by patients (p < 0.001). We conclude that warming propofol to 37 degrees C provides a simple and safe method of reducing the incidence of pain on injection without the addition of other agents.

Adolescent↗

PubMed1982

The lower limit to the size of small sonicated phospholipid vesicles.

The effective hydrodynamic radius of small sonicated phospholipid vesicles has been measured by photon correlation laser light scattering. It is found that the minimum radius obtained for these vesicles is within the range 10.25 +/- 0.55 nm independent of the phospholipid hydrocarbon chain length for synthetic phosphatidylcholines in the even numbered series of 12 to 18 carbons per hydrocarbon chain. The minimum radius of vesicles of egg yolk phosphatidylcholine is 10.7 +/- 0.3 nm.

Lasers↗

PubMed1981