Search PubMed⌕ Search

Biomedical subjects

G M Smith

Publications and source records attributed to G M Smith.

At least 73 records · Page 4Linked to original sources
PubMed1996

Evidence for a multi-domain structure for hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) is a multi-functional enzyme involved in several aspects of lipid metabolism. Limited tryptic digestion of HSL led to selective loss of activity against lipid substrates but not against the water-soluble substrate, p-nitrophenyl butyrate. Following labelling of the active site of HSL with either [3H]di-isopropylfluorophosphate or [14C]orlistat, tryptic digestion of HSL generated a stable radiolabelled domain of molecular mass approx. 17.6 kDa, consistent with this representing a catalytic domain of HSL capable of hydrolysing water-soluble but not lipid substrates. Following phosphorylation of HSL by cyclic AMP-dependent protein kinase, limited tryptic digestion produced a stable phosphorylated domain of molecular mass 11.5 kDa. Based on these experimental data a model for a domain structure of HSL is proposed.

Amino Acid Sequence↗
PubMed1996

Burst-firing sharpens frequency-tuning in primary auditory cortex.

Frequency-tuning in 46 high-firing single units from primary auditory cortex of ketamine-anaesthetized cats was studied separately for isolated-spike firing and burst spike firing. The tuning curve for burst firing was significantly narrower than for the overall firing rate condition and, consequently, much narrower than for the isolated spikes. The burst firing was found in excess of that predicted from a modulated Poisson process, so the results cannot be explained on basis of a change in firing rate criterion. We propose that burst firing in auditory cortex may selectively enhance frequency tuning dynamically controlled by the behavioural state of the animal as well as its stimulus history. Thus burst firing may have functional implications for cortical processing.

Acoustic Stimulation↗
PubMed1996

A phase I trial to assess the value of recombinant human granulocyte colony stimulating factor (R-MeTHuG-CSF, filgrastim) in accelerating the dose rate of chemotherapy for intermediate and high-grade non-Hodgkin's lymphoma (NHL). The Central Lymphoma Group.

In a multi-centre phase I study we investigated the possibility of reducing the interval between courses of standard CHOP (cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, vincristine 2 mgs day 1, and prednisolone 40 mg/m2 days 1-8) from 21 days to 15 days and then 10 days using granulocyte colony stimulating factor (r-MetHuG-CSF (Amgen)-filgrastim) to accelerate neutrophil recovery. Patients received CHOP followed by G-CSF 5 micrograms/kg s.c. from day 2 to the day before the next course (e.g. days 2-14 for the 15-day interval). A total of 28 patients with newly diagnosed intermediate grade or high grade NHL were studied. Four patients were studied at a 21-day interval, six patients were treated at a 15-day interval and subsequently six patients at a 10-day interval. Following analysis of this initial cohort, a further 12 patients were evaluated; four at the 15-day interval, and eight at the 10-day interval. No dose-limiting toxicity was seen in the four patients receiving 21-day CHOP. Dose-limiting toxicity was seen in 4/10 patients treated at the 15-day interval (M:F 7:3, median age 55.5, range 39-67 years). This consisted of infection in two patients, recurrent infection and debility in a third, and mucositis in a fourth. Seven patients experienced one or more infectious episodes requiring antibiotics (median number of episodes: 2, range 1-4). Fourteen patients (M:F 4:3, median age 47.5, range 25-63 years) were treated at the 10-day interval. Dose-limiting toxicity was seen in six patients. This consisted of severe mucositis in three patients, neutropenia and thrombocytopenia on two separate occasions in one patient, and steroid-induced gastritis in two patients. Nine patients had one or more documented infections (median: 2, range 1-3) requiring antibiotics, of which six were severe (WHO grade 3 or 4). One patient died of Pneumocystis carinii (PCP) pneumonia. In summary, G-CSF (filgrastim) will facilitate the shortening of the dosage interval between cycles of CHOP chemotherapy due to accelerated hematological recovery. However, non-hematological toxicity due to the shorter dosage interval is increased and infective episodes are frequent.

Adult↗
PubMed1996

Astrocytes infected with replication-defective adenovirus containing a secreted form of CNTF or NT3 show enhanced support of neuronal populations in vitro.

Neurotrophic factors have been shown to ameliorate neuronal death in several in vitro and in vivo models of neurodegenerative disease. However, delivery of polypeptide growth factors to compromised neurons in the CNS is problematic as the blood-brain barrier prevents systemic delivery, and chronic in-dwelling cannulae are required for intraparenchymal delivery. To circumvent these problems and specifically target neurotrophic factors to the environment surrounding degenerating neurons in the CNS, we have generated replication-defective adenovirus (Ad) vectors that contain a secretable form of ciliary neurotrophic factor (sCNTF) or neurotrophin-3 (NT-3). In this study, we demonstrate that sCNTF/Ad and NT-3/Ad can efficiently infect primary astrocytes, resulting in gene transcription and the production of functional protein. Using Northern blot analysis, dose-dependent expression of sCNTF or NT-3 mRNA was detected 7 days after infection. The levels of mRNA expressed in transgenic astrocytes was dependent on virus titer and increased with increasing virus concentration. sCNTF or NT-3 protein was also detected in astrocyte supernatants by immunoblot analysis and 2-site ELISA. ELISA indicated that astrocytes infected with sCNTF/Ad or NT-3/Ad secreted neurotrophic factors at a rate of approximately 120 pg/10(6) cells/h and 350 pg/10(6) cells/h, respectively. To test for secretion of bioactive sCNTF or NT-3 protein, E8 chick ciliary ganglion or nodose ganglion neurons were grown in medium conditioned by control astrocytes or astrocytes treated with sCNTF/Ad or NT-3/Ad, showing a robust and dose-dependent increase in neuronal survival when compared to control supernatant. In addition, motor neurons plated onto astrocyte monolayers pretreated with sCNTF/Ad showed a two- to fourfold increase in ChAT activity when compared to those grown on astrocytes pretreated with Lac-Z/Ad. This study demonstrates that, using replication-defective adenovirus, primary astrocytes can be efficiently engineered to secrete bioactive sCNTF or NT-3, resulting in enhanced survival of responsive peripheral and central neuronal populations.

Adenoviridae↗
PubMed1996

Neural connectivity only accounts for a small part of neural correlation in auditory cortex.

In order to allow the relation of functional connectivity patterns (inferred from cross-correlograms) to structural connectivity (the anatomical substrate), we analyzed cross-correlogram peaks for spontaneous and stimulated activity in the auditory cortex. It was assumed that the broad correlograms, usually encountered, represent neural connectivity as well as secondary effects such as intrinsic firing patterns, global synchrony related to the ongoing electroencephalographic activity, and stimulus-related effects. Data were collected from 604 neuron pairs recorded under spontaneous conditions in primary auditory cortex of seven juvenile (30-70 days) and nine adult cats. Three hundred and six pairs (51%) had a peak cross-correlation coefficient significantly different from zero. For 113 neuron pairs out of this subgroup, correlations were calculated also for spike trains recorded during click stimulation. After a combined burst-correction and deconvolution procedure was carried out, the correlation peak strengths were not significantly changed for spontaneous activity, but peak width was narrower for single-electrode pairs than for dual-electrode pairs, suggesting a better synchronization for neighboring neurons. Under click stimulation conditions, overall peak synchronization strength was independent of interelectrode distance, whereas, after correction for secondary and stimulus effects, peak synchronization was significantly lower for dual-electrode pairs. However, the primary peak width for single-electrode pairs under stimulus conditions was no longer different from that of dual-electrode pairs. This implies that both under spontaneous and stimulus conditions secondary effects largely obscure any underlying correlation produced by anatomical connectivity. The secondary effects may be the result of intrinsic as well as network properties in auditory cortex and may functionally be more important than the weak primary effects resulting from anatomical connections. Cross-interval analysis suggests that the correlations in auditory cortex are dynamic and may show random switching between states of stronger and weaker synchronization.

Acoustic Stimulation↗
PubMed1996

Diversity of the polar lipids of the food-borne pathogen Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive bacterium that can adapt to high salinity and cold. Because the membrane lipids may play a role in its survival and adaptation, we have examined the polar lipids of L. monocytogenes. Extraction of total lipids from L. monocytogenes yielded 7 +/- 1 mg/mL wet cells. Polar lipids represented 64% of total lipids and contained 9% lipid-phosphorus. Polar lipids were separated into 14 components by two-dimensional thin-layer chromatography. Eight components (88% of polar lipids) contained lipid-phosphorus; among these was one major component (34% of polar lipids). Two other phospholipids were ninhydrin-positive components and accounted for 15% of the polar lipids. Orcinol staining revealed two glyco- or sulfo-lipids accounting for 9% of polar lipids. Five components (4% of polar lipids) were amino components free of phosphorus. The major component contained 46% of its fatty acids as 15:0 anteiso, 24% as 17: 0 anteiso, and 11% as 15:0 iso. The fatty acid profile of the remaining polar lipids was variable, consisting primarily of 16:0, 18:0, 15:0 anteiso, and 17:0 anteiso. Their unsaturation level was < or = 20%; however, the major phosphoaminolipid component was 46% unsaturated. The ratios of 15:0 anteiso/17:0 anteiso and 15:0 anteiso/15:0 iso were similar in all classes, averaging 1.5 and 4.5, respectively. Since the adaptation process to stressful environments involves activation of a membrane transport system for the protectant glycine betaine, the membrane lipids may play a role in enabling transport.

Chromatography, High Pressure Liquid↗
PubMed1996

Mobilization of Philadelphia-negative peripheral blood mononuclear cells in chronic myeloid leukaemia using hydroxyurea and G-CSF (filgrastim).

A relatively simple and non-toxic out-patient-based regimen for the mobilization of Philadelphia-negative (Ph-ve) mononuclear cells in chronic myeloid leukaemia (CML) was evaluated in 10 patients, nine in stable chronic phase and one in accelerated phase. They received oral hydroxyurea at a mean dose of 3.5 g/m2 daily for 7 d, followed by 300 micrograms of G-CSF daily until the last day of harvesting. In the nine chronic-phase patients the mean number of days from the end of hydroxyurea to the commencement of harvesting was 14.5 (range 10-18). The patient in accelerated phase recovered and was harvested after 6 d. The mean number of aphereses performed was 3.4. Adequate numbers of stem cells were obtained in 9/10 patients judged by our usual criteria. Side-effects were mild in comparison to published intravenous schedules. No patients lost their hair. Five (50%) patients required admission with neutropenic fever which responded to antibiotics in all cases. Four (40%) patients developed a transient rash and four (40%) experienced mild oral mucostis. This level of toxicity enabled half of the patients to be treated entirely on an out-patient basis. The harvest products were analysed for cells belonging to the leukaemic clone by conventional cytogenetics, FISH and PCR. All were PCR positive. The mean Ph positivities by cytogenetics and FISH were comparable at 18.1% and 15% respectively. Half the patients had > 98% normal metaphases. We conclude that this approach is comparable in efficacy to published intravenous regimens and significantly less toxic. It can be safely used at diagnosis before interferon therapy commences.

Administration, Oral↗
PubMed1996

Sodium-driven, osmotically activated glycine betaine transport in Listeria monocytogenes membrane vesicles.

Transport of the osmoprotectant and cryoprotectant glycine betaine was investigated in membrane vesicles of Listeria monocytogenes. Uptake-driving transmembrane potentials ranging from 111 to 122 mV within the pH range of 5.5 to 7.5 could be generated by the electron donor system ascorbate-phenazine methosulfate but not by the electron donor system ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. Transport was dependent on both high concentrations of sodium ion and the presence of a hypertonic solute gradient. Arrhenius-type temperature activation was observed. Lineweaver-Burk plots indicated a Km of 4.4 microM for glycine betaine and a Vmax of 700 pmol/min x mg of protein. The Michaelis constant for NaCl depended on the solute used to maintain a constant hyperosmotic pressure, and the Km values were 200 and 75 mM when KCl and sucrose were employed, respectively. Transport was 65% lower in vesicles derived from cells grown under stress provided by KCI rather than NaCl and approximately 94% lower in vesicles derived from cells that were not grown under osmotic stress. This porter appears to be specific for glycine betaine, since neither proline, carnitine, nor choline inhibited uptake effectively. Kinetic studies using ionophores and artificial gradients indicate that glycine betaine is cotransported with sodium ion.

Betaine↗
PubMed1996

Assessment of IgH PCR strategies in multiple myeloma.

AIMS: To compare the ability of four commonly used PCR techniques to demonstrate clonal IgH rearrangements in multiple myeloma. METHODS: Bone marrow samples (containing a minimum of 10% plasma cells) were obtained from 127 patients with confirmed multiple myeloma. Framework 3 (Fr3) PCR was performed in all cases and the Framework 1 (Fr1f) PCR, which utilises six VH family specific primers, in 98 cases. In addition, 44 cases were assessed by Fr3, Fr1f, Framework 2 (Fr2) and Framework 1 consensus (Fr1 con) PCR techniques. JH primer selection was also assessed such that each PCR strategy was performed twice in each of the 44 cases, using the JH consensus primer (JH con) alone and then repeated with an equimolar mixture of JH con, JH3 and JH6 (JH mix). RESULTS: Clonal rearrangements were demonstrated in 71 (56%) of 127 cases with the Fr3 PCR and in 52 (53%) of 98 with the Fr1f PCR. However, by using both techniques it was possible to demonstrate clonal IgH rearrangements in 92 (75%) of 122 cases. Forty four cases were assessed by all four PCR techniques; in these cases the Fr3 and Fr1f PCRs demonstrated clonal rearrangements in 26 (59%) cases with a combined yield of 34 (77%). The Fr2 and Fr1 con PCR techniques had inferior pick up rates, demonstrating clonal rearrangements in 21 (48%) of 44 cases and a combined yield of 28 (63%). The Fr2 PCR did, however, demonstrate a clonal rearrangement in one case negative by both Fr3 and Fr1f. Two additional rearrangements were demonstrated by using JH mix; one became positive by Fr3, Fr1f and Fr2 and the other positive by Fr1f, Fr1 con and Fr2. CONCLUSIONS: By utilising both the Fr3 and Fr1f PCR techniques it is possible to demonstrate definitive clonal rearrangements in the majority of patients with multiple myeloma. The Fr1 con and Fr2 PCR techniques have inferior pick up rates but may detect some additional rearrangements.

Bone Marrow Examination↗
PubMed1996

Detection of clonal immunoglobulin gene rearrangements in the peripheral blood progenitor cells of patients with multiple myeloma: the potential role of purging with CD34 positive selection.

Aims-To determine the extent of clonal cell contamination of peripheral blood progenitor cell (PBPC) collections in patients with multiple myeloma (MM) and to assess the purging efficacy of CD34 positive selection.Methods-PBPC collections from 29 patients with MM were analysed for the presence of clonal immunoglobulin heavy chain (IgH) gene rearrangements with a fluorescence based PCR technique. In addition, the PBPC from eight of the 29 patients were "purged" by selection of CD34 positive haematopoietic progenitors with an avidin-biotin immunoabsorption column (Ceprate). In each case the unmanipulated PBPC, CD34 positive and waste fractions were all assessed for the presence of clonal IgH rearrangements.Results-Clonal IgH rearrangements (identical with those demonstrated in diagnostic bone marrow samples) were demonstrated in 10 (35%) of 29 cases and seemed to be confined to those with significant residual bone marrow disease. Clonal rearrangements were evident in the PBPC of two of the eight patients who underwent CD34 selection; in both instances a "clonal purge" was seen as it was not possible to demonstrate the clonal rearrangement in the CD34 positive fraction. In four of the six remaining cases the normal polyclonal fingerprint could not be demonstrated in the CD34 positive fraction, which is consistent with a significant reduction in contaminating B cells.Conclusions-Clonal cells contaminate PBPC collections in a significant proportion of patients with MM and may be eliminated by CD34 positive selection.

Journal Article↗
PubMed1996

Peripheral blood stem cell transplantation in myeloma using CD34 selected cells.

We have performed nine CD34 selection procedures on peripheral blood stem cells harvested from eight patients with myeloma using the Cellpro avidin-biotin immunoaffinity column (Ceprate). They all received CVAMP chemotherapy to maximum response prior to mobilisation. Six of the patients have been transplanted using these cells, one receiving successive autografts. Median absolute cell numbers processed and retrieved were: 31.1 x 10(9) pre-column, 2.07 x 10(8) in the final product and 30.4 x 10(9) in the column waste. Mean CD34 positivity in the product was 49% (range 18.4-98) with a median CD34+ yield of 31.4% (range 21-37.8). IgH PCR was performed and seven of the eight patients were amplifiable. Of these, two were positive in the pre-column product and both of these were successfully purged with a negative result in the final, post-column product. Patients were transplanted with a median of 2.0 x 10(6) CD34+ cells/kg (range 1.5-9.4) following conditioning with melphalan 200 mg/m2. The mean time to recovery of neutrophils to > 0.5 x 10(9)/l and platelets to > 20 x 10(9)/l was 16 and 17 days, respectively. At a mean follow-up of 9 months, four of the six patients transplanted are alive, three of them in complete remission and one in a clinically stable relapse. One has died of disease relapse and one of progressive neurological problems the aetiology of which was uncertain but there was no sign of progression of their myeloma. We conclude that PBSCT using CD34 selected cells is safe and practical in myeloma following remission induction with CVAMP chemotherapy.

Antigens, CD34↗
PubMed1995

Separating local from global effects in neural pair correlograms.

Cross-correlograms between spike trains of simultaneously recorded units in cortex show peaks that range in width from a few milliseconds to tens of milliseconds regardless of the distance between the units. The peak width reflects the degree of synchronization between the firings resulting from local connectivity and global synchronization. The local connectivity part may be hidden in the much stronger and broader correlations due to diffuse global synchronizing forces. This study proposes an estimator for such global synchronizing effects upon unit-pair correlations based on local field potentials (LFPs). It appears that the relative importance of the global component in the unit-pair correlograms as predicted by LFPs is largest in spontaneous activity and for units recorded on distant electrodes.

Acoustic Stimulation↗
PubMed1995

Rate covariance dominates spontaneous cortical unit-pair correlograms.

Correlations between the firings of simultaneously recorded single units in auditory cortex may result from neural connectivity as well as covariation in firing rate. We present a predictor for the rate covariance part of the correlogram based on spike counts in 50 ms. Data were collected from 604 neurone pairs recorded under spontaneous conditions in primary auditory cortex of seven juvenile (30-70 days) and nine adult cats. A total of 341 pairs (56%) had a peak correlation coefficient significantly different from zero. The rate covariance comprised on average 73% of the peak correlation for single electrode pairs and 67% of that for dual electrode pairs. The remaining event correlation was significantly different from zero in 150 of the pair correlograms tested.

Action Potentials↗
PubMed1995

Cloning and characterisation of a bovine P2Y receptor.

Using a chick P2Y1 receptor cDNA probe we have isolated a mammalian P2Y receptor clone from a bovine aortic endothelial cell library. The sequence has a high degree of similarity to the chick P2Y1 clone. When transfected into the Jurkat cell line, the cDNA conferred sensitivity to purinoceptor agonists. Using fura-2 loaded cells the potency order at the receptor was found to be 2-methylthioadenosine 5' triphosphate = adenosine 5' diphosphate > adenosine 5' triphosphate >> alpha,beta-methyleneadenosine 5' triphosphate and uridine 5' triphosphate. This corresponds to the agonist potency order expected for the bovine aortic endothelial cell P2Y receptor.

Adenosine Diphosphate↗
PubMed1995

Application of the free energy perturbation method to human carbonic anhydrase II inhibitors.

An analysis of the free energy perturbation (FEP) method is presented that attempts to evaluate the efficacy of the FEP method in the drug discovery process. To accomplish this we have evaluated whether the FEP technique can accurately predict energetic and structural quantities relating to the inhibition of human carbonic anhydrase II (HCAII) by sulfonamides. Three well-characterized (both structurally and energetically) sulfonamide inhibitors of HCAII were examined in this study, 1a, 1b, and 1c. Results from FEP simulations on these compounds indicate that the FEP method can predict energetic trends reasonably well; however, the FEP method was less successful in reproducing detailed structural data. In particular, an expected movement of His-64 when inhibitor 1c was bound did not occur. We conclude that the FEP method can be used to determine relative free energies of binding but cannot be relied upon to reproduce subtle geometric changes.

Carbonic Anhydrase Inhibitors↗
PubMed1995

Cytomegalovirus-induced T-cell proliferation and the development of progressive multifocal leucoencephalopathy following bone marrow transplantation.

Progressive multifocal leucoencephalopathy (PML) is a rare disorder characterized by progressive demyelination within the brain. It is usually fatal and occurs when there are defects in cell-mediated immunity. We report a case of PML which developed following allogeneic bone marrow transplantation. It was preceded by the development of a persistent polyclonal expansion of large granular lymphocytes (LGLs) which appeared to rise as a consequence of cytomegalovirus infection.

Adult↗
PubMed1995

Role of Glycine Betaine and Related Osmolytes in Osmotic Stress Adaptation in Yersinia enterocolitica ATCC 9610.

Yersinia enterocolitica is a gram-negative, food-borne pathogen that can grow in 5% NaCl and at refrigerator temperatures. In this report, the compatible solutes (osmolytes) which accumulate intracellularly and confer the observed osmotic tolerance to this pathogen were identified. In minimal medium, glutamate was the only detectable osmolyte that accumulated in osmotically stressed cells. However, when the growth medium was supplemented with glycine betaine, dimethylglycine, or carnitine, the respective osmolyte accumulated intracellularly to high levels and the growth rates of the osmotically stressed cultures improved from 2.4- to 3.5-fold. Chill stress also stimulated the intracellular accumulation of glycine betaine, but the growth rate was only slightly improved by this osmolyte. Both osmotic upshock and temperature downshock stimulated the rate of uptake of [(sup14)C]glycine betaine by more than 30-fold, consistent with other data indicating that the osmolytes are accumulated from the growth medium via transport.

Journal Article↗