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M Stubbs

Publications and source records attributed to M Stubbs.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effects of nicotinamide and carbogen on tumour oxygenation, blood flow, energetics and blood glucose levels.

Both host carbogen (95% oxygen/5% carbon dioxide) breathing and nicotinamide administration enhance tumour radiotherapeutic response and are being re-evaluated in the clinic. Non-invasive magnetic resonance imaging (MRI) and 31P magnetic resonance spectroscopy (MRS) methods have been used to give information on the effects of nicotinamide alone and in combination with host carbogen breathing on transplanted rat GH3 prolactinomas. Gradient recalled echo (GRE) MRI, sensitive to blood oxygenation changes, and spin echo (SE) MRI, sensitive to perfusion/flow, showed large signal intensity increases with carbogen breathing. Nicotinamide, thought to act by suppressing the transient closure of small blood vessels that cause intermittent tumour hypoxia, induced a small increase in blood oxygenation but no detectable change in perfusion/flow. Carbogen combined with nicotinamide was no more effective than carbogen alone. Both carbogen and nicotinamide caused significant increases in the nucleoside triphosphate/inorganic phosphate (betaNTP/Pi) ratio, implying that the tumour cells normally receive sub-optimal substrate supply, and is consistent with either increased glycolysis and/or a switch to more oxidative metabolism. The most striking observation was the marked increase in blood glucose (twofold) induced by both nicotinamide and carbogen. Whether this may play a role in tumour radiosensitivity has yet to be determined.

Animals↗
PubMed2000

Effect of intraarticular stainless steel implants on the health of the rabbit knee joint: an experimental study.

OBJECTIVE: This study was designed to investigate the histologic changes to the knee joints of rabbits after insertion of a metal implant in retrograde fashion. DESIGN: Eighteen rabbits had a modified stainless steel screw implanted in one knee, with the other knee serving as a sham-operated control. The animals were killed after two, six, or twelve months. OUTCOME MEASURES: The histologic status of the cartilage and synovium were graded by the Modified Mankin and Mirra criteria, respectively. RESULTS: At the time of killing, every insertion site was covered by fibrous tissue. Statistical analysis showed no significant differences in histologic scores between implanted and control knees. CONCLUSIONS: Insertion of a stable, well-fixed implant results in no deleterious effect to the knee joint.

Animals↗
PubMed2000

Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat).

The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.

Amino Acid Sequence↗
PubMed2000

Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.

Cell Line↗
PubMed1999

Evidence for glucose and sorbitol-induced nuclear export of glucokinase regulatory protein in hepatocytes.

Glucokinase is rapidly exported from the nucleus of hepatocytes in response to a rise in glucose or fructose 1-P. We demonstrate using confocal microscopy and quantitative imaging that in contrast to previous findings, the regulatory protein of glucokinase (GKRP) also translocates from the nucleus during substrate-induced translocation of glucokinase. However, the fractional decrease in nuclear GKRP is smaller than for glucokinase and is determined by the metabolic state and not by the distribution of glucokinase. Translocation of glucokinase and GKRP is not inhibited by leptomycin B, an inhibitor of exportin-1 function. These findings highlight the importance of quantitative imaging for determining nuclear export of proteins and suggest that GKRP may have a role in nuclear export or import of glucokinase.

Animals↗
PubMed1999

Measurement of the extracellular pH of solid tumours in mice by magnetic resonance spectroscopy: a comparison of exogenous (19)F and (31)P probes.

Precise measurement of pH(e) in vivo may be of clinical value for both diagnosis and selection of therapy. pH(e) measurements made by the (31)P probe 3-aminopropylphosphonate (3-APP) were compared with those made by the (19)F probe, 3-[N-(4-fluor-2-trifluoromethylphenyl)-sulphamoyl]-propionic acid (ZK-150471) in three solid tumour types, human HT29 xenografts, murine RIF-1 fibrosarcomas and Lettre tumours grown subcutaneously in mice. No significant differences were observed when probe measurements of pH(e) were compared at 20-60 min post-administration, although very low pH(e) values (ca. 6.0) were recorded in two out of eight Lettre tumours by ZK-150471. The more rapid pH(e) measurements possible using ZK-150471 showed that during the first 20 min post-administration significant increases occurred in pH(e) which were greatest in the more necrotic tumours. Since isolated cell experiments showed that ZK-150471 was non-toxic and did not enter the cells, this early increase in pH(e) may reflect gradual penetration by ZK-150471 of the reportedly alkaline necrotic space in the tumours. The wide chemical shift range, improved signal-to-noise and absence of signal overlap allowed a more rapid and precise measurement of pH(e) by ZK-150471 compared to 3-APP. These characteristics suggest that ZK-150471 is currently the preferred pH(e) probe for non-invasive MRS.

Animals↗
PubMed1999

Causes and consequences of acidic pH in tumors: a magnetic resonance study.

A consequence of metabolism in any tissue is the formation of hydrogen ions, which are actively transported out of the cell. However, although most solid tumors maintain their intracellular pH (pHi) within a narrow range to provide a favorable environment for various intracellular activities, their extracellular pH (pHe) is on average about 0.2 pH units more acid. It is important to understand the relationship between tumor metabolism and pH, and how it differs from that of normal tissue and to ask the question: How does an understanding of pH and tumor metabolism affect strategies for therapeutic approaches? Although, in vitro, isolated cell experiments have shown positive correlations between pHi and pHe the relationship is complex and somewhat dependent on experimental conditions. Measurement of pHi in solid tumors by non-invasive Magnetic Resonance Spectroscopy (MRS) has been possible for some time now and recently several specific markers for measuring pHe have become available. As a result we have been able to study the relationship between pHi and pHe in vivo in several different solid tumor types. In only one tumor type (HT29 xenografts) was there a significant correlation between pHi and pHe; in 3 other tumor types (RIF-1 in mice, GH3 prolactinomas and H9618a in rats) there was no correlation. A significant correlation between pHi and NTP/Pi ratios was seen across all tumor types. Theoretical considerations of causes of tumor acidity, hypotheses to explain extracellular acidity and the possibility that low pHe might be an intrinsic feature of the tumor phenotype and not merely the consequence of metabolic activity have been discussed. In addition the consquences for concepts of treatment based on pH are considered.

Adenosine Triphosphate↗
PubMed1999

Response of hepatoma 9618a and normal liver to host carbogen and carbon monoxide breathing.

The effects of hyperoxia (induced by host carbogen [95% oxygen/5% carbon dioxide breathing] and hypoxia (induced by host carbon monoxide [CO at 660 ppm] breathing) were compared by using noninvasive magnetic resonance (MR) methods to gain simultaneous information on blood flow/oxygenation and the bioenergetic status of rat Morris H9618a hepatomas. Both carbogen and CO breathing induced a 1.5- to 2-fold increase in signal intensity in blood oxygenation level dependent (BOLD) MR images. This was due to a decrease in deoxyhemoglobin (deoxyHb), which acts as an endogenous contrast agent, caused either by formation of oxyhemoglobin in the case of carbogen breathing, or carboxyhemoglobin with CO breathing. The results were confirmed by observation of similar changes in deoxyHb in arterial blood samples examined ex vivo after carbogen or CO breathing. There was no change in nucleoside triphosphates (NTP)/P(i) in either tumor or liver after CO breathing, whereas NTP/P(i) increased twofold in the hepatoma (but not in the liver) after carbogen breathing. No changes in tumor intracellular pH were seen after either treatment, whereas extracellular pH became more alkaline after CO breathing and more acid after carbogen breathing, respectively. This tumor type and the liver are unaffected by CO breathing at 660 ppm, which implies an adequate oxygen supply.

Animals↗
PubMed1999

Application of magnetic resonance techniques for imaging tumour physiology.

Magnetic resonance (MR) techniques have the unique ability to measure in vivo the biochemical content of living tissue in the body in a dynamic, non-invasive and non-destructive manner. MR also permits serial investigations of steady-state tumour physiology and biochemistry, as well as the response of a tumour to treatment. Magnetic resonance imaging (MRI), Magnetic resonance spectroscopy (MRS) and a mixture of the two techniques (spectroscopic imaging) allow some physiological parameters, for example pH, to be 'imaged'. Using these methods, information on tissue bioenergetics and phospolipid membrane turnover, pH, hypoxia, oxygenation, and various aspects of vascularity including blood flow, angiogenesis, permeability and vascular volume can be obtained. In addition, MRS methods can be used for monitoring anticancer drugs (e.g. 5FU, ifosfamide) and their metabolites at their sites of action. The role of these state-of-the-art MR methods in imaging tumour physiology and their potential role in the clinic are discussed.

Cell Hypoxia↗
PubMed1998

Encoding of human basic and glycosylated proline-rich proteins by the PRB gene complex and proteolytic processing of their precursor proteins.

Proline-rich proteins (PRPs) constitute a family of about 20 members in human saliva that are encoded by six genes. Assignment of genomic DNA coding regions is complicated because of the occurrence of many alleles and the great similarity of amino acid sequences of PRPs. To overcome these problems, the nucleotide sequences of the genes encoding basic and glycosylated PRPs from one person were determined and then aligned with her previously determined protein sequences. This, together with additional protein data, has also resolved various discrepancies between corresponding protein and DNA sequences. For the first time in one person it is now possible to account for all the regions in the PRB genes encoding basic and glycosylated PRPs, and the primary structures of all secreted basic and glycosylated PRPs have been determined. Each gene encodes a precursor protein that subsequently undergoes proteolytic cleavage, thereby giving rise to the secreted proteins. The results have allowed identification of all the proteolytic cleavage sites in the precursor proteins, which all conform to a consensus cleavage site for furin. To evaluate if furin is responsible for the precursor protein cleavages, a recombinant precursor protein was synthesized by in vitro transcription translation of a PRB1 allele. The protein was shown to be correctly cleaved by furin, giving rise to the expected secreted proteins.

Alleles↗
PubMed1998

Ligamento-muscular protective reflex in the lumbar spine of the feline.

A ligamento-muscular protective reflex in the lumbar spine was demonstrated in a feline model. Stimulating electrodes were applied to the supraspinous ligament between several lumbar vertebra (L1 to L6) while recording myoelectric discharge from the paraspinal muscles at the L3, L4 and L5, bilaterally. Electromyographic (EMG) activity was present in the paraspinal muscles bilaterally, upon stimulation of the supraspinous ligament, in six preparations. The EMG discharge was strongest in the muscles one level below that of the stimulated ligament, whereas weaker EMG signals were recorded from as far as two levels above and below. The mean time delay between the application of the stimulus to the ligament to the resulting EMG ranged from 2.52 to 2.77 ms at all levels. Stimulation of the supraspinous ligament in the L6 segment resulted in a weak reflex response, and stimulation in the L7 segment did not produce any EMG activity. It was concluded that mechanoreceptors in the supraspinous ligament at the L1/6 levels may initiate sensory signals upon strain of the ligament, during flexion. This, in turn, causes contraction of the paraspinal muscles, bilaterally, to extend the spine and prevent possible damage to the ligament while maintaining stability. The results may add to the understanding of low back pain, and to the formulation of surgical procedures which could spare the neural supply of the ligament, allowing advanced physiotherapeutic modalities to be implemented for post-surgical rehabilitation.

Animals↗
PubMed1998

Magnetic resonance imaging techniques for monitoring changes in tumor oxygenation and blood flow.

The application of functional magnetic resonance (MR) imaging techniques to the measurement of oxygenation and blood flow in tumors is described. Gradient recalled echo MR imaging (GRE-MRI) offers a real-time noninvasive method for monitoring tumor response to vasomodulators such as carbogen (95% O2/5% CO2) breathing in attempts to overcome tumor hypoxia and improve treatment efficacy. Although the response is tumor-type dependent, increases in signal intensity of up to 100% have been observed in several animal tumor types. Responses are also seen in human tumors. The observed increases in GRE-MRI signal intensity are due to a combination of a reduction of deoxyhemoglobin in the blood causing changes in the MR imaging relaxation time T2* and changes in blood flow and may also reflect the capillary density. Thus, the magnitude of the GRE image intensity change gives an indication of the potential response of an individual tumor to treatments that aim to improve tissue oxygenation and therefore how the tumor may respond to therapy. In addition, carbogen breathing by the host has been shown to increase the uptake and efficacy of chemotherapeutic agents in animal tumors.

Administration, Inhalation↗
PubMed1998

Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells.

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.

Adenocarcinoma↗
PubMed1998

Estimations of intra- and extracellular volume and pH by 31P magnetic resonance spectroscopy: effect of therapy on RIF-1 tumours.

Quantification of metabolite or drug concentrations in living tissues requires determination of intra- and extracellular volumes. This study demonstrates how this can be achieved non-invasively by 31P magnetic resonance spectroscopy (MRS) employing dimethyl methylphosphonate (DMMP) as a marker of total water space, 3-aminopropylphosphonate (3-APP) as a marker of extracellular space and P and 3-APP as markers of intracellular pH (pH) and extracellular pH (pHe) respectively. The MRS measurements of the tumour volumes were validated by classic radiolabelling methods using 3H2O and [14C]inulin as markers of total and extracellular space respectively. The extracellular volume fraction measured by radiolabelling of RIF-1 tumours was 23 +/- 0.83% (mean +/- s.e.m. n = 9), not significantly different (P > 0.1) from that found by MRS (27 +/- 2.9%, n = 9, London, and 35 +/- 6.7, n = 14, Baltimore). In untreated RIF-1 tumours, pH was about 0.2 units higher than pHe (P < 0.01). 5-Fluorouracil (5FU) treatment (165 mg kg(-1)) caused no significant changes in either pHe or per cent extracellular volume. However significant increases in pH, 48 h after treatment (P < 0.01) correlated with decreased tumour size and improved bioenergetic status [NTP/inorganic phosphate (Pi) ratio]. This study shows the feasibility of an MR method (verified by a 'gold standard') for studying the effects of drug treatment on intra- and extracellular spaces and pH in solid tumours in vivo.

Animals↗
PubMed1998

The effects of host carbogen (95% oxygen/5% carbon dioxide) breathing on metabolic characteristics of Morris hepatoma 9618a.

Characteristics of the tumour metabolic profile play a role in both the tumour-host interaction and in resistance to treatment. Because carbogen (95% oxygen/5% carbon dioxide) breathing can both increase sensitivity to radiation and improve chemotherapeutic efficacy, we have studied its effects on the metabolic characteristics of Morris hepatoma 9618a. Host carbogen breathing increased both arterial blood pCO2 and pO2, but decreased blood pH. A fourfold increase in tumour pO2 (measured polarographically) and a twofold increase in image intensity [measured by gradient recalled echo magnetic resonance (MR) imaging sensitive to changes in oxy/deoxyhaemoglobin] were observed. No changes were seen in blood flow measured by laser Doppler flowmetry. Tumour intracellular pH remained neutral, whereas extracellular pH decreased significantly (P < 0.01). Nucleoside triphosphate/inorganic phosphate (NTP/Pi), tissue and plasma glucose increased twofold and lactate decreased in both intra- and extracellular compartments, suggesting a change to a more oxidative metabolism. The improvement in energy status of the tumour was reflected in changes in tissue ions, including Na+, through ionic equilibria. The findings suggest that the metabolic profile of hepatoma 9618a is defined partly by intrinsic tumour properties caused by transformation and partly by tissue hypoxia, but that it can respond to environmental changes induced by carbogen with implications for improvements in therapeutic efficacy.

Administration, Inhalation↗
PubMed1997

In vivo detection of ifosfamide by 31P-MRS in rat tumours: increased uptake and cytotoxicity induced by carbogen breathing in GH3 prolactinomas.

The direct detection and monitoring of anti-cancer drugs in vivo by magnetic resonance spectroscopy (MRS) may lead to improved anti-cancer strategies. 31P-MRS has been used to detect and quantify ifosfamide (IF) in vivo in GH3 prolactinomas and N-methyl-N-nitrosourea (MNU)-induced mammary tumours in rats. The average concentration of IF in the GH3 prolactinoma over the first 2 h following a dose of 250 mg kg-1 i.v. was calculated to be 0.42 micromol g-1 wet weight, with a half-life of elimination (t1/2) of 2-4 h. Carbogen (95% oxygen/5% carbon dioxide) breathing increased the amount of IF taken up by the GH3 prolactinoma by 50% (P<0.01) to 0.68 micromol g-1 wet weight, although t1/2 elimination rates were unchanged. IF was also detected in the liver in vivo, with a t1/2 of about 1 h. Carbogen breathing did not affect the maximum peak area (Cmax) or the t1/2 in the liver. Most importantly, the carbogen-induced increase in IF uptake by the tumour caused significant growth delay at all time points in the GH3 tumour growth between day 5 and day 12 (P< 0.01) compared with IF alone. These findings show that carbogen breathing has potential for increasing the efficacy of anti-cancer drugs. Isolated GH3 cells were sensitive to the parent drug (IF) in vitro (IC50 = 1.3 +/- 0.2 mM) suggesting that the GH3 cells may be either expressing P450 enzymes or are sensitive to the parent drug per se.

Animals↗