PubMed Health⌕ Search

Biomedical subjects

T Robson

Publications and source records attributed to T Robson.

At least 37 records · Page 2Linked to original sources
PubMed1999

Expression of two ATP-gated ion channels, P2X5 and P2X6, in developing chick skeletal muscle.

Physiological and pharmacological studies have shown that ATP has potent effects on developing chick skeletal muscle. These effects have previously been shown to be developmentally regulated, and the responses were characteristic of activation of the P2X ligand-gated ion-channel family of ATP receptors. Here, using immunohistochemistry, we describe the expression patterns of two members of the P2X receptor family, P2X5 and P2X6, during development of skeletal muscle in the chick embryo. These receptors were first expressed at early stages of skeletal muscle development, and expression disappeared immediately before the stage at which fusion of myoblasts to form myotubes occurs. P2X5 was also demonstrated in nerves supplying developing skeletal muscle, in some dorsal root ganglion cells, and in dorsal and ventral spinal cord. No expression of the other five members of the P2X family were demonstrated in developing skeletal muscle.

Amino Acid Sequence↗
PubMed1999

Localisation of P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous epithelia.

In order to investigate whether purinoceptors are involved in the physiological renewal and regeneration of epithelia, we used immunohistochemical techniques on fresh frozen sections of various stratified squamous epithelial tissues (cornea, tongue, soft palate, oesophagus, vagina and footpad) of the rat and specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. Only two of the antibodies, anti-P2X5 and anti-P2X7, reacted with epithelial structures. P2X5 immunoreactivity was mainly associated with the membranes of the proliferating and differentiating cell layers (spinous and granular layer) in both keratinised and non-keratinised epithelia and growing hair follicles. In contrast, P2X7 immunoreactivity was clearly associated with the keratinisation process, the staining being most intense in the upper keratinised and the exfoliated layers. These findings suggest, for the first time, that P2X5 and P2X7 receptors play an important role in the physiological turnover of continuously regenerating cells, and further, raise the possibility that they represent novel targets for the development of pharmacological tools of potential benefit for diseases of epithelial dysfunction.

Animals↗
PubMed1999

P2X receptors in the rat duodenal villus.

Immunohistochemical techniques were performed on freshly frozen sections of the duodenum of the rat using specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. Of the antibodies to the seven known P2X receptor subtypes that mediate extracellular signalling by nucleotides, three reacted with discrete structures in the duodenal villus of the rat. Anti-P2X1 reacted with the capillary plexus in the intestinal villus, which did not extend to the crypt region, suggesting that nucleotides may be involved in the uptake and transport of metabolites. Anti-P2X5 immunostained the membranes of the narrow "stem" of villus goblet cells, where the nucleus and cell organelles reside, possibly influencing synthesis and release of mucins. P2X7 receptor immunoreactivity was only seen in the membranes of enterocytes and goblet cells at the tip of the villus, where cells are exfoliated into the lumen, consistent with earlier findings that P2X7 is involved in apoptotic events. Thus, in complex structures such as the intestinal villus, purinoceptors appear to participate in several and diverse signalling functions.

Amino Acid Sequence↗
PubMed1999

A novel human stress response-related gene with a potential role in induced radioresistance.

We have isolated a novel gene, DIR1, from L132 cells that is transiently repressed after exposure to low radiation doses and has a potential role in induced radioresistance. Molecular and cellular characterization of this gene reveals that it is unique but has similarities to a family of heat-shock-related proteins known as immunophilins. These have been implicated in various cellular functions including general stress responses and control of the cell cycle. Antisense strategies have demonstrated that the DIR1 gene also appears to have some involvement in the control of the cell cycle. Furthermore, there appears be a potential role for this gene product in the phenomenon of induced radioresistance through a mechanism that increases the rate of DNA repair in cells exposed to X rays and subsequently increases the cells' resistance to radiation. This is the first description of an immunophilin-like gene that has a possible role in adaptive/inducible responses to X rays in mammalian cells.

Amino Acid Sequence↗
PubMed1999

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation.

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 Km = 26.7 microM, Vmax = 0.43 microM/mg protein/min; RIF-1 Km = 33.5 microM, Vmax = 0.42 microM/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 Km = 37.5 microM; Vmax = 1.4 microM/mg protein/min; RIF-1 Km = 37.5 microM; Vmax = 1.2 microM/mg protein/ min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (Km = 4 microM, Vmax = 3.5 pmol/mg protein/min) and normal kidney (Km = 4 microM, Vmax = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

Animals↗
PubMed1998

Inhibition of human peripheral blood mononuclear cell proliferation by Streptococcus pyogenes cell extract is associated with arginine deiminase activity.

Streptococcus pyogenes (group A Streptococcus) cell extracts (CE) have a remarkably powerful and dose-dependent inhibitory effect on antigen, superantigen, or mitogen-stimulated human peripheral blood mononuclear cell (PBMC) proliferation in vitro. Purification of the inhibitory component present in S. pyogenes type M5 (Manfredo strain) CE by anion-exchange chromatography followed by gel filtration chromatography showed that the inhibitor had an approximate native molecular mass of 100 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inhibitory fractions followed by silver staining gave a single band with an approximate molecular mass of 47 kDa, indicating that the inhibitor is composed of two identical subunits. NH2-terminal sequencing of the protein revealed that it was identical to the previously characterized streptococcal acid glycoprotein (SAGP); this protein possesses between 31.5 and 39.0% amino acid identity with arginine deiminase (AD) from Mycoplasma hominis, Mycoplasma arginini, Pseudomonas putida, and Pseudomonas aeruginosa. AD enzyme activity was present in unfractionated CE prepared from a range of streptococcal strains, and partially purified inhibitory fractions of Manfredo CE also had high levels of activity. The inhibitory effect of Manfredo CE was overcome by the addition of L-arginine to proliferation assays in which human PBMC were stimulated with phytohemagglutinin. We conclude that SAGP, or its homolog, possesses AD activity and that the potent inhibition of proliferation of human T cells by streptococcal CE is due to activity of this enzyme.

Amino Acid Sequence↗
PubMed1997

Neurochemical coding in the small intestine of patients with Crohn's disease.

BACKGROUND: There have been conflicting results regarding the effect of Crohn's disease on the neurochemical composition of the enteric nervous system. AIMS: To examine the effect of Crohn's disease on the neurochemical composition of enteric nerve fibres and cell bodies using whole mount preparations of human ileum. METHODS: Whole wall ileum from seven normal subjects and nine patients with Crohn's disease was used to investigate the neurochemical composition of neurones and nerve fibres in the myenteric plexus, circular muscle, and serosa layer of ileum using immunohistochemical techniques. RESULTS: Increased tyrosine hydroxylase, 5-hydroxytryptamine, and neuropeptide Y immunoreactivity was exclusively seen in the myenteric plexus. There was increased neurofilament immunoreactivity in the myenteric plexus and nerve fibres of the circular muscle layer, and thick bundles of immunoreactive nerve fibres in the serosa layer. Increased vasoactive intestinal polypeptide, nitric oxide synthase, and pituitary adenylate cyclase activating peptide immunoreactivity was seen in the myenteric plexus and nerve fibres of the circular muscle layer, and aggregates of inflammatory cells in the serosa layer of the afflicted segment of Crohn's ileum. In addition, there was a chaotic display of nerve fibres containing some of the neuroactive substances with a high frequency of enlarged varicosities in the myenteric ganglia and/or nerve fibres of the circular muscle layer of Crohn's ileum. CONCLUSION: Results show quantitative as well as qualitative changes in the neurochemical composition of enteric nerve fibres and nerve cell bodies of Crohn's ileum. These changes and the presence of nitric oxide synthase and peptides immunoreactive inflammatory cells in the serosa layer suggest that nerve-immune interactions may have a significant role in the process of the inflammatory changes seen in Crohn's ileitis.

Adult↗
PubMed1996

Hypersensitivity to very-low single radiation doses: its relationship to the adaptive response and induced radioresistance.

There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.

Adaptation, Physiological↗
PubMed1996

Innervation of vasculature and microvasculature of the human vagina by NOS and neuropeptide-containing nerves.

The aims of the present study were to determine whether nerves that contain nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP) or substance P (SP) are present in the human vagina and, if so, to determine the pattern of innervation relative to that of other neurotransmitters, particularly vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Surgical specimens of vaginal tissue (n = 10) from pre- and postmenopausal women were fixed and processed for immunohistochemistry of peptides and NOS and for histochemistry of NADPH-diaphorase. SP-immunoreactive nerves were very sparse, being absent from 9 of the 10 tissue samples. For other peptides and NOS, the innervation of the deep arteries and veins was greater than that of blood vessels in the propria. Capillaries in the epithelial papillae also appeared to be innervated by nerves containing NOS, CGRP, NPY and VIP. Beneath the epithelium nerve fibres formed a subepithelial plexus; no nerve cell bodies were seen. The relative density of innervation by immunoreactive fibres was PGP-9.5 > NPY > VIP >> NOS > CGRP > SP. These results imply that nerves that utilise nitric oxide or NPY, VIP or CGRP as a neurotransmitter may play a role in controlling blood flow and capillary permeability in the human vagina. The origin and function of all these nerves is discussed.

Adult↗
PubMed1996

Radiation-induced changes in neuropeptides in the rat urinary bladder.

PURPOSE: To determine whether there was a change in innervation in the rat urinary bladder following x-ray irradiation. MATERIALS AND METHODS: The urinary bladders were obtained from rats irradiated 6 months previously with single doses of 15 Gy and 25 Gy x-radiation, and from nonirradiated (control) animals. They were examined immunohistochemically to localize neuropeptide Y (NPY), substance P (SP), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), met-enkephalin (m-ENK), leu-enkephalin (l-ENK), somatostatin (SOM) and the enzyme, tyrosine-hydroxylase (TH). Computer assisted image analysis was used to assess the density of immunoreactive nerve fibres. RESULTS: The greatest density of nerves observed in the bladder from control animals contained NPY, followed (in decreasing order) by CGRP, VIP, SP and TH. The nerves appeared to run predominantly along the longitudinal axis of the circular and longitudinal muscle fibres. SP-, CGRP-, TH- and occasionally VIP-immunoreactive nerves were observed in the lamina propria, at the base of the urothelium. Perivascular nerves containing neuropeptides and TH were observed throughout the bladder wall. There was an absence of m-ENK-, l-ENK- and SOM-immunoreactive nerves in the control and irradiated rat urinary bladders. In the rat urinary bladder irradiated with 25 Gy x-radiation, there was a significant increase (P < 0.05) in the density of NPY-, TH- and SP- but not CGRP- and VIP-immunoreactive nerves. There were regional differences within the bladder, that is, there was an increase in VIP-, CGRP- and SP-immunoreactive nerves around and within the urothelium. NPY-immunoreactive nerves were seen in the connective tissue and elastic fibres of the lamina propria for the first time. An increase in the density or fluorescence intensity of perivascular TH- but not neuropeptide-containing nerves was observed. CONCLUSIONS: The increase in the density of NPY-, SP- and TH-immunoreactive nerves in the irradiated bladders may be due to axonal sprouting which contributes to the symptoms of radiation injury.

Animals↗
PubMed1995

An increase of neuropeptide Y but not nitric oxide synthase-immunoreactive nerves in the bladder neck from male patients with bladder neck dyssynergia.

PURPOSE: To determine the distribution of neuropeptides in male patients with bladder neck dyssynergia and benign prostatic hyperplasia. MATERIALS AND METHODS: Bladder neck tissue, obtained from male patients with bladder neck dyssynergia (BND) and control patients with benign prostatic hyperplasia (BPH), was studied immunohistochemically for protein gene product 9.5 (a general neuronal marker), vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene-related peptide, substance P, growth associated protein 43 and nitric oxide synthase. RESULTS: In the bladder neck from control patients, the greatest density of nerves contained protein gene product 9.5, followed in decreasing order by neuropeptide Y; vasoactive intestinal polypeptide; calcitonin gene-related peptide; nitric oxide synthase; substance P and serotonin. The neuropeptides were found in the smooth muscle and were also associated with blood vessels. In patients with BND there was a statistically significant increase (P < 0.05) in the density of protein gene product 9.5- and neuropeptide Y-immunoreactive nerves in the smooth muscle and the base of the mucosa but not in blood vessels in the bladder neck, while the density of the other neuropeptides studied, nitric oxide synthase and serotonin did not significantly change from that of control tissue. Growth associated protein 43-immunoreactive nerves were absent from the bladder neck from both groups of patients. CONCLUSION: It is suggested that the increase in density of protein gene product 9.5- and neuropeptide Y-immunoreactive nerves, part of the sympathetic contractile system of the bladder neck, may exacerbate bladder outlet obstruction and thus play a role in the pathogenesis of BND.

Adult↗
PubMed1994

Increased DNA-repair capacity and the modulation of 2 proteins in and metallothionein overexpressing Chinese hamster cell line.

Elevated intracellular levels of metallothionein have been associated with resistance to the cytotoxic effects of some alkylating agents. In order to study the mechanisms responsible for this resistance, we used a pair of CHO cell lines consisting of normal K1-2 cells and their derivative K1-2MT, which overexpresses the human metallothionein II-A gene (Lohrer et al., 1989). K1-2MT cells were found to be resistant to cadmium chloride and the alkylating agents N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), but resistance did not extend to the alkylating agent, 1,3-bis(2-chloroethyl)- 1-nitrosourea, nor to adriamycin, an inhibitor of DNA synthesis. The DNA damage caused by MNNG, was only marginally less in resistant cells compared with the parental cell line, thus excluding drug scavenging as a possible mechanism for resistance. Also, glutathione S-transferases (GSTs) were present at equal levels in both cell lines (acidic and basic type GST) or slightly reduced in drug resistant K1-2MT cells (neutral type GST), thereby ruling out metabolic inactivation of the alkylating agents. However, the drug resistant phenotype was accompanied by a more efficient block of DNA synthesis after MNNG treatment and by a 3-h delay in the G2 phase of the cell cycle. Using two-dimensional gel electrophoresis of total protein extracts, we identified a 24-kDa protein (MIP1), which is only present in the resistant K1-2MT cells, and a 23.5-kDa protein (MIP2) which is 2-3 times over-synthesized in K1-2MT cells. MNNG treatment reduced the level of both proteins MIP1 and MIP2. These results suggest that the proteins MIP1 and possibly MIP2 may be responsible for the alkylating agent resistant phenotype and are probably modulated by the human metallothionein II-A protein.

Alkylating Agents↗
PubMed1992

Increased sensitivity of a Chinese hamster ovary cell line to alkylating agents after overexpression of the human metallothionein II-A gene.

The elevation of intracellular levels of metallothionein has been associated with the development of resistance to the cytotoxic effects of some alkylating agents. In order to study the mechanisms underlying metallothionein associated drug resistance we transfected the alkylating agent sensitive CHO cell line MMC-1 (Robson et al., 1985) with an episomally replicating plasmid coding for the human metallothionein II-A gene. Two transfectants were isolated which carried about 10 copies of the plasmid. The basal expression of metallothionein was increased from undetectable levels in the recipient cell line MMC-1 to between 22 and 26 micrograms metallothionein per 10(7) cells in the transfectants. Treatment with cadmium salts increased metallothionein levels a further 1.7-fold. Cytotoxicity assays revealed that MTII-A transfection increased the sensitivity of the parental MMC-1 cells to N-methyl-N'-nitro-N-nitrosoguanidine and N-nitroso-N-methylurea. These results suggest that metallothionein does not act as a simple scavenger of alkylating agents, but interacts with unknown factor(s) in host cells.

Alkylating Agents↗
PubMed1991

Immunoblotting human C4 bound to human erythrocytes in vivo and in vitro.

A study of C4 bound to human erythrocytes in vitro and in vivo has been made by immunoblotting with mouse monoclonal anti-C4c and anti-C4d and human polyclonal anti-C4d (Rodgers and Chido) following SDS-PAGE. Multi-banded patterns differentiated between C4A and C4B isotypes. Treatment of EC4b with trypsin eliminated immunoblotting but not agglutination reactions. Serum inactivation (factor I) of EC4b resulted in banding patterns similar to those obtained from patients' EC4d. Treatment of EC4b membranes with NH2OH affected many of the bands, two were lost, one was markedly reduced and others had altered SDS-PAGE mobility. Interpretation of the bands has been made in terms of C4-acceptor complexes and inactivation fragments of C4. A distinct difference in the banding of C4A and C4B isotypes has been detected.

Ammonium Hydroxide↗
PubMed1990

Differential effects on cell killing in metallothionein overexpressing CHO mutant cell lines.

An increased level of metallothionein (MT) within the cytosol has been associated with the development of resistance to the cytotoxic effects of alkylating agents, but the mechanism of this effect is unclear. To verify the possible role of MT as a direct scavenger of alkylating agents we transfected two alkylating agent sensitive CHO mutant cell lines, methyl methanesulphate-1 and -2 (MMS-1 and MMS-2), with the human metallothionein II-A (hMTII-A) gene. From each mutant two transfected cell clones were isolated which were found to bear episomally 50 to 70 copies of the plasmid pMTII-BPV coding for the hMTII-A gene. The transfected cell lines contained between 1.4 and 4.9 micrograms of MT per 10(7) cells. This could be increased 2- to 9-fold by heavy metal induction. Transfectants derived from mutant MMS-1 were equally sensitive as the parental cells to a set of alkylating agents. However, transfectants derived from mutant cell line MMS-2 gained significant resistance to both N-methyl-N'-nitro-N-nitrosoguanidine and N-nitroso-N-methylurea, whilst retaining sensitivity to MMS. The degree of resistance was not increased by MT induction after heavy metal ion exposure. These results indicate that although resistance to alkylating agents may be mediated by a process which involves MT expression, this is not due to simple scavenging of the cytotoxic agent by the MT protein.

Animals↗
PubMed1989

An epitope on C4 beta light (L) chains detected by human anti-Rg; its relationship with beta chain polymorphism and MHC associations.

Two out of ten Rg-specific antisera tested contain a third antibody specific for the beta chain of C4. Analysis of the beta chains of 66 unrelated individuals by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the epitope detected is located exclusively on the light (L) beta chain. A strong, but incomplete, association between the beta chain epitope and the expression of the Rg:2 determinant on the alpha chain of the same protein was also observed. While H (heavy) and L beta chains were not associated with a particular C4 isotype, previously unrecorded associations of beta chain polymorphism with the DR locus have been established.

Antibodies, Monoclonal↗
PubMed1989

Overexpression of metallothionein in CHO cells and its effect on cell killing by ionizing radiation and alkylating agents.

Metallothionein protein protects cells from the toxic effects of heavy metal ions. To establish its protective function against ionizing radiation and alkylating agents, a model system was created by transfecting two CHO cell lines (wild-type, K1-2 and X-ray sensitive, xrs-2 subclone Bc11) with the human metallothionein II-A (hMTII-A) gene integrated in a bovine papilloma derived autonomously replicating vector. The isolated transfectants are cadmium-resistant (Cdr), due to the overexpression of the hMTII-A gene. Their steady-state level of hMTII-A mRNA can be increased up to 40-fold after Cd treatment and 20-fold after induction with ionizing radiation. The transfected cell lines proved to be as sensitive as the recipient cell lines to ionizing radiation and bleomycin but the transfectants were significantly more resistant to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) and mitomycin C (MMC). These results lead to the conclusion that the MT protein does provide a defence mechanism to protect cells from monofunctional alkylating and cross-linking agents but not from free radicals.

Alkylating Agents↗