Robin Hesketh | University of Cambridge (original) (raw)

Papers by Robin Hesketh

Biochimica Et Biophysica Acta-biomembranes, 1976

1. Activation of adenylate cyclase in rat liver plasma membranes by fluoride or GMP-P(NH)P yielde... more 1. Activation of adenylate cyclase in rat liver plasma membranes by fluoride or GMP-P(NH)P yielded linear Arrhenius plots. Activation by glucagon alone, or in combination with either fluoride or GMP-P(NH)P resulted in biphasic Arrhenius plots with a well-defined break at .2. The competitive glucagon antagonist, des-His-glucagon did not activate the adenylate cyclase but produced biphasic Arrhenius plots in combination with fluoride or GMP-P(NH)P. The break temperatures and activation energies were very similar to those observed with glucagon alone, or in combination with either fluoride or GMP-P(NH)P.3. It is concluded that although des-His-glucagon is a potent antagonist of glucagon, it nevertheless causes a structural coupling between the receptor and the catalytic unit.

Biochimica Et Biophysica Acta-biomembranes, 1980

The membrane potential of mouse spleen lymphocytes has been assessed with two fluorescent probes.... more The membrane potential of mouse spleen lymphocytes has been assessed with two fluorescent probes. 3,3′-Dipropylthiadicarbocyanine (diS-C3-(5)) was used for most of the experiments. Solutions with high K+ concentrations depolarised the cells. Valinomycin, an ionophore which adds a highly K+-selective permeability to membranes, slightly hyperpolarised cells in standard (6 mM K+) solution, and in 145 mM K+ solution produced a slight additional depolarisation. These findings indicate a membrane whose permeability is relatively selective for K+. Very small changes in potential were seen when choline replaced Na+, or gluconate replaced Cl−, supporting the idea of K+ selectivity. The resting potential could be estimated from the K+ concentration gradient at which valinomycin did not change the potential — the ‘valinomycin null point’ — and under the conditions used the resting potential was approx. −60 mV.B cell-enriched suspensions were prepared either from the spleens of nu/nu mice or by selective destruction of T cells in mixed cell populations. The membrane potential of these cells was similar to that estimated for the mixed cells.In solution with no added K+, diS-C3-(5) itself appeared to depolarise the lymphocytes, in a concentration dependent manner. With the 100 nM dye normally used, the membrane potential in K+-free solution was around −45 mV, and 500 nM dye almost completely depolarised the cells. In standard solution quinine depolarised the cells. Valinomycin could still depolarise these cells indicating that depolarisation had not been due to dissipation of the K+ gradient. Since in K+-free solution diS-C3-(5) blocks the Ca2+-activated K+ channels in human red blood cell ghosts and quinine also blocks this K+ channel it is suggested that the resting lymphocyte membrane may have a similar Ca2+-activated K+ permeability channel.Because of the above mentioned effect of diS-C3-(5) and other biological side effects, such as inhibition of B cell capping, a chemically distinct fluorescent probe of membrane potential, bis(1,3-diethylthiobarbiturate)-trimethineoxonol was used to support the diS-C3-(5) data. This new probe proved satisfactory except that it formed complexes with valinomycin, ruling out the use of this ionophore. Results with the oxonol on both mixed lymphocytes and B cell-enriched suspensions gave confirmation of the conclusions from diS-C3-(5) experiments and indicated that despite its biological side effects, diS-C3-(5) could still give valid assessment of membrane potential.

Polymer Engineering and Science, 1980

Investigations of morphological changes which are induced in segmented elastomers by annealing an... more Investigations of morphological changes which are induced in segmented elastomers by annealing and quenching are reported. Four different polymers were studied each based on the same soft segment—1000 or 2000 molecular weight poly(tetramethylene oxide). The hard segments were 4,4′-diphenylmethane diisocyanate (MDI) chain extended with 1,4-butane diol (ET series), piperazine coupled with 1,4-butane diol bischloroformate (BN-1,4), or dimethyl terephthalate condensed with 1,4-butane diol (H-50). Following annealing at various temperatures (120, 150, 170, or 190°C), the polymers were quenched to ambient conditions, and their properties measured by differential scanning calorimetry (DSC) as a function of time following the quench. DSC measurements taken immediately after the quench show that the soft segment Tg is higher than that of the control, suggesting that the applied thermal history promoted increased mixing of hard and soft segments. As time passes after quenching, the Tg values decrease and approach an equilibrium value. This effect is much smaller for those samples having crystalline hard segments. Endotherms attributed to the disruption of long range ordering in the hard segment domains resulted from the annealing process. These endotherms appeared at higher temperatures for higher annealing temperatures. The positions of crystalline melting endotherms were independent of the annealing/quenching conditions investigated.

Febs Letters, 1998

Latent transforming growth factor-L L binding protein-1 (LTBP-1), plays an important role in cont... more Latent transforming growth factor-L L binding protein-1 (LTBP-1), plays an important role in controlling localisation and activation of transforming growth factor-L L (TGF-L L). We show that alternative splicing generates a form of mRNA which lacks bases 1277^1435 (termed LTBP-1v v53). The 53 amino acids encoded by these bases include the eighth cysteine of the first cysteine repeat and a consensus heparin binding sequence. Sequencing of genomic clones showed that alternative splicing resulted from the use of an intra-exonic 3P splice acceptor site. The loss of the heparin binding site implies that LTBP-1v v53 will bind to the extracellular matrix less efficiently than LTBP-1.

Biochemistry, 1976

ABSTRACT

Journal of Cell Biology, 1984

Stimulated histamine release was depressed at least tenfold in mitotic 2H3 rat basophilic cells w... more Stimulated histamine release was depressed at least tenfold in mitotic 2H3 rat basophilic cells when compared with interphase cells even though both contained comparable amounts of histamine . Antigen stimulation of IgE-sensitized interphase cells initiated an influx of Ca" that preceded secretion of histamine and a similar Ca" influx occurred in stimulated mitotic cells. This strongly suggests that during mitosis there is a dramatic inhibition of one or more of the steps on the pathway leading from elevated intracellular Ca" to the fusion of secretory granules with the plasma membrane .

Biochemical Journal, 2000

ABSTRACT The transforming growth factor beta (TGF beta) cytokines are a multifunctional family th... more ABSTRACT The transforming growth factor beta (TGF beta) cytokines are a multifunctional family that exert a wide variety of effects on both normal and transformed mammalian cells. The secretion and activation of TGF betas is regulated by their association with latency-associated proteins and latent TGF beta binding proteins (LTBPs). Over the past few years, three members of the LTBP family have been identified, in addition to the protoype LTBP1 first sequenced in 1990. Three of the LTBP family are expressed in a variety of isoforms as a consequence of alternative splicing. This review summarizes the differences between the isoforms in terms of the effects on domain structure and hence possible function. The close identity between LTBPs and members of the fibrillin family, mutations in which have been linked directly to Marfan's syndrome, suggests that anomalous expression of LTBPs may be associated with disease. Recent data indicating that differential expression of LTBP1 isoforms occurs during the development of coronary heart disease is considered, together with evidence that modulation of LTBP function, and hence of TGF beta activity, is associated with a variety of cancers.

Electrification in developed countries has progressively increased the mean level of extremely lo... more Electrification in developed countries has progressively increased the mean level of extremely low-frequency electromagnetic fields (ELF-EMFs) to which populations are exposed; these humanmade fields are substantially above the naturally occurring ambient electric and magnetic fields of Ç10 04 Vm 01 and Ç10 013 T, respectively. Several epidemiological studies have concluded that ELF-EMFs may be linked to an increased risk of cancer, particularly childhood leukemia. These observations have been reinforced by cellular studies reporting EMF-induced effects on biological systems, most notably on the activity of components of the pathways that regulate cell proliferation. However, the limited number of attempts to directly replicate these experimental findings have been almost uniformly unsuccessful, and no EMF-induced biological response has yet been replicated in independent laboratories. Many of the most well-defined effects have come from gene expression studies; several attempts have been made recently to repeat these key findings. This review analyses these studies and summarizes other reports of major cellular responses to EMFs and the published attempts at replication. The opening sections discuss quantitative aspects of exposure to EMFs and the incidence of cancers that have been correlated with such fields. The concluding section considers the problems that confront research in this area and suggests feasible strategies.-Lacy-Hulbert, A., Metcalfe, J. C., Hesketh, R. Biological responses to electromagnetic fields. FASEB J. 12, 395-420 (1998) Key Words: ELF-EMFs · protein kinase C · tumor development · growth factors · cell proliferation · geomagnetic field 1 This review is dedicated to Dr. Brian Maddock, who died suddenly in October 1997. Brian Maddock had been Administrator of the EMF Biological Research Trust in the U.K. since its inception and played a major role in coordinating and implementing the research programs funded by the Trust. Through his work in that position, he was well known to everyone involved in EMF research in the U.K., and also to many of our colleagues in the U.S. and in Europe. Throughout this community, he was regarded with much respect and affection.

American Journal of Pathology, 2002

Retinal neovascularization occurs in a variety of diseases including diabetic retinopathy, the mo... more Retinal neovascularization occurs in a variety of diseases including diabetic retinopathy, the most common cause of blindness in the developed world. There is accordingly considerable incentive to develop drugs that target the aberrant angiogenesis associated with these conditions. Previous studies have shown that a number of anti-angiogenic agents can inhibit retinal neovascularization in a well-characterized murine model of ischemia-induced proliferative retinopathy. Combretastatin-A4 (CA-4) is an anti-vascular tubulin-binding agent currently undergoing clinical evaluation for the treatment of solid tumors. We have recently shown that CA-4 is not tumor-specific but elicits anti-vascular effects in nonneoplastic angiogenic vessels. In this study we have examined the capacity of CA-4 to inhibit retinal neovascularization in vivo. CA-4 caused a dose-dependent inhibition of neovascularization with no apparent side effects. The absence of vascular abnormalities or remnants of disrupted neovessels in retinas of CA-4-treated mice suggests an anti-angiogenic mechanism in this model, in contrast to the anti-vascular effects observed against established tumor vessels. Importantly, histological and immunohistochemical analyses indicated that CA-4 permitted the development of normal retinal vasculature while inhibiting aberrant neovascularization. These data are consistent with CA-4 eliciting tissue-dependent anti-angiogenic effects and suggest that CA-4 has potential in the treatment of nonneoplastic diseases with an angiogenic component.

Members of the transforming growth factor ␤ (TGF-␤) family are potent inhibitors of the growth of... more Members of the transforming growth factor ␤ (TGF-␤) family are potent inhibitors of the growth of many epithelial cell types. Transmembrane signaling by TGF-␤ occurs via a complex of the serine/threonine kinases TGF-␤ type 1 receptor and TGF-␤ type 2 receptor (TG-FBR2), and inactivating mutations in the latter have recently been detected in some primary tumors and in several types of tumor-derived cell lines. The most common mutations that have been identified in TGFBR2 are frameshifts in a repetitive polyadenine region in replication error-positive colorectal carcinomas that result in a truncated protein and absence of receptor expression at the cell surface. A number of point mutations in the highly conserved serine/threonine kinase domain of TGFBR2 have also been reported, some of which have been correlated with either loss of trans-phosphorylation of TGF-␤ type 1 receptor or constitutive activation of trans-phosphorylation. No TGFBR2 mutations have been reported in human breast tumors, but anomalous expression of TGF-␤ in breast carcinomas suggests that TGF-␤ signaling may be defective. We have therefore systematically examined unmatched sets of 17 primary and 17 recurrent breast tumor samples for mutations in TG-FBR2, restricted to those regions of the gene in which mutations have previously been reported. None of the previously reported mutations was detected, but four novel mutations (V387M, N435S, V447A, and L452M) were found in the kinase domain in recurrent tumors. No mutations were detected in primary tumors. TGF-␤ signaling was significantly inhibited by each of the N435S, V447A, and L452M mutations.

British Journal of Cancer, 2001

Combretastatin-A4 phosphate (cis-CA-4) is a tubulin-binding agent currently undergoing clinical t... more Combretastatin-A4 phosphate (cis-CA-4) is a tubulin-binding agent currently undergoing clinical trials as an anti-tumour drug. We have investigated whether CA-4 functions as a tumour-specific anti-vascular agent using the hyperplastic thyroid as a novel in vivo model of neovascularization. CA-4 elicited pathological changes in normal tissue, manifested as the induction of multiple, discrete intravascular thrombi. These vascular-damaging effects indicate that CA-4P does not function as a tumour-specific agent but targets neovasculature irrespective of the primary angiogenic stimulus.

Lancet Oncology, 2001

The requirement for neovascularisation to permit the development of solid tumours beyond a thresh... more The requirement for neovascularisation to permit the development of solid tumours beyond a threshold size, has focused attention on the therapeutic potential of agents that prevent angiogenesis. The multistep nature of angiogenesis presents several targets for intervention, including the inhibition of the endothelial-cell migration or proliferation normally associated with developing vessels. Compounds that damage established tumour vasculature are also of potential clinical use. We review the development of one such antivascular drug, combretastatin A4. This tubulin-binding agent was originally isolated from an African shrub, Combretum caffrum. The disodium combretastatin A4 phosphate prodrug is currently undergoing phase I clinical trials in the UK and USA. This review assesses the in vitro and in vivo data for combretastatin and the prodrug, and the preliminary data that have emerged from the phase I clinical trials.