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Biomedical subjects

Andrew P Thomas

Publications and source records attributed to Andrew P Thomas.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of the cardiac L-type calcium channel in L6 cells by arginine-vasopressin.

L-type Ca2+ channel activity was measured in L6 cells as nifedipine-sensitive barium (Ba2+; 5 mM) influx in a depolarizing salt solution containing 140 mM KCl. Addition of AVP (arginine-vasopressin) during Ba2+ uptake reduced the rate of Ba2+ influx by 60-100%; this was followed by a gradual restoration of the initial rate of Ba2+ uptake. Blockade of PKC (protein kinase C) by pretreatment with 10 muM bisindolylmaleimide did not affect the initial inhibition of Ba2+ influx, but completely abolished the recovery phase. The effect of AVP was half-maximal at 10 nM AVP and was blocked by the V1a receptor antagonist d-(CH2)(5)-Tyr(Me)-AVP. Activation of G(alphas) by isoprenaline or cholera toxin antagonized the actions of AVP on Ba2+ uptake. This protection persisted in the presence of the PKA (protein kinase A) inhibitor KT5720, and was not mimicked by agents that increase cAMP. Inhibition of Ba2+ influx was also elicited by ATP and ET (endothelin 1) with an order of effectiveness ET<ATP<AVP. Each of these agents has been reported to act through G(q)-coupled receptors. We conclude that activation of G(q)-coupled receptors produces a rapid inhibition of the cardiac L-type Ca2+ channel, which is subsequently overcome by activation of PKC.

Adenosine Triphosphate↗
PubMed2006

Calcium signaling: double duty for calcium at the mitochondrial uniporter.

Uptake of Ca(2+) by mitochondria serves as a regulator of a number of important cellular functions, including energy metabolism, cytoplasmic Ca(2+) signals, and apoptosis. Recent findings reveal that the process of Ca(2+) uptake by the mitochondrial uniporter is itself regulated by Ca(2+) in a temporally complex manner.

Animals↗
PubMed2006

Ryanodine receptors in liver.

The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.

Animals↗
PubMed2006

Models of IP3 and Ca2+ oscillations: frequency encoding and identification of underlying feedbacks.

Hormones that act through the calcium-releasing messenger, inositol 1,4,5-trisphosphate (IP3), cause intracellular calcium oscillations, which have been ascribed to calcium feedbacks on the IP3 receptor. Recent studies have shown that IP3 levels oscillate together with the cytoplasmic calcium concentration. To investigate the functional significance of this phenomenon, we have developed mathematical models of the interaction of both second messengers. The models account for both positive and negative feedbacks of calcium on IP3 metabolism, mediated by calcium activation of phospholipase C and IP3 3-kinase, respectively. The coupled IP3 and calcium oscillations have a greatly expanded frequency range compared to calcium fluctuations obtained with clamped IP3. Therefore the feedbacks can be physiologically important in supporting the efficient frequency encoding of hormone concentration observed in many cell types. This action of the feedbacks depends on the turnover rate of IP3. To shape the oscillations, positive feedback requires fast IP3 turnover, whereas negative feedback requires slow IP3 turnover. The ectopic expression of an IP3 binding protein has been used to decrease the rate of IP3 turnover experimentally, resulting in a dose-dependent slowing and eventual quenching of the Ca2+ oscillations. These results are consistent with a model based on positive feedback of Ca2+ on IP3 production.

Animals↗
PubMed2006

Plant thorn tenosynovitis.

We report a case of plant thorn injury-induced tenosynovitis of the index finger of the left hand that was cured after surgical removal of the foreign body and partial synovectomy. We review current literature on this infrequently reported topic. Pathologically, thorn synovitis represents a foreign body reaction to retained plant material and is mostly aseptic. The presentation is delayed with the history of preceding injury often forgotten, and the diagnosis thus relies on careful elicitation of history. Imaging may be helpful to localize the plant material when the history is not clear. When possible, removal of the foreign material leads to complete remission, although some patients need partial or total synovectomy.

Foreign-Body Reaction↗
PubMed2006

The cellular phenotype of AZ703, a novel selective imidazo[1,2-a]pyridine cyclin-dependent kinase inhibitor.

Because the majority of cancers exhibit direct or indirect deregulation of cyclin-dependent kinase (CDK) function, members of the CDK family are attractive targets for the development of anticancer agents. As part of an ongoing program, novel imidazopyridines were identified and developed as potent and selective CDK inhibitors. Here, we describe data on the in vitro biological activities of one of these compounds, AZ703. The selectivity profile of AZ703 was investigated in kinase assays against a range of CDK enzymes as well as a panel of protein kinases in vitro. IC50s were assessed against different tumor cell lines in vitro. The mechanism of action of AZ703 was determined by observing changes in phosphorylation of CDK substrates and cell cycle effects on tumor and normal cells. In vitro studies revealed that AZ703 is a selective inhibitor of CDK1 and CDK2 and displays a mode of action consistent with the induction of G1-, S-, and G2-M-phase arrest. AZ703 also showed potent antiproliferative activity across a wide range of tumor cell lines in vitro. Moreover, AZ703 induced reversible blockade of normal cells while causing tumor cells to undergo apoptosis. We have identified AZ703 as a novel selective imidazo[1,2-a]pyridine CDK inhibitor that shows promising antitumor properties in vitro.

Apoptosis↗
PubMed2005

Calcium oscillations in type-1 astrocytes, the effect of a presenilin 1 (PS1) mutation.

Stimulation of type-1 astrocytes, by a number of agonists, has been shown to increase cytosolic Ca2+ concentrations in an oscillatory manner. However, it is unknown how these are driven or altered by aging, injury or disease. Therefore, we characterized the signaling properties of rat type-1 astrocytes in monolayer cultures. Ca2+ responses were recorded in astrocytes stimulated with ATP or glutamate. Oscillations were evident in cultures at 3 days in vitro (DIV 3) with a peak percentage (26%) of cells responding by DIV 7. The presence or absence of serum in the culture medium did not influence this percentage. Likewise, long-term culture (DIV 30+) did not increase the oscillating cell numbers. ATP was found to have a more potent effective dose (50 microM) than glutamate (10 mM). Membrane potential was recorded with fluorescent voltage-sensitive dye (membrane potential dye for FLIPR) and was similar regardless of the culture age and intracellular Ca2+ response. This suggests that mechanisms associated with the intracellular release of Ca2+ from endogenous Ca2+ stores, rather than ion fluxes across the plasma membrane, may contribute to the oscillations in the astrocytes. In order to identify a possible pathological significance of this response, we transfected astrocytes with wild-type presenilin (PS1) as well as PS1 harboring a mutation linked to familial Alzheimer's disease (FAD). PS mutation expressing astrocytes oscillated at lower ATP and glutamate concentrations when compared to wild-type PS1 expressing astrocytes. This indicates that PS1 mutation may introduce aberrations in the intracellular Ca2+ mobilization in astrocytes contributing to the accelerated pathogenesis in FAD.

Alzheimer Disease↗
PubMed2004

Imidazo[1,2-b]pyridazines: a potent and selective class of cyclin-dependent kinase inhibitors.

Modification of imidazo[1,2-a]pyridine CDK inhibitors lead to identification of less lipophilic imidazo[1,2-b]pyridazine series of CDK inhibitors. Although several equivalent compounds from these two series have similar structure and show similar CDK activity, the SAR of the two series differs significantly. Protein inhibitor structure determination has confirmed differences in binding mode and given some understanding of these differences in SAR. Potent and selective imidazo[1,2-b]pyridazine inhibitors of CDK2 have been identified, which show >1 microM plasma levels following a 2mg/kg oral dose to mice.

Animals↗
PubMed2004

Imidazo[1,2-a]pyridines. Part 2: SAR and optimisation of a potent and selective class of cyclin-dependent kinase inhibitors.

Exploration of SAR and optimisation of the imidazo[1,2-a]pyridine CDK inhibitors has lead to the discovery of novel, potent and selective inhibitors of the cyclin-dependent kinase CDK2. Understanding of SAR has identified positions of substitution, which allow modification of physical properties and offer the potential for in vivo optimisation.

Cyclin-Dependent Kinases↗
PubMed2004

Modified technique for arthroscopic Bankart repair using anchor sutures.

Bankart repair, or one of its modifications, is currently the gold standard procedure for treatment of anterior traumatic shoulder instability. It is now possible to perform the operation arthroscopically with the introduction of suture anchors. As described by Eugene Wolf, arthroscopic shoulder stabilization using the Mitek (Mitek Surgical Products, Ethicon, Edinburgh, U.K.) anchors requires two anterior portals and intra-articular knot tying. However, sliding the anchor on the inside limb of a suture loop could be challenging because the other limb could get tangled in the nitinol arc of the anchor. We describe a modification of the original technique to prevent that possibility and avoid any tension on the repair tissue during anchor passage. The proposed modification involves the use of a cheap, readily available silastic feeding tube to isolate the outside limb of the suture loop and stabilize labral tissues while the anchor is being passed. This tube also serves as a stent for knot tying. By allowing the whole operation to be performed through one anterior portal, the modified technique reduces possible morbidity associated with a second portal and further reduces cost.

Arthroscopy↗
PubMed2004

Trauma-hemorrhagic shock mesenteric lymph induces endothelial apoptosis that involves both caspase-dependent and caspase-independent mechanisms.

OBJECTIVE: To determine the mechanism by which gut-derived factors present in mesenteric lymph from rats subjected to trauma-hemorrhagic shock (T/HS) induce endothelial cell death. SUMMARY BACKGROUND DATA: Intestinal ischemia after hemorrhagic shock results in gut barrier dysfunction and the subsequent production of biologically active and tissue injurious factors by the ischemic gut. These factors are carried in the mesenteric lymph and reach the systemic circulation via the mesenteric lymph, thereby ultimately resulting in distant organ injury. Although studies have established that trauma-hemorrhagic (T/HS) shock but not trauma-sham-shock (T/SS) mesenteric lymph is cytotoxic to endothelial cells, whether T/HS lymph-induced endothelial cell death occurs via an apoptotic or a necrotic pathway is unknown. The mechanisms underlying T/HS lymph-induced cytotoxicity are likewise unknown. METHODS: Human umbilical vein endothelial cell (HUVEC) monolayers were incubated with medium, sham-shock, or post shock mesenteric lymph (5%) for 4 hours, after which the mode of cell death (ie, apoptosis versus necrosis) was determined using morphologic (confocal microscopy), biochemical (nucleosomal release), and DNA-based (gel electrophoresis) assays. To clarify the cellular pathways involved in T/HS lymph-induced HUVEC cell death, caspase-3, caspase-9, caspase-8, and BID activity was measured as was the ability of the pan-caspase inhibitor z-VAD-fmk to prevent T/HS lymph-induced cell death. RESULTS: T/HS, but not T/SS, mesenteric lymph or medium was cytotoxic and caused the appearance of the classic morphologic signs of apoptosis, including membrane blebbing, cell shrinkage, and apoptotic body formation. Nucleosomal release and a DNA laddering pattern was also observed in the HUVECs incubated with T/HS lymph. These signs of apoptosis were associated with increased caspase activity as reflected in activation of the pro-apoptotic caspases, caspase-8, -9, and -3, as well as the pro-apoptotic bcl-2-related protein BID. However, since the broad-spectrum caspase inhibitor z-VAD-fmk delayed T/HS lymph-induced HUVEC cell death, but did not prevent it fully, it appears that other factors besides caspases are involved in the endothelial cell toxicity of T/HS lymph. CONCLUSIONS: Gut-derived factors in T/HS, but not T/SS, mesenteric lymph cause endothelial cell death via an apoptotic mechanism that involves both caspase-dependent and caspase-independent pathways.

Animals↗
PubMed2003

Imidazo[1,2-a]pyridines: a potent and selective class of cyclin-dependent kinase inhibitors identified through structure-based hybridisation.

High-throughput screening identified the imidazo[1,2-a]pyridine and bisanilinopyrimidine series as inhibitors of the cyclin-dependent kinase CDK4. Comparison of their experimentally-determined binding modes and emerging structure-activity trends led to the development of potent and selective imidazo[1,2-a]pyridine inhibitors for CDK4 and in particular CDK2.

CDC2-CDC28 Kinases↗
PubMed2003

Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

Animals↗
PubMed2003

Glucagon-like peptide-1 mobilizes intracellular Ca2+ and stimulates mitochondrial ATP synthesis in pancreatic MIN6 beta-cells.

Glucagon-like peptide-1 (GLP-1) is a potent regulator of glucose-stimulated insulin secretion whose mechanisms of action are only partly understood. In the present paper, we show that at low (3 mM) glucose concentrations, GLP-1 increases the free intramitochondrial concentrations of both Ca(2+) ([Ca(2+)](m)), and ATP ([ATP](m)) in clonal MIN6 beta-cells. Suggesting that cAMP-mediated release of Ca(2+) from intracellular stores is responsible for these effects, increases in [ATP](m) that were induced by GLP-1 were completely blocked by the Rp isomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), or by chelation of intracellular Ca(2+). Furthermore, inhibition of Ins(1,4,5) P (3) (IP(3)) receptors with xestospongin C, or application of ryanodine, partially inhibited GLP-1-induced [ATP](m) increases, and the simultaneous blockade of both IP(3) and ryanodine receptors (RyR) completely eliminated the rise in [ATP](m). GLP-1 appeared to prompt Ca(2+)-induced Ca(2+) release through IP(3) receptors via a protein kinase A (PKA)-mediated phosphorylation event, since ryanodine-insensitive [ATP](m) increases were abrogated with the PKA inhibitor, H89. In contrast, the effects of GLP-1 on RyR-mediated [ATP](m) increases were apparently mediated by the cAMP-regulated guanine nucleotide exchange factor cAMP-GEFII, since xestospongin C-insensitive [ATP](m) increases were blocked by a dominant-negative form of cAMP-GEFII (G114E,G422D). Taken together, these results demonstrate that GLP-1 potentiates glucose-stimulated insulin release in part via the mobilization of intracellular Ca(2+), and the stimulation of mitochondrial ATP synthesis.

Adenosine Triphosphate↗
PubMed2002

ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration.

ZD6474 [N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine]is a potent, p.o. active, low molecular weight inhibitor of kinase insert domain-containing receptor [KDR/vascular endothelial growth factor receptor (VEGFR) 2] tyrosine kinase activity (IC(50) = 40 nM). This compound has some additional activity versus the tyrosine kinase activity of fms-like tyrosine kinase 4 (VEGFR3;IC(50) = 110 nM) and epidermal growth factor receptor (EGFR/HER1; IC(50) = 500 nM) and yet demonstrates selectivity against a range of other tyrosine and serine-threonine kinases. The activity of ZD6474 versus KDR tyrosine kinase translates into potent inhibition of vascular endothelial growth factor-A (VEGF)-stimulated endothelial cell (human umbilical vein endothelial cell) proliferation in vitro (IC(50) = 60 nM). Selective inhibition of VEGF signaling has been demonstrated in vivo in a growth factor-induced hypotension model in anesthetized rat: administration of ZD6474 (2.5 mg/kg, i.v.) reversed a hypotensive change induced by VEGF (by 63%) but did not significantly affect that induced by basic fibroblast growth factor. Once-daily oral administration of ZD6474 to growing rats for 14 days produced a dose-dependent increase in the femoro-tibial epiphyseal growth plate zone of hypertrophy, which is consistent with inhibition of VEGF signaling and angiogenesis in vivo. Administration of 50 mg/kg/day ZD6474 (once-daily, p.o.) to athymic mice with intradermally implanted A549 tumor cells also inhibited tumor-induced neovascularization significantly (63% inhibition after 5 days; P < 0.001). Oral administration of ZD6474 to athymic mice bearing established (0.15-0.47 cm(3)), histologically distinct (lung, prostate, breast, ovarian, colon, or vulval) human tumor xenografts or after implantation of aggressive syngeneic rodent tumors (lung, melanoma) in immunocompetent mice, produced a dose-dependent inhibition of tumor growth in all cases. Statistically significant antitumor activity was evident in each model with at least 25 mg/kg ZD6474 once daily (P < 0.05, one-tailed t test). Histological analysis of Calu-6 tumors treated with 50 mg/kg/day ZD6474 for 24 days showed a significant reduction (>70%) in CD31 (endothelial cell) staining in nonnecrotic regions. ZD6474 also restrained growth of much larger (0.9 cm(3) volume) Calu-6 lung tumor xenografts and induced profound regression in established PC-3 prostate tumors of 1.4 cm(3) volume. ZD6474 is currently in Phase I clinical development as a once-daily oral therapy in patients with advanced cancer.

Administration, Oral↗
PubMed2002

Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes.

Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation.

Animals↗