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Biomedical subjects

Denys N Wheatley

Publications and source records attributed to Denys N Wheatley.

At least 19 recordsLinked to original sources
PubMed2006

Action of Lovastatin (Mevinolin) on an in vitro model of angiogenesis and its co-culture with malignant melanoma cell lines.

BACKGROUND: Lovastatin and other statins may reduce the development of melanomas. The effects on melanoma cells and their ability to enhance angiogenesis in a co-culture system presented an opportunity to assess whether Lovastatin act on melanoma cells, HUVEC or both types of cells. RESULTS: Direct effects of co-culturing two different malignant melanoma cells (A375 and G361) on the process of angiogenesis in vitro was studied with our angiogenesis model, based on human dermal fibroblasts and human umbilical vein endothelial cells (HUVEC). Co-cultures were set up using "sland" and "dispersed seeding" techniques. A statistically significant increase in tubule formation in both cases was observed compared to controls. The effects of doses equivalent to therapeutic concentrations of Lovastatin were analysed. The drug inhibited the growth of all cell types, induced apoptosis, and markedly reduced the formation of tubules in the angiogenesis model at low concentrations. Its action was successfully reversed by the introduction of geranylgeranyl pyrophosphate. CONCLUSION: Lovastatin can reduce both tumour (melanoma) cell growth, and the angiogenic activity of these cells in co-cultures using an established 2-dimensional model angiogenesis system beyond that which would be seen by reduced proliferation alone.

Journal Article↗
PubMed2006

Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro.

The development of vessel-like structures in vitro to mimic as well as to realize the possibility of tissue-engineered small vascular networks presents a major challenge to cell biologists and biotechnologists. We aimed to establish a three-dimensional (3-D) culture system with an endothelial network that does not require artificial substrates or ECM compounds. By using human skin fibroblasts and endothelial cells (ECs) from the human umbilical vein (HUVECs) in diverse spheroid coculture strategies, we verified that fibroblast support and modulate EC migration, viability, and network formation in a 3-D tissue-like stromal environment. In mixed spheroid cultures consisting of human ECs and fibroblasts, a complex 3-D network with EC tubular structures, lumen formation, pinocytotic activity, and tight junction complexes has been identified on the basis of immunohistochemical and transmission electron microscopic imaging. Tubular networks with extensions up to 400 mum were achieved. When EC suspensions were used, EC migration and network formation were critically affected by the status of the fibroblast. However, the absence of EC migration into the center of 14-day, but not 3-day, precultured fibroblast spheroids could not be attributed to loss of F viability. In parallel, it was also confirmed that migrated ECs that entered cluster-like formations became apoptotic, whereas the majority of those forming vessel-like structures remained viable for >8 days. Our protocols allow us to study the nature of tubule formation in a manner more closely related to the in vivo situation as well as to understand the basis for the integration of capillary networks in tissue grafts and develop methods of quantifying the amount of angiogenesis in spheroids using fibroblast and other cells isolated from the same patient, along with ECs.

Blood Vessels↗
PubMed2005

Cell biology as the basis of a better understanding of cancer.

Clinicians will argue that cancer can only really receive the treatment that is needed through thorough understanding of medicine. However, even empirical approaches to therapy result in experimental analysis of the agencies involved on test cells, usually in culture. From the obverse perspective, cell biologists will argue that until we fully understand cell cycle regulation, tumour management will be too imprecise to make the best advances. A forum is needed whereby the fundamental studies on cells prior to, during and after transformation in vitro can be freely reported (open access) and discussed. The action of anticancer agents and cancer preventative substances can more easily be studied in vitro before the often excessive complexity of making similar studies in experimental and human cancers is tackled. Cancer Cell International is committed to providing such a forum. Ironically within a few months of launching this open access journal, Elsevier had much the same idea, and there one has to pay for the privilege of downloading vital papers in this biomedical field.

Editorial↗
PubMed2005

Integrity and stability of the citrulline-arginine pathway in normal and tumour cell lines.

Arginine catabolizing enzymes have been used on cancers for over 60 years. In the last 5 years the ability of arginine catabolizing enzymes, not only to inhibit proliferation, but to kill tumour cells has been reinvestigated. Selectivity of action lies in the inability of many tumours to circumvent arginine deprivation by recycling precursors through the urea cycle. While this offers an immediate window of opportunity to treat, e.g. melanomas and hepatocellular carcinomas (HCC) that have poor citrulline converting ability, it is possible that the deprivation can be applied to many other types of cancer. The problem of deficiency of the urea cycle enzymes in a wider range of normal and malignant cell lines has been addressed, and shown to be variable throughout several different tumour types. We also need to know how fickle recycling enzyme activity can be in both normal and tumour cells, and found to be remarkable stable. Increasing interest is shown in the amino acid (arginine) deprivation protocol because it has already moved into the clinic. Initial findings on a named-patient basis have been encouraging, and the development of a new rational approach to the systemic treatment of melanomas, HCCs and leukemias seems imminent. This is the more attractive because arginine deprivation protocols can also 'stage' tumour cells for combination therapy in cases where they might not be killed outright by deprivation alone.

Animals↗
PubMed2005

A new journal - "Theoretical Biology and Medical Modelling".

Biology has a conceptual basis that allows one to build models and theorize across many life sciences, including medicine and medically-related disciplines. A dearth of good venues for publication has been perceived during a period when bioinformatics, systems analysis and biomathematics are burgeoning. Steps have been taken to provide the sort of journal with a quick turnaround time for manuscripts which is online and freely accessible to all readers, whatever their persuasion or discipline. We have now been running for some time a journal which has had many good papers presented pre-launch, and a steady stream of papers thereafter. The value of this journal as a new venue has already been vindicated. Within a short space of time, we have founded a state-of-the-art electronic journal freely accessible to all in a much sort-after interdisciplinary field that will be of benefit to the thinking life scientist, which must include medically qualified doctors as well as scientists who prefer to build their new hypotheses on basic principles and sound concepts underpinning biology. At the same time, these principles are not sacrosanct and require critical analysis. The journal http://www.tbiomed.com promises to deliver many exciting ideas in the future.

Computational Biology↗
PubMed2005

Localization of transient receptor potential ion channels in primary and motile cilia of the female murine reproductive organs.

We have examined the subcellular localization of transient receptor potential (TRP) ion channels and the potential sensory role of cilia in murine female reproductive organs using confocal laser scanning microscopy analysis on ovary and oviduct tissue sections as well as on primary cultures of follicular granulosa cells. We show that the Ca2+ permeable cation channel, polycystin-2, as well as polycystin-1, a receptor that forms a functional protein complex with polycystin 2, distinctively localize to primary cilia emerging from granulosa cells of antral follicles in vivo and in vitro. Both polycystins are localized to motile oviduct cilia and this localization is greatly increased upon ovulatory gonadotropic stimulation. Further, the Ca2+ permeable cation channel, TRP vaniloid 4 (TRPV4), localizes to a sub-population of motile cilia on the epithelial cells of the ampulla and isthmus with high intensity in proximal invaginations of the epithelial folds. These observations are the first to demonstrate ciliary localization of TRP ion channels and their possible receptor function in the female reproductive organs. We suggest that polycystins 1 and 2 play an important role in granulosa cell differentiation and in development and maturation of ovarian follicles. In the oviduct both TRPV4 and polycystins could be important in relaying physiochemical changes in the oviduct upon ovulation.

Animals↗
PubMed2005

Landmarks in the first hundred years of primary (9+0) cilium research.

Primary cilia have had a long research history since 1898, but only in the last few years have investigations become intense. Only a few people took an interest in them until recently, when the consequences of their agenesis or dysfunction suddenly became apparent to all. Their function changed overnight from being speculative to being fully "acknowledged" as sensory, and the consequence of their lacking sensory function(s) led to a bonanza for cell and molecular biologists interested in the impact on clinical disease and disorder. Furthermore, once the medical profession had insight not just into the etiology of an extremely rare syndrome (situs inversus), but into one as prevalent as polycystic kidney disease, furious research activity sprang up in numerous centres, symposium were convened, special sessions arranged, and topical reviews written in all sorts of journals. Without an understanding of the history of research about this organelle, many reviews gave little credit to those who had persevered earlier to make key advances in primary cilium research, and most ignore the wealth of information on them that has relevance to fundamental (cell) biology as well as to medicine. Some of the "landmarks" presented here will hopefully provide a little guidance and a better perspective.

Biology↗
PubMed2005

Arginine deprivation and metabolomics: important aspects of intermediary metabolism in relation to the differential sensitivity of normal and tumour cells.

Arginine deprivation causes many types of tumour cells to die, often because they cannot recover or convert urea cycle intermediates into arginine. The powerful homeostatic mechanisms that kicks in to restore arginine levels in vivo are lacking in vitro, where there is no supply of citrulline. Comparison between cells deprived of arginine by direct elimination methods or indirectly via arginine degrading enzymes should show differences depending on their ability to handle alternative intermediates (ornithine, citrulline and argininosuccinate) of the urea cycle. The internal state of cells that can, versus those that cannot, use intermediates will metabolically be quite different. These differences should provide clear indicators regarding the sensitivity (susceptibility) of cells to arginine deprivation, from which we will be in a much better position to judge which tumours to treat, and possibly how to design the best treatment to eliminate them.

Arginine↗
PubMed2004

Metabolic scaling: consensus or controversy?

BACKGROUND: The relationship between body mass (M) and standard metabolic rate (B) among living organisms remains controversial, though it is widely accepted that in many cases B is approximately proportional to the three-quarters power of M. RESULTS: The biological significance of the straight-line plots obtained over wide ranges of species when B is plotted against log M remains a matter of debate. In this article we review the values ascribed to the gradients of such graphs (typically 0.75, according to the majority view), and we assess various attempts to explain the allometric power-law phenomenon, placing emphasis on the most recent publications. CONCLUSION: Although many of the models that have been advanced have significant attractions, none can be accepted without serious reservations, and the possibility that no one model can fit all cases has to be more seriously entertained.

Animals↗
PubMed2004

Arginine deiminase enhances dexamethasone-induced cytotoxicity in human T-lymphoblastic leukemia CCRF-CEM cells.

Since arginine deiminase (ADI; EC 3.5.3.6) inhibits cell proliferation by arresting cells in the G1 phase, we tested its synergistic effect on cell death induced by dexamethasone (DEX), which also induces apoptosis by G1 cell cycle arrest. ADI inhibited cell proliferation and induced apoptosis in human leukemic CEM cells in a dose-dependent manner. Simultaneous treatment with ADI and DEX showed synergistic effects on DNA fragmentation and LDH release. In addition, ADI exerted its anti-proliferative activity against DEX-resistant CEM cells. ADI suppressed expression of c-myc, a potential key regulator of cell proliferation and apoptosis, and increased expression of p27Kip1 cyclin-dependent kinase inhibitor. These results suggest that ADI efficiently increases the anti-cancer effect of DEX on human leukemic CEM cells through G1 cell cycle arrest involving downregulation of c-myc and upregulation of p27Kip1.

Antineoplastic Agents, Hormonal↗
PubMed2004

Controlling cancer by restricting arginine availability--arginine-catabolizing enzymes as anticancer agents.

Interest has recently been revived in enzymes that degrade essential amino acids. Arginine-catabolizing enzymes now predominate and are discussed in this review. Apart from reducing tumor load through cell death occurring as a result of deprivation alone, these catabolic enzymes conveniently leave the remaining malignant cells vulnerable to other therapeutic modalities through combinatorial treatments with cycle-dependent drugs, the timing of additional treatment after deprivation being crucial.

Animals↗
PubMed2004

Recovery of the Decorin-Enriched Fraction, Extract (D), From Human Skin: An Accelerated Protocol.

The original extraction procedure of Engel and Catchpole [1] has often been used to recover decorin-enriched material from the skin. This material has a strong inhibitory effect on fibroblast proliferation, and clearly suppresses it in skin except after the first 5-6 days of wounding when new scaffold material is required. The aim of our present study has been to find and evaluate the product of a faster recovery method, and to check its consistency as a more reliable means of regularly obtaining sufficient material for topical application in wounds that might become hypertrophic. Modifications of the original Toole and Lowther [2] extraction procedure have been carefully evaluated in an attempt to cut preparation time without compromising biological activity of the inhibitory extract. We have devised a faster recovery procedure without compromising biological activity, even if initial recovery has been somewhat reduced. The latter problem could be offset by repeated cycles of the final extraction step. The main inhibitory activity is shown to be within the decorin-enriched "extract D," as the core protein and DSPG II. Adjustment of the extract towards neutrality after dialysis against water keeps most of the extracted protein in solution and yielded a decorin-enriched preparation that had a specific activity equivalent to that of the old method. It also yielded a fraction that was readily lyophilised to give a small amount of material that could be stored indefinitely without loss of activity and readily redissolved in aqueous solution. A reliable and relatively quick method is presented for the production, from human skin, of a decorin-enriched preparation that has strong fibroblast inhibitory action. The value of the procedure is that it is inexpensive and can produce the quantities that might be used topically in reducing hypertrophic scarring of wounds.

Journal Article↗
PubMed2003

Polycystic kidney disease--the ciliary connection.

CONTEXT: "Cystic degeneration" of the kidneys was first described pathologically in 1841 and "polycystic kidneys" as a clinical syndrome in 1888. The heritable nature in some families was noted in 1899, and autosomal dominant and recessive patterns of inheritance of polycystic kidney disease (PKD) were later recognised. Autosomal dominant PKD is one of the most common human genetic diseases and results from mutations in PKD1 or PKD2. These genes encode two proteins, polycystin-1 and polycystin-2. STARTING POINT: Primary cilia are cellular organelles previously thought by some to be vestigial. New findings from several species, including algae, nematodes, and mice, implicate defects in structure or function of primary cilia as a possible common mechanism central to the development of some forms of recessive PKD. Two recent reports propose a causal link between ciliary dysfunction and autosomal dominant PKD. B Yoder and colleagues (J Am Soc Nephrol 2002; 13:2508-16) show that polycystin-1 and polycystin-2 are localised to primary cilia in cultured renal epithelial cells. S Nauli and colleagues (Nat Genet 2003; 33:129-37) show that polycystin-1 and polycystin-2 function as flow-sensitive mechanosensors in the same signal-transduction pathway. WHERE NEXT? Cystic epithelial cells show many altered cellular properties, including changes in proliferation, apoptosis, adhesion, differentiation, polarity, extracellular matrix synthesis, and fluid transport. The next important steps in PKD research will be to define the physiological roles of primary renal cilia and how defects in ciliary structure and function lead to the development of a cystic phenotype in different forms of PKD.

Animals↗
PubMed2003

Stochastic description of the ligand-receptor interaction of biologically active substances at extremely low doses.

Signalling molecules can be effective at extraordinarily low concentrations (down to attomolar levels). To handle such cases, probabilistic methods have been used to describe the formal kinetics of action of biologically active substances in these low doses, although it has been necessary to review what is meant by such a term. The mean numbers of transformed/degraded molecules and their dispersions were calculated for the possible range of ligand-receptor binding schemes. We used both analytical equations and numerical simulations to calculate the coefficients of variation (ratio of standard deviation to mean) and demonstrated that the distribution of the coefficient is highly dependent on the reaction scheme. It may, therefore, be used as an additional factor for discriminating between cooperative and noncooperative models of ligand-receptor interaction over extreme ranges of ligand dilution. The relevance to signalling behaviour is discussed.

Animals↗
PubMed2003

Diffusion, perfusion and the exclusion principles in the structural and functional organization of the living cell: reappraisal of the properties of the 'ground substance'.

The thesis is presented that only within very small microdomains of the cell internum might diffusion operate in the sorting of molecular affinities. Much of cell metabolism is guided and controlled in rate by the speed with which molecules that have to interact encounter one another. What is clear, however, is that the cell does not have a single 'modus operandi' but has the choice of many different strategies, each of which can contribute in different proportion to the rate of ongoing activity. It is probably our own desire to simplify things and use the most (or more) probable strategy that confines our appreciation of the overall robustness of the cell as a 'survival machine'. The main operative process at any given time (perfusion, diffusion or whatever) has always to be considered very carefully in relation to the organisational structure of the cell, which can be transient and fickle but nevertheless has been seen as involving an extensive cytomatrix, a ground substance, within an aqueous environment in which the degree of water structuring is even more fickle.

Biological Transport↗
PubMed2003

Arginine deprivation and tumour cell death: arginase and its inhibition.

Arginase treatment of cell cultures reduced arginine in the medium to approximately micromolar levels within 5-30 min, and proved as effective as arginine-free medium (AFM) prepared by formulation. The enzyme was heat stable and as active at pH 7.2 as at pH 9.9. It persisted in culture for at least 3 days with only a small diminution in its speed of action, and still actively destroyed arginine after 6 days, since arginine supplementation failed to rescue viable cells. Addition of L-norvaline, an inhibitor of arginase, rescued cells from arginase-induced deprivation. Its efficacy at low concentrations was short-lived (probably < 1 day), while at higher concentrations it did not appear to inhibit completely the enzyme. However, L-norvaline at these same levels also slowed the growth of positive non-enzyme treated controls receiving the normal arginine level. Thus the difference in this growth indicated that arginase was more inhibitory than cursory examination of initial kinetic data suggested. It also agreed with the inhibition of arginase in the ornithine assay used to measure biochemically enzyme activity. We conclude that norvaline partially but not completely antagonises arginase activity, which allows cell rescue in a dose-dependent manner between 0.4 and 4 mM, but cannot be used above about 2 mM without exhibiting a general non-specific interference of cell growth of its own, although no evidence of cell toxicity was observed in either AFM or arginine-containing medium. L-ornithine, the product of arginase that inhibits the enzyme by a feedback mechanism, had no inhibitory effect on arginase over a similar concentration range.

Animals↗
PubMed2002

Possible attenuation of the G2 DNA damage cell cycle checkpoint in HeLa cells by extremely low frequency (ELF) electromagnetic fields.

BACKGROUND: The issue remains unresolved as to whether low frequency magnetic fields can affect cell behaviour, with the possibility that they may be in part responsible for the increased incidence of leukaemia in parts of the population exposed to them. METHODS: Combined treatment of HeLa cells with gamma-irradiation (1, 3 and 5 Grays) and extra low frequency magnetic fields of ~50 Hz was carried out under rigorously controlled conditions. RESULTS: Synchronised cells progressing from S-phase arrived at mitosis on average marginally ahead of irradiation controls not exposed to ELF. In no instance out of a total of twenty separate experiments did this "double-insult" further delay entry of cells into mitosis, as had been anticipated. CONCLUSION: This apparently "non-genotoxic" agent (ELF) appears to be capable of affecting cells that would normally arrest for longer in G2, suggesting a weakening of the stringency of the late cycle (G2) checkpoint.

Journal Article↗