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Neuropathy is one of the most important complications of diabetes. According to recent advances, vitamin D deficiency might play a role in the development and progression of diabetic neuropathy. Moreover, therapeutic vitamin D supplementation has the potential to improve this condition. The aim of the present review was to summarize new data available in this area.The PubMed database was searched for articles written in English and published through September 2021, using combinations of the following key words: vitamin D, diabetes, diabetes mellitus, diabetic neuropathy, polyneuropathy, peripheral neuropathy, cardiac autonomic neuropathy, supplementation, and therapy.A number of studies have suggested that vitamin D deficiency can play a significant role in the development of peripheral neuropathy, diabetic foot ulcers, as well as cardiovascular autonomic neuropathy in patients with type 2 diabetes. Vitamin D supplementation might serve as an effective adjuvant therapy for neuropathic pain and may slow or stop the progression of neural damage.Vitamin D therapy for diabetic complications could be a reliable option; however, further studies are needed to confirm this notion.

Experiments and models were used to determine the extent to which aqueous bromine permeated into, and reacted with, the elastomer polydimethylsiloxane (PDMS). Thin films of PDMS were immersed in bromine water, and the absorbance of bromine in the aqueous phase was measured as a function of time. Kinetics were studied as a function of mass and thickness of the immersed PDMS films. We attribute the decrease of bromine in solution to permeation into PDMS, followed by a combination of diffusion, reversible binding, and an irreversible reaction with PDMS. In order to decouple the irreversible reaction from the reversible processes, kinetics were also studied for bromine-passivated PDMS films. Fits of the models to a variety of experiments yielded the partition coefficient of bromine between the water and PDMS phases, the diffusion constant of bromine in PDMS, the irreversible reaction constant between bromine and PDMS, the molar concentration of the reactive sites within PDMS, and the on and off rates of reversible binding of bromine to PDMS. Developing a quantitative reaction-diffusion model accounting for the transport of bromine through PDMS is necessary for the design of microfluidic devices fabricated using PDMS, which are used in experimental studies of the nonlinear dynamics of reaction-diffusion networks containing Belousov-Zhabotinsky chemical oscillators.

Inhibitory perturbations to the ferroin-catalyzed Belousov-Zhabotinsky (BZ) chemical oscillator operated in a continuously fed stirred tank reactor cause long term changes to the limit cycle: the lengths of the cycles subsequent to the perturbation are longer than that of the unperturbed cycle, and the unperturbed limit cycle is recovered only after several cycles. The frequency of the BZ reaction strongly depends on the acid concentration of the medium. By adding strong acid or base to the perturbing solutions, the magnitude and the direction of the frequency changes concomitant to excitatory or inhibitory perturbations can be controlled independently of the coupling strength. The dynamics of two BZ oscillators coupled through perturbations carrying a coupling agent (activator or inhibitor) and a frequency modulator (strong acid or base) was explored using a numerical model of the system. Here, we report new complex temporal patterns: higher order, partially synchronized modes that develop when inhibitory coupling is combined with positive frequency modulation (FM), and complex bursting patterns when excitatory coupling is combined with negative FM. The role of time delay between the peak and perturbation (the analog of synaptic delays in networks of neurons) has also been studied. The complex patterns found under inhibitory coupling and positive FM vanish when the delay is significant, whereas a sufficiently long time delay is required for the complex temporal dynamics to occur when coupling is excitatory and FM is negative.

We demonstrate that the dynamical behavior of strongly pulse-coupled Belousov-Zhabotinsky oscillators can be reproduced and predicted using a model that treats both the phase and the instantaneous frequency of the oscillators. Model parameters are extracted from the experimental data obtained using a single pulse-perturbed oscillator and are used to simulate the temporal dynamics of a system of two coupled oscillators. Our model exhibits the out-of-phase and anti-phase synchronization and the 1:N and N:M temporal patterns as well as the oscillator suppression that are observed in experiments when the inhibitory coupling is asymmetric. This approach may be adapted to other systems, such as coupled neurons, where the oscillatory dynamics is affected by strong pulses.

Existing models of the ferrocyanide-iodate-sulfite (FIS) reaction seek to replicate the oscillatory pH behavior that occurs in open systems. These models exhibit significant differences in the amplitudes and waveforms of the concentration oscillations of such intermediates as I(-), I3(-), and Fe(CN)6(3-) under identical conditions and do not include several experimentally found intermediates. Here we report measurements of sulfite concentrations during an oscillatory cycle. Knowing the correct concentration of sulfite over the course of a period is important because sulfite is the main component that determines the buffer capacity, the pH extrema, and the amount of oxidizer (iodate) required for the transition to low pH. On the basis of this new result and recent experimental findings on the rate laws and intermediates of component processes taken from the literature, we propose a mass action kinetics model that attempts to faithfully represent the chemistry of the FIS reaction. This new comprehensive mechanism reproduces the pH oscillations and the periodic behavior in [Fe(CN)6(3-)], [I3(-)], [I(-)], and [SO3(2-)]T with characteristics similar to those seen in experiments in both CSTR and semibatch arrangements. The parameter ranges at which stationary and oscillatory behavior is exhibited also show good agreement with those of the experiments.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can selectively trigger apoptosis in various cancer cell types. However, many cancer cells are resistant to death receptor-mediated apoptosis. Combination therapy with platinum complexes may affect TRAIL-induced signaling via modulation of various steps in apoptotic pathways. Here, we show that cisplatin or a more potent platinum(IV) complex LA-12 used in 20-fold lower concentration enhanced killing effects of TRAIL in human colon and prostate cancer cell lines via stimulation of caspase activity and overall apoptosis. Both platinum complexes increased DR5 surface expression in colon cancer cells. Small interfering RNA-mediated DR5 silencing rescued cells from sensitizing effects of platinum drugs on TRAIL-induced caspase-8 activation and apoptosis, showing the functional importance of DR5 in the effects observed. In addition, both cisplatin and LA-12 triggered the relocalization of DR4 and DR5 receptors to lipid rafts and accelerated internalization of TRAIL, which may also affect TRAIL signaling. Collectively, modulations of the initial steps of the extrinsic apoptotic pathway at the level of DR5 and plasma membrane are important for sensitization of colon and prostate cancer cells to TRAIL-induced apoptosis mediated by LA-12 and cisplatin.

In our previous study we have proved that colon cancer cells HT-29 pre-treated with specific 5-lipoxygenase inhibitor MK-886 became more susceptible to photodynamic therapy (PDT) with hypericin and we also found that this mutual combination induced cell cycle arrest and stimulated onset of apoptosis (Kleban et al., 2007. J. Photochem. Photobiol. B 84, 2). To further explain events associated with MK-886 mediated sensitization of tumor cells toward PDT with hypericin, more detailed study of signaling pathways leading to increase in apoptosis as well as cell cycle perturbations was performed and is presented herein. Intensive accumulation of HT-29 cells in G0/G1 phase of cell cycle led to expression analyses of several G0/G1 checkpoint molecules (cyclin A, cyclin E, cdk-2, pRb). Similarly, accumulation of apoptotic cells invoked analyses of key molecules involved in apoptotic signaling (caspase-3, -8, -9; PARP; Lamin B; Mcl-1; Bax) by Western blotting and caspase activity assay. Long term survival of cells was examined by clonogenicity test. As the effect of PDT is mediated by ROS production, levels of hydrogen peroxides and superoxide anion were monitored by flow cytometric analyses. In addition, an impact of MK-886 on LTB4 production and expression of 5-LOX was monitored. Massive G0/G1 arrest in the cell cycle accompanied by increase in cyclin E level and decrease/absention of cyclin A, cdk-2 and pRb expression indicated incapability for G1/S transition. Minimal changes in cleavage of procaspases observed in cells treated with non-toxic concentrations of either agent alone or their mutual combination were not quite in line with their activity (caspase-3, -8, -9) which was significantly increased mainly in combinations. Treatment with non-toxic concentration of MK-886 had minimal influence over ROS production compared to control cells. In contrast, hypericin alone markedly increased the level of ROS, but no additional effect of MK-886 pre-treatment was detected. Further analyses of particular ROS groups unveiled an impact of increasing MK-886 concentration on superoxide accumulation accompanied with depletion of hydrogen peroxide level within the cells. The clonogenicity test revealed disruption of colony formation after mutual combination of both agents as compared to MK-886 or PDT alone. In conclusion, we presume that stimulation of apoptosis in our experimental model was accomplished preferentially through the mitochondrial pathway, although caspase-8 activation was also noticed. Interestingly, pre-treatment with MK-886 modulated distribution of ROS production in mutual combination with PDT.

Objectives: This article is to study the role of G1/S regulators in differentiation of pluripotent embryonic cells. Materials and methods: We established a P19 embryonal carcinoma cell-based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G1/S regulators were analysed with respect to growth and differentiation parameters of the cells. Results and Conclusions: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E–CDK2 but not of cyclin D–CDK4/6–p27 complexes. In an exponentially growing P19 cell population, the cyclin D1–CDK4 complex is detected, which is replaced by cyclin D2/3–CDK4/6–p27 complex following density arrest. During endodermal differentiation kinase-inactive cyclin D2/D3–CDK4–p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2–CDK4–p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D–CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G1/S transition-regulating machinery in early embryonic cells.

The BrO3− — BrAc — Ru(bpy)32+ subsystem is shown to represent the core oscillator that serves as source of the long lasting temporal and spatial periodic behaviors observed in the BrO3− — H2PO2− — acetone — Mn2+ — Ru(bpy)32+ — acid “double substrate-double catalyst” oscillatory batch system. The BrAc — the substrate of the core oscillator — is formed and accumulated in the reactions taking place in the six-component system. BrAc was produced in a separate experiment with bromide, acetone, acid and excess bromate and the mixture was used for bringing about patterns in the thin solution layer after adding the Ru(bpy)32+ catalyst. The two-dimensional reaction-diffusion patterns that appear in the subsystem and its parent system are very similar in wave speed, wavelength, color and in the duration of the pattern evolution, therefore a common chemical origin is supposed to exist in their formation. The role that the BrAc may play in the mechanism of the BrO3− — reductant — acetone — catalyst type oscillators (∼ 30 variants) is also pointed out.

c-Jun is one of the major components of the activating protein-1 (AP-1), the transcription factor that participates in regulation of proliferation, differentiation, and apoptosis. In this study, we explored functional interactions of the c-Jun protein with several regulators of the G1/S transition in serum-deprived v-myb-transformed chicken monoblasts BM2. We show that the c-Jun protein induces expression of cyclin A, thus up-regulating activity of cyclin A-associated cyclin-dependent kinase 2 (CDK2), and causing massive programmed cell death of starved BM2cJUN cells. Specific inhibition of CDK2 suppresses frequency of apoptosis of BM2cJUN cells. We conclude that up-regulation of cyclin A expression and CDK2 activity can represent important link between the c-Jun protein, cell cycle machinery, and programmed cell death pathway in leukemic cells.

Coupled chemical oscillators are usually studied with symmetric coupling, either between identical oscillators or between oscillators whose frequencies differ. Asymmetric connectivity is important in neuroscience, where synaptic strength inequality in neural networks commonly occurs. While the properties of the individual oscillators in some coupled chemical systems may be readily changed, enforcing inequality between the connection strengths in a reciprocal coupling is more challenging. We recently demonstrated a novel way of coupling chemical oscillators, which allows for manipulation of individual connection strengths. Here we study two identical, pulse-coupled Belousov–Zhabotinsky (BZ) oscillators with unequal connection strengths. When the pulse perturbations contain KBr (inhibitor), this system exhibits simple out-of-phase and complex oscillations, oscillatory-suppressed states as well as temporally periodic patterns (N : M) in which the two oscillators exhibit different numbers of peaks per cycle. The N : M patterns emerge due to the long-term effect of the inhibitory pulse-perturbations, a feature that has not been considered in earlier works. Time delay was previously shown to have a profound effect on the system’s behaviour when pulse coupling was inhibitory and the coupling strengths were equal. When the coupling is asymmetric, however, delay produces no qualitative change in behaviour, though the 1 : 2 temporal pattern becomes more robust. Asymmetry in instantaneous excitatory coupling via AgNO3 injection produces a previously unseen temporal pattern (1 : N patterns starting with a double peak) with time delay and high [AgNO3]. Numerical simulations of the behaviour agree well with theoretical predictions in asymmetrical pulse-coupled systems.

It may be hypothesized that the lipoxygenase (LOX) metabolic pathway plays an important role in photodynamic therapy (PDT) of malignant tumours, and modification of this pathway may result in administration of lower doses of photodynamic active agents accompanied by reduced side effects. In this study, we examine in more detail the cytokinetic parameters of human colon adenocarcinoma HT-29 cells pre-treated for 48 or 24h with LOX inhibitor MK-886, followed by PDT induced by hypericin. Based on MTT assay the concentrations of both agents (MK-886 and hypericin) with relatively slight (non-significant) cytotoxic effects were selected. These concentrations were used for combined treatment, where MTT response, total cell number, floating cells quantification, viability, cell cycle progression and DNA synthesis were detected. Hoechst/PI staining, PARP fragmentation and mitochondrial membrane potential (MMP) were evaluated to determine the extent of apoptosis. While MK-886 alone caused mainly necrosis, 48h pre-treatment of cells with MK-886 followed by PDT with hypericin clearly shifted the type of cell death to apoptosis. PDT with hypericin alone caused apoptosis in 19% of the cell population. Some combined modalities significantly potentiated the apoptotic effect (31% of apoptotic cells; 2.5microM MK-886/0.1microM hypericin), i.e., by 60% more than after single treatment with hypericin. Increased apoptosis was confirmed by PARP (116kDa) cleavage to characteristic 89kDa fragments and changes in MMP. Increasing concentration of MK-886 was accompanied by massive changes in the cell cycle progression. Combined treatment with lower concentrations of MK-886 and hypericin increased accumulation of cells in the S phase, accompanied by inhibition of DNA synthesis. Increasing concentration of MK-886 in this combination caused the opposite effect, manifesting significant accumulation of cells in the G0/G1 phase. More pronounced effects were observed after the 48h pre-treatment schedule. This anti-proliferative effect was confirmed by BrdU incorporation. These results indicate that combined treatment involving PDT and LOX inhibitor MK-886 may improve the therapeutic effectiveness of PDT.

Objectives. The resistance of tumor cells to cisplatin remains a major cause of treatment failure in cancer patients. In this study, the ability of Pt(IV) complex with adamantylamine-LA-12 and its reduced counterpart with lower oxidation state Pt(II)-LA-9 to overcome intrinsic cisplatin resistance was investigated. Methods. The ovarian adenocarcinoma SK-OV-3 cells were exposed to cisplatin, LA-9, or LA-12 for 72 h and the effects of drug concentrations that caused 10% or 50% inhibition of cell proliferation were determined. After 24–72 h of sustained exposure viability, apoptosis and inhibition of proliferation were analyzed. DNA synthesis and cell cycle analysis were performed simultaneously in order to determine the modulation of cell cycle after platinum complexes treatment. Results. Lung Resistance-related Protein (LRP/MVP) was detected in SK-OV-3 cells but not in the other two ovarian cancer lines with different sensitivity to cisplatin. LRP/MVP overexpression may be an important factor contributing to intrinsic cisplatin resistance. Interestingly, Pt(IV) complex-LA-12 had approximately 2.7-fold lower IC 50 concentration than LA-9 or cisplatin in SK-OV-3 cells. Moreover, LA-12 caused persistent accumulation of cells in S-phase of the cell cycle while LA-9 and cisplatin treatment-induced S-phase arrest was transient and shifted to G 2 /M-phase at later intervals. Apoptosis seemed to be not the dominant type of cell death caused by such the derivatives, but it was the most intensive after LA-12 treatment. Conclusions. We found strong differences between effects of Pt(IV) complex-LA-12 and Pt(II) derivatives-LA-9 and cisplatin on cytokinetic parameters. Overall, LA-12 but not its reduced Pt(II) counterpart LA-9 is the compound effective in p53 null human ovarian cancer cells and it is able to overcome intrinsic cisplatin resistance in these cells.

The bromate-hypophosphite-acetone-Mn(II)-Ru(bpy)(3)(2+) batch oscillator was recently suggested for studying two-dimensional pattern formation. The system meets all major requirements that are needed for generation of good quality traveling waves in a thin solution layer. The serious drawback of using the system for studying temporal and spatial dynamical phenomena is its unknown chemical mechanism. In order to develop a mechanism that explains the observed long-lasting batch oscillations the bromate-hypophosphite-acetone-Mn(II)-Ru(bpy)(3)(2+) oscillator was revisited. We studied the dynamics both in the total system and in some composite reactions, and kinetic measurements were carried out in three subsystems. From the new experimental results we concluded that the two oscillatory sequences observed in the full system are originated from two oscillatory subsystems, the Mn(II)-catalyzed bromate-hypophosphite-acetone and the Ru(bpy)(3)(2+)-catalyzed bromate-bromoacetone reactions. Here we propose a mechanism which is capable of simulating the dynamical features that appeared in the complex system.

[(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], coded as LA-12, is an octahedral platinum(IV) complex containing a bulky hydrophobic ligand - adamantylamine. The use of bulky hydrophobic amines as non-leaving ligands, may increase uptake of the compound by the cancer cells. Therefore, the effects of LA-12 on sensitive (A2780) and cisplatin resistant (A2780cis) ovarian cancer cell lines were investigated and compared to those of cisplatin. IC(50) and IC(90) concentrations of LA-12 were 6- (A2780) or 18-fold (A2780cis) lower than those for cisplatin (MTT assay). Equitoxic concentrations (IC(50) or IC(90)) of both compounds caused a significant and similar time- and dose-dependent inhibition of cell proliferation and an increase in the number of floating cells which corresponded to the decrease of total cell viability. A different type and dynamics of cell cycle perturbation after cisplatin and LA-12 treatment were detected. Exposure to LA-12 resulted in transient accumulation of A2780 and A2780cis cells in S phase, while cisplatin caused G(2)/M arrest in sensitive and S phase arrest in resistant cells. A relatively low rate of apoptosis after exposure to IC(50) or IC(90) of both complexes was observed, markedly higher in resistant A2780cis cells. Western blot analysis indicated a concentration-dependent p53 level increase in both lines (higher after cisplatin treatment). PARP cleavage was observed only in A2780cis cells. In conclusion, LA-12 was found to be significantly more efficient than cisplatin, and it was able to overcome the acquired cisplatin resistance (showing resistance factor 2.84-fold lower than those for cisplatin). In spite of the low rate of apoptosis, LA-12 caused increase of p53 level and cell cycle perturbations in the ovarian cancer cell lines studied.

The aim of this study was to compare anti-tumor potency of platinum(IV) complexes with increasing hydrophobicity of their ligands. Cytotoxic potential of the new platinum(IV) complex, coded as LA-12 [(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], was compared within the series of complexes of the general formula (OC-6-43)-bis(acetato)(alkylamine)amminedichloroplatinum(IV). Alkylamine ligands with increasing hydrophobicity were: isopropylamine, cyclohexylamine, 1-adamantylamine and 3,5-dimethyl-1-adamantylamine. Particular platinum(IV) complexes were coded as LA-4, LA-2 (known as JM-216), LA-12 and LA-15, respectively. Cytotoxicity was tested with the microplate tetrazolium (MTT) assay on the panel of cancer cell lines and the results were verified by microscopy. HPLC was used to measure hydrophobicity, stability of complexes in various buffers and velocity constants for their reactivity with glutathione. Platinum(IV) complexes with bulky hydrophobic ligands (LA-12 and LA-15) demonstrated about one order higher velocity constant for pseudo-first-order reaction with glutathione in comparison to cisplatin, LA-4 and LA-2, whose velocity constants were close to those measured for cisplatin and related platinum(II) complexes. Cytotoxicities of LA-12 and LA-15 towards cisplatin-resistant epithelial carcinoma A2780/cisR were superior to cisplatin, LA-4 and LA-2 in both 24- and 72-h continuous exposure MTT tests. Rapid induction of apoptosis in the treated cancer cell lines and no cisplatin cross-resistance were found for LA-12, which is a candidate for clinical testing.

The dynamical behavior of the system comprising of the pH-dependent complex formation between histidine and Ni(II) ions coupled to the BrO3(-)-SO3(2-) pH oscillator was studied. The pH oscillator was demonstrated to be capable of forcing the pH-sensitive nickel ion-histidine equilibrium to alternate periodically between the unreacted and the fully complexed states. The periodic interconversions gave rise to an oscillatory distribution of the species that participate in the equilibrium and resulted in oscillations in the free [Ni(2+)], [NiHis(+)], and [Ni(His)2]. The preconditions of the successful coupling of metal ion-amino acid complexes to a primary pH oscillator are briefly discussed. Model calculations were performed to simulate the dynamics observed in the BrO3(-)-SO3(2-) - Ni(2+)-His CSTR system.

The neurohumoral modulation of the permeability increasing effect of histamine was studied in pigeon skin. Substances were administered through plasmapheresis capillaries inserted into the dorsal wing skin and the protein contents of the perfusates were determined by a quantitative method. The vascular labelling technique was also utilized to histologically identify leaky blood vessels. In the innervated skin histamine evoked a significant, dose-dependent plasma extravasation which was markedly augmented by the coadministration of a specific galanin receptor antagonist, galanin-1-16-bradykinin-2-9-amide (M35). Chronic cutaneous denervation per se resulted in a significant elevation of the permeability-enhancing effect of histamine. In the denervated skin this response was not affected by M35 but was significantly inhibited by galanin. It is concluded that in the normally innervated skin endogenous galanin may exert a neurogenic tonic inhibitory effect on histamine-induced plasma leakage. It is suggested that sensory nerves possess not only pro-inflammatory, but also anti-inflammatory (inhibitory) sensory-efferent functions.

Bioconvection occurs when upward swimming micro-organisms generate gravitational energy that initiates and maintains dissipative movement of the water in which they swim. Advection, and motion of the organisms relative to the fluid, generate patchiness in concentration that drives and shapes the geometry and rate of convection. This paper presents a method for quantitatively analyzing the development of self-organization, and numerical estimates that connect and interpret theory and experiment. While the oxygen consuming, oxgen-gradient-guided bacteria Bacillus subtilis are the sole subject here, the methods developed will find application to the analysis and modeling of other complex dynamic systems that ineluctably combine physics and biology. @S1063-651X~98!01210-0#

A new, rapid method is described which permits the genotyping of genetically modified animals from a microlitre volume of whole blood samples via one step polymerase chain reaction amplification. The major advantage of the presented method is the exclusion of a DNA preparation step, which significantly reduces the time expenditure and work load of the genetic testing. Pilot studies indicate, that this method is efficient and applicable also on tissue biopsies and larger amount of blood providing a rapid and reliable new technique over conventional genotyping approaches.

Finger on the pulse: in a system of two pulse-coupled Belousov-Zhabotinsky oscillators, introducing a time delay or increasing the coupling strength brings about novel dynamic features (see picture, the two oscillators are shown in different colors), such as reversal of the roles of excitatory and inhibitory coupling or fast anti-phase oscillation. These features are not observed in diffusively coupled systems, and shed light on how such pulse coupling occurs at synapses.

In situ hybridization, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis were applied to study the changes in expression of the major nociceptive ion channel transient receptor potential vanilloid type 1 receptor (TRPV1) after the perineural application of capsaicin or nerve transection. In control rats, quantitative morphometric and statistical analyses of TRPV1 protein and mRNA expression in L5 dorsal root ganglion cells revealed distinct populations of small (type C) and small-to-medium (type B) neurons, which showed very high and moderate levels of TRPV1, whereas larger (type A) neurons mostly did not express this receptor. After either transection or capsaicin treatment of the sciatic nerve, immunohistochemistry and Western blotting demonstrated a massive (up to 80%) decrease in the proportion of TRPV1-immunoreactive neurons and TRPV1 protein at all postoperative survival times. In situ hybridization indicated marked decreases (up to 85%) in the proportion of neurons that expressed TRPV1 mRNA after sciatic nerve transection. In contrast, although perineural treatment with capsaicin resulted in similar substantial decreases in the proportions of type B and C neurons of the L5 dorsal root ganglia 3 days postoperatively, a clear-cut tendency to recovery was observed thereafter. Hence, the proportions of both type B and C neurons expressing TRPV1 mRNA reached up to 70% of the control levels at 30 days postoperatively. In accord with these findings, quantitative RT-PCR revealed a marked and significant recovery in TRPV1 mRNA after perineural capsaicin but not after nerve transection. These observations suggest the involvement of distinct cellular mechanisms in the regulation of the TRPV1 mRNA expression of damaged neurons, specifically triggered by the nature of the injury. The present findings imply that the antinociceptive and anti-inflammatory effects of perineurally applied capsaicin involve distinct changes in neuronal TRPV1 mRNA expression and long-lasting alterations in (post)translational regulation.

All pH-oscillators reported to date function only under open (flow reactor) conditions. We describe an approach to generating pH-oscillations in a closed system by starting from an open system pH-oscillator, finding semibatch conditions under which it oscillates with an inflow of a single reactant to an otherwise closed reactor containing the remaining components, and replacing this inflow with a layer of silica gel impregnated with the key reactant. We present data showing the successful application of this technique to the BrO(3)(-)-Mn(2+)-SO(3)(2-), IO(3)(-)-Fe(CN)(6)(4-)-SO(3)(2-), and BrO(3)(-)-Fe(CN)(6)(4-)-SO(3)(2-) systems. In all three cases, sulfite ion is the species that is replenished via dissolution from the gel layer.

Experiments were carried out using a tensile testing machine to study the morphology of rupture lines in paper. Constant velocity strain was applied until the breaking process separated the sample into two pieces. The resulting quasi one-dimensional patterns show scaling correlations usually over two orders of magnitudes. The measured values of the Hurst exponent ζ are in the range 0.63 < ζ < 0.72. The relevance of models of statistical physics in the interpretation of the results is discussed.

LA-12 is a new platinum (IV) drug with promising cytotoxic effects in a wide range of cancer cell lines. Its confluence-dependent effects were compared with cisplatin (CDDP) and oxaliplatin (L-OHP) in HT-29 cells.Cytotoxicity was determined by MTT test, eosin exclusion assay, and cell number quantification. The cell cycle was analysed using propidium iodide DNA staining (flow cytometry), apoptosis by phosphatidylserine externalisation (annexin-V assay), mitochondrial membrane potential by flow cytometry, nuclear morphology by means of fluorescence microscopy, and PARP cleavage by Western blotting.While L-OHP and CDDP were practically inactive in the subconfluent cell population, LA-12 showed a similar toxicity in both subconfluent and growing populations. All compounds induced apoptosis, although with different potentials.LA-12 was able to overcome confluence-dependent resistance of HT-29 cells observed for other platinum compounds, which may have potential therapeutic use in slowly growing tumours.

Oscillations in the concentration of divalent ions Cd(2+), Ca(2+), Zn(2+), Co(2+) and Ni(2+) are induced by adding these species to the BrO(3)(-)-SO(3)(2-) chemical oscillator in a flow reactor. Producing periodic pulses in the concentrations of these non-redox ions extends our earlier approach to generating forced periodic behavior. Instead of driving pH-dependent equilibrium reactions of the target ion by a pH oscillator backward and forward, we now couple a redox core oscillating reaction to two consecutive reactions taking place between the components of the oscillator and the target element. In the systems examined here, the oscillatory reductant SO(3)(2-) binds the free metal ion in a MSO(3) precipitate, reducing its level to a minimal value when [SO(3)(2-)] is high, followed by release of the metal ion when the sulfite is oxidized by BrO(3)(-).

Chemosensitive afferent nerves expressing the capsaicin/TRPV1 (transient receptor potential vanilloid receptor-1) receptor are not only involved in the transmission of nociceptive impulses toward the central nervous system, but also play pivotal roles in the initiation and modulation of vascular, inflammatory, and immune reactions in a variety of organs, including the skin. These sensory nerves exert their efferent/local regulatory functions primarily via the release of vasoactive neuropeptides such as calcitonin gene-related peptide and substance P from their terminals upon antidromic or orthodromic stimulation. The chemical changes induced in the neural microenvironment by tissue injury or inflammation may promote the release of proinflammatory sensory neuropeptides and lead to augmentation of the inflammatory response. In turn, the release of anti-inflammatory peptides from sensory nerves or from inflammatory cells may result in an inhibition of cutaneous vascular reactions. The available experimental evidence implicates capsaicin-sensitive sensory nerves in the pathogenesis of certain skin diseases and maladies with cutaneous manifestations. The development of potent nonpeptide antagonists of sensory neuropeptides, the capsaicin/TRPV1 receptor, and the proteinase-activated receptors may offer new possibilities for the management of certain skin diseases and the itch and pain associated with altered sensory nerve functions.

The time evolution and geometry of rough interfaces observed in experiments on immiscible displacement of viscous fluids in porous media are analysed using the concepts of dynamic scaling and self-affine fractal geometry. The authors find that the development of the interface is governed by dynamic scaling and they have determined the corresponding surface exponents alpha and beta . The values of the exponents calculated for the experimental patterns are different from those obtained for a variety of two-dimensional models of marginally stable interfaces.

Sustained oscillations in the concentration of free fluoride ions can be generated when the BrO3--SO32--Mn(II) oscillator is coupled either to Al3+-F- complex formation or to the Ca2+-F- precipitation process in a flow reactor. By careful analysis of the relevant equilibria and optimization of the reactant concentrations, one can obtain [F-] oscillations of several orders of magnitude as measured with an ion-selective electrode. The BrO3--SO32--Mn(II)-Al(NO3)3-NaF system also exhibits bistability, that is, simultaneously stable steady states of high and low [F-].

In this paper the scaling behaviour of the surface of Escherichia coli and Bacillus subtilis colonies growing on agar plates are studied. We present the first experimental evidence for the self-affine fractal nature of the shape of a biological system. The value H ≃0.78 we obtained for the roughness exponent H describing the scaling of the surface width indicates that the development of the surface is subject to correlations due to the complexity of bacterial colony formation.

The authors introduce a new approach to the analysis of growing unstable interfaces in order to characterise the degree of their randomness. Highly ramified viscous fingering patterns are digitised and the related surface curvature data are evaluated using techniques common in the investigations of dynamical systems. Their results for the associated correlation dimension indicate that the experimentally observed deterministic patterns correspond to a spatial behaviour which is more complex than a low-dimensional chaotic geometry.

Adenosine has been observed to suppress the growth of MOLT-4 human leukemia cells in vitro. Changes in the cell cycle, especially increased percentage of cells in S phase, prolonged generation time, and induction of apoptosis at higher adenosine concentrations have been found to be responsible for the growth suppression. Dipyridamole, a drug inhibiting the cellular uptake of adenosine, reversed partially but significantly the adenosine-induced growth suppression. It follows from these results that the action of adenosine on the MOLT-4 cells comprises its cellular uptake and intracellular operation. These findings present new data on anticancer efficacy of adenosine.

One proposal to increase the efficiency of photodynamic therapy (PDT) is to accompany photosensitization with other treatment modalities, including modulation of arachidonic acid (AA) metabolism. The aim of this study was to evaluate the effectiveness of a combined modality approach employing 48 and 24 h pretreatment with various inhibitors of lipoxygenase (LOX; nordihydroguaiaretic acid, esculetin, AA-861, MK-886 and baicalein), cyclooxygenase (COX; diclofenac, flurbiprofen, ibuprofen, indomethacin, SC-560 and rofecoxib) and cytochrome P450-monooxygenase (proadifen) pathways, followed by hypericin-mediated PDT. Cytokinetic parameters like MTT assay, adherent and floating cell numbers, viability and cell cycle distribution analysis were examined 24 h after hypericin activation. Pretreatment of human colon cancer cells HT-29 prior to PDT with 5-LOX inhibitor MK-886 as well as 5, 12-LOX and 12-LOX inhibitors (esculetin and baicalein, respectively) resulted in significant and dose-dependent effects on all parameters tested. Pretreatment with diclofenac, flurbiprofen, ibuprofen and indomethacin, the nonspecific COX inhibitors, promoted hypericin-mediated PDT, but these effects were probably COX-independent. In contrast, application of SC-560 and rofecoxib, specific inhibitors of COX-1 and COX-2, respectively, attenuated PDT. Inhibition of P450 monooxygenase with proadifen implied also the significance of this metabolic pathway in cell survival and cell resistance to hypericin photocytotoxicity. In conclusion, our results testify that application of diverse inhibitors of AA metabolism may have different consequences on cellular response to hypericin-mediated PDT and that some of them could be considered for potentiation of PDT.

Photodynamic therapy (PDT) represents a new rapidly-developing anticancer approach based on administration of a non- or weakly-toxic photosensitizer and its activation with light of appropriate wavelength. Hypericin, one of the promising photosensitizers, is known to induce apoptosis with high efficiency in various cell line models. However, here we report the prevalence of necrosis accompanied by suppression of caspase-3 activation in colon adenocarcinoma HT-29 cells exposed to an extensive range of PDT doses evoked by variations in two variables -- hypericin concentration and light dose. Necrosis was the principal mode of cell death despite different PDT doses and the absence of anti-apoptotic Bcl-2 expression, even if the same condition induced caspase-3 activity at similar toxicity in HeLa cells. Introduction of Bcl-2 into HT-29 cells invoked caspase-3 activation, changed the Bcl-X(L) expression pattern, increased the apoptosis ratio with no effect on overall toxicity, and supported arrest in the G(2)/M-phase of cell cycle. Since it is known that Bcl-2 suppression in HT-29 is reversible and linked to the over-expression of mutated p53 and also considering our data, we suggest that the mutation in p53 and events linked to this feature may play a role in cell death signalling in HT-29 colon cancer cells.

Loss of programmed cell death pathways is one of the features of malignancy that complicate the response of cancer cells to a therapy. Activation of alternative cell death pathways offers a promising approach to enhance efficiency of cancer chemotherapy. We analysed programmed cell death pathways of v-myb-transformed BM2 monoblasts induced by arsenic trioxide, cycloheximide and camptothecin with U937 promonocytes as a reference cell line. We show that induced death of BM2 cells is not executed by caspases but rather by alternative cell death pathways. Camptothecin induces the lysosome-dependent cell death, arsenic trioxide induces autophagy, and most of cycloheximide-treated BM2 cells die by necrosis. The fact that alternative cell death pathways can be switched in cells with defects in activation and/or function of caspases suggests that understanding and targeting of these pathways could improve therapy of cancer cells suffering from defective apoptosis.

The p53 protein can control cell cycle progression, programmed cell death, and differentiation of many cell types. Ectopic expression of p53 can resume capability of cell cycle arrest, differentiation, and apoptosis in various leukemic cell lines. In this work, we expressed human p53 protein in v-Myb-transformed chicken monoblasts. We found that even this protein possessing only 53% amino acid homology to its avian counterpart can significantly alter morphology and physiology of these cells causing the G2-phase cell cycle arrest and early monocytic differentiation. Our results document that the species-specific differences of the p53 molecules, promoters/enhancers, and co-factors in avian and human cells do not interfere with differentiation- and cell cycle arrest promoting capabilites of the p53 tumor suppressor even in the presence of functional v-Myb oncoprotein. The p53-induced differentiation and cell cycle arrest of v-Myb-transformed monoblasts are not associated with apoptosis suggesting that the p53-driven pathways controlling apoptosis and differentiation/proliferation are independent.

Platinum (IV) derivative with adamantylamine-LA-12-represents a new generation of highly efficient anti-cancer drug derived from cisplatin and is currently in the final stage of phase I clinical trials. Understanding the specific mechanisms of its effects on cell cycle is necessary for defining the mode of action of LA-12. In this study, we characterized the ability of LA-12 to induce cell cycle perturbations in ovarian cancer cell line A2780 as compared to equitoxic cisplatin treatment. LA-12 induced a permanent accumulation of A2780 cells in S phase while cisplatin caused G2/M arrest at 24-h time point, where we also detected an increased expression of Gadd45alpha protein. Although both derivatives induced a rapid increase of p53 expression, this was not associated with a down-regulation of Mdm2 protein. Increased expression of p21(Cip1/WAF1) protein and its association with cyclins A and B1 suggested that this cyclin-dependent kinase inhibitor might contribute significantly to the observed perturbations of cell cycle. The results of this study provide insight into the mechanism of action of platinum-based derivative with adamantylamine on cell cycle in ovarian cancer cells. The differences between effects of LA-12 and cisplatin suggest that more attention should be paid to elucidation of modes of action of novel platinum(IV) complexes at cellular level.

Cultures of three-dimensional aggregates of embryonic stem cells (ESCs) called embryoid bodies (EBs) provide a valuable system for analyzing molecular mechanisms that regulate differentiation of this unique cell type. Cyclin-dependent kinase inhibitor p27Kip1 (p27) becomes elevated during the differentiation of mouse ESCs (mESCs). In this study, various aspects of differentiation of EBs produced from normal and p27-deficient mESCs were analyzed to address the biological significance of this elevation. It was found that EBs lacking p27 grew significantly bigger, but this was not accompanied by detect-able abnormalities in the activities of cyclin-dependent kinases (CDKs). In most EB cells, downregulation of activating cyclins rather than upregulation of inhibiting p27 is probably responsible for lowering the activity of their CDKs. Abnormalities in the development of specific cell lineages were also observed in p27-deficient EBs. These included elimination of cells positive for cytokeratin endo-A (TROMA-I) and increased proliferation and formation of cavities originating from cells positive for Lewis-X. Our data also suggest that although two different pools of Lewis-X-expressing cells, cluster forming (ESC-like) and cavity forming (neural progenitors), normally exist in EBs, the absence of p27 leads to the enhancement of only the neural pool. No failure was found when the neurogenic capacity of p27-deficient mESCs was tested using various protein markers. Together, our data point to a dual role of p27 in mESCs, with one role being in the regulation of proliferation and the other role in establishing some other aspects of a differentiated phenotype.

In mouse embryonic stem (mES) cells, the expression of p27 is elevated when differentiation is induced. Using mES cells lacking p27 we tested the importance of p27 for the regulation of three critical cellular processes: proliferation, differentiation, and apoptosis. Although cell cycle distribution, DNA synthesis, and the activity of key G1/S-regulating cyclin-dependent kinases remained unaltered in p27-deficient ES cells during retinoic acid-induced differentiation, the amounts of cyclin D2 and D3 in such cells were much lower compared with normal mES cells. The onset of differentiation induces apoptosis in p27-deficient cells, the extent of which can be reduced by artificially increasing the level of cyclin D3. We suggest that the role of p27 in at least some differentiation pathways of mES cells is to prevent apoptosis, and that it is not involved in slowing cell cycle progression. We also propose that the pro-survival function of p27 is realized via regulation of metabolism of D-type cyclin(s).

We report experimental results for the behavior of slow-combustion fronts in the presence of a columnar defect with excess or reduced driving, and compare them with those of mean-field theory. We also compare them with simulation results for an analogous problem of driven flow of particles with hard-core repulsion (ASEP) and a single defect bond with a different hopping probability. The difference in the shape of the front profiles for excess vs. reduced driving in the defect, clearly demonstrates the existence of a KPZ-type of nonlinear term in the effective evolution equation for the slow-combustion fronts. We also find that slow-combustion fronts display a faceted form for large enough excess driving, and that there is a corresponding increase then in the average front speed. This increase in the average front speed disappears at a non-zero excess driving in agreement with the simulated behavior of the ASEP model., 7 pages, 7 figures