Inhibition of ceruloplasmin and other copper oxidases by thiomolybdate

The dietary antagonism between copper and molybdate salts prompted a study of the inhibition of copper enzymes by thiomolybdate (TM). TM strongly inhibited the oxidase activity of five copper oxidase with I50% values in the 1-5 microM range. The mechanism of the TM effect on the copper oxidase, ceruloplasmin (Cp) (E.C. 1.16.3.1), was studied in detail. In Vmax vs. E plots, TM gave parallel data suggesting irreversibility but a large number of TM molecules per Cp were required. The inhibition of Cp by TM could not be reversed by dialysis. Isolation of TM-inhibited Cp on Sephadex G-10 did not yield any active Cp molecules. Cu(II) did not restore any inhibited oxidase activity. Gel electrophoresis supported the covalent binding of Cp by TM without any extensive change in protein structure. EPR results confirmed that Cu(II) is reduced to Cu(I) after reaction with TM. However, the Mo(VI) in MoS4(2-) did not change in oxidation number. Analysis of the TM-Cp compound accounted for all six Cu atoms as found in native Cp. The data suggest the covalent binding of sulfide to Cp copper. TM also inhibited the activity of ascorbate oxidase, cytochrome oxidase, superoxide dismutase, and tyrosinase. However, no inhibition of carbonic anhydrase, a zinc enzyme, was observed at 1 mM TM.     

Dissipative particle dynamics simulation of the soft micro actuator using polymer chain displacement in electro-osmotic flow

ABSTRACT

In this research, the numerical simulation of a soft polymer micro actuator performance has been investigated using the dissipative particle dynamics method in electro-osmotic flow. Effective factors including electro-osmotic flow and polymer chain parameters have been studied. First of all, considering a wide range of electro-osmotic parameters, the validation of analytical results is carried out in a simple micro channel. The electric field and zeta potential changes are linearly related to the flow rate, and the kh parameter behaves nonlinearly to around the kh?=?10. In the following, a convergent–divergent channel is used for the soft micro actuator simulation in which a polymer chain as a heart of actuation is embedded in the middle. As the main control parameter, the direction of the electric field is changed every 4?s, and it leads to a reciprocating motion. The numerical results indicate that the displacement of the soft polymer chain will be increased by enhancing the electric field, the number of beads, decreasing the harmonic bond coefficient and also exposing more length of a polymer chain in front of fluid flow. The results of this study may be useful for some future applications such as artificial fibres and muscles.     

Lipids on the move: Simulations of membrane pores, domains, stalks and curves

In this review we describe the state-of-the-art of computer simulation studies of lipid membranes. We focus on collective lipid-lipid and lipid-protein interactions that trigger deformations of the natural lamellar membrane state, showing that many important biological processes including self-aggregation of membrane components into domains, the formation of non-lamellar phases, and membrane poration and curving, are now amenable to detailed simulation studies.     

Space in systems biology of signaling pathways--towards intracellular molecular crowding in silico

How cells utilize intracellular spatial features to optimize their signaling characteristics is still not clearly understood. The physical distance between the cell-surface receptor and the gene expression machinery, fast reactions, and slow protein diffusion coefficients are some of the properties that contribute to their intricacy. This article reviews computational frameworks that can help biologists to elucidate the implications of space in signaling pathways. We argue that intracellular macromolecular crowding is an important modeling issue, and describe how recent simulation methods can reproduce this phenomenon in either implicit, semi-explicit or fully explicit representation.     

Role of labile iron in the toxicity of pharmacological ascorbate

Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At pharmacological concentrations the oxidation of ascorbate produces a high flux of H₂O₂ via the formation of ascorbate radical (Asc^•-). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H₂O₂ generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2 mM ascorbate decreased the flux of H₂O₂ generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe³⁺/Fe²⁺, have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo.     

A Neonate with Recurrent Vomiting and Generalized Hypotonia Diagnosed with a Deficiency of Dihydropyrimidine Dehydrogenase

Deficiency of dihydropyrimidine dehydrogenase (DPD) is a rare inborn error of pyrimidine metabolism. To date, only about 50 patients are known worldwide. The clinical picture is varied and is not yet fully described. Most patients are diagnosed at the age of 1–3 years. We present a patient diagnosed 8 weeks postpartum.

The female patient presented in the first 3 days after birth with agitation, choking, and vomiting. Six weeks later, the patient presented again with vomiting and insufficient weight gain. Metabolic screening of urine showed a strongly increased excretion of uracil and thymine, with no other abnormalities. This suggested a deficiency of DPD which was confirmed by enzyme analysis in peripheral blood mononucleair (PBM) cells (patient: activity <0.01 nmol/mg/h; controls: 9.9 ± 2.8 nmol/mg/h). The patient was homozygous for the IVS14+1G>A mutation.

MRI of the brain showed some cerebral atrophy; myelinization appeared normal. Many patients with DPD-deficiency suffer from convulsions and mental retardation, some show microcephaly, feeding difficulties, autism, and hypertonia. Our patient showed feeding difficulties and in the second half-year she developed slight motor retardation and generalized hypotonia. Further observation of the development of the patient may shed more light on the relationship between clinical symptoms and DPD deficiency. DPD deficiency may present in newborns with vomiting and hypotonia as the main symptoms.     

Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum

Zwf (code for glucose-6-phosphate dehydrogenase) and gnd (code for 6-phosphogluconate dehydrogenase) genes from Corynebacterium glutamicum were firstly cloned, and then site-directed mutagenesis was successfully introduced to remove allosteric inhibition by intracellular metabolites. Expression of the mutant zwf and gnd in Bacillus subtilis RH33 resulted in significant enhancement of riboflavin productivity, while the specific growth rate decreased slightly and the specific glucose uptake rate was unchanged. Introduction of the mutant zwf and gnd led to approximately 18% and 22% increased riboflavin production, respectively. An improvement by 31% and 39% of the riboflavin production was obtained by co-expression of the mutated dehydrogenases in shaker flask and fed-batch cultivation. Intracellular metabolites analysis indicated that metabolites detected in pentose phosphate pathway or riboflavin synthesis pathway of engineered strains showed higher concentration, while TCA cycle and glycolysis metabolites detected were lower abundance than that of parent strain.     

Simulation of Polymer Solutions by Dissipative Particle Dynamics

Abstract

Dissipative Particle Dynamics (DPD) is employed to model the dynamics and rheology of polymer solutions, and suspensions of spherical particles with adsorbed polymers. Static and dynamic scaling relationships for the variation of radius of gyration and relaxation time with polymer chain length are reviewed, demonstrating that the DPD polymer solution model correctly represents the effects of hydrodynamic interaction and excluded volume. Rheological simulations for both polymer solutions and polymer-sphere suspensions predict Newtonian viscosities at low shear rate followed by shear-thinning behavior as a reduced shear rate of unity is approached. Both the Newtonian viscosity and the extent of shear-thinning are greatly enhanced in the case of good solvents, compared to the viscosity curves for polymers and polymer-spheres structures dissolved in theta solvents and poor solvents.     

Targeting miR-21 enhances the sensitivity of human colon cancer HT-29 cells to chemoradiotherapy in vitro

5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for colorectal cancer treatment, but colorectal cancer cells are often resistant to primary or acquired 5-FU therapy. Several studies have shown that miR-21 is significantly elevated in colorectal cancer. This suggests that this miRNA might play a role in this resistance. In this study, we investigated this possibility and the possible mechanism underlying this role. We showed that forced expression of miR-21 significantly inhibited apoptosis, enhanced cell proliferation, invasion, and colony formation ability, promoted G1/S cell cycle transition and increased the resistance of tumor cells to 5-FU and X radiation in HT-29 colon cancer cells. Furthermore, knockdown of miR-21 reversed these effects on HT-29 cells and increased the sensitivity of HT-29/5-FU to 5-FU chemotherapy. Finally, we showed that miR-21 targeted the human mutS homolog2 (hMSH2), and indirectly regulated the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). These results demonstrate that miR-21 may play an important role in the 5-FU resistance of colon cancer cells.