The Promoting Effect of Radiation on Glucose Metabolism in Breast Cancer Cells under the Treatment of Cobalt Chloride.

We aimed to investigate the influence of radiation on hypoxia-treated breast cancers cells and its underlying mechanism. We mimicked the hypoxic response in MCF-7 cells by the treatment of CoCl2. Meanwhile, hypoxic MCF-7 cells induced by CoCl2 or untreated MCF-7 cells were treated with or without radiation, and then treated with or without hypoxia inducible factors-1α (HIF-1α) inhibitor. Subsequently, glucose update and lactate release rate were determined by commercial kits, as well as the expressions of HIF-1α and the glucose metabolic pathway related genes, including fructose biphoshatase 1 (FBP1), glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and isocitrate dehydrogenase 2 (IDH20) were detected by western blotting and/or RT-PCR. The results showed that glucose uptake rate and lactate release rate were increased in cells under hypoxia and/or radiation condition compared with untreated cells (p < 0.05), while the addition of HIF-1α inhibitor decreased these rates in hypoxia + radiation treated cells (p < 0.05). In addition, compared with untreated cells, the mRNA and protein levels of HIF-1α were significantly increased under hypoxia and radiation condition (p < 0.05), while which decreased after the addition of HIF-1α inhibitor (p < 0.05). Similar content changing trends (all p < 0.05) were observed in FBP1, IDH2, GLUT1, and LDHA but not HK2. In conclusion, the combination of radiation and hypoxia could promote the glucose metabolism. Furthermore, HIF-1α might inhibit the promoting effect of radiation on glycolysis in hypoxic MCF-7 cells by regulating the glucose metabolic pathway.

Physiological functions of beneficial elements.

Aluminum (Al), cobalt (Co), sodium (Na), selenium (Se), and silicon (Si) are considered beneficial elements for plants: they are not required by all plants but can promote plant growth and may be essential for particular taxa. These beneficial elements have been reported to enhance resistance to biotic stresses such as pathogens and herbivory, and to abiotic stresses such as drought, salinity, and nutrient toxicity or deficiency. The beneficial effects of low doses of Al, Co, Na and Se have received little attention compared to toxic effects that typically occur at higher concentrations. Better understanding of the effects of beneficial elements is important to improve crop productivity and enhance plant nutritional value for a growing world population.

Evidence for functional significance of the permuted C motif in Co2+-stimulated RNA-dependent RNA polymerase of infectious bursal disease virus.

Segment B of bisegmented infectious bursal disease virus (IBDV) encodes virus protein 1 (VP1), possessing RNA-dependent RNA polymerase (RdRp) activity. This multidomain protein includes an RdRp domain with a non-canonical order of three sequence motifs forming the active site: C-A-B. The A-B-C order of the motifs, as found in RdRps of the majority of viruses, was converted by relocation (permutation) of motif C to a C-A-B order. Due to the unusual location and unproven significance, the motif was named 'C?'. This motif includes an Ala-Asp-Asn tripeptide that replaces the C motif Gly-Asp-Asp sequence, widely considered a hallmark of RdRps. In this study, functional significance of the C? motif was investigated by using purified His-tagged VP1 mutants with either a double replacement (ADN to GDD) or two single-site mutants (ADD or GDN). All mutants showed a significant reduction of RdRp activity in vitro, in comparison to that of VP1. Only the least-affected GDN mutant gave rise to viable, albeit partially impaired, progeny using a reverse-genetics system. Experiments performed to investigate whether the C motif was implicated in the control of metal dependence revealed that, compared with Mn2+ and Mg2+, Co2+ stimulated RdRp unconventionally. No activity was observed in the presence of several divalent cations. Of two Co2+ salts with Cl- and SO4(2-) anions, the former was a stronger stimulant for RdRp. When cell-culture medium was supplemented with 50 microM Co2+, an increase in IBDV progeny yield was observed. The obtained results provide evidence that the unusual Co2+ dependence of the IBDV RdRp might be linked to the permuted organization of the motif.

Ionic effects on viral DNA packaging and portal motor function in bacteriophage phi 29.

In many viruses, DNA is confined at such high density that its bending rigidity and electrostatic self-repulsion present a strong energy barrier in viral assembly. Therefore, a powerful molecular motor is needed to package the DNA into the viral capsid. Here, we investigate the role of electrostatic repulsion on single DNA packaging dynamics in bacteriophage phi 29 via optical tweezers measurements. We show that ionic screening strongly affects the packing forces, confirming the importance of electrostatic repulsion. Separately, we find that ions affect the motor function. We separate these effects through constant force measurements and velocity versus load measurements at both low and high capsid filling. Regarding motor function, we find that eliminating free Mg(2+) blocks initiation of packaging. In contrast, Na(+) is not required, but it increases the motor velocity by up to 50% at low load. Regarding internal resistance, we find that the internal force was lowest when Mg(2+) was the dominant ion or with the addition of 1 mM Co(3+). Forces resisting DNA confinement were up to approximately 80% higher with Na(+) as the dominant counterion, and only approximately 90% of the genome length could be packaged in this condition. The observed trend of the packing forces is in accord with that predicted by DNA charge-screening theory. However, the forces are up to six times higher than predicted by models that assume coaxial spooling of the DNA and interaction potentials derived from DNA condensation experiments. The forces are also severalfold higher than ejection forces measured with bacteriophage lambda.

The RcnRA (YohLM) system of Escherichia coli: a connection between nickel, cobalt and iron homeostasis.

The transporter RcnA has previously been implicated in Ni(II) and Co(II) detoxification in E. coli probably through efflux. Here we demonstrate that the divergently described rcnA and rcnR gene products constitute a link between nickel, cobalt and iron homeostasis. Deletion of the rcnA gene resulted in increased cellular nickel, cobalt and iron concentrations. Expression of rcnA was induced by Ni(II) or Co(II). Overproduction of rcnR inhibited induction of rcnA by metal cations but RcnR did not bind to the rcnA promoter in vitro. When rcnR or fur, the gene of the global repressor of iron homeostasis, was deleted, expression of rcnA was also induced by iron. The promoter region of rcnA was positive in a Fur titration (FURTA) in vivo assay indicative of Fur binding. Thus, rcnA is part of the Fur regulon of E. coli. The implications of a connection between the homoeostasis of closely related transition metals are discussed.

A novel role of hypoxia-inducible factor in cobalt chloride- and hypoxia-mediated expression of IL-8 chemokine in human endothelial cells.

Tissue hypoxemia is common in several pathological diseases, including vaso-occlusion in sickle cell disease and myocardial infarction. One finds increased presence of leukocytes during lung injury and at sites of inflammation in vascular endothelium. In this study, we used human pulmonary microvascular endothelial cells and human dermal microvascular endothelial immortalized cell line to delineate the cellular signaling mechanism of hypoxia- and CoCl2 (a mimetic of hypoxia)-induced IL-8 expression, and the latter's role in chemotaxis of polmorphonuclear neutrophils. We show that hypoxia- and CoCl2-induced IL-8 mRNA and protein expression involved activation of PI3K/Akt and p38 MAPK, but not MEK kinase. Analysis of some transcription factors associated with IL-8 promoter revealed that hypoxia and CoCl2 increased DNA-binding activity of hypoxia-inducible factor-1alpha (HIF-1alpha), NF-kappaB, and AP-1. In addition, we show that hypoxia- and CoCl2-induced IL-8 expression requires activation of HIF as demonstrated by the following: 1) EMSA; 2) transfection studies with IL-8 promoter reporter constructs with mutation in HIF-1alpha binding site; 3) attenuation of IL-8 expression by both HIF-1alpha small interfering RNA and R59949; 4) augmentation of IL-8 expression by either transfection with HIF-prolyl hydroxylase-2 small interfering RNA or overexpression of HIF-1alpha; and 5) chromatin immunoprecipitation analysis. Moreover, conditioned medium from hypoxia-treated endothelial cells augmented chemotaxis of neutrophils, due to release of IL-8. These data indicate that hypoxia-induced signaling in vascular endothelium for transcriptional activation of IL-8 involves PI3K/Akt, p38 MAPK, and HIF-1alpha. Pharmacological agents, which inhibit HIF-1alpha, may possibly ameliorate inflammation associated with hypoxia in pathological diseases.

Analysis of corrinoids in ovine tissues.

Corrinoids from various ovine tissue samples (liver, blood, small intestinal fluid and faeces) were analysed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to estimate the distribution of corrinoids--the cobalamins hydroxocobalamin (OH-cbl), methylcobalamin (me-cbl) and 5'-deoxyadenosylcobalamin (ado-cbl), and cobalamin analogues--in these tissues. Samples were taken from either cobalt-deficient or cobalt-replete ewes, and ruminant and pre-ruminant lambs. In liver, ado-cbl predominated, followed by analogues, OH-cbl and me-cbl. Supplementation with either cobalt (ruminant) or vitamin B12 injections (pre-ruminant) increased the amount of ado-cbl and decreased analogues. In blood, OH-cbl predominated, followed by ado-cbl, analogues and me-cbl, respectively. In small intestinal fluid, the distribution from largest to smallest percentage was analogues, ado-cbl, OH-cbl and me-cbl. In faeces, analogues constituted the greatest proportion, followed by OH-cbl, ado-cbl and me-cbl, respectively. Owing to the small sample sizes only cautionary interpretations can be made. In contrast to humans, where me-cbl constitutes the highest proportion of corrinoids in plasma and ado-cbl in the liver, in sheep the amount of ado-cbl was consistently higher than me-cbl in all tissues. This may be due to the higher metabolic need of sheep for ado-cbl due to gluconeogenesis. Analogues and OH-cbl were found in each tissue, contrary to previous postulations. The much higher amount of vitamin B12 in small intestinal fluid compared with faeces indicates that a large proportion of the vitamin is absorbed by the gastro-intestinal tract.

Regulation of 2-deoxy-D-glucose transport, lactate metabolism, and MMP-2 secretion by the hypoxia mimetic cobalt chloride in articular chondrocytes.

Articular cartilage is an avascular tissue with significantly reduced levels of oxygen and nutrients compared to plasma and synovial fluid. Therefore, chondrocyte survival and cartilage homeostasis require effective mechanisms for oxygen and nutrient signaling. To gain a better understanding of the mechanisms responsible for oxygen and nutrient sensing in chondrocytes, we investigated the effects of hypoxic stimulation induced by cobalt chloride treatment (a hypoxia-mimetic) on glucose uptake and lactate production in chondrocytes. We also studied the effects of cobalt chloride and glucose deprivation on the expression and secretion of active MMP-2. Primary cultures of articular chondrocytes were either maintained in 20% O(2) (normoxia) or exposed to the hypoxia-mimetic cobalt chloride for up to 24 h at the following concentrations: 15 microM, 37.5 microM, and 75 microM. Glucose transport was determined by measuring the net uptake of nonmetabolizable 2-deoxy-D-[2, 6-(3)H] glucose into chondrocytes. Active MMP-2 secretion was assayed by gelatin zymography. Lactic acid production was assayed using a lactate kit. Exposure to cobalt chloride significantly increased the uptake of 2-deoxy-D-[2, 6-(3)H] glucose and the production of lactate. Glucose deprivation and cobalt chloride treatment increased levels of active MMP-2 in the culture medium. Our results suggest that these metabolic alterations are important events during adaptation to hypoxia. Upregulation of MMP-2 and the build-up of lactic acid will have detrimental effects on the extracellular matrix and may contribute to the pathogenesis and progression of osteoarthritis (OA).

Regulation of substrate specificity of plant alpha-mannosidase by cobalt ion: in vitro hydrolysis of high-mannose type N-glycans by Co2+-activated Ginkgo alpha-mannosidase.

In our previous study (Woo, K. K., et al., Biosci. Biotechnol. Biochem., 68, 2547-2556 (2004), we purified an alpha-mannosidase from Ginkgo biloba seeds; it was activated by cobalt ions and highly active towards high-mannose type free N-glycans occurring in plant cells. In the present study, we have found that the substrate specificity of Ginkgo alpha-mannosidase is significantly regulated by cobalt ions. When pyridylamino derivative of Man9GlcNAc2 (M9A) was incubated with Ginkgo alpha-mannosidase in the absence of cobalt ions, Man5GlcNAc2-PA (M5A) having no alpha1-2 mannosyl residue was obtained as a major product. On the other hand, when Man9GlcNAc2-PA was incubated with alpha-mannosidase in the presence of Co2+ (1 mM), Man3-1GlcNAc2-PA were obtained as major products releasing alpha1-3/6 mannosyl residues in addition to alpha1-2 mannosyl residues. The structures of the products (Man8-5GlcNAc2-PA) derived from M9A by enzyme digestion in the absence of cobalt ions were the same as those in the presence of cobalt ions. These results clearly suggest that the trimming pathway from M9A to M5A is not affected by the addition of cobalt ions, but that hydrolytic activity towards alpha1-3/6 mannosyl linkages is stimulated by Co2+. Structural analysis of the products also showed clearly that Ginkgo alpha-mannosidase can produce truncated high-mannose type N-glycans, found in developing or growing plant cells, suggesting that alpha-mannosidase might be involved in the degradation of high-mannose type free N-glycans.

Contrasting effects of Cd2+ and Co2+ on the blocking/unblocking of human Cav3 channels.

Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Ca(v)1 and Ca(v)2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Ca(v)3 family) is less documented. We have studied the blocking effect of Cd2+, Co2+ and Ni2+ on T-currents expressed by human Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca2+ (2 mM: ) currents from HEK-293 cells stably expressing recombinant T-type channels. Cd2+ and Co2+ block was 2- to 3-fold more potent for Ca(v)3.2 channels (EC50 = 65 and 122 microM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co2+ and Ni2+ shift the voltage dependence of Ca(v)3.1 and Ca(v)3.3 channels activation to more positive potentials. Interestingly, block of those two Ca(v)3 channels by Co2+ and Ni2+ was drastically increased at extreme negative voltages; in contrast, block due to Cd2+ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd2+ on Ca(v)3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.

Adrenaline diminishes K+ contractures and Ba2+-current in chicken slow skeletal muscle fibres.

The effects of adrenaline and the beta-adrenergic agonist isoprenaline on K+-evoked tension (K+-contracture) and Ba2+ current were investigated in chicken slow (anterior latissimus dorsi (ald)) muscle using isometric-tension measurements and current recording. Addition of adrenaline (10(-7) - 10(-5) M) or isoprenaline (10(-6) - 10(-5) M) to the bath reduced the amplitude of the K+-contractures. These effects were blocked by the beta-antagonist propranolol (5 x 10(-6) M). External application of a cAMP analogue (8-bromo cyclic AMP; 1 x 10(-4) M) also decreased the amplitude of the K+-contractures. To analyze the possible relationship between the induced tension reduction and effects on sarcolemmal Ca2+ channels, a slow action potential and a slow inward membrane current were studied in intact ald chicken muscle fibres. When the ald muscle was immersed in a Na+- and Cl--free solution containing Ba2+ and depolarizing pulses were delivered from a -80 mV holding potential, the muscle fibres exhibited a small, slow Ba2+-dependent potential (observed at about -26 mV, peak amplitude, around -10 mV). The response was blocked by the addition of Co2+ (5 mM) or Cd2+ (2 mM). Using the three-microelectrode voltage-clamp technique, a slow inward membrane current underlying the Ba2+ potential could be discerned. The current had a mean threshold of -60 mV, reached maximum at about -5 mV and ranged from ca. 9 to 19 pA/cm2 (depending on the external Ba2+ concentration). It had a mean reversal potential of +45 mV. The Ba2+ inward current was diminished when adrenaline or isoprenaline was added to the bath (1 x 10(-5) M); however, this decrease did not occur when propranolol was present (5 x 10-6 M). These results suggest that the decreases in the tension of K+-contractures induced by adrenaline and isoprenaline may occur through beta-adrenergic effects on sarcolemmal Ca2+ channels in ald chicken slow muscle fibres.

Regulation of carbonic anhydrase expression by zinc, cobalt, and carbon dioxide in the marine diatom Thalassiosira weissflogii.

TWCA1 is the major Zn-requiring isoform of carbonic anhydrase (CA) in the marine diatom Thalassiosira weissflogii. We have examined the roles that trace metals and CO(2) play in the regulation of TWCA1 expression over ranges of concentrations that bracket those encountered in the marine environment. Both steady-state levels of TWCA1 and the kinetics of induction were measured by western analysis. TWCA1 levels correlated well with cellular CA activity levels. TWCA1 was induced at a low CO(2) concentration but the level of induction, as determined by western analysis, was dependent on the availability of Zn. Co effectively substituted for Zn in regulating TWCA1 expression and promoting TWCA1 activity. Upon shift from low to high CO(2), the concentration of TWCA1 decreased. The expression of TWCA1 is diel cycle regulated, and cellular TWCA1 decreased during the dark phase. These results provide the basis for studying the expression of CA in field populations and, taken together with previous radiolabeling studies, provide strong evidence of in vivo metal substitution of Co for Zn in a CA. Our data also support the conclusion that TWCA1 plays a central role in carbon acquisition in T. weissflogii.

Development and evaluation of an indirect ELISA for detection of exfoliative toxin ExhA, ExhB or ExhC produced by Staphylococcus hyicus.

Immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) confirmed previous reports that the Staphylococcus hyicus exfoliative toxins ExhA and ExhB are metalloproteins, and further indicated that ExhC is also a metalloprotein. An indirect ELISA was developed for the detection of toxigenic strains as an alternative method to the use of phage typing for selection of S. hyicus isolates to be used in autogenous vaccine against exudative epidermitis in pigs. The indirect ELISA was evaluated by investigating the presence of toxin among a total of 655 S. hyicus isolates from 69 pig skin samples, one from each of the 69 pig herds with outbreak of exudative epidermitis. Toxigenic S. hyicus were detected in 74% of the cases by ELISA. From each of the five cases, in which initially no toxigenic S. hyicus were found, a further 40 S. hyicus-like colonies were tested in ELISA. Testing of this number of colonies has a >99% probability of disclosing toxigenic S. hyicus. Toxin-producing isolates were found in only two of the five cases investigated. This may indicate the existence of one or more variants of the exfoliative toxin of S. hyicus that are not detected in the indirect ELISA or that S. hyicus may be displaced from lesions of exudative epidermitis.

DNA cleavage by the EcoRV restriction endonuclease: roles of divalent metal ions in specificity and catalysis.

The roles of divalent metal ions in DNA cleavage by the EcoRV endonuclease were studied by using Co2+ or Mn2+ as substitutes for the natural cofactor Mg2+. In steady-state experiments with a 12 bp oligonucleotide substrate, Co2+ yielded a similar turnover rate to that with Mg2+, but Mn2+ gave a slower rate. Single turnovers of EcoRV on this substrate were analysed by stopped-flow and quench-flow methods, to determine the rates for the formation of the ternary enzyme-DNA-metal complex, the hydrolysis of the phosphodiester bonds and the dissociation of the cleaved DNA. With Co2+, all three steps had similar rates to those with Mg2+. In contrast, Mn2+ gave a faster rate for phosphodiester hydrolysis than either Mg2+ or Co2+, but a slower rate for product dissociation, thus accounting for its low turnover rate. Single turnovers on plasmids also yielded faster rates for substrate hydrolysis with Mn2+ compared to Mg2+ and Co2+. Since Mn2+ gave the most rapid rates for the hydrolytic step, despite being less electronegative than Co2+, the function of the metal ion at the active site of EcoRV cannot be just the polarisation of the scissile phosphate. Moreover, the minimal scheme for the Co2+-catalysed reaction requires two metal ions for DNA cleavage. The metal ions seem to be involved in the precise positioning of both the substrate and the water that acts as the attacking nucleophile and in activating that water molecule. A model is presented to account for how two metal ions might fulfil these functions.

Catalytic significance of the specificity of divalent cations as KS* and kcat* cofactors for secreted phospholipase A2.

Calcium is required for the substrate binding and for the chemical step of the interfacial catalytic turnover cycle of pancreatic phospholipase A2 (PLA2), but not for the binding of the enzyme to the interface. The role of calcium and other divalent cations (C) is analyzed for the effect on the substrate binding and kcat* for the chemical step. The cofactor role of 3d-cations(II) (C) for the hydrolysis of dimyristoylphosphatidylmethanol (DMPM) vesicles is characterized as an equilibrium dissociation constant for the interfacial binary (E*C) and ternary (E*CL) complexes of PLA2 and substrate mimics (L). Of the cations(II) that promote the binding of a mimic to the enzyme at the interface (E*), only a subgroup supports the chemical step. For example, Cd, Zn, and Cu form ternary E*CL complexes with kcat* of <1 s-1, compared to the rate of >100 s-1 with Ca, Fe, Mn, Co, and Ni. Oxygen exchange from H218O to the products of hydrolysis of DMPM incorporates one 18O in myristate. Incorporation of the first and second 18O occurs during the incubation of both the products of hydrolysis in H218O with PLA2 and Ca, but not with Zn. The cation-dependent changes in the UV difference spectrum, associated with the formation of E*C and E*CL, suggest that the changes are mainly due to catalytic His-48, and possibly Tyr-52 and Tyr-73, and are different with Ca as opposed to Zn. These results and simulations suggest considerable plasticity in the calcium binding and catalytic site environment. It is proposed that the higher ground state stability of the E*CS complex with the inhibitory cations increases the effective activation energy. For the chemical step, calcium coordinated with a nucleophilic water and the ester carbonyl oxygen facilitates the near-attack geometry in the E*CaS, and the His-48.Asp-99 pair acts as a proton acceptor. As a prelude to establishing the catalytic mechanism, factors controlling the energetically demanding transition state are also discussed.

Effects of a series of metalloporphyrins on adrenal, testicular and thyroid function in rats.

We have extended our earlier studies [Pharmacology 1986;34:9-16] on the effects of certain synthetic heme analogues and cobalt chloride (CoCl2) on endocrine functions mediated by the hypothalamic-pituitary axis to examine specifically the ability of Sn-protoporphyrin (SnPP) and Sn-mesoporphyrin (SnMP) to perturb adrenal, testicular and thyroid function since there is interest in the use of Sn(tin)-porphyrins in the treatment of hyperbilirubinemia of the newborn. SnPP and SnMP when administered to adult male rats did not alter serum corticosterone, testosterone, thyroxine or triiodothyronine levels when compared to control animals. In addition, administration of exogenous adrenocorticotrophic hormone produced an increase in serum corticosterone levels that was comparable in placebo-treated and SnPP- and SnMP-treated animals. These studies involved doses of both compounds substantially greater than those used clinically. The results clearly indicate that SnMP, presently the compound of choice for use in newborns, and SnPP do not in the doses studied impair adrenal, testicular and thyroid function in vivo.