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1.  A New Strategy to Stabilize Oxytocin in Aqueous Solutions: I. The Effects of Divalent Metal Ions and Citrate Buffer 
The AAPS Journal  2011;13(2):284-290.
In the current study, the effect of metal ions in combination with buffers (citrate, acetate, pH 4.5) on the stability of aqueous solutions of oxytocin was investigated. Both monovalent metal ions (Na+ and K+) and divalent metal ions (Ca2+, Mg2+, and Zn2+) were tested all as chloride salts. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by RP-HPLC and HP-SEC after 4 weeks of storage at either 4°C or 55°C. Addition of sodium or potassium ions to acetate- or citrate-buffered solutions did not increase stability, nor did the addition of divalent metal ions to acetate buffer. However, the stability of aqueous oxytocin in aqueous formulations was improved in the presence of 5 and 10 mM citrate buffer in combination with at least 2 mM CaCl2, MgCl2, or ZnCl2 and depended on the divalent metal ion concentration. Isothermal titration calorimetric measurements were predictive for the stabilization effects observed during the stability study. Formulations in citrate buffer that had an improved stability displayed a strong interaction between oxytocin and Ca2+, Mg2+, or Zn2+, while formulations in acetate buffer did not. In conclusion, our study shows that divalent metal ions in combination with citrate buffer strongly improved the stability of oxytocin in aqueous solutions.
doi:10.1208/s12248-011-9268-7
PMCID: PMC3085697  PMID: 21448747
citrate buffer; divalent metal ions; improved stability; oxytocin
2.  Aggregation Kinetics of Citrate and Polyvinylpyrrolidone Coated Silver Nanoparticles in Monovalent and Divalent Electrolyte Solutions 
Environmental science & technology  2011;45(13):5564-5571.
The aggregation kinetics of silver nanoparticles (AgNPs) that were coated with two commonly used capping agents—citrate and polyvinylpyrrolidone (PVP)—were investigated. Time-resolved dynamic light scattering (DLS) was employed to measure the aggregation kinetics of the AgNPs over a range of monovalent and divalent electrolyte concentrations. The aggregation behavior of citrate-coated AgNPs in NaCl was in excellent agreement with the predictions based on Derjaguin–Landau–Verwey–Overbeek (DLVO) theory, and the Hamaker constant of citrate-coated AgNPs in aqueous solutions was derived to be 3.7 × 10-20 J. Divalent electrolytes were more efficient in destabilizing the citrate-coated AgNPs, as indicated by the considerably lower critical coagulation concentrations (2.1 mM CaCl2 and 2.7 mM MgCl2 vs. 47.6 mM NaCl). The PVP-coated AgNPs were significantly more stable than citrate-coated AgNPs in both NaCl and CaCl2, which is likely due to steric repulsion imparted by the large, non-charged polymers. The addition of humic acid resulted in the adsorption of the macromolecules on both citrate- and PVP-coated AgNPs. The adsorption of humic acid induced additional electrosteric repulsion that elevated the stability of both nanoparticles in suspensions containing NaCl or low concentrations of CaCl2. Conversely, enhanced aggregation occurred for both nanoparticles at high CaCl2 concentrations due to interparticle bridging by humic acid clusters.
doi:10.1021/es200157h
PMCID: PMC3137917  PMID: 21630686
3.  Effect on Postpartum Hemorrhage of Prophylactic Oxytocin (10 IU) by Injection by Community Health Officers in Ghana: A Community-Based, Cluster-Randomized Trial 
PLoS Medicine  2013;10(10):e1001524.
Cynthia Stanton and colleagues conducted a cluster-randomized controlled trial in rural Ghana to assess whether oxytocin given by injection by community health officers at home births was a feasible and safe option in preventing postpartum hemorrhage.
Please see later in the article for the Editors' Summary
Background
Oxytocin (10 IU) is the drug of choice for prevention of postpartum hemorrhage (PPH). Its use has generally been restricted to medically trained staff in health facilities. We assessed the effectiveness, safety, and feasibility of PPH prevention using oxytocin injected by peripheral health care providers without midwifery skills at home births.
Methods and Findings
This community-based, cluster-randomized trial was conducted in four rural districts in Ghana. We randomly allocated 54 community health officers (stratified on district and catchment area distance to a health facility: ≥10 km versus <10 km) to intervention (one injection of oxytocin [10 IU] one minute after birth) and control (no provision of prophylactic oxytocin) arms. Births attended by a community health officer constituted a cluster. Our primary outcome was PPH, using multiple definitions; (PPH-1) blood loss ≥500 mL; (PPH-2) PPH-1 plus women who received early treatment for PPH; and (PPH-3) PPH-2 plus any other women referred to hospital for postpartum bleeding. Unsafe practice is defined as oxytocin use before delivery of the baby. We enrolled 689 and 897 women, respectively, into oxytocin and control arms of the trial from April 2011 to November 2012. In oxytocin and control arms, respectively, PPH-1 rates were 2.6% versus 5.5% (RR: 0.49; 95% CI: 0.27–0.88); PPH-2 rates were 3.8% versus 10.8% (RR: 0.35; 95% CI: 0.18–0.63), and PPH-3 rates were similar to those of PPH-2. Compared to women in control clusters, those in the intervention clusters lost 45.1 mL (17.7–72.6) less blood. There were no cases of oxytocin use before delivery of the baby and no major adverse events requiring notification of the institutional review boards. Limitations include an unblinded trial and imbalanced numbers of participants, favoring controls.
Conclusion
Maternal health care planners can consider adapting this model to extend the use of oxytocin into peripheral settings including, in some contexts, home births.
Trial registration
ClinicalTrials.gov NCT01108289
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Many women in low-and middle-income countries die unnecessarily during childbirth, even though the solutions to prevent or manage complications are well known. Maternal death rates are highest amongst poor women living in remote areas, as they are least likely to have access to adequate health care. One of the United Nation's Millennium Development Goals is to reduce maternal death rates by three-quarters by 2015. Between 1990 and 2010, these rates were nearly halved. So there is still some way to go to meet the target.
One of the major causes of maternal death is excessive bleeding after birth, known as postpartum hemorrhage (PPH). The highest rates of PPH are found in Africa (28% of births), with an overall global rate of 11%. PPH can be caused by the uterus not contracting after the baby is born, damage to tissues and blood vessels, retention of the placenta, and problems with blood-clotting.
PPH can be prevented by an injection of oxytocin (a hormone) or with tablets of the drug misoprostol immediately after birth. Other drugs exist but are used much less frequently in low-income countries. If the mother does bleed excessively, then these interventions can also be used to treat PPH in the hours following birth. These drugs cause the uterus to contract. Continued severe bleeding requires emergency treatment in hospital. The World Health Organization (WHO) recommends that in situations where women give birth without the assistance of a trained midwife, priority should be given to preventing PPH because access to emergency services may be limited.
Why Was This Study Done?
Of the two most common options for preventing PPH, oxytocin is generally the preferred choice. It has the advantage of having no side effects, whereas misoprostol can cause fever and shivering. A repeat injection of oxytocin can also be given if the mother continues to bleed excessively, whereas a dose of misoprostol after birth should only be given once. A major concern about both drugs is that the timing of administration must be precise. Giving a drug that causes the uterus to contract before birth can be harmful to both mother and baby. A disadvantage of oxytocin is that it requires someone trained and authorized to give an injection. For this reason, oxytocin has so far been generally limited to hospitals and clinics, where it can be administered by medically trained professionals. Another disadvantage is that oxytocin is weakened by heat, which means its storage and use may be impractical in hot countries.
The main aim of this study was therefore to find out whether health workers without midwifery skills are able to administer oxytocin safely when attending home births in poor, rural communities.
What Did the Researchers Do and Find?
The researchers carried out a cluster-randomized controlled trial in four rural districts in Ghana, working with community health officers (CHOs). CHOs are trained for two years in giving childhood immunizations and antenatal and postnatal care, but are not trained midwives. 54 CHOs were randomized to one of two groups. The CHOs in the first group gave a preventative oxytocin injection to the mother at every birth they attended. The oxytocin was administered using a pre-filled, disposable device called Uniject that is easier and quicker to use than a syringe and needle. The packaging also included a heat-sensitive label that indicated whether the oxytocin still met the manufacturer's criteria for an acceptably potent drug. CHOs in the second group acted as controls, and did not give any oxytocin injections to prevent PPH. The women seen by each CHO formed a cluster. Comparisons were made across the clusters of women that either received or did not receive the preventative intervention.
The researchers found that the women who were given a preventative oxytocin injection lost less blood after birth than the women who did not receive a preventative injection. There were also fewer cases of PPH amongst the women who received oxytocin for PPH prevention. 2.6% of the women who received a preventative oxytocin injection experienced PPH, compared to 5.5% of the controls. Therefore the risk of PPH was approximately halved. There were no cases of oxytocin use before delivery of the baby and no difference in the frequency of other birth complications between women in the intervention and control groups.
What Do These Findings Mean?
These findings show that under the trial conditions, CHOs can safely administer oxytocin injections when attending home births in poor, rural settings. This intervention also proved practical to use in the Uniject format.
The study therefore suggests that oxytocin should be considered for use in regions where maternal deaths from PPH are still unacceptably high. There are also several noteworthy limitations, such as unblinding and the imbalance between participant groups. The researchers emphasized that their findings do not mean that oxytocin is always a better choice than misoprostol for home births. Many factors will influence which intervention is the most feasible, such as the local availability of sufficiently skilled health professionals, the relative cost and availability of the two drugs, as well as ease of access to emergency health services.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001524.
This study is further discussed in a PLOS Medicine Perspective by João Paulo Souza
WHO Factsheet on Maternal Mortality
United Nations Population Fund's Goals to improve sexual and reproductive health
doi:10.1371/journal.pmed.1001524
PMCID: PMC3794862  PMID: 24130463
4.  The permeability of endplate channels to monovalent and divalent metal cations 
The Journal of General Physiology  1980;75(5):493-510.
The relative permeability of endplate channels to monovalent and divalent metal ions was determined from reversal potentials. Thallium is the most permeant ion with a permeability ratio relative to Na+ of 2.5. The selectivity among alkali metals is weak with a sequence, Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+, and permeability ratios of 1.4, 1.3, 1.1, 1.0, and 0.9. The selectivity among divalent ions is also weak, with a sequence for alkaline earths of Mg++ greater than Ca++ greater than Ba++ greater than Sr++. The transition metal ions Mn++, Co++, Ni++, Zn++, and Cd++ are also permeant. Permeability ratios for divalent ions decreased as the concentration of divalent ion was increased in a manner consistent with the negative surface potential theory of Lewis (1979 J. Physiol. (Lond.). 286: 417--445). With 20 mM XCl2 and 85.5 mM glucosamine.HCl in the external solution, the apparent permeability ratios for the alkaline earth cations (X++) are in the range 0.18--0.25. Alkali metal ions see the endplate channel as a water-filled, neutral pore without high-field-strength sites inside. Their permeability sequence is the same as their aqueous mobility sequence. Divalent ions, however, have a permeability sequence almost opposite from their mobility sequence and must experience some interaction with groups in the channel. In addition, the concentrations of monovalent and divalent ions are increased near the channel mouth by a weak negative surface potential.
PMCID: PMC2215258  PMID: 6247423
5.  Synergistic effects of metal ion and the pre-senile cataract-causing G98R αA-crystallin: self-aggregation propensities and chaperone activity 
Molecular Vision  2009;15:2050-2060.
Purpose
αA- and αB-crystallins are abundantly present in the eye lens, belong to the small heat shock protein family, and exhibit molecular chaperone activity. They are also known to interact with metal ions such as Cu2+, and their metal-binding modulates the structure and chaperone function. Unlike other point mutations in αA-crystallin that cause congenital cataracts, the G98R mutation causes pre-senile cataract. We have investigated the effect of Cu2+ on the structure and function of G98R αA-crystallin.
Methods
Fluorescence spectroscopy and isothermal titration calorimetry were used to study Cu2+ binding to αA- and G98R αA-crystallin. Circular dichroism spectroscopy was used to study secondary and tertiary structures, and dynamic light scattering was used to determine the hydrodynamic radii of the proteins. Chaperone activity and self-aggregation of the wild type and the mutant protein in the absence and the presence of the metal ions was monitored using light scattering.
Results
Our fluorescence quenching and isothermal titration calorimetric studies show that like αA-crystallin, G98R αA-crystallin binds Cu2+ with picomolar range affinity. Further, both wild type and mutant αA-crystallin inhibit Cu2+-induced generation of reactive oxygen species with similar efficiency. However, G98R αA-crystallin undergoes pronounced self-aggregation above a certain concentration of Cu2+ (above subunit to Cu2+ molar ratio of 1:3 in HEPES-NaOH buffer, pH 7.4). At concentrations of Cu2+ below this ratio, G98R αA-crystallin is more susceptible to Cu2+-induced tertiary and quaternary structural changes than αA-crystallin. Interestingly, Cu2+ binding increases the chaperone-like activity of αA-crystallin toward the aggregation of citrate synthase at 43 °C while it decreases the chaperone-like activity of G98R αA-crystallin. Mixed oligomer formation between the wild type and the mutant subunits modulates the Cu2+-induced effect on the self-aggregation propensity. Other heavy metal ions, namely Cd2+ and Zn2+ but not Ca2+, also promote the self-aggregation of G98R αA-crystallin and decrease its chaperone-like activity.
Conclusions
Our study demonstrates that unlike wild type αA-crystallin, G98R αA-crystallin and its mixed oligomers with wild type protein are vulnerable to heavy metal ions. Our study provides insight into aspects of how environmental factors could augment phenotype(s) in certain genetically predisposed conditions.
PMCID: PMC2768467  PMID: 19862354
6.  Lyophilization of a triply unsaturated phospholipid: Effects of trace metal contaminants 
As liquid liposomal formulations are prone to chemical degradation and aggregation, these formulations often require freeze drying (e.g. lyophilization) to achieve sufficient shelf-life. However, liposomal formulations may undergo oxidation during lyophilization and/or during prolonged storage. The goal of the current study was to characterize the degradation of 1, 2-dilinolenoyl-sn-glycero-3-phosphocholine (DLPC) during lyophilization, and to also probe the influence of metal contaminants in promoting the observed degradation. Aqueous sugar formulations containing DLPC (0.01 mg/ml) were lyophilized, and DLPC degradation was monitored using HPLC/UV and GC/MS methods. The effect of ferrous ion and sucrose concentration, as well as lyophilization stage promoting lipid degradation, was investigated. DLPC degradation increased with higher levels of ferrous ion. After lyophilization, 103.1% ± 1.1%, 66.9% ± 0.8%, and 28.7% ± 0.7% DLPC remained in the sucrose samples spiked with 0.0 ppm, 0.2 ppm and 1.0 ppm ferrous ion, respectively. Lipid degradation predominantly occurs during the freezing stage of lyophilization. Sugar concentration and buffer ionic strength also influence the extent of lipid degradation, and DLPC loss correlated with degradation product formation. We conclude that DLPC oxidation during the freezing stage of lyophilization dramatically compromises the stability of lipid-based formulations. In addition, we demonstrate that metal contaminants in sugars can become highly active when lyophilized in the presence of a reducing agent.
doi:10.1016/j.ejpb.2013.03.028
PMCID: PMC3797213  PMID: 23567484
DLPC; Lyophilization; liposomes; chemical stability; metal contamination; unsaturated lipids; oxidation; freezing; freeze-drying
7.  Enhanced Adsorption and Recovery of Uranyl Ions by NikR Mutant-Displaying Yeast 
Biomolecules  2014;4(2):390-401.
Uranium is one of the most important metal resources, and the technology for the recovery of uranyl ions (UO22+) from aqueous solutions is required to ensure a semi-permanent supply of uranium. The NikR protein is a Ni2+-dependent transcriptional repressor of the nickel-ion uptake system in Escherichia coli, but its mutant protein (NikRm) is able to selectively bind uranyl ions in the interface of the two monomers. In this study, NikRm protein with ability to adsorb uranyl ions was displayed on the cell surface of Saccharomyces cerevisiae. To perform the binding of metal ions in the interface of the two monomers, two metal-binding domains (MBDs) of NikRm were tandemly fused via linker peptides and displayed on the yeast cell surface by fusion with the cell wall-anchoring domain of yeast α-agglutinin. The NikRm-MBD-displaying yeast cells with particular linker lengths showed the enhanced adsorption of uranyl ions in comparison to the control strain. By treating cells with citrate buffer (pH 4.3), the uranyl ions adsorbed on the cell surface were recovered. Our results indicate that the adsorption system by yeast cells displaying tandemly fused MBDs of NikRm is effective for simple and concentrated recovery of uranyl ions, as well as adsorption of uranyl ions.
doi:10.3390/biom4020390
PMCID: PMC4101488  PMID: 24970221
cell surface engineering; arming yeast; bioadsorption; uranyl ions; NikR
8.  Chelating Agent Shock of Bacteriophage T5 
Journal of Virology  1968;2(9):944-950.
When two strains of phage T5 (heat-susceptible form T5st+ and its heat-resistant mutant T5st) were placed in solutions containing various high concentrations of chelating agents (sodium citrate and ethylenediaminetetraacetic acid) at room temperature, they could be effectively inactivated by rapid dilution in distilled water of relatively low temperatures (2 to 37 C). This phenomenon has been termed “chelating agent shock” (CAS). The susceptibility of phage T5 to CAS increased with an increase in the concentration of chelating agents and with an increase in temperature of the water used for rapid dilution. Under any given condition, T5st+ was much more sensitive to CAS than was T5st. Phage T5 was protected against inactivation by the addition of monovalent or divalent metal salts, but not by the addition of nonionic solutes, to the shocking water prior to CAS treatment. This finding is compatible with the view that cations combined with the phage protein are removed by the chelating agent, although no metal ion has been identified in the phage protein. Alternatively, since the chelating agents used are polyanions, they may bind relatively tightly to the protein subunits in the head of T5, thereby distorting the structure of the phage head. Rapid dilution of these distorted particles could lead to loss of phage DNA. No evidence for recovery of phage activity could be obtained by the addition of metal salts to the inactivated phage after CAS. The morphological properties of phage inactivated by CAS are similar to those of heat-inactivated T5 phage. Electron micrographs showed that most of the phage particles consisted of empty head membranes; some of the particles had lost their tails. Both heritable and nonheritable resistance to heat was accompanied by resistance to CAS in phage T5. The sensitive element detected by each test seemed to be the same.
Images
PMCID: PMC375716  PMID: 4972945
9.  Divalent Metal Ion Transport across Large Biological Ion Channels and Their Effect on Conductance and Selectivity 
Electrophysiological characterization of large protein channels, usually displaying multi-ionic transport and weak ion selectivity, is commonly performed at physiological conditions (moderate gradients of KCl solutions at decimolar concentrations buffered at neutral pH). We extend here the characterization of the OmpF porin, a wide channel of the outer membrane of E. coli, by studying the effect of salts of divalent cations on the transport properties of the channel. The regulation of divalent cations concentration is essential in cell metabolism and understanding their effects is of key importance, not only in the channels specifically designed to control their passage but also in other multiionic channels. In particular, in porin channels like OmpF, divalent cations modulate the efficiency of molecules having antimicrobial activity. Taking advantage of the fact that the OmpF channel atomic structure has been resolved both in water and in MgCl2 aqueous solutions, we analyze the single channel conductance and the channel selectivity inversion aiming to separate the role of the electrolyte itself, and the counterion accumulation induced by the protein channel charges and other factors (binding, steric effects, etc.) that being of minor importance in salts of monovalent cations become crucial in the case of divalent cations.
doi:10.1155/2012/245786
PMCID: PMC3449104  PMID: 23008773
10.  Activity, folding and Z-DNA formation of the 8-17 DNAzyme in the presence of monovalent ions 
The effect of monovalent ions on both the reactivity and global folding of the 8–17 DNAzyme is investigated and the results are compared with the hammerhead ribozyme, which has similar size and secondary structure. In contrast to the hammerhead ribozyme, the 8–17 DNAzyme activity is not detectable in the presence of 4 M K+, Rb+, and Cs+ and the complex, [Co(NH3)6]3+. Only Li+, NH4+ and to a lesser extent Na+ showed detectable activity. The observed rate constants (kobs ~10−3 min−1 for Li+ and NH4+) are ~1000-fold lower than that in the presence of 10 mM Mg2+, and ~200,000-fold slower than the estimated rate in the presence of 100 µM Pb2+. Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within ~10-fold of divalent metal ion-dependent activity, these results suggest that the 8–17 DNAzyme, obtained by in vitro selections has evolved to have a more stringent divalent metal ion requirement for high activity as compared to the naturally occurring ribozymes, making the 8–17 DNAzyme an excellent choice as a Pb2+ sensor with high selectivity. In contrast to the activity data, folding was observed in the presence of all the monovalent ions investigated, although those monovalent ions that do not support DNAzyme activity have weaker binding affinity (Kd ~0.35 M for Rb+ and Cs+), while those that confer DNAzyme activity possess stronger affinity (Kd ~0.22 M for Li+, Na+ and NH4 +). In addition, a correlation between metal ion charge density, binding affinity and enzyme activity was found among mono- and divalent metal ions except Pb2+; higher charge density resulted in stronger affinity and higher activity, suggesting that the observed folding and activity is at least partially due to electrostatic interactions between ions and the DNAzyme. Finally, circular dichroism (CD) study has revealed Z-DNA formation with the monovalent metal ions, Zn2+ and Mg2+; the Kd values obtained using CD were in the same range as those obtained from folding studies using FRET. However, Z-DNA formation was not observed with Pb2+. These results indicate that Pb2+-dependent function follows a different mechanism from the monovalent metal ions and other divalent metal ions; in the presence of latter metal ions, metal-ion dependent folding and structural changes, including formation of Z-DNA, play an important role in the catalytic function of the 8–17 DNAzyme.
doi:10.1021/ja8082939
PMCID: PMC2765493  PMID: 19326878
8–17 DNAzyme; Monovalent ions; Activity; Fluorescence Resonance Energy Transfer (FRET); Circular Dichroism (CD)
11.  Aggregation of recombinant human interferon alpha 2b in solution: Technical note 
AAPS PharmSciTech  2014;7(4):E118-E122.
Summary and Conclusions
Sodium phosphate buffer increased the aggregation of rhIFN-α2b in the range of 1.55 to 1.8103 day−1, as determined by SDS/PAGE under reduced and nonreduced conditions. In contrast, sodium citrate buffer decreased the aggregation rate of this cytokine, as compared with those samples in sodium phosphate buffer. Results from sodium citrate-phosphate buffer were very similar to those obtained with sodium citrate solutions.
On the other hand, EDTA Na2×2H2O reduced the aggregation rate of rhIFN-α2b, showing an aggregation kinetic constant in the range of 0.52 to 0.75×103 day−1. Polysorbates 20 and 80 were less effective than the chelating agent in preventing this degradation pathway.
Additionally, metal ions (Zn2+ and Cu2+) increased the aggregation kinetic constant of rhIFN-α2b, probably through undetermined metal-catalyzing reactions.
Taken together, these data can be useful for the development of new formulations containing rhIFN-α2b as an active ingredient.
doi:10.1208/pt070499
PMCID: PMC2750336  PMID: 17285753
12.  Ca2+-Citrate Uptake and Metabolism in Lactobacillus casei ATCC 334 
Applied and Environmental Microbiology  2013;79(15):4603-4612.
The putative citrate metabolic pathway in Lactobacillus casei ATCC 334 consists of the transporter CitH, a proton symporter of the citrate-divalent metal ion family of transporters CitMHS, citrate lyase, and the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Resting cells of Lactobacillus casei ATCC 334 metabolized citrate in complex with Ca2+ and not as free citrate or the Mg2+-citrate complex, thereby identifying Ca2+-citrate as the substrate of the transporter CitH. The pathway was induced in the presence of Ca2+ and citrate during growth and repressed by the presence of glucose and of galactose, most likely by a carbon catabolite repression mechanism. The end products of Ca2+-citrate metabolism by resting cells of Lb. casei were pyruvate, acetate, and acetoin, demonstrating the activity of the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Following pyruvate, the pathway splits into two branches. One branch is the classical citrate fermentation pathway producing acetoin by α-acetolactate synthase and α-acetolactate decarboxylase. The other branch yields acetate, for which the route is still obscure. Ca2+-citrate metabolism in a modified MRS medium lacking a carbohydrate did not significantly affect the growth characteristics, and generation of metabolic energy in the form of proton motive force (PMF) was not observed in resting cells. In contrast, carbohydrate/Ca2+-citrate cometabolism resulted in a higher biomass yield in batch culture. However, also with these cells, no generation of PMF was associated with Ca2+-citrate metabolism. It is concluded that citrate metabolism in Lb. casei is beneficial when it counteracts acidification by carbohydrate metabolism in later growth stages.
doi:10.1128/AEM.00925-13
PMCID: PMC3719530  PMID: 23709502
13.  Complementary Metal Ion Specificity of the Metal-Citrate Transporters CitM and CitH of Bacillus subtilis 
Journal of Bacteriology  2000;182(22):6374-6381.
Citrate uptake in Bacillus subtilis is stimulated by a wide range of divalent metal ions. The metal ions were separated into two groups based on the expression pattern of the uptake system. The two groups correlated with the metal ion specificity of two homologous B. subtilis secondary citrate transporters, CitM and CitH, upon expression in Escherichia coli. CitM transported citrate in complex with Mg2+, Ni2+, Mn2+, Co2+, and Zn2+ but not in complex with Ca2+, Ba2+, and Sr2+. CitH transported citrate in complex with Ca2+, Ba2+, and Sr2+ but not in complex with Mg2+, Ni2+, Mn2+, Co2+, and Zn2+. Both transporters did not transport free citrate. Nevertheless, free citrate uptake could be demonstrated in B. subtilis, indicating the expression of at least a third citrate transporter, whose identity is not known. For both the CitM and CitH transporters it was demonstrated that the metal ion promoted citrate uptake and, vice versa, that citrate promoted uptake of the metal ion, indicating that the complex is the transported species. The results indicate that CitM and CitH are secondary transporters that transport complexes of divalent metal ions and citrate but with a complementary metal ion specificity. The potential physiological function of the two transporters is discussed.
PMCID: PMC94783  PMID: 11053381
14.  Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme 
Nucleic Acids Research  2002;30(11):2374-2382.
The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn2+ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na+ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion–ribozyme complexes are converted to less desirable monovalent metal ion–ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley’s and DeRose’s two-phase folding model that was based on ground state structure analyses.
PMCID: PMC117202  PMID: 12034824
15.  Increasing Mechanical Strength of Gelatin Hydrogels by Divalent Metal Ion Removal 
Scientific Reports  2014;4:4706.
The usage of gelatin hydrogel is limited due to its instability and poor mechanical properties, especially under physiological conditions. Divalent metal ions present in gelatin such as Ca2+ and Fe2+ play important roles in the gelatin molecule interactions. The objective of this study was to determine the impact of divalent ion removal on the stability and mechanical properties of gelatin gels with and without chemical crosslinking. The gelatin solution was purified by Chelex resin to replace divalent metal ions with sodium ions. The gel was then chemically crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Results showed that the removal of divalent metal ions significantly impacted the formation of the gelatin network. The purified gelatin hydrogels had less interactions between gelatin molecules and form larger-pore network which enabled EDC to penetrate and crosslink the gel more efficiently. The crosslinked purified gels showed small swelling ratio, higher crosslinking density and dramatically increased storage and loss moduli. The removal of divalent ions is a simple yet effective method that can significantly improve the stability and strength of gelatin hydrogels. The in vitro cell culture demonstrated that the purified gelatin maintained its ability to support cell attachment and spreading.
doi:10.1038/srep04706
PMCID: PMC3988488  PMID: 24736500
16.  Use of Divalent Metal Ions in the DNA Cleavage Reaction of Human Type II Topoisomerases† 
Biochemistry  2009;48(9):1862-1869.
All type II topoisomerases require divalent metal ions in order to cleave and ligate DNA. In order to further elucidate the mechanistic basis for these critical enzyme-mediated events, the role of the metal ion in the DNA cleavage reaction of human topoisomerase IIβ was characterized and compared to that of topoisomerase IIα. The present study utilized divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that substituted a sulfur atom for the 3′-bridging oxygen or the non-bridging oxygens of the scissile phosphate. Based on time courses of DNA cleavage, cation titrations, and metal ion mixing experiments, we propose the following model for the use of divalent metal ions by human type II topoisomerases. First, both enzymes employ a two-metal-ion mechanism to support DNA cleavage. Second, an interaction between one divalent metal ion and the 3′-bridging atom of the scissile phosphate greatly enhances enzyme-mediated DNA cleavage, most likely by stabilizing the leaving 3′-oxygen. Third, there is an important interaction between a divalent second metal ion and a non-bridging atom of the scissile phosphate that stimulates DNA cleavage mediated by topoisomerase IIβ. If this interaction exists in topoisomerase IIα, its effects on DNA cleavage are equivocal. This last aspect of the model highlights a difference in metal ion utilization during DNA cleavage mediated by human topoisomerase IIα and IIβ.
doi:10.1021/bi8023256
PMCID: PMC2693261  PMID: 19222228
17.  Sorption of Heavy Metals to the Filamentous Bacterium Thiothrix Strain A1 
A study was undertaken to determine the ability of the filamentous bacterium Thiothrix strain A1 to sorb heavy metals from solution. Cells of Thiothrix strain A1 were harvested, washed, and suspended in solutions of metals. After an equilibration period, biomass was separated from solution and the metal content in acid-digested cells and/or filtrates was determined by atomic absorption spectrophotometry. Sorption of nickel and zinc was very rapid; most of the sorbed metal was bound in less than 10 min. The sorption data for copper fit the Freundlich isotherm, and nickel and zinc data fit biphasic Freundlich isotherms. Sorption of both nickel and zinc was dependent on cell age. Cells harvested 24 h after inoculation sorbed approximately one-half of the amount of metal per gram cell protein than did cells harvested after 48, 72, or 96 h. Calcium and magnesium effectively competed with zinc for binding sites, whereas potassium had only a slight effect on the capacity of cells to sorb zinc. The primary mechanism of metal sorption apparently was ion exchange, because 66 to 75% of nickel or zinc could be desorbed by placing metal-laden cells in a solution of 5 mM CaCl2. A competition experiment with nickel and zinc indicated that both metals occupied the same sorption sites. The strong chelating agents EDTA and NTA effectively prevented metal uptake, but lactate enhanced the uptake of nickel. Thiothrix strain A1 grown in nickel-containing medium had a relatively low uptake of nickel compared with uptake by resting cells suspended in a simple buffer solution.
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PMCID: PMC182077  PMID: 16348924
18.  Divalent Cu, Cd, and Pb Biosorption in Mixed Solvents 
Dead dried Chlorella vulgaris was studied in terms of its performance in binding divalent copper, cadmium, and lead ions from their aqueous or 50% v/v methanol, ethanol, and acetone solutions. The percentage uptake of cadmium ions exhibited a general decrease with decrease in dielectric constant values, while that of copper and lead ions showed a general decrease with increase in donor numbers. Uptake percentage becomes less sensitive to solvent properties the larger the atomic radius of the biosorbed ion, and uptake of copper was the most affected. FT-IR analyses revealed stability of the biomass in mixed solvents and a shift in vibrations of amide(I) and (II), carboxylate, glucose ring, and metal oxygen upon metal binding in all media. ΔνCOO values (59–69 cm−1) confirmed bidentate metal coordination to carboxylate ligands. The value of νasCOO increased slightly upon Cu, Cd, and Pb biosorption from aqueous solutions indicating lowering of symmetry, while a general decrease was noticed in mixed solvents pointing to the opposite. M–O stretching frequencies increased unexpectedly with increase in atomic mass as a result of solvent effect on the nature of binding sites. Lowering polarity of the solvent permits variations in metal-alga bonds strengths; the smaller the metal ion, the more affected.
doi:10.1155/2009/561091
PMCID: PMC2726428  PMID: 19688108
19.  Defining the buffering process by a triprotic acid without relying on stewart-electroneutrality considerations 
Upon the addition of protons to an aqueous solution, a component of the H+ load will be bound i.e. buffered. In an aqueous solution containing a triprotic acid, H+ can be bound to three different states of the acid as well as to OH- ions that are derived from the auto-ionization of H2O. In quantifying the buffering process of a triprotic acid, one must define the partitioning of H+ among the three states of the acid and also the OH- ions in solution in order to predict the equilibrium pH value. However, previous quantitative approaches that model triprotic acid titration behaviour and used to predict the equilibrium pH rely on the mathematical convenience of electroneutrality/charge balance considerations. This fact has caused confusion in the literature, and has led to the assumption that charge balance/electroneutrality is a causal factor in modulating proton buffering (Stewart formulation). However, as we have previously shown, although charge balance can be used mathematically as a convenient tool in deriving various formulae, electroneutrality per se is not a fundamental physicochemical parameter that is mechanistically involved in the underlying buffering and proton transfer reactions. The lack of distinction between a mathematical tool, and a fundamental physicochemical parameter is in part a reason for the current debate regarding the Stewart formulation of acid-base analysis. We therefore posed the following question: Is it possible to generate an equation that defines and predicts the buffering of a triprotic acid that is based only on H+ partitioning without incorporating electroneutrality in the derivation? Towards this goal, we derived our new equation utilizing: 1) partitioning of H+ buffering; 2) conservation of mass; and 3) acid-base equilibria. In validating this model, we compared the predicted equilibrium pH with the measured pH of an aqueous solution consisting of Na2HPO4 to which HCl was added. The measured pH values were in excellent agreement with the predictions of our equation. Our results provide further important evidence that one can mathematically model the chemistry of acid-base phenomenology without relying on electroneutrality (Stewart formulation) considerations.
doi:10.1186/1742-4682-8-29
PMCID: PMC3247848  PMID: 21849064
acid; base; proton; Stewart
20.  Quantitative and Comprehensive Decomposition of the Ion Atmosphere around Nucleic Acids 
Journal of the American Chemical Society  2007;129(48):14981-14988.
The ion atmosphere around nucleic acids critically affects biological and physical processes such as chromosome packing, RNA folding, and molecular recognition. However, the dynamic nature of the ion atmosphere renders it difficult to characterize. The basic thermodynamic description of this atmosphere, a full accounting of the type and number of associated ions, has remained elusive. Here we provide the first complete accounting of the ion atmosphere, using buffer equilibration and atomic emission spectroscopy (BE-AES) to accurately quantitate the cation association and anion depletion. We have examined the influence of ion size and charge on ion occupancy around simple, well-defined DNA molecules. The relative affinity of monovalent and divalent cations correlates inversely with their size. Divalent cations associate preferentially over monovalent cations; e.g., with Na+ in four-fold excess of Mg2+ (20 vs. 5 mM), the ion atmosphere nevertheless has three-fold more Mg2+ than Na+. Further, the dicationic polyamine putrescine2+ does not compete effectively for association relative to divalent metal ions, presumably because of its lower charge density. These and other BE-AES results can be used to evaluate and guide the improvement of electrostatic treatments. As a first step, we compare the BE-AES results to predictions from the widely-used nonlinear Poisson Boltzmann (NLPB) theory and assess the applicability and precision of this theory. In the future, BE-AES in conjunction with improved theoretical models, can be applied to complex binding and folding equilibria of nucleic acids and their complexes, to parse the electrostatic contribution from the overall thermodynamics of important biological processes.
doi:10.1021/ja075020g
PMCID: PMC3167487  PMID: 17990882
21.  INACTIVATION AND REACTIVATION OF B. MEGATHERIUM PHAGE 
The Journal of General Physiology  1955;39(2):225-249.
Preparation of Reversibly Inactivated (R.I.) Phage.— If B. megatherium phage (of any type, or in any stage of purification) is suspended in dilute salt solutions at pH 5–6, it is completely inactivated; i.e., it does not form plaques, or give rise to more phage when mixed with a sensitive organism (Northrop, 1954). The inactivation occurs when the phage is added to the dilute salt solution. If a suspension of the inactive phage in pH 7 peptone is titrated to pH 5 and allowed to stand, the activity gradually returns. The inactivation is therefore reversible. Properties of R.I. Phage.— The R.I. phage is adsorbed by sensitive cells at about the same rate as the active phage. It kills the cells, but no active phage is produced. The R.I. phage therefore has the properties of phage "ghosts" (Herriott, 1951) or of colicines (Gratia, 1925), or phage inactivated by ultraviolet light (Luria, 1947). The R.I. phage is sedimented in the centrifuge at the same rate as active phage. It is therefore about the same size as the active phage. The R.I. phage is most stable in pH 7, 5 per cent peptone, and may be kept in this solution for weeks at 0°C. The rate of digestion of R.I. phage by trypsin, chymotrypsin, or desoxyribonuclease is about the same as that of active phage (Northrop, 1955 a). Effect of Various Substances on the Formation of R.I. Phage.— There is an equilibrium between R.I. phage and active phage. The R.I. form is the stable one in dilute salt solution, pH 5 to 6.5 and at low temperature (<20°C.). At pH >6.5, in dilute salt solution, the R.I. phage changes to the active form. The cycle, active ⇌ inactive phage, may be repeated many times at 0°C. by changing the pH of the solution back and forth between pH 7 and pH 6. Irreversible inactivation is caused by distilled water, some heavy metals, concentrated urea or quanidine solutions, and by l-arginine. Reversible inactivation is prevented by all salts tested (except those causing irreversible inactivation, above). The concentration required to prevent R.I. is lower, the higher the valency of either the anion or cation. There are great differences, however, between salts of the same valency, so that the chemical nature as well as the valency is important. Peptone, urea, and the amino acids, tryptophan, leucine, isoleucine, methionine, asparagine, dl-cystine, valine, and phenylalanine, stabilize the system at pH 7, so that no change occurs if a mixture of R.I. and active phage is added to such solutions. The active phage remains active and the R.I. phage remains inactive. The R.I. phage in pH 7 peptone becomes active if the pH is changed to 5.0. This does not occur in solutions of urea or the amino acids which stabilize at pH 7.0. Kinetics of Reversible Inactivation.— The inactivation is too rapid, even at 0° to allow the determination of an accurate time-inactivation curve. The rate is independent of the phage concentration and is complete in a few seconds, even in very dilute suspensions containing <1 x 104 particles/ml. This result rules out any type of bimolecular reaction, or any precipitation or agglutination mechanism, since the minimum theoretical time for precipitation (or agglutination) of a suspension of particles in a concentration of only 1 x 104 per ml. would be about 300 days even though every collision were effective. Mechanism of Salt Reactivation.— Addition of varying concentrations of MgSO4 (or many other salts) to a suspension of either active or R.I. phage in 0.01 M, pH 6 acetate buffer results in the establishment of an equilibrium ratio for active/R.I. phage. The higher the concentration of salt, the larger proportion of the phage is active. The results, with MgSO4, are in quantitative agreement with the following reaction: See PDF for Equation Effect of Temperature.— The rate of inactivation is too rapid to be measured with any accuracy, even at 0°C. The rate of reactivation in pH 5 peptone, at 0 and 10°, was measured and found to have a temperature coefficient Q10 = 1.5 corresponding to a value of E (Arrhenius' constant) of 6500 cal. mole–1. This agrees very well with the temperature coefficient for the reactivation of denatured soy bean trypsin inhibitor (Kunitz, 1948). The equilibrium between R.I. and active phage is shifted toward the active side by lowering the temperature. The ratio R.I.P./AP is 4.7 at 15° and 2.8 at 2°. This corresponds to a change in free energy of –600 cal. mole–1 and a heat of reaction of 11,000. These values are much lower than the comparative one for trypsin (Anson and Mirsky, 1934 a) or soy bean trypsin inhibitor (Kunitz, 1948). Neither the inactivation nor the reactivation reactions are affected by light. The results in general indicate that there is an equilibrium between active and R.I. phage. The R.I. phage is probably an intermediate step in the formation of inactive phage. The equilibrium is shifted to the active side by lowering the temperature, adjusting the pH to 7–8 (except in the presence of high concentrations of peptone), raising the salt concentration, or increasing the valency of the ions present. The reaction may be represented by the following: See PDF for Equation The assumption that the active/R.I. phage equilibrium represents an example of native/denatured protein equilibrium predicts all the results qualitatively. Quantitatively, however, it fails to predict the relative rate of digestion of the two forms by trypsin or chymotrypsin, and also the effect of temperature on the equilibrium.
PMCID: PMC2147528  PMID: 13271723
22.  Regulation of the L-lactase dehydrogenase from Lactobacillus casei by fructose-1,6-diphosphate and metal ions. 
Journal of Bacteriology  1975;121(3):777-784.
The lactate dehydrogenase of Lactobacillus casei, like that of streptococci, requires fructose-1,6-diphosphate (FDP) for activity. The L. casei enzyme has a much more acidic pH optimum (pH 5.5) than the streptococcal lactate dehydrogenases. This is apparently due to a marked decrease in the affinity of the enzyme for the activator with increasing pH above 5.5; the concentration of FDP required for half-maximal velocity increase nearly 1,000-fold from 0.002 mM at pH 5.5 to 1.65 mM at 6.6. Manganous ions increase the pH range of activity particularly on the alkaline side of the optimum by increasing the affinity for FDP. This pH dependent metal ion activation is not specific for Mn2+. Other divalent metals, Co2+, Cu2+, Cd2+, Ni2+, Fe2+, Fe2+, and Zn2+ but not Mg2+, will effectively substitute for Mn2+, but the pH dependence of the activation differs with the metal ion used. The enzyme is inhibited by a number of commonly used buffering ions, particularly phosphate, citrate, and tris (hydroxymethyl) aminomethane-maleate buffers, even at low buffer concentrations (0.02 M). These buffers inhibit by affecting the binding of FDP.
PMCID: PMC246003  PMID: 234946
23.  Effect of Pressure-Induced Changes in the Ionization Equilibria of Buffers on Inactivation of Escherichia coli and Staphylococcus aureus by High Hydrostatic Pressure 
Applied and Environmental Microbiology  2013;79(13):4041-4047.
Survival rates of Escherichia coli and Staphylococcus aureus after high-pressure treatment in buffers that had large or small reaction volumes (ΔV°), and which therefore underwent large or small changes in pH under pressure, were compared. At a low buffer concentration of 0.005 M, survival was, as expected, better in MOPS (morpholinepropanesulfonic acid), HEPES, and Tris, whose ΔV° values are approximately 5.0 to 7.0 cm3 mol−1, than in phosphate or dimethyl glutarate (DMG), whose ΔV° values are about −25 cm3 mol−1. However, at a concentration of 0.1 M, survival was unexpectedly better in phosphate and DMG than in MOPS, HEPES, or Tris. This was because the baroprotective effect of phosphate and DMG increased much more rapidly with increasing concentration than it did with MOPS, HEPES, or Tris. Further comparisons of survival in solutions of salts expected to cause large electrostriction effects (Na2SO4 and CaCl2) and those causing lower electrostriction (NaCl and KCl) were made. The salts with divalent ions were protective at much lower concentrations than salts with monovalent ions. Buffers and salts both protected against transient membrane disruption in E. coli, but the molar concentrations necessary for membrane protection were much lower for phosphate and Na2SO4 than for HEPES and NaCl. Possible protective mechanisms discussed include effects of electrolytes on water compressibility and kosmotropic and specific ion effects. The results of this systematic study will be of considerable practical significance in studies of pressure inactivation of microbes under defined conditions but also raise important fundamental questions regarding the mechanisms of baroprotection by ionic solutes.
doi:10.1128/AEM.00469-13
PMCID: PMC3697583  PMID: 23624471
24.  A new class of inhibitors of peptide sorption and acylation in PLGA 
Acylation of peptides occurring within controlled-release depots prepared from copolymers of lactic and glycolic acid (PLGA) is a degradation reaction that may compromise product safety and efficacy. As peptide sorption to PLGA is believed to be a common precursor to peptide acylation, a new method to inhibit acylation is presented involving disruptors of peptide sorption, namely, inorganic divalent cations. Kinetics of sorption of a model peptide, octreotide acetate, to free-acid end-group PLGA was monitored in the presence and absence of water-soluble inorganic divalent cationic salts in HEPES buffer solution (pH 7.4, 37 °C). Sorption of cations and octreotide attained pseudo-equilibrium by 24 h. From 24-h sorption isotherms, all cations studied inhibited octreotide sorption to PLGA—the inhibiting effect of the cations increased in the order: Na+ < Mg2+ < Ca2+, Sr2+ < Ni2+ < Mn2+. Long-term inhibition of octreotide sorption in the presence of 15 mM CaCl2 and MnCl2 translated to decreased acylated octreotide present in solution by greater than 50% at 21 days incubation, i.e., from 32% in the cation-free control to 14 and 13% for CaCl2 and MnCl2, respectively. Over one month in vitro release, PLGA implants encapsulating octreotide acetate and CaCl2 or MnCl2 also showed substantial inhibition of acylation relative to no salt or NaCl controls, and similarly, strong inhibition of acylation upon divalent salt incorporation was observed during solvent extrusion of suspended peptide with polar organic carrier solvents. Hence, disrupting peptide sorption to PLGA with addition of inorganic divalent cations is a simple and viable strategy to inhibit acylation of peptides in PLGA delivery systems.
doi:10.1016/j.jconrel.2009.03.006
PMCID: PMC4269232  PMID: 19318114
acylation; octreotide acetate; sorption; divalent cations; PLGA
25.  Quantitative analysis of the ion-dependent folding stability of DNA triplexes 
Physical biology  2011;8(6):066006.
A DNA triplex is formed through binding of a third strand to the major groove of a duplex. Due to the high charge density of a DNA triplex, metal ions are critical for its stability. We recently developed the tightly bound ion (TBI) model for ion-nucleic acids interactions. The model accounts for the potential correlation and fluctuations of the ion distribution. We now apply the TBI model to analyze the ion-dependence of the thermodynamic stability for DNA triplexes. We focus on two experimentally studied systems: a 24-bp DNA triplex and a pair of interacting 14-bp triplexes. Our theoretical calculations for the number of bound ions indicate that the TBI model provides improved predictions for the number of bound ions than the classical Poisson-Boltzmann Equation (PB). The improvement is more significant for a triplex, which has a higher charge density, than a duplex. This is possibly due to the higher ion concentration around the triplex and hence stronger ion correlation effect for a triplex. In addition, our analysis for the free energy landscape for a pair of 14-mer triplexes immersed in an ionic solution shows that divalent ions could induced an attractive force between the triplexes. Furthermore, we investigate how the protonated cytosines in the triplexes affect the stability of the triplex helices.
doi:10.1088/1478-3975/8/6/066006
PMCID: PMC3427753  PMID: 22067830
folding stability; energy landscape; Triplex; TBI model

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