Ex vivo anti-microbial efficacy of various formaldehyde releasers against antibiotic resistant and antibiotic sensitive microorganisms involved in infectious keratitis.

BACKGROUND: Corneal infections with antibiotic-resistant microorganisms are an increasingly difficult management challenge and chemically or photochemically cross-linking the cornea for therapy presents a unique approach to managing such infections since both direct microbial pathogens killing and matrix stabilization can occur simultaneously. The present study was undertaken in order to compare the anti-microbial efficacy, in vitro, of 5 candidate cross-linking solutions against 5 different microbial pathogens with relevance to infectious keratitis. METHODS: In vitro bactericidal efficacy studies were carried out using 5 different FARs [diazolidinyl urea (DAU), 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (DMDM), sodium hydroxymethylglycinate (SMG), 2-(hydroxymethyl)-2-nitro-1,3-propanediol (NT = nitrotriol), 2-nitro-1-propanol (NP)] against 5 different microbial pathogens including two antibiotic-resistant species [methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Pseudomonas aeruginosa (PA), and Candida albicans (CA)]. Standard in vitro antimicrobial testing methods were used. RESULTS: The results for MSSA were similar to those for MRSA. DAU, DMDM, and SMG all showed effectiveness with greater effects generally observed with longer incubation times and higher concentrations. Against MRSA, 40 mM SMG at 120 min showed a > 95% kill rate, p < 0.02. Against VRE, 40 mM DAU for 120 min showed a > 94% kill rate, p < 0.001. All FARs showed bactericidal effect against Pseudomonas aeruginosa, making PA the most susceptible of the strains tested. Candida showed relative resistance to these compounds, requiring high concentrations (100 mM) to achieve kill rates greater than 50%. CONCLUSION: Our results show that each FAR compound has different effects against different cultures. Our antimicrobial armamentarium could potentially be broadened by DAU, DMDM, SMG and other FARs for antibiotic-resistant keratitis. Further testing in live animal models are indicated.

In vitro antimicrobial activity of a gel containing antimicrobial peptide AMP2041, chlorhexidine digluconate and Tris-EDTA on clinical isolates of Pseudomonas aeruginosa from canine otitis.

BACKGROUND: Pseudomonas aeruginosa (PA) may cause suppurative otitis externa with severe inflammation and ulceration in dogs. Multidrug resistance is commonly reported for this organism, creating a difficult therapeutic challenge. OBJECTIVE: The aim of this study was to evaluate the in vitro antimicrobial activity of a gel containing 0.5 µg/mL of antimicrobial peptide AMP2041, 0.07% chlorhexidine digluconate (CLX), 0.4% Tris and 0.1% EDTA on 30 clinical isolates of PA from canine otitis externa. MATERIALS AND METHODS: Antimicrobial activity was evaluated through minimal bactericidal concentration (MBC). Standardized bacterial suspensions were incubated with different concentrations of the gel at 37°C for 30 min and plated for colony forming unit (CFU) counts. Time-to-kill kinetics were evaluated with the undiluted product and at MBC for each PA strain at 30 s, 1, 5, 10, 15, 30 min, 24 and 48 h. RESULTS: The MBC was 1:64 for two of 30 strains, 1:128 for 15 of 30 strains and 1:256 for 13 of 30 strains. The geometric mean was 1:165, equivalent to a concentration of 0.003 µg/mL AMP2041 + 0.0004% CLX + 0.0024%Tris + 0.0006% EDTA. Time-to-kill assays with the undiluted product showed complete bactericidal effect within 30 s for all isolates, whereas at the MBC this effect was reached within 5 min for 20 of 30 isolates and within 30 min for all isolates. Bactericidal activity was maintained after 48 h for all isolates. CONCLUSION: This gel has shown rapid, complete and long-lasting activity against a panel of 30 PA isolates from cases of canine otitis externa.

Interaction of chlorhexidine with trisEDTA or miconazole in vitro against canine meticillin-resistant and -susceptible Staphylococcus pseudintermedius isolates from two UK regions.

BACKGROUND: Topical therapy is an important alternative to systemic antibacterial therapy for treatment of canine superficial pyoderma in light of the emergence of multidrug-resistant staphylococci. Chlorhexidine is widely used in shampoo products alone or in combination with miconazole or tromethamine-ethylenediaminetetraacetic acid (trisEDTA). Comparisons of these combinations have not been made. HYPOTHESIS/OBJECTIVES: To determine minimum inhibitory concentrations (MICs) of combinations of chlorhexidine/miconazole and chlorhexidine/trisEDTA in vitro in a collection of Staphylococcus pseudintermedius (SP) from northern (NUK) and southeastern (SEUK) United Kingdom (UK) sources. METHODS: MICs of chlorhexidine, miconazole, trisEDTA and combinations of chlorhexidine/miconazole (1:1) or chlorhexidine/trisEDTA (80:16:1 and 80:5:1) were determined for 196 canine SP isolates from NUK [49 meticillin-resistant (MRSP), 50 meticillin-susceptible (MSSP)] and fom SEUK (48 MRSP, 49 MSSP) using agar dilution. RESULTS: TrisEDTA alone did not inhibit growth. Chlorhexidine/miconazole MICs (median = 0.5 mg/L) were lower than those of either drug alone (P < 0.05) and lower than chlorhexidine/trisEDTA MICs (median = 1 mg/L; P < 0.0005) in each bacterial type and from both regions, except for miconazole in NUK MSSP. An additive interaction was noted between chlorhexidine and miconazole or trisEDTA (80:16:1 ratio) in 79 and 43 isolates, respectively, whereas antagonism between chlorhexidine and trisEDTA was noted for three isolates. NUK isolates were more susceptible than SEUK isolates (P < 0.05), except MRSP exposed to chlorhexidine and the chlorhexidine/trisEDTA (80:16:1) combination. CONCLUSIONS AND CLINICAL IMPORTANCE: These low MICs are likely to be exceeded by topical therapy. Evaluation of the mechanisms by which chlorhexidine combinations interact to reduce MICs is warranted, in view of increasing concerns of biocide tolerance in staphylococci.

Tuning Cerium(IV)-Assisted Hydrolysis of Phosphatidylcholine Liposomes under Mildly Acidic and Neutral Conditions.

With the goal of designing a lysosomal phospholipase mimic, we optimized experimental variables to enhance Ce(IV) -assisted hydrolysis of phosphatidylcholine (PC) liposomes. Our best result was obtained with the chelating agent bis-tris propane (BTP). Similar to the hydrolytic enzyme, Ce(IV) -assisted hydrolysis of PC phosphate ester bonds was higher at lysosomal pH (∼4.8) compared to pH 7.2. In the presence of BTP, the average cleavage yield at ∼pH 4.8 and 37 °C was: 67±1 %, 5.7-fold higher than at ∼pH 7.2 and roughly equivalent to the percent of phospholipid found on the metal-accessible exo leaflet of small liposomes. No Ce(IV) precipitation was observed. When BTP was absent, there was significant turbidity, and the amount of cleavage at ∼pH 4.8 (69±1 %) was 2.1-fold higher than the yield obtained at ∼pH 7.2. Our results show that BTP generates homogenous solutions of Ce(IV) that hydrolyze phosphatidylcholine with enhanced selectivity for lysosomal pH.

Electrogenerated chemiluminescence of tris(2,2' bipyridine)ruthenium(II) using common biological buffers as co-reactant, pH buffer and supporting electrolyte.

A series of aliphatic tertiary amines (HEPES, POPSO, EPPS and BIS-TRIS) commonly used to buffer the pH in biological experiments, were examined as alternative, non-toxic co-reactants for the electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(ii) ([Ru(bpy)3](2+)). These were found to be very attractive as "multi-tasking" reagents, serving not only as co-reactants, but also fulfiling the roles of pH buffer and supporting electrolyte within an aqueous environment; thus significantly simplifying the overall ECL analysis. Sub-nanomolar detection limits were obtained for [Ru(bpy)3](2+) in the presence of BIS-TRIS, making this species an valuable option for co-reactant ECL-based bioanalytical applications.

Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli.

Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human ß-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human ß-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.

Optimization of enrichment conditions on TiO2 chromatography using glycerol as an additive reagent for effective phosphoproteomic analysis.

Metal oxide affinity chromatography (MOAC) represented by titanium dioxide (TiO2) chromatography has been used for phosphopeptide enrichment from cell lysate digests prior to mass spectrometry. For in-depth phosphoproteomic analysis, it is important for MOAC to achieve high phosphopeptide enrichment efficiency by optimizing purification conditions. However, there are some differences in phosphopeptide selectivity and specificity enriched by various TiO2 materials and procedures. Here, we report that binding/wash buffers containing polyhydric alcohols, such as glycerol, markedly improve phosphopeptide selectivity from complex peptide mixtures. In addition, the elution conditions combined with secondary amines, such as bis-Tris propane, made it possible to recover phosphopeptides with highly hydrophobic properties and/or longer peptide lengths. To assess the practical applicability of our improved method, we confirmed using PC3 prostate cancer cells. By combining the hydrophilic interaction chromatography (HILIC) with the optimized TiO2 enrichment method prior to LC-MS/MS analysis, over 8300 phosphorylation sites and 2600 phosphoproteins were identified. Additionally, some dephosphorylations of those were identified by treatment with dasatinib for a kinase inhibitor. These results indicate that our method is applicable to understanding the profiling of kinase inhibitors such as anticancer compounds, which will be useful for drug discovery and development.

Prestorage in ovo injection of biological buffers: an approach to improve hatchability in long-term stored eggs.

A hypothesis was tested that the in ovo injection of biological buffers may reinforce the buffering capacity of albumen, thereby withstanding the increase in albumen pH during storage and improving hatchability and chick quality in long-term stored eggs. Hatching eggs (n = 2,420) were randomly assigned to 11 treatment groups (4 replicates of 55 eggs each) and injected (d 1) with distilled water, 25 or 50 mM HEPES (H25 and H50), Bicine (B25 and B50), Tris (T25 and T50), and Bis-Tris-propane (BTP25 and BTP50) solutions or were not injected (intact: control; or pricked with a needle: N). The eggs were then stored for 14 d during which the egg internal characteristics were evaluated at 1, 2, 3, 4, 5, 8, and 13 d of storage (n = 924 in total) and the remaining eggs (n = 1,496) were incubated. A decrease in albumen pH was found for H25, H50, B50, and BTP25 groups from 2 through 5 d postinjection. Eggs receiving H25, H50, and B50 recorded a higher albumen index (at 13 d of storage) and Haugh unit (between 8 and 13 d of storage) compared with the control. Interestingly, the hatchability of fertile eggs was influenced by the treatment effect (P = 0.0001) where the eggs receiving H25 (88.3%), H50 (88.9%), B50 (88.4%), and BTP25 (87.6%) recorded higher values than that of control (82.1%), associated with a decreased early embryonic mortality rate (P < 0.0001). In ovo injection of Tris buffer, however, profoundly decreased the hatchability (47.2 and 29.0% for T25 and T50, respectively) and percentage of first-grade chicks (67.5 and 63.6% for T25 and T50, respectively) compared with the control (90.1%). In conclusion, prestorage in ovo injection of H25, H50, B50, and BTP25 improved hatchability in long-term stored eggs in which a decreased albumen pH during the d 2 through 5 of storage period might be involved.

DCAP: a broad-spectrum antibiotic that targets the cytoplasmic membrane of bacteria.

Persistent infections are frequently caused by dormant and biofilm-associated bacteria, which often display characteristically slow growth. Antibiotics that require rapid cell growth may be ineffective against these organisms and thus fail to prevent reoccurring infections. In contrast to growth-based antimicrobial agents, membrane-targeting drugs effectively kill slow-growing bacteria. Herein we introduce 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), a potent broad-spectrum antibiotic that reduces the transmembrane potential of Gram-positive and Gram-negative bacteria and causes mislocalization of essential membrane-associated proteins, including MinD and FtsA. Importantly, DCAP kills nutrient-deprived microbes and sterilizes bacterial biofilms. DCAP is lethal against bacterial cells, has no effect on red blood cell membranes, and only decreases the viability of mammalian cells after ≥6 h. We conclude that membrane-active compounds are a promising solution for treating persistent infections. DCAP expands the limited number of compounds in this class of therapeutic small molecules and provides new opportunities for the development of potent broad-spectrum antimicrobial agents.

Crystallization and preliminary X-ray diffraction analysis of mevalonate kinase from Methanosarcina mazei.

Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.32 M MgCl2, 0.08 M bis-tris pH 5.5, 16%(w/v) PEG 3350. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a=97.11, b=135.92, c=46.03 Å. Diffraction data were collected to 2.08 Šresolution.

Comparison of oral nonsteroidal analgesic and intrauterine local anesthetic for pain relief in uterine fractional curettage: a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: We sought to investigate the analgesic efficacy of oral dexketoprofen trometamol and intrauterine lidocaine in patients undergoing fractional curettage. STUDY DESIGN: A randomized, double-blind, placebo-controlled trial was conducted on 111 women. Subjects were randomly assigned into 4 groups to receive either 25 mg of dexketoprofen or similar-appearing placebo tablets and either 5 mL intrauterine 2% lidocaine or saline. The main outcome measure was the intensity of pain measured by a 10-cm visual analog scale. Pain scoring was performed prior to, during, and 30 minutes after the procedure. RESULTS: No statistically significant difference was found among the mean pain scores of women during the procedure in the dexketoprofen and saline, placebo and lidocaine, and dexketoprofen and lidocaine groups. The mean pain scores in all 3 groups revealed significant reduction when compared with placebo and saline combination (P = .001). CONCLUSION: Administration of intrauterine lidocaine or oral dexketoprofen appears to be effective in relieving fractional curettage associated pain. However, a combination of them does not work better in further reduction of pain.

Acute coronary artery vasospasm associated with misoprostol for termination of pregnancy.

Prostaglandin E(1) analogues, gemeprost and misoprostol, are the most widely used drugs for medical termination of pregnancy within the first two trimesters of pregnancy. Gemeprost has been reported to be associated with acute cardiovascular accidents in a few cases, but no adverse cardiovascular events have been reported with misoprostol. For this reason, misoprostol has been considered a potentially safer drug and is widely recommended as a first-choice drug in combination with mifepristone. We report a case of a 32-year-old woman with active smoking and obesity, who developed an episode of transient acute coronary artery vasospasm following the administration of intravaginal misoprostol. This report illustrates that misoprostol can also be associated with acute coronary events, although it remains to be evaluated whether the risk is similar or lower to gemeprost.

Buffer interference with protein dynamics: a case study on human liver fatty acid binding protein.

Selection of suitable buffer types is often a crucial step for generating appropriate protein samples for NMR and x-ray crystallographic studies. Although the possible interaction between MES buffer (2-(N-morpholino)ethanesulfonic acid) and proteins has been discussed previously, the interaction is usually thought to have no significant effects on the structures of proteins. In this study, we demonstrate the direct, albeit weak, interaction between MES and human liver fatty acid binding protein (hLFABP). Rather than affecting the structure of hLFABP, we found that the dynamics of hLFABP, which were previously proposed to be relevant to its functions, were significantly affected by the binding of hLFABP with MES. Buffer interference with protein dynamics was also demonstrated with Bis-Tris buffer, which is quite different from MES and fatty acids in terms of their molecular structures and properties. This result, to our knowledge, is the first published report on buffer interference with protein dynamics on a microsecond to millisecond timescale and could represent a generic problem in the studies of functionally relevant protein dynamics. Although being a fortuity, our finding of buffer-induced changes in protein dynamics offers a clue to how hLFABP accommodates its ligands.

Free-metal ion depletion by "Good's" buffers.

Metal-ion affinity (formation) constants were determined for two "Good's" buffers, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) and N,N-bis(2-hydroxyethyl)glycine (bicine). The metal chelates formed undergo loss of an internal ligand (alcohol) proton (bicine) and undergo hydrolysis (bicine and TES) and dimerization reactions (TES). Bicine and TES buffer not only hydrogen ions but also metal ions. The metal complexes of "Good's" buffers also buffer hydrogen ions by secondary reactions. The consequences of these reactions are considered in relation to biomedical research.

Postmarketing cohort study to assess the safety profile of oral dexketoprofen trometamol for mild to moderate acute pain treatment in primary care.

Recently, new concerns on the safety profile of nonsteroidal anti-inflammatory drugs (NSAIDs) have been raised by the European Medicines Agency (EMEA) and other regulatory authorities. The safety profile of oral dexketoprofen trometamol for the treatment of acute mild to moderate pain of different causes in actual conditions of use in the primary care setting was assessed. A prospective cohort study was designed to evaluate the tolerability of dexketoprofen compared with other commonly prescribed analgesics. Medications were given according to specifications in the summary of product characteristics. The intensity of pain was assessed at baseline and at days 1 and 7 of drug treatment using a 100-mm visual analog scale (VAS). Adverse events (AEs) were recorded. A total of 7,337 patients (median age [IQR] = 46 [33-61] years) were included in the study comparing dexketoprofen (n = 5,429), diclofenac (n = 485), ibuprofen (n = 479), paracetamol (n = 459), metamizole (n = 207), aceclofenac (n = 103), naproxen (n = 74), piroxicam (n = 69) and dexibuprofen (n = 32). The reasons for use were: musculoskeletal disorders, headache, dysmenorrhea and odontalgia. Treatment compliance was very high. Metamizole-paracetamol and dexketoprofen showed the lowest prevalence of AEs (2.7% and 3.6%, respectively), while aceclofenac-diclofenac showed the highest prevalence (8.2%) (P < 0.0001). AEs most frequently observed during NSAID treatment were those related to the gastrointestinal tract (3.5% of subjects, 84% of all AEs), followed by AEs related to the nervous system (0.4%) and skin (0.1%). Most of the reported AEs (91.3%) were of mild to moderate intensity (303 of 332) and only 3.3% of them were considered severe (11 of 332). Risks for gastrointestinal AEs were adjusted for age, gender, history of previous NSAID intake, gastroprotective drugs and reason for prescription. Taking metamizole-paracetamol as the reference group, the odds ratios (OR, 95%) were: 1.30 (0.77-2.19) for dexketoprofen, 1.57 (0.79-3.13) for ibuprofen and dexibuprofen, 2.31 (0.64-8.27) for naproxen, 2.63 (0.85-8.15) for piroxicam and 3.37 (1.87-6.06) for aceclofenac-diclofenac. These results confirm the safety of oral treatment with dexketoprofen in patients with acute pain of various etiologies observed in previous studies and support the use of dexketoprofen as a first-line drug for the approved therapeutic indications.

[Dexketoprofen trometamol in the treatment of chronic prostatitis/chronic pelvic pain syndrome].

OBJECTIVE: To evaluate the clinical efficacy and safety of dexketoprofen trometamol in the treatment of patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). METHODS: A total of 115 patients with CP/CPPS were divided into a dexketoprofen trometamol group (n = 40), treated with dexketoprofen trometamol (25 mg, tid) and terazosin (2 mg, qn), an indometacin group (n = 40) given indometacin (25 mg, tid) and terazosin (2 mg, qn), and a terazosin group (n = 35) administered terazosin (2 mg, qn) only, all treated for 4 weeks. Scores on the NIH-chronic prostatitis symptom index (NIH-CPSI) were obtained before and after the treatment, and the efficacy and adverse events were observed and compared. RESULTS: The NIH-CPSI scores were significantly improved after the treatment in all the three groups. The clinical efficacy was significantly better in the dexketoprofen trometamol and indometacin groups than in the terazosin group (P < 0.05), but with no significant difference between the former two (P > 0.05). The rates of adverse events were 10.00%, 18.57% and 27.50% in the dexketoprofen trometamol, terazosin and indometacin groups, significantly lower in the former two than in the latter one (P < 0.05). CONCLUSION: The combination of dexketoprofen trometamol with terazosin could effectively improve the clinical symptoms of CP/CPPS, better than terazosin in therapeutic efficacy and than indometacin in drug tolerance.

In depth, thermodynamic analysis of Ca2+ binding to human cardiac troponin C: Extracting buffer-independent binding parameters.

BACKGROUND: Characterizing the thermodynamic parameters behind metal-biomolecule interactions is fundamental to understanding the roles metal ions play in biology. Isothermal Titration Calorimetry (ITC) is a "gold-standard" for obtaining these data. However, in addition to metal-protein binding, additional equilibria such as metal-buffer interactions must be taken into consideration prior to making meaningful comparisons between metal-binding systems. METHODS: In this study, the thermodynamics of Ca2+ binding to three buffers (Bis-Tris, MES, and MOPS) were obtained from Ca2+-EDTA titrations using ITC. These data were used to extract buffer-independent parameters for Ca2+ binding to human cardiac troponin C (hcTnC), an EF-hand containing protein required for heart muscle contraction. RESULTS: The number of protons released upon Ca2+ binding to the C- and N-domain of hcTnC were found to be 1.1 and 1.2, respectively. These values permitted determination of buffer-independent thermodynamic parameters of Ca2+-hcTnC binding, and the extracted data agreed well among the buffers tested. Both buffer and pH-adjusted parameters were determined for Ca2+ binding to the N-domain of hcTnC and revealed that Ca2+ binding under aqueous conditions and physiological ionic strength is both thermodynamically favorable and driven by entropy. CONCLUSIONS: Taken together, the consistency of these data between buffer systems and the similarity between theoretical and experimental proton release is indicative of the reliability of the method used and the importance of extracting metal-buffer interactions in these studies. GENERAL SIGNIFICANCE: The experimental approach described herein is clearly applicable to other metal ions and other EF-hand protein systems.

Elimination of ghost peaks by optimization of anion exchange chromatography method for determination of gamma-carboxyglutamic acid (Gla)-domainless impurity in recombinant activated clotting factor VII drug products.

Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories. Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73. To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises. By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks.

Structure of the two-subsite beta-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane.

The three-dimensional structure of the catalytically efficient beta-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3A resolution). Most H bonds between inhibitor and protein occur within subsite -1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. E186 (pK(a) 7.2) serves as catalytic acid. The pH (6-10) profile for 1/K(i)((BTP)) is bell-shaped with pK(a)'s 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.9 on the basic limb assigned to inhibitor. Mutation K99A eliminates pK(a) 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14(-)E186(-). A sedimentation equilibrium experiment estimates a K(d) ([dimer](2)/[tetramer]) of 7 x 10(-9)M. Similar k(cat) and k(cat)/K(m) values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms.

Separation of organic cations using novel background electrolytes by capillary electrophoresis.

A background electrolyte for capillary electrophoresis containing tris(-hydroxymethyl) aminomethane (THAM) and ethanesulfonic acid (ESA) gives excellent efficiency for separation of drug cations with actual theoretical plate numbers as high as 300,000. However, the analyte cations often elute too quickly and consequently offer only a narrow window for separation. The best way to correct this is to induce a reverse electroosmotic flow (EOF) that will spread out the peaks by slowing their migration rates, but this has always been difficult to accomplish in a controlled manner. A new method for producing a variable EOF is described in which a low variable concentration of tributylammonium- or triethylammonium ESA is added to the BGE. The additive equilibrates with the capillary wall to give it a positive charge and thereby produce a controlled opposing EOF. Excellent separations of complex drug mixtures were obtained by this method.