Iodoacetic Acid Research Articles

A New Strategy of Using O18-Labeled Iodoacetic Acid for Mass Spectrometry-Based Protein Quantitation

A new O(18) labeling protocol is designed to assist quantitation of cysteine-containing proteins using LC/MS. Unlike other O(18) labeling strategies, the labeling is carried out at the intact protein level (prior to its digestion) during reduction/alkylation of cysteine side chains using O(18)-labeled iodoacetic acid (IAA). The latter can be easily prepared by exchanging carboxylic oxygen atoms of commercially available IAA in O(18)-enriched water at low pH. Since incorporation of the O(18) label in the protein occurs at the whole protein, rather than peptide level, the quantitation results are not peptide-dependent. The excellent stability of the label in mild pH conditions provides flexibility and robustness needed of sample processing steps following the labeling. In contrast to generally costly isotope labeling reagents, this approach uses only two relatively inexpensive commercially available reagents (IAA and H(2)O(18)). The feasibility of the new method is demonstrated using an 80kDa human serum transferrin (hTf) as a model, where linear quantitation is achieved across a dynamic range spanning three orders of magnitude. The new approach can be used in quantitative proteomics applications and is particularly suitable for a variety of tasks in the biopharmaceutical sector, ranging from pharmacokinetic studies to quality control of protein therapeutics.

Mapping of sulfur metabolic pathway by LC Orbitrap mass spectrometry

For the first time a liquid chromatography method with high resolution mass spectrometric detection has been developed for the simultaneous determination all key metabolites of the sulfur pathway in yeast, including all thiolic (cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), γ-glutamyl-cysteine (Glu-Cys)) and non-thiolic compounds (methionine (Met), s-adenosyl-methionine (AdoMet), s-adenosyl-homocysteine (AdoHcy), and cystathionine (Cysta)). The developed assay also permits the speciation and selective determination of reduced, oxidized and protein bound fractions of all of the five thiols. Iodoacetic acid (IAA) was chosen as the derivatizing reagent. Thiols were extracted from sub-mg quantities of yeast using hot 75% ethanol. The detection limits were in the range of 1–12nmolL−1 for standard solution (high femotomole, absolute), except AdoMet (116nmolL−1), which was unstable. In freshly harvested yeast, most of the thiols were in the reduced forms and low levels of protein-bound GSH and Glu-Cys were found. In a selenium enriched yeast, the thiols were mainly in the oxidized forms, and a significant amount of protein-bound Cys, HCys, GSH, Cys-Gly and Glu-Cys were found. The method was also applied to the metabolic study of the adaptive response of Saccharomyces cerevisiae to hydrogen peroxide, cadmium, and arsenite, and the change in concentration of thiols in the sulfur pathway was monitored over a period of 4h.

Abstract 2324: Translational Stroke Research: Identification Of A Novel Family Of Natural Product Derived Flavonoid-Based Polyphenols With Potent Pleiotropic Neuroprotective Activity.

Multiple plant (fruits, vegetables) and spice (turmeric)-derived natural products, which have pleiotropic activities (anti-inflammatory, antioxidant, anti-apoptotic, neurogenic) are also neuroprotective in vitro against numerous chemical insults and promote behavioral recovery in vivo following embolic strokes. We have focused on 2 types of natural products: 1) curcumin-based compounds such as CNB-001 and 2) flavone-based compounds such as fisetin and baicalein. We have synthesized a new library of flavonoid molecules to improve their pharmacological characteristics and have selected candidates using a battery of vitro screening assays with stringent criteria: 1) HT22 hippocampal cell survival (in vitro ischemia model using 20µM iodoacetic acid); 2) primary mouse cortical neurons cell survival (in vitro excitotoxicity assay using 10µM glutamate); 3) HT22 hippocampal cell survival assay (oxytosis using 5 mM glutamate to induce non-NMDA mediated cell death due to the absence of NMDA receptors on HT22 cells). EC50 ≤ 100 nM and survival ≥80% in the IAA assay, ≥35% survival in the cortical neuron assay and EC 50 ≤ 300 nM and ≥50% survival in the oxytosis assay. All compounds were screened in triplicate using concentrations up to 10 µM. We chemically modified fisetin to improve its potency, increase blood brain barrier penetration (CLogP, an indicator of blood brain barrier penetration) and reduce tPSA (topographical polar surface area). We have developed a structure activity relationship (SAR) indicating that the absence of a hydroxyl group on the A ring of fisetin enhances its potency. Two compounds PM-001 and PM-004) are 6 fold more potent than fisetin. Altering a hydroxyl group at the 3-position of the B ring has no impact on activity. The addition of multiple hydrophobic groups to various positions on the A ring, such as the addition of methyl groups at the 6 and/or 7 position improved potency ∼10-100 fold over fisetin (e.g. compound PM-010) while the addition of a more hydrophobic naphthalene group (PM-002) and (PM-003) improved the potency ∼40 fold over fisetin and increased the ClogP and reduced the tPSA. SAR studies on the C ring suggested that hydrogen bond accepting groups such as a methoxy at the 4' position and a hydrogen bond donating group such as a hydroxyl at the 3' position further enhanced potency. For example compound CMS-064, CMS-069 and CMS-092 are some of the most potent compounds, with EC 50 s below 40 nM. These compounds also have CLogPs and tPSAs consistent with enhanced brain penetration. In conclusion, we have used a rational medicinal chemistry approach in combination with in vitro screening analysis to identify new flavonoid molecules to optimally promote neuroprotection. The molecules which will be discussed in detail are being further characterized and de-risked to treat stroke.

Iodoacetic acid, but not sodium iodate, creates an inducible swine model of photoreceptor damage

Our purpose was to find a method to create a large animal model of inducible photoreceptor damage. To this end, we tested in domestic swine the efficacy of two chemical toxins, known to create photoreceptor damage in other species: Iodoacetic Acid (IAA) and Sodium Iodate (NaIO3). Intravenous (IV) administration of NaIO3 up to 90 mg/kg had no effect on retinal function and 110 mg/kg was lethal. IV administration of IAA (5–20 mg/kg) produced concentration-dependent changes in visual function as measured by full-field and multi-focal electroretinograms (ffERG and mfERG), and 30 mg/kg IAA was lethal. The IAA-induced effects measured at two weeks were stable through eight weeks post-injection, the last time point investigated. IAA at 7.5, 10, and 12 mg/kg produce a concentration-dependent reduction in both ffERG b-wave and mfERG N1–P1 amplitudes compared to baseline at all post-injection times. Comparisons of dark- and light-adapted ffERG b-wave amplitudes show a more significant loss of rod relative to cone function. The fundus of swine treated with ≥10 mg/kg IAA was abnormal with thinner retinal vessels and pale optic discs, and we found no evidence of bone spicule formation. Histological evaluations show concentration-dependent outer retinal damage that correlates with functional changes. We conclude that NaIO3, is not an effective toxin in swine. In contrast, IAA can be used to create a rapidly inducible, selective, stable and concentration-dependent model of photoreceptor damage in swine retina. Because of these attributes this large animal model of controlled photoreceptor damage should be useful in the investigation of treatments to replace damaged photoreceptors.

Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots

The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a (15)N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (K(S)) and degradation (K(D)) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify K(S) and K(D) for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. K(S) and K(D) correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive (15)N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.

Key Role of Glycogen Storage in High K+-Induced Contraction of the Smooth Muscles of the Bovine Trachea

To elucidate the role of glycogen in the contraction of tracheal smooth muscle, we investigated the changes in the glycogen contents of the bovine trachea during contractions induced by high K(+) and hypoxia (achieved by bubbling N(2) instead of O(2)), either in a glucose-free condition or in the presence of iodoacetic acid (IAA), an inhibitor of glycolysis. Hyperosmotic addition of 65 mM KCl (H-65 K(+)) induced a sustained contraction. A glucose-free condition did not affect H-65 K(+)-induced contraction. However, hypoxia slightly inhibited the contraction, and glucose-free PSS with hypoxia or IAA remarkably inhibited the H-65 K(+)-induced contraction. H-65 K(+) induced a sustained increase in reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity. Hypoxia alone slightly enhanced PNred fluorescence, and when combined with a glucose-free condition, it remarkably enhanced the H-65 K(+)-induced PNred fluorescence. IAA inhibited PNred fluorescence. In the presence of H-65 K(+), a glucose-free condition, hypoxia and the combination of glucose-free PSS and hypoxia decreased the glycogen contents. However, IAA had no effect on glycogen contents. Although hypoxia or glucose-free PSS did not affect PCr and ATP contents, the combination of hypoxia and glucose-free PSS or IAA induced a gradual decrease of PCr content. In conclusion, we suggest that endogenous glycogen was utilized to increase the activity of glycolysis for maintaining high K(+)-induced contraction of the bovine trachea in the glucose -free and/or hypoxic condition.

Inhibitory Effect of Furosemide on Non-Selective Voltage-Independent Cation Channels in Human Erythrocytes

Background: Furosemide, a loop diuretic inhibiting the renal tubular Na⁺,K⁺,2Cl^- cotransporter, has been shown to decrease cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in platelets and erythrocytes. [Ca²⁺]_i in erythrocytes is a function of Ca²⁺ permeable cation channels. Activation of those channels e.g. by energy depletion or oxidative stress leads to increase of [Ca²⁺]_i, which in turn triggers eryptosis, a suicidal erythrocyte death characterized by cell membrane scrambling. The present study was performed to explore whether furosemide influences the cation channels and thus influences eryptosis. Methods: Cation channel activity was determined by whole-cell patch clamp, [Ca²⁺]_i utilizing Fluo3 fluorescence and annexin V binding to estimate cell membrane scrambling with phosphatidylserine exposure. Results: A 45 min exposure to furosemide (10 and 100 µM) slightly, but significantly decreased cation channel activity and [Ca²⁺]_i in human erythrocytes drawn from healthy individuals. ATP-depletion (> 3 hours, +37°C, 6 mM ionosine and 6 mM iodoacetic acid) enhanced the non-selective cation channel activity, increased [Ca²⁺]_i and triggered cell membrane scrambling, effects significantly blunted by furosemide (10 – 100 µM). Oxidative stress by exposure to tert-butylhydroperoxide (0.1 –1 mM) similarly enhanced the non-selective cation channels activity, increased [Ca²⁺]_i and triggered cell membrane scrambling, effects again significantly blunted by furosemide (10 – 100 µM). Conclusions: The present study shows for the first time that the loop diuretic furosemide applied at micromolar concentrations (10 – 100 µM) inhibits non-selective cation channel activity in and eryptosis of human erythrocytes.

Nontoxic Medical Imaging Agents Form Toxic DBPs

Iodinated X-ray contrast media (ICM), used medically to highlight soft tissues, are considered generally safe, and patients excrete 95% of the dose within 24 hours, making them a valuable tool in medical imaging. But a new study shows that excreted ICM react with chloramine and chlorine at water treatment plants to form toxic iodinated disinfection by-products (iodo-DBPs).1 “ICM are definitely a source of iodo-DBPs in drinking water,” says study coauthor Susan Richardson, a research chemist at the U.S. Environmental Protection Agency (EPA) in Athens, Georgia, although she says natural iodide salts from saltwater intrusion are probably the major source of these contaminants. In an earlier study of drinking water collected from 23 cities, Richardson and toxicologist Michael Plewa of the University of Illinois at Urbana–Champaign detected iodo-acids and iodo-trihalomethanes, both highly toxic iodo-DBPs.2 They assumed that the source of iodine was natural iodide salts from intrusion of ocean water or natural minerals in the source water. Yet surprisingly, some samples had high concentrations of iodo-DBPs despite little or no detectable natural iodide in the water. Their new study, done in collaboration with Stephen Duirk at the University of Akron and Thomas Ternes at the German Federal Institute of Hydrology, suggests that ICM, which contain three iodines per molecule, were an unexpected source of iodine in 10 of the original 23 cities. Further laboratory tests confirmed that ICM form iodo-DBPs in the presence of chlorine and chloramine, which are commonly used to treat drinking water.1 ICM are nearly impossible to remove from water by conventional treatment methods, says environmental engineer David Sedlak, codirector of the Berkeley Water Center at the University of California, Berkeley, and the compounds end up in rivers used for drinking water.3 Richardson’s team screened drinking water for five common ICM (io-pamidol, iopromide, diatrizoate, iomeprol, and iohexol). Iopamidol was detected in the highest concentrations—up to 2,700 ng/L, compared with less than 100 ng/L for the majority of the other ICM—and was chosen as the test ICM for controlled experiments. When iopamidol was dissolved in river water spiked with chlorine or chloramine, high levels of iodoacetic acid and iodo-trihalomethanes were produced in a dose-dependent manner. In comparison, no iodo-DBPs formed in the absence of iopamidol in river water containing the two disinfectants. Iodo-DBPs form best at pH 6.5–8.5, conditions common at water treatment plants.1 ICM are a major medical breakthrough—“We don’t want to stop using them,” Richardson says. Other types of ICM may form lower concentrations of iodo-DBPs than io-pamidol, and Richardson will test this hypothesis and seek insight into how ICM interact with chlorinated disinfectants. Better understanding of the reactions involved could ultimately prevent toxic by-products. More than 600 DBPs are known, and Plewa’s laboratory has systematically analyzed about 80 for genotoxicity and cytotoxicity in mammalian cells. Iodo-DBPs are more toxic than brominated and chlorinated analogs, and iodoacetic acid is the most genotoxic of all DBPs tested so far.4 Iodo-DBPs are being investigated for links to bladder cancer and birth defects.5,6 The EPA regulates some chlorinated and brominated DBPs in drinking water but not iodo-DBPs, whose toxicity is only just being recognized.7 “We don’t think about disinfection of drinking water modifying water contaminants and generating enhanced toxicity,” Plewa says. In this case, he says, “A pharmaceutical with absolutely no genotoxicity [i.e., ICM] forms highly genotoxic iodo-DBPs. How many other reactions with exogenous agents occur when water is disinfected for drinking?” Plewa points out that this toxic transformation of ICM differs from the impact of other pharmaceutical contaminants in water, such as estrogenic hormones, which directly cause adverse responses. It’s been assumed that the presence of ICM in drinking water poses no human health concerns. “But when ICM are transformed into compounds that are more toxic than the parent compound, it raises questions about assessing the potential risks associated with discharging these compounds into the environment,” Sedlak says. His research shows that ICM in wastewater are largely found near hospitals and imaging laboratories.8 “It would be straight-forward to collect and treat these compounds at the source,” Sedlak says. Plewa offers a practical solution: “Patients given ICM could urinate before leaving hospitals, and the collected urine could be safely disposed of at the hospital.”

12 ERK activation is necessary for temperature preconditioning in isolated adult rat ventricular myocytes

In the present study we investigated the molecular effects of temperature preconditioning (TP) in freshly isolated adult rat cardiac myocytes. TP consisted of incubating myocytes at 16°C for 2 min,...

Comparison of DNA damage in human-derived hepatoma line (HepG2) exposed to the fifteen drinking water disinfection byproducts using the single cell gel electrophoresis assay

Disinfection of drinking water reduces pathogenic infection, but generates disinfection by-products (DBPs) in drinking water. In this study, the effect of fifteen DBPs on DNA damage in human-derived hepatoma line (HepG2) was investigated by the single cell gel electrophoresis (SCGE) assay. These fifteen DBPs are: four trihalomethanes (THMs), six haloacetic acides (HAAs), three haloacetonitriles (HANs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and chloral hydrate (CH). Based on the minimal effective concentration (MEC) at which DBPs induced significant increase in olive tail moment (OTM), the rank order of DNA-damaging potency is: bromodichloromethane (BDCM) > dibromochloromethane (DBCM) > tribromomethane (TBM) > trichloromethane (TCM) of the four THMs; iodoacetic acid (IA) > bromoacetic acid (BA) > dibromoacetic acid (DBA) > dichloracetic acid (DCA) > trichloroacetic acid (TCA) of the five HAAs; dibromoacetonitrile (DBN) ≈ dichloroacetonitrile (DCN) > trichloroacetonitrile (TCN) of the three HANs. The DNA damaging potency of MX and CH is similar to TCA and DCA, respectively. IA is the most genotoxic DBP in the fifteen DBPs, followed by BA. Chloroacetic acid (CA) is not genotoxic in this assay. Our findings indicated that HepG2/SCGE is a sensitive tool to evaluate the genotoxicity of DBPs and iodinated DBPs are more genotoxic than brominated DBPs, but chlorinated DBPs are less genotoxic than brominated DBPs.

Purification and characterization of trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides

Trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides, was purified by fractionation with ammonium sulfate, ionic exchange, and affinity chromatography. The protein was purified 161.85-fold with a yield of 4%. Purified trypsin had an apparent molecular weight of 24kDa according to an SDS-PAGE analysis. Optimal profiles of temperature and pH of the enzyme were 50°C and 8-10, respectively, using Nα-benzoyl-L: -arginine ethyl ester as the substrate. The results of thermal and pH stability assays showed that the enzyme was stable at temperatures of up to 50°C and in the pH range of 6-8. Trypsin activity decreased with an increasing NaCl concentration (0-0.6M). The activity of purified trypsin was effectively inhibited by a soybean trypsin inhibitor and N-p-tosyl-L: -lysine chloromethyl ketone, and was slightly inhibited by iodoacetic acid, ethylenediaminetetraacetic acid, 1-(L: -trans-epoxysuccinyl-leucylamino)-4-guanidinobutane, and pepstatin A. Protein identification of the purified protease showed that the sequences of two peptides, LGEHNI and NLDNDIML, were highly homologous to other fish trypsins. The measurement of trypsin activity in different tissues showed that the highest activity was detected in pyloric ceca, followed by anterior intestine, middle intestine, hind intestine and spleen, but very low activities were found in other tissues. An inverse relationship between the trypsin activity in four tissues of pyloric ceca, anterior intestine, middle intestine and hind intestine and fish body weight as a result of increased pepsin in stomach indicated grouper growth status was increased.

Selective Rod Degeneration and Partial Cone Inactivation Characterize an Iodoacetic Acid Model of Swine Retinal Degeneration

PURPOSE. Transgenic pigs carrying a mutant human rhodopsin transgene have been developed as a large animal model of retinitis pigmentosa (RP). This model displays some key features of human RP, but the time course of disease progression makes this model costly, time consuming, and difficult to study because of the size of the animals at end-stage disease. Here, the authors evaluate an iodoacetic acid (IAA) model of photoreceptor degeneration in the pig as an alternative model that shares features of the transgenic pig and human RP. METHODS. IAA blocks glycolysis, thereby inhibiting photoreceptor function. The effect of the intravenous injection of IAA on swine rod and cone photoreceptor viability and morphology was followed by histologic evaluation of different regions of the retina using hematoxylin and eosin and immunostaining. Rod and cone function was analyzed by full-field electroretinography and multifocal electroretinography. RESULTS. IAA led to specific loss of rods in a central-to-peripheral retinal gradient. Although cones were resistant, they showed shortened outer segments, loss of bipolar cell synaptic connections, and a diminished flicker ERG, hallmarks of transition to cone dysfunction in RP patients. CONCLUSIONS. IAA provides an alternative rod-dominant model of retinal damage that shares a surprising number of features with the pig transgenic model of RP and with human RP. This IAA model is cost-effective and rapid, ensuring that the size of the animals does not become prohibitive for end-stage evaluation or therapeutic intervention.

Novel Cysteine Tags for the Sequencing of Non-Tryptic Disulfide Peptides of Anurans: ESI-MS Study of Fragmentation Efficiency

Mass spectrometry faces considerable difficulties in de novo sequencing of long non-tryptic peptides with S-S bonds. Long disulfide-containing peptides brevinins 1E and 2Ec from frog Rana ridibunda were reduced and alkylated with nine novel and three known derivatizing agents. Eight of the novel reagents are maleimide derivatives. Modified samples were subjected to MS/MS studies on FT-ICR and Orbitrap mass spectrometers using CAD/HCD or ECD/ETD techniques. Procedures, fragmentation patterns, and sequence coverage for two peptides modified with 12 tags are described. ECD/ETD and CAD fragmentation revealed complementary sequence information. Higher-energy collisionally activated dissociation (HCD) sufficiently enhanced y-ions formation for brevinin 1E, but not for brevinin 2Ec. Some novel tags [N-benzylmaleimide, N-(2,6-dimethylphenyl)maleimide] along with known N-phenylmaleimide and iodoacetic acid showed high total sequence coverage taking into account combined ETD and HCD fragmentation. Moreover, modification of long (34 residues) brevinin 2Ec with N-benzylmaleimide or N-(2,6-dimethylphenyl)maleimide yielded high sequence coverage and full C-terminal sequence determination with ECD alone.

Characterization of an enzyme capable of degrading the sheath of the Sphaerotilus natans from Terrabacter sp. FK3

A microorganism for biological control of sludge bulking was isolated from soil. The isolated microorganism was able to degrade the sheath of the Sphaerotilus natans causing a bulking of activated sludge. The isolated strain, FK3, was identified as a Terrabacter species by analysis of its 16S rRNA genetic sequence. Glucose (0.1%, w/v) and 0.1% yeast extract as carbon and nitrogen sources, respectively, were used in the medium for Terrabacter sp. FK3 growth and enzyme production. Sheath-degrading crude enzyme was purified from a Terrabacter sp. FK3 culture broth via ultrafiltration, followed by DEAE-cellulose chromatography. Although the enzyme activity was optimal at pH 8 and 30°C, the enzyme maintained stable activity below 50°C and in the pH range from 5–10. Sheath-degrading enzyme activity was accelerated by addition of Mg2+, Ca2+, and Fe2+, but inhibited by addition of Ag2+ or Hg2+. Catalysis was additionally promoted by addition of hydroxyurea, sodium azide, and iodoacetic acid, but inhibited by EDTA.

English

A novel melanoidin decolorizing enzyme (MDE) produced by Geotrichum sp. No. 56, which exhibits decolorization activity against synthetic melanoidin and molasses containing wastewater, was purified and characterized. The purification process was performed using ammonium sulfate fractionation, DEAE-Cellulose and Sephadex G-150 column chromatography. The melanoidin decolorization activity of the MDE was improved up to 7.3 U mg-1, the equivalent to 8.3-fold increase from the initial protein preparation, with an overall yield of 9.1%. The results of gel filtration and SDS-PAGE revealed that the purified MDE was a dimmer with 127.5 kDa, and both subunits were alike with 63.5 kDa each. The optimum pH and temperature for the purified MDE were 6.5 and 45°C, respectively. The MDE activity was highly specific for β-D-glucose, but completely inhibited by cysteine, iodoacetic acid, 2-mercaptoethanol, gluconic acid, HgCl2 or AgNO3. The MDE also decolorized the spent broth of monosodium glutamate fermentation. TheGeotrichum sp. No. 56 producing MDE, which shares various characteristics with glucose oxidase (GOx), could be useful for decolorizing fermented wastewater. Key words: Decolorization, Geotrichum sp., glucose oxidase, melanoidin, molasses wastewater.

Characterization and detergent compatibility of purified protease produced from Aspergillus niger by utilizing agro wastes

A study was conducted to purify and characterize a novel protease produced from Aspergillus niger using different lignocellulosic agro-based by-products including corncobs, wheat bran, and rice bran as substrates under SSF. Maximum protease activity was recorded on wheat bran fermented culture media after the 3rd day of incubation. The optimal conditions found for protease production using wheat bran were cultivation period (3 days), substrate concentration (10 g), pH (7), incubation temperature (45oC), inoculum size (4 mL), and 3% surfactant Tween-80 (2 mL). A purification fold of 2.41 with 29 U/mg specific activity and 70.73 % recovery was achieved after purification. Purified protease from A. niger had a molecular weight of 47 kDa on SDS-PAGE. The enzyme activity profile showed that purified protease was optimally active at pH 7 and 45oC as optimum values. A. niger protease was reasonably stable in the pH range 5-8 and 35-60oC for up to 1 h incubation. Protease was activated by various metal ions/inhibitors tested, Mn2+, Cd2+, Mg2+, Cu2+, PMSF, Pepstatin and Iodoacetic acid at 1 mM, proving the enzyme as metalloprotease, whereas an inhibitory effect was shown by certain agents including EDTA and SDS. The purified protease was compatible with five local detergents with up to 25 days of shelf life at room temperature. The maximum production of protease in the presence of a cheaper substrate at low concentration and its potential as a detergent additive for improved washing makes the strain and its enzymes potentially useful for industrial purposes, especially for the detergent and laundry industry.

Purification and characterization of alkaline-stable β-amylase in malted African finger millet (Eleusine coracana) seed

Alkaline-stable β-amylase (EC 3.2.1.2) was purified to apparent homogeneity from malted African finger millet (Eleusine coracana) seed by ammonium sulfate fractionation and anion exchange and affinity chromatographies. Gel filtration chromatography together with SDS-PAGE revealed that the enzyme is monomeric, with a molecular weight of 59.1kDa. It has a pI value of 5.2 and is optimally active at pH 5.0 and 50°C. 2DE-MS isolated and identified two isoforms of the enzyme with unique amino acid sequences. The purified enzyme was highly selective for soluble potato starch, amylose and amylopectin, and α-cyclodextrin was shown to be a competitive inhibitor with a Gibbs free energy (ΔG°′) of binding of 18.11kJ/mol. The enzyme was stable at a pH range of 4.0–10.0 and temperature range of 30–70°C. It was irreversibly inactivated by heating to 60°C and 70°C, which was often related to aggregation. The apparent KM of the enzyme for p-nitrophenyl maltopentaoside (PPNG-5) was 2.1mM. NH4+, Hg2+, Al3+, EDTA, ascorbic acid, iodoacetamide, iodoacetic acid and gibberellic acid all inhibited the enzyme. The enzyme unfolded after treatment with 4.0M guanidine–HCl and regained 68% of its original activity upon dilution at 4°C in the presence of 5mM trehalose. The kinetic parameters of renaturation were first order. This enzyme could be important in the malting of African finger millet seeds, based on the kinetics and properties reported in this study.

Taurine Biosynthesis by Neurons and Astrocytes

The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capability as reported by incorporation of radioactivity from [(35)S]cysteine into taurine, in primary murine astrocytes and neurons, and in several transformed cell lines (human (SH-SY5Y) and murine (N1E-115) neuroblastoma, human astrocytoma (U-87 MG and 1321 N1), and rat glioma (C6)). Extensive incorporation of radioactivity from [(35)S]cysteine into taurine was observed in rat glioma cells as well as in primary mouse astrocytes and neurons, establishing the presence of an intact taurine synthesis pathway in these cells. Interestingly, exposure of cells to cysteine or cysteamine resulted in elevated intracellular hypotaurine without a corresponding increase in taurine levels, suggesting that oxidation of hypotaurine limits taurine synthesis in cells. Consistent with its role as an organic osmolyte, taurine synthesis was stimulated under hypertonic conditions in neurons.

Purification and characterization of cathepsin B from the gut of the sea cucumber (Stichopus japonicas)

Cathepsin B from the gut of sea cucumber (Stichopus japonicas) was purified 81-fold with a 3% recovery by ammonium sulfate fractionation and a series chromatography on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-Gel 3000 SWxl. The purified protein appeared as a single band on Native-PAGE but showed 2 bands of 23 and 26 kDa on SDS-PAGE. The optimum activity was found at pH 5.5 and 45°C. The enzyme was stable at pH 4.5–6.0 and the thermal stability was up to 50oC. The enzyme was strongly inhibited by E-64, iodoacetic acid, and antipain, demonstrating it is a cysteine protease containing sulfhydryl groups. Cu2+, Ni2+, and Zn2+ could strongly inhibit the enzyme activity. The amino acid sequences of the purified enzyme were acquired by mass spectrometer, which did not show any homology with previously described cathepsins, suggesting it may be a novel member.

Formation of Toxic Iodinated Disinfection By-Products from Compounds Used in Medical Imaging

Iodinated X-ray contrast media (ICM) were investigated as a source of iodine in the formation of iodo-trihalomethane (iodo-THM) and iodo-acid disinfection byproducts (DBPs), both of which are highly genotoxic and/or cytotoxic in mammalian cells. ICM are widely used at medical centers to enable imaging of soft tissues (e.g., organs, veins, blood vessels) and are designed to be inert substances, with 95% eliminated in urine and feces unmetabolized within 24 h. ICM are not well removed in wastewater treatment plants, such that they have been found at elevated concentrations in rivers and streams (up to 100 μg/L). Naturally occurring iodide in source waters is believed to be a primary source of iodine in the formation of iodo-DBPs, but a previous 23-city iodo-DBP occurrence study also revealed appreciable levels of iodo-DBPs in some drinking waters that had very low or no detectable iodide in their source waters. When 10 of the original 23 cities' source waters were resampled, four ICM were found--iopamidol, iopromide, iohexol, and diatrizoate--with iopamidol most frequently detected, in 6 of the 10 plants sampled, with concentrations up to 2700 ng/L. Subsequent controlled laboratory reactions of iopamidol with aqueous chlorine and monochloramine in the absence of natural organic matter (NOM) produced only trace levels of iodo-DBPs; however, when reacted in real source waters (containing NOM), chlorine and monochloramine produced significant levels of iodo-THMs and iodo-acids, up to 212 nM for dichloroiodomethane and 3.0 nM for iodoacetic acid, respectively, for chlorination. The pH behavior was different for chlorine and monochloramine, such that iodo-DBP concentrations maximized at higher pH (8.5) for chlorine, but at lower pH (6.5) for monochloramine. Extracts from chloraminated source waters with and without iopamidol, as well as from chlorinated source waters with iopamidol, were the most cytotoxic samples in mammalian cells. Source waters with iopamidol but no disinfectant added were the least cytotoxic. While extracts from chlorinated and chloraminated source waters were genotoxic, the addition of iopamidol enhanced their genotoxicity. Therefore, while ICM are not toxic in themselves, their presence in source waters may be a source of concern because of the formation of highly toxic iodo-DBPs in chlorinated and chloraminated drinking water.