Activation versus inhibition of microsomal glutathione S-transferase activity by acrolein. Dependence on the concentration and time of acrolein exposure.

The toxicity of acrolein, an α,ß-unsaturated aldehyde, is due to its soft electrophilic nature and primarily involves the adduction of protein thiols. The thiol glutathione (GSH) forms the first line of defense against acrolein. The present study confirms that acrolein added to isolated rat liver microsomes can increase microsomal GSH transferase (MGST) activity 2-3 fold, which can be seen as a direct adaptive increase in the protection against acrolein. At a relatively high exposure level, acrolein appeared to inhibit MGST. The activation is due to adduction of thiol groups, and the inactivation probably involves adduction of amino groups in the enzyme by acrolein. The preference of acrolein to react with thiol groups over amino groups can explain why the enzyme is activated at a low exposure level and inhibited at a high exposure level of acrolein. These opposite forms of direct adaptation on the level of enzyme activity further narrow the thin line between survival and promotion of cell death, governed by the level of exposure.

Multiparametric protocol for the determination of thiol redox state in living matter.

Thiol redox state (TRS) evaluation is mostly restricted to the estimation of GSH and GSSG. However, these TRS parameters can estimate the GSSG/GSH potential, which might be useful for indicating abnormalities in redox metabolism. Nonetheless, evaluation of the multiparameric nature of TRS is required for a more accurate assessment of its physiological role. The present protocol extends the partial assessment of TRS by current methodologies. It measures 15 key parameters of TRS by two modular subprotocols: one for the glutathione (GSH)- and cysteine (CSH)-based nonprotein (NP) thiols/mixed disulfides (i.e., GSH, GSSG, GSSNP, CSH, CSSNP, NPSH, NPSSNP, NP(x)SH(NPSSNP), NP(x)SH(NPSH)), and the other for their protein (P) thiols/mixed disulfides (i.e., PSH, PSSG, PSSC, PSSNP, PSSP, NP(x)SH(PSSNP)). The protocol eliminates autoxidation of GSH and CSH (and thus overestimation of GSSG and CSSNP). Its modularity allows the determination GSH and GSSG also by other published specific assays. The protocol uses three assays; two are based on the photometric reagents 4,4'-dithiopyridine (DTP) and ninhydrin (NHD), and the third on the fluorometric reagent o-phthaldialdehyde (OPT). The initial assays employing these reagents have been extensively modified and redesigned for increased specificity, sensitivity, and simplicity. TRS parameter values and their standard errors are estimated automatically by sets of Excel-adapted algebraic equations. Protocol sensitivity for NPSH, PSH, NPSSNP, PSSP, PSSNP, CSH, CSSNP, PSSC, NP(x)SH(NPSSNP), and NP(x)SH(NPSH) is 1 nmol -SH/CSH, for GSSNP 0.2 nmol, for GSH and GSSG 0.4 nmol, and for PSSG 0.6 nmol. The protocol was applied on human plasma, a sample of high clinical value, and can be also applied in any organism.

Nonthermal atmospheric argon plasma jet effects on Escherichia coli biomacromolecules.

Nonthermal atmospheric plasma jet, a promising technology based on ionized gas at low temperatures, can be applied for disinfection of contaminated surfaces. In this study, Escherichia coli cells and their macromolecules were exposed to the nonthermal atmospheric argon plasma jet for different time durations. Total protein, genomic DNA, and malondialdehyde (MDA) levels of E. coli were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining; agarose gel electrophoresis; and measurement of absorbance at 534 nm, respectively. After exposure, the spectroscopic results of liquid samples indicated that the survival reduction of E. coli can reach to 100 % in an exposure time of 600 s. Moreover, inactivation zones of E. coli, DNA degradation, and MDA levels were significantly increased. Additionally, banding patterns of total protein were changed and amino acid concentrations increased following ninhydrin test. The experimental results suggest that the nonthermal plasma could serve as an effective instrument for both sterilizing E. coli and degrading macromolecules from the surface of the objects being sterilized.

Critical evaluation of ninhydrin for monitoring surgical instrument decontamination.

BACKGROUND: New Department of Health (England) Choice Framework for Local Policies and Procedures guidance (CFPP 0101) still states that ninhydrin can be used to check for efficient protein removal from surgical instruments processed in sterile services departments (SSDs). AIM: With the potential transfer of variant Creutzfeldt-Jakob disease (vCJD) via surgical procedures it is necessary to re-evaluate recommended methods for protein detection. METHODS: This paper reports studies on the sensitivity and applicability of ninhydrin for detecting proteins in laboratories and SSDs. The efficiency of protein removal by swabbing was also evaluated. FINDINGS: Ninhydrin showed poor sensitivity toward proteins. Limits of detection for bovine serum albumin (BSA) in solution were 205 µg/mL compared with arginine 6 µg/mL. A commercial kit could detect neither rat brain homogenate nor BSA at <1000 µg protein pipetted directly into the vials. Swabbing with water-wetted rayon swabs was inefficient at removing protein (50 µg) from instruments (N = 6) with 32 ± 4% BSA and 61 ± 5% fibrinogen remaining bound. Swabs dipped in 0.5% detergent (Triton X-100) solution had slightly better removal efficiency with 20 ± 3% BSA and 24 ± 2.8% fibrinogen remaining. CONCLUSIONS: Ninhydrin kits, currently used in SSDs, are ineffective at detecting residual proteins due not only to the insensitivity of ninhydrin towards proteins but also to the poor desorption of adhered proteins by swabbing. Overall ninhydrin, either as a laboratory reagent or as supplied in protein detection kits, does not provide sensitive detection of proteins and generates high numbers of false negatives when used in decontamination practices.

Locally released small (non-protein) ninhydrin-reacting molecules underlie developmental differences of cultured medullary versus spinal dorsal horn neurons.

Neurons located in the trigeminal subnucleus caudalis (Vc) play crucial roles in pain and sensorimotor functions in the orofacial region. Because of many anatomical and functional similarities with the spinal dorsal horn (SDH), Vc has been termed the medullary dorsal horn--analogous to the SDH. Here, we report that when compared with embryonic SDH neurons in culture, neurons isolated from the Vc region showed significantly slower growth, lower glutamate receptor activity, and more cells undergoing cell death. SDH neuron development was inhibited in co-cultures of SDH and Vc tissues while Vc neuron development was promoted by co-culture with SDH tissues. Furthermore, we identified that small (non-protein) ninhydrin-reacting molecules purified from either embryonic or post-natal Vc-conditioned medium inhibited neuronal growth whereas ninhydrin-reacting molecules from SDH-conditioned medium promoted neuronal growth. These findings suggest the involvement of locally released factors in the region-specific regulation of neuronal development in Vc and SDH, central nervous system regions playing critical roles in pain, and point to novel avenues for investigating central nervous system regionalization and for designing therapeutic approaches to manage neurodegenerative diseases and pain.

Surgical repair of a 30 mm long human median nerve defect in the distal forearm by implantation of a chitosan-PGA nerve guidance conduit.

This paper describes a clinical case study in which a chitosan/polyglycolic acid nerve guidance conduit (chitosan-PGA NGC) was utilized to repair a 30 mm long median nerve defect in the right distal forearm of a 55 year-old male patient. Thirty-six months after the nerve repair, the palm abduction of the thumb and the thumb-index digital opposition recovered, facilitating the patient to accomplish fine activities, such as handling chopsticks. Static two-point discrimination measured 14, 9 and 9 mm in the thumb, index and middle fingers of the right hand. Reproducible compound muscle action potentials were recorded on the right abductor pollicis. The ninhydrin test, a classical method for assessing sympathetic nerve function, showed partial recovery of the perspiration function of the injured thumb, index and middle fingers. This repair case suggested a possible strategy for the clinical reconstruction of extended defects in human peripheral nerve trunks by the implantation of chitosan-PGA NGCs.

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase.

AIMS: 1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. METHODS AND RESULTS: A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat-resistant polypropylene chimney-top 96-well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC-utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC-utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. CONCLUSIONS: Determination of bacterial ACC consumption by the PCR-plate ninhydrin-ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-plate ninhydrin-ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates.

A colorimetric microplate assay method for high-throughput analysis of arginase activity in vitro.

Several analytical methods have been developed for the determination of arginase (l-arginine amidinohydrolase) activity in physiological samples. These methods are limited by the considerable effort and time required to obtain reliable and reproducible measurements. Here we describe a simple high-throughput colorimetric assay for the determination of arginase activity based on the ornithine-ninhydrin reaction. This method is an improvement over the original single cuvette assay developed by Chinard in that no boiling step is required. The turnaround time has been reduced, with improved precision and reproducibility. The method was extended to the determination of arginase activity in human leukemic (K562) cells and sickle erythrocytes. We believe that the method will find applications for routine analysis as well as for characterizing the action of novel and potent inhibitors on arginase activity.

Application of metabolomic principles to disorders of nucleotide metabolism reveals new metabolic perturbations.

A metabolomic analysis of plasma amino acids and acylcarnitines was applied to four disorders of nucleotide metabolism. Multivariate analysis gave score plots that show segregation of hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase deficient plasma from controls with equivocal results for adenosine deaminase and dihydropyrimidine dehydrogenase deficiencies. Loadings plots revealed the principal metabolites responsible for the discrimination between these classes. There were increases for HPRT in C4-, C6-, and C3-DC (malonyl)-carnitines, and decreased serine. For APRT there were increases in C4- to C10- and C3-DC to C6-DC-carnitines, urea, 1-methylhistidine, 3-methylhistidine, and decreased tryptophan. For ADA deficiency there were increases in C4- and C6-carnitines, taurine, and isoleucine.

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities.

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.

Changes in some nitrogenous compounds in the blood and tissues of freshwater pikeperch (Sander lucioperca) during salinity acclimation.

The effect of ambient salinity changes (0.9, 6 and 12 psu) on the levels of dissolved ammonia (DA), ninhydrin positive substances (NPS), trimethylamine (TMA) and trimethylamine oxide (TMAO) in the blood and tissue of medium-acclimated Sander lucioperca L. (also Stizostedion lucioperca) were investigated. In freshwater, blood and tissue total free amino acid levels (measured as NPS) were 3.62 mM and 60.61 mM, respectively. The NPS content increased significantly (P<0.05) in the tissue and blood on acclimation to 6 and 12 psu salinities. The mass-specific tissue TMAO concentration of pikeperch acclimated to normal freshwater was 0.413+/-0.084 micromol TMAO g(-1). Results reveal that TMAO levels are positively influenced by the external salinity medium where significant differences in mean levels occurred between the groups (P<0.05). The calculated p[NH(3)] and [NH(4)(+)] gradients reveal that the [NH(3)] gradient was consistently low (cf. the [NH(4)(+)] gradient). The gradient of p[NH(3)] decreased with the medium increased salinities. The results suggest that freshwater pikeperch may be able to resist salinity changes by manipulation of nitrogen metabolism. Free amino acids and TMAO are involved in mediating response to salinity exposure in freshwater pikeperch.

A novel one-pot, four component synthesis of some densely functionalized pyrroles.

A new one-pot four component procedure for synthesis of densely functionalized pyrroles using commercially available ninhydrin with phosphorane intermediates produced in the reaction between triphenylphosphine, ammonium thiocyanate (or ammonium acetate) and various dialkyl acetylenedicarboxylates was developed.

The effect of heat shock on amino acid transport and cell volume in 3T3 cells.

In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44 degrees C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response.

Chitosan as an enabling excipient for drug delivery systems. I. Molecular modifications.

Chitosan was physicochemically modified for its potential use as a matrix for an implantable antibiotic delivery system that could sustain bactericidal concentrations in the vicinity of an implant or prosthesis. Deacetylation and depolymerization of chitosan were implemented in order to increase the number or accessibility of the reactive amino groups on the polymer backbone for better polymer-drug interaction. The deacetylation process involved reaction of particulate chitosan/depolymerized chitosan with alkali. The rate of deacetylation of chitosan was directly proportional to the reaction temperature up to 80 degrees C; beyond 80 degrees C, rapid degradation of the polymer occurred. The depolymerization of chitosan involved acid digestion of the polymer followed by application of mechanical agitation. This depolymerized product, although water insoluble, possessed a molecular weight that was one to two orders of magnitude lower than that of commercially available chitosans. These products not only exhibited improved reactivity, but also showed increased crystallinity when compared with the parent chitosan. The reactivity was found to be inversely proportional to chitosan's molecular weight. The depolymerization and deacetylation treatments afforded formation of chitosan having a greater number of amino groups available for interactions with the anionic actives.

Similarities in the metabolism of alloxan and dehydroascorbate in human erythrocytes.

The beta-cell toxin alloxan is reduced within cells to dialuric acid, which may then decompose to release damaging reactive oxygen species. We tested whether such redox cycling of alloxan occurs in the human erythrocyte, a cell with stronger antioxidant defenses than beta-cells. Erythrocytes incubated with increasing concentrations of alloxan progressively accumulated dialuric acid, as measured directly by HPLC with electrochemical detection. At concentrations up to 2 mM, alloxan decreased cellular GSH slightly, but did not affect erythrocyte contents of ascorbate or alpha-tocopherol. Intracellular H2O2 generation, measured as inhibition of endogenous catalase activity in the presence of 3-amino-1,2,4-triazole (aminotriazole), was decreased by alloxan. Despite its failure to induce significant oxidant stress in erythrocytes, 2 mM of alloxan doubled the activity of the hexose monophosphate pathway (HMP). This likely reflected consumption of reducing equivalents during reduction of alloxan to dialuric acid. Alloxan pretreatment enhanced the ability of erythrocytes to reduce extracellular ferricyanide while protecting alpha-tocopherol in the cell membrane from oxidation by ferricyanide. Ninhydrin, a hydrophobic derivative of alloxan, showed similar effects, but caused progressive GSH depletion and cell lysis at concentrations above 50 microM. The ability of alloxan to enhance ferricyanide reduction and to spare alpha-tocopherol suggests that dialuric acid or other reducing species within the cells can protect or recycle alpha-tocopherol and donate electrons to a transmembrane transfer process. This behavior resembles that observed for the dehydroascorbate (DHA)/ascorbate pair, and leads to the unexpected conclusion that alloxan increases the reducing capacity of the erythrocyte.