Activity of Hospital Disinfectants against Vegetative Cells and Spores of Clostridioides difficile Embedded in Biofilms.

Clostridioides difficile spores can survive in the environment in either mono- or mixed-species biofilms. However, no previous studies have investigated chemical disinfection of C. difficile spores embedded in biofilms. Thus, the purpose of this study was to assess the in vitro effectiveness of hospital disinfectants against C. difficile spores embedded within biofilms. Five unique C. difficile strains embedded in three different biofilm types grown for 72 or 120 h were exposed to seven different hospital disinfectants. C. difficile abundance [as log(number of CFU/milliliter)] was calculated after manufacturer-determined contact times along with biofilm biomass and microscopy. The primary analysis compared differences between C. difficile vegetative cell and spore counts as well as amounts of biomass after exposure to disinfectants. C. difficile vegetative cells and spores were recovered from biofilms regardless of the type of biofilm growth or biofilm growth time. No disinfectant was able to completely eliminate C. difficile from the biofilms. Overall, Clorox, ortho-phthalaldehyde (OPA), and Virex were most effective at killing C. difficile spores regardless of biofilm age, ribotype, or wash conditions (whether biofilms are washed or unwashed) (P = 0.001, each). Clorox and OPA were also effective at killing total vegetative cell growth (P = 0.001, each), but Virex was found to be ineffective against vegetative cell growth in biofilms (P = 0.77). Clorox and Virex were most effective in reducing biomass, followed by Nixall, OPA, and Vital Oxide. No disinfectant was able to completely eliminate C. difficile embedded within biofilms although differences among disinfectants were noted. Future research will be required to determine methods to eradicate this persister reservoir.

An exception to the rule "no association between antibiotic resistance and decreased disinfectant microbicidal efficacy": Orthophthalaldehyde (OPA) and Pseudomonas aeruginosa isolated from ICU and paraplegic patients.

BACKGROUND: Antibiotic resistance and decreased susceptibility to disinfectants are not usually associated in microorganisms, but we have found an exception to this rule: P. aeruginosa versus orthophthalaldehyde (OPA). METHODS: Bactericidal effect of OPA was measured at 10 minutes on endodoncy files contaminated with an ATCC strain (control) or 206 strains of P. aeruginosa recently isolated from 206 ICU and paraplegic patients in a tertiary university hospital, in two consecutive years. RESULTS: Differences in bactericidal effect of OPA were found between the strains isolated each year (decreased susceptibility in the first period), but in both years the statistical differences (p < 0.05) were maintained according to whether the strains were "susceptible" to antibiotics, "resistant" (to one family of antibiotics) or "multi-resistant" (resistant to more than one family of antibiotics), exhibiting a reduction in their OPA susceptibility in parallel to an increase of their antibiotic resistance. In contrast, there were no differences depending on the type of sample (sputum, urine, faeces, pharynx) or of patient (paraplegic or ICU: adult, newborn, burn). Finally we selected 15 strains with an OPA effect below 3.5 log10 at 10 minutes and repeated the study with an OPA exposure of 15 minutes. In these conditions OPA showed a total bactericidal effect on these P. aeruginosa strains. CONCLUSIONS: There was an association between antibiotic resistance and decreased OPA susceptibility. This normally does not require an increase in disinfection time, but, for endoscope disinfection or instruments from colonized/infected patients with resistant/multiresistant P. aeruginosa, we consider it better to use 15 min of OPA. Regular tests (e.g., once every 12 months) with germ-carriers, should be performed to assess ecological changes in susceptibility to high level disinfectants and must include not only ATCC strains, but also recently isolated microorganisms with different antibiotic sensitivities (susceptible, resistant and multi-resistant).

Synergistic sporicidal effect of ethanol on a combination of orthophthalaldehyde and didecyldimethylammonium chloride.

UNLABELLED: The objective of this study was to evaluate the potential synergistic effect of ethanol on a combination of orthophthalaldehyde (OPA) and didecyldimethylammonium chloride (DDAC) against the spores of Bacillus subtilis var. Niger. The quantitative carrier test for sporicidal testing of high-level disinfectants according to the guideline of China (Technical Standard for Disinfection 2002) was used as method. Considerable synergistic effect was observed after a 30-min treatment at 20°C. There was an augment in mean log reduction as the concentration of DDAC was increased ranging from 0·2 to 3 g l(-1) in combination with 6 g l(-1) OPA. Ten and 20% ethanol in combination with 6 g l(-1) OPA and 2 g l(-1) DDAC caused more than a 3-log reduction while either 6 g l(-1) OPA, 2 g l(-1) DDAC and 20% ethanol alone or a combination of two of the three agents produced less than a 1-log reduction. Further, 40-min exposure time of combination of OPA, DDAC and 20% ethanol led to greater than a 5-log reduction in spores, and no spore growth was observed following 60- and 90-min exposures. SIGNIFICANCE AND IMPACT OF THE STUDY: Orthophthalaldehyde (OPA) is very effective at concentrations far lower than its recommended in-use concentration of 0·5% (w/v) and is equally effective against both the gram-negative and gram-positive bacteria. However, it shows lower activity against spores. The synergistic sporicidal effect exhibited by ethanol on a combination of OPA and DDAC can be considered to enhance sporicidal activity for using in situations of sterilization, to reduce in-use concentration of OPA used alone, which may minimize its side effect. OPA may be a more satisfactory and the first-choice agent to replace glutaraldehyde (GTA) as a high-level disinfectant for medical devices.

Injectable antibacterial tissue-adhesive hydrogel based on biocompatible o-phthalaldehyde/amine crosslinking for efficient treatment of infected wounds.

Injectable antibacterial hydrogels have attracted considerable attention in wound management. However, the development of injectable hydrogels with excellent antibacterial activity, good biocompatibility, and strong tissue adhesion remains a challenge. In this study, an antibacterial tissue-adhesive hydrogel was developed based on a catalyst-free o-phthalaldehyde (OPA)/amine reaction by simply mixing OPA-terminated four-arm poly(ethylene glycol) (4aPEG-OPA) and ε-poly-l-lysine (ε-PLL) solutions. The hydrogel showed tunable gelation time, storage moduli, and degradation rate depending on the polymer concentration and 4aPEG-OPA/ε-PLL mass ratio. The hydrogel exhibited nearly 100% bacterial inhibition rates in-vitro against Gram-negative E. coli and Gram-positive S. aureus, while maintaining good biocompatibility. The hydrogel matched well in shape and tightly adhered to the tissue after in-situ formation at the wound sites. Following the treatment of rat models of full-thickness skin incisions and round wounds, the hydrogel effectively closed the wounds and promoted wound healing. Moreover, after administering to S. aureus infected full-thickness skin wounds, the hydrogel exhibited remarkable efficacy in inhibiting wound infection with a bacterial inhibition rate over 99.94%, achieving a significantly accelerated wound healing compared with the commercially available Prontosan® gel. Therefore, the hydrogel exhibits great potential as a wound dressing for infection prevention and promotion of healing.

Persister cells in a biofilm treated with a biocide.

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.

Bactericidal effects and cytotoxicity of new aromatic dialdehyde disinfectants (ortho-phthalaldehyde).

We investigated the bactericidal effects and cytotoxicity of an ortho-phthalaldehyde product in comparison with those of its predecessor glutaraldehyde products. Bactericidal effects ware examined on Mycobacterium terrae, a standard organism used for investigating the bactericidal effect of high-level disinfectants. Cytotoxicity as determined by the MTT assay was examined by using four cell lines. The colony forming test, a method to examine residual toxicity, and the evaporation test, a newly developed method to examine the toxicity of the evaporated ingredients, were performed. Test solutions were 2.25% and 3.5% glutaraldehyde (GA) products and a 0.55% ortho-phthalaldehyde (OPA) product, and glutaraldehyde itself. All the disinfectants showed sufficient bactericidal effects on M. terrae. Meanwhile, the OPA product was less toxic than GA products and GA itself to all the cell lines tested. The colony forming test showed that GA products and GA itself exerted residual cytotoxicity more potently than did the OPA product. The evaporation test showed that GA products and GA itself exerted cytotoxicity via evaporation more potently than did the OPA product. In conclusion, OPA appears to be less cytotoxic than GA even though bactericidal effects were comparable. This may be due to the lower concentration of the active ingredient (ortho-phthalaldehyde) in the OPA product.

Azacoccones F-H, new flavipin-derived alkaloids from an endophytic fungus Epicoccum nigrum MK214079.

Three new flavipin-derived alkaloids, azacoccones F-H (1-3), along with six known compounds (4-9) were isolated from the endophytic fungus Epicoccum nigrum MK214079 associated with leaves of Salix sp. The structures of the new compounds were established by analysis of their 1D/2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS) data. The absolute configuration of azacoccones F-H (1-3) was determined by comparison of experimental electronic circular dichroism (ECD) data with reported ones and biogenetic considerations. Epicocconigrone A (4), epipyrone A (5), and epicoccolide B (6) exhibited moderate antibacterial activity against Staphylococcus aureus ATCC 29213 with minimal inhibitory concentration (MIC) values ranging from 25 to 50 µM. Furthermore, epipyrone A (5) and epicoccamide A (7) displayed mild antifungal activity against Ustilago maydis AB33 with MIC values of 1.6 and 1.8 mM, respectively. Epicorazine A (8) showed pronounced cytotoxicity against the L5178Y mouse lymphoma cell line with an IC50 value of 1.3 µM.

Role of porins in the susceptibility of Mycobacterium smegmatis and Mycobacterium chelonae to aldehyde-based disinfectants and drugs.

Nosocomial outbreaks attributable to glutaraldehyde-resistant, rapidly growing mycobacteria are increasing. Here, evidence is provided that defects in porin expression dramatically increase the resistance of Mycobacterium smegmatis and Mycobacterium chelonae to glutaraldehyde and another aldehyde disinfectant, ortho-phthalaldehyde. Since defects in porin activity also dramatically increased the resistance of M. chelonae to drugs, there is thus some concern that the widespread use of glutaraldehyde and ortho-phthalaldehyde in clinical settings may select for drug-resistant bacteria.

Anticancer potential of NF-κB targeting apoptotic molecule "flavipin" isolated from endophytic Chaetomium globosum.

BACKGROUND: Anticancer compounds from natural sources have drawn attention due to their structural diversity and relatively lesser side effects. Endophytic fungi are one such natural resource from, which plethoras of anticancerous compounds have been isolated. PURPOSE: The objective of the study was to isolate and characterize the bioactive metabolite from Chaetomium globosum that exhibits astonishing antiproliferative activity against cancerous cell lines. METHODS: Flavipin was isolated by bioassay-guided fractionation and identified using FT-IR, EI-MS and NMR studies. MTT assay was used to determine the cytotoxicity. Fluorescent staining (AO/EB) and DNA fragmentation studies confirmed the occurrence of apoptosis. Real time PCR and Western blotting were used to analyze the expression of apoptosis related genes and its proteins, respectively. RESULTS: Flavipin inhibited proliferation of A549, HT-29 and MCF-7 cancer cells in dose dependent manner with an IC50 concentration of 9.89 µg/ml, 18 µg/ml and 54 µg/ml, respectively, whereas it was comparatively less sensitive (IC50 = 78.89 µg/ml) against normal cell line (CCD-18Co). At IC50 concentration cancerous cells exhibited cell shrinkage and fragmentation of DNA, which indicated that flavipin induced apoptotic cell death. In treated cells there is an up-regulation of p53 gene and its associated protein, whereas reciprocal expression was observed in BCL-2 gene and its protein. Furthermore, western blotting results also showed down-regulation of NFκB. CONCLUSION: This is the first report on the antiproliferative activity of flavipin isolated from endophytic C. globosum and also proposed that interaction of flavipin with NFкB could be a possible mechanism for this activity. Flavipin induced apoptosis at low concentrations in cancer cell lines (A549, HT-29) and exhibited itself as a potential anticancer agent.

Comparison of the fixative properties of five disinfectant solutions.

Following a French circular published in 2001, the use of glutaraldehyde for the disinfection of reusable medical devices was abandoned in favour of non-fixative disinfectants such as peracetic-acid-based solutions. Data published regarding the fixative properties of alternative disinfectants remain contradictory. We compared the effect of repetitive treatments of polytetrafluoroethylene (PTFE) tubes, contaminated by a liquid medium inoculated with Pseudomonas aeruginosa, using five different disinfectant solutions: two peracetic acid solutions (with and without an activator), glutaraldehyde, ortho-phthaldehyde and succine dialdehyde. The results confirmed that repeated treatments of a PTFE tube with a 2% glutaraldehyde solution induce an important accumulation and/or fixation of protein, compared to peracetic-acid-based disinfectants, for which the accumulation and/or fixation of proteins remain low and vary from one formulation to another.

The effects of a biocide and a surfactant on the detachment of Pseudomonas fluorescens from glass surfaces.

Application of antimicrobial chemicals is a general procedure in the cleaning and disinfection of food-contacting surfaces. Adhesion to glass surfaces and chemically induced detachment of Pseudomonas fluorescens ATCC 13525(T) were studied in situ, under flow conditions, in a well-controlled parallel plate flow chamber (PPFC). Ortho-phthalaldehyde (OPA) and cetyltrimethyl ammonium bromide (CTAB) were applied separately, at several concentrations, to attached bacteria and their subsequent detachment was monitored. Following treatments the remaining adhered bacteria were characterized in terms of viability and cell size. Simultaneously, the planktonic cell surface was characterized in order to correlate PPFC results with thermodynamic approaches for adhesion evaluation, and surface free energy of chemically treated cells with adhesion strength. About 2.8x10(6) cells/cm(2) adhered to the glass surface after 30 min of bacterial flow, although thermodynamic analyses evidenced unfavourable adhesion. The independent application of OPA and CTAB promoted bacterial detachment to a small extent (16% of total cells). The remaining adhering bacteria were totally non-viable for OPA> or =0.75 mM and CTAB> or =0.25 mM, showing a lack of correlation between bacterial viability and detachment. The cellular size decreased as attachment proceeded and with chemical treatment. Both chemicals altered the cell surface properties, increasing the cell-glass adhesion strength, and promoting the emergence of polar characteristics. The overall results emphasize that OPA and CTAB were markedly ineffective in removing glass-attached P. fluorescens, demonstrating that bacteria can be non-viable but remain strongly attached to the adhesion surface.

Hydroxymethyl bioisosteres of phenolic GluN2B-selective NMDA receptor antagonists: Design, synthesis and pharmacological evaluation.

Antagonists addressing selectively NMDA receptors containing the GluN2B subunit are of particular interest for the treatment of various neurological disorders including neurodegenerative diseases. With the aim to bioisosterically replace the metabolically labile phenol of 7-amino-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-ols, several analogs were docked into the ifenprodil binding site leading to the hydroxymethyl derivatives 4 as promising candidates. They display the same binding pose as Ro 25-6981 and the same H-bond interactions with Gln110 and Glu236 within the GluN2B subunit. The phenylalkyl moieties occupy the hydrophobic pocket formed predominantly by Pro78 (GluN2B), Phe114 (GluN2B), and Tyr109 (GluN1b). Starting from o-phthalaldehyde, the hydroxymethyl derivatives 4 were prepared in a 7-step synthesis with a haloform reaction of trichloroacetophenone 7 as key step. In receptor binding studies, the phenylpropyl derivative 4a shows promising GluN2B affinity (Ki = 101 nM) and high selectivity over the PCP binding site and both σ receptor subtypes. 4a was able to inhibit the glutamate/glycine induced cytotoxicity at mouse fibroblasts with an IC50 value of 5.2 µM. It is assumed that the hydroxymethyl moiety of 4a stabilizes the closed channel conformation by an H-bond with Glu236 as does the phenolic OH moiety of 3, Ro 25-6981 and ifenprodil.

Biochemical characterization of a novel beta-1--3, 1--4 glucan 4-glucanohydrolase from Thermomonospora sp. having a single active site for lichenan and xylan.

A bifunctional high molecular weight (Mr, 64,500 Da) beta-1-3, 1-4 glucan 4-glucanohydrolase was purified to homogeneity from Thermomonospora sp., exhibiting activity towards lichenan and xylan. A kinetic method was used to analyze the active site that hydrolyzes lichenan and xylan. The experimental data was in agreement with the theoretical values calculated for a single active site. Probing the conformation and microenvironment at active site of the enzyme by fluorescent chemo-affinity label, OPTA resulted in the formation of an isoindole derivative with complete inactivation of the enzyme to hydrolyse both lichenan and xylan confirmed the results of kinetic method. OPTA forms an isoindole derivative by cross-linking the proximal thiol and amino groups. The modification of cysteine and lysine residues by DTNB and TNBS respectively abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating the participation of cysteine and lysine in the formation of isoindole complex.

Rapid communication: effects of Steris 1 sterilization and Cidex ortho-phthalaldehyde high-level disinfection on durability of new-generation flexible ureteroscopes.

BACKGROUND AND PURPOSE: The effects of commonly used reprocessing methods on flexible ureteroscope longevity have never been examined. We prospectively studied the effects of Steris 1 sterilization and Cidex ortho-phthalaldehyde (OPA) high-level disinfection (HLD) on the image quality, physical structure, and deflective properties of two new flexible ureteroscopes. MATERIALS AND METHODS: Two identical "out-of-the-box" Storz 11278AU1 flexible ureteroscopes (Karl Storz Endoscopy, Tuttlingen, Germany) were sterilized individually using the Steris 1 system (Steris Mentor, Ohio) or disinfected with Cidex OPA (Advanced Sterilization Products, J&J, Irvine, CA) for 100 trials followed by a crossover to the other method for another 100 trials over a period of 1 year. After every five trials, optical quality, angle of deflection, and fiber damage were analyzed in the laboratory. Throughout the study, neither of these ureteroscopes was used clinically. RESULTS: After 100 trials, ureteroscope 1, which was sterilized initially in the Steris system, had a 12-mm tear on its shaft (noted after the 17th trial), 297 damaged fibers, and a 37% drop in resolution (loss of 3.75 lines/mm). There was no change in deflection from baseline. In contrast, after 100 cycles, ureteroscope 2, which was subjected to HLD with Cidex OPA, had no visible external damage, a 0% change in resolution, 10 damaged fibers, and no change in deflection. After the crossover, ureteroscope 2 developed a semilunar defect that obscured the endoscopic view, whereas there was no further significant damage to ureteroscope 1. CONCLUSION: After 100 cycles, the Steris 1 system rendered the flexible ureteroscope unusable, whereas HLD with Cidex OPA had minimal adverse impact.

Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols.

Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259-269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine-lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.

Comparison of the microbicidal efficacy on germ carriers of several tertiary amine compounds with ortho-phthalaldehyde and Perasafe.

Several tertiary amine formulations have been marketed as high-level disinfectants (HLDs). This study compared some of these formulations with two accepted HLDs [ortho-phthalaldehyde (OPA) and Perasafe] by determining the bactericidal effect on 52 micro-organisms using a metallic germ carrier, determining the sporicidal effect using a commercial germ carrier (3M spores), and performing a corrosion test on surgical blades with human blood. OPA and Perasafe were significantly more effective than all the tertiary amines tested, and acted within a contact time of 10 min compared with 20 min for the other products. For Gram-negative micro-organisms, Instrunet FA showed no significant differences at 20 min compared with OPA and Perasafe at 10 min. The amines tested did not differ significantly in global bactericidal efficacy. Unlike the tertiary amines, OPA and Perasafe were effective against mycobacteria (15-min contact period), but were not sporicidal. All agents (except one tertiary amine) passed the corrosion test. In conclusion, OPA and Perasafe can be considered as HLDs. However, 15-20 min of contact is required and both products have disadvantages.

Plasma level of a 5-fluorouracil metabolite, fluoro-beta-alanine correlates with dihydropyrimidine dehydrogenase activity of peripheral blood mononuclear cells in 5-fluorouracil treated patients.

5-fluorouracil (5-FU) is mostly metabolized after administration, and the metabolizing enzyme, dihydropyrimidine dehydrogenase (DPD), seems to be the rate-limiting factor. However, there are few reports on the final metabolite, fluoro-beta-alanine (FBAL). We report here the results of determination of the FBAL level in 5-FU treated patients and the correlation between the FBAL level and the DPD activity in peripheral blood mononuclear cells (PBMCs). Blood samples were collected from 20 patients, who had received continuous intravenous infusion (CIV) of 5-FU (320 mg/m2/24 hr) after resection of colorectal cancer, and the FBAL level was determined by high performance liquid chromatography (HPLC), after derivatizing into o-phthalaldehyde (OPA) and detecting fluorescence. DPD activity was measured in cytosol prepared from PBMCs using HPLC radioassay. The average FBAL plasma level during CIV of 5-FU was 911.0 ng/ml (521.0 to approximately 1834.6 ng/ml) and that of DPD activity in PBMCs was 282.6 pmol/min/mg-protein (145.0 to approximately 568.0 pmol/min/mg-protein). There was a significant correlation between the FBAL level and the DPD activity (r=0.805, p<0.0001). FBAL level in plasma may be useful in predicting the DPD activity in PBMCs, however, further studies are required considering the small number of cases in this study.

Biochemical and cytotoxic actions of 3,6-dihydroxy-4,5-dimethylphthalaldehyde in sarcoma 180 cells.

The replication of Sarcoma 180 cells in culture was inhibited by 3,6-dihydroxy-4,5-dimethylphthalaldehyde (HMPA). The inhibition of growth caused by HMPA was evident after treatment of cells with drug for only 15 min. This exposure period caused decreased in (a) cloning efficiency, (b) transport and/or phosphorylation of [3H]thymidine and [3H]uridine, (c) incorporation of radioactive nucleosides into acid-insoluble material, and (d) incorporation of [3H]leucine into protein. Examination of the cytotoxicities of the model compounds 2,3,5,6-tetramethyl-1,4-dihydroquinone (durohydroquinone) and o-phthalaldehyde indicated that the dialdehyde portion of the molecule was responsible for the cytocidal effects of HMPA. The ratio of adenosine triphosphate to adenosine diphosphate in the acid-soluble fraction of Sarcoma 180 cells incubated in vitro with HMPA for 45 min was reduced in a concentration-dependent manner. The reduction in the ATP pool size produced by HMPA contrasts with the action of the periodate oxidation product of cytidine dialdehyde, which has been reported to increase the intracellular concentration of adenosine triphosphate.

Effects of immersion disinfection of agar-alginate combined impressions on the surface properties of stone casts.

This study investigated the effects of disinfection of agar-alginate combined impressions on the surface properties of the resulting stone casts. Two brands of cartridge-form agar impression material and one alginate impression material were used. Agar-alginate combined impressions of smooth glass plates were prepared. The impressions were immersed in 0.55% ortho-phthalaldehyde solution or 0.5% sodium hypochlorite solution for 1, 3, 5 and 10 min. A stone cast made with an impression that had not been immersed was prepared as a control. The surface roughness (Ra) of the stone casts was measured, and the cast surfaces were observed by SEM. Immersion of agar-alginate combined impressions in 0.5% sodium hypochlorite solution for up to 10 min had no serious adverse effects on the surface properties of the stone casts. In contrast, even 1 min of immersion in 0.55% ortho-phthalaldehyde solution caused deterioration of the cast surface properties.

Inhibition of camel lens zeta-crystallin/NADPH:quinone oxidoreductase by pyridoxal-5'-phosphate.

Camel lens zeta-crystallin was inhibited by pyridoxal-5'-phosphate (PAL-P) and o-phthalaldehyde. PAL-P inactivated zeta-crystallin in a time- and concentration-dependent manner. The initial rate of inactivation followed pseudo-first-order kinetics with the second-order rate constant of 91 M-1 s-1. The modified enzyme showed the characteristic absorption peak at 325 nm indicative of the formation of phosphopyridoxallysine. Quantitative analysis suggested the incorporation of 1 mole of PAL-P/subunit of enzyme. NADPH was able to substantially protect zeta-crystallin against PAL-P inactivation, whereas the substrate 9,10-phenanthrenequinone (PQ) did not provide any protection. Inhibition of zeta-crystallin by PAL-P was uncompetitive with NADPH (Ki=37 microM) and non-competitive with respect to the substrate (Ki=57 microM). Inhibition of zeta-crystallin by o-phthalaldehyde was used to establish the location of an essential lysine residue. Incubation of zeta-crystallin with o-phthalaldehyde resulted in the formation of an isoindole derivative that had a characteristic fluorescence spectrum. This suggested that a lysine residue is located within 3 A of a cysteine residue at the NADPH binding region. SDS-PAGE showed the o-phthalaldehyde-modified enzyme remained largely monomer (approx. 80%), although bands corresponding to dimer and tetramer forms were also present. These results suggested that an essential lysine residue is located in the vicinity of the NADPH binding site. This residue may simply ensure the proper binding of NADPH to the active site of zeta-crystallin.