Nontoxic combretafuranone analogues with high in vitro antibacterial activity.

A library of thirty two 3,4-diphenylfuranones related to both combretastatin A-4 and antifungal 5-(acyloxymethyl)-3-(halophenyl)-2,5-dihydrofuran-2-ones was prepared. Cytotoxic effects on a panel of cancer and normal cell lines and antiinfective activity were evaluated, and the data were complemented with tests for the activation of caspase 3 and 7. High cytotoxicity was observed in some of the halogenated analogues, eg. 3-(3,4-dichlorophenyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one with IC50 0.12-0.23 µM, but the compounds were also highly toxic against non-malignant control cells. More importantly, notable antibacterial activity indicating G+ selectivity has been found in the 3,4-diarylfuranone class of compounds for the first time. Hydroxymethylation of furanone C5 knocked out cytotoxic effects (up to 40 µM) while maintaining significant activity against Staphylococcus strains in some derivatives. MIC95 of the most promising compound, 3-(4-bromophenyl)-5,5-bis(hydroxymethyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one against S. aureus strain ATCC 6538 was 0.98 µM (0.38 µg/mL) and 3.9 µM (1.52 µg/mL) after 24 and 48 h, respectively.

Leaching of N-nitrosodimethylamine (NDMA) in turfgrass soils during wastewater irrigation.

N-nitrosodimethylamine (NDMA) is a carcinogenic by-product of chlorination that is frequently found in municipal wastewater effluent. NDMA is miscible in water and negligibly adsorbed to soil, and therefore may pose a threat to ground water when treated wastewater is used for landscape irrigation. A field study was performed in the summer months under arid Southern California weather conditions to evaluate the leaching potential of NDMA in turfgrass soils during wastewater irrigation. Wastewater was used to irrigate multiple turfgrass plots at 110 to 160% evapotranspiration rate for about 4 mo, and leachate was continuously collected and analyzed for NDMA. The treated wastewater contained relatively high levels of NDMA (114-1820 ng L(-1); mean 930 ng L(-1)). NDMA was detected infrequently in the leachate regardless of the soil type or irrigation schedule. At a method detection limit of 2 ng L(-1), NDMA was only detected in 9 out of 400 leachate samples and when it was detected, the NDMA concentration was less than 5 ng L(-1). NDMA was relatively persistent in the turfgrass soils during laboratory incubation, indicating that mechanisms other than biotransformation, likely volatilization and/or plant uptake, contributed to the rapid dissipation. Under conditions typical of turfgrass irrigation with wastewater effluent it is unlikely that NDMA will contaminate ground water.

Loss pathways of N-nitrosodimethylamine (NDMA) in turfgrass soils.

N-nitrosodimethylamine (NDMA) is a potent carcinogen that is often present in municipal wastewater effluents. In a previous field study, it was observed that NDMA did not leach through turfgrass soils following 4 mo of intensive irrigation with NDMA-containing wastewater effluent. To better understand the loss pathways for NDMA in landscape irrigation systems, a mass balance approach was employed using in situ lysimeters treated with 14C-NDMA. When the lysimeters were subjected to irrigation and field conditions after NDMA application, very rapid dissipation of NDMA was observed for both types of soil used in the field plots. After only 4 h, total 14C activity in the lysimeters decreased to 19.1 to 26.1% of the applied amount, and less than 1% of the activity was detected below the 20-cm depth. Analysis of plant materials showed that less than 3% of the applied 14C was incorporated into the plants, suggesting only a minor role for plant uptake in removing NDMA from the vegetated soils. The rapid dissipation and limited downward movement of NDMA in the in situ lysimeters was consistent with the negligible leaching observed in the field study, and suggests volatilization as the only significant loss pathway. This conclusion was further corroborated by rapid NDMA volatilization found from water or a thin layer of soil under laboratory conditions. In a laboratory incubation experiment, prolonged wastewater irrigation did not result in enhanced NDMA degradation in the soil. Therefore, although NDMA may be present at relatively high levels in treated wastewater, gaseous diffusion and volatilization in unsaturated soils may effectively impede significant leaching of NDMA, minimizing the potential for ground water contamination from irrigation with treated wastewater.

Surface NADH oxidase of HeLa cells lacks intrinsic membrane binding motifs.

Disulfide-thiol interchange proteins with hydroquinone (NADH) oxidase activities (designated NOX for plasma membrane-associated NADH oxidases) occur as extrinsic membrane proteins associated with the plasma membrane at the outer cell surface. The cancer-associated NOX protein, designated tNOX, has been cloned. The 34-kDa plasma membrane-associated form of the protein contains no strongly hydrophobic regions and is not transmembrane. No myristoylation or phosphatidylinositol anchor motifs were discovered. Evidence for lack of involvement of a glycosylphosphatidylinositol-linkage was derived from the inability of treatment with a phosphatidylinositol-specific phospholipase C or with nitrous acid at low pH to release the NOX protein from the surface of HeLa cells or from plasma membranes isolated from HeLa cells. Binding of NOX protein to the plasma membrane via amino acid side chain modification or by attachment of fatty acids also is unlikely based on use of specific fatty acid antisera to protein bound fatty acids and as a result of binding to the cancer cell surface of a truncated form of recombinant tNOX. Incubation of cells or plasma membranes with 0.1 M sodium acetate, pH 5, at 37 degrees C for 1 h, was sufficient to release tNOX from the HeLa cell surface. Release was unaffected by protease inhibitors or divalent ions and was not accelerated by addition of cathepsin D. The findings suggest dissociable receptor binding as a possible basis for their plasma membrane association.

Analysis of estrogenic hormones in municipal wastewater effluent and surface water using enzyme-linked immunosorbent assay and gas chromatography/tandem mass spectrometry.

Although the estrogenic hormones 17 beta-estradiol and 17 alpha-ethinyl estradiol can be quantified in polluted waters by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/tandem mass spectrometry (GC/MS/MS), the compounds often are present at concentrations below detection limits. Enzyme-linked immunosorbent assays (ELISAs) provide a sensitive and robust means of quantifying estrogenic hormones in wastewater effluents and surface waters. Results from ELISA analysis of estrogenic hormones in secondary wastewater effluent indicate concentrations comparable to those that cause vitellogenesis in fish. Confirmatory analyses by GC/MS/MS are consistent with ELISA results. Effluent filtration, using sand filtration or microfiltration, removes approx. 70% of the hormones from secondary effluent, while advanced treatment, using reverse osmosis, removes more than 95% of hormones. The detection limits for estrogenic hormones are approx. 0.1 ng/L in wastewater effluent and 0.05 ng/L in surface water. The ELISA technique provides a relatively simple and practical method of assessing the fate of estrogenic hormones in engineered and natural systems.

A drug-unresponsive and protease-resistant CNOX protein from human sera.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ammonium sulfate fractionation were employed in series to purify and concentrate a 12.5-kDa protein fragment with a periodic (24-min period) proteinase K-resistant and drug-unresponsive NADH oxidase (CNOX) activity from pooled sera from healthy volunteers. The activity was unresponsive to capsaicin to distinguish it from the previously isolated cancer-associated NOX form (tNOX). Polyclonal antisera generated to the CNOX fragment cross-reacted with 20.5- to 24-kDa proteins of human sera, human lymphocytes, and plasma membranes from Escherichia coli with the molecular weight depending on source and conditions of treatment with proteinase K.

Determination of metal complexes of ethylenediaminetetraacetate in the presence of organic matter by high-performance liquid chromatography.

A high-performance liquid chromatography method is described here for the determination of the Cd(II), Co(II), Cu(II), Pb(II), and Zn(II) complexes of ethylenediaminetetraacetate (EDTA) in municipal wastewaters and surface waters. The method involves separation by ion-exchange chromatography on a reversed-phase C18 column coated with ion-pair reagent, followed by post-column conversion to FeEDTA and subsequent detection by UV absorbance. Although Co(II) and Cu(II) coelute, they can be quantified by analyzing absorbance by CuEDTA2- prior to post-column conversion. The method detection limit of 6-8 x 10(-8) M (5-7 ng) is an order of magnitude improvement over previous UV absorbance post-column reaction methods. The technique can be used in the presence of organic matter encountered in matrices such as untreated wastewater without pre-concentration or sample cleanup.

Mutations in activation-induced cytidine deaminase in patients with hyper IgM syndrome.

Recent studies have shown that mutations in a newly described RNA editing enzyme, activation-induced cytidine deaminase (AID), can cause an autosomal recessive form of hyper IgM syndrome. To determine the relative frequency of mutations in AID, we evaluated a group of 27 patients with hyper IgM syndrome who did not have defects in CD40 ligand and 23 patients with common variable immunodeficiency. Three different mutations in AID were identified in 18 patients with hyper IgM syndrome, including 14 French Canadians, 2 Lumbee Indians, and a brother and sister from Okinawa. No mutations were found in the remaining 32 patients. In the group of patients with hyper IgM syndrome, the patients with mutations in AID were older at the age of diagnosis, were more likely to have positive isohemagglutinins, and were less likely to have anemia, neutropenia, or thrombocytopenia. Lymphoid hyperplasia was seen in patients with hyper IgM syndrome and normal AID as well as the patients with hyper IgM syndrome and defects in AID.

Peer reviewed: understanding microcontaminants in recycled water.

Increasing attention is being given to the detection, treatment, and removal of problematic effluent-derived contaminants.

Polyhalogenated dibenzo-p-dioxins and dibenzofurans in the exhaust air during textile processings.

5 textile finishing processes were investigated for polychlorinated dibenzo-p-dioxins/furans (PCDD/F), polybrominated dibenzo-p-dioxins/furans (PBDD/F) and mixed polychlorinated-brominated dibenzo-p-dioxins/furans (PBCDD/F). For the purpose of these investigations the complete balance--dioxins input from the raw textiles, output over the finished textiles and output through the air path--was done. In the exhaust air only small PXDD/F concentration were detected. The textiles contain before and after the process nearly the same PCDD/F-concentrations. The concentrations of PBCDD/F arises after the process. Only the chimney depositions contain higher PCDD/F-concentrations up to 1,806.1 ng I-TE/kg, PBDD/F-concentrations up to 1,572.6 ng/kg and PBCDD/F-concentrations up to 40,801.4 ng/kg.

Capsaicin-responsive NADH oxidase activities from urine of cancer patients.

NADH oxidases of low specific activities from urine of cancer patients were found to be inhibited or stimulated by the vanilloid capsaicin (8-methyl-N-vanillyl-6-noneamide). Similar activities, inhibited or stimulated by capsaicin, were reported previously for sera of cancer patients but not for sera of normal volunteers or for patients with disorders other than cancer. Like those from sera, the activities from urine were resistant to heat and to digestion with proteinase K. Two different fractions with capsaicin-responsive NADH oxidase activities were obtained by FPLC. One fraction in which the 33-kDa band was the major component exhibited NADH oxidase activity stimulated by capsaicin. Another fraction in which 66-kDa and 45-kDa bands were major components exhibited NADH oxidase activities inhibited by capsaicin. A monoclonal antibody generated to a ca 34-kDa form of the NADH oxidase from sera reacted with a urine protein of a ca 33-kDa band in the capsaicin-stimulated fraction. The 33-kDa protein was of low abundance and was estimated to be present in amounts between 5 and 100 microgram/L, depending on the particular patient.

A drug-responsive and protease-resistant peripheral NADH oxidase complex from the surface of HeLa S cells.

Our laboratory described a ca. 34-kDa protein of the HeLa S cell surface that bound an antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea (LY181984) with high affinity and that exhibited NADH oxidase and protein disulfide-thiol interchange activities also inhibited by LY181984. The quinone site inhibitor 8-methyl-N-vanillyl-6-noneamide (capsaicin) also blocked these same enzymatic activities. Using capsaicin inhibition as the criterion, the drug-responsive oxidase was released from the surface of HeLa S cells and purified. The activity of the released capsaicin-inhibited oxidase was resistant to heating at 50 degrees C and to protease digestion. After heating and proteinase K digestion, the activity was isolated in >90% yield by FPLC as an apparent 50- to 60-kDa multimer. Final purification by preparative SDS-PAGE yielded a capsaicin-inhibited NADH oxidase activity of a specific activity indicative of >500-fold purification relative to the plasma membrane. The final activity correlated with a ca. 34-kDa band on SDS-PAGE. Matrix-assisted laser desorption mass spectroscopy as well as reelectrophoresis of the 34-kDa band indicated that the ca. 34-kDa material was a stable mixture of 22-, 17-, and 9.5-kDa components which occasionally migrated as a ca. 52-kDa complex. The purified complex tended to multimerize and formed insoluble 10- to 20-nm-diameter amyloid rods. The components of the purified 34-kDa complex were blocked to N-terminal amino acid sequencing and were resistant to further protease digestion. After multimerization into amyloid rods, the protein remained resistant to proteases even under denaturing conditions and to cyanogen bromide either with or without prior alkylation.

Rapid infusion of Sandoglobulin in patients with primary humoral immunodeficiency.

We studied 16 patients with primary disorders of humoral immunity to determine the practicality of infusing intravenous gamma globulin at rates of infusion and concentrations higher than the 4 mg/kg/min and 6% currently recommended. In the first portion of the study, the concentration of Sandoglobulin was increased from 6% to 12%. In the second portion, the flow rate was increased to 5 mg/kg/min, and if no reactions occurred, the time of each successive infusion was decreased by 10 minutes until infusions were completed in 15 to 20 minutes or vasomotor reactions occurred. Thirteen of the 16 patients completed the study; six patients achieved reaction-free rates greater than 15 mg/kg/min, and the other patients achieved rates ranging from 7.1 to 12 mg/kg/min. Seven patients had infusion times less than 30 minutes, with four patients completing infusions in 15 minutes. In the 13 patients who completed the study, there were 14 reactions in 159 infusions, mostly fever and chills, and often at the end or after the infusion. Only one infusion could not be completed because of an adverse reaction. Three patients were not able to complete the study because of adverse reactions; there were seven reactions in 11 infusions in these three patients, although none of the reactions were considered serious. Overall, in this study, most immunodeficient patients (13/16) were able to tolerate infusion rates of Sandoglobulin two to 10 times higher than the standard rates now recommended. The maximal rate of infusion must be individualized, but for carefully selected patients, infusions of 400 mg/kg can be completed in 1 hour or less.(ABSTRACT TRUNCATED AT 250 WORDS)