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[Comparative studies on the antimicrobial activity of S-alkylthiuroniumhalides (author's transl)].

S-alkylthiuroniumhalides according to formula I (table 1) have been tested under standardized conditions with respect to their antimicrobial activity against bacteria, yeasts and fungi. In table 2 the bacteriostatic and fungistatic efficiency and in table 3 the microbicidal efficiency of 8 different S-alkylthiuronium compounds are compared with the efficiency of N,N-dimethyl-coconutoilalkyl-benzyl-ammonium-chloride. The germ inhibitory effect of the quaternary ammonium compound has been superior to the S-alkylthiuronium salts in almost all respects. The germicidal effect of the S-alkylthiuroniumhalides depends on the alkyl chain length bound to the S-atom and gets the optimum with S-dodecylthiuronium-chloride. Already a 0.0005% concentration is sufficient to kill all test germs within 30 minutes. S-dodecylthiuroniumchloride proved to be just as effective as the known well effective quaternary ammonium compounds. It seems to be possible to increase the microbicidal efficiency by additional alkylating - specially methylating - of both nitrogen atoms.

Arthrodermataceae↗

Indoor air quality during renovation actions: a case study.

A temporary renovation activity releases considerably high concentrations of particulate matter, viable and non-viable, into air. These pollutants are a potential contributor to unacceptable indoor air quality (IAQ). Particulate matter and its constituents lead, sulfate, nitrate, chloride, ammonium and fungi as well as fungal spores in air were evaluated in a building during renovation action. Suspended dust was recorded at a mean value of 6.1 mg m(-3) which exceeded the Egyptian limit values for indoor air (0.15 mg m(-3)) and occupational environments (5 mg m(-3)). The highest particle frequency (23%) of aerodynamic diameter (dae) was 1.7 microm. Particulate sulfate (SO(4)(2-)), nitrate (NO(3)(-)), chloride (Cl(-)), ammonium (NH(4)(+)) and lead components of suspended dust averaged 2960, 28, 1350, 100 and 13.3 microg m(-3), respectively. Viable fungi associated with suspended dust and that in air averaged 1.11 x 10(6) colony forming unit per gram (cfu g(-1)) and 92 colony forming unit per plate per hour (cfu p(-1) h(-1)), respectively. Cladosporium(33%), Aspergillus(25.6%), Alternaria(11.2%) and Penicillium(6.6%) were the most frequent fungal genera in air, whereas Aspergillus(56.8%), Penicillium(10.3%) and Eurotium(10.3%) were the most common fungal genera associated with suspended dust. The detection of Aureobasidium, Epicoccum, Exophiala, Paecilomyces, Scopulariopsis, Ulocladium and Trichoderma is an indication of moisture-damaged building materials. Alternaria, Aureobasidium, Cladosporium, Scopulariopsis and Nigrospora have dae > 5 microm whereas Aspergillus, Penicillium and Verticillium have dae < 5 microm which are suited to penetrate deeply into lungs. Particulate matter from the working area infiltrates the occupied zones if precautionary measures are inadequate. This may cause deterioration of IAQ, discomfort and acute health problems. Renovation should be carefully designed and managed, in order to minimize degradation of the indoor and outdoor air quality.

Air Pollution, Indoor↗

Role of urease in megasome formation and Helicobacter pylori survival in macrophages.

Previous studies have demonstrated that Helicobacter pylori (Hp) delays its entry into macrophages and persists inside megasomes, which are poorly acidified and accumulate early endosome autoantigen 1. Herein, we explored the role of Hp urease in bacterial survival in murine peritoneal macrophages and J774 cells. Plasmid-free mutagenesis was used to replace ureA and ureB with chloramphenicol acetyltransferase in Hp Strains 11637 and 11916. ureAB null Hp lacked detectable urease activity and did not express UreA or UreB as judged by immunoblotting. Deletion of ureAB had no effect on Hp binding to macrophages or the rate or extent of phagocytosis. However, intracellular survival of mutant organisms was impaired significantly. Immunofluorescence microscopy demonstrated that (in contrast to parental organisms) mutant Hp resided in single phagosomes, which were acidic and accumulated the lysosome marker lysosome-associated membrane protein-1 but not early endosome autoantigen 1. A similar phenotype was observed for spontaneous urease mutants derived from Hp Strain 60190. Treatment of macrophages with bafilomycin A1, NH4Cl, or chloroquine prevented acidification of phagosomes containing mutant Hp. However, only ammonium chloride enhanced bacterial viability significantly. Rescue of ureAB null organisms was also achieved by surface adsorption of active urease. Altogether, our data indicate a role for urease and urease-derived ammonia in megasome formation and Hp survival.

Adsorption↗

Biochemical responses of two erythrinidae fish to environmental ammonia.

The non-ionized form of ammonia is very toxic to many aquatic species. It is especially important in several aspects of fish biology. A large range of organismal strategies for coping with environmental stressors is usually observed in living organisms. Among those, the responses for managing chemical stressors are well studied. The present work compares biochemical responses of two evolutionarily close species, Hoplias malabaricus and Hoplerythrinus unitaeniatus, exposed to environmental ammonia. Adult fish were submitted to 1.0 mg/L of ammonium chloride for 24 hours, and plasma ammonia and urea levels were determined. The activities of OUC enzymes OCT and ARG, and the accessory enzyme GS, were quantified in liver extract and are expressed below in mumol/min/mg of wet tissue. Increases in OUC enzymes (GS from 1.14 to 2.43, OCT from 0.81 to 1.72, and ARG from 3.15 to 4.23), plasma ammonia (from 0.95 to 1.42 mmol/L), and plasma urea (from 0.82 to 1.53 mmol/L) were observed (p < 0.05) in H. malabaricus exposed to 1 mg/L of ammonia chloride. The GS in H. unitaeniatus increased from 1.43 to 1.84, however the OCT, ARG, and plasma urea from H. unitaeniatus did not change. These data indicate that each species responds differently to the same environmental stressor.

Adaptation, Physiological↗

Mediation of reduction of spontaneous and experimental pulmonary metastases by ricin A-chain immunotoxin 45-2D9-RTA with potentiation by systemic monensin in mice.

We developed a model to assess the therapeutic effects of the 45-2D9-ricin A-chain immunotoxin (RTA) on pulmonary metastases. The 45-2D9 mouse monoclonal antibody recognizes a Mr 74,000 glycoprotein highly expressed by rat fibroblasts transformed with the Kirsten sarcoma virus (transformed rat fibroblasts). These cells metastasize spontaneously and form lung colonies in nu/nu and irradiated BALB/c mice. Injection i.v. of 45-2D9-RTA specifically reduced formation of spontaneous pulmonary metastases and lung colonies originating from freshly disaggregated tumor cells or cultured cells. Antibody alone or mixed with unconjugated ricin A chain and an immunotoxin that recognizes a melanoma-associated antigen were ineffective. Unconjugated 45-2D9 antibody specifically blocked the 45-2D9-RTA activity in vivo. Administration of the lysosomotrophic agents ammonium chloride and chloroquine in vivo did not potentiate immunotoxin-mediated reduction in lung colonies although they were effective in vitro. Monensin potentiated 45-2D9-RTA activity in vitro and in vivo.

Ammonium Chloride↗

Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes.

The biosynthesis of lysosomal cysteine proteases, cathepsins B and H, was investigated by using pulse-chase experiments in vivo in primary cultures of rat hepatocytes. Cathepsins B and H were isolated from either total cell extracts or culture medium labeled with [35S]methionine by immunoprecipitation and analyzed for their molecular forms. Within 60 min of chase, cellular proforms of cathepsins B of 39 kDa and H of 41 kDa were converted to single-chain form cathepsins B of 29 kDa and H of 28 kDa, respectively, and persisted as these forms even after 12-h chase periods. The proforms of cathepsins B and H derived from pulse-labeling experiments showed complete susceptibility to endoglycosidase H treatment, indicating that these proenzymes bear high-mannose-type oligosaccharides at the stage of initial events of biosynthesis. In the presence of tunicamycin, unglycosylated proenzymes of cathepsins B of 35 kDa and H of 34 kDa were found to be secreted into the extracellular medium without undergoing proteolytic processing. Furthermore, in the presence of swainsonine, a potent inhibitor of Golgi mannosidase II, considerable amounts of the proenzymes were secreted and accumulated in the medium during chasing periods. These results suggest that the oligosaccharide moiety of these enzymes would be necessary for the intracellular sorting mechanism. In monensin-treated cells, the conversion of intracellular proenzymes to mature enzymes was significantly inhibited and the proenzymes were secreted into the medium. In the presence of chloroquine or ammonium chloride, proteolytic processing of the proenzymes was completely prevented and the enhanced secretion of proenzymes was observed. These results suggest that in the presence of lysosomotropic amines the intracellular sorting of proenzymes might not occur properly during biosynthesis.

Alkaloids↗

125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts.

125I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37 degrees C, the cell-bound 125I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound 125I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0 degrees C, cell-bound 125I-hEGF is not degraded but slowly dissociates from the cell. The data are consistent with a mechanism in which 125I-hEGF initially is bound to the cell surface and subsequently is internlized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-CH2Cl, benzyl guanidobenzoate), a lysosomotropic agent (chloroquine) various local anesthetics (cocaine, lidocaine, procaine), and ammonium chloride. After the binding and degradation of 125I-hEGF the fibroblasts are no longer able to rebind fresh hormone. The binding capacity of these cells is restored by incubation in a serum-containing medium; this restoration is inhibited by cycloheximide or actinomycin D.

Ammonium Chloride↗

Internalization and intracellular fate of anti-CD5 monoclonal antibody and anti-CD5 ricin A-chain immunotoxin in human leukemic T cells.

We have investigated the entry and subsequent intracellular fate of T101 monoclonal antibody (MoAb) and T101-ricin A-chain (RTA) immunotoxin (IT) directed against the CD5 antigen (Ag) expressed on human leukemic CEM cells. We provide direct evidence for the internalization of T101 MoAb and the corresponding IT. Both the MoAb and IT were internalized at a relatively low rate. This slow internalization process could be related to the partial recycling of the MoAb/Ag or IT/Ag complexes. Analysis of the internalized molecules showed that their molecular weight was only partially altered after internalization and that no free A-chain could be found inside the cells, indicating that lysosomal degradation and cleavage of disulfide-linked conjugates is a quantitatively minor phenomenon compared with the localization of internalized anti-CD5 ITs in an endosomo-Golgi compartment, followed by their recycling to the cell surface. We believe that this is the major factor explaining the low efficacy of anti-CD5 IT when assayed in the absence of potentiating substances. Finally, we showed that the presence of ammonium chloride and monensin, which both dramatically enhance the kinetics of IT activity, did not affect the rate of internalization or the intracellular localization of the conjugate, suggesting that these activators could act at the postendocytotic level on a limited number of IT molecules.

Ammonium Chloride↗

Mechanism of lysis of Trypanosoma brucei gambiense by human serum.

Resistance to lysis by human serum (HS) is an important parameter used to distinguish Trypanosoma brucei brucei from both Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Neither the exact nature of the trypanolytic factor (TLF) nor the mechanism of action by which HS lyses susceptible trypanosomes is well understood. This report tries to elucidate the role played by the variable surface glycoprotein (VSG) coat and trypanosome surface-related processes in the mechanism of HS lysis of HS-sensitive (HSS) and HS-resistant (HSR) trypanosomes. Procyclic forms of T. brucei gambiense transformed from either HSS or HSR bloodstream stages were found to be HSR. These procyclic forms were shown to have lost their VSG coat. However, the addition of excess soluble VSG from HSS trypanosomes did not block lysis of HSS trypanosomes. Human serum lysis was significantly inhibited if the trypanosomes were incubated with membrane stabilizers, i.e., including cytochalasins (B, D, and E specifically), zinc acetate, vinblastine, and benzyl alcohol, or with the lysosomotropic agents ammonium chloride and chloroquine. The inhibition exerted by these compounds was always reversible. The results in this report, taken together, strengthen the hypothesis that the lytic factor interacts with and moves along the trypanosome surface to be internalized eventually.

Ammonium Chloride↗

Pseudo arylsulfatase A deficiency. Biosynthesis of an abnormal arylsulfatase A.

Pseudo arylsulfatase A deficiency, an asymptomatic condition, and metachromatic leukodystrophy, a severe neurodegenerative disease, are both associated with profound reductions of arylsulfatase A activity in man. We now report that with metabolic labelling, cultured pseudo deficient cells synthesized about 20% of the normal amount of arylsulfatase A at a reduced rate of apparent synthesis and increased rate of degradation. However, in the presence of ammonium chloride which stimulated secretion of lysosomal enzymes, these cells synthesized about 80% of the normal amount of enzyme protein. Hence, the defect in pseudo arylsulfatase A deficiency is associated with labile arylsulfatase A molecules which can be stabilized if they are diverted from intracellular storage.

Ammonium Chloride↗

Method for the determination of 15NH3 enrichment in biological samples by gas chromatography/electron impact ionization mass spectrometry.

An alternative method for the determination of [15N]ammonia enrichment in biological fluids was developed. It is based on the use of glutamate dehydrogenase of bovine liver (EC 1.4.1.2.) with 2-oxopentanoic acid as substrate, to convert the ammonia present in the sample into norvaline, the enrichment of which can be measured by gas chromatography/mass spectrometry as its tertiary butyldimethylsilyl (TBDMS) derivative under electron impact selective ion recording (SIR) conditions. The principal advantage of the present approach is that it is simpler and quicker than the previously described methods, because the synthetic product, norvaline, is not present in biological fluids and pre-processing of the sample is unnecessary. The procedure includes a pre-incubation stage which allows removal of contaminant ammonia present in the reagents used for the enzyme reaction. The contributions of other sources of nitrogen to norvaline production have been checked and quantified: these may provide limitations of the technique when samples for analysis are low in ammonia (e.g. arterial or hepatic venous blood). To reduce these contributions, short times of incubation are proposed. The results from two experiments in vivo in which two sheep were infused with [15N]ammonium chloride in the mesenteric vein are presented and the biological implications which arise from the results are discussed. The validity of the procedures was demonstrated by the quantitative recovery from the mesenteric and portal veins of [15N]ammonia infused.

Ammonia↗

A simple method for short-term culturing bone marrow and unstimulated blood from acute leukemias.

It has become routine practice to culture bone marrow and/or unstimulated peripheral blood for cytogenetic analysis due to associations of consistent chromosomal abnormalities within specific subgroups of leukemias. Unfortunately, two leukemias for which numerous chromosomal aberrations have been recorded, acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL), frequently present with very low cell counts often resulting in unsuccessful cultures. Furthermore, when cultures are successful, chromosomes are often contracted and metaphase spreads can be of poor quality. In the present investigation, by first treating blood or bone marrow from AML and ALL patients with ammonium chloride (1.5 mM) we were able to obtain chromosomal preparations often similar in quality to those obtained from normal stimulated blood cultures. With this improved culturing method it is also possible to generate high-quality chromosomes and DNA from the same sample.

Ammonium Chloride↗

Acidic compartments and rhoptry formation in Toxoplasma gondii.

DAMP (3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine), which differentially accumulates in acidic compartments, was used to identify such compartments in Toxoplasma gondii tachyzoites at the electron microscope level. In both free tachyzoites and dividing intracellular parasites the only sites of DAMP accumulation were mature and forming rhoptries. No labelling of other secretory organelles (micronemes and dense granules), the ER, Golgi or any other membrane-bounded organelles or anything resembling a lysosomal system was observed. Labelling of the forming rhoptries was higher and more homogenous than in mature rhoptries in which labelling was confined to the expanded ends of each organelle. The acid pH-dependent accumulation of DAMP in the forming and mature rhoptries was blocked by ammonium chloride and monensin, reagents known to abolish intracellular pH gradients. Estimates of rhoptry pH, based on the level of DAMP accumulation, show that the intralumenal pH of forming rhoptries is more acidic (pH 5.5-3.5) than the mature rhoptries (pH 7.0-5.0).

Ammonium Chloride↗

Distinct mechanisms of entry by envelope glycoproteins of Marburg and Ebola (Zaire) viruses.

Since the Marburg (MBG) and Ebola (EBO) viruses have sequence homology and cause similar diseases, we hypothesized that they associate with target cells by similar mechanisms. Pseudotype viruses prepared with a luciferase-containing human immunodeficiency virus type 1 backbone and packaged by the MBG virus or the Zaire subtype EBO virus glycoproteins (GP) mediated infection of a comparable wide range of mammalian cell types, and both were inhibited by ammonium chloride. In contrast, they exhibited differential sensitivities to treatment of target cells with tunicamycin, endoglycosidase H, or protease (pronase). Therefore, while they exhibit certain functional similarities, the MBG and EBO virus GP interact with target cells by distinct processes.

Ammonium Chloride↗

Measurement of secretory vesicle pH reveals intravesicular alkalinization by vesicular monoamine transporter type 2 resulting in inhibition of prohormone cleavage.

1. The acidic interior of neuroendocrine secretory vesicles provides both an energy gradient for amine-proton exchangers (VMATs) to concentrate small transmitter molecules, for example catecholamines, and an optimal pH for the prohormone convertases which cleave hormone precursors. There is evidence that VMAT activity modulates prohormone cleavage, but in the absence of measurements of pH in secretory vesicles in intact cells, it has not been possible to establish whether these effects are attributable to raised intravesicular pH due to proton transport through VMATs. 2. Clones were generated of the hamster insulinoma cell line HIT-T15 expressing a pH-sensitive form of green fluorescent protein (GFP-F64L/S65T) targeted to secretory vesicles, with and without co-expression of VMAT2. In order to study prohormone cleavage, further clones were generated that expressed preprogastrin with and without co-expression of VMAT2. 3. Confocal microscopy of GFP fluorescence indicated that the pH in the secretory vesicles was 5.6 in control cells, compared with 6.6 in cells expressing VMAT2; the latter was reduced to 5.8 by the VMAT inhibitor reserpine. 4. Using a pulse-chase labelling protocol, cleavage of 34-residue gastrin (G34) was found to be inhibited by co-expression with VMAT2, and this was reversed by reserpine. Similar effects on vesicle pH and G34 cleavage were produced by ammonium chloride. 5. We conclude that VMAT expression confers the linked abilities to store biogenic amines and modulate secretory vesicle pH over a range influencing prohormone cleavage and therefore determining the identity of regulatory peptide secretory products.

Alkalies↗

Occurrence of peptide and clavine ergot alkaloids in tall fescue grass.

Evidence is presented that ergot alkaloids are ubiquitous in tall fescue pastures infected with the clavicipitaceous fungal endophyte Sphacelia typhina (or Acremonium coenophialum). Ergopeptide alkaloids, predominantly ergovaline, constituted 10 to 50 percent of the total ergot alkaloid concentration, which was as high as 14 milligrams per kilogram in sheaths and 1.5 milligrams per kilogram in blades. Ergot alkaloid concentrations were substantially increased by application of large amounts (10 millimoles per liter) of potassium nitrate or ammonium chloride to infected plants in the greenhouse. The results indicate that ergot alkaloids are probably responsible for the toxicity to cattle of this common pasture and lawn grass and that ergotism-like toxicoses may be caused by clavicipitaceous fungi other than Claviceps.

Ammonium Chloride↗

Biological differences between vesicular stomatitis virus Indiana and New Jersey serotype glycoproteins: identification of amino acid residues modulating pH-dependent infectivity.

We previously generated recombinant vesicular stomatitis viruses (VSV) based on the Indiana serotype genome which contained either the homologous glycoprotein gene from the Indiana serotype (VSIV-GI) or the heterologous glycoprotein gene from the New Jersey serotype (VSIV-GNJ). The virus expressing the GNJ gene was more pathogenic than the parental VSIV-GI virus in swine, a natural host (26). For the present study, we investigated the biological differences between the GI and GNJ proteins that may be related to the differences in pathogenesis between VSIV-GI and VSIV-GNJ. We show that the capacities of viruses with either the GNJ or GI glycoprotein to infect cultured cells differ depending on the pH. VSIV-GNJ could infect cells at acidic pHs, while the infectivity of VSIV-GI was severely reduced. VSIV-GNJ infection was also more sensitive to inhibition by ammonium chloride, indicating that the GNJ protein had a lower pH threshold for membrane fusion. We applied selective pressure to VSIV-GI by growing it at successively lower pH values and isolated variant viruses in which we identified amino acid changes that conferred low-pH-resistant infectivity. Repeated passage in cell culture at pH 6.8 resulted in the selection of a VSIV-GI variant (VSIV-6.8) that was similar to VSIV-GNJ regarding its pH- and ammonium chloride-dependent infectivity. Sequence analysis of VSIV-6.8 revealed that it had a single amino acid substitution in the amino-terminal region of the glycoprotein (F18L). This alteration was shown to be responsible for the observed phenotype by site-directed mutagenesis of a VSIV-GI full-length cDNA and analysis of the recovered engineered virus. A further adaptation of VSIV-6.8 to pHs 6.6 and 6.4 resulted in additional amino acid substitutions in areas of the glycoprotein that were not previously implicated in attachment or fusion.

Amino Acid Substitution↗