Fluoromethylcyclopropylamine derivatives as potential in vivo toxicophores - A cautionary disclosure.

Fluorination of metabolic hotspots in a molecule is a common medicinal chemistry strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.

Safety evaluation of ß-agarase preparations from Streptomyces coelicolor A3(2).

Recent studies on neoagarooligosaccharides prepared by hydrolyzing agar with ß-agarase DagA produced from Streptomyces coelicolor A3(2) have enhanced our knowledge about the enzymatic utility of S. coelicolor. For safety evaluation, a crude extracellular protein containing DagA (crDagA) was prepared from the culture broth of S. coelicolor A3(2) M22-2C43, a highly productive strain of DagA. All genotoxicity tests, such as bacterial reverse mutation assay, eukaryotic chromosomal aberration assay, and in vivo micronucleus assay in mice showed no mutagenic activity of crDagA. No abnormalities were found in the appearance or behavior upon single oral administration up to 20,000 mg/kg body weight (BW) [318 mg TOS (Total Organic Solids)/kg BW] and long-term repeated oral administration toxicity tests up to 10,000 mg/kg BW/day (159 mg TOS/kg BW/day) in Sprague Dawley®™ rats. In addition, there were no statistically significant differences in the body weight change, food intake, hematology, blood biochemistry, organ weight, and clinical signs between the crDagA-administered and non-administered groups during the experimental period. This result showed that crDagA produced from S. coelicolor A3(2) is a safe, non-toxic substance, and therefore, can be used safely for manufacturing neoagarooligosaccharide, a functional substance effective in improving metabolic syndrome.

Efficient fructose production from plant extracts by immobilized inulinases from Kluyveromyces marxianus and Helianthus tuberosus.

The enzymatic hydrolysis of poly- and oligosaccharides from plants seems like an advantageous approach for sugars production. Two inulinases producing fructose from plant oligosaccharides were isolated from yeast Kluyveromyces marxianus and plant Helianthus tuberosus. Both enzymes were immobilized on polymeric carriers by using the static adsorption approach. We could save 80.4% of the initial catalytic activity of plant inulinase immobilized on KU-2 cation-exchange resin and 75.5% of yeast enzyme activity adsorbed on AV-17-2P anion-exchange resin. After immobilization, the Km values increased 1.5 and 6 times for enzymes from K. marxianus and H. tuberosus, respectively. The optimal temperatures for catalysis of both enzymes were increased from 48-50 °C up to 70 °C. The activities of both immobilized enzymes remained unchanged after the 10 cycles of 20-min hydrolysis reaction at 70 °C model batch reactor. Sorbents, native and immobilized enzymes did not exhibit any mutagenic or cytotoxic activity.

PSA modification of NCAM supports the survival of injured retinal ganglion cells in adulthood.

Neural cell adhesion molecule (NCAM) is known as the cell surface glycoprotein, and it belongs to the immunoglobulin superfamily of adhesion molecules. Polysialic acid (PSA) is a carbohydrate attached to NCAM via either of two specific sialyltransferases: ST8SiaII and ST8SiaIV. Polysialylated neural cell adhesion molecule (PSA-NCAM) mediates cell interactions, plays a role in axon growth, migration, synaptic plasticity during development and cell regeneration. Some evidence has shown that PSA-NCAM supports the survival of neurons. It was demonstrated that PSA-NCAM is present in abundance in the retina during development and in adulthood. The aim of this study was to investigate whether PSA-NCAM promotes retinal ganglion cell (RGC) survival in transgenic mice with deficiencies in sialyltransferases or NCAM or after the administration of endoneuraminidase (Endo-N). RGC injury was induced by intravitreal administration of kainic acid (KA). These studies showed that injection of Endo-N after 14 days enhances the toxicity of KA to RGCs in wild-type (WT) mice by 18%. In contrast, in knockout mice (ST8SiaII-/-, ST8SiaIV-/-, NCAM-/-), survival of RGCs after KA injury did not change. Deficiencies of either ST8SiaII or ST8SiaIV did not influence the level of PSA-NCAM in the adult retina, however, in neonatal animals, decreased levels of PSA-NCAM were observed. In knockout ST8SiaII-/- adults, a reduced number of RGCs was detected, whereas in contrast, increased numbers of RGCs were noted in NCAM-/- mice. In conclusion, these data demonstrate that PSA-NCAM supports the survival of injured RGCs in adulthood. However, the role of PSA-NCAM in the adult retina requires further clarification.

Enhancement of immunoregulatory effects of Lactobacillus gasseri TMC0356 by heat treatment and culture medium.

AIMS: The aim of this study was to investigate the influence of heat treatment and culture media on the immunoregulatory effects of a probiotic strain, Lactobacillus gasseri TMC0356 (TMC0356). METHODS AND RESULTS: TMC0356 cultured in deMan-Rogosa-Sharpe and same food grade (FG) media were inactivated with the heat treatment at 70 and 90°C. Viable and heat-killed TMC0356 were tested for their ability to induce interleukin (IL)-12 production in the murine macrophage cell line J774.1. These TMC0356 were examined for their resistance to N-acetylmuramidase. Their morphology was observed by scanning electron microscopy. The heat-killed TMC0356 significantly induced IL-12 production in J774.1 cells and exhibited enhanced resistance to N-acetylmuramidase compared with viable TMC0356. Morphological changes were observed in TMC0356 when cultured in FG medium. Cell morphology and induction of IL-12 production in J774.1 cells were also associated. CONCLUSIONS: These results suggest that heat treatment and culture medium composition modified the immunoregulatory effects of TMC0356 to induce IL-12 production in macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that probiotic immunoregulatory effects may be modified by the processing technology of cell preparation.

Subchronic oral toxicity study with regular and enzymatically depolymerized sodium carboxymethylcellulose in rats.

Enzymatically depolymerized sodium carboxymethylcellulose (CMC-ENZ) is a new functional food ingredient which has a lower molecular weight and viscosity than regular sodium carboxymethylcellulose (CMC). Both compounds are known not to be absorbed to a significant extent, and the human safety of CMC as a thickening agent and stabilizer in food is well established. In the present study, the subchronic oral toxicity of CMC-ENZ was examined and compared with that of CMC in Wistar rats. Seven groups of 20 rats/sex were fed diets with 0 (controls), 2.5, 5 and 10% CMC and 2.5, 5 and 10% CMC-ENZ for a 3-month period. There was only one death that was unrelated to the treatment. Water intake, urine production and urinary sodium excretion increased with increasing doses of CMC and CMC-ENZ due to their sodium content of about 7-8%. The treatment-related occurrence of diarrhoea and caecal enlargement in the mid- and high-dose groups, a slight increase of plasma alkaline phosphatase, and increased urinary calcium and citrate excretions were considered to be generic effects that typically are observed in rodent studies with low digestible carbohydrates. The increased occurrence of nephrocalcinosis and hyperplasia of the urothelial epithelium in some of the treated groups was interpreted as an indirect consequence of a more alkaline urine coupled with an increased calcium excretion. As the frequency and severity of all these changes did not differ between corresponding CMC and CMC-ENZ dose groups, it is concluded that the two products have a similar toxicological profile.

Safety evaluation of pullulanase enzyme preparation derived from Bacillus licheniformis containing the pullulanase gene from Bacillus deramificans.

Pullulanase enzyme is an amylopectin debranching enzyme used in starch hydrolysis. This article describes studies conducted to investigate the safety of a pullulanase enzyme preparation produced by a strain of Bacillus licheniformis that has been transformed by introduction of genetic material from another Bacillus species, B. deramificans. A 4-week dietary toxicity study in rats was conducted in which test animals received pullulanase in the feed at concentrations of 0.2, 1.0, and 5.0%. No adverse treatment-related effects were observed. Lack of genetic toxicity potential was demonstrated by the results of a bacterial mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, in an in vitro histidine forward mutation study in mouse lymphoma cells, and in in vivo mouse bone marrow chromosome aberration and micronucleus assays. The enzyme preparation also has been shown to be a nonirritant in eye and primary dermal irritation tests in rabbits and is nontoxic by inhalation exposure. Finally, the genetically altered B. licheniformis has been demonstrated to be nonpathogenic upon single intraperitoneal injection to rats of both live and killed cells at doses up to 10(11) cells/kg. The results of these studies demonstrate that the enzyme preparation may be considered safe when employed in starch processing.

Light and electron microscopic study of m2 muscarinic acetylcholine receptor in the basal forebrain of the rat.

The m2 muscarinic acetylcholine receptor gene is expressed at high levels in basal forebrain, but the paucity of information about localization of the encoded receptor protein has limited the understanding of cellular and subcellular mechanisms involved in cholinergic actions in this region. The present study sought to determine the cellular localization of m2 protein, its relationship to cholinergic neurons, and its pre- and postsynaptic distribution in the rat medial septum-diagonal band complex using immunocytochemistry with polyclonal rabbit antibodies and a newly developed rat monoclonal antibody specific to the m2 receptor. Light microscopic colocalization studies demonstrated that m2 was present in a subset of choline acetyltransferase immunoreactive neurons, in choline acetyltransferase-negative neurons, and in more neuropil elements than was choline acetyltransferase. Intraventricular injections of 192 IgG-saporin, an immunotoxin directed to the low-affinity nerve growth factor receptor, resulted in depletion of choline acetyltransferase-immunoreactive neurons in the medial septum-diagonal band complex, whereas m2 immunoreactivity in neurons and in the neuropil was unchanged. By electron microscopy, m2 receptor in medial septum-diagonal band complex was localized to the plasmalemma of a small population of small to medium-sized neurons, and it was also found in dendrites, axons, and axon terminals in the neuropil. Neurons expressing m2 immunoreactivity received synaptic contacts from unlabelled axon terminals. A small distinct subpopulation of large neurons, unlabelled by m2 immunoreactivity, received synaptic contacts from m2-immunoreactive terminals. Thus, m2 receptor is situated to mediate the local effects of acetylcholine on basal forebrain cholinergic and noncholinergic neurons and, also, at both pre- and postsynaptic sites.

Purification and partial characterization of single-chain ribosome-inactivating proteins from the seeds of Phytolacca dioica L.

Three ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe et al. (1992) Bio/Technology 10, 405-412) were purified from the seeds of Phytolacca dioica. These proteins, called Phytolacca dioica RIPs (PD-S1, PD-S2 and PD-S3 RIPs), are glycoproteins, with M(r) approx. 30,000, inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and depurinate rat liver rRNA in an apparently identical manner as the A-chain of ricin and other RIPs (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Part of the purified rat liver ribosomes appeared resistant to the action of PD-S RIPs. The most abundant protein, PD-S2 RIP, gave a weak or nil cross-reaction with sera against various other RIPs, including a pokeweed antiviral protein from the roots of Phytolacca americana. PD-S2 RIP was linked to a monoclonal antibody (Ber-H2) against the CD30 human lymphocyte antigen and the resulting immunotoxin was selectively toxic to the CD30 + Hodgkin's lymphoma-derived L540 cell line.

[Studies of the chronic toxicity of mold pectinase]. / Prouchvaniia vurkhu khronichnata toksichnost na plesennata perktinaza.

Studied was the action of mould pectinase produced in this country, using Asp. niger. Rats and guinea pigs were subject to the testing through continuous oral treatment. The preparation was mixed with feed at 0.1 and 0.2 ratio percents (1000 and 2000 ppm)--with the rats for a period of 90 days, and with the guinea pigs--in the course of 180 days. It was found that the enzyme preparation stimulates the growth (more strongly expressed in rats), showing no adverse effect on the appetite and behaviour as well as on the tested clinical and biochemical indices of the blood and urine and the structure and development of viscera and the reproductive capacity of the test animals. The positive effect on the growth of animals is explained by the better utilization of the nutrient components of the ration, and the harmlessness of the preparation--by its weak resorption in the digestive tract (resorption index 13) and the fact that it is not able to attack animal tissues having no pectin substances.