The FIKK kinase of Toxoplasma gondii is not essential for the parasite's lytic cycle.

FIKK kinases are a novel family of kinases unique to the Apicomplexa. While most apicomplexans encode a single FIKK kinase, Plasmodium falciparum expresses 21 and piroplasms do not encode a FIKK kinase. FIKK kinases share a conserved C-terminal catalytic domain, but the N-terminal region is highly variable and contains no known functional domains. To date, FIKK kinases have been primarily studied in P. falciparum and Plasmodium berghei. Those that have been studied are exported from the parasite and associate with diverse locations in the infected erythrocyte cytosol or membrane. Deletion of individual P. falciparum FIKK kinases indicates that they may play a role in modification of the infected erythrocyte. The current study characterises the single FIKK gene in Toxoplasma gondii to evaluate the importance of the FIKK kinase in an apicomplexan that has a single FIKK kinase. The TgFIKK gene encoded a protein of approximately 280kDa. Endogenous tagging of the FIKK protein with Yellow Fluorescent Protein showed that the FIKK protein exclusively localised to the posterior end of tachyzoites. A Yellow Fluorescent Protein-tagged FIKK and a Ty-tagged FIKK both co-localised with T. gondii membrane occupation and recognition nexus protein to the basal complex and were localised apical to inner membrane complex protein-5 and Centrin2. Deletion of TgFIKK, surprisingly, had no detectable effect on the parasite's lytic cycle in vitro in human fibroblast cells or in acute virulence in vivo. Thus, our results clearly show that while the FIKK kinase is expressed in tachyzoites, it is not essential for the lytic cycle of T. gondii.

Broccoli seed extract: Genotoxicity and subchronic toxicity studies.

Potential health benefits have been attributed to broccoli consumption. Hence, there is potential for use of broccoli seed extract (BSE) in food or for use as a dietary supplement. To assess the potential safety of a BSE product, three genotoxicity experiments, including an Ames, in vivo mouse micronucleus, and in vivo mouse sperm abnormality assay, were carried out. BSE was subject to an acute oral toxicity test and was evaluated in a 30-day feeding study in rats. BSE showed no mutagenic activity in the Ames assay and no evidence of genotoxic potential in the in vivo assays at doses up to 10 g/kg body weight (bw). The LD50 of BSE in rats was >10 g/kg bw/d. In the 30-day feeding study, in which BSE was administered in the diet to provide doses of 0, 0.3, 1.0, or 3.0 g/kg bw/d, no toxicological significant effects were noted on body weight, body weight gain, organ weights, or on the results of hematological, clinical chemistry and histopathological evaluations. The no-observed-adverse-effect level was considered to be 3.0 g/kg bw/d, the highest dose tested. Collectively, these results support the safe use of BSE as a food ingredient or product.

Subchronic toxicity evaluation of potato protein isolates.

The protein content of potatoes has a high nutritional value on par with eggs and soybeans. As a result, processed potato protein isolates may have commercial value for addition to other food products to increase protein content. A manufacturing process has been developed to produce total potato (TP), as well as low (LMW) and high molecular (HMW) weight, protein isolates as food ingredients. To assess the safety of these isolates, groups of 10 Wistar rats/sex were administered dietary admixtures containing 15% HMW, 7.5% LMW or 15% TP protein isolates for a period of 90days. There was no effect of treatment on clinical signs, mortality, body weight and body weight gain. No biologically significant changes occurred in hematological and clinical chemistry parameters. No statistically significant changes in organ weights were recorded. Histopathological analyses revealed no clear, treatment-related changes. A slight increase in the incidence, but not severity, of vacuolation of the zona fasciculate of the adrenal gland was noted in males of the 15% HMW and 7.5% LMW groups. The finding was not considered adverse or ascribed any toxicological significance. Overall, HMW, LMW, and TP protein isolates were well-tolerated and without adverse effect. These data support the safety of potato protein isolates.

Effect of dietary seaweed extracts and fish oil supplementation in sows on performance, intestinal microflora, intestinal morphology, volatile fatty acid concentrations and immune status of weaned pigs.

A 2x2 factorial experiment (ten sows per treatment) was conducted to investigate the effect of maternal dietary supplementation with a seaweed extract (SWE; 0 v. 10·0 g/d) and fish oil (FO; 0 v. 100 g/d) inclusion from day 109 of gestation until weaning (day 26) on pig performance post-weaning (PW) and intestinal morphology, selected microflora and immune status of pigs 9 d PW. The SWE contained laminarin (10 %), fucoidan (8 %) and ash (82 %) and the FO contained 40 % EPA and 25 % DHA. Pigs weaned from SWE-supplemented sows had higher daily gain (P=0·063) between days 0 and 21 PW and pigs weaned from FO-supplemented sows had higher daily gain (P<0·05) and gain to feed ratio (P<0·01) between days 7 and 14 PW. There was an interaction between maternal SWE and FO supplementation on caecal Escherichia coli numbers (P<0·05) and the villous height to crypt depth ratio in the ileum (P<0·01) and jejunum (P<0·05) in pigs 9 d PW. Pigs weaned from SWE-supplemented sows had lower caecal E. coli and a higher villous height to crypt depth ratio in the ileum and jejunum compared with non-SWE-supplemented sows (P<0·05). There was no effect of SWE on E. coli numbers and villous height to crypt depth ratio with FO inclusion. Maternal FO supplementation induced an increase in colonic mRNA abundance of IL-1α and IL-6 (P<0·05), while SWE supplementation induced an increase in ileal TNF-α (P<0·01) and colonic TFF3 mRNA expression (P<0·05). In conclusion, these results demonstrate that SWE and FO supplementation to the maternal diet influenced the gastrointestinal environment and performance of the weaned pig.

Effect of maternal fish oil and seaweed extract supplementation on colostrum and milk composition, humoral immune response, and performance of suckled piglets.

An experiment with a 2 x 2 factorial arrangement of treatments (n = 10 sows/treatment) was conducted to investigate the effect of maternal dietary supplementation with seaweed extract (SWE: 0 vs. 10.0 g/d) and fish oil (FO) inclusion (0 vs. 100 g/d) from d 109 of gestation until weaning (d 26) on sow colostrum and milk composition, humoral immune response on d 5 and 12 of lactation, and suckling piglet performance. Furthermore, the influence of dietary treatment on the phagocytic activity of whole blood white cells at weaning was examined. The SWE (10 g) contained laminarin (1 g), fucoidan (0.8 g), and ash (8.2 g) and was extracted from a Laminaria spp. The FO contained approximately 40% eicosapentaenoic acid and 25% docosahexaenoic acid. The SWE-supplemented sows had greater colostrum IgG (P < 0.01) and milk protein (P < 0.05) concentrations on d 12 of lactation compared with non-SWE-supplemented sows. Piglets suckling SWE-supplemented sows had greater serum IgG (P < 0.01) and IgA (P < 0.05) concentrations on d 5 and IgG concentrations on d 12 (P < 0.05) of lactation compared with those suckling non SWE-supplemented sows. In contrast, FO supplementation exerted a suppressive effect on piglet serum IgA concentrations on d 5 of lactation (P < 0.05) compared with non-FO-supplemented diets. Dietary FO supplementation enhanced the n-3 PUFA proportion of sow milk (P < 0.001) and piglet serum at weaning (P < 0.001). Piglets suckling SWE-supplemented sows had a greater percentage of Escherichia coli phagocytizing leukocytes (P < 0.05) and a reduced percentage of E. coli phagocytizing lymphocytes (P < 0.01) compared with non-SWE-supplemented sows. Piglets suckling FO-supplemented sows had a greater percentage of leukocytes (P < 0.05) and lymphocytes (P < 0.05) phagocytizing E. coli compared with non-FO-supplemented sows. However, total leukocyte, lymphocyte, monocyte, and neutrophil numbers were not influenced by sow dietary treatment. Average piglet weaning weight and ADG between birth and weaning were not influenced by sow dietary treatment. In conclusion, the current study demonstrates that SWE supplementation from d 109 of gestation until weaning enhanced colostral IgG concentrations and circulatory IgG concentrations in suckled piglets on d 5 and 12 of lactation. Furthermore, the percentage of leukocytes and lymphocytes phagocytizing E. coli at weaning increased in piglets suckling FO-supplemented sows, indicating an enhancement of immune function against presenting pathogens. However, the combination of SWE and FO bestowed no positive effect on immune responses investigated in the current study.

Safety studies of l-alanyl-l-glutamine (l-AG).

The safety of l-alanyl-l-glutamine (l-AG) derived by fermentation using a recombinant Escherichia coli strain containing the l-amino acid alpha-ligase gene from Bacillus subtilis, was assessed in acute and subchronic toxicity studies in the rat. l-AG was tested in vitro in a bacterial reverse mutation assay and in a chromosome aberration assay. l-AG was not acutely toxic when administered to Sprague-Dawley rats by gavage at 2000mg/kg bw. In a 14-day range-finding study, l-AG at up to 5% in the diet was without effect. In the 13-week dietary study, there were no toxicologically significant differences between the treated groups (1.0, 3.0 and 5.0% l-AG) and the controls (0% and 5% l-AG produced via a different method) with respect to body weight gain, feed consumption, feed efficiency, or the results of ophthalmological, haematological, clinical chemistry, and urinalysis evaluations. Three of 10 high-dose males had mild testicular changes, however, these were of exactly the same nature and severity as those that occur spontaneously, and were considered unlikely to be treatment-related. The NOAEL in both males and females was established as the highest dose tested at 3129 and 3601mg/kg bw/day, respectively (5.0% in the diet). There was no evidence of genotoxicity of l-AG in the Ames assay or in the in vitro CHL cell chromosome aberration study.

Long-term safety of alpha-lipoic acid (ALA) consumption: A 2-year study.

Alpha-lipoic acid (ALA) (CAS RN 1077-28-7), also referred to as thioctic acid, has been demonstrated to exhibit strong anti-oxidant properties. In order to test the long-term toxicity of ALA, groups of 40-50 male and female, 5-6-week-old, Sprague-Dawley rats were subjected to oral administration of 20, 60, or 180 mg/kg body weight (bw)/day ALA for 24 months. There was no significant difference between control animals and treated animals at 20 or 60 mg/kg bw/day with respect to body weight gain, food consumption, behavioural effects, haematological and clinical chemistry parameters, and gross and histopathological findings. In all treatment groups, mortality was slightly lower as compared to the control. The absolute weights of the heart (high-dose males), thymus (high-dose males), and left adrenal (mid-dose males), liver (high-dose females), and lungs (high-dose females) were decreased in comparison to controls. These changes were of no toxicological significance. The only notable finding in rats of both sexes dosed at 180 mg/kg bw/day was a reduction in food intake relative to the controls and a concomitant decrease in body weight. This decrease in body weight led to significant differences between the control and high-dose rats with respect to the absolute weights of certain organs. However, no gross or histopathological changes were associated with these findings. The no-observed-adverse-effect level (NOAEL) is considered to be 60 mg/kg bw/day.

Safety evaluation of alpha-lipoic acid (ALA).

The safety of the antioxidant alpha-lipoic acid (racemic form) (ALA), also called thioctic acid (CAS RN 1077-28-7) was assessed in acute and subchronic toxicity studies as well as in in vitro and in vivo mutagenicity/genotoxicity studies. ALA was not acutely toxic to rats (LD(50)>2000mg/kg bw, OECD method 425). Administration of 31.6 or 61.9mg ALA/kg bw/day for 4 weeks to male/female Wistar rats did not show any adverse effects. Specifically, there was no significant difference between control and treated animals at 31.6 or 61.9mg ALA/kg bw with regard to body weight gain, feed consumption, animal behaviour, or haematological and clinical chemistry parameters. Only the high-dose of 121mg ALA/kg bw was associated with slight alterations in liver enzymes as well as histopathological effects on the liver and mammary gland. ALA did not possess any mutagenic activity in the Ames assays conducted with various bacterial strains of Salmonella typhimurium. Moreover, there was no evidence of genotoxic activity in a mouse micronucleus assay. The results of these studies support the safety of ALA. The no-observed-adverse-effect level (NOAEL) is considered to be 61.9mg/kg bw/day.

A fluorescence polarization based Src-SH2 binding assay.

The tyrosine kinase pp60c.src has been implicated as being a potential therapeutic target in several human diseases including cancer and osteoporosis. An important region within this kinase is the SH2 domain (Src homology 2) which binds to phosphorylated tyrosine residues contained within specific peptide sequences. Homologous domains are found in a variety of cytoplasmic proteins and have been shown to be essential for controlling many important signaling pathways. Developing specific inhibitors of SH2 interactions would therefore be extremely useful for modulating a variety of signaling pathways and potentially be useful for the treatment of human disease. Current methodology for the development of organic molecules as drug leads requires the ability to test thousands of individual compounds or natural product extracts in biochemical assays. Such tests must be reproducible, simple, and versatile. This paper describes an assay based on fluorescence polarization for measuring the binding of compounds to the Src-SH2 domain. The assay is insensitive to changes in fluorescence intensity working even in solutions with moderate optical density and functions in the presence of up to 20% dimethyl sulfoxide. These features make it especially useful for high-throughput screening of both natural and synthetic compound libraries.

Effect of colostral fat level on fat deposition and plasma metabolites in the newborn pig.

The effects of colostral fat level on fat deposition and plasma concentrations of glucose, insulin, and free fatty acids (FFA) were determined in 28 newborn pigs during the first postnatal day. Soon after birth, pigs were allotted to four treatments groups. Group 1 was killed at birth. The remaining pigs were fed intragastrically sow colostrum that contained high (10.2%; HFC), normal (4.8%; NFC) or low (1.0%; LFC) levels of total fat at the rate of 15 to 18 g/kg birth weight at 65- to 70-min intervals. A total of 21 feedings was provided and pigs were killed 1 h after the last feeding. Body fat deposition increased linearly (P less than .01) with the amount of ingested fat by .32 (+/- .04) g per 1-g increase in fat intake. Fatty acid composition of the pigs changed toward that of the colostrum with increased fat in colostrum. More liver glycogen was lost (P less than .01) in pigs given LFC. Plasma concentrations of glucose and insulin were similar in pigs fed HFC and NFC. After the 11th feeding (14 h postnatal), LFC resulted in lower plasma glucose concentrations (P less than .05) than HFC or NFC. Plasma insulin concentrations also were lower in pigs fed LFC. Plasma FFA concentrations remained unchanged in pigs fed LFC but increased with both fat content in colostrum (P less than .05) and time (P less than .05) in the other two groups. Colostral fat plays a major role in the supply of energy and in glucose homeostasis in the neonatal pig.