Effects of extended transition milk feeding on blood metabolites of female Holstein dairy calves at 3 weeks of age: a liquid chromatography with tandem mass spectrometry-based metabolomics approach.

Transition milk (TRM) is a rich source of bioactive components that promotes intestinal development and growth, and reduces the susceptibility to diarrhoea in calves. The objective of this study was to characterise the effects of replacing pasteurised waste milk (none-saleable milk containing antibiotic and/or drug residues) with pasteurised TRM for 3 wk on blood metabolites of dairy calves at 21 d of age. A total of 84 healthy newborn female Holstein calves was blocked by birth order and assigned randomly to four treatment groups with partial replacement of pasteurised waste milk by TRM (second milking after parturition) at 0 (0 L/day TRM + 6 L/day milk), 0.5 (0.5 L/day TRM + 5.5 L/day milk), 1 (1 L/day TRM + 5 L/day milk), or 2 L (2 L/day TRM + 4 L/day milk) for a 21-day period. Serum metabolome was determined by liquid chromatography with tandem mass spectrometry-based metabolomics analysis on a subset of 26 randomly selected individuals from calves fed pasteurised waste milk (CON, 6 L/d milk; n = 13) or TRM (2 L/d TRM + 4 L/d milk; n = 13) at 21 d of age. The identified metabolites (194 out of 265) were categorised according to chemical class and the number of metabolites per class in the serum, amongst which glycerophospholipids 16% (n = 43), fatty acyls 7% (n = 19), organic acids 7% (n = 18), organic heterocyclic compounds 5% (n = 13), benzenoids 5% (n = 12), sphingolipids 5% (n = 12), organic oxygen compounds 4% (n = 11), and nucleic acids 3% (n = 9), were the predominant types. Significant differences in metabolites were determined by the volcano plot. Applying the volcano plot, only two metabolites (ceramide and phosphatidylserine) were significantly different between CON and TRM. Overall, our results suggested that prolonged TRM feeding for 3 wk had little effect on the serum metabolome of the dairy calves. We speculate that the potential effects of feeding TRM for 3 wk compared with waste milk were spatially limited to affect the composition of the local gut microbial community and the growth or function of the intestinal epithelium, not allowing detection of the likely effects in the serum through a metabolomic approach.

Identification of polcalcin as a novel allergen of Amaranthus retroflexus pollen.

INTRODUCTION: Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients' sera. METHODS: Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract. RESULTS: Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10kDa protein in crude extract. These results were confirmed by inhibition methods, too. CONCLUSION: The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart.

Molecular identification and antifungal susceptibility testing of Candida species isolated from dental plaques.

OBJECTIVE: The aim of the present study was to provide insight into the prevalence and susceptibility profiles of Candida species isolated from the dental plaque of Iranian immunocompetent patients. As a biofilm, Candida species are responsible for several disorders common to the oral cavity including gingivitis, dental caries, periodontitis, and the less common severe systemic infections specifically in immunosuppressed individuals. METHOD: PCR-RFLP was performed to identify yeasts isolated from the dental plaques of 40 immunocompetent patients. Moreover, antifungal susceptibility testing was performed in according to CLSI guidelines (M27-A3). RESULTS: Among 40 yeasts isolated from the dental plaques of immunocompetent patients, Candida albicans was the most common species (92.5%), followed by P. kudriavzevii (7.5%). It is the first isolation of P. kudriavzevii from dental plaques and the first evaluation of antifungal effect of the new imidazole, luliconazole and echinocandins against these samples worldwide. Luliconazole, voriconazole, amphotericin B and anidulafungin showed the best activity with the lowest geometric mean (GM) 0.03, 0.06, 0.08 and 0.09µg/ml, respectively, followed by miconazole (0.14µg/mL), caspofungin (0.24µg/mL) fluconazole (0.38µg/mL) and itraconazole (0.5µg/mL). CONCLUSION: The current study demonstrated luliconazole and echinocandins displayed excellent activity against all Candida isolates from dental plaques, presenting promising and potent alternative for all oral Candidiasis.

Radon exhalation rate and natural radionuclide content in building materials of high background areas of Ramsar, Iran.

Radon exhalation rates from building materials used in high background radiation areas (HBRA) of Ramsar were measured using an active radon gas analyzer with an emanation container. Radon exhalation rates from these samples varied from below the lower detection limit up to 384 Bq.m(-2) h(-1). The (226)Ra, (232)Th and (40)K contents were also measured using a high resolution HPGe gamma- ray spectrometer system. The activity concentration of (226)Ra, (232)Th and (40)K content varied from below the minimum detection limit up to 86,400 Bq kg(-1), 187 Bq kg(-1) and 1350 Bq kg(-1), respectively. The linear correlation coefficient between radon exhalation rate and radium concentration was 0.90. The result of this survey shows that radon exhalation rate and radium content in some local stones used as basements are extremely high and these samples are main sources of indoor radon emanation as well as external gamma radiation from uranium series.

Epidemiology of HTLV-1 in Neyshabour, Northeast of Iran.

BACKGROUND: HTLV-1 infection is endemic in Mashhad, Northeast of Iran. This study was designed to assess the epidemiology of HTLV-1 and potential risk factors in Neyshabour, Northeastern Iran. METHODS: All those who referred to Iranian Academic Center for Education, Culture and Research (ACECR)- Mashhad Laboratory in Neyshabour for evaluation of HTLV-I by ELISA, were tested using WB if the ELISA result was positive. A questionnaire about risk factors of infection was completed for all cases. RESULTS: HTLV-1 infection was positive in 7.2% (35/483) of the participants according to the results of enzymelinked immunosorbent assay (ELISA) and western blot (WB) tests. Infection was significantly associated with age, family size, income and blood transfusion. However, gender, education, birthplace, race, marital status, and history of surgery, dentistry, traditional cupping, and hospitalization showed no significant relation. CONCLUSION: It seems that HTLV-I infection is highly endemic in Neyshabour and it is seems that more effective treatment strategies are needed.

Determination of aluminum and zinc in Iranian consumed tea.

To determine aluminum and zinc in Iranian consumed tea, 31 tea samples were analysed for Al and Zn concentration of tea leaves and tea infusion. The results show that average concentration of Al and Zn in tea leaves was 326 and 50.7 mg. Kg(-1) respectively in this regard Nemoneh and Shahrzad show the highest and lowest concentration respectively in term of Al, also Debsh and Alkozi follow same situation in Zn concentration. Also the solubility of metals in the first infusion was significantly (Al, Zn: P < 0.01) higher than the second infusion and the solubility in the second infusion was also significantly higher than the third infusion (Al: P < 0.01; Zn: P < 0.05). Al leachate in the first infusion (2 min) for Mosama and Seilan tea was highest and lowest respectively. Also in the second and third infusion (5 and 10 min) Golkis and Nemoneh tea show the highest and lowest figures respectively. On the other hand, data from Zn transfer in tea infusion in the first infusion show that Mosama and Ahmad Atry tea have the highest and lowest leachate percentage respectively, while in the second and third infusion Mosama and Golkis tea follow the same situation. Calculation of percentage 'available' Al and Zn to the human system showed that 1 l of tea can provide 37.2% of the daily dietary intake of Al, the percentage 'available' for absorption in the intestine is only 1.78% for overall mean Al concentration. Also daily dietary intake of Zn was 2.13% while percentage available for absorption in the intestine was 0.72%.

Effect of Diabecon on sugar-induced lens opacity in organ culture: mechanism of action.

Cataract is the leading cause of blindness worldwide. Apart from ageing, diabetes has been considered to be one of the major risk factors of cataract. The high sugar levels in diabetes may cause tissue disruption and intumescences by osmotic changes induced via aldose reductase (AR) mediated polyol pathway. Therefore, agents that can inhibit AR and prevent sorbitol accumulation may be helpful to combat sugar-induced cataract. In the present study, AR inhibitory activity of Diabecon (an herbal drug used for diabetes) was studied together with its effect against sugar-induced lens opacity in organ culture. Diabecon aqueous extract (DAE) showed potential inhibitory activity with an IC50 value of 10 microg/ml against rat lens AR. Incubation of goat lens with supraphysiological concentrations of glucose (100 mM) led to the loss of lens transparency associated with increased AR activity, decreased soluble protein and increased protein carbonyls and glycation. Addition of DAE (0.3 mg/ml) to the medium preserved transparency and ameliorated the decrease in lens soluble protein due to hyperglycemia and also prevented the formation of glycated protein. Interestingly DAE inhibited aldose reductase activity in lens incubated with 100 mM glucose. DAE decreased protein carbonyls, prevented the loss of beta(L)-crystallin against 100 mM of glucose. We have also demonstrated here that most of these effects are mainly due to Gymnema sylvestre, one of the constituent herbs of Diabecon. These results suggest that Diabecon protect the lens against sugar-induced cataract by multiple mechanisms.

Preclinical evaluation of a prostate-targeted gene-directed enzyme prodrug therapy delivered by ovine atadenovirus.

Gene-directed enzyme prodrug therapy (GDEPT) based on the Escherichia coli enzyme, purine nucleoside phosphorylase (PNP), provides a novel strategy for treating slowly growing tumors like prostate cancer (CaP). PNP converts systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine, that kills PNP-expressing and nearby cells by inhibiting DNA, RNA and protein synthesis. Reporter gene expression directed by a hybrid prostate-directed promoter and enhancer, PSMEPb, was assayed after plasmid transfection or viral transduction of prostate and non-CaP cell lines. Androgen-sensitive (AS) LNCaP-LN3 and androgen-independent (AI) PC3 human CaP xenografts in nude mice were injected intratumorally with an ovine atadenovirus vector, OAdV623, that carries the PNP gene under PSMEPb, formulated with cationic lipid for enhanced infectivity. Fludarabine phosphate was then given intraperitoneally for 5 days at 75 mg/m2/day. PNP expression was evaluated by enzymic conversion of its substrate using reverse phase HPLC. OAdV623 showed excellent in vitro transcriptional specificity for CaP cells. In vivo, expression of PNP persisted for > 6 days after OAdV623 injection and a single treatment provided 100% increase in tumor doubling time and > 50% inhibition of tumor growth for both LNCaP-LN3 and PC3 lines, with increased tumor necrosis and apoptosis and decreased tumor cell proliferation. OAdV623 significantly suppressed the growth of AS and AI human CaP xenografts in mice.

Development of an in situ toxicity assay system using recombinant baculoviruses.

A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or more cloned enzymes. The ability of these enzymes to prevent or enhance the toxicity of drugs and xenobiotics is then measured in situ. Initial parameters for the system were developed and optimized using baculoviruses engineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1. SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several other toxic epoxides including: cis-stilbene oxide, 1,2,7,8-diepoxyoctane, allylbenzene oxide, and estragole oxide. The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to [3H]allylbenzene oxide or [3H]estragole oxide. On the other hand, 9,10-epoxyoctadecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to cells expressing sEH, suggesting that the corresponding fatty acid diols were cytotoxic. This was confirmed by showing that chemically synthesized diols of these fatty acid epoxides were toxic to control SF-21 cells at the same concentration as were the epoxides to cells expressing sEH. A recombinant baculovirus containing a chimeric cDNA formed between the rat P4501A1 and the yeast NADPH-P450 reductase was also constructed and expressed in this system. A model compound, naphthalene, was toxic to SF-21 infected with the rat P4501A1/reductase chimeric co-infecting SF-21 cells with either a human or a rat microsomal EH virus along with P4501A1/reductase virus. These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health concern.

Effects of dietary pyrrolizidine (Senecio) alkaloids on vitamin A metabolism in rats.

In two experiments, the effect of feeding the pyrrolizidine alkaloid (PA)-containing plant tansy ragwort (Senecio jacobaea) on the metabolism of vitamin A in rats was examined. In Experiment 1, dietary levels of 0, 5 and 10% tansy ragwort and 0, 25,000 and 100,000 IU supplementary vitamin A/kg diet were used. In rats fed tansy ragwort, both plasma and liver concentrations of vitamin A were depressed (P less than 0.05). Plasma values were decreased to about 50% of control values. In Experiment 2, rats fed 5% tansy ragwort had depressed plasma and liver vitamin A concentrations 48 h after oral dosing with vitamin A. Fecal excretion of vitamin A was decreased in tansy ragwort-fed rats. In control rats, most fecal vitamin A was excreted in the first 24 h post-dosing, while in tansy ragwort-fed animals, the excretion was delayed, suggesting a possible effect of PA on gut motility. The results indicate that PA causes reductions in liver and plasma vitamin A concentrations. Postulated mechanisms include an effect of PA on hepatic retinol-binding protein synthesis, and impaired biliary excretion depressing vitamin A absorption.