Accurate analysis of urea in milk and milk powder by isotope dilution gas chromatography–mass spectrometry

A high order method for measuring urea concentrations in milk and milk powder was developed. The method can be applied to certify the concentration of urea in some new milk and milk powder CRMs. This high accurate method for analysis of milk is valuable given the inherent challenges associated with the complexity of the sample matrix. A measurement procedure based on gas chromatography/isotope dilution mass spectrometry (GC/IDMS) was developed. Samples were pre-treated with acetonitrile to remove proteins and the method was applied to determine urea concentrations in milk and milk powder. Excellent precision was obtained, with within- and between-set coefficients of variation of 0.15–0.46 and 0.18–0.65%, respectively. The measurement uncertainty is evaluated. The method can trace to mass.     

Monitoring of genotoxic damage in smokeless tobacco ((Maraş powder) consumers using micronucleated exfoliated oral cells

Mara? powder is a kind of smokeless tobacco used in the south-eastern region of Turkey. The present study was carried out to assess possible DNA damage in exfoliated oral cells of Mara? powder users by analysing the frequencies of micronuclei (MN), which is a simple and reliable biomarker for genotoxic damage and to screen for the detection of site-specific differences in the frequencies of MN. The mean (±SD) MN frequency in the inner lip mucosa site was 1.27(±0.55) % for Mara? powder users and 0.88(±0.47) % for non-smoking control subjects (p < 0.05) and 0.82(±0.40) % for the buccal site of Mara? powder users (p < 0.01). There was no significant site-specific difference between the inner lip site and the buccal mucosa site 0.73(±0.43) % in the MN frequency of non-smoking control subjects (p > 0.05). There was no significant effect of daily consumption of Mara? powder, and duration of usage on MN frequencies. The present study suggests that the oral use of smokeless tobacco represents a genotoxic hazard and also that use of MN from a single site may be misleading as a marker of genotoxic exposure. Habitual use of Mara? powder should be taken into account and could be considered unsafe.     

Spray-drying of Dunaliella salina to produce a β-carotene rich powder

Powders of Dunaliella salina biomass were obtained by spray drying a cell concentrate under different drying regimes. A three-factor, two-level experimental design was employed to investigate the influence of inlet temperature, outlet temperature and feed solids on β-carotene recovery. The effect of microencapsulation in a polymer matrix of maltodextrin and gum arabic was also studied. All powders were stored under specific conditions to assess the stability of the native β-carotene. There was a trend indicating that lower outlet temperature yielded higher carotenoid recoveries, β-carotene recovery varying between 57% and 91%. Microencapsulated biomass yielded 100% recoveries. All non-microencapsulated powders were unstable in terms of β-carotene content in the presence of natural light and oxygen showing 90% degradation over a 7-day period. The incorporation of a microencapsulating agent had a significant increase in the storage stability. Results indicated a first-order degradation of the β-carotene in microencapsulated powders with kinetic constants of 0.06 day⁻¹ and 0.10 day⁻¹. HPLC analysis showed no effect of drying processes on isomer composition (9-cis-β-carotene and all-trans-β-carotene ratio). This behaviour was also observed during storage of the microencapsulated powders. Received 16 October 1996/ Accepted in revised form 13 November 1997     

Variability in secondary structure of the antimicrobial peptide Cateslytin in powder,solution, DPC micelles and at the air–water interface

Cateslytin (bCGA ³⁴⁴RSMRLSFRARGYGFR³⁵⁸), a five positively charged 15 amino-acid residues arginine-rich antimicrobial peptide, was synthesized using a very efficient procedure leading to high yields and to a 99% purity as determined by HPLC and mass spectrometry. Circular dichroism, polarized attenuated total reflectance fourier transformed infrared, polarization modulation infrared reflection Absorption spectroscopies and proton two-dimensional NMR revealed the flexibility of such a peptide. Whereas being mostly disordered as a dry powder or in water solution, the peptide acquires a α-helical character in the “membrane mimicking” solvent trifuoroethanol. In zwitterionic micelles of dodecylphophatidylcholine the helical character is retained but to a lesser extent, the peptide returning mainly to its disordered state. A β-sheet contribution of almost 100% is detected at the air–water interface. Such conformational plasticity is discussed regarding the antimicrobial action of Cateslytin. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

One-step synthesis of n-octyl β-d-xylotrioside, xylobioside and xyloside from xylan and n-octanol using acetone powder of Aureobasidium pullulans in supercritical fluids

n-Octyl -d-xylotrioside, xylobioside and xyloside, which are effective next-generation surfactants, can be prepared by the one-step reaction of xylan and n-octanol using the acetone powder (acetone-dried cells) of Aureobasidium pullulansas the enzyme source of xylanase in supercritical carbon dioxide (CO₂) and fluoroform (CHF₃) fluids. Yields of the n-octyl glycosides were significantly increased using supercritical CHF₃, and the total yields of the n-octyl -d-glycosides exceeded 250 mg g^–1 xylan. n-Octyl -d-xylotrioside was produced only by using supercritical fluids.     

Grape powder extract attenuates tumor necrosis factor α-mediated inflammation and insulin resistance in primary cultures of human adipocytes

Grapes are rich in phenolic phytochemicals that possess anti-oxidant and anti-inflammatory properties. However, the ability of grape powder extract (GPE) to prevent inflammation and insulin resistance in human adipocytes caused by tumor necrosis factor α (TNFα), a cytokine elevated in plasma and white adipose tissue (WAT) of obese, diabetic individuals, is unknown. Therefore, we examined the effects of GPE on markers of inflammation and insulin resistance in primary cultures of newly differentiated human adipocytes treated with TNFα. We found that GPE attenuated TNFα-induced expression of inflammatory genes including interleukin (IL)-6, IL-1β, IL-8, monocyte chemoattractant protein (MCP)-1, cyclooxygenase (COX)-2 and Toll-like receptor (TLR)-2. GPE attenuated TNFα-mediated activation of extracellular signal-related kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) and activator protein-1 (AP-1, i.e., c-Jun). GPE also attenuated TNFα-mediated IκBα degradation and nuclear factor-kappa B (NF-κB) activity. Finally, GPE prevented TNFα-induced expression of protein tyrosine phosphatase (PTP)-1B and phosphorylation of serine residue 307 of insulin receptor substrate-1 (IRS-1), which are negative regulators of insulin sensitivity, and suppression of insulin-stimulated glucose uptake. Taken together, these data demonstrate that GPE attenuates TNFα-mediated inflammation and insulin resistance in human adipocytes, possibly by suppressing the activation of ERK, JNK, c-Jun and NF-κB.     

Tomato powder impedes the development of azoxymethane-induced colorectal cancer in rats through suppression of COX-2 expression via NF-κB and regulating Nrf2/HO-1 pathway

Cancer is one of the leading causes of death worldwide. Since dietary factors have been connected to a reduced risk of a diversity of human cancers, in this study we investigated the effects of tomato powder (TP) on the development of azoxymethane (AOM)-induced colorectal cancer in Wistar rats, and possible mechanism(s) by which TP shows its chemopreventive activity. Here we show that TP added to feed at 5% rate decreases the rate of aberrant crypt foci (ACF) and reduces the development of adenocarcinoma and growth of AOM-induced colorectal cancer in rats. In addition, we demonstrate that TP supplementation shows its chemopreventive activities through inhibition of cyclooxygenase-2 (COX-2) expression via NF-κB pathway and promotion of apoptosis, as well as regulating Nrf2/HO-1 signaling pathway in colorectal tissue of AOM-treated rats. Our findings identify an intimate connection between dietary supplementation of TP and the decreased risk of colorectal cancer in rats, and suggest that consumption of TP would be a natural candidate for the prevention of colorectal cancer in men.     

Crystal structure determination of the β-cyclodextrin-p-aminobenzoic acid inclusion complex from powder X-ray diffraction data

The 1:1 inclusion complex of β-cyclodextrin and p-aminobenzoic acid was prepared and characterized by TG-DTA. The crystal structure of the complex was solved directly from powder X-ray diffraction data using the direct space approach and refined using Rietveld refinement techniques. The complex crystallizes in monoclinic P2₁ space group, with unit cell parameters a = 20.7890 ?, b = 10.2084 ?, c = 15.1091 ?, β = 110.825°, V = 2997 ?³. The amino group is located at the wide side of the β-cyclodextrin cavity, forming hydrogen bonds with β-cyclodextrin, and the carboxyl group is located at the narrow side. The crystallographic data obtained from powder diffraction data were compared with the single crystallographic data, and the result shows that solving crystal structure of cyclodextrins inclusion complexes of such complexity is accessible to powder diffractionists to some extent.     

Biotransformation of Digitoxin in Cell Cultures of Capsicum frutescens in the Presence of β-cyclodextrin

Cell suspension cultures of Capsicum frutescens accumulated digoxin, purpureaglycoside A and other unknown derivatives when digitoxin, a cardiac glycoside, was used as a precursor. The feeding of digitoxin complexed with &#103 -cyclodextrin increased the accumulation of digoxin, purpureaglycoside A and other unknown derivatives. Control cultures (without digitoxin) did not produce any of these metabolites. The growth of cells was affected by both digitoxin as well as digitoxin- &#103 -cyclodextrin. The accumulation of purpureaglycoside A and digoxin reached a maximum of 1241 and 374 &#119 g 100 ml &#109 1 culture on the 6th and 2nd day, respectively, which was 3.9 and 4.5 fold higher than cultures treated with digitoxin alone (sampled on the 13th day). The other unknown derivatives formed in digitoxin- &#103 -cyclodextrin fed cultures were 15 times higher than digitoxin alone fed C. frutescens cultures. The addition of glucose to digitoxin- &#103 -cyclodextrin treated cultures increased the accumulation of purpureaglycoside A which reached a maximum of 3589 &#119 g 100 ml &#109 1 culture after 12 h incubation, which was a 2.9 fold increase over cultures treated with digitoxin- &#103 -cyclodextrin alone.     

Evolutionary Advantages of Participation of Vasopressin instead of Vasotocin in Regulation of Water–Salt Balance in Mammals

In experiments on non-anesthetized Wistar white rats there was studied reaction of kidney to an intramuscular injection of arginine vasotocin or arginine vasopressin at doses from 0.001 to 0.05 µg/100 g body mass on the background of a water load. Water (5 ml/100 g body mass) was administered through a catheter into stomach to suppress secretion of endogenous antidiuretic hormone (ADH). In experiments with water administration, diuresis increased due to a decrease of osmotic permeability of renal tubules and to excretion of osmotically free water, with the constant clearance of sodium ions. Injection of 0.05 µg arginine vasopressin led to a marked decrease of diuresis due to a rise of reabsorption of osmotically free water without elevation of excretion of osmotically active substances. Injection of the same dose of arginine vasotocin resulted in no increase of diuresis; however, reabsorption of osmotically free water and excretion of osmotically active substances including sodium ions were more pronounced. Hence, both vasotocin and vasopressin increased osmotic permeability of the tubular epithelium, but vasotocin, unlike vasopressin, promoted reduction of reabsorption of sodium ions and their loss with urine. A suggestion is made that one of the reasons for replacement in mammals of the molecular ADH forms (vasotocin by vasopressin) was the absence of the pronounced natriuretic effect in arginine vasopressin. This was of crucial significance to preserve sodium ions in the organism, to maintain water–salt balance in animals adapted to the terrestrial life, and to provide not only osmo-, but also volumoregulation.     

Alteration in virulence characteristics of biofilm cells of Pseudomonas aeruginosa in presence of Tamm-Horsfall protein

Summary Tamm-Horsfall glycoprotein is the most abundant protein in human urine. The present investigation was planned to study the effect of Tamm-Horsfall protein (THP) on elaboration of virulence factors by biofilm cells of Pseudomonas aeruginosa. It was observed that with increase in concentration of THP from 10 to 50 μg/ml there was significant enhancement in elaboration of all the virulence factors by biofilm cells of P. aeruginosa. However, with further increase in concentration of THP from 50 to 70 μg/ml, significant decrease in elaboration of all the virulence traits was observed. Implications of these findings in relation to urinary tract infections caused by P. aeruginosa have been discussed.     

Glycosylation ofα 1 glycoprotein in septic shock: Changes in degree of branching and in expression of sialyl Lewisx groups

The occurrence of differences in acute-phase response, with respect to concentration and glycosylation of ₁-acid glycoprotein (AGP) was studied in the sera of patients surviving or not from septic shock. Crossed affino-immunoelectrophoresis was used with concanavalin A andAleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of sialyl Lewis^x (SLeX) groups on AGP. Septic shock apparently induced an acute-phase response as indicated by the increased serum levels and changed glycosylation of AGP. In the survivor group a transient increase in diantennary glycan content was accompanied by a gradually increasing fucosylation and SLeX expression, comparable to those observed in the early phase of an acute-inflammatory response. Remarkably, in the non-survivor group a modest increase in diantennary glycan content was accompanied by a strong elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical for the late phase of an acute-phase response. Our results suggest that these changes in glycosylation of AGP can have a prognostic value for the outcome of septic shock.Abbreviations AAL Aleuria aurantia lectin - AGP ₁-acid glycoprotein - CAIE crossed affinoimmunoelectrophoresis - Con A Concanavalin A - HSPC human serum protein calibrator - IL-1 interleukin 1 - IL-6 interleukin 6 - LIF leukaemia inhibitory factor - LPS lipopolysaccharide - SLeX sialyl Lewis^x - TNF tumour necrosis factor     

Conservation of Orchids in China

China, as one of the most important Range States of orchids in the world, has about 1300 species withabundant varieties of orchids. However, owing to over, abuse harvest and illegal trades, the wild populations ofthese species have declined dramatically, even bringing some species to the edge of extinction. In response to such challenges, the Chinese government has put conservation of orchids as a priority agenda, taking a series of Innovative and effective measures to protect the orchids in the wild.[第一段]     

Mode of action of sulfoxides in preventing loss of activity of 11β-hydroxylase in cultured bovine adrenocortical cells

Previously, loss of 11β-hydroxylase activity when adrenocortical cells are incubated with the pseudosubstrate cortisol was found to be reduced when the concentration of oxygen was lowered, or when butylated hydroxyanisole (BHA) or dimethyl sulfoxide (Me₂SO) were included in the medium. In the present experiments, we tested the hypothesis that Me₂SO protects 11β-hydroxylase by scavenging OH^? radicals. Substances known to react with OH^? at high rates and non-toxic enough to be used at concentrations of 10–100 mM, including several alcohols, benzoate and radioprotectant thiols, did not prevent loss of activity of 11β-hydroxylase in the presence of 50 μM cortisol. Two of the alcohols, ethanol and glycerol, as well as Me₂SO, were radioprotective in cultured bovine adrenocortical cells. Therefore free OH^? radicals do not appear to be involved in loss of 11β-hydroxylase activity. When sulfoxides other than dimethyl sulfoxide were tested for their ability to protect 11β-hydroxylase in the presence of cortisol, several aryl sulfoxides, particularly dibenzyl sulfoxide, as well as dipropyl sulfoxide, were active at concentrations to $\text{1}\text{200}$ of that required for Me₂SO. Previously, we have demonstrated that 11β-hydroxylase inhibitors, particularly metyrapone, effectively protect against loss of 11β-hydroxylase activity in the presence of pseudosubstrates and therefore we examined whether sulfoxides may act by directly inhibiting 11β-hydroxylase. Me₂SO showed an ED₅₀ for inhibition of 11β-hydroxylase activity of > 1 M, in contrast to its ED₅₀ for protection of 34 mM. For metyrapone, however, the ED₅₀ for inhibition of the enzyme (250 nM) was close to that for protection of activity (270 nM). The other sulfoxides showed ED₅₀-values for inhibition of 11β-hydroxylase that were substantially higher than the ED₅₀-values for protection. Sulfoxides may have a mixed mode of action in protection of 11β-hydroxylase activity, as previously shown for phenols; they may protect by radical scavenging, but may also need to bind close to the active site of the enzyme where destructive radicals may be formed.     

Histoenzymorphology of β-D-glucuronidase in cortical granules in oocytes of some representative vertebrates

Summary Histoenzymorphologic investigations on -D-glucuronidase in cortical granules in oocytes of some representative vertebrates revealed interesting differences in localisation of the enzyme in the two techniques employed using 8-hydroxyquinoline -D-glucosiduronic acid and naphthol AS-BI -D-glucosiduronic acid as substrates. In the former a nonspecific localisation in the entire cytoplasm of the oocytes excepting nucleus was evident whereas in the latter the cortical granules alone exhibited intense activity. To some extent this difference could be accounted for on the basis of yolk content of the eggs. -D-glucoronidase-rich lysosomal cortical granules were found in the amphibian, reptilian and avian oocytes whereas mammalian oocyte cortical granules were devoid of this enzyme. -D-glucuronidase in cortical granules was sensitive to low concentrations of Hg⁺⁺, Cu⁺⁺, glucuronic and mucic acids. Other histochemical evidence for the lysosomal nature of the cortical granules is presented and discussed. -D-glucuronidase seems to have a role in ovum-nutrition and activation which, thus, make a new addition to the existing knowledge of physiological role of -D-glucuronidase and lysosomes.Dr. A. T. Varute — Research Project guide.