In vivo digestomics of milk proteins in human milk and infant formula using a suckling rat pup model

Human milk is the optimal mode of infant feeding for the first several months of life, and infant formulas serve as an alternative when breast-feeding is not possible. Milk proteins have a balanced amino acid composition and some of them provide beneficial bioactivities in their intact forms. They also encrypt a variety of bioactive peptides, possibly contributing to infant health and growth. However, there is limited knowledge of how milk proteins are digested in the gastrointestinal tract and bioactive peptides are released in infants. A peptidomic analysis was conducted to identify peptides released from milk proteins in human milk and infant formula, using a suckling rat pup model. Among the major milk proteins targeted, α-lactalbumin and β-casein in human milk, and β-lactoglobulin and β-casein in infant formula were the main sources of peptides, and these peptides covered large parts of the parental proteins’ sequences. Release of peptides was concentrated to specific regions, such as residues 70–92 of β-casein in human milk, residues 39–55 of β-lactoglobulin in infant formula, and residues 57–96 and 145–161 of β-CN in infant formula, where resistance to gastrointestinal digestion was suggested. In the context of bioactive peptides, release of fragments containing known bioactive peptides was confirmed, such as β-CN-derived opioid and antihypertensive peptides. It is therefore likely that these fragments are of biological significance in neonatal health and development.     

Effect of high bromide levels in the organism on the biological half-life of iodine in the rat

In experiments on rats, a significant influence of an extraordinarily high bromide intake on the whole-body biological half-life of iodine was established. Very high bromide intake (1) decreased the amount of radioiodide accumulated in the thyroid, (2) changed the proportion between the amount of iodine retained in the thyroid and the total amount of absorbed iodine, (3) significantly shortened the biological half-life of iodine in the thyroid from approximately 101 h to 33 h in animals maintained on an iodine-sufficient diet and from 92 h to about 30 h in rats fed a low-iodine diet, and (4) changed the time-course (added a further phase) of iodine elimination from the body. These changes were caused, with high probability, by an increase of iodine elimination by kidneys due to an excess of bromide. The overall picture of iodine elimination in animals fed the low-iodine diet was similar to that in animals maintained on iodine-sufficient diet. Presented in part at the 20th Workshop on Macro and Trace Elements held in Jena (Germany) on December 1–2, 2000.     

High bromide intake affects the accumulation of iodide in the rat thyroid and skin

The effect of a high bromide intake on the kinetics of iodide uptake and elimination in the thyroid and skin of adult male rats was studied. In rats fed a diet with sufficient iodine supply (>25 μg I/d), the iodide accumulation in the skin predominated during the first hours after ¹³¹I -iodide application. From this organ, radioiodide was gradually transferred into the thyroid. A high bromide intake (>150 mg Br⁻/d) in these animals led to a marked decrease in iodide accumulation, especially by the thyroid, because of an increase in iodide elimination both from the thyroid and from the skin. In rats kept under the conditions of iodine deficiency (<1 μ I/d), the iodide accumulation in the thyroid, but not in the skin, was markedly increased as a result of a thyrotropic stimulation. The effect of a high bromide intake (>100 mg Br⁻/d) in these animals was particularly pronounced because the rates of iodide elimination were most accelerated both from their thyroid and from their skin. Presented in part at the 20th Workshop on Macro and Trace Elements held in Jena (Germany) on December 1–2, 2000.     

外源性精胺对缺氧诱导乳鼠心肌细胞凋亡的影响及其机制

目的：观察外源性精胺对缺氧所致的乳鼠心肌细胞凋亡的影响,并探讨其机制。方法：复制原代培养乳鼠心肌细胞缺氧损伤模型（使用pH=6.8的Hank's平衡盐溶液作为细胞培养基,排出氧气,然后在缺氧箱中培养24 h）,细胞随机分为正常对照（Control）组、缺氧（Hypoxia）组和精胺干预（Hypoxia+Sp）组。Western blot检测心肌细胞多胺代谢关键酶（ODC、SSAT）蛋白质表达;CCK-8,Hoechst 33342染色观察细胞凋亡情况;光吸收法检测细胞（或培养液）内T-SOD和Caspase-3/-9活性,MDA、GSH含量;DCFH-DA染色观察细胞内活性氧（ROS）生成。结果：与正常组相比,Hypoxia组SSAT蛋白质表达、细胞凋亡率、MDA含量以及细胞内ROS生成增加,而ODC蛋白质表达、SOD活性、GSH含量降低;与Hypoxia组比较,Sp处理可减轻上述指标的变化。结论：外源性精胺可减轻缺氧引起的乳鼠心肌细胞损伤和凋亡,其机制与恢复多胺稳态和清除活性氧有关。     

Effects of olive and fish oil Ca soaps in ewe diets on milk fat and muscle and subcutaneous tissue fatty-acid profiles of suckling lambs

Enhancing healthy fatty acids (FAs) in ewe milk fat and suckling lamb tissues is an important objective in terms of improving the nutritional value of these foods for the consumer. The present study examined the effects of feeding-protected lipid supplements rich in unsaturated FAs on the lipid composition of ewe milk, and subsequently in the muscle and subcutaneous adipose tissues of lambs suckling such milk. Thirty-six pregnant Churra ewes with their new-born lambs were assigned to one of three experimental diets (forage/concentrate ratio 50 : 50), each supplemented with either 3% Ca soap FAs of palm (Control), olive (OLI) or fish (FO) oil. The lambs were nourished exclusively by suckling for the whole experimental period. When the lambs reached 11 kg BW, they were slaughtered and samples were taken from the Longissimus dorsi and subcutaneous fat depots. Although milk production was not affected by lipid supplementation, the FO diet decreased fat content (P<0.001), whereas the OLI milk FA profile resembled that of the Control diet. In contrast, although FO drastically diminished the contents of stearic and oleic acids (P<0.001), all the saturated even-numbered carbon FAs from 6:0 to 14:0 increased (P<0.05). FO also produced the highest levels of c9,t11-18:2 (2.21%) and n-3 FAs, 20:5 n-3 (0.58%), 22:5 n-3 (0.48%) and 22:6 n-3 (0.40%). The high levels of trans-11 18:1 (7.10%) obtained from the FO diet would suggest that Ca soaps only confer partial protection in the rumen. In contrast, the lack of significant differences in trans-10 18:1 levels (P>0.05) and other trans-FAs between Control and FO treatments would indicate that FO treatment does not alter rumen biohydrogenation pathways under the assayed conditions. Changes in dam milk FA composition induced differences in the FA profiles of meat and fat depots of lambs, preferentially incorporated polyunsaturated FAs into the muscle rather than storing them in the adipose tissue. In the intramuscular fat of the FO treatment, all the n-3 FAs reached their highest concentrations: 0.97 (18:3 n-3), 2.72 (20:5 n-3), 2.21 (22:5 n-3) and 1.53% (22:6 n-3). In addition, not only did FO intramuscular fat have the most cis-9, trans-11 18:2 (1.66%) and trans-11 18:1 (3.75%), but also the lowest n-6/n-3 ratio (1.80) and saturated FA content were not affected. Therefore, FO exhibited the best FA profile from a nutritional point of view.     

Biological half-lives of bromide and sodium in the rat are connected and dependent on the physiological state

The parallel course of the excretion rates of sodium and bromide ions was demonstrated in adult male rats administered simultaneously with ²⁴Na-sodium chloride and ⁸²Br-bromide. These excretion rates were inversely proportional to the magnitude of sodium intake in the animals. The biological half-life of bromide, as a substitute for sodium or chloride, was investigated with the aid of the radionuclide ⁸²Br in animals situated in very different physiological states (i.e., in lactating and nonlactating female rats as well as in young rats of varying ages [2,4,6, and 10 wk of age]). The ⁸²Br radioactivity retained in mothers and in whole litters was measured in vivo at appropriate time intervals (up to 240 h) after the application of ⁸²Br-bromide to the mothers. The time-course of the changes in the ⁸²Br radioactivity of the young was calculated as the difference between the rate of ⁸²Br intake in the mother's milk and the ⁸²Br excretion through the kidneys into the urine. The rate of ⁸²Br excretion through the kidneys of the dam could be calculated also. Nonweaned young rats (12 d) had the highest half-life (269 h) and lactating dams had the lowest (44 h). The determined values demonstrated that nonweaned young apparently conserve sodium, because of its relatively low concentration in mother's milk, whereas lactating dams, because of their large food intake, waste sodium. Presented in part at the 4th International Symposium on Trace Elements in Human: New Perspectives held in Athens (Greece) on 9–11 October 2003     

On the fission interference correction and its dependence on the epithermal to thermal neutron flux ratio in thermal naa of molybdenum

The present work aims at the following: (1) analyzing the experimental fission interference factor for molybdenum, F_mo, obtained by the authors, who have described the irradiation conditions used as concerns the epithermal to thermal neutron flux ratio, Φ_epi/Φ₀; (2) establishing a simple calculation model that describes the dependence ofF _mo on Φ_epi/Φ₀ in an adequate way, to provide a satisfactory basis to explain the scatter found in the existing experimental data; and (3) clearly indicating the basic recommendations to take into account in order to obtain with high accuracy the concentration of molybdenum in samples containing uranium.     

Effects of reactor temperature and sample mass on the activation of biological and geological materials with a SLOWPOKE reactor

Neutron activation analysis with a SLOWPOKE reactor relies on the stability of the neutron flux in the irradiation sites. Flux monitors were irradiated to measure the flux variation with the reactor temperature and with the amount of moderator in the irradiation vial. The thermal flux decreased by 2.7% for a 10‡C increase in reactor temperature. The thermal flux increased by up to 8% and the fast flux decreased by up to 13% depending on sample size and hydrogen content.     

History of development and applications of nuclear analytical methods in the Czech Republic

This article presents the evolution of the field of nuclear analytical methods, as well as its inventions and applications from the 1960s to the present in the territory of the former Czechoslovakia.     

Up- and down-regulation of membrane receptors as possible mechanisms related to the antiulcer actions of milk in rat gastric mucosa

The effects of a cow's milk diet on receptor activity and histamine metabolism in gastric glands and mucosa isolated from adult rats were examined. The milk diet was associated with (1) a decreased mobilization of H₂ receptors by histamine and (2) an increased mobilization of PGE₂ (prostaglandin E₂) receptors in mucous cells (cytoprotective effect) and parietal cells (antiacid effect). These changes are not observed for the receptors reducing pentagastrin- and histamine-induced gastric acid secretion (pancreatic/enteroglucagons, somatostatin) and stimulating mucus, bicarbonate and pepsin secretions in the rat (secretin). Cimetidine produced a parallel displacement of the histamine dose-response curve, suggesting competitive inhibition between this classical H₂ receptor antagonist and histamine in the two experimental groups. Prostaglandins and other components in milk such as EGF (epidermal growth factor) and somatostatin might therefore protect gastric mucosa by a differential control of PGE₂ and histamine H₂ receptor activity eitherdirectly (PGE₂ in milk) orindirectly (inhibition of endogeneous histamine synthesis/release and stimulation of PGE-I synthesis/release).     

Effect of fermented milk intake on plasmid transfer and on the persistence of transconjugants in the digestive tract of gnotobiotic mice

Plasmid transfer occurs in the digestive tract and the transconjugants may become durably established. The aim of the present work is to investigate the effect of probiotics on plasmid transfer and on establishment of transconjugants in the gut. Plasmid transfers were carried out in the digestive tract of germ free mice associated with an E. coli K12 donor strain harboring three plasmids (R388, self-transmissible, pCE325 and pUB2380, mobilisable,) and an E. coli recipient strain, PG1, of human origin (Duval-Iflah et al., 1994). Milks fermented with either Lactobacillus bulgaricus or Streptococcus thermophilus or symbiosis, S85, of both strains were given daily as 1/3 of food diet. Fermented milks have no effect on the transfer of R388 and pUB2380 except a slight increase of TC(R388) with milk fermented with S85. Long term ingestion of milk fermented with S85 inhibited the formation and the establishment of transconjugants TC(pCE325). Milk fermented with L. bulgaricus lowered the population density of TC(pCE325) in animals where they were already established. This phenomenon was reversible, since the density of TC(pCE325) increased in the same animals after cessation of supplementation. Bacterial cultures obtained in MRS broth and given in state of drinking water were compared with fermented milks. Bacterial cultures with L. bulgaricus and with S85 favoured the establishment of TC(pCE325). These results indicate for the first time that probiotics have various effects on the formation and/or establishment of transconjugants in the gut of axenic mice. The effects depend on whether the probiotics were cultivated in milk or in MRS, indicating that bacterial metabolites and viable bacteria can be involved.     

The application of preirradiation combustion and neutron activation analysis technique for the determination of iodine in food and environmental reference materials

The pre-irradiation combustion (PC) of samples to liberate iodine, followed by trapping the iodine on charcoal and quantifying the element by neutron activation analysis (NAA), has been used at the National Institute of Standards and Technology for the determination of iodine in biological materials. The applicability of this technique to numerous environmental and dietary matrices is illustrated by analysis of a range of certified reference materials (CRMs) and a powdered grass material that was prepared as an in-house reference material (RM). Because of the combustion step involved, samples with low or no fat content (e.g., cereal products, selected botanical specimens, and nonfat milk powder) and inorganic materials (e.g., coal fly ash and dried sediments) are more suited for analysis by this method. In general, the results for several types of samples obtained by this method agreed with those obtained by a second radiochemical (R) NAA, as well as by a third method using inductively coupled plasma mass spectrometry (ICP-MS). PC-NAA is a useful technique for determining iodine in biological and environmental samples, especially for verification of iodine results obtained from other methods.     

Effect of FASN gene on milk yield and milk composition in the Chinese Holstein dairy population

Fatty acid synthase (FASN) is a multifunctional protein that catalyzes de novo synthesis of fatty acids in cells. It plays a key role in the lipid biosynthesis as well as in the general metabolism of all living animals. We herein investigated polymorphisms of FASN. As a result, six single nucleotide polymorphisms (SNPs) were found and then genotyped in 752 Chinese Holstein cows. It was found that g.17924A>G was non‐synonymous, g.13965 C>T, g.16907 T>C and g.18663T>C were synonymous mutations and two other two SNPs, g.8948 C>T (ss491228481) and g.14439T>C (rs133498277), were in intronic sequences of the gene. All such identified SNPs were found to be associated with milk yield and composition traits (P = 0.0441 to <0.0001). Significant additive and allele substitution effects were observed for three yield traits at all six loci as well (P < 0.05 to <0.01). Complete linkage disequilibrium among the five SNPs, with the exception of g.8948 C>T, was observed.     

Vanadium transport across placenta and milk of rats to the fetus and newborn

The transport of vanadium across placenta and milk rats was studied by iv injection of low doses of 48V-labeled pentavanadate (0.1 micrograms V/rat) to pregnant and nursing rats. Significant concentrations of vanadium were found in the liver, intestine, and kidneys of the fetuses, showing that vanadium is capable to pass the placental barrier and, thus, being metabolized in the fetuses. Two days after injection of 0.1 micrograms V/rat as 48V-labeled vanadate to nursing rats, 34 ng V/g milk were found, decreasing to 4 ng V/g at the d 12 after dosing. The corresponding suckling rats showed a significant absorption of vanadium taken up by the milk, as suggested by the presence in their intestine, where 48V is easily absorbed in form of low molecular weight components. Vanadium in milk may be transported in the form of a biocomplex with lactoferrin, since at 12 d after injection, the 48V in the rat milk was mainly found in fractions corresponding to proteins. In weanling rats, 7 d postlactation and 18 d after administration of vanadium to the mother, only very small amounts of 48V were still present in the organs. Excretion studies on weanling rats for 7 d showed that vanadium is rapidly released mainly via feces when earlier taken up by the milk of iv injected mothers and having the same elimination pattern as the milk, which lost about 80% of its vanadium concentration after 12 d.     

In vitro measurement of the impact of human milk oligosaccharides on the faecal microbiota of weaned formula-fed infants compared to a mixture of prebiotic fructooligosaccharides and galactooligosaccharides

Aims: To investigate the impact of human milk oligosaccharides (HMOs) from a single donor (SO), HMOs from multiple donors (PO), a fructooligosaccharides and galactooligosaccharides mixture (FG) on the composition of a batch culture inoculated with faecal microbiota from formula‐fed infants. Methods and Results: Three substrates were compared using 24‐h pH‐controlled anaerobic batch cultures inoculated with infant faecal slurries. Changes in bacterial populations, short‐chain fatty acids (SCFA) production and bacterial 16S rRNA gene profiles were determined. All three substrates significantly increased numbers of bifidobacteria, bacteroides and those aligning with the clostridial cluster XIVa. Neither the FG nor the HMOs substrates supported the growth of the Clostridium perfringens–histolyticum group. SCFA production corresponded to changes observed in bacterial populations. Denaturing gradient gel electrophoresis fingerprint analysis showed a distinct profile of faecal bacteria present in each infant. Conclusions: HMOs modulated infant faecal culture composition in a similar manner to the prebiotic mixture FG in vitro. Significance and Impact of the Study: This is the first demonstration of the impact of pure HMOs on the mixed culture of infant faecal bacteria. HMOs induced the growth of several saccharolytic bacterial groups and may thus play a role in the health‐promoting attributes of human breast milk and have an extended significance in infant diet during/after weaning.     

右美托咪定对大鼠海马神经元生长发育的影响及其机制

目的:探讨右美托咪定(DEX)对新生大鼠海马神经元发育过程及脑源性神经营养因子(BDNF)-酪氨酸受体激酶B(Trk B)信号通路分子表达的影响。方法:从新生的大鼠分离出海马神经元细胞进行体外培养,将细胞接种于96孔板,实验分为4组(对照组、1μmol/L DEX处理组、5μmol/L DEX处理组、50μmol/L DEX处理组),每组设置6复孔,分别给予不同浓度的右美托咪定1、5和50μmol/L处理,于处理后2、4、6、8、10 d检测细胞活性,于处理后10 d检测细胞凋亡情况、q-PCR检测突触素(SYN)和突触后密度蛋白95(PSD95)的mRNA表达水平,分析BDNF、Trk B及N-甲基-D-天冬氨酸受体(NMDA)蛋白表达情况。结果:与对照组相比,不同剂量DEX处理组的神经元细胞活力无显著差异,1μmol/L和5μmol/L DEX处理组中SYN和PSD95 mRNA的表达和Trk B蛋白均无显著性差异(P>0.05),而50μmol/L DEX处理组中SYN和PSD95 mRNA的表达显著升高(P<0.01),BDNF蛋白表达水平显着上调(P<0.01),p-N-甲基-D-天冬氨酸受体的表达增加(P<0.01)。结论:50μmol/L DEX对大鼠海马神经元有一定的作用,其可能通过上调BDNF的表达和N-甲基-D-天冬氨酸受体的磷酸化水平来实现。     

Studies on the uptake of benzanthrone by rat skin and its efflux through serum

The uptake of benzanthrone by rat skin showed saturation kinetics and was dependent upon the weight of skin and time, temperature and pH of the incubation medium. Heating of segments above 50‡C caused significant lowering of the uptake. The uptake was irreversibly inhibited by HgCl² and not by sodium arsenate, KCN, NaF,p-chloromercuriben zoate, N-ethyl-maleimide, cycloheximide, iodoacetic acid and 2,4-dinitrophenol suggesting that the uptake was not energy-dependent. Lipid micelles of the skin accounted for a part of the binding. Most of the benzanthrone taken up by the skin was effluxed through serum proteins.     

Stereoselective effect of kynurenine enantiomers on the excretion of serotonin and its metabolite in rat urine

A solution of optically pure kynurenine (KYN), i.e., D ‐KYN or L ‐KYN, was administered intravenously to male Sprague‐Dawley rats (10 mg kg^?1 ml^?1). The time‐course of changes in the concentrations of urinary monoamines and their metabolites such as 5‐hydroxytryptamine (5‐HT), 5‐hydroxyindole acetic acid (5‐HIAA), dopamine, and 3‐methoxytyramine were investigated by reversed‐phase high‐performance liquid chromatography with electrochemical detection after precolumn derivatization with (2R)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (NPCA). We observed a stereoselective difference in the effects of the KYN enantiomers. Only D ‐KYN, not L ‐KYN, caused a significant increase in urinary 5‐HT levels within 30 min after its administration. With regard to the metabolites, urinary 3‐MT level was increased by D ‐KYN administration. On the other hand, no significant change in the DA level was observed after administration of either D ‐KYN or L ‐KYN. These results suggest that D ‐KYN could affect the activity of neuroactive amines, especially 5‐HT, in vivo. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Actions of avermectin B1a on the γ-aminobutyric AcidA receptor and chloride channels in rat brain

The interaction of avermectin B_1a (AVM) with the γ-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [³H]flunitrazepam and inhibited the binding of both [³H]muscimol and [³⁵S]t-butylbicyclo-phosphorothionate to the GABA_A receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of ³⁶chloride (Cl) flux were studied. The ³⁶Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, ³⁶Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced ³⁶Cl influx into these microsacs in a dose–dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced ³⁶Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABA_A-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage–dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

The effects of dehydroepiandrosterone (DHEA) and its metabolites on the polycystic ovarian condition (PCO): cystogenic changes of rat granulosa cells in vitro

During mammalian folliculogenesis, granulosa cells (GCs) are initially steroidogenically quiescent, later proliferate, and subsequently commence to hormonally differentiate, first producing estrogen and later, in the preovulatory stage, secreting both estrogen and progesterone. In this study and elsewhere, we have used follicle-stimulating hormone with a combination of growth factors in vitro to simulate the above in vivo conditions. In a previous study, we used dehydroepiandrosterone (DHEA) to accomplish the polycystic ovary condition (PCO) in rats. In the latter model, there were high circulating levels of DHEA and its metabolite, androstenedione. In the present study, we investigated the effects of high levels of DHEA (10⁻⁵M) and its metabolites, androstenedione, androstenediol and dehydroepiandrosterone sulfate on the quiescent, proliferative, and steroidogenically differentiating stages of GCs cultured in a serum-free medium for up to 10 days. In addition to possessing the regularly occurring organelles, when cultured with the aforementioned androgens, the GCs acquired endoplasmic reticulum of the smooth variety which is associated with steroidogenesis. The radioimmunoassay data showed that GCs cultured in the quiescent and proliferative stages in the presence of the androgens, no longer remain in these stages but proceed to differentiate in a preovulatory direction by producing both estrogen and progesterone. This study supports our hypothesis that high circulating levels of DHEA and/or its metabolites have most effect during the quiescent and proliferative stages of granulosa cells, with regard to their structure and their steroidogenic activities.