Gastrointestinal metabolism of phytoestrogens in lactating dairy cows fed silages with different botanical composition.

Dietary phytoestrogens are metabolized or converted in the gastrointestinal tract of ruminants, only limited knowledge exists on the extent and location of this conversion in vivo. The objective of this study was to quantify the gastro-intestinal metabolism of phytoestrogens in lactating dairy cows fed silages with different botanical composition. Four lactating rumen cannulated Norwegian Red cattle were assigned to a 4 × 4 Latin square with 1 cow per treatment period of 3 wk. The 4 treatment silages were prepared from grasslands with different botanical compositions: organically managed short-term timothy (Phleum pratense L.) and red clover (Trifolium pratense L.) ley (2 yr old: ORG-SG); organically managed long-term grassland with a high proportion of unsown species (6 yr old; ORG-LG); conventionally managed perennial ryegrass (Lolium perenne L.) ley (CON-PR); and conventionally managed timothy ley (CON-TI). The herbages were cut, wilted, and preserved with additive in round bales, fed as a mix of the first and third cut at 90% of ad libitum intake, and contributed to 70% of the total dry matter intake. Milk, feed, omasal digesta, urine, and feces were collected at the end of each period and analyzed for the concentrations of phytoestrogens by using a liquid chromatography-tandem mass spectrometry technique. Concentration of total isoflavones was highest in ORG-SG and lowest in CON-TI silage, whereas the content of total lignans was highest in the grass silages. The isoflavones were extensively metabolized in the rumen on all diets, and the recovery of formononetin and daidzein in omasum, mainly as equol, averaged 0.11 mg/mg. The apparent intestinal metabolism was less severe as, on average, 0.29 mg/mg of the omasal flow was recovered in feces. The plant lignans were also strongly degraded in the rumen. However, the flow of lignans to omasum and excretion in feces were, on average, 7.2- and 5.2-fold higher, respectively, than the intake of the plant lignans matairesinol and secoisolariciresinol, known as precursors of mammalian lignans. Thus, excretion to milk could not be directly related to intake, implying that plant lignans other than matairesinol and secoisolariciresinol in forage are precursors for enterolactone production in the rumen and for its content in milk. Equol followed mainly the flow of large particles out of the rumen, whereas the mammalian lignans were distributed between phases proportional to dry matter flow. The main metabolism of phytoestrogens occurred in the rumen and the main route of excretion was through feces and urine, with only a small part being excreted in milk. The concentration of phytoestrogens in milk can be manipulated through intake but the intermediate transfer capacity to milk appears to be limited by saturation.

Effect of short-term versus long-term grassland management and seasonal variation in organic and conventional dairy farming on the composition of bulk tank milk.

Bulk tank milk from 28 dairy farms was sampled every second month for 2 yr to assess the effects of grassland management, production system and season on milk fatty acid (FA) composition, concentrations of fat-soluble vitamins, Se, and milk sensory quality. Grassland management varied in terms of time since establishment. Short-term grassland management (SG) was defined as establishment or reseeding every fourth year or more often, and long-term grassland management (LG) was defined as less frequent establishment or reseeding. Fourteen organic (ORG) dairy farms with either short-term or long-term grassland management were paired with 14 conventional (CON) farms with respect to grassland management. Within ORG farms, SG farms differed from LG farms in herbage botanical composition, but not in concentrate FA concentrations, dry matter intake, or milk yield. Within CON farms, herbage composition, concentrate FA concentrations, dry matter intake, and milk yield showed no or insignificant variations. The ORG farms differed from CON farms in herbage botanical composition, concentrate FA concentrations, concentrate intake, and milk yield. Compared with ORG-LG farms, ORG-SG farms produced milk fat with higher proportions of C10:0 and C12:0 associated with higher herbage proportions of legumes (Fabaceae) and lower proportions of other dicotyledon families. Compared with milk from CON farms, milk fat from ORG farms had higher proportions of most saturated FA and all n-3 FA, but lower proportions of C18:0 and C18:1 cis-9 associated with higher forage proportion and differences in concentrations of FA in concentrates. Compared with the outdoor-feeding periods, the indoor feeding periods yielded milk fat with higher proportions of most short-chain and medium-chain FA and lower proportions of most C18-FA associated with grazing and higher forage proportions. Milk concentrations of α-tocopherol and ß-carotene were lower during the grazing periods. Inclusion of fishmeal in organic concentrates may explain higher Se concentrations in organically produced milk. Milk sensory quality was not affected in this study. In conclusion, grassland management had minor effects on milk composition, and differences between ORG farms and CON farms may be explained by differences in concentrate intake and concentrate FA concentrations. Milk produced on ORG farms versus CON farms and milk produced during the outdoor versus indoor feeding periods had potential health benefits due to FA composition. In contrast, the higher milk-fat proportions of saturated FA in milk from ORG farms may be perceived as negative for human health.

Effect of silage botanical composition on ruminal biohydrogenation and transfer of fatty acids to milk in dairy cows.

Ruminal biohydrogenation and transfer of fatty acids (FA) to milk were determined for 4 silages with different botanical compositions using 4 multiparous Norwegian Red dairy cows [(mean ± SD) 118 ± 40.9 d in milk, 22.5 ± 2.72 kg of milk/d, 631 ± 3.3 kg of body weight, 3.3 ± 0.40 points on body condition score at the start of the experiment] fitted with rumen cannulas. Treatments consisted of 4 experimental silages: a mix of the first and third cut of organically managed short-term grassland with timothy (Phleum pratense L.) and red clover (Trifolium pratense L.; 2 yr old; ORG-SG); organically managed long-term grassland with a high proportion of unsown species (6 yr old; ORG-LG); conventionally managed ley with perennial ryegrass (Lolium perenne L.; CON-PR); and conventionally managed ley with timothy (CON-TI). The herbages were cut, wilted, and preserved with additive in round bales and fed at 0.90 of ad libitum intake. A barley (Hordeum vulgare L.) concentrate constituted 300 g/kg of dry matter of the total feed offered. A Latin square design (4 × 4) with 3-wk periods and the last week in each period used for sampling was implemented. Omasal flows of FA were measured using Yb acetate, Cr-EDTA, and the indigestible neutral detergent fiber fraction as indigestible markers. The composition of FA was analyzed in feed, omasal digesta, and milk. Compared with ORG-LG, ORG-SG had a higher herbage proportion of red clover (0.36 vs. 0.01) and lower proportions of timothy (0.42 vs. 0.18), smooth meadowgrass (Poa pratensis L.), meadow fescue (Festuca pratensis Huds.), white clover (Trifolium repens L.), dandelion (Taraxacum spp.), and creeping buttercup (Ranunculus repens L.). The silages were well preserved. The concentration of neutral detergent fiber was higher and the concentration of Kjeldahl-N was lower for CON-TI than for the other silages. Silage type had no effect on dry matter intake, but milk yield was lower for CON-TI than for the other silages. Apparent biohydrogenation of C18:3n-3 was lower for ORG-SG (932 g/kg) than for ORG-LG (956 g/kg), CON-PR (959 g/kg), and CON-TI (958 g/kg). Compared with the grass-based silages, ORG-SG and ORG-LG resulted in higher omasal flows of C18:1 trans FA and higher milk fat proportions of C18:1 trans FA and C18:2 cis-9,trans-11. Apparent recovery of C18:3n-3 in milk was higher for ORG-SG (61 g/kg) than for ORG-LG (33 g/kg), CON-PR (34 g/kg), and CON-TI (38 g/kg), and milk fat proportion of C18:3n-3 was higher for ORG-SG than for CON-TI. Milk fat proportions of C16:0 were lower for ORG-SG and ORG-LG compared with those for CON-PR and CON-TI. It was concluded that high proportions of red clover and other dicotyledons in the silages affected ruminal biohydrogenation and increased milk fat proportions of beneficial FA.

Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas.

Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrin-containing fibrous structures associated with the Chlamydomonas centrosome.

In vitro pepsin digestibility and amino acid composition in soluble and residual fractions of hydrolyzed chicken feathers.

Beta-keratin in poultry feathers is a structural protein that is resistant to degradation due to disulfide and hydrogen bonds. Feather meal can be a valuable feed compound if the digestibility can be increased. The objective of the present study was to analyze the effects of chemical, enzymatic, and pressure-thermic treatments for chicken feathers on solubility, in vitro protein digestibility (IVPD), and amino acid composition of solubilized and residual fractions. Two experiments were conducted. In experiment 1, models for solubility and IVPD were developed including the above factors applying a central composite face-centered design. Addition of sodium hydroxide (NaOH) and sodium sulfite (Na2SO3), and autoclaving time affected solubility and IVPD of the feather hydrolysates, but not addition of keratinolytic enzyme. In experiment 2, 7 combinations of the hydrolysis factors NaOH, Na2SO3, and autoclaving time with a predicted IVPD of 900 g/kg of DM, calculated for the sum of solubilized and residual feather fractions, were included to measure effects on IVPD and amino acid composition in each fraction. The IVPD values were higher for solubilized than residual fractions when treated with NaOH and autoclaving, but no differences were found when treated with Na2SO3 and autoclaving. Losses of cystine were substantial for all treatments, but lower for Na2SO3 than for NaOH. Furthermore, use of lower Na2SO3 concentration and longer autoclaving time reduced losses of cystine. Compared with NaOH treatments, Na2SO3 gave lower losses of threonine, arginine, serine, and tyrosine. With reference to the ideal protein profile for Atlantic salmon (Salmo salar L.), the treatments with 60 or 90 min autoclaving and 0.36 or 0.21% Na2SO3 had the highest chemical scores. The scores were generally higher for amino acids in residual than solubilized fractions, but with 90 min autoclaving and 0.21% Na2SO3 differences were small. In conclusion, hydrolysis of chicken feathers with low concentrations of Na2SO3 combined with autoclaving results in feather meal with high nutritional value for Atlantic salmon; separation of solubilized and residual fractions is not necessary.

CD-tagging: a new approach to gene and protein discovery and analysis.

We describe a new method for gene discovery and analysis, CD-tagging, that puts specific molecular tags on a gene, its transcript and its protein product. The method has been successfully tested in two organisms, the haploid unicellular alga Chlamydomonas reinhardtii and the metazoan Drosophila melanogaster. The method utilizes a specially designed DNA molecule, the CD-cassette, that contains splice acceptor and donor sites surrounding a short open reading frame. Insertion of the CD-cassette into an intron in a target gene introduces a new exon, represented by the open reading frame of the CD-cassette, surrounded by two functional hybrid introns. As a result (i) the gene is tagged by a specific nucleotide sequence, (ii) the mRNA is tagged by a specific nucleotide sequence and (iii) the protein is tagged by a specific peptide sequence. Because these tags are unique, specific nucleotide or antibody probes can be used to obtain and/or analyze the gene, transcript or protein. As a gene discovery technology, CD-tagging has two unique advantages: 1) Genes can be identified through a primary screen at the protein level, and so the very process by which a gene is identified provides specific empirical information about its biological function. 2) The cassette arms, which are spliced out of the transcript of the target gene, are available to carry a wide variety of DNA sequences, such as genes encoding drug resistance that can be used to select for the presence of the CD-cassette in the genome.

Platelet MAO activity: relationships to clinical and psychometric variables.

Platelet monoamine oxidase (MAO) activity was measured in a group of hospitalized male psychiatric patients. The findings of this study confirm previous reports of: (1) reduced platelet MAO activity in patients with auditory hallucinations, (2) reduced platelet MAO activity in patients with alcoholism, and (3) a positive correlation between platelet MAO activity and a psychometric index of social introversion.

The memorability and discriminability of primitive perceptual units in infancy.

Three-month-olds were operantly trained to kick to move a block mobile that displayed a horizontal and a vertical line ("textons") arranged as L, T, or + on each side. Delayed recognition was tested either 0, 1, 3, 5, 7, or 9 days later with either the same or a different display. Infants failed to discriminate between Ls and Ts after delays longer than 1 hr but discriminated both from +s, which contains an additional texton (the line crossing), after delays as long as 7 days but not 9. Also, they remembered +s longer than Ls and Ts. These data indicate that the same primitive perceptual units that mediate adult texture segregation are differentially discriminated and differentially memorable in early infancy.

Effects of directional expectations on motion perception and pursuit eye movements.

Expectations about future motions can influence both perceptual judgements and pursuit eye movements. However, it is not known whether these two effects are due to shared processing, or to separate mechanisms with similar properties. We have addressed this question by providing subjects with prior information about the likely direction of motion in an upcoming random-dot motion display and measuring both the perceptual judgements and pursuit eye movements elicited by the stimulus. We quantified the subjects' responses by computing oculometric curves from their pursuit eye movements and psychometric curves from their perceptual decisions. Our results show that directional cues caused similar shifts in both the oculometric and psychometric curves toward the expected motion direction, with little change in the shapes of the curves. Prior information therefore biased the outcome of both eye movement and perceptual decisions without systematically changing their thresholds. We also found that eye movement and perceptual decisions tended to be the same on a trial-by-trial basis, at a higher frequency than would be expected by chance. Furthermore, the effects of prior information were evident during pursuit initiation, as well as during pursuit maintenance, indicating that prior information likely influenced the early processing of visual motion. We conclude that, in our experiments, expectations caused similar effects on both pursuit and perception by altering the activity of visual motion detectors that are read out by both the oculomotor and perceptual systems. Applying cognitive factors such as expectations at relatively early stages of visual processing could act to coordinate the metrics of eye movements with perceptual judgements.

Short-term culture of exfoliated cells from the urine of patients with bladder tumors.

This report concerns the short-term culture of urothelial cells from the urine sediment of over 100 patients with bladder tumors. Primary cell outgrowth was obtained in approximately 60% of the cultures initiated. Culture outcome was not related to tumor grade, patient age, or volume of the urine sample. Around 85% of the proliferating cultures were successfully transferred into multi-compartment chamber/slides. These results suggest that the culture system may be a useful tool for the study of urothelial cells using patient material obtained by non-invasive means.

Substituting new details for old? Effects of delaying postevent information on infant memory.

How that which we remember is selectively distorted by new information was studied in 3-month-old infants who learned to move a particular crib mobile by operant foot kicking. Infants who were passively exposed to a novel mobile 1, 2, or 3 days later subsequently treated the novel mobile as if they had actually been trained with it. Also, after the longest exposure delay, they no longer recognized the original mobile. Likewise, when the novel mobile was exposed after the longest delay, it could prime the forgotten training memory in a reactivation paradigm, but the original mobile no longer could. These data reveal that what we remember about an event is selectively distorted by what we encounter later. Moreover, the later in the retention interval we encounter new postevent information, the greater is its impact on retention.

Levels-of-processing effects in infant memory?

In 3 experiments, we manipulated 3-month-olds' attention to different components of a training display and assessed the effect on retention of those components. Attention was manipulated via a pop-out display (one target amidst 6 distractors) that enhances selective attention to the target relative to the distractors, and retention was assessed with displays composed entirely of targets or distractors. In Experiment 1, infants recognized both target and distractors after 1 day, confirming that both are initially encoded at some level. In Experiment 2, infants recognized a target L and distractor Ls after delays longer and shorter, respectively, than infants trained with Ls in a homogeneous display. Experiment 3 replicated the preceding pattern of results with a + stimulus. Thus, increasing or decreasing attention to an item during encoding produces a corresponding increase or decrease in its memorability. This finding is consistent with a levels of processing account and is inconsistent with accounts that deny a capacity for explicit memory to prelinguistic infants.

Reinstatement versus reactivation effects on active memory in infants.

Reinstatement and reactivation are procedurally different reminder paradigms used with infants and children, but most developmental psychologists do not distinguish between them. In 4 experiments with 102 three-month-olds, we asked if they differ functionally as well. Independent groups of infants received either a reactivation or a reinstatement reminder 3 days after training, when the memory is active, but its specific details have been forgotten. In Experiment 1, we measured retention after increasing delays until infants forgot altogether. A single reinstatement protracted retention twice as long after training as a single reactivation. In Experiments 2-4, whether the reminder was the original training stimulus or a novel one differentially affected the duration and specificity of memory in the 2 procedures as well. These data demonstrate that the distinction between reinstatement and reactivation is not artificial. In addition to differing procedurally, reinstatement and reactivation differ functionally, with different memory-preserving effects.

A dissociation in infants' memory for stimulus size: evidence for the early development of multiple memory systems.

Adults' memory performance on recognition (explicit memory) tests is sensitive to stimulus size, but their performance on priming (implicit memory) tests is not. This memory dissociation is taken as evidence for two, functionally distinct memory systems. Young infants, however, are thought to possess only a single representational system that supports implicit memory; the system that supports explicit memory is thought not to mature before 8-9 months of age. In two experiments with 54 infants, we asked if 3-month-olds exhibit a memory dissociation for stimulus size on recognition and priming tests. All infants learned to move a mobile displaying +s of a given size. In Experiment 1, infants recognized +s in the original size but not 33% smaller or larger. In Experiment 2, +s were effective memory primes in a reactivation task, irrespective of size. The finding that young infants exhibit a memory dissociation for stimulus size adds to growing evidence that two memory systems are functional from early in development.

Infants' eyewitness testimony: effects of postevent information on a prior memory representation.

In eyewitness testimony research, postevent information impairs retention of the original event and increases the probability that interpolated information will be identified as part of the original event. The present experiments studied these effects with 3-month-olds. Infants learned to kick to move a particular crib mobile and then were briefly exposed to information about a novel mobile. The novel postevent information impaired recognition of the original mobile when it immediately followed training but not when it was delayed by 1 day. Like adults, infants treated the postevent information as part of the original training event, continuing to do so for at least 2 weeks. We propose that postevent information displaces conflicting information coactive with it in primary memory and creates a new, updated memory token of the event. Once the new token leaves primary memory, however, it is protected; only a copy can be retrieved and modified in the future.

Effect of hyperosmolality on alkaline phosphatase and stress-response protein 27 of MCF-7 breast cancer cells.

MCF-7, a continuous cell line derived from a human breast carcinoma, exhibits very low alkaline phosphatase (ALP) activity. The enzyme is heat-stable and is inhibited by L-phenylalanine and L-phenylalanylglycylglycine, but not by L-homoarginine, 1-bromotetramisole, or levamisole. These data indicate that MCF-7 produces term-placental ALP, the oncodevelopmental enzyme form inappropriately expressed by a variety of human tumors. In contrast to human cancer cells that produce this enzyme monophenotypically, ALP activity of MCF-7 cells is not significantly increased by glucocorticoids or sodium butyrate. By comparison, exposure to hyperosmolality causes a striking increase in enzyme activity. Cycloheximide blocks this effect. The results obtained with cell-free assays were confirmed by cytochemical and immunocytochemical assays on whole cells. Because some of the agents tested in the enzyme modulation experiments affect cell proliferation, their possible effect on two stress-response proteins (srp 27 and srp 72) was also examined; specific immunocytochemical assays were used. These tests revealed that neither protein is affected by glucocorticoids; that sodium butyrate has no effect on srp 27, but alters the intracellular distribution of srp 72; and that hyperosmolality, while not significantly affecting srp 72, causes an increase in srp 27.

Nucleus-basal body connector in Chlamydomonas: evidence for a role in basal body segregation and against essential roles in mitosis or in determining cell polarity.

In the unicellular biflagellate green alga Chlamydomonas reinhardtii each basal body is linked to the nucleus by a fibrous nucleus-basal body connector (NBBC) that contains the calcium-binding protein centrin. (Wright et al.: Journal of Cell Biology 101:1903-1912.; Salisbury et al.: Journal of Cell Biology 107:635-642; Huang et al.: Journal of Cell Biology 107:121-131). In order to explore the cellular function of the NBBC we used antiserum directed against centrin to examine a number of mutants known to be defective for basal body assembly and/or localization. Of three variable flagella-number mutants examined, one, vfl-2, is dramatically defective with respect to the NBBC in that 1) the union between basal bodies and nucleus is very labile, 2) there is no detectible centrin in the NBBC region, and 3) total cellular centrin levels are reduced 75-80% relative to wild type. The existence of these defects in a mutant incapable of maintaining normal flagellar number supports the view that the NBBC plays an important role in determining proper basal body localization and/or segregation. In contrast to vfl-2, the mutants vfl-1, vfl-3, uni-1, and bald-2 contain approximately normal levels of centrin and possess stable NBBCs. The observation of NBBCs in the mutant bald-2, which lacks all but very rudimentary basal bodies, indicates that the assembly of the NBBC does not require fully formed basal bodies and that such assembly may not require basal bodies at all. Finally, the possibility that the NBBC is required for induction of gene expression following deflagellation was tested by examining vfl-2 for such induction. Results indicate that the connector does not play a necessary role in the induction process.