Glycerin and lecithin inclusion in diets for brown egg-layinghens: Effects on egg production and nutrient digestibility

The effects of the inclusion of raw glycerin (GLYC) and lecithin in the diet on egg production,egg quality and total tract apparent retention (TTAR) of dietary components was studied inbrown egg-laying hens from 23 to 51 wk of age. The experimental design was completelyrandomized with six diets combined as a 2 × 3 factorial with two levels of GLYC (0 vs.70 g/kg) and three animal fat to lecithin ratios (40:0, 20:20 and 0:40 g/kg). Each treatmentwas replicated eight times and the experimental unit was a cage with ten hens. Productionwas recorded by replicate every 28-d period and cumulatively. For the entire experiment,the inclusion of GLYC in the diet hindered feed conversion ratio per kilogram of eggs (2.071vs. 2.039; P < 0.05) but did not affect any of the other production or egg quality traits studied.The replacement of animal fat by lecithin (40:0, 20:20 and 0:40 g/kg) increased egg weight(60.1, 60.7 and 61.8 g, respectively; P < 0.001) and egg mass production (56.8, 57.5 and58.8 g/d, respectively; P < 0.01) and improved yolk color as measured by the DSM colorfan (9.2, 9.2 and 9.5, respectively; P < 0.001) and feed conversion ratio per kilogram of eggs(2.072, 2.068 and 2.027, respectively; P < 0.05). Feed intake, egg production and body weightgain, however, were not affected. The inclusion of GLYC in the diet did not affect nutrientretention but lecithin inclusion improved TTAR of dry matter (P < 0.05), organic matter(P < 0.05), ether extract (P < 0.001) and gross energy (P < 0.001). In summary, the inclusionof 70 g glycerol/kg diet hindered feed conversion ratio per kilogram of eggs but did notaffect any other production or digestibility trait. The replacement of animal fat by lecithinimproved egg weight, egg yolk color and nutrient digestibility. Consequently, lecithin canbe used as a lipid source in laying hen diets with beneficial effects on egg production

Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and batch cultures of rumen microorganisms

The objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.

Influence of glycerin and lecithin inclusion in the diet on liver characteristics and lipid fraction in the serum of brownegg laying hens at 55 week of age

The effects of the inclusion of raw glycerin (GLYC) and raw lecithin, in the diet (23 to 55 wk) on liver characteristics and various serum lipid fractions were studied in brown egg-laying hens at 55 wk of age. The control diets were based on corn, soybean meal, and 4% supplemental fat and contained 2,750 kcal AMEn/kg, 16.5% CP, and 0.73% digestible Lys. The diets were arranged as a 2 × 3 factorial with 2 levels of GLYC (0 and 7%) and 3 animal fat to lecithin ratios (4:0, 2:2, and 0:4%). Each treatment was replicated 8 times and the experimental unit was a cage with 10 hens. At 55 wk of age, 2 hens per cage replicate were randomly selected, weighed individually, and slaughtered by CO2 inhalation. Liver was immediately removed and weighed and the color recorded by spectrophotometry. In addition, blood samples from one bird per replicate were collected from the wing vein and the concentration of total cholesterol, low and high density lipoprotein cholesterol, and triglycerides were determined. The data were analyzed as a completely randomized design and the main effects of GLYC and lecithin content of the diet and the interactions were determined. No interactions between GLYC and lecithin content of the diets were detected for any of the variables studied. Liver characteristics and serum lipid traits were not affected by the inclusion of GLYC in the diet. The substitution of animal fat by lecithin, however, reduced the redness (a* 14.9 to 13.8) and yellowness (b* 8.60 to 7.20) values of the liver (P < 0.05) but did not affect the content of serum lipid fractions. It is concluded that the inclusion of GLYC and lecithin in the diet did not affect liver size or serum lipid fraction. However, the inclusion of lecithin reduced the a* and b* value of the liver

In vivo evidence that erythropoietin protects neurons from ischemic damage

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-d-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.

Absence of cytokine receptor-dependent specificity in red blood cell differentiation in vivo

Erythropoietin (EPO) is required for red blood cell development, but whether EPO-specific signals directly instruct erythroid differentiation is unknown. We used a dominant system in which constitutively active variants of the EPO receptor were introduced into erythroid progenitors in mice. Chimeric receptors were constructed by replacing the cytoplasmic tail of constitutively active variants of the EPO receptor with tails of diverse cytokine receptors. Receptors linked to granulocyte or platelet production supported complete erythroid development in vitro and in vivo, as did the growth hormone receptor, a nonhematopoietic receptor. Therefore, EPOR-specific signals are not required for terminal differentiation of erythrocytes. Furthermore, we found that cellular context can influence cytokine receptor signaling.

Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by Gsα

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the α subunit of the stimulatory GTP-binding protein of AC (Gsα) to stimulate enzyme activity. Concentration–response curves with Gsα suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by Gsα interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572–F683). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsα-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L1042–T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsα concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L1042–T1051) in the C2 domain of the enzyme to modulate Gsα-elicited stimulation of activity.

Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.

Uridine Diphosphate–Glucose Transport into the Endoplasmic Reticulum of Saccharomyces cerevisiae: In Vivo and In Vitro Evidence

It has been proposed that synthesis of β-1,6-glucan, one of Saccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose–dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP–glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER–containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP–glucose was temperature dependent and saturable with an apparent Km of 46 μM and a Vmax of 200 pmol/mg protein/3 min. Transport was substrate specific because UDP–N-acetylglucosamine did not enter these vesicles. Demonstration of UDP–glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP–glucose:glycoprotein glucosyltransferase (GT) in S. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected in alg6 or alg5 mutant cells, which transfer Man9GlcNAc2 to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Δ, lack cell wall β-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Δ mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.

Rapid transition in the structure of a coral reef community: The effects of coral bleaching and physical disturbance

Coral reef communities are in a state of change throughout their geographical range. Factors contributing to this change include bleaching (the loss of algal symbionts), storm damage, disease, and increasing abundance of macroalgae. An additional factor for Caribbean reefs is the aftereffects of the epizootic that reduced the abundance of the herbivorous sea urchin, Diadema antillarum. Although coral reef communities have undergone phase shifts, there are few studies that document the details of such transitions. We report the results of a 40-month study that documents changes in a Caribbean reef community affected by bleaching, hurricane damage, and an increasing abundance of macroalgae. The study site was in a relatively pristine area of the reef surrounding the island of San Salvador in the Bahamas. Ten transects were sampled every 3–9 months from November 1994 to February 1998. During this period, the corals experienced a massive bleaching event resulting in a significant decline in coral abundance. Algae, especially macroalgae, increased in abundance until they effectively dominated the substrate. The direct impact of Hurricane Lili in October 1996 did not alter the developing community structure and may have facilitated increasing algal abundance. The results of this study document the rapid transition of this reef community from one in which corals and algae were codominant to a community dominated by macroalgae. The relatively brief time period required for this transition illustrates the dynamic nature of reef communities.

Methylation is not the main force repressing the retrotransposon MAGGY in Magnaporthe grisea

We have introduced the LTR-retrotransposon MAGGY into a naive genome of Magnaporthe grisea and estimated the copy number of MAGGY in a cell by serial isolation of fungal protoplasts at certain time intervals. The number of MAGGY elements rapidly increased for a short period following introduction. However, it did not increase geometrically and reached equilibrium at 20–30 copies per genome, indicating that MAGGY was repressed or silenced during proliferation. De novo methylation of MAGGY occurred immediately following invasion into the genome but the degree of methylation was constant and did not correlate with the repression of MAGGY. 5-Azacytidine treatment demethylated and transcriptionally activated the MAGGY element in regenerants but did not affect transpositional frequency, suggesting that post-transcriptional suppression, not methylation, is the main force that represses MAGGY proliferation in M.grisea. Support for this conclusion was also obtained by examining the methylation status of MAGGY sequences in field isolates of M.grisea with active or inactive MAGGY elements. Methylation of the MAGGY sequences was detected in some isolates but not in others. However, the methylation status did not correlate with the copy numbers and activity of the elements.

Differential Expression of Three Members of the 1-Aminocyclopropane-1-Carboxylate Synthase Gene Family in Carnation1

We investigated the expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes in carnation (Dianthus caryophyllus cv White Sim) under conditions previously shown to induce ethylene biosynthesis. These included treatment of flowers with 2,4-dichlorophenoxyacetic acid, ethylene, LiCl, cycloheximide, and natural and pollination-induced flower senescence. Accumulation of ACC synthase transcripts in leaves following mechanical wounding and treatment with 2,4-dichlorophenoxyacetic acid or LiCl was also determined by RNA gel-blot analysis. As in other species, the carnation ACC synthase genes were found to be differentially regulated in a tissue-specific manner. DCACS2 and DCACS3 were preferentially expressed in styles, whereas DCACS1 mRNA was most abundant in petals. Cycloheximide did not induce increased accumulation of ACC synthase transcripts in carnation flowers, whereas the expression of ACC synthase was up-regulated by auxin, ethylene, LiCl, pollination, and senescence in a floral-organ-specific manner. Expression of the three ACC synthases identified in carnation did not correspond to elevated ethylene biosynthesis from wounded or auxin-treated leaves, and there are likely additional members of the carnation ACC synthase gene family responsible for ACC synthase expression in vegetative tissues.

Cadherin Sequences That Inhibit β-Catenin Signaling: A Study in Yeast and Mammalian Cells

Drosophila Armadillo and its mammalian homologue β-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/β-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/β-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or β-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/β-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited β-catenin–mediated signaling. This suggests that the interaction between β-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

Evolution and divergence of sodium channel genes in vertebrates

Invertebrate species possess one or two Na+ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na+ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na+ channel gene followed by tandem duplications of Na+ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na+ channel genes in a teleost fish. Using an approach designed to clone all of the Na+ channel genes in a genome, we found six Na+ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na+ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na+ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na+ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na+ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than {D[A(BC)]}.

The Role of Cytosolic Potassium and pH in the Growth of Barley Roots1

In an earlier paper we showed that in fully developed barley (Hordeum vulgare L.) root epidermal cells a decrease in cytosolic K+ was associated with an acidification of the cytosol (D.J. Walker, R.A. Leigh, A.J. Miller [1996] Proc Natl Acad Sci USA 93: 10510–10514). To show that these changes in cytosolic ion concentrations contributed to the decreased growth of K+-starved roots, we first measured whether similar changes occurred in cells of the growing zone. Triple-barreled ion-selective microelectrodes were used to measure cytosolic K+ activity and pH in cells 0.5 to 1.0 mm from the root tip. In plants growing from 7 to 21 d after germination under K+-replete conditions, the mean values did not change significantly, with values ranging from 80 to 84 mm for K+ and 7.3 to 7.4 for pH. However, in K+-starved plants (external [K+], 2 μm), the mean cytosolic K+ activity and pH had declined to 44 mm and 7.0, respectively, after 14 d. For whole roots, sap osmolality was always lower in K+-starved than in K+-replete plants, whereas elongation rate and dry matter accumulation were significantly decreased after 14 and 16 d of K+ starvation. The rate of protein synthesis in root tips did not change for K+-replete plants but declined significantly with age in K+-starved plants. Butyrate treatment decreased cytosolic pH and diminished the rate of protein synthesis in K+-replete roots. Procaine treatment of K+-starved roots gave an alkalinization of the cytosol and increased protein synthesis rate. These results show that changes in both cytosolic pH and K+ can be significant factors in inhibiting protein synthesis and root growth during K+ deficiency.

Identification of the Binding and Inhibition Sites in the Calmodulin Molecule for Ophiobolin A by Site-Directed Mutagenesis1

Ophiobolin A, a fungal toxin that affects maize and rice, has previously been shown to inhibit calmodulin by reacting with the lysine (Lys) residues in the calmodulin. In the present study we mutated Lys-75, Lys-77, and Lys-148 in the calmodulin molecule by site-directed mutagenesis, either by deleting them or by changing them to glutamine or arginine. We found that each of these three Lys residues could bind one molecule of ophiobolin A. Normally, only Lys-75 and Lys-148 bind ophiobolin A. Lys-77 seemed to be blocked by the binding of ophiobolin A to Lys-75. Lys-75 is the primary binding site and is responsible for all of the inhibition of ophiobolin A. When Lys-75 was removed, Lys-77 could then react with ophiobolin A to produce inhibition. Lys-148 was shown to be a binding site but not an inhibition site. The Lys-75 mutants were partially resistant to ophiobolin A. When both Lys 75 and Lys-77 or all three Lys residues were mutated, the resulting calmodulins were very resistant to ophiobolin A. Furthermore, Lys residues added in positions 86 and/or 143 (which are highly conserved in plant calmodulins) did not react with ophiobolin A. None of the mutations seemed to affect the properties of calmodulin. These results show that ophiobolin A reacts quite specifically with calmodulin.