Development of selected bacterial groups of the rectal microbiota of healthy calves during the first week postpartum.

AIMS: The intestinal microbiota of newborn calves is largely unexplored even if it is of great significance for their future health. Therefore, the aim of the study was to gain a better insight into the development dynamics of certain bacterial groups during the first week of life. METHODS AND RESULTS: Faecal samples of healthy Simmental calves (dual-purpose breed; n = 80), bottle fed and raised in a dairy farm were taken immediately after birth and at 6/12/24/48/72/168 h (h) after birth. Samples were analysed using cultural, biochemical and molecular-biological methods. The aerobe, anaerobe, Enterobacteriaceae and Enterococcus counts of healthy calves increased significantly between 6 and 24 h postpartum (P <0·05). Apart from the anaerobes, bacterial counts decreased after reaching a plateau at 24-48 h. Enterococcus faecalis was detected in significantly higher counts compared to E. faecium (P <0·05). Lactobacilli developed more slowly and increased until day 7 after birth to a mean value of 6·8 × 107  CFU per g. MALDI-TOF analysis of 2338 lactobacilli isolates resulted in 36 different species. CONCLUSIONS: Lactobacillus reuteri became the most common Lactobacillus sp. during the first week of life. SIGNIFICANCE AND IMPACT OF THE STUDY: This fact seems to be very important for the calf's intestinal health because L. reuteri is known to show in vitro bactericidal effects against bacterial pathogens and anti-infective activities against rotaviruses and Cryptosporidium parvum.

The integrity of the alpha-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes.

Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the alphaI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the alphaI-helix of LFABP (alpha(I)LbetaIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from alpha(I)LbetaIFABP compared to IFABP. The results indicate that the alphaI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the alphaII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the alphaI helix of IFABP in its physical interaction with membranes.

Listening to nurses' moral voices: building a quality health care environment.

In this paper we describe a research project in nursing ethics aimed at exploring the meaning of ethics for nurses providing direct care with clients. This was a practice-based project in which participants who were staff nurses, nurses in advanced practice, and students in nursing were asked to tell us (or describe to us) how they thought about ethics in their practice, and what ethical practice meant to them. We then undertook to analyze, describe and understand the enactment of ethical practice, the opportunities for and barriers to such enactment, as well as the resources nurses need for ethical practice. We drew out implications of these findings for nursing leaders. We identified practice realities that create a climate for ethical or moral distress, and the way in which nurses attempt to maintain their moral agency. Practice realities included nurses' ethical concerns about policies guiding care; the financial, human and temporal resources available for care; and the power and conflicting loyalties nurses encounter inproviding good care. Maintaining moral agency involved use of a variety of ethical resources and the identification of resources needed to provide good care, as well as the processes used to enact moral agency. Nurse leaders are also moral agents. Important implications of these findings for nursing leaders are that they need moral courage to be self-reflective, to name their own moral distress, and to act so that their nursing staff are able to be moral agents. Nurse leaders need to be the moral compass for nurses, using their power as a positive force to promote, provide and sustain quality practice environments for safe, competent and ethical practice.

Role of macrophage-expressed adipocyte fatty acid binding protein in the development of accelerated atherosclerosis in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease process associated with elevated levels of plasma cholesterol, especially low-density lipoproteins. The latter become trapped within the arterial wall and are oxidized and taken up by macrophages to form foam cells. This process is an initiating event for atherosclerosis. Fatty acid binding proteins (FABP) are involved in fatty acid metabolism and cellular lipid transport, and adipocyte FABP (aP2) is also expressed in macrophages. We recently generated mice lacking both apolipoprotein (Apo)E and aP2 (ApoE-/-aP2-/-) and found that these mice, compared with ApoE-/- mice, developed markedly smaller atherosclerotic lesions that contained fewer macrophages. Here we investigated the mechanism(s) responsible for this prevention of atherosclerotic lesion formation. Bone marrow transplantations were performed in ApoE-/- mice, receiving cells from either ApoE-/- or ApoE-/-aP2-/- mice. The lack of aP2 in donor marrow cells led to the development of smaller (5.5-fold) atherosclerotic lesions in the recipient mice. No differences were found in plasma cholesterol, glucose, or insulin levels between recipients of bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice. However, the expression of chemoattractant and inflammatory cytokines was decreased in macrophages from ApoE-/-aP2-/- mice compared with ApoE-/- mice, which may contribute to the decrease in atherosclerotic lesion formation. Taken together, we demonstrate the importance of macrophage aP2 in the development of atherosclerotic lesions.

Common mechanisms of monoacylglycerol and fatty acid uptake by human intestinal Caco-2 cells.

Free fatty acids (FFA) and sn-2-monoacylglycerol (sn-2-MG), the two hydrolysis products of dietary triacylglycerol, are absorbed from the lumen into polarized enterocytes that line the small intestine. Intensive studies regarding FFA transport across the brush-border membrane of the enterocyte are available; however, little is known about sn-2-MG transport. We therefore studied the kinetics of sn-2-MG transport, compared with those of long-chain FFA (LCFA), by human intestinal Caco-2 cells. To mimic postprandial luminal and plasma environments, we examined the uptake of taurocholate-mixed lipids and albumin-bound lipids at the apical (AP) and basolateral (BL) surfaces of Caco-2 cells, respectively. The results demonstrate that the uptake of sn-2-monoolein at both the AP and BL membranes appears to be a saturable function of the monomer concentration of sn-2-monoolein. Furthermore, trypsin preincubation inhibits sn-2-monoolein uptake at both AP and BL poles of cells. These results suggest that sn-2-monoolein uptake may be a protein-mediated process. Competition studies also support a protein-mediated mechanism and indicate that LCFA and LCMG may compete through the same membrane protein(s) at the AP surface of Caco-2 cells. The plasma membrane fatty acid-binding protein (FABP(pm)) is known to be expressed in Caco-2, and here we demonstrate that fatty acid transport protein (FATP) is also expressed. These putative plasma membrane LCFA transporters may be involved in the uptake of sn-2-monoolein into Caco-2 cells.

Collision-mediated transfer of long-chain fatty acids by neural tissue fatty acid-binding proteins (FABP): studies with fluorescent analogs.

Mammalian fatty acid-binding proteins (FABP) are a family of intracellular proteins (approx 15 kDa) that bind long-chain fatty acids (FA) with high affinity. They are believed to serve as cytoplasmic transporters of FA and to target FA to specific cellular sites of utilization. Several different FABPs are expressed in neural tissue, including brain FABP (B-FABP), myelin FABP (M-FABP), and heart FABP (H-FABP). We have previously shown that H-FABP transfers FAvia direct collisional interactions with acceptor model membranes. In the present studies, we use a fluorescence resonance energy transfer (FRET) assay to examine the rate and mechanism of transfer of a fluorescent long-chain fatty acid from B-FABP to phospholipid vesicles. The rate of transfer is shown to be independent of buffer ionic strength and dramatically enhanced by the presence of specific anionic phospholipids. These results are consistent with a mechanism by which FA are transferred from B-FABP to phospholipid membranes by a transient collision-based mechanism.

Role of surface lysine residues of adipocyte fatty acid-binding protein in fatty acid transfer to phospholipid vesicles.

The tertiary structure of murine adipocyte fatty acid-binding protein (AFABP) is a flattened 10-stranded beta-barrel capped by a helix-turn-helix segment. This helical domain is hypothesized to behave as a "lid" or portal for ligand entry into and exit from the binding cavity. Previously, we demonstrated that anthroyloxy-labeled fatty acid (AOFA) transfer from AFABP to phospholipid membranes occurs by a collisional process, in which ionic interactions between positively charged lysine residues on the protein surface and negatively charged phospholipid headgroups are involved. In the present study, the role of specific lysine residues located in the portal and other regions of AFABP was directly examined using site-directed mutagenesis. The results showed that isoleucine replacement for lysine in the portal region, including the alphaI- and alphaII-helices and the beta C-D turn, resulted in much slower 2-(9-anthroyloxy)palmitate (2AP) transfer rates to acidic membranes than those of native AFABP. An additive effect was found for mutant K22,59I, displaying the slowest rates of FA transfer. Rates of 2AP transfer from "nonportal" mutants on the beta-G and I strands were affected only moderately; however, a lysine --> isoleucine mutation in the nonportal beta-A strand decreased the 2AP transfer rate. These studies suggest that lysines in the helical cap domain are important for governing ionic interactions between AFABP and membranes. Furthermore, it appears that more than one distinct region, including the alphaI-helix, alphaII-helix, beta C-D turn, and the beta-A strand, is involved in these charge-charge interactions.

Deletion of the helical motif in the intestinal fatty acid-binding protein reduces its interactions with membrane monolayers: Brewster angle microscopy, IR reflection-absorption spectroscopy, and surface pressure studies.

Intestinal fatty acid binding protein (IFABP) appears to interact directly with membranes during fatty acid transfer [Hsu, K. T., and Storch, J. (1996) J. Biol. Chem. 271, 13317-13323]. The largely alpha-helical "portal" domain of IFABP was critical for these protein--membrane interactions. In the present studies, the binding of IFABP and a helixless variant of IFABP (IFABP-HL) to acidic monolayers of 1,2-dimyristoylphosphatidic acid (DMPA) has been monitored by surface pressure measurements, Brewster angle microscopy (BAM), and infrared reflection-absorption spectroscopy (IRRAS). Protein adsorption to DMPA exhibited a two phase kinetic process consisting of an initial slow phase, arising from protein binding to the monolayer and/or direct interfacial adsorption, and a more rapid phase that parallels formation of lipid-containing domains. IFABP exhibited more rapid changes in both phases than IFABP-HL. The second phase was absent when IFABP interacted with zwitterionic monolayers of 1,2-dipalmitoylphosphatidylcholine, revealing the important role of electrostatics at this stage. BAM images of DMPA monolayers with either protein revealed the formation of domains leading eventually to rigid films. Domains of DMPA/IFABP-HL formed more slowly and were less rigid than with the wild-type protein. Overall, the IRRAS studies revealed a protein-induced conformational ordering of the lipid acyl chains with a substantially stronger ordering effect induced by IFABP. The physical measurements thus suggested differing degrees of direct interaction between the proteins and DMPA monolayers with the IFABP/DMPA interaction being somewhat stronger. These data provide a molecular structure rationale for previous kinetic measurements indicating that the helical domain is essential for a collision-based mechanism of fatty acid transfer to phospholipid membranes [Corsico, B., Cistola, D. P., Frieden, C. and Storch, J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 12174-12178].

Organizational, Nonorganizational, and Intrinsic religiosity and academic dishonesty.

The present study was a preliminary examination of the relations among the Organizational, Nonorganizational, and Intrinsic dimensions of religiosity and academic dishonesty. 244 college students completed the Duke Religion Index and nine questions assessing academic dishonesty. Analysis indicated that (1) regardless of sex, High Nonorganizational and Intrinsic religiosity was associated with lower reported rates of academic dishonesty, and (2) there was an interaction between Organizational religiosity and sex, with High Organizational women and men reporting similar rates of academic dishonesty. Furthermore, the frequency of academic dishonesty reported by High Organizational women was higher than the rates reported by Moderate and Minimal Organizational women.

Unravelling the significance of cellular fatty acid-binding proteins.

Cellular long-chain fatty acid (FA) transport and metabolism are believed to be regulated by membrane-associated and soluble proteins that bind and transport FAs. Several different classes of membrane proteins have been proposed as FA acceptors or transmembrane FA transporters. New evidence from in-vitro and whole-animal studies supports the existence of protein-mediated transmembrane transport of FAs, which is likely to coexist with passive diffusional uptake. The trafficking of FAs by intracellular fatty acid-binding proteins may involve their interaction with specific membrane or protein targets. Evidence is also emerging for concerted actions between the membrane and cytoplasmic fatty acid-binding proteins that allow for efficient regulation of FA transport and metabolism.

Nutrition: a reservoir for integrative science.

In the last twenty years, powerful new molecular techniques were introduced that made it possible to advance knowledge in human biology using a reductionist approach. Now, the need for scientists to deal with complexity should drive a movement toward an integrationist approach to science. We propose that nutritional science is one of the best reservoirs for this approach. The American Society for Nutritional Sciences can play an important role by developing and delivering a cogent message that convinces the scientific establishment that nutrition fills this valuable niche. The society must develop a comprehensive strategy to develop our image as the reservoir for life sciences integration. Our efforts can start with our national meeting and publications, with the research initiatives for which we advocate, with our graduate training programs and with the public relations image we project for ourselves. Defining the image and future directions of nutrition as the discipline that can integrate scientific knowledge from the cell and molecule to the whole body and beyond to populations can be the most important task that our society undertakes. If we do not effectively meet this challenge, a golden opportunity will pass to others and nutritional scientists will be left to follow them.

The fatty acid transport function of fatty acid-binding proteins.

The intracellular fatty acid-binding proteins (FABPs) comprise a family of 14-15 kDa proteins which bind long-chain fatty acids. A role for FABPs in fatty acid transport has been hypothesized for several decades, and the accumulated indirect and correlative evidence is largely supportive of this proposed function. In recent years, a number of experimental approaches which more directly examine the transport function of FABPs have been taken. These include molecular level in vitro modeling of fatty acid transfer mechanisms, whole cell studies of fatty acid uptake and intracellular transfer following genetic manipulation of FABP type and amount, and an examination of cells and tissues from animals engineered to lack expression of specific FABPs. Collectively, data from these studies have provided strong support for defining the FABPs as fatty acid transport proteins. Further studies are necessary to elucidate the fundamental mechanisms by which cellular fatty acid trafficking is modulated by the FABPs.

Adipocyte metabolism in adipocyte fatty acid binding protein knockout mice (aP2-/-) after short-term high-fat feeding: functional compensation by the keratinocyte [correction of keritinocyte] fatty acid binding protein.

Mice null for adipocyte fatty acid binding protein (AFABP) compensate by increasing expression of keratinocyte fatty acid binding protein (KFABP) (Hotamisligil et al. Science 274:1377-1379, 1996). In the present study, AFABP knockout (KO) and wild-type (WT) mice became equally obese on a high-fat diet, as judged by fat pad weights, adipocyte size, and body composition analysis. High-fat feeding led to moderate insulin resistance in both WT and AFABP knockout mice, as indicated by an approximately 2-fold increase in plasma insulin. However, in the high fat-fed mice, plasma glucose levels were approximately 15% lower in the AFABP-KO mice. Adipocytes isolated from AFABP-KO and WT mice fed high- or low-fat diets exhibited similar rates of basal and norepinephrine-stimulated lipolysis and insulin-stimulated rates of glucose conversion to fatty acids and glyceride-glycerol. However, basal glucose conversion to fatty acids was higher in adipocytes of AFABP-KO mice. Adipocyte tumor necrosis factor-alpha release was similarly increased by high-fat diet-induced obesity in both WT and AFABP-KO mice. As assessed by Western blot analysis, the level of KFABP protein in AFABP-KOs was approximately 40% of the level of AFABP in WT controls. The binding affinities of KFABP for long-chain fatty acids were 2- to 4-fold higher than those of AFABP, but the relative affinities for different fatty acids were similar. As for AFABP, the rate of fatty acid transfer from KFABP to model phospholipid vesicles was increased with acceptor membrane concentration and by inclusion of acidic phospholipids, indicating a similar mechanism of transfer. We conclude KFABP can functionally compensate for the absence of AFABP, resulting in no major alterations in adipocyte metabolism or fat accumulation in response to short-term feeding of high-fat diets that result in moderate hyperinsulinemia.

Liver and intestinal fatty acid-binding proteins obtain fatty acids from phospholipid membranes by different mechanisms.

Intestinal enterocytes contain high concentrations of two cytosolic fatty acid-binding proteins (FABP), liver FABP (L-FABP) and intestinal FABP (I-FABP), which are hypothesized to play a role in cellular fatty acid trafficking. The mechanism(s) by which fatty acids move from membranes to each of these proteins is not known. Here we demonstrate that fluorescent anthroyloxy fatty acid analogues (AOFA) are transferred from phospholipid vesicles to L-FABP versus I-FABP by different mechanisms. For L-FABP a diffusion-mediated transfer process is demonstrated. The AOFA transfer rate from phosphatidylcholine-containing vesicles (POPC) to L-FABP is similar to that observed with another diffusional process, namely inter-membrane AOFA transfer. Furthermore, the AOFA transfer rate was modulated by buffer ionic strength and AOFA solubility, while the transfer rate remained relatively unchanged by the presence of anionic phospholipids in vesicles. In contrast, the data for I-FABP suggest that a transient collisional interaction of I-FABP with the phospholipid membrane occurs during AOFA extraction from the vesicles by the protein. In particular, the presence of the anionic phospholipid cardiolipin in donor vesicles increased the rate of AOFA transfer to I-FABP by 15-fold compared with transfer to POPC vesicles. The effects of ionic strength on transfer suggest that the interaction of I-FABP with cardiolipin-containing vesicles is likely to contain an electrostatic component. Finally, based on the regulation of AOFA transfer to I-FABP compared with transfer from I-FABP, it is hypothesized that apo- and holo-I-FABPs adopt conformations which may differentially promote I-FABP-membrane interactions. In summary, the results suggest that I-FABP, but not L-FABP, can directly extract fatty acids from membranes, supporting the concept that I-FABP may increase the cytosolic flux of fatty acids via intermembrane transfer.

The adipocyte fatty acid-binding protein binds to membranes by electrostatic interactions.

The adipocyte fatty acid-binding protein (AFABP) is believed to transfer unesterified fatty acids (FA) to phospholipid membranes via a collisional mechanism that involves ionic interactions between lysine residues on the protein surface and phospholipid headgroups. This hypothesis is derived largely from kinetic analysis of FA transfer from AFABP to membranes. In this study, we examined directly the binding of AFABP to large unilamellar vesicles (LUV) of differing phospholipid compositions. AFABP bound LUV containing either cardiolipin or phosphatidic acid, and the amount of protein bound depended upon the mol % anionic phospholipid. The K(a) for CL or PA in LUV containing 25 mol % of these anionic phospholipids was approximately 2 x 10(3) M(-1). No detectable binding occurred when AFABP was mixed with zwitterionic membranes, nor when acetylated AFABP in which surface lysines had been chemically neutralized was mixed with anionic membranes. The binding of AFABP to acidic membranes depended upon the ionic strength of the incubation buffer: >/=200 mM NaCl reduced protein-lipid complex formation in parallel with a decrease in the rate of FA transfer from AFABP to negatively charged membranes. It was further found that AFABP, but not acetylated AFABP, prevented cytochrome c, a well characterized peripheral membrane protein, from binding to membranes. These results directly demonstrate that AFABP binds to anionic phospholipid membranes and suggest that, although generally described as a cytosolic protein, AFABP may behave as a peripheral membrane protein to help target fatty acids to and/or from intracellular sites of utilization.

The Calgary Conjoint Nursing Program. Part II: Successful political action.

When three calgary institutions decided to develop a collaborative nursing program to prepare for the transition to baccalaureate nursing education by the year 2000, the planners found that they had to overcome a broad range of political and institutional hurdles. Faculty from Mount Royal College, Foothills Hospital School of Nursing and the University of Calgary spent six years developing the curriculum and planning for the implementation of the Calgary Conjoint Nursing Program (CCNP). (See The Calgary Conjoint Nursing Program, Part I: Spirit of Collaboration, in the March 1999 issue of this journal.)