Magnetically Aligned Lipid Bilayers with High Cholesterol for Solid-State NMR of Membrane Proteins.

Phospholipid bicelles are valuable membrane model systems to study membrane proteins by NMR and other physicochemical techniques. The range of bicelle compositions that are compatible with uniaxial alignment of the lipid bilayers in a magnetic field is still limited with regard to the addition of large amounts (>20%) of cholesterol and/or sphingolipids. Here, we demonstrate that n-dodecyl-ß-D-melibioside (DDMB), which was recently introduced as a detergent to produce sphingolipid-cholesterol-rich isotropic bicelles for solution NMR studies, can also be used to produce magnetically alignable lipid bilayers with high cholesterol content that are well suited for solid-state NMR of membrane proteins. Remarkably, DDMB enables the preparation of high q bicelles that contain 50% mol cholesterol while retaining their ability to form a stable, well-aligned liquid crystalline bilayer phase in a magnetic field. We show that the intact 46-residue membrane-bound form of Pf1 bacteriophage coat protein and a truncated construct of the membrane protein Vpu from HIV-1 (residues 2-30) in DDMB bicelles are well aligned and undergo fast and uniaxial rotational diffusion about the bilayer normal, similarly to what is observed in other bicelle and macrodisc systems. We also demonstrate a spectroscopic method that measures the increase in the thickness of DMPC bilayers that results from the addition of cholesterol, using the PISA-wheel spectral patterns of trans-membrane helices as a molecular goniometer. For example, we find that the hydrophobic thickness of DMPC bilayers is increased by approximately 2.5 Å in the presence of 35% mol cholesterol.

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease.

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically.

A Multipurpose Leguminous Plant for the Mediterranean Countries: Leucaena leucocephala as an Alternative Protein Source: A Review.

In tropical and subtropical regions, as well as in the internal and/or marginal Mediterranean areas, one of the most important problems related to animal production is represented by the inadequate nutritional supplies. The low productivity of the animals, often connected to reduced annual growth, is, in fact, not infrequently attributable to the low nitrogen content and the high fiber content of the local plant species and crop residues that constitute the base ingredients of the rations commonly adopted by farmers. The use of the supplementation with arboreal and shrub fodder, although often containing anti-nutritional factors and toxins that limit its use, could be a profitable way to alleviate the nutritional deficiencies of the basic diets. Leucaena leucocephala (Lam.) De Wit is native to Central America and widely naturalized in the majority of Latin American countries. It is a legume suitable for tropical and subtropical environments including the countries of the Mediterranean area. Moreover, its spread is desirable if we consider the multiple uses to which it is suitable, the considerable amount of biomass produced, and its role in preserving the environment. The aim of this work was to highlight the characteristics of Leucaena that can justify its wide diffusion. A structured analysis of strengths and weaknesses was performed accordingly. Being a good protein source for feeding livestock, it could be a species to be introduced in the inland areas of the Mediterranean countries as an alternative protein source; the limit represented by the presence of anti-nutritional factors could be overcome by feed processing and by launching targeted research programs.

Serum iPTH range in a reference population: From an integrated approach to vitamin D prevalence impact evaluation.

BACKGROUND: The iPTH upper reference limit (URL) reported by our laboratory provider (Abbott Laboratories) at Tor Vergata University Hospital was evaluated by internal verification procedures as not representative of our population and resulting as underestimated. In this study, a new reference interval has been investigated and established by comparing a direct and an indirect method based on a statistical reduction from results stored in the laboratory database. METHODS: For reference interval calculation from the healthy population, we analyzed a cohort of 100 blood donors (84% males and 16% females) screened with no bone-related and malabsorption diseases. We analyzed a cohort of 495 patients retrieved from more than 800 iPTH results by excluding subjects with pathological measurement for calcium, phosphorus, and creatinine for the reference interval evaluation. Patients with vitamin D results were included in the analysis. Vitamin D sufficiency status during the period from January to September 2020 was also evaluated by investigating 3,050 patients. RESULTS: The iPTH reference interval of a healthy blood donor population was measured as 25.2-109.1 pg/mL (2.7-11.6 pmol/L) at 2.5 and 97.5 distribution percentile. The iPTH reference interval from data stored in the laboratory database was 19.3-112.5 pg/mL (2.0-11.9 pmol/L). Furthermore, 60% of the whole population had prevalently insufficient vitamin D concentration (<30 ng/dL; <75 nmol/L). The impact of vitamin D concentration on the iPTH reference interval was measured for insufficient vitamin D (<30 ng/dL; <75 nmol/L) as 15.2-127.7 pg/mL (1.6-13.5 pmol/L), desirable vitamin D (30-40 ng/ml; 75-100 nmol/L) as 25.6-105 pg/mL (2.7-10.7 pmol/L) and optimal vitamin D (>40 ng/ml; >100 nmol/L) as 26.2-89.2 pg/mL (2.8-9.4 pmol/L), respectively. CONCLUSIONS: The URL reported in manufacturer datasheets likely refers to a normal population with non-pathological vitamin D levels. On the contrary, the considered population was mostly vitamin D insufficient, resulting in a URL shift. On this basis, we suggest describing in medical reports the iPTH range for vitamin D deficiency for diagnosis of primary hyperparathyroidism even when a specific vitamin D request is lacking. On the other hand, reporting optimal vitamin D-based iPTH reference interval could be clinically relevant in supplemented patients as a marker of treatment efficacy.

Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity.

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

Effect of GH p.L127V Polymorphism and Feeding Systems on Milk Production Traits and Fatty Acid Composition in Modicana Cows.

Growth hormone participates in the regulation of lactation and lipid metabolism. A trial study was conducted to evaluate the effects of genetic polymorphism at GH p.L127V and its interaction with feeding system (extensive, EX; semi-intensive, SI) on milk traits and fatty acids composition in Modicana cows. In the semi-intensive farm (SI) diet consisted of hay, concentrate and 2 h of grazing. In the extensive farm (EX) feeding consisted in 8 h of grazing and hay. The frequencies of LL, LV and VV genotypes were, respectively: 0.64, 0.34, 0.02. GH polymorphism and its interaction with feeding system did not influence milk yield and composition. Cows carrying LL genotype produced milk with lower 6:0 and 8:0 and higher 16:1 c9 and 18:1 c9, total UFA and total MUFA. Feeding significantly affected fatty acids: in EX cows lower SFA and higher PUFA and UFA were found, compared to SI cows. The lower, more favorable atherogenic index of milk from EX system was coherent with the improved healthy characteristics of milk from animals fed almost exclusively on pasture. A significant interaction genotype x feeding system was evident for 18:1, higher in the LL cows only in the EX system, but not in the SI system.

Leptin Gene Polymorphism in Goats Fed with Diet at Different Energy Level: Effects on Feed Intake, Milk Traits, Milk Fatty Acids Composition, and Metabolic State.

The study investigated the effects of a polymorphism at the LEP gene intron 1 microsatellite region and its interaction with diet energy level on feed intake, milk traits, milk fatty acid composition, and metabolic state in goats. Sixteen Girgentana lactating goats at mid-lactation, selected on the basis of their genotype (8 goats homozygous 266 bp/266 bp, L genotype; 8 goats heterozygous 266 bp/264 bp, H genotype), were fed ad libitum according to a change-over design, with two diets at different energy levels reached with different hay inclusion: low energy diet (LE)-100% of hay; and high energy diet (HE)-65% of hay. No differences in milk yield and composition or in dry matter intake were found between leptin genotypes or between diets. Leptin genotype had no effect on plasma metabolite concentrations. The differences between diets were recorded for plasma ß-hydroxybutyric acid (BHBA) concentrations with higher (p = 0.01) values for the HE compared to the LE diet (0.44 vs. 0.24 mmol/L, respectively). Nonesterified fatty acid (NEFA) values seem to indicate a positive energy balance in goats. No interaction genotype per diet was evident for most of the studied parameters. Fatty acid composition was strongly influenced by LEP genotype: L goats, compared to H goats, showed higher levels of monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and 14:1/14:0 desaturation index; lower levels of saturated fatty acids (SFA); and a more favorable atherogenic index. These results seem to suggest an improvement of health characteristics of milk with the L genotype.

Metal-ion Binding to Host Defense Peptide Piscidin 3 Observed in Phospholipid Bilayers by Magic Angle Spinning Solid-state NMR.

Cationic antimicrobial peptides (AMPs) are essential components of the innate immune system. They have attracted interest as novel compounds with the potential to treat infections associated with multi-drug resistant bacteria. In this study, we investigate piscidin 3 (P3), an AMP that was first discovered in the mast cells of hybrid striped bass. Prior studies showed that P3 is less active than its homolog piscidin 1 (P1) against planktonic bacteria. However, P3 has the advantage of being less toxic to mammalian cells and more active on biofilms and persister cells. Both P1 and P3 cross bacterial membranes and co-localize with intracellular DNA but P3 is more condensing to DNA while P1 is more membrane active. Recently, we showed that both peptides coordinate Cu2+ through an amino-terminal copper and nickel (ATCUN) motif. We also demonstrated that the bactericidal effects of P3 are linked to its ability to form radicals that nick DNA in the presence of Cu2+ . Since metal binding and membrane crossing by P3 is biologically important, we apply in this study solid-state NMR spectroscopy to uniformly 13 C-15 N-labeled peptide samples to structurally characterize the ATCUN motif of P3 bound to bilayers and coordinated to Ni2+ and Cu2+ . These experiments are supplemented with density functional theory calculations. Taken together, these studies refine the arrangement of not only the backbone but also side chain atoms of an AMP simultaneously bound to metal ions and phospholipid bilayers.

Control of Transmembrane Helix Dynamics by Interfacial Tryptophan Residues.

Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW5,19ALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific 2H- and 15N-labeled residues. GW5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as 2H quadrupolar or 15N-1H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW5,19ALP23, the modified GW4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the 2H and 15N NMR signals confirm that the extent of dynamic averaging, particularly rotational "slippage" about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional 2H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment.

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR.

The human chemokine interleukin-8 (IL-8; CXCL8) is a key mediator of innate immune and inflammatory responses. This small, soluble protein triggers a host of biological effects upon binding and activating CXCR1, a G protein-coupled receptor, located in the cell membrane of neutrophils. Here, we describe 1H-detected magic angle spinning solid-state NMR studies of monomeric IL-8 (1-66) bound to full-length and truncated constructs of CXCR1 in phospholipid bilayers under physiological conditions. Cross-polarization experiments demonstrate that most backbone amide sites of IL-8 (1-66) are immobilized and that their chemical shifts are perturbed upon binding to CXCR1, demonstrating that the dynamics and environments of chemokine residues are affected by interactions with the chemokine receptor. Comparisons of spectra of IL-8 (1-66) bound to full-length CXCR1 (1-350) and to N-terminal truncated construct NT-CXCR1 (39-350) identify specific chemokine residues involved in interactions with binding sites associated with N-terminal residues (binding site-I) and extracellular loop and helical residues (binding site-II) of the receptor. Intermolecular paramagnetic relaxation enhancement broadening of IL-8 (1-66) signals results from interactions of the chemokine with CXCR1 (1-350) containing Mn2+ chelated to an unnatural amino acid assists in the characterization of the receptor-bound form of the chemokine.

Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy.

The structure of monomeric human chemokine IL-8 (residues 1-66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1-72), with the main differences being the location of the N-loop (residues 10-22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67-72). NMR was used to analyze the interactions of monomeric IL-8 (1-66) with ND-CXCR1 (residues 1-38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1-72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.

Herbage intake and milk yield in Comisana ewes as effect of 4 vs 7 h of grazing during late lactation.

Thirty-two Comisana ewes at late lactation were used in two trials carried out during late spring in 2 consecutive years, with the aim to evaluate the effect of the duration of grazing on herbage intake and performance. In each trial, 16 pluriparous Comisana lactating ewes were equally divided into two groups which grazed in two separate areas of natural pasture from 11:00 to 15:00 h (group 4H) or from 10:00 to 17:00 (group 7H). A concentrate mixture (500 g/day) was also offered to each ewe. The mean maximum temperature was, respectively, 23.5 ± 3.8 °C during experiment 1 and 27.0 ± 3.1 °C during experiment 2. Probably as a consequence of the differences in climatic conditions, the results on herbage intake and milk production were different during the two trials. Herbage dry matter intake was not affected by the duration of grazing during trial 1, whereas it was significantly lower in 4H group compared to that in 7H group (0.67 vs 1.02 kg/day; P < 0.001) during trial 2. It could be hypothesised that while with lower environmental temperature (trial 1), the 4H ewes were able to reach good intake levels despite grazing during the hottest hours; with higher temperatures throughout the trial (trial 2), the 4H ewes reduced ingestion. Milk production was higher in 4H group during trial 1 (778 vs 707 g/day; P = 0.006), whereas it was not affected by the number of hours of grazing during trial 2, despite the higher intake levels reached by the 7H group. In conclusion, 3 extra hours of grazing for ewes at late lactation on a low quality pasture could be nullified in terms of yield response.

Communicating systems in the body: how microbiota and microglia cooperate.

Microglia are tissue macrophages of the central nervous system (CNS). Their key tasks are immune surveillance as well as responding to infections or other pathological states such as neurological diseases or injury. In recent years it has been discovered that microglia are additionally crucial for the maintenance of brain homeostasis during development and adulthood by adjusting the neuronal network and phagocytosing neuronal debris. Microglia persist in the CNS throughout the life of the organism and self-renew without engraftment of bone-marrow-derived cells. Until recently it remained unknown what controls their maturation and activation under homeostatic conditions. In this review we discuss new aspects of the interaction between host microbiota and brain function with special focus on the brain-resident innate immune cells, the microglia.

Host microbiota constantly control maturation and function of microglia in the CNS.

As the tissue macrophages of the CNS, microglia are critically involved in diseases of the CNS. However, it remains unknown what controls their maturation and activation under homeostatic conditions. We observed substantial contributions of the host microbiota to microglia homeostasis, as germ-free (GF) mice displayed global defects in microglia with altered cell proportions and an immature phenotype, leading to impaired innate immune responses. Temporal eradication of host microbiota severely changed microglia properties. Limited microbiota complexity also resulted in defective microglia. In contrast, recolonization with a complex microbiota partially restored microglia features. We determined that short-chain fatty acids (SCFA), microbiota-derived bacterial fermentation products, regulated microglia homeostasis. Accordingly, mice deficient for the SCFA receptor FFAR2 mirrored microglia defects found under GF conditions. These findings suggest that host bacteria vitally regulate microglia maturation and function, whereas microglia impairment can be rectified to some extent by complex microbiota.

Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA).

The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.

Magic angle Lee-Goldburg frequency offset irradiation improves the efficiency and selectivity of SPECIFIC-CP in triple-resonance MAS solid-state NMR.

The efficiency and selectivity of SPECIFIC-CP, a widely used method for selective double cross-polarization in triple-resonance magic angle spinning solid-state NMR, is improved by performing the tangential-shaped (13)C irradiation at an offset frequency that meets the Lee-Goldburg condition (LG-SPECIFIC-CP). This is demonstrated on polycrystalline samples of uniformly (13)C, (15)N labeled N-acetyl-leucine and N-formyl-Met-Leu-Phe-OH (MLF) at 700MHz and 900MHz (1)H resonance frequencies, respectively. For the single (13)Cα of N-acetyl-leucine, relative to conventional broad band cross-polarization, the SPECIFIC-CP signal has 47% of the intensity. Notably, the LG-SPECIFIC-CP signal has 72% of the intensity, essentially the theoretical maximum. There were no other changes in the experimental parameters. The three (13)Cα signals in MLF show some variation in intensities, reflecting the relatively narrow bandwidth of a frequency-offset procedure, and pointing to future developments for this class of experiment.

Structure of the chemokine receptor CXCR1 in phospholipid bilayers.

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.

Morning versus afternoon cutting time of Berseem clover (Trifolium alexandrinum L.) affects feed intake, milk yield and composition in Girgentana goats.

Twenty lactating Girgentana goats were used to evaluate the effect of morning v. afternoon cutting time of Berseem clover (Trifolium alexandrinum L.) on feed intake, milk yield and milk composition. Goats were randomly divided into two groups of ten animals, receiving 10 kg of fresh Berseem clover cut at 9.00 (AM group) or 16.00 (PM group), respectively; 500 g of concentrate was given individually to goats before offering forage. Feed intake increased (P<0·01) in the PM group (30·5 v. 25·3 g dry matter/kg body weight), associated with the different nutrient content of diets: lower crude protein but higher dry matter, neutral detergent fibre, water soluble carbohydrates (WSC) and total fatty acids in the PM-harvested forage. Milk production, protein and casein content were higher (P<0·05) in the PM group (1415 g/d, 3·25% and 2·42% v. 1277 g/d, 3·15% and 2·33%, respectively), whereas no differences between groups were detected for milk fat, lactose or urea content. Body weight slowly decreased from the start to the end of the experiment, without differences between groups. This study showed an important milk yield responses in Girgentana goats offered afternoon-cut compared with morning-cut Berseem clover, due to a marked increase in WSC in the afternoon-cut forage.

Amphipathic antimicrobial piscidin in magnetically aligned lipid bilayers.

The amphipathic antimicrobial peptide piscidin 1 was studied in magnetically aligned phospholipid bilayers by oriented-sample solid-state NMR spectroscopy. (31)P NMR and double-resonance (1)H/(15)N NMR experiments performed between 25 °C and 61 °C enabled the lipid headgroups as well as the peptide amide sites to be monitored over a range of temperatures. The α-helical peptide dramatically affects the phase behavior and structure of anionic bilayers but not those of zwitterionic bilayers. Piscidin 1 stabilizes anionic bilayers, which remain well aligned up to 61 °C when piscidin 1 is on the membrane surface. Two-dimensional separated-local-field experiments show that the tilt angle of the peptide is 80 ± 5°, in agreement with previous results on mechanically aligned bilayers. The peptide undergoes fast rotational diffusion about the bilayer normal under these conditions, and these studies demonstrate that magnetically aligned bilayers are well suited for structural studies of amphipathic peptides.

Mechanically, magnetically, and "rotationally aligned" membrane proteins in phospholipid bilayers give equivalent angular constraints for NMR structure determination.

The native environment for membrane proteins is the highly asymmetric phospholipid bilayer, and this has a large effect on both their structure and dynamics. Reproducing this environment in samples suitable for spectroscopic and diffraction experiments is a key issue, and flexibility in sample preparation is essential to accommodate the diverse size, shape, and other physical properties of membrane proteins. In most cases, to ensure that the biological activities are maintained, this means reconstituting the proteins in fully hydrated planar phospholipid bilayers. The asymmetric character of protein-containing bilayers means that it is possible to prepare either oriented or unoriented (powder) samples. Here we demonstrate the equivalence of mechanical, magnetic, and what we refer to as "rotational alignment" of membrane proteins in phospholipid bilayer samples for solid-state NMR spectroscopy. The trans-membrane domain of virus protein "u" (Vpu) from human immunodeficiency virus (HIV-1) and the full-length membrane-bound form of fd bacteriophage coat protein in phospholipid bilayers are used as examples. The equivalence of structural constraints from oriented and unoriented (powder) samples of membrane proteins is based on two concepts: (1) their alignment is defined by the direction of the bilayer normal relative to the magnetic field and (2) they undergo rapid rotational diffusion about the same bilayer normal in liquid crystalline membranes. The measurement of angular constraints relative to a common external axis system defined by the bilayer normal for all sites in the protein is an essential element of oriented sample (OS) solid-state NMR.