Skeletal malformation in growing milk sheep.

INTRODUCTION: Two six-month old female Lacaune lambs with severe skeletal malformations of both front limbs were presented to the Department of Farm Animals, University of Zurich. The clinical examination showed alert animals with a high body weight and body condition score as well as a valgus deformation without pain or swelling. Radiographic examination showed severe irregularities in the epiphysial plate of the metacarpal bones in both lambs. Delayed growth in the lateral aspects of the physis was evident and resulted in valgus deformation. Nutritional causes were considered as the main reason for this presentation and a nutritional consultation was performed by the Institute of Animal Nutrition and Dietetics, University of Zurich. The estimated energy intake of these lambs was 65 % higher than the recommended maximum for growing sheep and the estimated vitamin D content of the diet was 71 % below the recommended allowance. Both animals were euthanized, and peripheral quantitative computed tomography (pQCT) was performed postmortem to measure total bone mineral density (BMD), trabecular bone mineral density (tBMD) and cortical bone mineral density (cBMD) of the left and right metatarsal bone of both animals. The BMD and the tBMD at 10 % of bone length were below the reference values and the BMD at 50 % was above the reference values. In addition, postmortem examination revealed a Salter-Harris-Typ-1 facture in the right caput humeri of one lamb. Histological evaluation showed defects in the articular cartilage with an eburnation in the metacarpal region and a disrupted area of columnar cartilage. This case report supports the fact that a high dietary energy intake leads to damage to the cartilage and the epiphyseal zone in sheep. In addition, insufficient dietary vitamin D intake contributed to the incomplete bone mineralization, as well as delayed growth and skeletal malformation.

INTRODUCTION: Deux agneaux Lacaune femelles de six mois présentant de graves malformations squelettiques des deux membres antérieurs ont été présentés au Département des animaux de rente de l'Université de Zurich. L'examen clinique a montré des animaux alertes avec un poids corporel et une note d'état corporel élevés ainsi qu'une déformation en valgus sans douleur ni gonflement. L'examen radiographique a montré de graves irrégularités dans la plaque épiphysaire des os métacarpiens chez les deux agnelles. Un retard de croissance du côté latéral de la physis était évident, entraînant une déformation en valgus. Des causes nutritionnelles ont été considérées comme étant la principale raison de cette situation et une consultation nutritionnelle a été effectuée par l'Institut de nutrition animale et de diététique de l'Université de Zurich. L'apport énergétique estimé de ces agneaux était supérieur de 65 % au maximum recommandé pour les moutons en croissance et la teneur estimée en vitamine D du régime alimentaire était inférieure de 71 % à l'apport recommandé. Les deux animaux ont été euthanasiés et une tomographie quantitative périphérique a été réalisée post mortem pour mesurer la densité minérale osseuse totale (DMO), la densité minérale osseuse trabéculaire (DMOt) et la densité minérale osseuse corticale (DMOc) des métatarses gauche et droit des deux animaux. La DMO et la DMOt à 10 % de la longueur de l'os étaient inférieures aux valeurs de référence et la DMO à 50 % était supérieure aux valeurs de référence. En outre, l'examen post-mortem a révélé une facture de Salter-Harris-Typ-1 dans le caput humeri droit d'un agneau. L'évaluation histologique a montré des défauts dans le cartilage articulaire avec une éburnation sousjacente dans la région métacarpienne et une zone de destruction du cartilage colonnaire. Ce rapport de cas confirme le fait qu'un apport énergétique alimentaire élevé entraîne des lésions du cartilage et de la zone épiphysaire chez les ovins. En outre, un apport alimentaire insuffisant en vitamine D a contribué à une minéralisation osseuse incomplète, ainsi qu'à un retard de croissance et à une malformation du squelette.

[Deer farming in Switzerland - Current epidemiological situation with focus on husbandry, management and nutrition]. / Gehegewild in der Schweiz ­ Aktuelle epidemiologische Situation mit Schwerpunkt Haltung, Management und Fütterung.

INTRODUCTION: Between September 2016 and February 2017 a survey in Swiss deer farms were conducted to gain information about their husbandry. Questions about the business, feeding, management, health and deworming strategies were asked. 98 (19%) out of 527 registered farms (2016) participated in the survey. The farms were often run on a sideline business, had an average used agricultural area of 7.3 ha with an average of 38 deer. Pasture access was the preferred feeding strategy followed by offering first and second cut hay. Between 2013-2015 the most common causes of death were sudden death and injuries. Parasites were classified as no or rather small problem by 91 out of 102 deer owner. Fecal parasitological examinations of fecal samples were conducted in 36 (35%) of the responding farms. Gastrointestinal roundworms (Trichostrongylidae) were identified as the most common pathogens (in 42-59% of sampled farms), in addition large lungworms (Dictyocaulus sp.) and coccidia were detected. 45% of the participating farmers conducted at least one treatment against parasites between 2013 and 2015.

INTRODUCTION: Dans le but d'avoir une vue d'ensemble sur la détention du gibier d'élevage en Suisse, une enquête a été menée entre septembre 2016 et février 2017, comprenant des questions relatives à l'exploitation, à l'alimentation, à la situation sanitaire et aux stratégies en matière de vermifugation. 98 des 527 exploitations annoncées en 2016 (19%) ont participé à cette étude. Ces exploitations, qui constituent fréquemment un gain accessoire, avaient une surface agricole d'en moyenne 7,3 ha avec 38 cervidés. En matière d'alimentation, c'est le foin et le regain qui étaient le plus souvent utilisés en complément du pâturage. Les causes de pertes dans les troupeaux entre 2013 et 2015 étaient principalement les cas de mort subite ainsi que les blessures. 91 de 102 détenteurs de cervidés considéraient les parasites comme n'étant pas un problème ou n'étant qu'un faible problème. Des échantillons de selles, prélevés dans 36 (35%) des exploitations ayant répondu au questionnaire, montraient que les nématodes gastro-intestinaux (Trichostrongylidae) étaient les plus fréquents (présents dans 42-59% des exploitations testées); des vers pulmonaires (Dictyocaulus sp.) et des coccidies ont également été trouvés. Environ 45 % des détenteurs de cervidés ayant participé à l'enquête avaient effectué, dans la période comprise entre 2013 et 2015 au moins un traitement antiparasitaire.

Correlations of structure and rates of energy transfer for through-bond energy-transfer cassettes.

Fluorescent DNA-labeling cassettes are designed to have a common absorbing chromophore matched to a single exciting laser wavelength, but up to four different emitters. Experiments reported here have examined the energy-transfer rates and fluorescence polarization characteristics for two different types of cassette, involving three distinct relative orientations of the donor and acceptor transition moments and the axis of the rigid linker. Energy-transfer times range from <200 fs to approximately 20 ps, the fastest transfer times occurring when the transition moments of the donor and acceptor species are aligned parallel to the linker axis. Experimental evidence is presented that supports a through-bond energy-transfer mechanism, in contrast with a commercial DNA-labeling agent, which exhibits much slower transfer times controlled by FRET. These rigid cassettes also exhibit polarized fluorescence from the acceptor species, so that this particular type of DNA-labeling probe has some of the advantages of single-molecule probes such as rhodamine and coumarin dyes.

Antifreeze proteins in Alaskan insects and spiders.

Prior to this study, antifreeze proteins (AFPs) had not been identified in terrestrial arthropods from the Arctic or anywhere in Alaska. The hemolymph of 75 species of insects and six spiders from interior and arctic Alaska were screened for thermal hysteresis (a difference between the freezing and melting points), characteristic of the presence of AFPs. Eighteen species of insects and three spiders were shown to have AFPs. Ten of the insects with AFPs were beetles including the first species from the families Chrysomelidae, Pythidae, Silphidae and Carabidae. In addition, the first Neuropteran to have AFPs was identified, the lacewing Hemerobius simulans together with the second and third Diptera (the first Tipulids) and the second and third Hemiptera, the stinkbug Elasmostethus interstinctus (the first Pentatomid), and the water strider Limnoporus dissortis (the first Gerrid). Prior to this study, 33 species of insects and three spiders had been reported to have AFPs. Most AFP-producing terrestrial arthropods are freeze avoiding, and the AFPs function to prevent freezing. However, some of the AFP- producing insects identified in this study are known to be freeze tolerant (able to survive freezing) to very low temperatures (-40 to -70 degrees C).

Conformational preferences and vibrational frequency distributions of short peptides in relation to multidimensional infrared spectroscopy.

Molecular dynamics simulations of the structural distributions and the associated amide-I vibrational modes are carried out for dialanine peptide in water and carbon tetrachloride. The various manifestations in nonlinear-infrared spectroscopic experiments of the distributions of conformations of solvated dialanine are examined. The two-dimensional infrared (2D-IR) spectrum of dialanine exhibits the coupling between the amide oscillators and the correlations of the frequency fluctuations. An internally hydrogen-bonded conformation exists in CCl(4) but not in H(2)O where two externally hydrogen-bonded forms are preferred. Simulations of solvated dialanine show how the 2D-IR spectra expose the underlying structural distributions and dynamics that are not deducible from linear-infrared spectra. In H(2)O the 2D-IR shows cross-peaks from large coupling in the alpha-helical conformer and an elongated higher frequency diagonal peak, reflecting the broader distribution of structures for the more flexible acetyl end. In CCl(4), the computed cross-peak portion of the 2D-IR shows evidence of two amide-I transitions in the high-frequency region which are not apparent from the diagonal peak profile. The vibrational frequency inhomogeneity of the amide-I band arises from fluctuations of the instantaneous normal modes of these conformers rather than the shifts induced by hydrogen bonding. The simulation shows that there are correlations between fluctuations of the acetyl and amino end frequencies in H(2)O that arise from mechanical coupling and not from hydrogen bonding at the two ends of the molecule. The angular relationships between the two amide units which also show up in 2D-IR were computed, and spectral manifestations of them are discussed. The simulations also permit a calculation of the rate of energy transfer from one side of the molecule to the other. From these calculations, 2D-IR spectroscopy in conjunction with simulations is seen to be a promising tool for determining dynamics of structure changes in dipeptides.

Tris-benzimidazole derivatives: design, synthesis and DNA sequence recognition.

Two tris-benzimidazole derivatives have been designed and synthesized based on the known structures of the bis-benzimidazole stain Hoechst 33258 complexed to short oligonucleotide duplexes derived from single crystal X-ray studies and from NMR. In both derivatives the phenol group has been replaced by a methoxy-phenyl substituent. Whereas one tris-benzimidazole carries a N-methyl-piperazine at the 6-position, the other one has this group replaced by a 2-amino-pyrrolidine ring. This latter substituent results in stronger DNA binding. The optimized synthesis of the drugs is described. The two tris-benzimidazoles exhibit high AT-base pair (bp) selectivity evident in footprinting experiments which show that five to six base pairs are protected by the tris-benzimidazoles as compared to four to five protected by the bis-benzimidazoles. The tris-benzimidazoles bind well to sequences like 5'-TAAAC, 5'-TTTAC and 5'-TTTAT, but it is also evident that they can bind weakly to sequences such as 5'-TATGTT-3' where the continuity of an AT stretch is interrupted by a single G*C base pair.

Two-dimensional IR spectroscopy can be designed to eliminate the diagonal peaks and expose only the crosspeaks needed for structure determination.

The power of two-dimensional (2D) IR spectroscopy as a structural method with unprecedented time resolution is greatly improved by the introduction of IR polarization conditions that completely eliminate diagonal peaks from the spectra and leave only the crosspeaks needed for structure determination. This approach represents a key step forward in the applications of 2D IR to proteins, peptides, and other complex molecules where crosspeaks are often obscured by diagonal peaks. The technique is verified on the model compound 1,3-cyclohexanedione and subsequently used to clarify the distribution of structures that the acetylproline-NH(2) dipeptide adopts in chloroform. In both cases, crosspeaks are revealed that were not observed before, which, in the case of the dipeptide, has led to additional information about the structure of the amino group end of the peptide.

Two-dimensional infrared spectroscopy: a promising new method for the time resolution of structures.

Recently, new methods for determining time-evolving structures using infrared analogs of NMR spectroscopy have been introduced that have outstanding potential in structural biology. Already, within the past two years, structures of dipeptides, tripeptides and pentapeptides have been determined on much faster timescales than the conformational dynamics. Also, two-dimensional infrared correlation spectra of some proteins and isotopically edited alanine-rich helices have been examined.

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy.

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

Two-dimensional infrared spectroscopy of peptides by phase-controlled femtosecond vibrational photon echoes.

Two-dimensional infrared spectra of peptides are introduced that are the direct analogues of two- and three-pulse multiple quantum NMR. Phase matching and heterodyning are used to isolate the phase and amplitudes of the electric fields of vibrational photon echoes as a function of multiple pulse delays. Structural information is made available on the time scale of a few picoseconds. Line narrowed spectra of acyl-proline-NH(2) and cross peaks implying the coupling between its amide-I modes are obtained, as are the phases of the various contributions to the signals. Solvent-sensitive structural differences are seen for the dipeptide. The methods show great promise to measure structure changes in biology on a wide range of time scales.

Intense terahertz pulses by four-wave rectification in air.

We describe a new four-wave rectification method for the generation of intense, ultrafast terahertz (THz) pulses from gases. The fundamental and second-harmonic output of an amplified Ti:sapphire laser is focused to a peak intensity of ~5x10(14)W/cm (2) . Under these conditions, peak THz fields estimated at 2 kV/cm have been observed; the measured power spectrum peaks near 2 THz. Phase-dependent measurements show that this is a coherent process and is sensitive to the relative phases of the fundamental and second-harmonic pulses. Comparable THz signals have been observed from nitrogen and argon as well as from air.

The dynamics of structural deformations of immobilized single light-harvesting complexes.

Single assemblies of the intact light-harvesting complex LH2 from Rhodopseudomonas acidophila were bound to mica surfaces at 300 K and examined by observing their fluorescence after polarized light excitation. The complexes are generally not cylindrically symmetric. They act like elliptic absorbers, indicating that the high symmetry found in crystals of LH2 is not present when the molecules are immobilized on mica. The ellipticity and the principal axes of the ellipses fluctuate on the time scale of seconds, indicating that there is a mobile structural deformation. The B850 ring of cofactors shows significantly less asymmetry than B800. The photobleaching strongly depends on the presence of oxygen.

Heme protein dynamics revealed by geminate nitric oxide recombination in mutants of iron and cobalt myoglobin.

Nitric oxide myoglobin (MbNO) at 300 K was photodissociated with 405 nm pulses. The NO recombination in several mutants of iron and cobalt myoglobins was investigated at a time resolution of ca. 70 fs. The geminate recombination of NO was nonexponential on sub-nanosecond time scales. For both metals, the change of the detailed structure of the heme pocket (position 68 mutations) caused significant changes in the rates of recombination; however, the metal substitution influenced the recombination much less than did amino acid substitution. The results indicate a primary role of the heme pocket structure in the dynamics, and they suggest that proximal protein relaxation is not the limiting factor in the geminate recombination process. Recombination in cobalt derivatives is somewhat more efficient on the sub-nanosecond time scales than in corresponding iron myoglobins, consistent with other results that show a greater intrinsic reactivity toward the NO of cobalt compared with the iron heme. A comparison of results using Soret band excitation with previous Q-state excitation studies demonstrates that the ligand dissociates with a similar kinetic energy in both cases, suggesting fast intramolecular energy redistribution before dissociation.

The two-dimensional IR nonlinear spectroscopy of a cyclic penta-peptide in relation to its three-dimensional structure.

A form of two-dimensional (2D) vibrational spectroscopy, which uses two ultrafast IR laser pulses, is used to examine the structure of a cyclic penta-peptide in solution. Spectrally resolved cross peaks occur in the off-diagonal region of the 2D IR spectrum of the amide I region, analogous to those in 2D NMR spectroscopy. These cross peaks measure the coupling between the different amide groups in the structure. Their intensities and polarizations relate directly to the three-dimensional structure of the peptide. With the help of a model coupling Hamiltonian, supplemented by density functional calculations, the spectra of this penta-peptide can be regenerated from the known solution phase structure. This 2D-IR measurement, with an intrinsic time resolution of less than 1 ps, could be used in all time regimes of interest in biology.

Expression and intracellular localisation of odorant receptors in mammalian cell lines using Semliki Forest virus vectors.

Odorant receptors are members of the G protein-coupled receptor superfamily. They are expressed on the surface of cilia of olfactory neurons, where they bind ligand (odorant). Studies of the molecular mechanisms of olfaction are complicated by the extremely large number of receptor genes, and difficulties in pairing a particular mammalian receptor to a specific odorant ligand in vivo. Here we report expression and localisation studies of two rat odorant receptor genes (17 and OR5), and C. elegans odr-10, using the Semliki Forest virus (SFV) system. All receptors were epitope-tagged at the N- or C-terminus in order to facilitate their detection in infected cells, and determine the localisation and membrane-orientation of recombinant proteins. The immortalised mouse olfactory neuronal cell line OLF 442, rat cortical and striatal primary neuron cultures, and the baby hamster kidney (BHK) cells, were infected and tested. Immunofluorescence and confocal microscopy studies performed on permeabilised, non-permeabilised and native cells revealed that in BHK cells the rat receptors 17 and OR5 were not targeted to the plasma membrane and remained in the endoplasmic reticulum. In contrast, in the mouse olfactory cell line OLF 442 both rat receptors were correctly inserted into the plasma membrane. Similar results were obtained using primary neurons, indicating that like mature neurons, the immortalised OLF 442 cells are capable of providing for correct odorant receptor processing and targeting.

cDNA cloning and molecular characterization of human brain metalloprotease MP100: a beta-secretase candidate?

Metalloprotease MP100 was originally isolated as a beta-secretase candidate from human brain using a beta-amyloid precursor protein (beta-APP)-derived p-nitroanilide (pNA) peptide substrate. Peptide sequences from purified MP100 were now found to resemble sequences reported for a puromycin-sensitive aminopeptidase (PSA) highly enriched in brain, and cDNA cloning revealed nearly complete homology of MP100 to PSA, with only a single bp difference resulting in an amino acid change at position 184. Another MP100 cDNA encoded a protein with a 36-amino acid deletion (positions 180-217) and a two-amino acid insertion after Val533. Purified recombinant human MP100 cleaved the original pNA substrate as well as a free beta-site-spanning amyloid beta (A beta) peptide (A beta(-10/+10)), generating A beta(1-10). The latter substrate, however, remained uncleaved, if N- and C-terminally blocked, and also purified beta-APP was not cleaved. Double immunoimaging revealed partial, patchy, colocalization of beta-APP and MP100 in doubly transfected human embryonic kidney cells (HEK cells) and in normal neuroblastoma cells, and both proteins could be coimmunoprecipitated from rat brain extracts, suggesting their close vicinity in vivo. Coexpression of MP100 and beta-APP695, however, did not boost A beta levels in HEK cells, although active enzyme was produced. Thus, MP100 does not exert true beta-secretase-like function in cells, although it may well act as a secondary exoprotease in a complex beta-APP/A beta metabolism.

Peptidyl transferase center activity observed in single ribosomes.

We demonstrate the functional activity of single ribosomal complexes, opening the way for detailed studies of the trajectories of protein synthesis. Our approach employs a single-molecule detection system, capable of picoseconds to minutes resolution, to observe a growing peptide labeled at its N terminus with the fluorophore tetramethylrhodamine (TMR). Single complexes of mRNA-programmed ribosomes with TMR-Met-tRNAMetf or TMR-Met-Phe-tRNAPhe are immobilized on mica and observed by fluorescence. Immobilized ribosome.mRNA.TMR-Met-tRNAMetf complexes form peptide bonds with puromycin. Single-molecule detection reveals dynamics on the scale of seconds at the ribosomal peptidyl transferase center.

Protein fluctuations are sensed by stimulated infrared echoes of the vibrations of carbon monoxide and azide probes.

The correlation functions of the fluctuations of vibrational frequencies of azide ions and carbon monoxide in proteins are determined directly from stimulated photon echoes generated with femtosecond infrared pulses. The asymmetric stretching vibration of azide bound to carbonic anhydrase II exhibits a pronounced evolution of its vibrational frequency distribution on the time scale of a few picoseconds, which is attributed to modifications of the ligand structure through interactions with the nearby Thr-199. When azide is bound in hemoglobin, a more complex evolution of the protein structure is required to interchange the different ligand configurations, as evidenced by the much slower relaxation of the frequency distribution in this case. The time evolution of the distribution of frequencies of carbon monoxide bound in hemoglobin occurs on the approximately 10-ps time scale and is very nonexponential. The correlation functions of the frequency fluctuations determine the evolution of the protein structure local to the probe and the extent to which the probe can navigate those parts of the energy landscape where the structural configurations are able to modify the local potential energy function of the probe.

Fluorescence and photobleaching dynamics of single light-harvesting complexes.

Single light-harvesting complexes LH-2 from Rhodopseudomonas acidophila were immobilized on various charged surfaces under physiological conditions. Polarized light experiments showed that the complexes were situated on the surface as nearly upright cylinders. Their fluorescence lifetimes and photobleaching properties were obtained by using a confocal fluorescence microscope with picosecond time resolution. Initially all molecules fluoresced with a lifetime of 1 +/- 0.2 ns, similar to the bulk value. The photobleaching of one bacteriochlorophyll molecule from the 18-member assembly caused the fluorescence to switch off completely, because of trapping of the mobile excitations by energy transfer. This process was linear in light intensity. On continued irradiation the fluorescence often reappeared, but all molecules did not show the same behavior. Some LH-2 complexes displayed a variation of their quantum yields that was attributed to photoinduced confinement of the excited states and thereby a diminution of the superradiance. Others showed much shorter lifetimes caused by excitation energy traps that are only approximately 3% efficient. On repeated excitation some molecules entered a noisy state where the fluorescence switched on and off with a correlation time of approximately 0.1 s. About 490 molecules were examined.

Nonexponential kinetics of a single tRNAPhe molecule under physiological conditions.

The fluorescence decay functions of individual, specifically labeled tRNAPhe molecules exhibit nonexponential character as a result of conformational dynamics occurring during the measurement on a single molecule. tRNAPhe conformational states that interchange much more slowly are evidenced by the distribution of lifetimes observed for many individual molecules. A structural model for the nonexponential decay indicates that the tRNAPhe-probe adduct fluctuates between two states, one of which provides conditions that quench the probe fluorescence.