Quantitative phase measurements of tendon collagen fibres

Collagen is the main component of structural mammalian tissues. In tendons, collagen is arranged into fibrils with diameters ranging from 30 nm to 500 nm. These fibrils are further assembled into fibres several micrometers in diameter. Upon excessive thermal or mechanical stress, damage may occur in tendons at all levels of the structural hierarchy. At the fibril level, reported damage includes swelling and the appearance of discrete sites of plastic deformation that are best observed at the nanometer‐scale using, for example, scanning electron microscopy. In this paper, digital in‐line holographic microscopy is used for quantitative phase imaging to measure both the refractive index and diameter of collagen fibres in a water suspension in the native state, after thermal treatments, and after mechanical overload. Fibres extracted from tendons and subsequently exposed to 70 °C for 5, 15, or 30 minutes show a significant decrease in refractive index and an increase in diameter. A significant increase in refractive index is also observed for fibres extracted from tendons that were subjected to five tensile overload cycles.

     

Reaction rates of oxygen with hemoglobin measured by non-equilibrium facilitated oxygen diffusion through hemoglobin solutions

The purpose of this study was to verify the concept of non-equilibrium facilitated oxygen diffusion. This work succeeds our previous study, where facilitated oxygen diffusion by hemoglobin was measured at conditions of chemical equilibrium, and which yielded diffusion coefficients of hemoglobin and of oxygen. In the present work chemical non-equilibrium was induced using very thin diffusion layers. As a result, facilitation was decreased as predicted by theory. Thus, this work presents the first experimental demonstration of non-equilibrium facilitated oxygen diffusion. In addition, association and dissociation rate parameters of the reaction between oxygen and bovine and human hemoglobin were calculated and the effect of the homotropic and heterotropic interactions on each rate parameter was demonstrated. The results indicate that the homotropic interaction--which leads to increasing oxygen affinity with increasing oxygenation--is predominantly due to an increase in the association rate. The heterotropic interaction--which leads to decreasing oxygen affinity by anionic ligands--appears to be effected in two ways. Cl- increases the dissociation rate. In contrast, 2,3-diphosphoglycerate decreases the association rate.     

Digestive and metabolic utilization of lauric, myristic and stearic acid in cows, and associated effects on milk fat quality

In an experiment with 3 x 6 Brown Swiss cows, the effects of dietary supplementations (40 g/kg) of non-esterified lauric (12 : 0), myristic (14 : 0) and stearic acid (18 : 0) on digestibility, metabolisability, milk fat composition and melting properties were investigated. The diet consisted of forage and concentrate in a ratio of 3 : 2. Cows were fed the C18 : 0 supplemented diet for 10 days before treatment feeding started for a 15-day experimental period where, at the end, excreta were quantitatively collected and gaseous exchange was measured. The DM intake averaged 17.9 kg/d for the C14 : 0 and C18 : 0 diets and was reduced (P < 0.05) by 18% in the C12 : 0 diet. The realised intakes of total C12 : 0, C14 :0 and C18 : 0 amounted to 368, 391 and 617 g/d in the respective groups. The efficiency of ME utilization for lactation was higher (P < 0.001) in the C12 : 0 group than in the two other groups indicating differences in metabolism of C12 : 0 in comparison with C14 : 0 and C18 : 0. Shifts in dietary fatty acid supplementation were clearly reflected in the milk fat composition. Associated changes were elevated CLA and C18 : 1 trans when supplementing C12 : 0, and a high C18 : 1 to C16 : 0 ratio (P < 0.05) in the C12 : 0and C18 : 0 groups which resulted in an easier melting milk fat than with supplementary C14 : 0. Despite certain favourable effects of C12 : 0 in metabolic energy utilization and milk fat melting properties (relative to C14 : 0), more research is needed on how to improve its palatability for dairy cows.     

Silages containing buckwheat and chicory: quality, digestibility and nitrogen utilisation by lactating cows

The suitability of silages containing buckwheat (Fagopyrum esculentum) and chicory (Cichorium intybus) for the nutrition of dairy cows was determined. Buckwheat and chicory were sown in mixture with ryegrass (Lolium multilorum), and a pure ryegrass culture served as a control forage. Swards were harvested 55 d after sowing and were ensiled after wilting, without additives in small round bales. Finally, buckwheat and chicory made up the dietary dry matter (DM) proportions of 0.46 and 0.34, respectively. Concentrates were restricted to 2 kg/d. Diets were fed to 3 x 6 late-lactating cows for 15 d at ad libitum access. During the collection period (days 10-15) amounts of feed intake and faeces, urine and milk were recorded and samples were taken. Ensilability was good for buckwheat and ryegrass swards, but was so less for the chicory sward, which was rich in total ash. The buckwheat silage was rich in acid detergent fibre (445 g/kg DM) and lignin (75.7 g/kg DM) and contained less crude protein (135 g/kg DM) and ether extract (15.8 g/kg DM) than the other silages. Consistent with that, the apparent digestibility of the organic matter and fibre were lowest when feeding this silage. The potassium concentrations in the chicory and ryegrass silages were high (61 g/ kg) and lower in buckwheat (47 g/kg). No significant treatment effects on intake, body weight, milk yield or milk composition as well as plasma beta-hydroxybutyrate and non-esterified fatty acids occurred. Being lowest in nitrogen (N) content, the buckwheat silage resulted in the lowest urine N losses and the most efficient N utilisation for milk protein synthesis, but this at cost of body N retention. The results show that silages containing buckwheat and chicory may be used as components of the forage part of dairy cows' diets even though they were found to have a lower feeding value than ryegrass silage.     

The role of the rumen ciliate protozoa for methane suppression caused by coconut oil

Diets containing either coconut oil or rumen-protected fat (54 g kg⁻¹ dry matter each) were supplied to Rumen Simulation Technique fermenters filled with faunated and defaunated rumen fluid in a 2 × 2 factorial design. Defaunation immediately reduced methane formation by about 40% with each diet. With coconut oil, methane gradually declined in faunated and defaunated rumen fluid. Finally, the extent of methane suppression was similar, both with coconut oil and with defaunation. Independently of the status of protozoa, the population of methanogens in rumen fluid was significantly reduced by coconut oil. The results suggest that defaunation and coconut oil independently and additively suppress rumen methanogenesis.     

Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded breaks in vivo

Antibacterial quinolones inhibit type II DNA topoisomerases by stabilizing covalent topoisomerase-DNA cleavage complexes, which are apparently transformed into double-stranded breaks by cellular processes such as replication. We used plasmid pBR322 and two-dimensional agarose gel electrophoresis to examine the collision of replication forks with quinolone-induced gyrase-DNA cleavage complexes in Escherichia coli. Restriction endonuclease-digested DNA exhibited a bubble arc with discrete spots, indicating that replication forks had been stalled. The most prominent spot depended upon the strong gyrase binding site of pBR322, providing direct evidence that quinolone-induced cleavage complexes block bacterial replication forks in vivo. We differentiated between stalled forks that do or do not contain bound cleavage complex by extracting DNA under different conditions. Resealing conditions allow gyrase to efficiently reseal the transient breaks within cleavage complexes, while cleavage conditions cause the latent breaks to be revealed. These experiments showed that some stalled forks did not contain a cleavage complex, implying that gyrase had dissociated in vivo and yet the fork had not restarted at the time of DNA isolation. Additionally, some branched plasmid DNA isolated under resealing conditions nonetheless contained broken DNA ends. We discuss a model for the creation of double-stranded breaks by an indirect mechanism after quinolone treatment.     

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain

Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn²⁺ cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn²⁺ is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision.     

Presence of a plasma membrane targeting sequence in the amino-terminal region of the rat somatostatin receptor 3

Although peptide hormone receptors commonly exert their actions at the plasma membrane the cellular mechanisms that route the receptor proteins to the cell surface during biosynthesis are not well characterized. Here we report on the identification of a plasma membrane targeting sequence of rat somatostatin receptor subtype 3. While type 3 somatostatin receptors are present almost exclusively at the cell surface, type 1 receptors localize in addition largely in intracellular vesicular compartments. Chimeric receptors were constructed between rat somatostatin receptors 3 and 1. They were tagged by recombinant DNA techniques with a herpes simplex virus glycoprotein D epitope at the carboxyl-termini to facilitate their detection using fluorescence microscopic methods. Following transfection of the constructs in human embryonic kidney and rat insulinoma cells the chimeric receptors were analyzed by indirect immunofluorescence using anti-epitope monoclonal antibody and confocal laser scanning microscopy. The results demonstrate that the amino-terminal domain of somatostatin receptor 3 suffices to guide chimeric receptors to the cell surface. In marked contrast, chimeric receptors that lack this sequence but contain instead the amino-terminus of somatostatin type 1 receptor localize in an intracellular vesicular compartment.     

Genetic Analysis of Regulatory Mutants of Alkaline Phosphatase of E. COLI

A fine structure map of the phoR region of E. coli , mutations of which affect the rate of alkaline phosphatase synthesis, was constructed by Hfr x F^- crosses. Mutations causing three different phenotypes (previously reported as phoRa, phoRb, phoRc (Garen and Echols 1962a, b)) are clustered in three closely linked genetic loci. PhoR mutants of all three types, including the phoRb type not previously tested, are recessive to wild-type phoR⁺. In addition, phoRa and phoRc complement each other, while phoRa and phoRb do not. Our results support the hypothesis of Morris et al. (1974) that phoRc mutants represent a cistron (phoB) different from phoR.