Dietary β-alanine enhances brain, but not muscle, carnosine and anserine concentrations in broilers

Carnosine (β‐alanyl‐L‐histidine) and its derivative anserine (β‐alanyl‐1‐methyl‐L‐histidine) are present in high concentrations in the muscle and brain of chickens. They are known as antioxidants and putative neurotransmitters in the brain. If administration of β‐alanine (β‐Ala), one of the constituents of carnosine, could increase the concentrations of these dipeptides in the brain and muscles, it could improve brain function and increase the commercial value of the meat in chickens. As an early step in investigating this hypothesis, in the present study, the effect of dietary β‐Ala on these dipeptide concentrations in the brain, Musculus pectoralis superficialis, Musculus pectoralis profundus and Musculus biceps femoris was investigated in broilers. Four‐week‐old broilers were given a commercial diet or diet containing 0.5, 1 or 2%β‐Ala for 4 weeks. At the end of the experiment, concentrations of both dipeptides were increased in the brain, while taurine concentration was decreased. In the muscles, concentrations of these dipeptides were unchanged. These results indicate that dietary β‐Ala might influence brain function, but is ineffective in increasing the concentrations of carnosine and anserine in the muscles of broilers.     

Changes in walking parameters of milking cows after hoof trimming

We examined the walking parameters of milking cows in relation to hoof trimming. The hooves of eight Holstein cows were trimmed. Two days before and after the hoof trimming, walking behavior, limb angles, back posture and vertical movement of the back while walking were measured. Walking rate (1.27 vs 0.87 m/s; P = 0.003), step length (1.46 vs 1.33 m; P = 0.009) and stepping rate (0.78 vs 0.65 steps/s; P = 0.046) were found to increase significantly after hoof trimming. Limb angles at the start and end of the support phase changed significantly after hoof trimming, thus indicating an improvement in the ‘on tiptoe’ appearance. The parameters relating to backline indicated that the marked arched shape of the back diminished after hoof trimming. Before hoof trimming, the positions of the withers varied widely and irregularly while walking; however, it changed after hoof trimming as the sinusoid curve and the range of vertical movement of withers decreased significantly. Overall, it has been quantitatively indicated that walking characteristics are improved after hoof trimming. The parameters used in the present study are expected to be useful for monitoring the walking characteristics of cows.     

Optimal conditions for visualizing water-conducting pathways in a living tree by the dye injection method

To elucidate the water-conducting pathways in living trees by the dye injection method, suitable sample preparation procedures are needed. We evaluated quantitatively the properties and concentrations of three dyes (acid fuchsin, basic fuchsin and safranin) widely used for this purpose, and determined the optimal conditions required to avoid artifacts after dye injection into the sap stream of Pieris japonica D. Don. Among the dyes tested, an aqueous solution of acid fuchsin at a concentration of 0.1% or more was the most useful for delineating water movement. In non-transpiring stem segments, the vertical movement of acid fuchsin by capillarity and diffusion from the dye injection site was limited. However, acid fuchsin moved rapidly in the horizontal direction by capillarity and diffusion, and most xylem cells were stained within 2 h. A delay of more than 2 h between dye injection and examination of the tissues greatly reduces the precision of the method. Use of the dye injection method without appropriate, well-defined experimental procedures may give rise to misleading information about the functional water-conducting pathway in living trees.     

An in vitro screening assay to discover novel inhibitors of 4-coumarate:CoA ligase

4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) exists only in plants and plays an important role in the phenylpropanoid pathway. Identification of inhibitors targeting 4CL provides a novel approach for developing effective plant growth inhibitors (PGIs). The full-length gene of tobacco 4CL (Nt4CL1) was cloned and expressed in Escherichia coli Cast & Chalm. The recombinant 4CL protein was extracted and purified by several purification steps including gel-filtration and anion-exchange chromatography. 4CL activity assay was miniaturized and optimized using a 96-well microplate and a reader. Among 28 existing herbicides, propanil and swep strongly inhibited in vitro 4CL enzyme activity, and they were selected for further studies. The process of this assay can be developed into a high-throughput screening system of PGI targeting 4CL in the phenylpropanoid pathway.     

Verification of a phenotypic discrimination method for hybrid larch seedlings using DNA markers

The hybrid between Kuril larch (Larix gmelinii var. japonica) and Japanese larch (L. kaempferi) is an important afforestation tree species in Hokkaido, Japan, because of its rapid juvenile growth, straight stem, and resistance to bark gnawing by voles. To produce desirable hybrid seedlings, precise seedling discrimination is essential. However, continuous variations in morphological and phenological traits occur across L. gmelinii var. japonica × L. kaempferi and L. gmelinii var. japonica × L. gmelinii var. japonica seedlings. Therefore, we used DNA markers to verify and improve the morphological and phenological discrimination method. We collected seeds from an interspecific seed orchard and, using chloroplast DNA analysis, we showed that the hybridization rates of 1-year-old seedlings were different between years (2004, 23.2 %; 2005, 53.6 %) and between mother trees (2004, 5.8–39.4 %; 2005, 20.0–81.0 %). Discriminant analyses revealed that the root collar diameter of 2-year-old seedlings, number of sylleptic branches of 2-year-old seedlings, and day of terminal bud set in 1- and 2-year-old seedlings are traits that aid in discriminating hybrid seedlings. The proportions of correctly discriminated individuals were found to be 81.7–88.2 % when using improved phenotypic discrimination methods. These methods adopted traits with high discrimination abilities during discriminant analyses and determined the selection intensity according to the hybridization rate estimated from the DNA markers. In contrast, the proportions of correctly discriminated individuals obtained using the current method were lower, 72.7–78.5 %, because this method was based on seedling height and adopted a constant selection intensity.     

MicroRNA expression profiling of cat and dog kidneys

MicroRNAs (miRNAs) play a role in the pathogenesis of certain diseases and may serve as biomarkers. Here, we present the first analysis of miRNA expression in the kidneys of healthy cats and dogs. Kidneys were divided into renal cortex (CO) and medulla (MD), and RNA sequence analysis was performed using the mouse genome as a reference. A total of 277, 276, 295, and 297 miRNAs were detected in cat CO, cat MD, dog CO, and dog MD, respectively. By comparing the expression ratio of CO to MD, we identified highly expressed miRNAs in each tissue as follows: 41 miRNAs including miR-192-5p in cat CO; 45 miRNAs including miR-323-3p in dog CO; 78 miRNAs including miR-20a-5p in cat MD; and 11 miRNAs including miR-132-5p in dog MD. Further, the target mRNAs of these miRNAs were identified. These data provide veterinary medicine critical information regarding renal miRNA expression.     

Solvent tuning of electrochemical potentials in the active sites of HiPIP versus ferredoxin

A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fd) active sites. Despite this structural similarity, HiPIPs react oxidatively at physiological potentials, whereas Fds are reduced. Sulfur K-edge x-ray absorption spectroscopy uncovers the substantial influence of hydration on this variation in reactivity. Fe-S covalency is much lower in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HiPIP covalency decreases when unfolding exposes an otherwise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a correlation of Fe-S covalency with ease of oxidation and therefore suggest that hydration accounts for most of the difference between Fd and HiPIP reduction potentials.