Identification of lucerne stem cell wall traits related to in vitro neutral detergent fibre digestibility

Genetic improvement of forage digestibility, especially utilizing marker assisted selection and recombinant DNA techniques, requires identification of specific biochemical traits and associated genes that impact digestibility. We undertook a study to identify cell wall (CW) traits of lucerne (Medicago sativa L.) stems that were consistently and strongly correlated with in vitro neutral detergent fibre (NDF) digestibility, a measurement that has been shown to correlate with animal performance. Spring and summer harvested lucerne stem material, for 2 years, from 24 individual plants in each of two germplasm sources were analyzed for 16 and 96 h in vitro NDF digestibility, and cell wall concentration and composition (monosaccharide constituents of cellulose, hemicellulose, and pectin; and Klason lignin (KL)) by the Uppsala dietary fibre method using near-infrared reflectance spectroscopy (NIRS). Pearson correlation coefficients were calculated for the relationships among these cell wall traits and with in vitro NDF digestibility. Concentrations of the pectin monosaccharide components were all negatively correlated (r=−0.73 to −0.94) with total cell wall concentration. In contrast, the three most abundant cell wall components glucose (Glc), xylose (Xyl) and Klason lignin were not correlated, or only weakly positively correlated (r<0.35), with cell wall concentration. Cell wall concentration was consistently negatively correlated (r=−0.60 to −0.94) with both 16 and 96 h in vitro NDF digestibility. In contrast, Klason lignin concentration was only marginally correlated (r<0.30) with 16 h in vitro NDF digestibility, but strongly negatively correlated (r=−0.71 to −0.74) with 96 h in vitro NDF digestibility. This is consistent with previous reports which show that lignin affects potential extent of digestion, but not rate. Cell wall glucose and xylose concentrations were inconsistently correlated with fibre digestibility. The monosaccharide components of pectin were consistently positively correlated (r=0.54–0.90) with in vitro NDF digestibility, except for 96 h in vitro NDF digestibility of spring harvested stems. Growth environment (year) and germplasm source had only minor impacts on the preceding correlation patterns, whereas spring versus summer harvests accounted for the inconsistencies observed among correlations for cell wall traits. The results of this study indicate that genetic improvement of fibre digestibility of lucerne stems should target genes that reduce total cell wall concentration, perhaps by reducing the rate of xylem tissue deposition during maturation, and reduce Klason lignin and increase pectin concentrations in the cell wall to improve potential extent and rate of fibre digestibility, respectively.     

Toward functional glycomics by localization of tissue lectins: immunohistochemical galectin fingerprinting during diethylstilbestrol-induced kidney tumorigenesis in male Syrian hamster

The current study focused on galectins (-1, -3, -4, -7, and –8) and deliberately performed immunohistochemical fingerprinting to explore their complexity in a context of experimental renal carcinogenesis. The diethylstilbestrol (DES)-induced renal tumors in male Syrian hamster kidney (SHKT) represent a unique animal model for the study of estrogen-dependent renal malignancies. Kidney sections of DES-treated hamsters (3 days to 11 months of DES exposure) were analyzed by immunohistochemistry using a panel of non-crossreactive antibodies raised against galectins-1, -3, -4, -7, and -8. Levels of expression were quantitatively determined by using computer-assisted microscopy on immunostained tissue sections. Except for galectin-4, all above mentioned galectins were expressed in kidney tumors. Small clusters of galectin-1-positive, most likely preneoplastic cells at the corticomedullary junction were already evident 1 week after DES administration. Galectin-1 and -3 expression was apparently associated with the first steps of the neoplastic transformation, because small tumorous buds were found to be positive after 1 month of treatment. In contrast, galectins-7 and -8 were detected in large tumors and medium-sized tumors, respectively, thereby indicating an involvement in later stages of DES-induced SHKT. Galectins-1, -3, -7, and -8 were also detected by immunofluorescence staining in the HKT-1097 cell line established from SHKT, thus illustrating the stability of galectin expression in tumor cells. Our data document the presence and differential regulation of galectins in the course of renal tumorigenesis in the model of DES-induced SHKT.     

Pentabromopseudilin: a myosin V inhibitor suppresses TGF-β activity by recruiting the type II TGF-β receptor to lysosomal degradation

Pentabromopseudilin (PBrP) is a marine antibiotic isolated from the marine bacteria Pseudomonas bromoutilis and Alteromonas luteoviolaceus. PBrP exhibits antimicrobial, anti-tumour, and phytotoxic activities. In mammalian cells, PBrP is known to act as a reversible and allosteric inhibitor of myosin Va (MyoVa). In this study, we report that PBrP is a potent inhibitor of transforming growth factor-β (TGF-β) activity. PBrP inhibits TGF-β-stimulated Smad2/3 phosphorylation, plasminogen activator inhibitor-1 (PAI-1) protein production and blocks TGF-β-induced epithelial–mesenchymal transition in epithelial cells. PBrP inhibits TGF-β signalling by reducing the cell-surface expression of type II TGF-β receptor (TβRII) and promotes receptor degradation. Gene silencing approaches suggest that MyoVa plays a crucial role in PBrP-induced TβRII turnover and the subsequent reduction of TGF-β signalling. Because, TGF-β signalling is crucial in the regulation of diverse pathophysiological processes such as tissue fibrosis and cancer development, PBrP should be further explored for its therapeutic role in treating fibrotic diseases and cancer.     

Towards a phylogenetic framework for the evolution of shakes, rattles, and rolls in Myiarchus tyrant-flycatchers (Aves: Passeriformes: Tyrannidae)

A phylogeny of 19 of the 22 currently recognized species of Myiarchus tyrant-flycatchers is presented. It is based on 842bp of mitochondrial DNA (mtDNA) sequences from the ATPase subunit 8 and ATPase subunit 6 genes. Except for the morphologically distinct M. semirufus, mtDNAs of the remaining 18 species fall into either of two clades. One comprises predominantly Caribbean and Central and North American taxa (Clade I), and the other is of predominantly South American taxa (Clade II). The phylogeny is only very broadly concordant with some vocal characters and also with the limited morphological diversity for which the group is well known. Paraphyly in several species (M. swainsoni, M. tuberculifer, M. ferox, M. phaeocephalus, M. sagrae, M. stolidus) suggests that morphological evolution, albeit resulting in limited morphological diversity, has been more rapid than that of mtDNA, or that current taxonomy is faulty, or both. A South American origin for Myiarchus is likely. Dispersal and vicariance both appear to have been involved in generating the present-day distribution of some species. Relatively recent dispersal events out of South America are inferred to have brought species of Clades I and II into broad sympatry. Jamaica has been colonized independently at least twice by members of Clades I and II. The phylogeny brings a historical perspective that in turn suggests that ecological study of closely related species from within each major clade where they are sympatric will be especially rewarding.     

Non-classical monocytes contribute to innate immune training in cattle

Innate immune training is defined as a property of innate immune cells to react stronger to a secondary contact with pathogens. Induction of innate immune training has been reported for a variety of pathogens and selected pattern recognition receptor-ligands, such as β-glucans (βG). We examined whether Saccharomyces cerevisiae cell wall component βG induces training in bovine monocytes in vitro based on a heightened TNF secretion after stimulation by trained monocyte-derived macrophages with Escherichia coli LPS. Sorted CD14-expressing monocytes (classical and intermediate monocytes), as well as single populations of sorted classical, intermediate and non-classical monocytes could not be trained by βG, whereas macrophages derived from plastic-adherent mononuclear cell preparations displayed features of a trained function. The hypothesis, that non-classical monocytes need to be present in a mixed monocyte population in order to be trained by βG could be verified by a successful training of positively sorted whole monocyte populations (CD14CD16/M) containing all three monocyte subpopulations. The trainability depended on conditions favoring M1 polarization of macrophages. Altogether, innate immune training of bovine monocytes seems to depend on the presence of non-classical monocytes. This adds new information to the role of this monocyte subpopulation in the bovine immune system.     

Adaptation-dependent plasticity of rod bipolar cell axon terminal morphology in the rat retina

We chose synaptic terminals of rat rod bipolar cells as a model system to study activity-related changes in the overall morphology and the fine structure of synaptic sites. Using confocal laser scanning microscopy in conjunction with three-dimensional reconstruction and electron microscopy, we examined the effect of light and dark adaptation on axon terminals identified by protein kinase C (PKC) immunoreactivity. Rod bipolar cell axon terminals consisted of 2–3 polymorphic boutons situated close to the ganglion cell layer and a single ovoid swelling located more distally. Both components of the terminal complex showed adaptation-dependent differences in the distribution of PKC immunoreactivity and in their morphology. In light-adapted rod bipolar cell axon terminals, PKC immunoreactivity was homogeneously distributed throughout the cytoplasm, whereas terminals from dark-adapted animals showed PKC immunoreactivity preferentially localised in the submembrane compartment and a reduced staining of the more central cytoplasm. In three-dimensional reconstructions of optical sections and at the ultrastructural level, the shape of light-adapted axon terminals was round and smooth and exhibited more convexly curved synaptic membranes. In contrast, dark-adapted terminals had irregular contours, numerous dimples and a concave synaptic curvature. No spinules of bipolar cell terminals were observed in dark-adapted material. These observations are discussed in the context of activity-related morphological plasticity of central nervous system synapses and of the functions of PKC in the cycle of vesicle fusion and retrieval at the tonically active ribbon synapses of the rod bipolar axon terminal. Received: 9 April 1998 / Accepted: 23 June 1998     

Transition metal complexes IX. Nickel complexes with a chiral azaphosphole ligand

The reaction of (−)-(R)-myrtenal and (+)-(R)-phenylethylamine gave a Schiff base 1 which was reacted with MePBr₂ in the presence of a base to give under dehydrohalogenation of an intermediate McCormack product a salt 2. Treatment of 2 with sodium led to the formation of the azaphosphole 4. η³-C₃H₅NiCl and 4 gave a 1:1 adduct 5 and nickel(0) gave a 1:4 complex 6. Compounds 4–6 were characterized by NMR spectroscopy as well as by single crystal X-ray structure determination.     

Optimizing Mouse Surgery with Online Rectal Temperature Monitoring and Preoperative Heat Supply. Effects on Post-Ischemic Acute Kidney Injury

Body temperature affects outcomes of tissue injury. We hypothesized that online body core temperature recording and selective interventions help to standardize peri-interventional temperature control and the reliability of outcomes in experimental renal ischemia reperfusion injury (IRI). We recorded core temperature in up to seven mice in parallel using a Thermes USB recorder and ret-3-iso rectal probes with three different protocols. Setup A: Heating pad during ischemia time; Setup B: Heating pad from incision to wound closure; Setup C: A ventilated heating chamber before surgery and during ischemia time with surgeries performed on a heating pad. Temperature profile recording displayed significant declines upon installing anesthesia. The profile of the baseline experimental setup A revealed that <1% of the temperature readings were within the target range of 36.5 to 38.5°C. Setup B and C increased the target range readings to 34.6 ± 28.0% and 99.3 ± 1.5%, respectively. Setup C significantly increased S3 tubular necrosis, neutrophil influx, and mRNA expression of kidney injury markers. In addition, using setup C different ischemia times generated a linear correlation with acute tubular necrosis parameters at a low variability, which further correlated with the degree of kidney atrophy 5 weeks after surgery. Changing temperature control setup A to C was equivalent to 10 minutes more ischemia time. We conclude that body temperature drops quickly in mice upon initiating anesthesia. Immediate heat supply, e.g. in a ventilated heating chamber, and online core temperature monitoring can help to standardize and optimize experimental outcomes.     

Hypersensitive lignification response as the mechanism of non-host resistance of wheat against oat crown rust

Seven-day-old seedlings of the near-isogenic wheat ( Triticum aestivum L.) lines Prelude and Prelude-Sr5, susceptible and resistant to wheat stem rust, respectively, were inoculated with uredospores of the oat crown rust fungus Puccinia coronata Cda. f. sp. avenae Fraser & Led. Fluorescence microscopy revealed that the majority of colonies developed intercellular infection structures including haustorial mother cells and haustoria after penetration of wheat mesophyll cells. All penetrated cells became necrotic, and exhibited bright yellow autofluorescence. This autofluorescence was not extractable with alkali, and fluorescent cells stained positively with phloroglucinol/HCI, suggesting that hypersensitive cell death was correlated with cellular lignification. Accordingly, the lignin biosynthetic enzymes phenylalanine ammonia-lyase (EC4.3.1.5). 4-coumarate:CoA ligase (EC6.2.1.12), cinnamyl-alcohol dehydrogenase (EC1.1.1.149), and peroxidases (EC1.11.1.7) increased in activity during the expression of resistance. The induced pattern of peroxidase iso/ymes closely resembled that observed for highly incompatible wheat/wheat stem rust interactions. Furthermore, an elieitor was extracted from oat crown rust germlings. which induces lignification when injected into the intercellular space of wheat leaves. This elieitor appears to be functionally similar to that isolated from wheat stem rust germlings. The results suggest that the non-host resistance of wheat to the xenopara-site oat crown rust closely resembles the race/cullivar-speeific resistant mechanism of highly resistant wheat varieties to wheat stem rust.