Plexin-B1/RhoGEF-mediated RhoA activation involves the receptor tyrosine kinase ErbB-2

Plexins are widely expressed transmembrane proteins that mediate the effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still rather unclear. Plexin-B1 has recently been shown to mediate activation of RhoA through a stable interaction with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. However, it is unclear how the activity of plexin-B1 and its downstream effectors is regulated by its ligand Sema4D. Here, we show that plexin-B family members stably associate with the receptor tyrosine kinase ErbB-2. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. A dominant-negative form of ErbB-2 blocks Sema4D-induced RhoA activation as well as axonal growth cone collapse in primary hippocampal neurons. Our data indicate that ErbB-2 is an important component of the plexin-B receptor system and that ErbB-2-mediated phosphorylation of plexin-B1 is critically involved in Sema4D-induced RhoA activation, which underlies cellular phenomena downstream of plexin-B1, including axonal growth cone collapse.     

PCR detection of uncultured rumen bacteria

16S rRNA sequences of ruminal uncultured bacterial clones from public databases were phylogenetically examined. The sequences were found to form two unique clusters not affiliated with any known bacterial species: cluster of unidentified sequences of free floating rumen fluid uncultured bacteria (FUB) and cluster of unidentified sequences of bacteria associated with rumen epithelium (AUB). A set of PCR primers targeting 16S rRNA of ruminal free uncultured bacteria and rumen epithelium adhering uncultured bacteria was designed based on these sequences. FUB primers were used for relative quantification of uncultured bacteria in ovine rumen samples. The effort to increase the population size of FUB group has been successful in sulfate reducing broth and culture media supplied with cellulose.     

Long-Term Consumption of High-Fat Diet in Rats: Effects on Microglial and Astrocytic Morphology and Neuronal Nitric Oxide Synthase Expression

Obesity in humans is associated with cognitive decline and elevated risk of neurodegenerative diseases of old age. Variations of high-fat diet are often used to model these effects in animal studies. However, we previously reported improvements in markers of memory and learning, as well as larger hippocampi and higher metabolite concentrations in Wistar rats fed high-fat, high-carbohydrate diet (HFCD, 60 % energy from fat, 28 % from carbohydrates) for 1 year; this diet leads to mild ketonemia (Setkowicz et al. in PLoS One 10:e0139987, 2015). In the present study, we follow up on this cohort to assess glial morphology and expression of markers related to gliosis. Twenty-five male Wistar rats were kept on HFCD and twenty-five on normal chow. At 12 months of age, the animals were sacrificed and processed for immunohistochemical staining for astrocytic (glial fibrillary acidic protein), microglial (Iba1), and neuronal (neuronal nitric oxide synthetase, nNOS) markers in the hippocampus. We have found changes in immunopositive area fraction and cellular complexity, as studied by a simplified Sholl procedure. To our knowledge, this study is the first to apply this methodology to the study of glial cells in HFCD animals. GFAP and Iba1 immunoreactive area fraction in the hippocampi of HFCD-fed rats were decreased, while the mean number of intersections (an indirect measure of cell complexity) was decreased in GFAP-positive astrocytes, but not in Iba1-expressing microglia. At the same time, nNOS expression was lowered after HFCD in both the cortex and the hippocampus.     

The composition of the autumn and winter diets in two Polish populations of fallow deer

The diets of fallow deerDama dama (Linnaeus, 1758) were analysed on the basis of the rumen contents of 69 individuals harvested between October and January in two areas of Poland: Iława, where animals are fed artificially in winter and Pszczyna, where almost no winter feeding is applied. The composition of the diet was found to be similar in both areas, with graminoids — as the staple food — accounting for 43.1% in Iława and 45.9% in Pszczyna. Further similarities between the areas were noted in regard to the other main foods eaten, ie the foliage of broadleaved deciduous trees and Scots pine, as well as forbs. Together, these four categories of food accounted for 73.1% and 73.0% of the diet in Iława and Pszczyna, respectively. Browse and bark were eaten in very small amounts, an observation suggesting that the contribution made by fallow deer to overall cervid-induced damage at least in some Polish forests is relatively small. Sample size limitations confined analysis of monthly food composition to the data from Iława only. These revealed graminoids as the major food each month, though shares of the diet ranged between 56.4% in November and 30.3% in January. Maximum levels of consumption of broadleaved deciduous foliage (21.9%) and forbs (10.3%) were recorded in October, and afterwards decreased. In turn, the role of pine foliage share increased from October (7.8%) through January (14.5%). The maximum availability of artificial forage occurred in January and its share in the diet was then as high as 21.2%.     

On the phylogenetic position of the palaeopteran Syntonopteroidea (Insecta: Ephemeroptera), with a new species from the Upper Carboniferous of England

A new syntonopterid, Anglolithoneura magnifica gen. et sp. n., is described from a siderite concretion (nodule) from the Late Carboniferous (Langsettian) of Lancashire County (UK). The new genus is diagnosed on hind wing venation and compared with other syntonopterid genera. The new species is the first syntonopterid formally described from the Late Carboniferous of Europe. The systematic positions of other potential Syntonopteroidea (Miracopteron mirabile, Bojophlebia prokopi, and specimens described in 1985 by J. Kukalová-Peck from Obora in the Czech Republic) are reconsidered. Wing venation synapomorphies are proposed for the Syntonopteroidea (sensu novo), and for a potential clade ((Ephemeroptera+Syntonopteroidea)+Odonatoptera) separated from the Palaeodictyopterida. The close relations of the new species with Lithoneura lameerei Carpenter, 1938 from Mazon Creek (Illinois, USA) provide additional support for a Euramerican connection during the Late Carboniferous.     

Mesenchymal stromal cells from donors varying widely in age are of equal cellular fitness after in vitro expansion under hypoxic conditions

Background aimsMesenchymal stromal cells (MSC) are gaining in popularity as an experimental therapy for a number of conditions that often require expansion ex vivo prior to use. Data comparing clinical-grade MSC from various ages of donors are scant. We hypothesized that MSC from older donors may display differences in cellular fitness when expanded for clinical use.MethodsWe evaluated the expression of several markers of aging, oxidative stress and growth kinetics, and telomere length, in MSC obtained from a wide age range (8 months to 58 years).ResultsTo evaluate cellular fitness we compared MSC expanded from younger (8 months–6 years) versus older (38–58 years) donors in terms of selected cell-surface markers, lipofuscin, migration ability, telomere length and expression of iNOS, PGE₂, p16INK and SOD. Results did not differ between these groups. Neither SOD activity (0.025 versus 0.028 U/mL) nor death after oxidative challenge was significantly different (1% versus 1.5%, P = 0.14). We did find that, although MSC from older individuals produced slightly fewer cells over a 28-day culture period and had a slightly longer doubling time (54 h versus 42 hr, a satisfactory clinical product could still be obtained regardless of age cohort.ConclusionsCollectively, these data show that MSC can be expanded without significant alterations in expansile properties or obvious changes in parameters associated with senescence. Because cellular fitness was equivalent in these cohorts, MSC from donors up to age 58 years can be used as a source of cells for cellular therapy.     

Cyclic GMP-dependent protein kinase and soluble guanylyl cyclase disappear in elicited rat neutrophils

The nitric oxide/soluble guanylyl cyclase/cGMP-dependent protein kinase (NO/sGC/PKG) cascade has been shown to affect important functions of circulating neutrophils. We demonstrate that neutrophils isolated from rats treated intraperitoneally with peptone protease cannot use this signaling pathway. Although PKG was detected at both the mRNA and protein levels in peripheral blood neutrophils (PBNs) of control rats, it was expressed neither in PBNs nor in peritoneal exudate neutrophils (PENs) of provoked rats. Also, mRNA of the alpha and beta chains of heterodimeric sGC was present in PBNs, but absent in PENs. Consistently, PBNs responded to activators of sGC with cGMP synthesis, while PENs did not. These results showed that neutrophils recruited by a provoking agent lost PKG and, in the case of PENs, also sGC and thus the capacity to respond to NO with cGMP signaling. We speculate that such downregulation of the sGC/PKG pathway is likely a result of the high activity of inducible NO synthase observed in inflammatory neutrophils.