The Glutamatergic Neurons in the Spinal Cord of the Sea Lamprey: An In Situ Hybridization and Immunohistochemical Study

Glutamate is the main excitatory neurotransmitter involved in spinal cord circuits in vertebrates, but in most groups the distribution of glutamatergic spinal neurons is still unknown. Lampreys have been extensively used as a model to investigate the neuronal circuits underlying locomotion. Glutamatergic circuits have been characterized on the basis of the excitatory responses elicited in postsynaptic neurons. However, the presence of glutamatergic neurochemical markers in spinal neurons has not been investigated. In this study, we report for the first time the expression of a vesicular glutamate transporter (VGLUT) in the spinal cord of the sea lamprey. We also study the distribution of glutamate in perikarya and fibers. The largest glutamatergic neurons found were the dorsal cells and caudal giant cells. Two additional VGLUT-positive gray matter populations, one dorsomedial consisting of small cells and another one lateral consisting of small and large cells were observed. Some cerebrospinal fluid-contacting cells also expressed VGLUT. In the white matter, some edge cells and some cells associated with giant axons (Müller and Mauthner axons) and the dorsolateral funiculus expressed VGLUT. Large lateral cells and the cells associated with reticulospinal axons are in a key position to receive descending inputs involved in the control of locomotion. We also compared the distribution of glutamate immunoreactivity with that of γ-aminobutyric acid (GABA) and glycine. Colocalization of glutamate and GABA or glycine was observed in some small spinal cells. These results confirm the glutamatergic nature of various neuronal populations, and reveal new small-celled glutamatergic populations, predicting that some glutamatergic neurons would exert complex actions on postsynaptic neurons.     

Mendelian randomization supports causality between maternal hyperglycemia and epigenetic regulation of leptin gene in newborns

Leptin is an adipokine that acts in the central nervous system and regulates energy balance. Animal models and human observational studies have suggested that leptin surge in the perinatal period has a critical role in programming long-term risk of obesity. In utero exposure to maternal hyperglycemia has been associated with increased risk of obesity later in life. Epigenetic mechanisms are suspected to be involved in fetal programming of long term metabolic diseases. We investigated whether DNA methylation levels near LEP locus mediate the relation between maternal glycemia and neonatal leptin levels using the 2-step epigenetic Mendelian randomization approach. We used data and samples from up to 485 mother-child dyads from Gen3G, a large prospective population-based cohort. First, we built a genetic risk score to capture maternal glycemia based on 10 known glycemic genetic variants (GRS₁₀) and showed it was an adequate instrumental variable (β = 0.046 mmol/L of maternal fasting glucose per additional risk allele; SE = 0.007; P = 7.8 × 10⁻¹¹; N = 467). A higher GRS₁₀ was associated with lower methylation levels at cg12083122 located near LEP (β = −0.072 unit per additional risk allele; SE = 0.04; P = 0.05; N = 166). Direction and effect size of association between the instrumental variable GRS₁₀ and methylation at cg12083122 were consistent with the negative association we observed using measured maternal glycemia. Lower DNA methylation levels at cg12083122 were associated with higher cord blood leptin levels (β = −0.17 log of cord blood leptin per unit; SE = 0.07; P = 0.01; N = 170). Our study supports that maternal glycemia is part of causal pathways influencing offspring leptin epigenetic regulation.     

Interleukin-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates interleukin-17A and RORγt in γδ T cells

Introduction  

Interleukin (IL)-23 is essential for the development of various experimental autoimmune models. However, the role of IL-23 in non-autoimmune experimental arthritis remains unclear. Here, we examined the role of IL-23 in the non-autoimmune antigen-induced arthritis (AIA) model. In addition, the regulatory potential of IL-23 in IL-17A and retinoic acid-related orphan receptor gamma t (RORγt) expression in CD4⁺ and TCRγδ⁺ T cells was evaluated systemically as well as at the site of inflammation.     

New derivatives of the Streptomyces temperate phage phi C31 useful for the cloning and functional analysis of Streptomyces DNA

The thiostrepton resistance gene (tsr) of Streptomyces azureus, and a synthetic oligonucleotide adapter sequence, were introduced into the DNA of attP-site-deleted phage phi C31-based cloning vectors. The DNA of two of the new derivatives, KC515 and KC516, contains single sites for the enzymes BamHI, BglII, PstI, PvuII, SstI (two sites close together) and XhoI, available for the insertion of DNA of up to 4 kb. The two vectors also contain a cloned, promoterless viomycin phosphotransferase gene (vph) from Streptomyces vinaceus. When an internal segment of the Streptomyces coelicolor glycerol (gyl) operon was inserted at the appropriate position and in the correct orientation next to vph, it could bring about in vivo recombination leading to fusion of vph of the chromosomally located gyl operon, resulting in glycerol-regulated expression of viomycin resistance. Two other new phi C31 derivatives, KC505 and KC518, are PstI and BamHI replacement vectors, respectively, for 2-8-kb DNA fragments, and allow simple screening for the presence of inserted DNA.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Cell cycle -dependent proteolysis in plants. Identification Of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor mg132

It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable.     

Role of calcium in signal transduction during the hypersensitive response caused by basidiospore-derived infection of the cowpea rust fungus

The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea-cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection.     

Identification and characterisation of seed storage protein transcripts from Lupinus angustifolius

Background  

In legumes, seed storage proteins are important for the developing seedling and are an important source of protein for humans and animals. Lupinus angustifolius (L.), also known as narrow-leaf lupin (NLL) is a grain legume crop that is gaining recognition as a potential human health food as the grain is high in protein and dietary fibre, gluten-free and low in fat and starch.