Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

A series of non-nucleoside-based 2,4-dinitrophenyl (DNP) phosphoramidites have been prepared and used in the multiple labelling of oligonucleotides during solid-phase synthesis. The length of spacer arm between the DNP label and the oligonucleotide phosphate backbone, and the number of attached DNP groups have both been varied in order to determine the optimum conditions for anti-DNP antibody binding. Detection using enzyme-linked colorimetric techniques showed sensitivity equivalent to that obtainable using biotinylated oligonucleotides.     

An adaptive, object oriented strategy for base calling in DNA sequence analysis.

An algorithm has been developed for the determination of nucleotide sequence from data produced in fluorescence-based automated DNA sequencing instruments employing the four-color strategy. This algorithm takes advantage of object oriented programming techniques for modularity and extensibility. The algorithm is adaptive in that data sets from a wide variety of instruments and sequencing conditions can be used with good results. Confidence values are provided on the base calls as an estimate of accuracy. The algorithm iteratively employs confidence determinations from several different modules, each of which examines a different feature of the data for accurate peak identification. Modules within this system can be added or removed for increased performance or for application to a different task. In comparisons with commercial software, the algorithm performed well.     

The 11p15.5 ribonucleotide reductase M1 subunit locus is not imprinted in Wilms' tumour and hepatoblastoma

We have utilized the polymerase chain reaction (PCR) to amplify a transcribed Taq1 polymorphism in the ribonucleotide reductase M1 subunit (RRM1) gene at chromosome 11p15.5, to investigate whether this locus is subjected to imprinting in embryonal tumours. The polymorphism was amplified from cDNA from 6 Wilms' tumours, one hepatoblastoma and corresponding samples of adjacent kidney or liver from individuals who were constitutionally heterozygous for the polymorphism. Taq1 digestion of PCR products revealed that both alleles were transcribed in all samples where both were present at the genomic level, indicating that the RRM1 locus is not subjected to imprinting in Wilms' tumour or hepatoblastoma.     

Cover crops do not increase soil organic carbon stocks as much as has been claimed: What is the way forward?

When compared to virgin land (forest and grassland), croplands store significantly lower amounts of organic carbon (OC), mainly as a result of soil tillage, and decreased plant inputs to the soil over the whole year. Doubts have been expressed over how much reduced and zero tillage agriculture can increase OC in soils when the whole soil profile is considered. Consequently, cover-crops that are grown in-between crops instead of leaving soils bare appear as the “last man standing” in our quest to enhance cropland OC stocks. Despite the claim by numerous meta-analyses of a mean carbon sequestration rate by cover crops to be as high as 0.32 ± 0.08 ton C ha⁻¹ year⁻¹, the present analysis showed that all of the 37 existing field studies worldwide only sampled to a depth of 30 cm or less and did not compare treatments on the basis of equivalent soil mass. Thirteen studies presented information on OC content only and not on OC stocks, had inappropriate controls (n = 14), had durations of 3 years or lower (n = 5), considered only one to two data points per treatment (n = 4), or used cover crops as cash crops (i.e., grown longer that in-between two crops) instead of catch crops (n = 2), which in all cases constitutes shortcomings. Of the remaining six trials, four showed non-significant trends, one study displayed a negative impact of cover crops, and one study displayed a positive impact, resulting in a mean OC storage of 0.03 ton ha⁻¹ year⁻¹. Models and policies should urgently adapt to such new figure. Finally, more is to be done not only to improve the design of cover-crop studies for reaching sound conclusions but also to understand the underlying reasons of the low efficiency of cover crops for improved carbon sequestration into soils, with possible strategies being suggested.     

Phylogeny of the insulin-like growth factors (IGFS) and receptors: A molecular approach

The IGFs (IGF-I and IGF-II) are essential for normal mammalian growth and development. Their actions are mediated primarily by their interactions with the type I IGF receptor (IGF-I receptor), a transmembrane tyrosine kinase. The ligands and the IGF-I receptor are structurally related to insulin and to the insulin receptor, respectively. Analysis of evolutionary conservation has often provided insights into essential regions of molecules such as hormones and their receptors. The genes for insulin and IGFs have been partially characterized in a number of vertebrate species extending evolutionarily from humans as far back as fish. The sequences of the exons encoding the mature insulin and IGF peptides are highly conserved among vertebrate species, and IGF-I-Iike molecules are found in species whose origins extend back as much as 550 million years. The insulin receptor is also highly conserved in vertebrate species, and an insulinreceptor-like molecule has been characterized in Drosophila. In contrast, IGF-I receptors have only been characterized in mammalian species and partially studied in Xenopus, in which the tyrosine kinase domain is highly conserved. Studies are presently being undertaken to analyze in more detail the regulation of the genes encoding this important family of growth factors and the structure/function relationships in the gene products themselves. © 1993 Wiley-Liss, Inc.     

Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

The influence of GABA on cells in the gustatory region of the hamster solitary nucleus

A brainstem slice preparation was used to investigate GABA-inducedresponses in the gustatory region of the nucleus of the solitarytract (NST) of the hamster. The baseline activities of 91 cellsin the rostral NST were examined extracellularly; 59 cells werelocated in the rostral central (RC), 21 in the rostral lateral(RL), six in the ventral (V) and five in the medial (M) subdivisionof the NST. Of the 80 cells in the gustatory region of the NST(RC and RL subdivisions), application of GABA produced dose-dependentinhibition in 55 (69%), excitation in 9 (11%) and no effectin 16 cells (22%). In contrast, only nine cells were responsiveto baclofen, a GABA^B agonist. In all subdivision of the rostralNST, 57 cells were inhibited by GABA and the responses of 48of these were blocked by the specific GABA^A antagonist, bicucullinemethiodide (BICM). Application of BICM alone often yielded anexcitatory burst of impulses; this effect was eliminated whensynaptic release was blocked by perfusion with a high magnesiumphysiological saline solution (PSS/Mg⁺⁺). The GABA^A-responsivecells were distributed predominantly within the RC subdivision,whereas the GABA^B-responsive neurons were mostly in the RL subdivisionof the NST. The influences of GABA on the membrane properties of cells withinthe gustatory region (RC and RL subdivisions) of the NST wererecorded using conventional intracellular (16 cells) or whole-cellpatch (17 cells) recording methods. Intracellular recordingrevealed that GABA produced hyperpolarisation of the membrane,decreased the firing frequency, and increased the membrane conductance.In the patch-clamp experiments, the application of GABA evokedboth inward and outward currents, and an increase in membraneconductance. The reversal potential produced by GABA was closeto the Cl– equilibrium potential. The effects of GABAwere blocked by BICM. These results suggest that (i) GABA hasa strong inhibitory influence on rostral NST neurons, whichin the majority of cells is mediated through GABA^A, receptors;and (ii) the gustatory region of the NST may contain a tonicallyactive GABAergic netw     

Circadian rhythms and glial cells of the central nervous system

Glial cells are the most abundant cells in the central nervous system and play crucial roles in neural development, homeostasis, immunity, and conductivity. Over the past few decades, glial cell activity in mammals has been linked to circadian rhythms, the 24-h chronobiological clocks that regulate many physiological processes. Indeed, glial cells rhythmically express clock genes that cell-autonomously regulate glial function. In addition, recent findings in rodents have revealed that disruption of the glial molecular clock could impact the entire organism. In this review, we discuss the impact of circadian rhythms on the function of the three major glial cell types – astrocytes, microglia, and oligodendrocytes – across different locations within the central nervous system. We also review recent evidence uncovering the impact of glial cells on the body's circadian rhythm. Together, this sheds new light on the involvement of glial clock machinery in various diseases.     

Bifidobacterium longum supplementation improves age-related delays in fracture repair

Age-related delays in bone repair remains an important clinical issue that can prolong pain and suffering. It is now well established that inflammation increases with aging and that this exacerbated inflammatory response can influence skeletal regeneration. Recently, simple dietary supplementation with beneficial probiotic bacteria has been shown to influence fracture repair in young mice. However, the contribution of the gut microbiota to age-related impairments in fracture healing remains unknown. Here, we sought to determine whether supplementation with a single beneficial probiotic species, Bifidobacterium longum (B. longum), would promote fracture repair in aged (18-month-old) female mice. We found that B. longum supplementation accelerated bony callus formation which improved mechanical properties of the fractured limb. We attribute these pro-regenerative effects of B. longum to preservation of intestinal barrier, dampened systemic inflammation, and maintenance of the microbiota community structure. Moreover, B. longum attenuated many of the fracture-induced systemic pathologies. Our study provides evidence that targeting the gut microbiota using simple dietary approaches can improve fracture healing outcomes and minimize systemic pathologies in the context of aging.