NMR T1 relaxation time measurements and calculations with translational and rotational components for liquid electrolytes containing LiBF4 and propylene carbonate.

Longitudinal relaxation (T1) measurements of (19)F, (7)Li, and (1)H in propylene carbonate/LiBF4 liquid electrolytes are reported. Comparison of T1 values with those for the transverse relaxation time (T2) confirm that the measurements are in the high temperature (low correlation time) limit of the T1 minimum. Using data from pulsed field gradient measurements of self-diffusion coefficients and measurements of solution viscosity measured elsewhere, it is concluded that although in general there are contributions to T1 from both translational and rotational motions. For the lithium ions, this is mainly translational, and for the fluorine ions mainly rotational.

Two cytokine signaling molecules co-operate to promote axonal transport and growth.

The neuropoietic cytokines and their cytoplasmic signaling molecules contribute to axotomy-induced events in the nerve cell body that are beneficial to axonal regeneration. Previous studies have revealed a paradox in that, in vivo, suppressor of cytokine signaling (SOCS3) is induced in axotomized primary sensory neurons which are in a growth mode but, in vitro, SOCS3 strongly inhibits neurite growth from the same neurons. The present studies in cell lines with immuno-precipitation and western blotting, and Förstner resonance energy transfer showed that SOCS3 binds to the C terminus of C-Jun N-terminal kinase-interacting protein-1 (JIP1), increases its serine phosphorylation, and increases its binding to kinesin. Axonal transport was studied in vitro in adult rat primary sensory neurons by analyses of recovery of fluorescence after photobleaching and of the velocity and direction of movement of organelles. Over-expression of SOCS3 in addition to JIP1 had two consequences. First, recovery of fluorescence after photobleaching was more rapid and, second, JIP1-containing organelles moved more quickly and more frequently in retrograde direction. With respect to neurite outgrowth, SOCS3 alone was, as expected, strongly inhibitory but, in the presence of excess JIP1 augmented the stimulatory activity of the latter. The observations indicate that interactions between JIP1 and SOCS3 influence favorably axonal transport and growth in vitro.

Posttraumatic activity of signal pathways of nuclear factor κB in mature sensory neurons.

The aim of this study was to clear out whether injury to the peripheral nerve leads to activation of nuclear factor κB in mature spinal ganglia. Analysis of matrix RNA of nuclear factor κB-dependent genes (monocyte chemoattractant protein MCP-1 and inhibitor of nuclear factor κB IκBα) showed different levels of expression of these genes in the spinal ganglia in vivo after axotomy and in vitro after TNF-α stimulation. On the other hand, DNA-binding activity of nuclear factor κB increased in the spinal ganglia 6 h after axotomy and after 10-min incubation of sensory neuron culture with TNF-α. These data attest to possible involvement of nuclear factor κB in the posttraumatic regulation of gene transcription in spinal ganglion cells.

The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome.

Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.

Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth.

BACKGROUND: Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut. RESULTS: To further the understanding of genetic attenuation that may be occurring in bifidobacteria cultures, we obtained the complete genome sequence of an intestinal isolate, Bifidobacterium longum DJO10A that was minimally cultured in the laboratory, and compared it to that of a culture collection strain, B. longum NCC2705. This comparison revealed colinear genomes that exhibited high sequence identity, except for the presence of 17 unique DNA regions in strain DJO10A and six in strain NCC2705. While the majority of these unique regions encoded proteins of diverse function, eight from the DJO10A genome and one from NCC2705, encoded gene clusters predicted to be involved in diverse traits pertinent to the human intestinal environment, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production. Seven of these unique regions were suggested by a base deviation index analysis to have been precisely deleted from strain NCC2705 and this is substantiated by a DNA remnant from within one of the regions still remaining in the genome of NCC2705 at the same locus. This targeted loss of genomic regions was experimentally validated when growth of the intestinal B. longum in the laboratory for 1,000 generations resulted in two large deletions, one in a lantibiotic encoding region, analogous to a predicted deletion event for NCC2705. A simulated fecal growth study showed a significant reduced competitive ability of this deletion strain against Clostridium difficile and E. coli. The deleted region was between two IS30 elements which were experimentally demonstrated to be hyperactive within the genome. The other deleted region bordered a novel class of mobile elements, termed mobile integrase cassettes (MIC) substantiating the likely role of these elements in genome deletion events. CONCLUSION: Deletion of genomic regions, often facilitated by mobile elements, allows bifidobacteria to adapt to fermentation environments in a very rapid manner (2 genome deletions per 1,000 generations) and the concomitant loss of possible competitive abilities in the gut.

The Calyptogena magnifica chemoautotrophic symbiont genome.

Chemoautotrophic endosymbionts are the metabolic cornerstone of hydrothermal vent communities, providing invertebrate hosts with nearly all of their nutrition. The Calyptogena magnifica (Bivalvia: Vesicomyidae) symbiont, Candidatus Ruthia magnifica, is the first intracellular sulfur-oxidizing endosymbiont to have its genome sequenced, revealing a suite of metabolic capabilities. The genome encodes major chemoautotrophic pathways as well as pathways for biosynthesis of vitamins, cofactors, and all 20 amino acids required by the clam.

Nature of signals that initiate the immune response during Wallerian degeneration of peripheral nerves.

Monocyte chemoattractant protein-1 is produced by Schwann cells during Wallerian degeneration of a peripheral nerve and contributes to a selective accumulation of macrophages in the degenerating segment. An in vitro preparation has been developed to analyze the molecules from axons and non-neuronal cells in nerves that stimulate an increased production of monocyte chemoattractant protein-1 mRNA by Schwann cells. For this purpose, Schwann cells obtained from neonatal rats were maintained in culture, exposed to putative molecular stimuli and analyzed for their content of monocyte chemoattractant protein-1 mRNA. Under basal conditions, the concentration of monocyte chemoattractant protein-1 in Schwann cells was low. Freeze-killed fragments or homogenates of nerve (or brain) but not viable nerve or freeze-killed muscle were effective in inducing monocyte chemoattractant protein-1 mRNA. The inductive activity was abolished by heating. Results of dialysis of supernatants of nerve homogenates indicate that a protein or proteins of 1-10 kDa were capable of stimulating synthesis of monocyte chemoattractant protein-1 by Schwann cells. Also, the activity in nerve homogenates was partially inhibited by antibodies to Toll-like receptor-4. The observations suggest that a non-secreted protein is released from disintegrating axons to initiate the innate immune response that characterizes Wallerian degeneration.

The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies.

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, alpha-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7).

Presence of Ras guanyl nucleotide-releasing protein in striosomes of the mature and developing rat.

Ras signal transduction pathways have been implicated as key regulators in neuroplasticity and synaptic transmission in the brain. These pathways can be modulated by Ras guanyl nucleotide exchange factors, (GEF) which activate Ras proteins by catalysing the exchange of GDP for GTP. Ras guanyl nucleotide-releasing protein (RasGRP), a recently discovered Ras GEF, that links diacylglycerol and probably calcium to Ras signaling pathways, is expressed in brain as well as in T-cells. Here, we have used a highly selective monoclonal antibody against RasGRP to localize this protein within the striatum and related forebrain structures of developing and adult rats. RasGRP immunolabeling was found to be widespread in the mature and developing rat forebrain. Most notably, it presented a prominent patchy distribution throughout the striatum at birth and at all postnatal ages examined. These patches were found to correspond with the striosomal compartment of the striatum, as identified by micro-opioid receptor labeling in the adult. RasGRP-immunoreactivity was also observed in the matrix-like compartment surrounding these patches/striosomes but appeared later in development and was always weaker than in the patches. In both striatal compartments, RasGRP was exclusively expressed by medium-sized spiny neurons and showed no preference for neurons that project either directly or indirectly to the substantia nigra. At the ultrastructural level, immunogold labeling of RasGRP was confined to the cell bodies and dendritic shafts of these output neurons. We conclude that the prominent expression of RasGRP in striosomes may be of significance for diacylglycerol signaling in the striatum, and could be of importance for the processing of limbic-related activity within the basal ganglia.

Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus.

Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function. Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.

High-throughput plasmid purification for capillary sequencing.

The need for expeditious and inexpensive methods for high-throughput DNA sequencing has been highlighted by the accelerated pace of genome DNA sequencing over the past year. At the Joint Genome Institute, the throughput in terms of high-quality bases per day has increased over 20-fold during the past 18 mo, reaching an average of 18.3 million bases per day. To support this unprecedented scaleup, we developed an inexpensive automated method for the isolation and purification of double-stranded plasmid DNA clones for sequencing that is tailored to meet the more stringent needs of the newer capillary electrophoresis DNA sequencing machines. The protocol is based on the magnetic bead method of solid phase reversible immobilization that has been automated by using a CRS-based robotic system. The method described here has enabled us to meet our increases in production while reducing labor and materials costs significantly.

Synthesis of leukemia inhibitory factor in injured peripheral nerves and their cells.

The time and site of induction of leukemia inhibitory factor mRNA in injured rat sciatic nerves and its regulation in Schwann cells and fibroblasts from neonatal rat nerves were investigated. Leukemia inhibitory factor mRNA is induced at the lesion site within 6 h of sciatic nerve transection but only after 24 h in the more distal segments. In vitro, interleukin-1beta increases the concentration of leukemia inhibitory mRNA in nerve fibroblasts but not in Schwann cells. Changes in leukemia inhibitory factor mRNA concentration in injured nerves and peripheral nerve cells are similar to those for nerve growth factor mRNA.

Influence of injury and cytokines on synthesis of monocyte chemoattractant protein-1 mRNA in peripheral nervous tissue.

The signals and the source of the signals for monocyte/macrophage entry into the injured peripheral nervous tissue are not yet defined. This study was undertaken to determine the distribution of the chemokine monocyte chemoattractant protein-1 mRNA in injured rat and mouse nerves and to investigate the mechanisms that regulate its synthesis in rat Schwann cells. Results from RNase protection assays showed that, following sciatic nerve transection in rats, mRNA for monocyte chemoattractant protein-1 was induced at the site of lesion within 3 h of surgery and in more distal segments from 24 h for at least 8 days. In cultured Schwann cells, tumour necrosis factor-alpha but not interleukin-1 beta, interleukin-6, transforming growth factor-beta 1, platelet-derived growth factor-BB or nerve growth factor induced monocyte chemoattractant protein-1 mRNA in a time- and dose-dependent fashion. The induction of monocyte chemoattractant protein-1 mRNA in Schwann cells treated with tumour necrosis factor-alpha was reduced by inhibitors of nuclear factor-kappa B and the p38 mitogen-activated protein kinase. In mice that lack the two receptors for tumour necrosis factor, the message for JE, a murine homologue of monocyte chemoattractant protein-1, was still induced within 6 h of injury at the lesion site. However, in more distal segments 4 days after transection the concentration of JE mRNA was lower than that of control mice. Tumor necrosis factor-alpha is the only cytokine that was shown to induce monocyte chemoattractant protein-1 mRNA in cultured Schwann cells and is one of the factors that regulate the synthesis of monocyte chemoattractant protein-1 in injured nerves.

The gene encoding TBC1D1 with homology to the tre-2/USP6 oncogene, BUB2, and cdc16 maps to mouse chromosome 5 and human chromosome 4.

TBC1D1 is the founding member of a family of related proteins with homology to tre-2/UPS6, BUB2, and cdc16 and containing the tbc box motif of 180-220 amino acids. This protein family is thought to have a role in differentiation and in regulating cell growth. We set out to map the TBC1D1 gene in mouse and human. Segregation analysis of a TBC1D1 RFLP in two independent mouse RI (recombinant inbred) lines reveals that mouse Tbc1d1 is closely linked to Pgm1 on chromosome 5. The human TBC1D1 gene was assigned to human chromosome 4p15.1-->4q21 using Southern blot analyses of genomic DNAs from rodent-human somatic cell lines. A human-specific genomic fragment was observed in the somatic cell lines containing human chromosome 4 or the 4p15.1-->4q21 region of the chromosome. TBC1D1 maps to the region containing the ortholog of mouse Pgm1 adding another locus to this long region of conserved synteny between mouse and man.

Reciprocal actions of interleukin-6 and brain-derived neurotrophic factor on rat and mouse primary sensory neurons.

In low-density, serum-free cultures of neurons from embryonic rat dorsal root ganglia, interleukin-6 supports the survival of less than one third of the neurons yet virtually all of them bear interleukin-6 alpha-receptors. A finding that might explain this selectivity is that interleukin-6 acts on sensory neurons in culture through a mechanism requiring endogenous brain-derived neurotrophic factor. Antibodies or a trkB fusion protein that block the biological activity of brain-derived neurotrophic factor synthesized by dorsal root ganglion neurons also block the survival-promoting actions of interleukin-6 on these neurons. Two results indicate that interleukin-6 influences synthesis of brain-derived neurotrophic factor in adult dorsal root ganglion neurons. Intrathecal infusion of interleukin-6 in rats increases the concentration of brain-derived neurotrophic factor mRNA in rat lumbar dorsal root ganglia. The induction of brain-derived neurotrophic factor in dorsal root ganglion neurons that is seen after nerve injury in rats or wild-type mice is severely attenuated in mice with null mutation of the interleukin-6 gene. In brief, the ability of interleukin-6 to support the survival of embryonic sensory neurons in vitro depends upon the presence of endogenous brain-derived neurotrophic factor and the induction of brain-derived neurotrophic factor in injured adult sensory neurons depends upon the presence of endogenous interleukin-6.

Distribution of ras guanyl releasing protein (RasGRP) mRNA in the adult rat central nervous system.

In the nervous system, Ras signal transduction pathways are involved in cellular differentiation, neuronal survival and synaptic plasticity. These pathways can be modulated by Ras guanyl nucleotide exchange factors (Ras GEFs), which activate Ras protein by catalyzing the exchange of GDP for GTP. RasGRP, a recently discovered Ras GEF is expressed in brain as well as in T cells. In addition to the catalytic domain which catalyzes dissociation of Ras-GDP, RasGRP has a pair of calcium-binding EF hands and a diacylglycerol binding domain. The structure of RasGRP suggests that it serves to link calcium and lipid messengers to Ras signaling pathways. We have used an RNase protection assay to detect RasGRP mRNA in various regions of the rat brain and we have determined the cellular distribution of RasGRP mRNA by in situ hybridization. RasGRP mRNA is widely distributed and is present in both interneurons and projection neurons but not confined to any neuronal type or neurotransmitter phenotype. The presence of RasGRP mRNA in archicortical neurons suggests that this pathway may be important in phylogenetically older regions of the CNS. The restriction of RasGRP mRNA to subsets of neurons suggests that activation of Ras by RasGRP has a specific function in certain neuronal types. We did not detect RasGRP in glial cells.

Endogenous interleukin-6 contributes to hypersensitivity to cutaneous stimuli and changes in neuropeptides associated with chronic nerve constriction in mice.

Partial nerve injury is a potential cause of distressing chronic pain for which conventional analgesic treatment with opiates or anti-inflammatory agents is not very effective. Constriction nerve injury, widely used to study neuropathic pain, was shown here to induce interleukin-6 (IL-6) mRNA in a subset of rat primary sensory neurons. When we inflicted chronic nerve constriction on mice with null mutation of the IL-6 gene, the hypersensitivity to cutaneous heat and pressure that is induced in wild-type mice was not evident, the loss of substance P in sensory neurons was excessive and the induction of galanin in central sensory projections was reduced. In additional experiments, intrathecal infusion of IL-6 in rats was shown to stimulate synthesis of galanin in approximately one-third of lumbar dorsal root ganglion neurons. The results of these experiments indicate that endogenous IL-6 mediates some of the hypersensitive responses that characterize peripheral neuropathic pain, and influences two neuropeptides that have been implicated in pain transmission.

Nature of the retrograde signal from injured nerves that induces interleukin-6 mRNA in neurons.

In previous studies, interleukin-6 was shown to be synthesized in approximately one-third of lumbar dorsal root ganglion neurons during the first week after nerve transection. In present studies, interleukin-6 mRNA was found to be induced also in axotomized facial motor neurons and sympathetic neurons. The nature of the signal that induces interleukin-6 mRNA in neurons after nerve injury was analyzed. Blocking of retrograde axonal transport by injection of colchicine into an otherwise normal nerve did not induce interleukin-6 mRNA in primary sensory neurons, but injection of colchicine into the nerve stump prevented induction of interleukin-6 mRNA by nerve transection. Therefore, it was concluded that interleukin-6 is induced by an injury factor arising from the nerve stump rather than by interruption of normal retrograde trophic support from target tissues or distal nerve segments. Next, injection into the nerve of a mast cell degranulating agent was shown to stimulate interleukin-6 mRNA in sensory neurons and systemic administration of mast cell stabilizing agents to mitigate the induction of interleukin-6 mRNA in sensory neurons after nerve injury. These data implicate mast cells as one possible source of the factors that lead to induction of interleukin-6 mRNA after nerve injury. In search of a possible function of inducible interelukin-6, neuronal death after nerve transection was assessed in mice with null deletion of the interleukin-6 gene. Retrograde death of neurons in the fifth lumbar dorsal root ganglion was 45% greater in knockout than in wild-type mice. Thus, endogenous interleukin-6 contributes to the survival of axotomized neurons.