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.

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.

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.

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.

Systemic administration of ciliary neurotrophic factor induces cachexia in rodents.

Ciliary neurotrophic factor (CNTF) has previously been shown to promote the survival of several classes of neurons and glial. We report here that in addition to its effects on the nervous system, CNTF can induce potent effects in extra-neural tissues. Implantation of C6 glioma cells engineered to secrete CNTF either subcutaneously or into the peritoneal cavity of adult mice, or systemic injections of purified rat or human recombinant CNTF, resulted in a rapid syndrome of weight loss resulting in death over a period of 7-10 d. This weight loss could not be explained by a reduction in food intake and involved losses of both fat and skeletal muscle. CNTF also induced the synthesis of acute phase proteins such as haptoglobin. Implantation of C6 lines expressing a nonsecreted form of CNTF, or the parental C6 line itself, did not result in wasting effects. Analysis of this CNTF-induced wasting indicates similarities with the previously described cachectins, tumor necrosis factor, interleukin 6, and leukemia inhibitory factor, but does not involve the induction of these cytokines.

Neurotrophic factors in regeneration.

Nerve growth factor, fibroblast growth factor, and ciliary neurotrophic factor can protect selected populations of neurons from some of the degenerative changes that otherwise follow axonal injury or other insults. The function of diffusible neurotrophic factors in axonal regeneration is still unclear, however. Knowledge of the nerve growth factor congeners, brain-derived neurotrophic factor and neurotrophin-3, is advancing rapidly as is the identification of neurotrophin receptors, several of which are membrane-bound tyrosine kinases.

Molecular cloning of a cDNA with a novel domain present in the tre-2 oncogene and the yeast cell cycle regulators BUB2 and cdc16.

In an effort to identify genes that are differentially regulated during mast cell development, subtracted cDNA prepared from wild-type murine P815 mastocytoma cells and a P815 subline that exhibits properties of mast cell differentiation was used to screen mast cell cDNA libraries. Several known mast cell-specific cDNAs were isolated including mast cell carboxypeptidase A (MC-CPA), murine mast cell protease-5 (MMCP-5), and gp49. A novel cDNA, designated Tbc1, was identified that showed differential expression in the two mast cell lines. The amino acid sequence predicted from the cDNA contains a 200 amino acid domain that is homologous to regions in the tre-2 oncogene and the yeast regulators of mitosis, BUB2 and cdc16. The N-terminal region contains a number of cysteine and histidine residues, potentially encoding a zinc finger domain. Tbc1 is a nuclear protein and is expressed in highest levels in hematopoietic cells, testis and kidney. Within these tissues, expression of Tbc1 is cell- and stage-specific. Based on sequence similarity, pattern of expression and subcellular localization, Tbc1 may play a role in the cell cycle and differentiation of various tissues.

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.

Ciliary neurotrophic factor: a review.

Ciliary neurotrophic factor (CNTF) is a 22-kDa protein predicted to share with leukemia inhibitory factor (LIF) and interleukin-6 a common amphipathic helical domain. Consistent with this prediction, the CNTF receptor complex is composed of the CNTF alpha receptor, the LIF beta receptor and gp130 a signalling molecule for LIF and interleukin-6. The major sources of synthesis of CNTF are Schwann cells and astrocytes, but it remains unclear how much CNTF is released from these glial cells and by what mechanism. In vitro, CNTF supports the survival of all classes of peripheral nervous system neurons plus many CNS neurons, induces neurite outgrowth, promotes a cholinergic phenotype in sympathetic neurons and arrests division of neuronal precursor cells. Several cell lines also respond to CNTF. In vivo, CNTF rescues several types of neurons from axotomy-induced death. The functions of CNTF in the development and maintenance of the nervous system remain enigmatic.

Nerve growth factor induces functional nicotinic acetylcholine receptors on rat sensory neurons in culture.

Neonatal sensory neurons from rat nodose ganglia express nicotinic acetylcholine receptors when grown in tissue culture without other cell types. The present study investigates the role of nerve growth factor in inducing these receptors. Nerve growth factor has little effect on the growth and survival of nodose neurons in culture, although most neurons were found by quantitative radioautography to have high-affinity nerve growth factor receptors. Nerve growth factor strongly influenced the expression of nicotinic receptors on these neurons: the proportion of acetylcholine-sensitive neurons was approximately 60% in cultures with nerve growth factor compared with 15% in cultures grown without nerve growth factor. The proportion of acetylcholine-sensitive neurons increased over the first week, plateaued by day 12 and remained high for at least three weeks. In contrast, without NGF, the proportion of acetylcholine-sensitive neurons was low throughout the three-week period. The results indicate that nerve growth factor is an important factor in promoting nicotinic receptors on these neurons in culture.

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.

Galanin expression in neuropathic pain: friend or foe?

We investigated a possible link between galanin expression and evoked pain accompanying painful partial sciatic nerve lesions. Increased galanin immunoreactivity (IR) in the dorsal horn, in gracile nucleus, and in sensory neurons following chronic constriction injury (CCI) compared to complete sciatic transection suggested a facilitatory role in thermal and mechanical hypersensitivity (allodynia). We therefore investigated the effects of endogenous interleukin-6 (IL-6) and nerve growth factor (NGF) on allodynia and neuropeptide expression. IL-6 knockout mice showed decreased allodynia and galanin-IR compared to wild-type mice, but also decreased substance P (SP)-IR in the dorsal horn. Anti-NGF-treated rats with CCI also showed decreased allodynia and SP-IR, but increased galanin-IR in the dorsal horn. These results suggest that evoked pain is more tightly linked to SP than to galanin expression. If galanin's effects are inhibitory as the bulk of the literature suggests, its effects are subordinate to those of SP and to other changes following CCI.

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.

Nerve growth activities in rat peripheral nerve.

Nerve growth activities in rat sciatic nerves were assayed by recording the neuritic outgrowth from chick embryonic ganglia cultured in collagen gels beside nerve fragments for two days. Living nerve explants released activity that resembled nerve growth factor (NGF) in its effect on sympathetic ganglia and that was almost totally blocked by an antiserum to 2.5 S mouse NGF. Frozen and thawed specimens from normal nerves elicited responses from sympathetic ganglia that were only partially suppressed by anti-NGF and also induced neuritic outgrowth from ciliary ganglia. Thus, from observations on normal nerves, at least two agents promoting axonal extension in vitro were deduced to exist; one substance similar to NGF plus another, non-NGF factor. The level of NGF-like activity was low in killed segments of normal nerves but higher in autologous nerve grafts and degenerating nerves two days after grafting or cutting. However, one or two weeks after nerve transection, distal nerve segments contained little nerve growth activity of either kind. Furthermore, when endoneurial fragments from chronically denervated stumps were cultured, they appeared to have lost some of their capacity to produce NGF-like activity in vitro although the production of activity had, if anything, increased in the perineurial region. In summary, rat peripheral nervous tissue releases two or more soluble substances that stimulate neuritic outgrowth. The level of one or both activities in the endoneurium can be altered by manipulation of nerves in vivo.

Transplantation of embryonic spinal and cerebral tissue to sciatic nerves of adult rats.

Neurons and glia in spinal or cerebral tissue removed from young rat embryos survived for months after subperineurial implantation in the sciatic nerve of isogenous rats. With HRP tracing, axons from a few grafted neurons were demonstrated to grow distally into host nerves.

Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection.

Regeneration of crushed axons in rat dorsal spinal roots was measured to investigate the transganglionic influence of an additional peripheral axonal injury. The right sciatic nerve was cut at the hip and the left sciatic nerve was left intact. One week later, both fifth lumbar dorsal roots were crushed and subsequently, regeneration in the two roots was assessed with one of two anatomical techniques. By anterograde tracing with horseradish peroxidase, the maximal rate of axonal regrowth towards the spinal cord was estimated to be 1.0 mm/day on the left and 3.1 mm/day on the right. Eighteen days after crush injury, new, thinly myelinated fibers in the root between crush site and spinal cord were 5-10 times more abundant ipsilateral to the sciatic nerve transection. The central axons of primary sensory neurons regenerate more quickly if the corresponding peripheral axons are also injured.

Studies of axonal regeneration in C57BL/6J and A/J mice.

The genetic and biological nature of a deficiency in axonal regeneration in C57BL/6J mice was investigated. From analysis of recombinant inbred strains, the genetic basis for the deficient regeneration behaviours of C57BL/6J and A/J mice is deduced to involve multiple loci. The defect in axonal regeneration in C57BL/6J mice appears to be a delay rather than permanent impairment and appears to involve sensory more than motor axons.

Decreased synthesis of ciliary neurotrophic factor in degenerating peripheral nerves.

The mRNA for ciliary neurotrophic factor in normal and injured sciatic nerves has been assayed by an RNase protection assay. The endoneurial concentration of ciliary neurotrophic factor mRNA is diminished in the distal nerve stump within three days of sciatic nerve transection, is less than one tenth of normal after one week, and remains low if regeneration does not occur. The synthesis of ciliary neurotrophic factor in injured peripheral nerves is decreased when and where the synthesis of nerve growth factor is increased.