Constitutive and heat-inducible expression of HSP105 in neurons and glial cells in culture

The constitutive and heat-inducible expression of HSP105 was investigated in newborn mouse brain cell cultures by Northern blotting, Western blotting and immunocytochemistry. HSP105 was expressed most abundantly in the brain among the various tissues examined. HSP105 mRNA and protein were both present at substantial levels in brain cell cultures under unstressed conditions and up-regulated greatly during 3-48 h following exposure to heat stress (43 degrees C/20 min). HSP105 was expressed in nearly all neurons, oligodendrocytes, microglia and astrocytes with its location of both cytoplasmic and nuclear regions under unstressed and heat-stressed conditions. HSP105 expression was significantly down-regulated in astrocytes following treatment with IL-beta or TNF-alpha (50 ng/ml for 6 days), both of which are known growth-stimulatory cytokines for astrocytes. These results indicate that HSP105 is constitutive and heat-inducible HSP in neurons and glial cells in which its expression is under the control of both stressful stimuli and growth-regulatory factors.     

Regulation of galanin in rat sympathetic neurons in vitro

The neuropeptide galanin is induced in sensory and autonomic neurons after peripheral nerve lesion. Leukemia inhibitory factor (LIF) has been suggested to be involved in the up-regulation of galanin. A direct effect of LIF on galanin content in pure sympathetic neuron cultures dissociated from newborn rat superior cervical ganglia was investigated by radioimmunoassay and immunohistochemistry. Galanin increases in sympathetic neurons during a 12 day culture period in the presence of NGF (10 ng/ml). Five days after addition of LIF (10 ng/ml) a 7-fold elevation is observed when compared to control cultures. Furthermore, galanin increases significantly in the presence of non-neuronal cells and in response to potassium-induced depolarization. The proportion of galanin-immunoreactive neurons in mixed cultures is similar to that found in adult rat superior cervical ganglia after transection of the major postganglionic branches. The results corroborate the hypothesis that LIF, presumably released from ganglionic satellite cells, induces galanin in a subpopulation of sympathetic neurons in vivo and in vitro.     

Retinal FABP principally localizes to neurons and not to glial cells

The presence of fatty acid-binding protein (FABP) in the embryonic chick retina may be linked to the demand for polyunsaturated fatty acids in this developing neural tissue. There is a decline in the overall level of FABP as the retina matures, suggesting a role for FABP in cellular differentiation. However, this pattern is not present in the chick brain, indicating a unique function for FABP in the retina. Immunohistochemical staining of paraffin sections of chick retina from embryonic day 21 revealed immunopositive photoreceptor inner segments, outer nuclear layer, 'radial processes' in the inner nuclear layer, a subpopulation of cells in the ganglion cell layer, and inner limiting membrane. This pattern suggested that FABP positive cells were photoreceptors, Müller (glial) cells, and possibly ganglion cells. Staining of sections for glutamine synthetase, an enzyme specific for Müller cells, was similar but not identical to the pattern observed with FABP; thus identification of these cells as FABP-positive was not conclusive. However, in retinal cells dissociated from day E14 embryos and cultured for one week, staining with FABP was more intense in the neurons than in the 'flat' cells (presumed to be derived from the Müller cells). Retinal FABP thus appears to be localized predominantly in neurons, and may serve to sequester fatty acids in preparation for neurite outgrowth as the retinal cells differentiate.     

Interferon regulatory factor 1 is induced by interferon-gamma equally in neurons and glial cells

Class I MHC protein is induced in glia but not mature neurons by IFN-gamma. We have compared IFN-gamma signal transduction in these populations. There were identical levels of STAT1 homodimers and IRF-1 by gel-shift and IRF-1 mRNA was induced equally. However class I MHC, beta2-microglobulin and interleukin 1-beta converting enzyme mRNA levels were greatly reduced in neurons. These experiments show that there is no defect in expression of IRF-1 in response to IFN-gamma in mature mouse neurons but that insufficient class I MHC gene expression is induced for detectable cell surface protein expression.     

Endothelial cell conditioned media mediated regulation of glutamine synthetase activity in glial cells

The responsiveness of late passage C-6 glial cells to human retinal endothelial cell-conditioned medium (HREC-CM) was examined using glutamine synthetase (GS) activity as test parameter. Treatment with 50% or 100% HREC-CM for 4-5 days slightly affected the morphology but significantly increased GS activity. Increased glial GS activity induced by vascular endothelial cells is of relevance in preventing extracellular glutamate toxicity and regulating the brain/retinal blood barrier.     

Parallel induction of nitric oxide and tetrahydrobiopterin synthesis by cytokines in rat glial cells

Activation of monocyte-derived macrophages with cytokines leads to the induction of nitric oxide synthase. Much less is known about the effects of cytokines on microglia, resident brain macrophages, or on astrocytes. In this study, we compared the induction by lipopolysaccharide, interferon-gamma, and tumor necrosis factor-alpha of nitric oxide production and synthesis of tetrahydrobiopterin, the required cofactor for nitric oxide synthase, in microglia and peritoneal macrophages. Activation of microglia induced parallel increases in nitric oxide and intracellular tetrahydrobiopterin levels, although induction of the latter appears to be somewhat more sensitive to diverse stimulators. As with macrophages, inducible nitric oxide production in microglia was blocked by inhibitors of tetrahydrobiopterin biosynthesis. Interleukin-2, an important component of the neuroimmunomodulatory system, was only a weak activator of microglia by itself but potently synergized with interferon-gamma to stimulate production of both nitric oxide and tetrahydrobiopterin. Astrocytes were also activated by lipopolysaccharide and combinations of cytokines but showed a somewhat different pattern of responses than microglia. Biopterin synthesis was increased to higher levels in astrocytes than in microglia, but maximal induction of nitric oxide production required higher concentrations of cytokines than microglia and the response was much lower. These results suggest that tetrahydrobiopterin synthesis in glial cells is a potential target for therapeutic intervention in acute CNS infections whose pathology may be mediated by overproduction of nitric oxide.     

Ultrastructure of the glial cells after spinal cord compression in rabbits

Sump syndrome is a rare complication of biliary-enteric anastomosis. Classically, the distal bile duct becomes obstructed by gastrointestinal debris after choledochoduodenostomy, resulting in cholangitis or, less commonly pancreatitis. Obstruction of the biliary tree by gastrointestinal contents after Roux-en-Y choledochojejunostomy or hepaticojejunostomy has not been described in the English-language literature. This report details the diagnostic and operative management of the first patient with sump syndrome after hepaticojejunostomy. The presumed pathophysiology was reflux of vegetable matter up the efferent limb, resulting in hepatic duct obstruction and cholangitis. The patient ultimately required complex choledochoscopic drainage of the intrahepatic biliary tree and revision of the previous Roux-en-Y hepaticojejunostomy.     

Anatomical and behavioral effects of colchicine administration to rats late in utero

Offspring from pregnant rats injected with 4mg/kg body weight colchicine on Embryonic Days 18, 19, and 20 were found to have isocortical and hippocampal structures greatly reduced in mass when examined at birth. Cells with pyknotic nuclei were found in Layers 5, 4, and 3 of the cerebral isocortex, the habenula, and anterior medial nuclei of the thalamus. Brains taken at Postnatal Days 22 and 132 were reduced in overall size, and had a 20-30% reduction of cells at the vertex of the neocortex with up to 50% reduction in the thickness of the corpus callosum. A decrease in activity, an increase in fearfulness and/or decreased tendency to explore, reduced error scores on the Hebb-Williams maze, poor performance on the Maier elevated maze, and a lessened sensitivity to sound-induced seizures were correlated with these anatomical changes.     

Multihormonal regulation of progesterone synthesis in cultured human midluteal cells

The objectives of this investigation were to examine in vivo insulin like-growth factor-I (IGF-I) secretion by the human midcorpora lutea (mid-CL) and the effects of IGF-I, hCG, FSH, and human GH on progesterone (P) production by CL cells obtained from patients at laparotomy. We first examined whether the CL produces IGF-I by measuring IGF-I levels in the ovarian vein from the ovary bearing the CL. The IGF-I concentration in the ovarian vein bearing the CL (206 +/- 31 ng/mL) was significantly increased compared to the concentration in the contralateral ovarian vein (179.2 +/- 32 ng/mL; P < 0.05). Luteal cells isolated from mid-CL were cultured in serum-free medium 199 in the presence and absence of hCG, FSH, GH, and graded concentrations of IGF-I. At the end of the incubation period (24 h), P levels in the medium were measured by RIA. The treatment with IGF-I (0.1-10 ng/mL) showed a dose-dependent stimulatory action of IGF-I on P synthesis in the luteal cell system, being maximal between 5-10 ng/mL. The treatment with hCG (10 IU/mL), IGF-I (5 ng/mL), and GH (1000 ng/mL) increased basal P synthesis by 300%, 80%, and 30%, respectively (P < 0.001 and P < 0.05). FSH (100 ng/mL), either alone or in combination with IGF-I, failed to stimulate P synthesis. Treatment with IGF-I monoclonal antibody (1:5000) completely reduced P synthesis induced by 5 ng/mL IGF-I and slightly reduced basal P synthesis as well as GH-stimulated P synthesis by human midluteal cells. To further evaluate the specific role of IGF-I on luteal steroidogenesis, IGF-I receptor was identified by chemical cross-linking of [125I]IGF-I to mid-CL membranes. Experiments conducted in the absence and presence of unlabeled IGF-I (500 ng) revealed proteins with characteristics of the type I IGF receptor. These results are consistent with multihormonal regulation of P synthesis by the human mid-CL. hCG and IGF-I play a major role in the stimulation of P synthesis and, to a lesser extent, human GH. These in vivo and in vitro data suggest that the CL is a site of secretion, action, and reception of IGF-I during the midluteal phase.     

Thyroid hormone induces protein secretion and morphological changes in astroglial cells with an increase in expression of glial fibrillary acidic protein

Degradation and utilization of protein by Prevotella ruminicola B1(4), a proteolytic bacterium that is prominent in the rumen, was examined. In preliminary experiments, proteinaceous N sources produced faster growth rates than did NH4Cl, based on changes in optical density over time. However, ammonium chloride produced a greater maximum cell density than did proteinaceous N sources. Of the proteinaceous N sources, an enzymatic hydrolysate of soybean protein with a relative peptide size of 3 AA residues produced a greater growth rate and maximum cell density compared with the other proteinaceous N sources. Further experiments revealed that P. ruminicola B1(4) grew faster and to a greater final dry weight with soybean protein than with casein. Degradation of both proteins was low as was indicated by the slow disappearance of soluble protein, low concentrations of free AA and peptides, and the decrease in ammonia concentrations over time. Patterns of degradation did differ between the two proteins, however. Accumulation of peptides and free AA from soybean protein peaked 2 h earlier than those from casein, and concentrations of free AA and peptides from soybean protein were lower on average than those from casein. Prevotella ruminicola B1(4) preferentially utilized Asp, Ile, Leu, Lys, and Arg from soybean protein compared with casein. The relative size of peptides that accumulated from both proteins, as determined by the ratio of ninhydrin reaction after HCl hydrolysis to ninhydrin reaction before HCl hydrolysis, suggested that part of the proteolytic activity of P. ruminicola B1(4) is a dipeptidase. Our findings suggest that P. ruminicola may have a greater impact on peptide degradation than on protein degradation in the rumen.     

Role of glial filaments in cells and tumors of glial origin: a review

In the adult human brain, normal astrocytes constitute nearly 40% of the total central nervous system (CNS) cell population and may assume a star-shaped configuration resembling epithelial cells insofar as the astrocytes remain intimately associated, through their cytoplasmic extensions, with the basement membrane of the capillary endothelial cells and the basal lamina of the glial limitans externa. Although their exact function remains unknown, in the past, astrocytes were thought to subserve an important supportive role for neurons, providing a favorable ionic environment, modulating extracellular levels of neurotransmitters, and serving as spacers that organize neurons. In immunohistochemical preparations, normal, reactive, and neoplastic astrocytes may be positively identified and distinguished from other CNS cell types by the expression of the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). Glial fibrillary acidic protein is a 50-kD intracytoplasmic filamentous protein that constitutes a portion of, and is specific for, the cytoskeleton of the astrocyte. This protein has proved to be the most specific marker for cells of astrocytic origin under normal and pathological conditions. Interestingly, with increasing astrocytic malignancy, there is progressive loss of GFAP production. As the human gene for GFAP has now been cloned and sequenced, this review begins with a summary of the molecular biology of GFAP including the proven utility of the GFAP promoter in targeting genes of interest to the CNS in transgenic animals. Based on the data provided the authors argue cogently for an expanded role of GFAP in complex cellular events such as cytoskeletal reorganization, maintenance of myelination, cell adhesion, and signaling pathways. As such, GFAP may not represent a mere mechanical integrator of cellular space, as has been previously thought. Rather, GFAP may provide docking sites for important kinases that recognize key cellular substrates that enable GFAP to form a dynamic continuum with microfilaments, integrin receptors, and the extracellular matrix.     

Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis

Normal erythrocytes are under physiological conditions characterized by low cation and high anion conductance. However, in the case of sickle cell anemia the erythrocytes contain a modified haemoglobin, HbS, which under low oxygen tension gives rise to sickling. This condition is preceded by an increase in cation conductance, especially due to the Ca2+-activated K+-channels, leading to net-efflux of KCI and thereby decreased cellular volume, which is part of the pathological condition.A possible symptomatic treatment could be application of conductance blockers, targeting the Ca2+-activated K+-channel or the anion conductance in order to minimize the passive transport of ions and solvent. It has been argued, that due to the high anion conductance, solute loss depended at moderately increased cation conductances on the cation only. Consequently the Ca2+-activated K+-conductance should be the target for attempts to modify solute loss.It is shown that: knowledge of mean conductances (time averages) for pathways showing fluctuations are insufficient to predict the quantitative effect of conductance inhibitors, since inhibition is strongly dependent on the kinetics of the mechanisms mediating the translocation and a block of the high conductance anion pathway can be as effective as inhibition of the Ca+-activated K+-conductance with regard to net salt loss.     

Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon

A three-dimensional cell culture system was used as a model to study the influence of low levels of mercury in the developing brain. Aggregating cell cultures of fetal rat telencephalon were treated for 10 days either during an early developmental period (i.e., between days 5 and 15 in vitro) or during a phase of advanced maturation (i.e., between days 25 and 35) with mercury. An inorganic (HgCl2) and an organic mercury compound (monomethylmercury chloride, MeHgCl) were examined. By monitoring changes in cell type-specific enzymes activities, the concentration-dependent toxicity of the compounds was determined. In immature cultures, a general cytotoxicity was observed at 10(-6) M for both mercury compounds. In these cultures, HgCl2 appeared somewhat more toxic than MeHgCl. However, no appreciable demethylation of MeHgCl could be detected, indicating similar toxic potencies for both mercury compounds. In highly differentiated cultures, by contrast, MeHgCl exhibited a higher toxic potency than HgCl2. In addition, at 10(-6) M, MeHgCl showed pronounced neuron-specific toxicity. Below the cytotoxic concentrations, distinct glia-specific reactions could be observed with both mercury compounds. An increase in the immunoreactivity for glial fibrillary acidic protein, typical for gliosis, could be observed at concentrations between 10(-9) M and 10(-7) M in immature cultures, and between 10(-8) M and 3 x 10(-5) M in highly differentiated cultures. A conspicuous increase in the number and clustering of GSI-B4 lectin-binding cells, indicating a microglial response, was found at concentrations between 10(-10) M and 10(-7) M. These development-dependent and cell type-specific effects may reflect the pathogenic potential of long-term exposure to subclinical doses of mercury.     

The protein tyrosine phosphatase SHP-2 is expressed in glial and neuronal progenitor cells, postmitotic neurons and reactive astrocytes

In this study we examined the distribution and developmental profile of the src homology 2 (SH2) domain-containing protein tyrosine phosphatase SHP-2 in the mouse brain. We found that SHP-2 is present in both mitotically active and postmitotic cells in the forebrains of embryonic day 12 (E12) mice. In a developmental study extending from embryonic day 12 to adulthood, Western blotting analysis demonstrated equivalent levels of SHP-2 protein at all of the ages examined. Expression of SHP-2 paralleled the level of enzymatic activity at the different developmental periods. In the adult brain SHP-2 was restricted to diverse classes of neurons, while the majority of glial cells did not express detectable levels of protein. However, reactive astrocytes in response to an ischemic brain injury showed SHP-2 immunolabelling. Our data suggest that SHP-2 may play a role in pathways of neuronal and glial progenitor cells, in a broad spectrum of neuronal responses in the adult brain and in the gliotic response to the injury.     

The use of transplanted glial cells to reconstruct glial environments in the CNS

Transplantation studies have demonstrated that glia-depleted areas of the CNS can be reconstituted by the introduction of cultured cells. Thus, the influx of Schwann cells into glia-free areas of demyelination in the spinal cord can be prevented by the combined introduction of astrocytes and cells of the O-2A lineage. Although Schwann cell invasion of areas of demyelination is associated with destruction of astrocytes, the transplantation of rat tissue culture astrocytes ("type-1") alone cannot suppress this invasion, indicating a role for cells of the O-2A lineage in reconstruction of glial environments. By transplanting different glial cell preparations and manipulating lesions so as to prevent meningeal cell and Schwann cell proliferation it is possible to demonstrate that the behaviour of tissue culture astrocytes ("type-1") and astrocytes derived from O-2A progenitor cells ("type-2") is different. In the presence of meningeal cells, tissue culture astrocytes clump together to form cords of cells. In contrast, type-2 astrocytes spread throughout glia-free areas in a manner unaffected by the presence of meningeal cells or Schwann cells. Thus, progenitor-derived astrocytes show a greater ability to fill glia-free areas than tissue culture astrocytes. Similarly, when introduced into infarcted white matter in the spinal cord, progenitor-derived astrocytes fill the malacic area more effectively than tissue culture astrocytes, although axons do not regenerate into the reconstituted area.     

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cgamma1 (PLCgamma1). We used recombinant glutathione-S-transferase (GST)-PLCgamma1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCgamma1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology-2 (SH2) domain of PLCgamma1, but there was enhanced binding with tandemly expressed amino- and carboxy-terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen-activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.     

Dermorphin and deltorphin glycosylated analogues: synthesis and antinociceptive activity after systemic administration

In the present paper we describe the synthesis of some dermorphin and deltorphin analogues beta-O- and alpha-C-glycosylated on the C-terminal amino acid residue and report their opioid receptor affinity and selectivity as well as their analgesic potency after subcutaneous injection in mice.