Opioid Effects on [3H]Norepinephrine Release from Dissociated Embryonic Locus Coeruleus Cell Cultures

Abstract: The acute and chronic effects of opioid exposure on [³H]norepinephrine ([³H]NE) release were examined in cell cultures of embryonic rat locus coeruleus (LC). Initial morphological and biochemical characterization of the cultures indicated that the cells exhibited properties similar to those observed in situ. Specific [³H]NE uptake was saturable with a K _m value of 222 ± 52 n M . [³H]NE accumulated by LC cells was released in response to 20 m M K⁺ stimulation, in a calcium-dependent manner. Both components of neurotransmitter release, spontaneous and K⁺ evoked, were significantly inhibited by β-endorphin, with the latter being maintained in the presence of tetrodotoxin. The pharmacology of the opioid effect was consistent with that of μ-receptor activation. The effect of chronic exposure to the μ-selective agonist fentanyl (1 μ M ) was examined following 4 days of drug treatment. Although there was no significant effect of fentanyl on K⁺-evoked [³H]NE release, these cells were tolerant to the acute inhibitory effect of β-endorphin. These results indicate that this is an appropriate system for examining the effects of acute and chronic opioid treatment on noradrenergic cells in vitro. In addition, this system may be useful as a CNS model for examining mechanisms that underlie tolerance and dependence following chronic opioid exposure.     

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.     

Evidence for κ- and μ-Opioid Receptor Expression in C6 Glioma Cells

Abstract: The astrocytoma cell line rat C6 glioma has been used as a model system to study the mechanism of various opioid actions. Nevertheless, the type of opioid receptor(s) involved has not been established. Here we demonstrate the presence of high-affinity U69,593, endomorphin-1, morphine, and β-endorphin binding in desipramine (DMI)-treated C6 cell membranes by performing homologous and heterologous binding assays with [³H]U69,593, [³H]morphine, or ¹²⁵I-β-endorphin. Naive C6 cell membranes displayed U69,593 but neither endomorphin-1, morphine, nor β-endorphin binding. Cross-linking of ¹²⁵I-β-endorphin to C6 membranes gave labeled bands characteristic of opioid receptors. Moreover, RT-PCR analysis of opioid receptor expression in control and DMI-treated C6 cells indicate that both κ- and μ-opioid receptors are expressed. There does not appear to be a significant difference in the level of μ nor κ receptor expression in naive versus C6 cells treated with DMI over a 20-h period. Collectively, the data indicate that κ- and μ-opioid receptors are present in C6 glioma cells.     

In Human Fetal Astrocytes Exposure to Interleukin-1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division

Abstract: In human astrocyte cultures established from second-trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3⁺/glial fibrillary acidic protein (GFAP)⁺. The GD3⁺ cells were always GFAP⁺ and grew as flat, highly spread cells but changed to process-bearing cells after interleukin-1β (IL-1β) stimulation. It is interesting that IL-1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [³H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3⁺ population, however, consistently increased in absolute number after IL-1β stimulation, in a dose- and time-dependent manner. The IL-1β-mediated increase in number of GD3⁺ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL-1β enhanced both the mean fluorescence intensity and the percentage of GD3⁺ cells. To investigate whether the increase in GD3⁺ astrocyte cell number was due to proliferation of preexisting GD3⁺ astrocytes or due to conversion of GD3⁻ to GD3⁺ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double-positive cells were extremely rare in both control and IL-1β-stimulated cultures. Moreover, an increase in number of GD3⁺ astrocytes was still observed in control and IL-1β-stimulated cultures where GD3⁺ cells had been initially eliminated by cell sorting. These results indicate that GD3⁺ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.     

Interaction between superantigen and T-cell receptor Vβ element determines levels of superantigen-dependent cell-mediated cytotoxicity of CD8+ T cells in induction and effector phases

Specific superantigens activate different T-cell fractions with distinct TCR Vβ elements in association with MHC class II molecules and also induce SDCC against MHC class II⁺ target cells. In the present study, to determine whether the responsiveness of each CD8⁺ T-cell fraction expressing a different TCR Vβ element is primarily determined by the TCR Vβ, we compared the levels of proliferation and SDCC in Vβ3⁺ and Vβ11⁺ T cells upon stimulation with SEA. Upon stimulation with SEA_wt, the levels of proliferation were higher in Vβ3⁺ T cells than in Vβ11⁺ T cells. The levels of SDCC were also higher for the combination of Vβ3⁺ T cells and SEA_wt than for the combination of Vβ11⁺ T cells and SEA_wt during both the induction phase and the effector phase. In addition, upon stimulation with SEA_m, the levels of proliferation were higher in Vβ11⁺ T cells than in Vβ3⁺ T cells. And then, the levels of SDCC were also higher for the combination of Vβ11⁺ T cells and SEA_m than for the combination of Vβ3⁺ T cells and SEA_m during both the induction phase and the effector phase. These results suggest that the SAG-responsiveness of each CD8⁺ T-cell fraction expressing a different TCR Vβ element is primarily determined by the interaction between the TCR Vβ element and the SAG.     

The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli

In Escherichia coli , lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5 mM EGTA was compensated for by the addition of Zn²⁺. In the presence of 0.5 mM EGTA, only the parental strain was able to take up ⁶⁵Zn²⁺. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI ) and localized at 42 min on the genetic map of E. coli . At high Zn²⁺ concentrations, the znu mutants took up more ⁶⁵Zn²⁺ than the parental strain. The high-affinity ⁶⁵Zn²⁺ uptake was repressed by growth in the presence of 10 μM Zn²⁺. A znuA–lacZ operon fusion was repressed by 5 μM Zn²⁺ and showed a more than 20-fold increase in β-galactosidase activity when Zn²⁺ was bound to 1.5 μM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn²⁺-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli . A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity ⁶⁵Zn²⁺ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB .     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Characterization of amplified esterase Estβ12 associated with organophosphate resistance in a multi-resistant population of the mosquitoCulex quinquefasciatus from Cuba

Esterase amplification is the major organophosphorus (OP) insecticide resistance mechanism in Culex mosquitoes. The amplified Estα2 ^1\ Estβ2 ¹ esterases are found in > 90% of resistant populations worldwide, whereas amplified DNAs (amplicons) containing Estβ1s are much rarer. Individuals with the Estβ1 amplicons appear to be at a selective disadvantage in competition with those carrying the Estα2 ¹\ Estβ2 ¹ amplicons. To test the hypothesis that this is because Estβ1 is less able to bind insecticide than the common amplified esterases, Estβ1² was purified from the multi-resistant Habana strain of Culex quinquefasciatus , from Cuba. In its native form Estβ1 is a monomeric enzyme of 66 kDa, with a pI of 4.8. The bimolecular rate constants for interaction of Estβ1² with several OP insecticides were similar to those for the commonly elevated esterases Estα2¹ and Estβ2¹, and much higher than for the electrophoretically identical non-elevated Estβ1³ and Estα3. Hence the apparent selective advantage of the Estα2 ¹\ Estβ2 ¹ amplicon is not due to its greater efficiency of insecticide binding, as OP insecticides are significantly better inhibitors of all the amplified esterases than of their non-amplified counterparts and therefore should be equally effective at conferring resistance.     

Estradiol Secretion by Tadpole Ovaries Masculinized by Implanted Capsules of Cyanoketone

Female tadpoles of Rana catesbeiana were laparotomized at metamorphic stages XI-XIII and an empty capsule or one containing cyanoketone (CK), which is an inhibitor of Δ⁵-3β-hydroxysteroid dehydrogenase (Δ⁵-3β-HSD), was implanted intraperitoneally. Ovarian activity of Δ⁵-3β-HSD was examined histochemically 2 months later, estradiol-17β (E₂) secretion by the ovaries was measured by RIA 4 months later and histological changes of the ovaries were examined 6 months later. The Δ⁵-3β-HSD activity of the CK-treated ovaries was much lower than that of controls. E₂ secretion per froglet by CK-treated ovaries was about one third that of controls (p<0.001). Histological examination showed various degrees of masculinization of the ovaries, about 28% of which were totally transformed into testis-like structures.
As a result of suppressed Δ⁵-3β-HSD activity, dehydroepiandrosterone would have accumulated, resulting in deficient E₂ secretion and, therefore, ovarian masculinization. In tadpoles, this effect does not depend on the pituitary, whereas interrenal hyperplasia and hyperactivity do, indicating that interrenal function is not essential for ovarian masculinization. From these findings and our previous results, we suggest that disturbance of steroidogenesis by CK in the ovaries results in their masculinization.     

Molecular characterization of major histocompatibility complex (B) haplotypes in broiler chickens

In Leghorn (laying) chickens, susceptibility to a number of infectious diseases is strongly associated with the major histocompatibility ( B ) complex. Nucleotide sequence data have been published for six class I ( B-F ) alleles and for class II ( B-Lβ ) alleles or isotypes from 17 Leghorn haplotypes. It is not known if classical B-L or B-F alleles in broilers are identical, at the sequence level, to any Leghorn alleles. This report describes molecular and immunogenetic characterization of two haplotypes from commercial broiler breeder chickens that were originally identified by serology as a single haplotype, but were differentiated serologically in the present work. The two haplotypes, designated B ^A4 and B ^A4variant, shared identical B-G restriction fragment length polymorphism patterns, but differed in one B-Lβ fragment that cosegregated with the serological B haplotype. Furthermore, the nucleotide sequences of the highly variable exons of an expressed B-LβII family gene and B-F gene from the two haplotypes were markedly different from each other. Both the B-LβII family and B-F gene sequences from the B ^A4 haplotype were identical to the sequences obtained from the reference B ²¹ haplotype in Leghorns; however, in the B ^A4 haplotype the B-Lβ ²¹ and B-F ²¹ alleles were in linkage with B-G alleles that were not G ²¹. The nucleotide sequences from B ^A4variant were unique among the reported chicken B-LβII family and B-F alleles.     

Change of specific T cells in an emerging neonatal infectious disease induced by a bacterial superantigen

A new epidemic, NTED, has recently occurred in Japan. The cause of NTED is a bacterial superantigen, TSST-1. The aim of the present study was to analyze the change in Vβ2⁺ T cells reactive to TSST-1 in NTED in order to establish T-cell-targeted diagnostic criteria for NTED. Blood samples from 75 patients with clinically diagnosed NTED were collected from 13 neonatal intensive care units throughout Japan. We investigated the percentages of Vβ2⁺, Vβ3⁺ and Vβ12⁺ T cells and their CD45RO expressions in the samples using flow cytometry. In 18 of the 75 patients, we conducted multiple examinations of the T cells and monitored serial changes. The Vβ2⁺ T-cell population rapidly changed over three phases of the disease. Whereas the percentage of Vβ2⁺ T cells was widely distributed over the entire control range, CD45RO expression on Vβ2⁺ T cells in CD4⁺ in all 75 patients was consistently higher than the control range. Patients cannot necessarily be diagnosed as having NTED based on expansion of Vβ2⁺ T cells alone in the early acute phase. Instead, CD45RO expression on specific Vβ2⁺ cells is a potential diagnostic marker for a rapid diagnosis of NTED. We present three diagnostic categories of NTED. Fifty patients (66.7%) were included in the category 'definitive NTED'. It is important to demonstrate an increase of Vβ2⁺ T cells in the following phase in cases of 'probable NTED' or 'possible NTED'.     

α2-Macroglobulin Complexes with and Mediates the Endocytosis of β-Amyloid Peptide via Cell Surface Low-Density Lipoprotein Receptor-Related Protein

Abstract: A primary histopathological feature of Alzheimer's disease is the accumulation of β-amyloid (Aβ) in the brain of afflicted individuals. However, Aβ is produced continuously as a soluble protein in healthy individuals where it is detected in serum and CSF, suggesting the existence of cellular clearance mechanisms that normally prevent its accumulation and aggregation. Here, we demonstrate that Aβ forms stable complexes with activated α₂-macroglobulin (α₂M^⋆), a physiological ligand for the low-density lipoprotein receptor-related protein (LRP) that is abundantly expressed in the CNS. These α₂M^⋆/¹²⁵I-Aβ complexes are immunoreactive with both anti-Aβ and anti-α₂M IgG and are stable under various pH conditions, sodium dodecyl sulfate, reducing agents, and boiling. We demonstrate that α₂M^⋆/¹²⁵I-Aβ complexes can be degraded by glioblastoma cells and fibroblasts via LRP, because degradation is partially inhibited by receptor-associated protein (RAP), an antagonist of ligand interactions with LRP. In contrast, the degradation of free ¹²⁵I-Aβ is not inhibited by RAP and thus must be mediated via an LRP-independent pathway. These results suggest that LRP can function as a clearance receptor for Aβ via a physiological ligand.     

THE SELECTIVE NEURONAL UPTAKE AND RELEASE OF [3H]DL-2,4-DIAMINOBUTYRIC ACID BY RAT CEREBRAL CORTEX

Abstract— At 25°C the accumulation of [³H] dl -2,4-diaminobutyric acid (DABA) into small rat cortical slices was linear with time and a tissue: medium ratio of 35:1 was attained after 60 min. At 37°C the uptake was no longer linear and the tissue: medium ratio at 60 min was 66:1. Uptake was unaffected by the addition of 10 μ m -AOAA and dependent on the presence of Na⁺ in the incubation media. The uptake was shown to have a high affinity component with a K _m of 20.7 μ m and a V _max of 28.6 nmol/g/min. IC50's for the inhibition of [³H]DABA uptake by dl -DABA, l -DABA and GABA were 80, 40 and 17 μ m respectively. Two m m β -alanine, however, caused less than 13% inhibition of [³H]DABA uptake. Electron microscopic autoradiographs showed the [³H]DABA to be accumulated by 22% of the identifiable nerve terminals and, after 14 days exposure, the density of silver grains over nerve terminals was 36–38 times higher than that over the rest of the electron micrograph. On the other hand, [³H]DABA was not taken up into rat sensory ganglia and light level autoradiography showed the small amount of [³H]DABA accumulated by the ganglia to be evenly distributed throughout the tissue. Both electrical stimulation for 30 s and exposure of the tissue to a medium containing 47 m m -K⁺ for 2 min caused a marked increase in the efflux of [³H]DABA from the tissue. Both these effects were abolished by a reduction in Ca²⁺ concentration and an increase in the Mg²⁺ concentration of the superfusing medium. These results suggest that l -DABA acts as a 'false transmitter' for the neuronal uptake, storage and release of GABA.     

Nerve Growth Factor, Epidermal Growth Factor, and Insulin Differentially Potentiate ATP-Induced [Ca2+]i Rise and Dopamine Secretion in PC12 Cells

Abstract: To study how growth factors affect stimulus-secretion coupling pathways, we examined the effects of nerve growth factor (NGF), epidermal growth factor (EGF), and insulin on ATP-induced [Ca²⁺]_i rise and dopamine secretion in PC12 cells. After a 4-day incubation of cells, all three factors increased ATP-induced dopamine secretion significantly. We then examined which step of ATP-induced secretion was affected by the growth factors. Cellular levels of dopamine-β-hydroxylase and catecholamines were increased by NGF treatment but were not affected by EGF or insulin. The ATP-induced [Ca²⁺]_i rise was also enhanced after growth factor treatment. The EC₅₀ of ATP for inducing [Ca²⁺]_i rise and dopamine secretion was increased by NGF treatment but not by treatment with EGF or insulin. Accordingly, the dependence on [Ca²⁺]_i of dopamine secretion was increased significantly only in NGF-treated cells. Our results suggest that for EGF- and insulin-treated PC12 cells, the increase in secretion is mainly due to increased potency of ATP in inducing [Ca²⁺]_i rise. NGF treatment not only increased the potency of ATP but also decreased the Ca²⁺ sensitivity of the secretory pathway, which as a result becomes more tightly regulated by changes in [Ca²⁺]_i.     

Neuronal Regulation of Interleukin 6 Secretion in Murine Spleen: Adrenergic and Opioidergic Control

Abstract: The PNS was anticipated to be involved in the modulation of immune responses. To study aspects of this neuronal-immune communication, a recently developed tissue slice method was used to study the effects of adrenergic and opioidergic transmitters on interleukin 6 (IL-6) secretion in the spleen. The α₂-adrenergic agonist p -aminoclonidine (10⁻⁷ M ) inhibited IL-6 secretion (control vs. p -aminoclonidine, 100.0 ± 4.76 vs. 59.3 ± 6.6% of control values; p < 0.001). The α₁-adrenergic agonist methoxamine (10⁻⁸ M ) also inhibited IL-6 secretion (100.0 ± 4.8 vs. 71.5 ± 3.8%; p < 0.001). The endogenous opioids β-endorphin (10⁻¹⁰ M ), methionine-enkephalin (10⁻⁹ M ), and leucine-enkephalin (10⁻⁹ M ) inhibited IL-6 secretion as well ( p = 0.0051, p = 0.0337, and p = 0.0226, respectively). Electrical stimulation of spleen slices inhibited IL-6 secretion (100.0 ± 4.3 vs. 56.7 ± 4.6% of control values; p < 0.001). The involvement of α-adrenergic and opioidergic molecules in this electrically induced inhibition was shown by the use of antagonists. Electrical inhibition of IL-6 secretion was attenuated by phentolamine (10⁻⁷ M ; p = 0.0345), by naloxone (10⁻⁶ M ; p = 0.0046), by cyprodime (10⁻⁸ M ; p = 0.0014), and by the combination of cyprodime (10⁻⁷ M ) plus phentolamine (10⁻⁸ M ; p < 0.0001). We conclude from the complementary studies that the inhibition of IL-6 secretion induced by electrical pulses was mostly mediated by α-adrenergic and μ-opioidergic endogenous transmitters.