Delayed but sustained induction of mitogen-activated protein kinase activity is associated with beta-adrenergic receptor-mediated morphological differentiation of astrocytes

Astroglial beta-adrenergic receptors (beta-ARs) are functionally linked to regulate cellular morphology. In primary cultures, the beta-AR agonist isoproterenol (ISP) can transform flat polygonal astrocytes into process-bearing, mature stellate cells by 48 h, an effect that can be blocked by the beta-AR antagonist, propranolol. ISP induced immediate activation of protein kinase A (PKA) which persisted up to 2 h, with no visible change in cell morphology. However, activation of PKA was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. In addition to PKA inhibitors, the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 also blocked ISP-induced morphological transformation. ISP treatment resulted in a biphasic response of cellular phosphorylated MAPK (phosphorylated extracellular signal-regulated kinase; p-ERK) level: an initial decline in p-ERK level followed by a sustained induction at 12-24 h, both of which were blocked by PKA inhibitor. The induction in pERK level coincided with initiation of morphological differentiation of the astrocytes and nuclear translocation of p-ERK. A long-lasting activation of p-ERK activity by ISP, at a later stage, appears to be critical for the transformation of astrocytes.     

Inhibition of Orientia tsutsugamushi infection by a truncated recombinant 56‐kDa outer membrane protein

Aims: The objective of this study was to evaluate recombinant 56‐kDa outer membrane protein as a potential inhibitor to infection from Orientia tsutsugamushi. Methods and Results: The 56‐kDa protein was cloned and expressed in an Escherichia coli system, and the degree of target cell attachment to immobilized 56‐kDa protein was measured in a cell adhesion assay. The results showed that the 56‐kDa protein has an ability to attach HeLa cells. Furthermore, treatment of target cells with a truncated 56‐kDa 1–418 (amino acid residues) protein inhibited target cell infection by O. tsutsugamushi, demonstrated with an indirect immunofluorescence antibody assay. Conclusions: The truncated 56‐kDa protein (a.a. 1–418) plays an important role in O. tsutsugamushi infection, and the 56‐kDa protein could be useful and effective in the inhibition of O. tsutsugamushi attachment and infection. Significance and Impact of the Study: The attachment of the 56‐kDa protein to target cells was directly determined by in vitro adherence test, and the invasion of target cells by O. tsutsugamushi was inhibited by treating the target cells with a truncated 56‐kDa protein.     

Inhibition of EGF-induced ERK/MAP kinase-mediated astrocyte proliferation by μ opioids: integration of G protein and β-arrestin 2-dependent pathways

Although μ, κ, and δ opioids activate extracellular signal‐regulated kinase (ERK)/mitogen‐activated protein (MAP) kinase, the mechanisms involved in their signaling pathways and the cellular responses that ensue differ. Here we focused on the mechanisms by which μ opioids rapidly (min) activate ERK and their slower (h) actions to inhibit epidermal growth factor (EGF)‐induced ERK‐mediated astrocyte proliferation. The μ‐opioid agonists ([d‐ ala², mephe⁴, gly‐ol⁵] enkephalin and morphine) promoted the phosphorylation of ERK/MAP kinase within 5 min via G_i/o protein, calmodulin (CaM), and β‐arrestin2‐dependent signaling pathways in immortalized and primary astrocytes. This was based on the attenuation of the μ‐opioid activation of ERK by pertussis toxin (PTX), the CaM antagonist, W‐7, and siRNA silencing of β‐arrestin2. All three pathways were shown to activate ERK via an EGF receptor transactivation‐mediated mechanism. This was disclosed by abolishment of μ‐opioid‐induced ERK phosphorylation with the EGF receptor‐specific tyrosine phosphorylation inhibitor, AG1478, and μ‐opioid‐induced reduction of EGF receptor tyrosine phosphorylation by PTX, and β‐arrestin2 targeting siRNA in the present studies and formerly by CaM antisense. Long‐term (h) treatment of primary astrocytes with [d ‐ala²,mephe⁴,gly‐ol⁵] enkephalin or morphine, attenuated EGF‐induced ERK phosphorylation and proliferation (as measured by 5′‐bromo‐2′‐deoxy‐uridine labeling). PTX and β‐arrestin2 siRNA but not W‐7 reversed the μ‐opioid inhibition. Unexpectedly, β‐arrestin‐2 siRNA diminished both EGF‐induced ERK activation and primary astrocyte proliferation suggesting that this adaptor protein plays a novel role in EGF signaling as well as in the opioid receptor phase of this pathway. The results lend insight into the integration of the different μ‐opioid signaling pathways to ERK and their cellular responses.     

Inhibition of β-Amyloid Production by Activation of Protein Kinase C

The cellular factors regulating the generation of β-amyloid from the amyloid precursor protein (APR) are unknown. Activation of protein kinase C (PKC) by phorbol ester treatment inhibited the generation of the 4-kDa β-amyloid peptide in transfected COS cells, a human glioma cell line, and human cortical astrocytes. An analogue of diacylglycerol, the endogenous cellular activator of PKC, also inhibited the generation of β-amyloid. Activation of PKC increased the level of secreted APP in transfected COS cells but did not significantly affect the level of secreted APP in primary human astrocytes or in the glioma cell line. Cell-associated APP and the secreted APP derivative, but not β-amyloid, were phosphorylated on serine residues. Activation of PKC did not increase the level of APP phosphorylation, suggesting that PKC modulates the proteolytic cleavage of APP indirectly by phosphorylation of other substrates. These results indicate that PKC activation inhibits β-amyloid production by altering APP processing and suggest that β-amyloid production can be regulated by the phospholipase C-diacylglycerol signal transduction pathway.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

β-Amyloid-Induced Neurotoxicity of a Hybrid Septal Cell Line Associated with Increased Tau Phosphorylation and Expression of β-Amyloid Precursor Protein

Abstract: Recent evidence suggests that β-amyloid peptide (β-AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which β-AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated β-AP_1–40 treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF-1, Tau-1, and Tau-5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF-1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser^199/202 and Ser³⁹⁶. In association with the increase in tau phosphorylation, the immunoreactivity of cell-associated and secreted β-amyloid precursor protein (β-APP) was markedly elevated. Application of antisense oligonucleotide to β-APP reduced expression of β-APP and immunoreactivity of phosphorylated tau. Control peptide β-AP_1–28 did not produce significant effects on tau phosphorylation, although it slightly increased cell-associated β-APP. These results suggest that βAP_1–40-induced tau phosphorylation may be associated with increased β-APP expression in degenerated neurons.     

Genetic typing of the 56-kDa type-specific antigen gene of contemporary Orientia tsutsugamushi isolates causing human scrub typhus at two sites in north-eastern and western Thailand

Orientia tsutsugamushi is the causative agent of scrub typhus, a major cause of febrile illness in the rural areas of Southeast Asia. Twenty-three strains of O. tsutsugamushi were isolated from patients with scrub typhus in north-east (Udorn Thani province) and western Thailand (Tak province) between 2003 and 2005. The isolates were characterized by sequencing the entire ORF of the 56-kDa-type-specific antigen gene, followed by phylogenetic analysis. The majority (15/23) of isolates clustered with the Karp-type strain, six with a Gilliam-type strain and one each with the TA716- and TA763-type strains. Overall, there was considerable diversity in sequence, comparable to that seen in strains from across the rest of the scrub typhus-endemic world. There was no significant difference in the distributions of strains between the two provinces (P=0.08, Fisher's exact) nor a temporal change in distribution with year of isolation (P=0.80, Fisher's exact). Within this diversity there were also examples of isolates with identical 56-kDa genotypes that were cultured from patients from the same geographical areas.     

Incorporation of Glycosylated β-Galactosidase into Bovine Brain Synaptosomes

Using a synthesized glycoprotein, beta-galactosidase modified with p-aminophenyl beta-D-galactopyranoside (beta-D-Gal beta-gal), the incorporation of the glycoprotein into bovine brain synaptosomes was studied. The uptake was mediated by a specific receptor to beta-D-galactoside, and was inhibited by GM1 ganglioside. The uptake was found to require energy and to be sensitive to metabolic inhibitors. Kinetic studies on beta-D-Gal beta-gal uptake indicated the presence of a saturable, carrier-mediated transport system in synaptosomes. By subcellular fractionation the beta-D-Gal beta-gal taken up was found in the fractions corresponding to the nucleus and membrane fragments, the soluble cytosomal fractions, and the mitochondria and lysosomes. The uptake was markedly increased by addition of Ca2+ to the incubation medium. The maximal uptake was obtained at pH 8.0 in the presence of 10 mM Ca2+ at 37 degrees C. By addition of a Ca2+ ionophore A23187, beta-D-Gal beta-gal uptake was increased in a dose-dependent way parallel to the increase in the intrasynaptosomal concentration of Ca2+. Preincubation of synaptosomes with calmodulin antagonists such as trifluoperazine and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7) was found to inhibit the uptake markedly, and diazepam, an inhibitor of Ca2+/calmodulin-dependent protein kinase, also inhibited the uptake. At a concentration between 1 and 10 microM, 50% inhibition of the uptake was observed with either inhibitor. On the other hand, the addition of dibutyryl cyclic AMP did not affect the uptake of the glycoprotein into synaptosomes. These results suggest that the incorporation of this macromolecule is dependent on a Ca2+/calmodulin-dependent protein kinase.     

Chronic Morphine Administration Increases β-Adrenergic Receptor Kinase (βARK) Levels in the Rat Locus Coeruleus

Abstract: Based on the established role of β-adrenergic receptor kinase (βARK) and β-arrestin in the desensitization of several G protein-coupled receptors, we investigated the effect of chronic morphine administration on βARK and β-arrestin levels in selected brain areas. Levels of βARK were measured by blot immunolabeling analysis using antibodies specific for two known forms of βARK, i.e., βARK1 and βARK2. It was found that chronic morphine treatment produced an ∼35% increase in levels of βARK1 immunoreactivity in the locus coeruleus, but not in several other brain regions studied. In contrast, chronic morphine treatment failed to alter levels of βARK2 immunoreactivity in any of the brain regions studied. Levels of β-arrestin immunoreactivity, measured using an antiserum that recognizes two major forms of this protein in brain, were also found to increase (by ∼20%) in the locus coeruleus. It is proposed that chronic morphine regulation of βARK1 and β-arrestin levels may contribute to opioid-receptor tolerance that is known to occur in this brain region.     

Structure-Activity Analyses of β-Amyloid Peptides: Contributions of the β25–35 Region to Aggregation and Neurotoxicity

Abstract: The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of β-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of β-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of β-amyloid, β25–35, and the full-length protein, β1–42. We examine the effects of amino acid residue deletions and substitutions on the ability of β-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant β-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of β25–35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the β33–35 region. In β1–42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of β-sheet secondary structure in aggregating, toxic β-amyloid peptides but not in nonaggregating, nontoxic β-amyloid peptides. Together, these data further define the primary and secondary structures of β-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.     

Sequence analysis of the hypervariable regions of the 56 kDa immunodominant protein genes of Orientia tsutsugamushi strains in Malaysia

The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95-99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.     

β-Synuclein occurs in vivo in lipid-associated oligomers and forms hetero-oligomers with α-synuclein

α-synuclein (αS) and β-synuclein (βS) are homologous proteins implicated in Parkinson's disease and related synucleinopathies. While αS is neurotoxic and its aggregation and deposition in Lewy bodies is related to neurodegeneration, βS is considered as a potent inhibitor of αS aggregation and toxicity. No mechanism for the neuroprotective role of βS has been described before. Here, we report that similar to αS, βS normally occurs in lipid-associated, soluble oligomers in wild-type (WT) mouse brains. We partially purified βS and αS proteins from whole mouse brain by size exclusion followed by ion exchange chromatography and found highly similar elution profiles. Using this technique, we were able to partially separate βS from αS and further separate βS monomer from its own oligomers. Importantly, we show that although αS and βS share high degree of similarities, βS oligomerization is not affected by increasing cellular levels of polyunsaturated fatty acids (PUFAs), while αS oligomerization is dramatically enhanced by PUFA. We show the in vivo occurrence of hetero-oligomers of αS and βS and suggest that βS expression inhibits PUFA-enhanced αS oligomerization by forming hetero-oligomers up to a quatramer that do not further propagate.     

Effect of GM-CSF on cytokine induction by soluble β-glucan SCG in vitro in β-glucan-treated mice

SCG is a 6-branched 1,3-β- d -glucan, which are major cell wall structural components in fungi. Leukocytes from DBA/1 and DBA/2 mice are highly sensitive to SCG, producing cytokines such as GM-CSF, IFN-γ, TNF-α and IL-12p70, but not IL-6. GM-CSF plays a key biological role in this activity. In the present study, we examined the effect of giving i.p. SCG to DBA/2 mice on cytokine production in vitro . SCG was given i.p. to DBA/2 mice on day 0. Splenocytes were prepared on day 7 and cultured in the presence of SCG in vitro . The levels of cytokine production induced by SCG in vitro were lower in the cells from SCG-treated mice than in control mice. Expression of the β-glucan receptor, dectin-1, in SCG-treated mice was comparable with that shown in control mice. However, the consumption of exogenously added rmGM-CSF in vitro was observed in SCG-treated mice. The addition of a large amount of rmGM-CSF to the culture medium resulted in larger amounts of TNF-α and IL-6 in SCG-treated mice than in normal mice. These results suggested that GM-CSF was closely related with the reactivity of β-glucan. Giving SCG increased the number of macrophages and granulocytes in the spleen. These results suggested that in SCG-treated mice, a change of cell population would be related to modulation of the profile of cytokine production induced by SCG in vitro .     

β-Amyloid Protein Precursor and τ mRNA Levels Versus β-Amyloid Plaque and Neurofibrillary Tangles in the Aged Human Brain

Abstract: To learn whether or not the levels of β-amyloid protein precursor (APP) and τ mRNAs are related to the formation of β-amyloid and neurofibrillary tangles, we quantified these mRNA levels in three cortical regions of 38 aged human brains, which were examined immunocyto-chemically for β-amyloid and tangles. Marked individual variabilities were noted in APP and τ mRNA levels among elderly individuals. The mean APP mRNA level was slightly reduced in the β-amyloid plaque (++) group, but not in the plaque (+) group, compared to the plaque (−) group. Some brains in the plaque (−) group showed increased APP expression, the extent of which was not seen in the plaque (+)or(++) group. The differences in the mean τ mRNA levels were not statistically significant among the tangle (−), (+), and (++) groups. These results show that β-protein and τ deposition do not accompany increased expression of the APP and τ genes, respectively, and thus suggest that factors other than gene expression may be at work in the progression of β-amyloid and/or tangle formation in the aged human brain.     

β-Amyloid1–40 Increases Expression of β-Amyloid Precursor Protein in Neuronal Hybrid Cells

Abstract: Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for β-amyloid peptide (β-AP), a 40–43-amino-acid peptide derived from a larger membrane glycoprotein, β-amyloid precursor protein (β-APP). Previous experiments have demonstrated that β-AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that β-APP mRNA content is increased 3.5-fold in 24 h after treatment with β-AP_1–40. Accompanying β-AP_1–40-induced cell injury, levels of cell-associated β-APP and a C-terminal intermediate fragment are increased up to 15-fold, and levels of secreted forms of β-APP and 12- and 4-kDa fragments are also increased. Application of β-APP antisense oligodeoxynucleotide reduces both cytotoxicity and β-APP expression. 6-Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase β-APP expression. These results suggest a selective positive feedback mechanism whereby β-AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of β-APP with the net consequence of further neuronal damage.     

S100β Increases Levels of β-Amyloid Precursor Protein and Its Encoding mRNA in Rat Neuronal Cultures

Abstract: S100β has been implicated in the formation of dystrophic neurites, overexpressing β-amyloid precursor protein (βAPP), in the β-amyloid plaques of Alzheimer's disease. We assessed the effects of S100β on cell viability of, neurite outgrowth from, and βAPP expression by neurons in primary cultures from fetal rat cortex. S100β (1–10 ng/ml) enhanced neuronal viability (as assessed by increased mitochondrial activity and decreased lactic acid dehydrogenase release) and promoted neurite outgrowth. Higher levels of S100β (100 ng/ml, but not 1 µg/ml) produced qualitatively similar, but less marked, effects. S100β also induced increased neuronal expression of the microtubule-associated protein MAP2, an effect that is consistent with trophic effects of S100β on neurite outgrowth. S100β (10 and 100 ng/ml) induced graded increases in neuronal expression of βAPP and of βAPP mRNA. These results support our previous suggestion that excessive expression of S100β by activated, plaque-associated astrocytes in Alzheimer's disease contributes to the appearance of dystrophic neurites overexpressing βAPP in diffuse amyloid deposits, and thus to the conversion of these deposits into the diagnostic neuritic β-amyloid plaques.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

The negative regulator of β-lactamase induction AmpD is a N-acetyl-anhydromuramyl-L-alanine amidase

Abstract Construction of a malE-ampD gene fusion allowed purification of biologically active fusion protein by affinity chromatography. The cloned malE-ampD gene fusion complemented a chromosomal ampD mutation. Purified MalE-AmpD fusion protein was found to have murein amidase activity with a pronounced specificity for 1,6-anhydromuropeptides, the characteristic murein turnover products in Escherichia coli . Being a N-acetyl-anhydromuramyl-L-alanine amidase AmpD is likely to be involved in recycling of the turnover products. It is suggested that the negative regulatory effect of AmpD is due to the hydrolysis of anhydro-muropeptides which may function as signals for β-lactamase induction.