Acrylamide Alters Cytoskeletal Protein Level in Rat Sciatic Nerves

Occupational exposure and experimental intoxication with acrylamide (ACR) produce neuropathy characterized by nerve degeneration. To investigate the mechanism of ACR-induced neuropathy, male adult Wistar rats were given ACR (20, 40 mg/kg i.p. 3 days/week) for 8 weeks. Sciatic nerves were Triton-extracted and centrifuged at a high speed (100,000 × g) to yield pellet and supernatant fractions. The contents of six cytoskeletal proteins (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results showed that the three neurofilament (NF) subunits (NF-L, NF-M, NF-H) in both the pellet and the supernatant fraction decreased significantly (P < 0.01) in the high-dosing group, except for NF-M in the pellet. α-tubulin, β-tubulin, and β-actin increased significantly in the supernatant (P < 0.01), whereas both α-tubulin and β-tubulin decreased significantly in the pellet (P < 0.01). However, β-actin was not altered significantly in the sciatic nerves pellet. These findings suggest that ACR altered the cytoskeletal protein level in sciatic nerve, which may be one of the molecular mechanisms of ACR-induced peripheral neuropathy.     

Time-dependent Alteration of Cytoskeletal Proteins in Cerebral Cortex of Rat During 2,5-Hexanedione-induced Neuropathy

To investigate the mechanisms of the axonopathy induced by 2,5-hexanedione (2,5-HD), male Wistar rats were administered at a dosage of 400 mg/kg/day 2,5-HD (five times per week). The rats produced a slightly, moderately, or severely abnormal neurological changes, respectively, after 2, 4, or 8 weeks of treatment. The cerebrums were Triton-extracted and ultracentrifuged to yield a pellet fraction and a corresponding supernatant fraction. The relative levels of six cytoskeletal proteins (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by immunoblotting. The results showed that NFs content in HD-treated rats demonstrated a progressive decline as the intoxication of HD continued. As for microtubule proteins, the levels of α-tubulin and β-tubulin demonstrated some inconsistent changes. The content of α-tubulin kept unchangeable, while the content of β-tubulin increased significantly at the late stage of HD exposure. Furthermore, the content of β-actin in both fractions remained unaffected throughout the study. These findings suggest that HD intoxication resulted in a progressive decline of NFs, which was highly correlated with the development of HD-induced neuropathy.     

2,5-Hexanedione Induced Decrease in Cytoskeletal Proteins of Rat Sciatic–tibial Nerve

Exposure chronically to n-hexane produces peripheral–central axonopathy mediated by 2,5-hexanedione (HD). Previous studies have demonstrated decreases in neurofilament (NF) contents of peripheral and central nervous regions from rats intoxicated with HD, and recent analysis has demonstrated that axonal atrophy, instead of NF-filled swellings, is a specific component of morphologic alterations. To deeply investigate the alterations of cytoskeletal proteins in HD peripheral neuropathy, the relative levels of NF-L, NF-M, NF-H, -tubulin, -tubulin and -actin of rat sciatic–tibial nerves were determined by SDS-PAGE and immunoblotting. HD was administrated to Wistar rats by intraperitoneal injection at dosage of 200 or 400 mg/kg/day (five-times per week). Rats were sacrificed after 6 weeks of treatment, and sciatic–tibial nerves were dissected, homogenized, and used for the determination of cytoskeletal proteins. Except for supernatant NF-L that could not be assayed, the results showed HD intoxication was associated with significant decreases in NF subunits in both of the supernatant and the pellet fractions of sciatic–tibial nerve homogenates (P<0.01), and obvious reductions in -tubulin, -tubulin and -actin only in the supernatant (P<0.05 or P<0.01). Among these alterations, the falls in the levels of NF subunits tended to be greater compared to those of the other cytoskeletal proteins in all HD-exposed groups, and the trend for decrements in NF-M was greater than those in the other NF subunits. Thus, HD intoxication was associated with significant declines in cytoskeletal protein contents in rat sciatic–tibial nerves, and the decreases might be related to the involvement of the peripheral axonopathy induced by HD.     

The Changes of Cytoskeletal Proteins in Plasma of Acrylamide-Induced Rats

Acrylamide (ACR) is a known industrial neurotoxic chemical that can induce neurodegeneration. Cytoskeletal protein aggregation is a pathological hallmark of neurodegenerative disorders. This study was an initial exploration on cytoskeletal proteins in plasma as potential biomarkers of ACR neurotoxicity. Low and high ACR groups received 20 mg/kg and 40 mg/kg ACR by intraperitoneal injection in adult Wistar rats and control group received physiological saline. Rats were all killed after 8 weeks to evaluate the levels of neurofilament(NF)-L, NF-M, NF-H, β-actin, α-tubulin, β-tubulin, tau, MAP₂ proteins in plasma using both SDS-PAGE and western blotting. Compared with the control, the levels of NF-L, NF-M, NF-H, β-actin, tau, MAP₂ proteins decreased and the level of α-tubulin increased in high ACR group, the levels of α-tubulin, β-tubulin and MAP₂ increased in low ACR group. The results suggested that the changes of these proteins might be relevant to the neurotoxicity of ACR. Some of the cytoskeletal proteins in plasma might be used as marker of biological effect in ACR induced neuropathy.     

Acrylamide-Induced Changes in the Neurofilament Protein of Rat Cerebrum Fractions

Acrylamide (ACR) is known to produce central–peripheral distal axonopathy, which is characterized by distal swellings and secondary degeneration both in experimental animals and human. Ultrastructurally, excessive accumulation of neurofilaments (NFs) in the distal swollen axon is a major pathological hallmark. However, the mechanisms of ACR axonopathy remain unknown. Twenty seven male Wistar rats were randomly divided into three groups. Lower and higher ACR groups were received 20 and 40 mg/kg ACR by i.p. injection respectively. The control group received physiological saline. All rats were sacrificed after 8 weeks of treatment and their cerebrums were dissected, homogenized and used for the determination of the NF proteins. In general, the levels of light NF (NF-L) and medium NF (NF-M) subunits increased consistently in the supernatant, whereas they decreased consistently in the pellet from rats treated with ACR. Compared to that of the control group, the levels of NF-L increased respectively by 104% and 45% (P<0.01) in the supernatant and decreased by 16% and 11% (P<0.01) in the pellet of rat cerebrums in lower and higher groups. The enhancement of NF-M was 76% and 147% (P<0.05, P<0.01) in supernatant, and the reduction was 26% and 36% (P<0.01) in pellet in lower and higher group respectively. The heavy NF (NF-H) level changed slightly. The present results suggested that the change of NF-L and NF-M levels in cerebrum might be relevant to the mechanisms of the neurofilamentous axonopathies induced by ACR.     

Change of Morphology and Cytoskeletal Protein Gene Expression during Dibutyryl cAMP-induced Differentiation in C6 Glioma Cells

Elevation of the intracellular cAMP level induces morphological changes of astrocyte-like differentiation in C6 glioma cells. Such changes may be accompanied with expression of cytoskeletal protein genes. We therefore analyzed morphological changes after a treatment with dibutyryl cAMP (dbcAMP) and then assessed the expression of cytoskeletal protein genes by a quantitative real-time polymerase chain reaction. The cell number remained unaltered upon incubation with 1 mM dbcAMP in medium supplemented with 0.1% fetal bovine serum (FBS), whereas the number and lengths of processes increased, when compared with those of cells incubated in medium supplemented with 0.1% or 10% FBS only. The amounts of β-actin, γ-actin, and β-tubulin mRNAs in C6 cells, but not α-tubulin mRNA, increased during the early proliferation in DMEM containing 10% FBS. The expression of cytoskeletal protein genes decreased when incubated with 0.1% FBS or 1 mM dbcAMP in 0.1% FBS, compared with those of cells cultured in 10% FBS. These results indicated that, during the early proliferation in normal culture condition, the expression of cytoskeletal protein genes in C6 cells, except α-tubulin, increased, while in differentiating or differentiated C6 glioma cells, cAMP-induced morphological changes were not accompanied with elevation of gene expression for cytoskeletal proteins, such as actin and tubulin.     

Regulation of proliferation of UM-SCV-1A and UM-SCV-6 vulvar carcinoma cells by cytokines

 The biology and pathogenesis of vulvar carcinoma are poorly understood at present. In order to understand this disease better, we have used recently developed squamous cell carcinoma lines of the vulva as models. Two cell lines originating from two individuals (UM-SCV-1A and UM-SCV-6) were cultured in vitro in 10% fetal calf serum. The effects of interleukins 10 and 13, interferons α and γ, granulocyte/macrophage-growth-stimulating factor (GM-CSF), tumor necrosis factor α (TNFα), and transforming growth factor β (TGFβ) on the proliferation of the cells was investigated by using radioactively labelled uridine as tracer. In addition, an investigation on the molecular structure of extracted cellular DNA was carried out to investigate whether programmed cell death (apoptosis) would be inducible by any of the factors. In UM-SCV-1A cells, interleukin-10 (IL-10) and interleukin-13 (IL-13) caused an approximately 12-fold decrease in DNA synthesis in cells cultured for 72 h (P<0.001), while GM-CSF had no significant effect. TGFβ showed a significant inhibitory effect on deoxyuridine incorporation (P<0.001), which was 2.0- and 4.2-fold at 48 h and 72 h, respectively. TFGα showed a 1.2-fold inhibitory effect on DNA synthesis at 48 h (P<0.01) and a 1.5-fold inhibition at 72 h (P<0.05). Interferon γ (IFNγ) showed an inhibitory effect on DNA synthesis (1.3-fold; P<0.01). In UM-SCV-6 cells, both IL-10 and IL-13 showed inhibitory effects on deoxyuridine incorporation (1.3- and 1.4-fold at 48 h, respectively; P<0.001) that were even more pronounced at 72 h (2.4- and 2.5-fold respectively; P<0.001). IFNγ caused a 3.6-fold inhibition of DNA synthesis by UM-SCV-6 cells at 72 h (P<0.001). Both TFGβ and TNFα inhibited uridine incorporation (3.0- and 1.6-fold at 48 h, respectively; 2.7-fold at 72 h for both factors). GM-CSF inihibited DNA synthesis by UM-SCV-6 cells 1.3- 2.0-fold at 48 h and 72 h, respectively. In dose/response analyses, the effect of INFα on DNA synthesis was inhibitory in both cell lines at 48 h, while stimulatory effects were observed at 72 h. Electrophoretic analyses of DNA isolated from cells cultured in the presence or absence of different factors did not reveal DNA fragmentation. All cytokines, with the exception of IFNα, showed inhibitory effects on DNA synthesis by vulvar carcinoma cells. Of the factors studied, the recently described interleukins 10 and 13 showed potent inhibition of cell growth, encouraging further investigation on the molecular mechanisms of the observed inhibition. Apoptosis does not seem to be induced in the two vulvar carcinoma cell lines by any of the cytokines studied. Received: 26 March 1996 / Accepted: 5 December 1996     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

Effects of castration on aggression and levels of serum sex hormones and their central receptors in mandarin voles (Microtus mandarinus)

Aggression in socially monogamous mandarin vole (Microtus mandarinus) was observed after castration. Levels of serum sex hormones and their central receptors were also measured using enzyme-linked immunosorbent assay and immunohistochemistry methods. The data indicate that adult males showed higher levels of aggression after castration. However, castration significantly reduced levels of serum testosterone, and the number of androgen receptor immunoreactive neurons in the anterior hypothalamus, bed nucleus of the stria terminalis, medial amygdaloid nucleus (P < 0.01) and lateral septal nucleus (P < 0.05). In addition, levels of estrogen receptor β in the anterior hypothalamus and medial amygdaloid nucleus (P < 0.05), bed nucleus of the stria terminalis and lateral septal nucleus (P < 0.01) declined to varying degrees at weekly intervals. In contrast, serum 17β-estradiol concentrations were up-regulated by castration and castration did not change levels of estrogen receptor α in the medial amygdaloid nucleus and lateral septal nucleus, but increased it in the anterior hypothalamus 3 weeks after castration (P < 0.05). We suggest that higher levels of aggression induced by castration may be independent of serum testosterone and androgen receptors, and may be associated with high serum 17β-estradiol concentrations, stable estrogen receptor α immunoreactive neurons in some brain regions and the relative ratio of the two estrogen receptors.     

Local treatment with the selective IκB kinase β inhibitor NEMO-binding domain peptide ameliorates synovial inflammation

Nuclear factor (NF)-κB is a key regulator of synovial inflammation. We investigated the effect of local NF-κB inhibition in rat adjuvant arthritis (AA), using the specific IκB kinase (IKK)-β blocking NF-κB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-β-induced IκBα phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-α and IL-1-β in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-β-induced TNF-α production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-α-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-κB blockade using a small peptide inhibitor of IKK-β has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-β-targeted NF-κB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.     

Regulation of b- and a-Glycolytic Activities in the Sediments of a Eutrophic Lake

Temporal changes in α-and β-glucosidase activities, dissolved organic matter content, and bacterial biomass were studied in the superficial sediment layer of a eutrophic lake during the period of anoxia. The mean α-and β-glucosidase activities were 30.7±11.0 and 15.1±6.2 nmol h⁻¹ g⁻¹ of dry sediment, respectively. The specifc β-glucosidase activity seemed to be stimulated by carbohydrates (r=0.80, P<0.05), whereas the specifc α-glucosidase activity was negatively correlated with the dissolved protein concentration (r=−0.72, P<0.10). To test the effect of organic matter on hydrolytic activities under controlled conditions, changes in specific activities were studied in relation to the concentrations of different types of organic matter: phytoplankton, polymers (proteins, cellobiose, and starch) and monomers (glucose and amino acids). The specifc α-and β-glucosidase activities were strongly induced by their natural substrates (starch and cellobiose, respectively) (P<0.05) and were not inhibited by glucose. Proteins inhibited these activities (P<0.05), whereas supplementation with amino acids had no effect on specifc glycolytic activities.     

Fibroblast spreading induced by connective tissue stretch involves intracellular redistribution of α- and β-actin

Mechanical stretching of connective tissue occurs with normal movement and postural changes, as well as treatments including physical therapy, massage and acupuncture. Connective tissue fibroblasts were recently shown to respond actively to short-term mechanical stretch (minutes to hours) with reversible cytoskeletal remodeling, characterized by extensive cell spreading and lamellipodia formation. In this study, we have examined the effect of tissue stretch on the distribution of α- and β-actin in subcutaneous tissue fibroblasts ex vivo. Normal fibroblasts uniformly exhibited α-smooth muscle actin (α-SMA) immunoreactivity. Unlike cultured fibroblasts and smooth muscle cells, α-SMA in these fibroblasts was not in F-actin form (indicated by lack of phalloidin co-localization) nor was it organized into distinct stress fibers. The lack of stress fibers and fibronexus was confirmed by electron microscopy, indicating that these cells were not myofibroblasts. In unstretched tissue, the pattern of α-actin was diffuse and granular. With tissue stretch (30 min), α-actin formed a star-shaped pattern centered on the nucleus, while β-actin extended throughout the cytoplasm including lamellipodia and cell cortex. This dual response pattern of α- and β-actin may be an important component of cellular mechanotransduction mechanisms relevant to physiologic and therapeutic mechanical forces applied to connective tissue.     

Effects of amino-acid substitutions in β tubulin on benomyl sensitivity and microtubule functions inCoprinus cinereus

The sensitivity of the homobasidiomyceteCoprinus cinereus to the benzimidazole fungicide benomyl allowed us to isolate β-tubulin mutants as strains resistant to benomyl. To understand the molecular basis for the interaction between benomyl and β tubulin and for cellular defects in the β-tubulin mutants, we first analyzed the wild-type β1-tubulin gene (benA) ofC. cinereus, revealing thatbenA contains eight introns and encodes a 445 amino-acid protein. We then characterized 16 β1-tubulin mutants. The 16 mutations involved 11 different amino-acid substitutions at 10 different residues in β1 tubulin. The mutated residues were widely distributed along the primary sequence of β1 tubulin, from residue 3 in the N-terminal domain to residue 350 in the intermediate domain, but half of them appeared to be close to the αβ intradimer interface in an atomic model determined by electron crystallography. The benomyl resistant strain BEN 193, which exhibits clear heat sensitivity for hyphal growth and defects in various cellular processes, had a novel mutation, i.e., the Leu to Phe substitution at residue 350. Benomyl resistance and the heat sensitivity in BEN 193 were suppressed by additional amino-acid substitutions at various residues in β1 tubulin, suggesting that conformational changes of β1 tubulin are involved in the alterations. The DDBJ/GeneBank/EMBL accession number for the sequence reported in this paper is AB000116.     

Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex

Summary. Many of the highly organized microtubular arrangements in ciliates are located in the cortical area containing membrane vesicles and vacuoles. In Tetrahymena thermophila and Paramecium caudatum, immunofluorescence microscopy with the monoclonal antibody TU-06, directed against β-tubulin, revealed distinct staining of this cortical region alone, while the cilia and other microtubular structures were unstained. The specificity of the antibody was confirmed by immunoblotting and by preabsorption of the antibody with purified tubulin. Double-label immunofluorescence with antibodies against γ-tubulin, detyrosinated α-tubulin, and centrin showed that the TU-06 epitope is localized outside the basal body region. This was also confirmed by immunogold electron microscopy of thin sections. Proteolytic digestion of porcine brain β-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the β-tubulin region β81–95, a region which is phylogenetically highly conserved. As known posttranslational modifications of β-tubulin are located outside this area, the observed staining pattern cannot be interpreted as evidence of subcellular sequestration of modified tubulin. The limited distribution of the epitope could rather reflect the dependence of TU-06 epitope exposition on conformations of tubulin molecules in microtubule arrangements or on differential masking by interacting proteins. Correspondence and reprints: Institute of Molecular Genetics, Czech Academy of Sciences, Vídeňská 1083, 14220 Prague, Czech Republic.     

Acyl-biotinyl Exchange Chemistry and Mass Spectrometry-Based Analysis of Palmitoylation Sites of In Vitro Palmitoylated Rat Brain Tubulin

Research has shown that the palmitoyl group of α-tubulin mediates the hydrophobic interaction between microtubules and intracellular membranes and that palmitoylated tubulin plays a role in signal transduction. There are 20 cysteine residues per α/β tubulin heterodimer. C376 of α-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of α-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on α-tubulin was based on ³H-labeling, enzymolysis, purification and sequencing. This approach, although efficient, is laborious. Mass spectrometry (MS), especially tandem MS, has been shown to be a successful method for identification of various post-translational modifications of proteins. We report here a convenient MS-based method to comprehensively analyze the palmitoylation sites of the α/β tubulin heterodimer. Acyl-biotinyl exchange chemistry and streptavidin agarose affinity purification were applied to enrich palmitoylated peptides from tubulin. After nano-LC-MS/MS analysis, database searching and manual analysis of the spectra revealed that 11 cysteine residues of the α/β tubulin heterodimer were palmitoylated.