Neuron-immune interactions in the sensitized thalamus induced by mustard oil application to rat molar pulp.

We have reported that mustard oil application to the rat dental pulp induces neuronal activation in the thalamus. To address the mechanisms involved in the thalamic changes, we performed neuronal responsiveness recording, immunohistochemistry, and molecular biological analysis. After mustard oil application, neuronal responsiveness was increased in the mediodorsal nucleus. When MK801 (an N-methyl-D-aspartate receptor antagonist) was applied to the mediodorsal nucleus, the enhanced responsiveness was decreased. N-methyl-D-aspartate receptor 2D, glial fibrillary acidic protein, and antigen-presenting cell-related gene mRNAs in the contralateral thalamus were up-regulated at 10 minutes after mustard oil application, but were down-regulated within 10 minutes after the antagonist application. OX6-expressing microglia and glial fibrillary acidic protein-expressing astrocytes did not increase until 60 minutes after mustard oil application. These results suggested that the thalamic neurons play some roles in regulating the glial cell activation in the mediodorsal nucleus via N-methyl-D-aspartate receptor 2D during pulp inflammation-induced central sensitization.

Activation of p38 mitogen-activated protein kinase is required for in vivo brain-derived neurotrophic factor production in the rat hippocampus.

Several lines of evidence strongly suggest that brain-derived neurotrophic factor (BDNF) is associated with the formation, storage and recall of memory in the hippocampus and that it is important to maintain a considerable level of hippocampal BDNF in order to keep normal functions. BDNF can be synthesized in an activity-dependent manner. In fact, kainic acid or AMPA enhances BDNF levels in hippocampal granule neurons. However, the mechanisms of BDNF production are largely unclear. Recently, we have found that riluzole, which blocks voltage-gated sodium channels and thereby reduces glutamate release, actually strengthens immunoreactivity of BDNF in hippocampal granule neurons of rats. Therefore, we examined the riluzole-activated signaling pathways for BDNF production. Riluzole increased levels of phospho-p38 mitogen-activated protein kinase (p38 MAPK), as well as BDNF levels. Inhibition of p38 MAPK by SB203580 reduced riluzole effects, while activation of p38 MAPK by anisomycin increased levels of BDNF, suggesting that p38 MAPK can mediate BDNF production. Riluzole-induced elevation of phospho-activating transcription factor-2, a transcription factor downstream of p38 MAPK, was also observed. A blocker of N-type voltage-gated calcium channels reduced the effects of riluzole on BDNF production and p38 MAPK activation. We also examined a possible involvement of the adenosine A1 receptor in BDNF production because riluzole can influence ecto-nucleotide levels. An A1 receptor agonist inhibited riluzole-induced elevation of BDNF levels, whereas an antagonist not only increased levels of BDNF and active p38 MAPK but also augmented riluzole effects. These results indicate that, in the rat hippocampus, there is an in vivo signaling pathway for BDNF synthesis mediated by p38 MAPK, and that N-type voltage-gated calcium channels and/or adenosine A1 receptors contribute to p38 MAPK activation.

Antigen-presenting cells in human radicular granulomas.

Substantial numbers of dendritic cells have been detected in radicular granulomas. To test the hypothesis that local antigen presentation from dendritic cells to T-cells is involved critically in immunological responses within radicular granulomas, we compared characteristics of dendritic cells and macrophages by morphological and biological analyses. Under light microscopy, HLA-DR+ and CD68+ cells showed diverse profiles, including dendritic-shaped cells. Transmission electron microscopy revealed that HLA-DR+ dendritic cells, with long cytoplasmic processes and lacking distinct phagosomes, were concentrated in the lymphocyte-rich area. HLA-DR alpha-chain, CD83, and CD86 mRNAs from HLA-DR+ dendritic cells, and CD28 mRNA from CD28+ T-cells were up-regulated in lymphocyte-rich area. Scanning electron microscopy demonstrated that the density of gold particles on dendritic cells was higher than that on HLA-DR+ macrophages. These results suggest that dendritic cells in radicular granulomas are associated with local defense reactions as stronger antigen-presenting cells, as compared with macrophages.

CD30+ large cell transformation of mycosis fungoides after psoralen plus ultraviolet A photochemotherapy.

Psoralen plus ultraviolet A (PUVA) photochemotherapy is widely used for the therapy of mycosis fungoides (MF). Clinical progression of MF is often associated with an increase in the size of tumour cells known as transformation. We report two patients with CD30+ large cell transformation that appeared after low-dose PUVA therapy for MF. Clinical data, histopathology, immunohistopathology and T-cell receptor gene rearrangement were studied. Nodules consisted of atypical large cells that expressed CD30. Monoclonal rearrangement of T-cell receptors was observed in one case. Low-dose PUVA therapy may be associated with CD30+ large cell transformation in patients with MF.

Mechanisms underlying estrogen-induced sexual differentiation in the hypothalamus.

Estrogen plays critical roles in the sexual differentiation of the developing brain and gender-specific regulation of reproductive neuroendocrinology. Of the different regions of the brain, it is well known that hypothalamic areas contain key sexually differentiated neuronal circuits. Estrogen receptor (ER) proteins localized in the nucleus affect the expression of target genes when bound to their ligand estrogen. However, recent studies suggest that this may not be the only mechanism of estrogen action. Instead, estrogen can influence various cellular events through regulating different signaling pathways. Cross-talk between direct effects of estrogen on gene transcription and its effects on signaling pathways should be examined in future to elucidate mechanisms underlying sexual differentiation in the hypothalamus.

Idiopathic acquired generalized anhidrosis due to occlusion of proximal coiled ducts.

Idiopathic acquired generalized anhidrosis is a very rare disease of unknown pathogenesis. We report a 25-year-old man with acquired generalized anhidrosis due to occlusion of the coiled ducts. He did not have sweat secretion over the entire surface of the body, including the palms and soles. Sweat-inducing stimuli provoked tingling pain on the skin. Pilocarpine iontophoresis on the forearm did not induce sweat secretion. Neurological examination did not reveal any abnormality in the central or peripheral nervous system. Skin biopsy showed that the coiled ducts were occluded by an amorphous eosinophilic substance. This amorphous eosinophilic substance was positive with periodic acid-Schiff (PAS) staining and was resistant to digestion by diastase. Electron microscopy demonstrated that the coiled ducts were completely occluded by an amorphous substance. The substance occluding the coiled ducts contained fibrous structures. These findings suggested that the acquired generalized anhidrosis in this patient was caused by occlusion of the coiled ducts by a PAS-positive substance probably derived from dark cell granules.

Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen.

BACKGROUND: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B- and T-cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti-TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex. RESULTS: The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two-hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co-immunoprecipitation, both in vitro and in vivo. TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation. CONCLUSION: TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.

Curative treatment of central hemangioma in the mandible by direct puncture and embolisation with n-butyl-cyanoacrylate (NBCA).

Management of central hemangioma in the mandible is difficult because of the abundant vascular network in this region. One of the most common signs of these patients, especially in the mixed dentition period, is hypermobility of the teeth with spontaneous hemorrhage from the surrounding gingival sulcus. Various therapeutic modalities have been considered, but surgery is the most frequently used. In cases of a large extensive lesion, however, intralesional injections of sclerosing agents have often been successful. A case of central hemangioma of the mandible with arteriovenous malformations in a 10-year-old girl is reported. She was treated with direct injection of an embolic material, n-butyl-cyanoacrylate, which brought satisfactory results. Preoperative embolisation of feeder vessels with Gelfoam and Avitene soaked in thrombin together with this direct injection is a safe treatment modality that is as effective as surgery.

Terminal deoxynucleotidyltransferase directly interacts with a novel nuclear protein that is homologous to p65.

BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B- and T-cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT. RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 1 (TdIF1). TdIF1 has a high degree of homology to the transcription factor p65, which belongs to the nuclear receptor superfamily. TdIF1 contains HMG-I and HMG-Y DNA binding domains (AT-hooks) and can bind to single- and double-stranded DNA. TdT and TdIF1 were co-eluted at position 232 kDa by gel filtration of MOLT4 lysate. TdIF1 can enhance TdT activity fourfold in vitro assay system using oligo(dT)16 as primers. CONCLUSIONS: TdIF1 binds directly to TdT, both in vitro and in vivo. TdIF1 and TdT exist as the members of a 232 kDa protein complex. TdIF1 can enhance TdT activity maximum fourfold in vitro assay system, suggesting that it positively regulates the synthesis of the N region during V(D)J recombination in the Ig and TcR genes.

Melatonin, a pineal secretory product with antioxidant properties, protects against cisplatin-induced nephrotoxicity in rats.

In an attempt to define the role of the pineal secretory melatonin and an analogue, 6-hydroxymelatonin (6-OHM), in limiting oxidative stress, the present study investigated the cisplatin (CP)-induced alteration in the renal antioxidant system and nephroprotection with the two indolamines. Melatonin (5 mg/kg), 6-OHM (5 mg/kg), or an equal volume of saline were administered intraperitoneally (i.p.) to male Sprague Dawley rats 30 min prior to an i.p. injection of CP (7 mg/kg). After CP treatment, the animals each received indolamine or saline every day and were sacrificed 3 or 5 days later and plasma as well as kidney were collected. Both plasma creatinine and blood urea nitrogen increased significantly following CP administration alone; these values decreased significantly with melatonin co-treatment of CP-treated rats. In the kidney, CP decreased the levels of GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio, an index directly related to oxidative stress. When animals were treated with melatonin, the reduction in the GSH/GSSG ratio was prevented. Treatment of CP-enhanced lipid peroxidation in the kidney was again prevented in animals treated with melatonin. The activity of the antioxidant enzyme, glutathione peroxidase (GSH-Px), decreased as a result of CP administration, which was restored to control levels with melatonin co-treatment. Upon histological analysis, damage to the proximal tubular cells was seen in the kidneys of CP-treated rats; these changes were prevented by melatonin treatment. 6-OHM has been shown to have some antioxidative capacity, however, the protective effects of 6-OHM against CP-induced nephrotoxicity were less than those of melatonin. The residual platinum concentration in the kidney of melatonin co-treated rats was significantly lower than that of rats treated with CP alone. It is concluded that administration of CP imposes a severe oxidative stress to renal tissue and melatonin confers protection against the oxidative damage associated with CP. This mechanism may be reasonably attributed to its radical scavenging activity, to its GSH-Px activating property, and/or to its regulatory activity for renal function.

Developmental changes in the localization of activated C-JUN N-terminal kinase (JNK/SAPK) in the chick spinal cord.

To examine the role of c-Jun N-terminal kinase (JNK/SAPK) in the developing nervous system of vertebrates, the localization of an active form of JNK, phosphorylated JNK (p-JNK), was studied in the lumbosacral spinal cord of the chick embryo. We also examined the localization of phosphorylated neurofilaments (NFs, potential targets of p-JNK) and cyclin-dependent kinase 5 (Cdk5), which is known to phosphorylate cytoskeletal proteins, including NFs, and compared their expression with that of p-JNK. Additionally, the localization of phosphorylated forms of c-Jun and ATF-2 was compared with that of p-JNK. On embryonic day 3 (E3), the expression of p-JNK was observed in regions containing early-projecting axons. Axons in these regions also expressed phosphorylated NFs. Subsequently, on E5 and E8, the expression of both p-JNK and phosphorylated NFs increased concomitantly in the axonal tracts in the spinal white matter. Thus, white matter expressed both p-JNK and phosphorylated NFs, whereas there was only weak expression of Cdk5. By E13, the spinal cord expression pattern of p-JNK and phosphorylated NFs had changed compared to earlier ages. Although phosphorylated NFs were still expressed in the white matter, the expression of p-JNK was decreased in axons in the white matter, whereas strong p-JNK expression appeared in cell nuclei in the gray matter. In summary, the present study revealed that the localization of p-JNK in the spinal cord changes dramatically from axons to cell nuclei during development, suggesting multiple roles of p-JNK, depending on the developmental age.

Accumulation of phosphorylated neurofilaments and increase in apoptosis-specific protein and phosphorylated c-Jun induced by proteasome inhibitors.

The ubiquitin-proteasome system has been regarded as being important in the progression of neurodegenerative diseases, although its exact role remains uncertain. This in vitro study using PC12h cell cultures examined whether interference with the ubiquitin-proteasome system by proteasome inhibitors induces the neuropathological features of neurodegenerative diseases. Perikaryal accumulation of phosphorylated neurofilaments and an increase in c-Jun as well as phosphorylated form of c-Jun and apoptosis-specific protein were induced by the proteasome inhibitors lactacystin and N-carbobenzoxy-leucyl-leucyl-leucinal. These changes were not observed when only calpain was inhibited. The present study therefore suggests the possibility that a perturbation of the ubiquitin-proteasome system may be one of the causes that result in the development of neuropathological features. Additionally, activity assays showed that the proteasome inhibitor caused an increase in the activity of c-Jun N-terminal kinase (JNK/SAPK), which can phosphorylate neurofilaments and c-Jun, suggesting the possible involvement of JNK in phosphorylation of these proteins.

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin alpha and beta chains.

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.

Tandem mass spectrometric analysis of (13)C-containing ions from a mixture of homologous peptides differing by one mass unit at a residue.

Tandem mass spectrometry of a mixture of two peptides that differ from each other by a single mass unit due to mutation is presented. The mutant beta-globin of hemoglobin Hoshida is present along with the normal counterpart, and the amino acid substitution of glutamine for glutamic acid is located within tryptic peptide T5 of M(r) 2057. 9. The mass of the mutated peptide is 1 u lower. In the isotopic cluster for the doubly charged ion of the peptide T5, the resolved ion with mass of 1030.0 represents the normal peptide with 93 (12)C atoms and the mutated one with 92 (12)C and one (13)C atoms. Collision-induced dissociation (CID) of this composite ion identified the mutation by presenting a key fragment derived from the (12)C-only mutant peptide, as reported in a previous study. Similarly, when an ion containing multiple (13)C atoms was selected as a precursor for CID, the mutation could be identified, even in large fragments, by a marked change in the shape of the isotopic cluster for the consecutive product ions. This study demonstrates the merit of selecting a resolved ion rather than the whole isotopic cluster as a precursor in the CID measurements of large peptides or proteins for characterizing heterozygous mutations.