Effect of interleukin-1beta and dehydroepiandrosterone on the expression of lumican and fibromodulin in fibroblast-like synovial cells of the human temporomandibular joint.

Several epidemiological studies have reported that temporomandibular disorders (TMDs) are more prevalent in women than in men. It has recently been proposed that sex hormones such as estrogen, testosterone and dehydroepiandrosterone (DHEA) are involved with the pathogenesis of TMDs. Although studies have investigated the relationship between estrogen and testosterone and the restoration of TMDs, the relationship between DHEA and TMDs is unknown. The synovial tissue of the temporomandibular joint (TMJ) is made up of connective tissue with an extracellular matrix (ECM) composed of collagen and proteoglycan. One proteoglycan family, comprised of small leucine-rich repeat proteoglycans (SLRPs), was found to be involved in collagen fibril formation and interaction. In recent years, the participation of SLRPs such as lumican and fibromodulin in the internal derangement of TMJ has been suggested. Although these SLRPs may contribute to the restoration of the synovium, their effect is still unclear. The purpose of this study was to investigate the effect of DHEA, a sex hormone, on the expression of lumican and fibromodulin in human temporomandibular specimens and in cultured human TMJ fibroblast-like synovial cells in the presence or absence of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). In the in vivo study, both normal and osteoarthritic (OA) human temporomandibular synovial tissues were immunohistochemically examined. In the in vitro study, five fibroblast-like synoviocyte (FLS) cell lines were established from human TMJ synovial tissue of patients with osteoarthritis. The subcultured cells were then incubated for 3, 6, 12 or 24 h with/without IL-1beta (1 ng/mL) in the presence or absence of DHEA (10 µM). The gene expression of lumican and fibromodulin was examined using the real-time polymerase chain reaction (PCR) and their protein expression was examined using immunofluorescent staining. We demonstrated that the expression of lumican significantly differs from that of fibromodulin in synovial tissue in OA and furthermore, that IL-1beta induced a significant increase in lumican mRNA and immunofluorescent staining in FLS compared to cells without IL-1beta. DHEA plus IL-1beta induced a significant increase in fibromodulin, but not in lumican mRNA, compared to DHEA alone, IL-1beta alone and in the absence of DHEA and IL-1beta. In immunofluorescent staining, weaker fibromodulin staining of FLS cells was observed in cells cultured in the absence of both DHEA and IL-1beta compared to fibromodulin staining of cells cultured with DHEA alone, with DHEA plus IL-1beta, or with IL-1beta alone. These results indicate that DHEA may have a protective effect on synovial tissue in TMJ by enhancing fibromodulin formation after IL-1beta induced inflammation. DHEA enhancement of fibromodulin expression may also exert a protective effect against the hyperplasia of fibrous tissue that TGF-beta1 induces. In addition lumican and fibromodulin are differentially expressed under different cell stimulation conditions and lumican and fibromodulin may promote regeneration of the TMJ after degeneration and deformation induced by IL-1beta.

Adaptive changes of extracellular amino acid concentrations in mouse dorsal striatum by 4-AP-induced cortical seizures.

The striatum is a major target of cerebral cortical output. The cortico-striatal projection has been well described, however, the neurochemical changes that occur in the striatum after prolonged cortical hyperactivation remain to be investigated. In this study, extracellular levels of glutamate, GABA, and alanine levels were measured in the dorsal striatum using microdialysis in anesthetized mice at resting condition and during 4-aminopyridine (4-AP)-induced cortical seizures. After topical application of 4-AP on the primary motor cortex that induced cortical seizures, the extracellular level of striatal GABA increased by 40% in 60 min. By contrast, the extracellular level of striatal glutamate decreased by 20%. Moreover, the surface amounts of striatal glutamate/aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1), the major astrocytic high-affinity glutamate transporters, tended to increase by cortical seizures in 60 min, suggesting a recruitment of the glutamate transporters from internal stores. 4-AP also resulted in a steady increase of alanine levels which are thought to reflect glutamate and pyruvate metabolism in neurons and astrocytes. These observations possibly delineate adaptive changes of striatal metabolism by severe cortical seizures.

Shosaikoto increases calprotectin expression in human oral epithelial cells.

BACKGROUND AND OBJECTIVE: Oral epithelial cells help to prevent against bacterial infection in the oral cavity by producing antimicrobial peptides (AMPs). A broad-spectrum AMP, calprotectin (a complex of S100A8 and S100A9 proteins), is expressed by oral epithelial cells and is up-regulated by interleukin-1alpha (IL-1alpha). Shosaikoto (SST) is a traditional Japanese herbal medicine that has immunomodulatory effects and is reported to enhance the levels of IL-1alpha in epithelial cells. The purpose of this study was to investigate the effect of SST on the expression of calprotectin and other AMPs through the regulation of IL-1alpha in oral epithelial cells. MATERIAL AND METHODS: Human oral epithelial cells (TR146) were cultured with SST (at concentrations ranging from 10 to 250 microg/mL) in the presence or absence of anti-IL-1alpha or IL-1 receptor antagonist. The expression of S100A8- and S100A9-specific mRNAs was examined by northern blotting. Calprotectin expression and IL-1alpha secretion were investigated by immunofluorescent staining or ELISA. The expression of other AMPs and IL-1alpha was analyzed by RT-PCR and by quantitative real-time PCR. RESULTS: Shosaikoto (25 microg/mL) significantly increased the expression of S100A8- and S100A9-specific mRNAs and calprotectin protein. Shosaikoto increased S100A7 expression, but had no effect on the expression of other AMPs. The expression of IL-1alpha-specific mRNA and its protein were slightly increased by SST. A neutralizing antibody against IL-1alpha or IL-1 receptor antagonist inhibited SST up-regulated S100A8/S100A9 mRNA expression. CONCLUSION: These results suggest that SST increases the expression of calprotectin and S100A7 in oral epithelial cells. In response to SST, up-regulation of calprotectin may be partially induced via IL-1alpha.

Regulation of calprotectin expression by interleukin-1alpha and transforming growth factor-beta in human gingival keratinocytes.

BACKGROUND AND OBJECTIVE: Calprotectin, a heterodimer of S100A8 and S100A9 with antimicrobial properties, is expressed in gingival keratinocytes and plays an important role in innate immunity. Because calprotectin expression is localized in the spinous cell layer of the gingival epithelium, we hypothesized that the expression of calprotectin in keratinocytes is related to the differentiation stage. The aim of the present study was to investigate the relationship between calprotectin expression and keratinocyte differentiation using some factors that regulated its differentiation. MATERIAL AND METHODS: Normal human gingival keratinocytes were isolated from gingival tissues obtained at the extraction of wisdom teeth, and were cultured in serum-free keratinocyte medium supplemented with interleukin-1alpha or calcium, which promote keratinocyte differentiation, and transforming frowth factor-beta (TGF-beta) or retinoic acid, which suppress its differentiation. The expression of S100A8/A9 mRNA and the production of calprotectin in normal human gingival keratinocytes were examined by northern blotting and enzyme-linked immunosorbent assay, respectively. The expression of cytokeratin 14, involucrin and filaggrin (marker proteins of keratinocyte differentiation) was investigated by immunohistochemical staining, and the DNA-binding activity of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor, was examined by electrophoretic mobility shift assay. RESULTS: The expression of S100A8/A9 mRNA and the production of calprotectin were increased by interleukin-1alpha and calcium, but decreased by TGF-beta. RA inhibited the expression of S100A8/A9 and keratinocyte differentiation, which were induced by interleukin-1alpha. C/EBPalpha DNA-binding activity in normal human gingival keratinocytes was enhanced by interleukin-1alpha and calcium, but suppressed by TGF-beta. CONCLUSION: The present study suggests that calprotectin expression is related to keratinocyte differentiation and that C/EBPalpha is a regulator of calprotectin expression in keratinocytes.

Muscle metabolism in patients with polymyositis simultaneously evaluated by using 31P-magnetic resonance spectroscopy and near-infrared spectroscopy.

Simultaneous measurements of muscle energy metabolism using (31)P-magnetic resonance spectroscopy ((31)P-MRS) and the kinetics of muscular oxygen metabolism using near-infrared spectroscopy (NIRS) were conducted in polymyositis (PM) patients. The subjects were 12 PM patients (age 45 +/- 12 years) and 12 normal controls (age 41 +/- 12 years). The muscle phosphocreatine (PCr) index and intracellular pH (pHi) were determined with (31)P-MRS and the changes in intramuscular oxygenated (oxy-Hb), deoxygenated (deoxy-Hb), and total haemoglobin (total Hb) were evaluated with NIRS . The pHi and PCr index before steroid therapy in PM patients were significantly lower during exercise than in normal controls, and their recovery was statistically significantly delayed compared with the controls. The pattern of changes in NIRS over time before steroid therapy in PM patients differed from that in normal controls. There were smaller changes in deoxy-Hb and oxy-Hb during exercise, and total Hb decreased during exercise. In contrast, the kinetics of muscular metabolism after steroid therapy showed changes similar to those seen in normal controls. Simultaneous (31)P-MRS and NIRS measurements to determine the kinetics of muscular metabolism are expected to be useful as a noninvasive approach for the evaluation of treatment effects in PM patients.

Novel expression of equivocal messages containing both regions of choline/ethanolamine kinase and muscle type carnitine palmitoyltransferase I.

For characterization of the detailed gene structure of human muscle type carnitine palmitoyltransferase I (M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts. As a result, we found a cDNA clone containing a nucleotide sequence unexpected from the reported M-CPTI gene structure in the upstream region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase-beta (CK/EK-beta) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the CK/EK-beta gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of CK/EK-beta and M-CPTI. Furthermore, cDNAs containing both exons of these genes were detected by the polymerase chain reaction using the cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these results, the production and organization of these overlapping transcripts are discussed.

Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure.

To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of beta-adrenergic receptor (beta-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of beta(1)-AR and C/EBPbeta in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.

Suppression and enhancement of the Freund's incomplete adjuvant-induced writhing reaction by sodium ascorbate in mice.

We noticed that an intraperitoneal injection of Freund's incomplete adjuvant (FIA) into mice could stimulate the induction of a writhing reaction. The FIA emulsion-induced writhing reaction was found to be remarkably inhibited by preadministration of oral indomethacin, a non-steroidal anti-inflammatory and analgesic drug. The induction of the writhing reaction was also inhibited by intravenous preadministration of sodium ascorbate (SAs) in saline. In the experiments where SAs was added to FIA, it was demonstrated that SAs had dual activity of suppression and enhancement. At lower concentrations SAs functioned as a suppressor of the writhing reaction, while at concentrations higher than about 1 mg/50 microl/mouse it acted as an enhancer of the reaction. Furthermore, this writhing reaction induced by FIA+SAs emulsion was also inhibited by preadministraion of SAs itself as well as indomethacin. These results suggested that the mechanism of the writhing reaction induced by FIA was concerned with the production of prostaglandins (PGs), and SAs might be involved in regulation of the writhing reaction. In this paper, we propose a mouse writhing model induced by FIA or FIA+SAs emulsion as a novel pain model useful for assessment of analgesic and anti-inflammatory agents.

Magnetic resonance imaging and 11C-N-methylspiperone/positron emission tomography studies in a patient with the interval form of carbon monoxide poisoning.

Magnetic resonance (MR) and (11)C-N-methylspiperone ((11)C-NMSP)/positron emission tomography (PET) imagings were repeatedly performed in a 50-year-old man with the interval form of carbon monoxide (CO) poisoning. In MR images obtained when delayed neuropsychiatric symptoms developed (two months after poisoning), the inner segments of the bilateral globus pallidus appeared as high signal intensities in the T1-weighted and low signal intensities in the T2-weighted images, suggesting prior focal hemorrhage in these areas. A PET study with (11)C-NMSP performed at that time showed an increase in dopamine D2 receptor binding in the caudate and putamen. Treatment with bromocriptine was very effective and five months after the poisoning, MR and (11)C-NMSP/PET images showed improvement, concomitantly with the disappearance of the neuropsychiatric symptoms.

The brainstem and thalamic lesions in dentatorubral-pallidoluysian atrophy: an MRI study.

We studied the frequency and characteristics of brainstem and thalamic lesions in dentatorubral-pallidoluysian atrophy using MRI. Of 15 subjects diagnosed by DNA analysis, 13 had lesions in the pontine base, nine in the midbrain, and five in the thalamus. Lesions were correlated positively with the patient's age, but not with neurologic features or numbers of CAG repeats. Patients with Machado-Joseph disease or spinocerebellar ataxia 1 did not show these characteristic lesions.

Structural features of the gene encoding human muscle type carnitine palmitoyltransferase I.

We isolated a human muscle type of carnitine palmitoyltransferase I (CPTI-M) genomic clone and determined its entire nucleotide sequence. By comparison of the nucleotide sequence of the genomic clone with that of cDNA, we determined the intron/exon junctions. For detection of the exon(s) in the 5'-region of the CPTI-M gene, we isolated cDNA clones corresponding to the 5'-region of its transcript by 5'-rapid amplification of cDNA ends (5'-RACE method). Results showed two alternative exons, 1A and 1B, that do not encode amino acids in the 5'-region of the human CPTI-M gene. The gene encoding human CPTI-M was found to consist of two 5'-non-coding exons, 18 coding exons and one 3'-non-coding exon spanning approximately 10 kbp. Furthermore, on analysis of the 5'-flanking region, a putative gene encoding a 'choline kinase homologue' was found to be located only about 300 bp upstream from exon 1A of the human CPTI-M gene. Comparison of the gene structure of human CPTI-M with the reported partial gene structure of human liver type CPTI (CPTI-L) showed that the intron insertion sites were completely conserved in these two genes.

Modulation of extracellular glutamate concentration by nitric oxide synthase inhibitor in rat transient forebrain ischemia.

The purpose of the present study was to clarify the effect of topical administration of a nitric oxide synthase inhibitor on extracellular glutamate concentration in transient forebrain ischemia. Two microdialysis probes were inserted into the bilateral striata of Wistar rats. NG-Nitro-L-arginine (L-NNA) with or without L-arginine was topically administered into the unilateral striatum through one of the microdialysis probes, while Ringer's solution was perfused into the contralateral striatum as the control, and 14 minutes of forebrain ischemia was applied. The extracellular glutamate concentration during ischemia and subsequent reperfusion was statistically significantly higher on the 100 microM L-NNA-perfused side than on the control side, but 1 mM L-NNA was ineffective. When 100 microM L-NNA was perfused together with 500 microM L-arginine, the glutamate concentration did not differ from that on the control side. Moreover, administration of 500 microM L-arginine significantly suppressed the glutamate elevation after reperfusion. The fact that the lower dose of L-NNA increased the accumulation of glutamate during ischemia and reperfusion without altering blood flow may indicate that nitric oxide affords protection against ischemia neuronal damage. However, since the higher dose of L-NNA did not affect the glutamate concentration, it appears that the effect of nitric oxide on extracellular glutamate concentration in forebrain ischemia differs, depending on the degree of the inhibition of NOS activity.

Reversible magnetic resonance imaging lesions in Wilson's disease: clinical-anatomical correlation.

Described herein is a patient with Wilson's disease who had tremor as a prominent neurological manifestation. T2-weighted magnetic resonance imaging showed abnormal high signal intensities in the bilateral lenticular nuclei, thalami, and red nuclei of the midbrain. Improvement of tremor with copper chelating agents was well correlated with a decrease of the abnormal signals in the thalami and the red nuclei.

Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I.

With a cDNA probe encoding rat muscle type carnitine palmitoyltransferase I (CPTI), we isolated cDNA and genomic clones encoding the human homologue and deduced the primary structure of human muscle type CPTI. By Northern analysis, we confirmed the dominant expression of this isoform in heart and skeletal muscle.

A new approach to determine the effect of mismatches on kinetic parameters in DNA hybridization using an optical biosensor.

We have demonstrated a simple yet direct method for determining the kinetic parameters in DNA-DNA interactions using biosensor technology based on the surface plasmon resonance phenomenon; a technique that does not require complex DNA labeling. To determine the effect of mismatches on the kinetics involved in DNA-DNA interactions, DNA hybridization kinetics were monitored in real time using synthetic oligonucleotides less than 20 bases in length which contained either a complementary sequence or mismatched bases. Upon analysis of the kinetic parameters obtained in oligonucleotide hybridization, we found that they were significantly affected by the presence of mismatches as well as by their number and location in a DNA duplex. In addition, the presented biosensor method is sensitive enough to detect kinetic effects caused by the presence of a single-mismatched base pair. Our findings strongly suggest that analysis of kinetic parameters involved in DNA-DNA interactions is advantageous for detecting the presence of mismatch base pairs in a DNA duplex.

High expression of a novel carnitine palmitoyltransferase I like protein in rat brown adipose tissue and heart: isolation and characterization of its cDNA clone.

To characterize energy metabolism in rat brown adipose tissue (BAT), we carried out differential screening of a cDNA library of BAT with a cDNA probe of white adipose tissue (WAT) and isolated one cDNA clone. It contained a single open reading frame of 2,316 bases which encodes a protein of 88.2 kDa. The predicted amino acid sequence showed the highest homology (62.6%) with that of rat carnitine palmitoyltransferase I (CPTI). The transcript corresponding to this cDNA was found to be abundantly expressed in BAT and heart. Therefore, the isolated clone is concluded to encode a CPTI like protein expressed in BAT and heart.

[Chemosensitivity testing using stem cell assay in urological and other malignancies].

We have attempted to grow several kinds of malignant tumors using human tumor stem cell assay. Formation of colonies in vitro was seen in 65 of 132 primary tumors (49%), including 25 of 41 (61%) uroepithelial cancers, 12 of 19 (63%) renal cancers, five of 12 (42%) testicular cancers, five of 21 (24%) gastrointestinal malignancies, five of 12 (42%) lung cancers, five of 11 (45%) hematopoietic malignancies and five of 16 (31%) other malignancies. Growth sufficient for in vitro chemosensitivity tests of CDDP developed from seven cases of uroepithelial cancer, three of them (43%) were sensitive to 2.5 micrograms X hour/ml of CDDP. The specimens from a metastatic testicular tumors that received several courses of PVB chemotherapy resulted in the resistance of the in vitro chemosensitivity test at a higher dosage of CDDP. Nine cases of renal cancer had sufficient growth for an in vitro chemosesitivity test of interferon. One of them was sensitive for alpha 2 type interferon. Three of seven cases were sensitive for alpha type of interferon. To predict clinical correlation, 19 patients were tested with the same drugs used in the in vitro chemosensitivity test. The predictability resulted in more than 60% of true positive, 91% of true negative and 86% of overall predictability.

Pharmacokinetics and pharmacodynamics of 4-aminopyridine in anesthetized dogs.

The pharmacokinetics and pharmacodynamics of 4-aminopyridine (4-AP), a drug which antagonizes nondepolarizing neuromuscular blockade, were studied in seven anesthetized dogs. Using a constant infusion of pancuronium, force of the anterior tibialis contraction in response to stimulation of the sciatic nerve was depressed to 10% of the control tension (90% depression of twitch tension). After 20 min of steady state, 4-AP (1.0 mg/kg) was administered i.v. Serum, urine and bile samples were analyzed for 4-AP concentration at several intervals for 10 hr after administration of 4-AP, using a sensitive high-performance liquid chromatographic assay (1 ng/ml). Serum data best fit a three-compartment pharmacokinetic model. The volume of the central compartment was 412 +/- 352 ml/kg (mean +/- S.D.) and the volume of distribution at steady state was 2517 +/- 363 ml/kg. Initial half-lives were 1.1 +/- 0.7 and 25.4 +/- 11 min. The terminal elimination half-life was 125 +/- 23 min and total clearance was 21 +/- 4 ml/kg/min. Of the injected dose, 60 +/- 9% was recovered in the urine and only 0.01 +/- 0.01% of the dose was recovered in the bile in 10 hr. Inasmuch as renal clearance of 4-AP exceeded glomerular filtration rate we conclude that 4-AP undergoes tubular secretion into the urine. The pharmacodynamic results included an onset time of 14 +/- 8 min, peak effect (maximum percentage of antagonism of twitch tension depression) 97 +/- 27% and duration of action 219 +/- 54 min. We conclude that 4-AP has a longer serum elimination half-life and a longer and more variable duration of action than other antagonists (i.e., neostigmine and pyridostigmine) of nondepolarizing neuromuscular blockade.

Efferent projection of the nucleus praeopticus medialis to the median eminence in rats.

Efferent projection from the medial preoptic nucleus, especially to the median eminence, was studied in the rat by light and electron microscopy, in combination with 3H-leucine labelling and electrical coagulation of the nucleus. In the light microscopy autoradiographs of the brain, an ipsilateral distribution of silver grains was followed caudally and dorsally in the periventricular area, into the triangular area between the ventromedial nucleus and the arcuate nucleus, and, further, into the paraventricular and supraoptic nuclei and median eminence. After electrical coagulation of the medial preoptic area, electron microscopy showed numerous degenerated nerve terminals in the external layer of the median eminence. Occasionally, cored vesicles (90 nm mean diameter with a core of 70 nm mean diameter) and clear vesicles (40-50 nm diameter) were encountered in the axoplasm of the degenerated nerve terminals. These findings indicate that the axons of the nerve cells of the medial preoptic nucleus extend caudally, terminating in the external layer of the median eminence.