Styrene monomer primarily induces CYP2B1 mRNA in rat liver.

To determine the cytochrome P450 (CYP) primarily expressed after styrene exposure, seven forms of hepatic CYP mRNA in rats treated with 600 mg kg(-1) styrene were examined. CYP1A2, CYP2B1/2, CYP2E1 and CYP3A2 mRNA were observed using real-time LightCycler PCR. The amount of CYP2B1 mRNA was significantly increased, 47-fold compared with controls, suggesting that this CYP is the primary cytochrome P450 in rats exposed to styrene. Significant increases in the amount of CYP2E1, CYP1A2 and CYP2B2 mRNA were also observed after styrene exposure, and their increase levels were 3.1-, 1.7- and 1.7-fold higher than controls, respectively. Western blot analysis also indicated that the protein levels of CYP2B1, CYP2B2, CYP2E1 and CYP1A2 showed clear increases after styrene treatment, corresponding to their mRNA expression. CYP2C11 mRNA decreased significantly in rats after styrene exposure. CYP1A1 was detected at the mRNA level in rat liver, but it was not detected at the protein level. The expression of epoxide hydrolase (EH), involved in Phase I drug metabolism, was also examined. EH mRNA increased 2-fold compared with controls after styrene exposure. Styrene thus appears to be a chemical compound that induces multiple CYPs. The results demonstrate that CYP2B1 is the primarily induced CYP form by styrene treatment to rats at acute toxic level.

A glycoside of Nicotina tabacum affects mouse dopaminergic behavior.

Climbing in the forced swimming test is considered a dopaminergic-specific behavior. A substance of Nicotina tabacum affecting dopamine neuronal activity was investigated using the mouse behavioral system. The substance was found to be a glycoside with the peripheral sugar chain structures Fuc alpha 1-2Gal, Gal beta 1-4GlcNAc and GalNAc alpha 1-3GalNAc and with basic polymannoses. The glycoside dose-dependently increased behavior via D2 neuronal activity, but not D1 activity. This suggests that smoking can affect human brain function not only via the nicotinic cholinergic neuron, but also via the D2 neuron.

Urinary copper excretion in type 2 diabetic patients with nephropathy.

BACKGROUND/AIMS: The aim of this study was to examine the relationship between the degree of urinary copper excretion and stages of diabetic nephropathy. METHODS: Copper, ceruloplasmin and albumin concentrations were measured in serum and urine samples from 41 type 2 diabetic outpatients with different stages of nephropathy and from 10 healthy controls. The copper/albumin and copper/ceruloplasmin ratios in serum and urine were determined. Furthermore, we examined whether free copper ions are dissociated from ceruloplasmin under various pH conditions. RESULTS: Urinary copper concentrations significantly increased only in macroalbuminuric patients. The copper/ceruloplasmin and copper/albumin ratios in urine were consistently greater than those in serum which were not different between patients and healthy controls except the copper/albumin ratio in macroalbuminuric patients. The ratios in urine decreased in parallel with the progression of nephropathy. Copper was found to be released from ceruloplasmin under acidic conditions. CONCLUSION: Urinary copper excretion in healthy controls may be the result of dissociation from the albumin-copper complex of serum during its passage through the kidney. In diabetic patients with advanced nephropathy, urinary copper excretion may be due to dissociations from both copper-albumin and ceruloplasmin-copper complexes filtered through the damaged glomerulus. Overloading of urinary copper to damaged renal tubules may play some roles in the progression of nephropathy in patients with advanced nephropathy.

Pancytopenia in tuberous sclerosis.

A 46-year-old woman with edema and pancytopenia was referred for further evaluation. She was diagnosed as tuberous sclerosis with clinical manifestations such as facial adenoma sebaceous, ungual and periungual fibroma, subependymal nodules and renal angiomyolipoma. Her edema seemed due to hypercardiac function induced by massive anemia. X-ray revealed extraordinary thickening of the cortex of long bones of the extremities as well as patchy osteosclerotic findings in vertebra, suggesting that hematopoietic space was significantly reduced. Pancytopenia improved after splenectomy. Histological examination revealed several intrasplenic hemangiomas but its relationship to hypersplenism was not clear. It seemed that her massive pancytopenia was induced by a combination of hypersplenism and significant reduction in hematopoetic space. In tuberous sclerosis, various systemic complications sometimes induce severe hematological abnormalities. According to previous literatures, the present case of tuberous sclerosis manifested the most outstanding hematological complications.

Refolding of target proteins from a "rigid" mutant chaperonin demonstrates a minimal mechanism of chaperonin binding and release.

One of the most interesting facets of GroEL-facilitated protein folding lies in the fact that the requirement for a successful folding reaction of a given protein target depends upon the refolding conditions used. In this report, we utilize a mutant of GroEL (GroEL T89W) whose domain movements have been drastically restricted, producing a chaperonin that is incapable of utilizing the conventional cyclic mechanism of chaperonin action. This mutant was, however, still capable of improving the refolding yield of lactate dehydrogenase in the absence of both GroES and ATP hydrolysis. A very rapid interconversion of conformations was detected in the mutant immediately after ATP binding, and this interconversion was inferred to form part of the target release mechanism in this mutant. The possibility exists that some target proteins, although dependent on GroEL for improved refolding yields, are capable of refolding successfully by utilizing only portions of the entire mechanism provided by the chaperonins.

Attenuation of luminol-amplified chemiluminescent intensity and lipid peroxidation in the livers of quercetin-fed mice.

We assessed the antioxidative effects of quercetin-feeding on ddY strain male mice by measuring luminol-amplified chemiluminescence that was enhanced by a hydrophilic free radical initiator 2, 2'-azobis(2-amidinopropane) dihydrochloride. Quercetin suppressed chemiluminescent intensity in a dose-dependent manner in vitro with a half-inhibition concentration (IC(50)) of 3x10(-8) M, which was lower than the value of synthetic antioxidant 2, 6-di-tert-butyl-p-cresol (IC(50): 7x10(-7) M). Lysosomal (12000xg pellet) and supernatant fractions obtained from the livers of mice fed a diet containing 0.2% quercetin for 7 days showed more inhibition of chemiluminescent intensity than those from non-treated mice. Quercetin feeding also resulted in the inhibition of lipid peroxidation as determined by a thiobarbituric acid reactive substance test which detected suppression of the release of lysosomal enzymes induced by lipophilic free radical initiator 2, 2'-azobis(2,4-dimethylvaleronitrile). These results probably reflect radical quenching activity of quercetin, indicating that the measurement of chemiluminescence is a useful tool for the assessment of total antioxidant activity in biological systems.

A case of Sjögren's syndrome complicated by nephrogenic diabetes insipidus and renal tubular acidosis.

Abstract We describe the case of a 46-year-old woman with Sjögren's syndrome (SS) presenting with a 6-year history of polyuria and polydipsia. Laboratory data revealed hyperchloremic metabolic acidosis, a normal anion gap, and an inability to acidify urine following an acid loading test and to concentrate the urine in response to water deprivation and antidiuretic hormone administration. Lymphocyte infiltration in the interstitium was found on renal biopsy. These findings allowed us to diagnose distal renal tubular acidosis (RTA) and nephrogenic diabetes insipidus (NDI). Steroid pulse therapy resulted in normalization of the blood pH, but failed to remit the inability to concentrate the urine. These observations suggest therapeutic applications for RTA in SS, and that further investigation is required to design a therapeutic strategy for NDI in SS.

[Expression of brain-derived neurotrophic factor gene in retina following vitreous tap].

PURPOSE: Mechanical injuries to the retina following vitreous tap are reported to protect photoreceptor cells in a rat model of the retinal degeneration and enhance the survival rate of retinal ganglion cells in the optic nerve transection. Neurotrophic factors are presumably involved in the protective mechanisms. In order to see whether neurotrophic factors are synthesized in the retina, we studied the expression of neurotrophic factors in the retina following vitreous tap in rats. MATERIALS AND METHODS: One eye each of 20 mature rats received transscleral vitreous taps at three points of entry and retinal injury. The retinas were removed and examined at day 0 to 14 of treatment. RESULTS: Following injury, the retina showed increased expression of glial fibrillary acidic protein (GFAP) mRNA and brain-derived neurotrophic factor (BDNF) mRNA. There was no enhancement of neurotrophin-3 (NT-3) mRNA when examined by reverse transcription-polymerase chain reaction (RT-PCR). CONCLUSIONS: Activated retinal glial cells may produce BDNF which prevents retinal neuronal cell damage following injury.

The fragment of complement 3a lacking the primary 9 amino acids induces promoting activity on mouse voluntary running.

We have newly found that interleukin-2 (IL-2) increases mouse voluntary running 24 hours, but not 30 minutes, after the injection. We suspected that IL-2 induced a substance increasing the voluntary running for 24 hours after injection. Serum obtained from mice 24 hours after the IL-2 treatment was fractioned with the use of an ion-exchanger and an ultra-filtration method, and the amino acid sequence analysis indicated that the substance purified from the effective fraction was a fragment of mouse complement 3a (C3a) lacking the primary 9 amino acids. The 20 amino acid peptide synthesized according to the fragment showed the activity increasing the voluntary running, but the 20 amino acid peptide synthesized according to the C3a itself did not. The effect of the synthesized peptide was demuted by haloperidol but not by a specific dopamine 2 antagonist (-)sulpiride. The present findings clearly indicate that IL-2 produces the C3a fragment lacking the primary 9 amino acids which directly promotes the voluntary running, and that the effect of the fragment is mediated by an activity of haloperidol on the neurons, except for the dopamine 2 antagonism.

Functional communications between the apical and equatorial domains of GroEL through the intermediate domain.

The Escherichia coli GroEL subunit consists of three domains with distinct functional roles. To understand the role of each of the three domains, the effects of mutating a single residue in each domain (Y203C at the apical, T89W at the equatorial, and C138W at the intermediate domain) were studied in detail, using three different enzymes (enolase, lactate dehydrogenase, and rhodanese) as refolding substrates. By analyzing the effects of each mutation, a transfer of signals was detected between the apical domain and the equatorial domain. A signal initiated by the equatorial domain triggers the release of polypeptide from the apical domain. This trigger was independent of nucleotide hydrolysis, as demonstrated using an ATPase-deficient mutant, and, also, the conditions for successful release of polypeptide could be modified by a mutation in the apical domain, suggesting that the polypeptide release mechanism of GroEL is governed by chaperonin-target affinities. Interestingly, a reciprocal signal from the apical domain was suggested to occur, which triggered nucleotide hydrolysis in the equatorial domain. This signal was disrupted by a mutation in the intermediate domain to create a novel ternary complex in which GroES and refolding protein are simultaneously bound in a stable ternary complex devoid of ATPase activity. These results point to a multitude of signals which govern the overall chaperonin mechanism.

Submerged culture of Tricholoma matsutake mycelium in bubble column fermentors.

Submerged culture of Tricholoma matsutake mycelium was carried out using two bubble column fermentors, a standard bubble column and an external-loop airlift column. The effects of the aeration rate and column type on culture performance in terms of the mycelia morphology, glucose consumption, cell yield, and growth rate were investigated. Morphologically, three types of pellets-large spherical, small spherical and filamentous-were observed depending on the aeration rate. On the whole, the standard bubble column gave a higher cell yield and a better growth rate than the airlift type. The maximum cell yield and growth rate attained at a superficial air velocity of 0.38 cm/s were superior to those obtained in a flask culture, suggesting that the bubble column fermentor has the potential to be used for submerged culture of T. matsutake.

Platelet-derived growth factor B-chain homodimer suppressing a convulsion of epilepsy model mouse El.

El mouse is a mutant which has epileptic convulsions after tossing-up stimulations and has a hippocampal dysfunction. Platelet-derived growth factor B-chain homodimer has been reported to be a trophic factor of hippocampal neurons. We found that a recombinant PDGF-BB suppressed the convulsions of El mice in a dose-dependent manner. Furthermore, thrombin-treated mice manifested no convulsions, but thrombin receptor activating peptide-treated ones had convulsions. These findings suggest that an abnormality in PDGF-BB release may make El mice susceptible to tonic-clonic convulsions.

Interleukin-1 alpha stimulates interstitial collagenase gene expression in human dental pulp fibroblast.

Interstitial collagenase (matrix metalloprotease-1) is a member of a family of matrix metalloproteases and is thought to play a role in degradation of the extracellular components, such as collagens in normal extracellular matrix remodeling and in many disease processes. We examined interstitial collagenase mRNA expression in human dental pulp fibroblast cultures by Northern blot analysis. These cells did not express interstitial collagenase mRNA in an unstimulated condition. Inflammatory cytokines, such as interleukin-1 alpha, induced interstitial collagenase mRNA expression in these cells. The interstitial collagenase mRNA levels began to increase after 2-h exposure, reaching a maximum after 8 h, then dropping to the unstimulated level at 48 h. These effects were observed in a dose-dependent manner in a dose range of 0.1 to 10 ng/ml. Transforming growth factor-beta reduced the levels of interstitial collagenase mRNA expression that were induced by interleukin-1 alpha. These observations suggest that interstitial collagenase mRNA expression in human dental pulp fibroblasts is regulated by the inflammatory cytokines and that interstitial collagenase may play a role in tissue degradation in inflamed dental pulp.

Interleukin-8 gene expression by human dental pulp fibroblast in cultures stimulated with Prevotella intermedia lipopolysaccharide.

Interleukin (IL)-8 mRNA expression was investigated in human dental pulp fibroblast cultures after stimulation with lipopolysaccharide (LPS) prepared from Prevotella intermedia and inflammatory cytokines. The expression of IL-8 mRNA and the release of IL-8 induced by P. intermedia LPS in pulpal fibroblast cultures were detected by Northern blot analysis and ELISA, respectively. The sufficient concentration of P. intermedia LPS on the IL-8 mRNA expression was 0.1 microgram/ml in pulpal fibroblast cultures. IL-8 mRNA levels began to increase after 2 h of exposure, reached a maximum at 4 to 8 h, and declined after 48 h, reaching the unstimulated level by 60 h. IL-8 production by the pulpal fibroblasts began to increase after 8 h of exposure upon stimulation with 10 microgram/ml of P. intermedia LPS. By contrast Salmonella LPS and synthetic lipid A did not increase IL-8 mRNA concentrations in pulpal fibroblast cultures. Recombinant human IL-1 alpha, beta, and tumor necrosis factor-alpha were capable of stimulating these cells to express IL-8 mRNA but natural human interferon-beta, gamma, and recombinant human IL-6 were incapable in our assay. These results suggest that pulpal fibroblasts are immunoresponsive cells and can elaborate IL-8 upon stimulation with P. intermedia LPS.

Microbial induction of dentinal caries in human teeth in vitro.

The ability of Lactobacillus casei, Streptococcus sobrinus, Actinomyces viscosus, and Streptococcus salivarius to induce dental caries was determined in vitro. A class I cavity (depth: 2mm) was prepared in extracted human caries-free premolars to make dentin blocks. The blocks were inoculated with these four bacterial strains in a monoinfective fashion and were incubated under anaerobic conditions. In addition to the monoinfection groups, mixed-infection groups of L. Casei with S. sobrinus or A. viscosus were also prepared. Half of the culture medium was renewed every 3 days, and the pH of the medium was measured. After 4 or 12 wk, these dentin blocks were prepared by Brown-Brenn staining and by contact microradiography for light microscopic observation and for immunohistochemical staining. The final pH of the S. salivarius group was the highest among the experimental groups, at approximately 5.1; that of the others was approximately 4.3. Bacterial invasion into the dentinal tubules was observed in all but the S. salivarius group. Among the monoinfection groups, the S. sobrinus group showed the highest invasion rate, followed by the A. Viscosus group and the L. casei group. The invasion rate was also high in the mixed-infection groups. Immunohistochemical staining revealed invasion only by L. casei, and not by S. sobrinus and A. viscosus. The invasion rate by L. casei was higher in the mixed-infection group with either S. sobrinus or A. viscosus than in the monoinfection groups. These findings suggest that lactobacillus might play an important role in the initiation and progress of dentinal caries, and that this bacterial species might exhibit a cooperative cariogenicity when it coexists with other bacterial species that surpasses its individual cariogenicity.

Novel function of guanidine hydrochloride in reverse micellar extraction of lysozyme from chicken egg white.

The efficiency of guanidine hydrochloride (GuHCl) addition in the suppression of gel formation and the extraction of lysozyme during reverse micellar extraction from chicken egg white was investigated. A low concentration of GuHCl in the feed permitted the successful extraction of lysozyme in its native form without gel formation, which is perceived as a novel function of GuHCl. The highest recovery and specific activity of lysozyme were obtained at a GuHCl concentration of 0.06 M in 25 mM AOT reverse micellar extraction from 20-fold-diluted natural chicken egg white. Lysozyme and ovalbumin CD spectra in the corresponding GuHCl aqueous solutions revealed no changes in the higher order structures of the proteins. Furthermore, the specific activity of lysozyme in the feed was well preserved in the GuHCl system.

Immunopathological activities of extracellular products of Streptococcus mitis, particularly a superantigenic fraction.

Previously, we prepared extracellular products, fractions F-1 and F-2 of Streptococcus mitis 108, an isolate from the tooth surface of an infant, and showed that F-1 exhibited inflammatory cytokine-inducing activities. In the present study, we present evidence that fraction F-2 induced human T-cell proliferation in the presence of irradiated human peripheral blood mononuclear cells and selectively activated T cells bearing V beta 2 and V beta 5.1 in the T-cell receptor. F-1, on the other hand, stimulated human gingival fibroblasts to support the T-cell proliferation in the same way as human gamma interferon or Prevotella intermedia lipopolysaccharide (LPS). Fraction F-1 also primed gingival fibroblasts to support the production of interleukin-2 and gamma interferon by the T cells upon stimulation with F-2. Human gingival fibroblasts stimulated with fraction F-1, like those stimulated by P. intermedia LPS and human gamma interferon, exhibited human leukocyte antigen (HLA)-DR mRNA expression and cell surface HLA-DR molecules as detected by enzyme-linked immunosorbent assay. An anti-HLA-DR monoclonal antibody inhibited T-cell proliferation in response to F-2, probably through inactivating the accessory function of HLA-DR-bearing fibroblasts. T cells activated with F-2 in the presence of irradiated peripheral blood mononuclear cells exhibited definite cytotoxic effects against fibroblasts and squamous carcinoma cells originating from human oral tissues. These findings are strongly suggestive of an association of extracellular products of viridans streptococci with pathogenesis of oral mucosal diseases, particularly those disorders in gingiva which are accompanied by heavy infiltration of T cells.

Bacterial invasion into dentinal tubules of human vital and nonvital teeth.

The difference in resistance to bacterial invasion into the dentinal tubules between vital and nonvital teeth has not been determined. This study was conducted to clarify the effect of vital pulp on bacterial invasion into the dentinal tubules. The specimens were 19 intact pairs of bilateral upper third molars of 19 healthy, young adult male volunteers. In each case, 30 or 150 days before extraction, pulpectomies and root canal fillings were carried out unilaterally and a class V cavity involving the dentin was made on the palatal surface of both the pulpectomized tooth and the nonpulpectomized opposite tooth. The cavities were left unprotected to expose them to oral flora until the extractions were done, and the extracted teeth were examined histologically. When extraction followed 150-day exposure to the oral flora, there was a statistically significant difference in the bacterial invasion rate between the vital and nonvital teeth. It was postulated that vital teeth were much more resistant to bacterial invasion into the dentinal tubules than were nonvital teeth, thereby suggesting that the vital pulp plays some important role in this process.

Identification of antigenic epitopes in a surface protein antigen of Streptococcus mutans in humans.

The reactivities of antibodies in human serum and saliva to a cell surface protein antigen (PAc) of Streptococcus mutans and synthetic peptides covering the PAc molecule were examined. Both an enzyme-linked immunosorbent assay (ELISA) and Western blotting (immunoblotting) showed that all the serum samples from five adult subjects harboring serotype c S. mutans in their oral cavity reacted with recombinant PAc (rPAc). On the other hand, the serum from a 4-month-old infant did not react with rPAc in ELISA. The immunoglobulin A (IgA) antibodies in saliva samples from the five adult subjects reacted with rPAc. However, in saliva samples from these subjects, the titers of IgA antibody to rPAc did not correlate with the titers of serum antibody to the antigen. To map continuous antigenic epitopes in the PAc molecule, we synthesized 153 decapeptides covering the entire mature PAc molecule, 121 overlapping decapeptides covering the alanine-rich repeating region (A-region) of the PAc molecule, and 21 overlapping decapeptides covering the middle region (residues 824 to 853) according to multiple pin-coupled peptide synthesis technology. Of 153 decapeptides covering the mature PAc, 27 decapeptides showed a strong reaction with the antibodies in serum from the adult subjects. The epitope-scanning patterns in the serum samples from these subjects were also very similar to each other. The antigenic epitope patterns in the saliva resembled those in the serum. However, the ELISA titers of salivary IgA antibodies to these decapeptides differed from the titers of the serum antibody. Of the 121 overlapping decapeptides covering the A-region, 27 decapeptides showed a positive reaction with the antibodies in serum from the adult subjects. All of these 27 decapeptides had either one or two of the five common sequences YQAXL, NADAKA, VQKAN, NNAKNA, and IKKRNA. Six decapeptides of the 21 overlapping decapeptides covering the middle region reacted strongly with the serum antibodies from a high PAc responder, and each of the six decapeptides had one of the two common sequences KVTKEKP and VKPTAPTK. These epitopes might therefore be relevant to the humoral responses against the PAc protein during natural infection with S. mutans in humans.

Increased endothelin-1 production in fibroblasts derived from patients with systemic sclerosis.

OBJECTIVES: To determine whether fibroblasts from patients with systemic sclerosis (SSc) produce excessive amounts of endothelin-1 (ET-1), which is recognised as having vasoconstrictive properties and as having a potent mitogenic effect on fibroblasts. METHODS: Dermal fibroblasts were removed from 11 patients with SSc and from five normal controls (NC). The assay of ET-1 protein was measured by an ELISA that used two anti-ET-1 antibodies. The gene expression of prepro ET-1 mRNA was evaluated by a reverse-transcriptase polymerase chain reaction (RT-PCR) method. RESULTS: Levels of ET-1 protein were significantly higher in SSc fibroblast cultures than in those of normal fibroblasts (p < 0.01). The expression of prepro ET-1 mRNA was also higher in SSc fibroblasts than in normal fibroblasts. The addition of interleukin-1 beta (IL-1 beta) increased the production of ET-1 by fibroblasts. CONCLUSION: The findings indicate that the overproduction of ET-1 is a novel abnormal function in SSc fibroblasts, and that ET-1 induced by fibroblasts may play a role in the fibrosis and Raynaud's phenomenon of SSc.