Continuous partially hydrolyzed guar gum intake reduces cold-like symptoms: a randomized, placebo-controlled, double-blinded trial in healthy adults.

OBJECTIVE: Partially hydrolyzed guar gum (PHGG), a water-soluble dietary fiber produced by the controlled partial enzymatic hydrolysis of guar gum beans, has various physiological roles. PHGG is expected to influence the immune function and prevent infections. The objective of this study was to examine the effect of continuous ingestion of PHGG for 12 weeks on the development of cold-like symptoms. PATIENTS AND METHODS: A placebo-controlled, double blind, randomized, parallel-group comparative study was conducted. 96 healthy Japanese adults received 5.2 g PHGG or placebo daily for 12 weeks. Cold-like symptoms were assessed based on patient diary, and the levels of short-chain fatty acids (SCFAs) in stool and blood immune markers at baseline and at weeks 6 and 12. RESULTS: The cumulative number of "no symptoms" days for all symptoms was significantly larger in the PHGG than in the placebo group. The result of the analysis by severity of cold-like symptoms also showed significant differences, with the PHGG group having a lower severity of cold-like symptoms. Propionic acid at weeks 6 and 12 and n-butyric acid and total SCFAs at week 12 were significantly higher in the PHGG than in the placebo group. The Interferon-γ level was significantly lower at week 6 in the PHGG than in the placebo group. CONCLUSIONS: PHGG intake may affect immune function and suppress cold-like symptoms through the production of SCFAs in healthy adults.

Expression of the T cell receptor Vbeta repertoire in a human T cell resistant to asbestos-induced apoptosis and peripheral blood T cells from patients with silica and asbestos-related diseases.

To explore the effects of asbestos and silica on the human immune system, an experimental model of low-dose and long-term exposure was established using a human HTLV-1-immortalized polyclonal T cell line, MT-2 (MT-2Org). MT-2 cells were continuously exposed to asbestos at a concentration (10 microg/ml) which does not induce complete cell death during short-term exposure. After acquiring resistance to CB-induced apoptosis (designated MT-2Rst), an immunological comparison was made between the MT-2Org and MT-2Rst lines in terms of T cell receptor-Vbeta (TcR-Vbeta) expression. MT-2Rst cells showed excess expression of various TcR-Vbeta, although TcR-Vbeta-overpresenting cells were characterized as undergoing apoptosis due to first contact with CB. Patients with asbestos-related diseases (ARD), such as asbestosis and malignant mesothelioma, were compared with silicosis (SIL) patients as a disease control and with healthy donors (HD). SIL and ARD not only differed in their causative materials, silica and asbestos as mineral silicates, but also in terms of complications; autoimmune disorders in SIL and tumors in ARD. ARD patients showed a restricted overpresentation of TcR-Vbeta without clonal expansion, whereas SIL patients revealed significant overpresentation of TcR-Vbeta 7.2. These experimental and clinical analyses indicate the superantigenic and dysregulation of autoimmunity-inducing effects of asbestos and silica, respectively.

Reduced function of CD4+25+ regulatory T cell fraction in silicosis patients.

The quality and quantity of CD4+25+ regulatory T cells (Treg) in silicosis patients (SIL) were examined and compared with results from healthy donors (HD) because SIL often develop autoimmune diseases along with pulmonary disorders. Peripheral blood mononuclear cells from 57 SIL and 50 HD were analyzed for Treg. Treg frequency and clinical parameters were subjected to a factor analysis. Treg and CD4+25- T cells (Tneg) from five HD and five SIL, sorted by flow-cytometer, were used for functional assays of Treg, the expression pattern of Treg specific genes (FoxP3, GITR and CTLA-4) and activation-related genes (CD122 and CD123). Although the actual frequency of Treg did not differ between SIL and HD, the age-corrected level was reduced in SIL. The factor analysis showed that Treg frequency was positively associated with the serum level of IL-2. The inhibitory effect of Treg on Tneg activation was decreased when the Treg:Tneg ratio was 1:1/4 to 1/2. In addition, Treg dominancy of FoxP3 and CTLA-4 expression and Tneg dominancy of CD132 expression found in HD were lost in SIL. These results indicated that the Treg fraction in SIL may be substituted with chronically activated T cells due to recurrent exposure to silica, resulting in a reduction in the frequency and function of Treg. Since the reduction of Treg may precede the clinical manifestation, as silicosis may be a pre-clinical status for autoimmune diseases, control of Treg function using cell and/or gene therapy may be a good way to manage autoimmune disease.

Inhibitory effects of anti-oxidants on apoptosis of a human polyclonal T-cell line, MT-2, induced by an asbestos, chrysotile-A.

To clarify the effects of silica and silicates on cellular features of lymphocytes, a human T-lymphotropic virus type-1-immortalized polyclonal T-cell line, MT-2, was exposed to various concentrations of chrysotile-A, an asbestos classified as silicate. MT-2 cells underwent apoptosis in a dose- and time-dependent manner. The mitochondrial apoptotic pathway was activated during chrysotile-A-induced apoptosis of MT-2 cells, because of the phosphorylation of JNK and p38, increase of BAX and release of cytochrome-c from mitochondria to cytoplasma. In addition, anti-oxidants such as hydroxyl-radical excluders and capturers of superoxide and inhibitors of superoxide production effectively reduced the size of the apoptotic fraction in MT-2 cells cultured with chrysotile-A. These results indicate that the activation of reactive oxygen species may play a central role in asbestos-induced T-cell apoptosis, and anti-oxidants may help to prevent complications of pneumoconiosis.

Effects of calponin on force generation by single smooth muscle cells.

Although the actin-binding and actomyosin adenosinetriphosphatase (ATPase) inhibitory properties of calponin are well documented in vitro, its function in the smooth muscle cell has not been elucidated. To address this question, we utilized the ferret aortic smooth muscle cell, which shows a protein kinase C-dependent contraction even at pCa (-log [Ca2+]) 9.0 in the absence of a change in myosin light chain phosphorylation. Force was recorded from single, briefly permeabilized cells stimulated via a Ca(2+)-independent pathway by either phenylephrine or the epsilon isoenzyme of protein kinase C. Treatment of stimulated cells with wild-type recombinant calponin reduced steady-state contractile force by 45-60%. When calponin application preceded protein kinase C epsilon treatment, contraction was completely suppressed. On the other hand, calponin phosphorylated at Ser175 or mutant calponin with a Ser175 --> Ala replacement had no effect on contractile force. A peptide corresponding to Leu166-Gly194 of calponin, which included an actin-binding domain but excluded the actomyosin ATPase inhibitory region, was synthesized. Treatment of aortic smooth muscle cells with this peptide triggered a concentration-dependent contraction, presumably by alleviating the inhibitory effect of endogenous calponin. A control peptide with a scrambled sequence of the same residues produced no detectable contractile response. Although other interpretations are possible, these results are consistent with the view that calponin participates in thin filament-mediated regulation of smooth muscle contraction and that it may be part of a Ca(2+)-independent pathway downstream of protein kinase C epsilon.

A novel cancer predisposition syndrome in the Eker rat model.

Hereditary renal carcinomas (RCs) develop in virtually all Eker rats by the age of one year. Investigation of extrarenal primary tumors occurring in Eker rats late in life (at 2 years) additionally revealed pituitary adenomas (17/31 = 54.8% (carrier) vs 7/32 = 21.9% (non-carrier siblings of affected animals), P < 0.01), sarcomas of the spleen (21/31 = 67.7% vs. 0/32 = 0%, P < 0.001) and sarcomas (of probable stromal origin) of the uterus (8/17 = 47.1% vs. 0/15 = 0%, P < 0.01). Thus, the Eker rat might provide a novel animal model of cancer predisposition syndromes.

Brain metallothionein gene expression and regulation.

Metallothionein (MT) gene expression in the brain has been most thoroughly studied using rodents. Although MT is considered to be a 'housekeeping' protein even in the brain, the basal MT mRNA expression level is not always high. Differences in the responses of rats and mice have made it difficult to interpret the data. Moreover, the response to inducers is not always apparent, probably because the brain is protected by the blood-brain barrier and initial responses to inducers in peripheral tissues modulate their accumulation in the brain. A relatively high content of MT protein in the brain might be sufficient to elicit minute alterations in the level of inducers. Nonetheless, regulation of MT gene expression in the brain seems to be important in e.g. maintaining the levels of trace elements and controlling redox potentials. The localization and utilization of trans elements such as MTF-I and MEP-I in the brain will provide new aspects for study. The high homology among MT isoforms with respect to nucleotide as well as amino acid sequences has made it difficult to obtain cDNA probes or antibodies capable of distinguishing MT isoforms. Thus, their cross-reactivity might make changes in MT mRNAs appear minimal when MT isoforms are differently regulated. The rapid developments in methodology permitting sensitive, rapid, high-resolution analysis could clarify the background of tissue- and cell-specific gene regulation as well as differential induction.

A novel renal cell carcinoma susceptibility gene maps on chromosome 10 in the Eker rat.

Hereditary renal cell carcinoma (RC) in the rat, originally reported by Eker in 1954, is an example of Mendelian dominant predisposition to a specific cancer in an experimental animal. We previously reported that this predisposing inherited gene is a tumor suppressor gene fitting Knudson's "two-hit" model. This study was designed to map the RC susceptibility gene in the Eker rat using backcross animals. Our present data clearly show that the RC gene is genetically linked to the protamine-1 gene (Lod score = 11.65) and the interleukin-3 gene (Lod score = 4.13), both of which are located on the proximal part of rat chromosome 10. Rat chromosome 10 is currently believed to have no syntenic relationship to human chromosome 3p, the presumed site of the putative tumor suppressor gene for human RC and the locus of von Hippel Lindau disease (affected patients develop multiple RCs). Thus, the Eker rat might have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.

Effects of methylmercury on protein kinase A and protein kinase C in the mouse brain.

The effects of methylmercury administration on adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (protein kinase A) and protein kinase C were investigated by determining their second messenger bindings ([3H]cAMP binding for protein kinase A and [3H]PDBu for protein kinase C) and enzymatic activities in the brains of methylmercury-treated mice. After single administrations of methylmercury (10 mgHg/kg, sc), no neurological symptoms were observed, while the mercury concentration in the brain reached 5.6 ppm. Neither second messenger bindings nor enzymatic activities of either protein kinase displayed significant changes. When methylmercury was administered repeatedly (10 mg Hg/kg x 5), the mercury concentration was 11.7 ppm and the enzymatic activity of protein kinase C was reduced to 75% of the control level without significant change in [3H]PDBu binding. Significant change has not been observed in either [3H]cAMP binding or enzymatic activity of protein kinase A. The reduction of enzymatic activity of protein kinase C was reversed by the simultaneous administration of selenite (0.5 mgSe/kg x 5). However, the fact that selenite administration alone displayed not a significant but about a 20% increase in [3H]PDBu binding suggested that selenite itself could affect the level of protein kinase C despite having no apparent effects on protein kinase C in vitro. Further investigation is necessary to assess whether protein kinase C is involved in the detoxication mechanism of selenite with respect to methylmercury. Since the mercury concentration in the brain was higher than the IC50s for both protein kinase A and protein kinase C observed in vitro even after single administration, methylmercury might inhibit both protein kinases, which might impair intracellular signal transduction. This might in part conceal the symptoms during the early stages of methylmercury toxicity.

Mechanisms of Ca(2+)-independent contraction in single permeabilized ferret aorta cells.

The mechanisms by which prostaglandin F2 alpha (PGF2 alpha) can cause contractions at constant intracellular Ca2+ were investigated by the direct measurement of force from single saponin-permeabilized smooth muscle cells from the ferret aorta. The size of PGF2 alpha contractions did not change between pCa 9.0 and pCa 6.6. The remainder of the experiments were carried out at pCa 7.0. At pCa 7.0, PGF2 alpha (0.1-100 microM) induced sustained force in a dose-dependent manner, reaching a maximum (2.61 +/- 0.20 microN, n = 14) in 10 minutes. Both protein kinase C pseudosubstrate inhibitor (3 microM) and staurosporine (1 microM) significantly inhibited PGF2 alpha (100 microM)-induced contractions, but staurosporine was more effective. Staurosporine caused 88.8 +/- 13.3% inhibition, whereas protein kinase C pseudosubstrate inhibitor inhibited 62.3 +/- 9.6% of the PGF2 alpha-induced contraction. An inhibitor of type-1 and type-2A protein phosphatases, microcystin-LR, at a concentration of 1 microM induced a gradual and sustained contraction (1.53 +/- 0.21 microN). A lower concentration of microcystin-LR (100 nM) also induced a small but significant contraction (0.36 +/- 0.26 microN). Pretreatment with both 1 microM and 100 nM microcystin-LR caused significant inhibition of the PGF2 alpha-induced contraction from 2.61 +/- 0.20 microN (n = 14) to 0.32 +/- 0.20 microN (n = 6) (p < 0.01) and 1.52 +/- 0.21 microN (n = 6) (p < 0.01), respectively. These results indicate that the part of the PGF2 alpha-induced contraction that occurs at a constant, low intracellular Ca2+ is the combined result of activation of protein kinase C and phosphatase inhibition.

Regulation of vascular smooth muscle tone by caldesmon.

Caldesmon is an actin-binding protein present in smooth muscle cells that also inhibits actin-activated myosin ATPase activity. To assess the possible role of caldesmon in the regulation of smooth contraction, we investigated the effects of synthetic peptides on force directly recorded from single hyperpermeable smooth muscle cells of ferret aorta and portal vein. GS17C, a peptide that contains the residues from Gly651 to Ser667 of the caldesmon sequence plus an added cysteine at the C terminus, binds calmodulin in a Ca(2+)-dependent manner and also binds to F-actin but does not inhibit actomyosin ATPase activity (Zhan, Q., Wong, S.S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814). In cells in which Ca2+ was clamped at pCa 7.0, GS17C induced a dose-dependent contraction (EC50 = 0.92 microM) in aorta cells, whereas it evoked little or no contraction in portal vein cells. The GS17C-induced contraction in aorta cells was inhibited at higher Ca2+ concentrations (above pCa 6.6) and by pretreatment with calmodulin. Another peptide, C16AA, which contains the residues from Ala594 to Ala609 and does not bind actin or calmodulin, did not induce contraction. Our results strongly suggest that GS17C induces contraction by the displacement of the inhibitory region of endogenous caldesmon and, furthermore, that caldesmon present in these smooth muscle cells regulates contraction by providing a basal resting inhibition of vascular tone.

Effects of ryanodine on acetylcholine-induced Ca2+ mobilization in single smooth muscle cells of the porcine coronary artery.

To study the essential features of acetylcholine (ACh)- and caffeine-sensitive cellular Ca2+ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca2+ mobilization were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i) using Fura 2 in Ca(2+)-containing or Ca(2+)-free solution. The resting [Ca2+]i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca2+, 10 microM ACh or 128 mM K+ produced a phasic, followed by a tonic, increase in [Ca2+]i but 20 mM caffeine produced only a phasic increase. In Ca(2+)-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca2+]i rapidly decreased to 102 nM within 5 min, and 10 microM ACh or 20 mM caffeine (but not 128 mM K+) transiently increased [Ca2+]i. Ryanodine (50 microM) greatly inhibited the phasic increase in [Ca2+]i induced by 10 microM ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca2+. By contrast, ryanodine (50 microM) enhanced the tonic increase in [Ca2+]i induced by 128 mM K+ and also by 10 microM ACh in Ca(2+)-containing solution. In Ca(2+)-free solution containing 0.5 mM EGTA, ACh (10 microM) failed to increase [Ca2+]i following application of 20 mM caffeine. The level of [Ca2+]i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 microM ACh in Ca(2+)-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotransmission in the urinary bladder of rabbits and guinea pigs.

Alpha, beta-Methyleneadenosine 5'-triphosphate (alpha, beta-mATP) produced transient contraction of strips of bladder taken from rabbits or guinea pigs, and mechanical responses to field stimulation at 5-100 Hz were reduced by this drug by 5-20%. Atropine reduced responses by approximately 50%, and both drugs together by 80-95%. In double sucrose gap experiments on the rabbit bladder, alpha, beta-mATP selectively reduced but did not abolish an initial excitatory junction potential (ejp), and atropine selectively abolished a late depolarization. In the guinea pig, a single ejp was partially inhibited by either alpha,beta-mATP or atropine. Residual responses were further reduced by tetrodotoxin in both species. The initial ejp and late depolarization in the rabbit were reduced in parallel by hemicholinium over 2 h, suggesting that release of acetylcholine (ACh) and the second transmitter by nerves may be coupled. ACh but not ATP produced an increase in intracellular concentration of inositol trisphosphate in dispersed smooth muscle cells from the rabbit bladder; ATP but not carbachol produced a small transient current across the cell membrane in this species. It is concluded that ACh mobilizes intracellular Ca2+ for contraction, whereas the effect of ATP is dependent on extracellular Ca2+.

Action of mercurials on 3H-cAMP binding to the regulatory subunit-II of cAMP-dependent protein kinase.

Binding of the regulatory subunit type II (RII) of adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinase was inhibited by Hg2+ with an IC50 value of 0.31 microM. Methyl mercury, p-chloromercuribenzoic acid (PCMB), and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) also inhibited cAMP binding with IC50 values of 70-80 microM for organic mercurials and 130 microM for DTNB. Addition of 1 mM 2-mercaptoethanol and 1 M cysteine to the assay mixture reversed these inhibitions. N-ethylmaleimide (NEM) showed little effect on the binding. On the other hand, Hg2+ and methyl mercury markedly suppressed enzymatic activity of the catalytic subunit. The IC50 value was 0.13 microM for Hg2+ and 0.15 microM for organic mercurials. Scatchard plots of kinetic analysis data for the cAMP binding revealed a noncompetitive type of inhibition by mercurials and DTNB. It is suggested that blockade of sulfhydryl groups resulted in the inhibition of cAMP binding to the RII subunit, which might result in preserving the association of the RII subunit and the catalytic subunits and in preventing further inactivation of the catalytic subunit by Hg2+.

Actions of second messengers synthesized by various spasmogenic agents and their relation to mechanical responses in dog tracheal smooth muscle.

1. The effects of the spasmogenic agents, carbachol (CCh), histamine, 5-hydroxytryptamine (5-HT) and 9,11-epithio-11,12-methano-thromboxane A2 (STA2) were investigated on smooth muscle tissues of the dog trachea. 2. CCh (10 microM) produced a larger contraction than high K (128 mM), 10 microM histamine, 5-HT or STA2. Histamine and 5-HT produced the same amplitude of contraction as each other. In Ca-free solution containing 0.2 mM EGTA, only a phasic contraction was evoked by the above agents (except for K which induced no contraction at all). 3. In skinned muscle tissues, the maximum amplitude of contraction that could be induced by Ca (10 microM) was slightly larger than the maximum CCh-induced contraction (also at 10 microM) evoked in intact muscle tissues. Caffeine and inositol 1,4,5-trisphosphate (IP3) both produced contraction. 4. CCh, histamine and 5-HT (10 microM) produced a sustained contraction for over 30 min and also increased phosphorylation of the 20 kD protein of myosin light chain (MLC20) for over 30 min with no attenuation. Greater concentrations of the above agents caused more phosphorylation of MLC20. 5. CCh (above 1 nM), histamine (above 10 nM) and 5-HT (above 100 nM) increased the amount of IP3, in a concentration-dependent manner. Synthesis of IP3 induced by the above agents reached its peak value within 30 s and lasted for about 3 min. The potencies for the synthesis of IP3 were in the following order: CCh greater than histamine greater than 5-HT greater than STA2. 6. Isoprenaline (10 microM) markedly enhanced but CCh (10 microM) slightly reduced the amount of cyclic AMP. 5-HT (10 microM) and STA2 (10 microM) reduced, but histamine (10 microM) and CCh (1O microM) increased the amount of cyclic GMP. 7. Using fura 2, cytosolic Ca was measured by monitoring the ratio of the fluorescent signal excited at 340 and 380nm wavelengths in the presence of extracellular Ca. CCh (1O microM) increased the Ca transient from 182nm to 1.42 um. When the CCh-induced peak Ca transient (1O microM) was normalised, 10 microM histamine, 5-HT and STA2 showed smaller values such as 0.49, 0.53 and 0.04 times the control, respectively, and these values corresponded well with the amplitudes of contraction evoked by each of the stimulants. 8. The results can be summarized as follows: stronger spasmogenic responses occur on application of CCh than on application of 5-HT or histamine, and STA2 may have a minor role as a spasmogenic agent. The maximum amplitudes (peaks) of contraction evoked by the above spasmogenic agents are closely related to the maximum increase in cytosolic Ca, but sustained contraction and increased phosphorylation of myosin cannot be explained by the increased amount of Ca. In the case of 5-HT and histamine, synthesized cyclic nucleotides may interact with the action of 1P3 for the regulation of contraction in a positive or negative manner, respectively.

A widespread intramucosal naevus associated with hypertrophy of oral mucosa and alveolar bone. A case report.

A case of widespread intramucosal naevus with unusual clinical expression associated with hypertrophy of the oral mucosa and maxilla is presented. Hypertrophy was seen in the alveolar bone and all layers of the oral mucosa just beneath the pigmented area. The diagnosis and further examination is discussed.