All 40 tested isolates of M-type 57 group A streptococci gave the same, highly characteristic inhibitory pattern (P-type 614) when tested by deferred antagonism using a set of nine indicator strains. One component of this inhibitory activity was attributed to the production of a bacteriocinlike substance, streptococcin A-M57 (SA-M57). Production of SA-M57 was enhanced by the presence of blood and also by growth at an alkaline pH. Partially purified SA-M57 was obtained from culture supernatants by a combination of ammonium sulphate fractionation and column chromatography. On Sephadex G-100 two different molecular weight forms (SA-M57 alpha, greater than 100 000; SA-M57 beta, 33 000) were demonstrated. Both SA-M57 forms were protease and heat sensitive and had identical inhibitory activity against a collection of indicator bacteria. The adsorption to and rate of kill of sensitive cells by SA-M57 was enhanced in the presence of human plasma. Partially purified SA-M57 preparations were devoid of M-type 57 protein and curing studies showed that loss of SA-M57 did not correlate with loss of M protein production.

The effects of 20 mM aspirin (ASA), 20 mM sodium salicylate (SA), or 10(-4) M indomethacin placed in the nutrient solution (N) to stimulate systemic administration were investigated at pHN 7.3 in Ussing-chambered amphibian gastric mucosae. In histamine-stimulated tissues, the initial rise and subsequent rapid fall in potential difference, rise in resistance, and inhibition of hydrogen ion (H+) secretion induced by SAN did not occur with ASAN unless hydrolysis of ASAN produced a SAN of greater than 3 mM. In metiamide-treated tissues, 20 mM SAN caused an immediate fall in potential difference and an increase in resistance; 2 mM SAN and 20 mM ASA produced similar qualitative electrical changes, but only those induced by ASA were reversible. IndomethacinN caused no significant changes in potential difference, resistance, or H+ secretion in histamine- or metiamide-treated tissues. Despite producing highly significant reductions in generation of prostaglandin E2, and prostaglanndin F2 alpha and 6-keto prostaglandin F1 alpha, ASAN and indomethacin caused no surface ulceration. Sodium salicylate placed in the nutrient solution caused only a small reduction in prostaglandin F2 alpha, without change in the other prostaglandins, and produced extensive edema in the lamina propria, histologically. We conclude the following: (a) The inhibition of H+ secretion and electrical changes caused by SAN in histamine-treated gastric fundus are not observed with ASAN unless there is hydrolysis to [SAN] greater than 3 mM. (b) Our data strongly implicate the SAN in ASAN-containing solutions as being responsible for the electrical effects and inhibition of H+ secretion. (c) There is no correlation in vitro between inhibition of prostaglandin synthesis and the electrical or morphologic changes produced by nutrient exposure to ASA, SA, or indomethacin.

Several in vivo studies demonstrated that tumor metastasis depend on the expression of carbohydrate Lewis structures. Lewis antigens and their derivatives such as Lewis b (Leb), Lewis X (LeX), sialyl Lewis X (SA-LeX), sialyl Lewis a (SA-Lea), and Lewis Y (LeY) were identified as tumor-associated structures approximately 20 years ago by Koprowski et al. using hybridoma technology and showed that upregulation and/or de novo expression of these determinants on the tumor cell surface is associated with a poor prognosis. LeX and SA-LeX are ligands for selectin adhesion molecules; E- and P-selectins are vascular receptors expressed on activated endothelial cells (ECs) and L-selectin is expressed on leukocytes. Leukocytes also express on their surface LeX and SA-LeX determinants, which are involved in the initial steps of extravasation, that is, rolling, which is alpha step mediated by interaction with E-selectin on ECs. We hypothesized that the tumor cells transmigration from the bloodstream to metastatic sites is similar to lymphocyte extravasation and that adhesion of cancer cells in analogy with the lymphocyte rolling is mediated by interaction of carbohydrate determinants on tumor cells with selectins on ECs. To assess the role of interaction of carbohydrate structures with E-selectin in metastatic process in vivo, we demonstrated that the peptides mimicking SA-Lea blocked colonization of tumor cells in experimental model of lung metastasis in vivo. Furthermore, the metastases formation was completely attenuated in E-selectin-knock out (KO) mice demonstrating the importance of selectin-mediated interaction in this process. We also showed that a peptide mimicking SA-Lea E-selectin ligand has an ability to significantly reduce neutrophil recruitment into peritoneal cavity in acute inflammatory conditions. These studies support the hypothesis that the interaction of tumor cells via the carbohydrate SA-Lea determinant and E-selectin constitutes the important step in the metastatic process in analogy with lymphocyte extravasation and that carbohydrate antigen mimics have a potential as anti-inflammatories and anti-adhesive tumor therapeutics.

OBJECTIVE

To evaluate the reliability and validity of the Chinese version of addiction severity index (ASI)-5th version (ASI-C-5), in illegal drug users receiving methadone maintenance treatment (MMT) in China.

METHODS

One hundred and eighty-six heroin addicts (144 men and 42 women) receiving MMT at three clinics in Guizhou province, southwest China, were recruited. They were all interviewed with a questionnaire of ASI-C-5 and 35 were re-interviewed at an interval of seven days to assess its test-retest reliability.

RESULTS

Cronbach's alpha for internal consistency of CSs varied from 0.60 to 0.81 in all domains. Test-retest reliability of composite scores (CSs) of ASI-C-5 were satisfactory (r=0.38-0.97). Based on item analysis and expert's suggestions, five items were deleted and one item was modified in ASI-C-5. Criterion validity of ASI-C-5 was found acceptable, as compared to addicts' self-rating anxiety scale (SAS) and self-rating depression scale (SDS) (r=0.59 and 0.45) except for social support rating scale (SSRS).

CONCLUSIONS

ASI-C-5 can be used for heroin addicts receiving MMT with acceptable reliability and validity.

Fumonisins are fungal metabolites and suspected human carcinogens. They inhibit ceramide synthase in vitro, enhance tumor necrosis factor alpha (TNFalpha) production, and cause apoptosis. Fumonisin B1 (FB1) was fed to rats and mice for 2 years or, in separate studies, given to rats or mice for up to 4 weeks. Kidney tubule adenomas and carcinomas were found in male rats fed>> or = 50 ppm, whereas liver adenomas and carcinomas were found in female mice fed>> or = 50 ppm for 2 years. In the short-term studies, increases in tissue concentration of the ceramide synthase substrate sphinganine (Sa) and the Sa to sphingosine (So) ratio were correlated with apoptosis. Further, hepatotoxicity was ameliorated in mice lacking either the TNFR1 or the TNFR2 TNFalpha receptors. Thus, FB1 was carcinogenic to rodents and thefindings support the hypothesis that disrupted sphingolipid metabolism and TNFalpha play important roles in its mode of action.

Autoreactivity plays a major role in the pathogenesis of RA. The rheumatoid factor has been and still is for now more than 50 years the only autoreactivity that is clinically applied in the diagnosis of RA. This well reflects the current way of thinking that a single antigen or a single cause drives an individual into disease. Although by now many other autoantigens and autoreactivities have been described, their discovery was always on the search for the one and only autoreactivity that causes RA. This includes also immune reactivities directed against xenogenic antigens. But, none of the known RA-associated autoreactivities is present in all RA patients and none of them occurs exclusively in RA. Thus, the observed sensitivities and specificities are well below 100%. Therefore, RA has often been postulated to consist of various immunological subentities with similar clinical symptoms. Nevertheless, none of the autoreactivities correlates with a distinct clinical feature or course of disease. It is about time to say good-bye to the idea that a single antigen or immunoreactvity causes and maintains rheumatoid arthritis. In this paper we present RA as the clinical outcome of an immune system that has shifted from a healthy to an autoimmune steady state. This is accomplished by many different reactivities and autoreactivities that occur either in parallel or one after the other. The entirety of the known RA-associated reactivities and (auto)antigens is presented in detail. The major RA-relevant autoantigens comprise BiP, citrulline, the Sa-antigen, hnRNP A2, p205, IgG, calpastatin, calreticulin, collagen and the shared HLA-DR epitope. The accumulation of factor--involving autoreactivities, cytokines, environmental and genetic factors--that challenge the normal regulatory mechanisms of the immune system lead to a regulatory catastrophe. In individuals developing the clinical features of RA the immune system has been regulated to a new--autoimmune--steady state. This attractor "rheumatoid arthritis" has many features of what has originally been described by Irun Cohen as the immunological homunculus: The healthy immune system is configured such as to direct its attention to major self-antigens. Thus it creates an autoreactivity to many autoantigens as a prerequisite for regulatory mechanisms that are sufficient to control them. The shift from the normal to rheumatoid attractor involves the inflammatory cytokines TNF-alpha, IL-1 and IL-6, autoreactive T- and B cells directed at a variety of synovial and systemic antigens, activated dendritic cells and macrophages, tissue destruction and genetic factors such as the association with shared epitope. Environmental factors involved may also, but do not necessarily, include infection. With the appearance of clinical features of RA, naive, potentially autoreactive T cells infiltrate the synovial compartment and become activated by dendritic cells and other APCs. The autoantigenic peptides that are presented to these T cells are derived from inflammatory cell and tissue destruction as well as from tissue repair and remodeling processes. These T cells proliferate and either provide help to B cells with the specificity to the same antigens or cause direct cytopathic tissue damage. Thereby, more and novel antigens are generated, released and presented again to naive or primed autoreactive T cells. These processes involving cytokines, tissue destruction and autoreactive T cells are sufficient to maintain RA even without the permanent presence of a triggering agent. The recursive autoimmune processes are well consistent with the finding of the many different autoreactivities in RA and their respective sensitivities and specificities. The massive influx of T cells into the arthritic joint is accompanied by the anergization of over 90% of T cells in this compartment--which further substantiates the concept of the RA attractor within the self-regulating immune system. Thereby, the RA-attracted immune system is not able to completely downregulate the inflammation and the local tissue damage/repair. Thus, the immune system is permanently stimulated and suddenly by chance shifts to a stable state different from the healthy system--reaching the wide fields of rheumatoid arthritis which in itself is self-sustaining as the healthy state before disease onset.

Leukotrienes (LTs) are lipid mediators derived from arachidonic acid (AA) involved in a number of autoimmune/inflammatory disorders including asthma, allergic rhinitis and cardiovascular diseases. Salvinorin A (SA), a diterpene isolated from the hallucinogenic plant Salvia divinorum, is a well-established analgesic compound, but its anti-inflammatory properties are under-researched and its effects on LT production is unknown to date. Here, we studied the possible effect of SA on LT production and verified its actions on experimental models of inflammation in which LTs play a prominent role. Peritoneal macrophages (PM) stimulated by calcium ionophore A23187 were chosen as in vitro system to evaluate the effect of SA on LT production. Zymosan-induced peritonitis in mice and carrageenan-induced pleurisy in rats were selected as LT-related models to evaluate the effect of SA on inflammation as well as on LT biosynthesis. SA inhibited, in a concentration-dependent manner, A23187-induced LTB4 biosynthesis in isolated PM. In zymosan-induced peritonitis, SA inhibited cell infiltration, myeloperoxidase activity, vascular permeability and LTC4 production in the peritoneal cavity without decreasing the production of prostaglandin E2. In carrageenan-induced pleurisy in rats, a more sophisticated model of acute inflammation related to LTs, SA significantly inhibited LTB4 production in the inflammatory exudates, along with reducing the phlogistic process in the lung. In conclusion, SA inhibited LT production and it was effective in experimental models of inflammation in which LTs play a pivotal role. SA might be considered as a lead compound for the development of drugs useful in LTs-related diseases.

Hypoxia inducible factor-1α (HIF-1α) is an essential regulator of the cellular response to low oxygen concentrations, activating a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1α is overexpressed in various cancers and therefore represents a considerable chemotherapeutic target. Salternamide A (SA), a novel small molecule that is isolated from a halophilic Streptomyces sp., is a potent cytotoxic agent against a variety of human cancer cell lines. However, the mechanisms by which SA inhibits tumor growth remain to be elucidated. In the present study, we demonstrate that SA efficiently inhibits the hypoxia-induced accumulation of HIF-1α in a time- and concentration-dependent manner in various human cancer cells. In addition, SA suppresses the upstream signaling of HIF-1α, such as PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3 signaling under hypoxic conditions. Furthermore, we found that SA induces cell death by stimulating G2/M cell cycle arrest and apoptosis in human colorectal cancer cells. Taken together, SA was identified as a novel small molecule HIF-1α inhibitor from marine natural products and is potentially a leading candidate in the development of anticancer agents.

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has proven to be as sweet as native two-chain monellin. SCM is more stable than the native monellin for both heat and acidic environments. Data from gel filtration HPLC and NMR indicate that the SCM exists as a monomer in aqueous solution. The solution structure of SCM has been determined by nuclear magnetic resonance (NMR) spectroscopy and dynamical simulated annealing calculations. A stable alpha-helix spanning residues Phe11-Ile26 and an antiparallel beta-sheet formed by residues 2-5, 36-38, 41-47, 54-64, 69-75, and 83-88 have been identified. The sheet was well defined by backbone-backbone NOEs, and the corresponding beta-strands were further confirmed by hydrogen bond networks based on amide hydrogen exchange data. Strands beta2 and beta3 are connected by a small bulge comprising residues Ile38-Cys41. A total of 993 distance and 56 dihedral angle restraints were used for simulated annealing calculations. The final simulated annealing structures ((SA)k) converged well with a root-mean-square deviation (rmsd) between backbone atoms of 0.49 A for secondary structural regions and 0.70 A for backbone atoms excluding two loop regions. The average restraint energy-minimized (REM) structure exhibited root-mean-square deviations of 1.19 A for backbone atoms and 0.85 A for backbone atoms excluding two loop regions with respect to 20 (SA)k structures. The solution structure of SCM revealed that the long alpha-helix was folded into the concave side of a six-stranded antiparallel beta-sheet. The side chains of Tyr63 and Asp66 which are common to all sweet peptides showed an opposite orientation relative to H1 helix, and they were all solvent-exposed. Residues at the proposed dimeric interface in the X-ray structure were observed to be mostly solvent-exposed and demonstrated high degrees of flexibility.

alpha-Conotoxin MII, isolated from Conus magus, is a potent peptidic toxin which specifically targets the mammalian neuronal nicotinic acetylcholine receptor, alphaalpha-conotoxin MII in aqueous solution has been determined by two-dimensional 1H NMR spectroscopy. NOE-derived distances, refined by an iterative relaxation matrix approach, as well as dihedral and chirality restraints were used in high-temperature biphasic simulated annealing calculations. Fourteen minimum energy structures out of 50 subjected to the SA simulations were chosen for evaluation; these 14 structures have a final RMS deviation of 0.76 +/- 0.31 and 1.35 +/- 0.34 A for the backbone and heavy atoms, respectively. The overall structure is unusually well-defined due to a large helical component around the two disulfide bridges. The principal backbone folding motif may be common to a subclass of alpha-conotoxins. There are two distinct surfaces on the molecule almost at right angles to one another. One entirely consists of the hydrophobic residues Gly1, Cys2, Cys3, Leu15, and Cys16. The second comprises the hydrophilic residues Glu11, His12, Ser13, and Asn14. These surfaces on the ligand could be essential for the subtype-specific recognition of the receptor.

OBJECTIVE

Sleep disturbances are prevalent but often overlooked or underestimated. We suspected that sleep disorders might be particularly common among pharmacy customers, and that they could benefit from counselling. Therefore, we described the prevalence and severity of symptoms associated with sleep and wakefulness disorders among Swiss pharmacy customers, and estimated the need for counselling and treatment.

METHODS

In 804 Swiss pharmacies (49% of all community pharmacies) clients were invited to complete the Stanford Sleep Disorders Questionnaire (SDQ), and the Epworth Sleepiness Scale (EPW). The SDQ was designed to classify symptoms of sleep and wakefulness into the four most prevalent disorders: sleep apnoea syndrome (SAS), insomnia in psychiatric disorders (PSY), periodic leg movement disorders/restless legs (RLS) and narcolepsy (NAR). Data were entered into an internet-linked database for analysis by an expert system as a basis for immediate counselling by the pharmacist.

RESULTS

Of 4901 participants, 3238 (66.1%) were female, and 1663 (33.9%) were male. The mean age (SD) of females and males was 52.4 (18.05), and 55.1 (17.10) years, respectively. The percentages of female and male individuals above cut-off of SDQ subscales were 11.4% and 19.8% for sleep apnoea, 40.9% and 38.7% for psychiatric sleep disorders, 59.3% and 46.8% for restless legs, and 10.4% and 9.4% for narcolepsy respectively. The prevalence of an Epworth Sleepiness Scale score >11 was 16.5% in females, and 23.9% in males. Reliability assessed by Cronbach's alpha was 0.65 to 0.78 for SDQ subscales, and for the Epworth score.

CONCLUSIONS

Symptoms of sleep and wakefulness disorders among Swiss pharmacy customers were highly prevalent. The SDQ and the Epworth Sleepiness Scale score had a satisfactory reliability to be useful for identification of pharmacy customers who might benefit from information and counselling while visiting pharmacies. The internet-based system proved to be a helpful tool for the pharmacist when counselling his customers in terms of diagnostic classification and severity of symptoms associated with the sleeping and waking state.

The Revised Social Anhedonia Scale (SAS) with 40 items (Eckblad et al., 1982) which studies the social dimension of anhedonia has been validated in the United-States (Mishlove & Chapman, 1985). However, no french translation and validation of this scale has been made to date. This work presents the french translation of the Social Anhedonia Scale and its validation. After a back-translation and final adjustment, it has been submitted to a sample of 126 control subjects from the general population. Furthermore, they were asked to fill two other scales: the Chapman Physical Anhedonia (PAS) with 61 items and the Fawcett Pleasure Scale (36 items), both of them exploring the subjects answer in terms of anhedonia/hedonia towards social, sensorial and/or physical experiences. The internal validity has been determined on the one hand by the Cronbach alpha coefficient which showed a strong unidimensional characteristic (0.80) and the other hand by the correlation of each item with the total score using the point biserial coefficient which ranged from .204 to .559. The concurrent validation has been determined by the Pearson correlation coefficient between the french version of social anhedonia scale and the french version of physical anhedonia scale. The values were .42, p = .001. Furthermore, this two scales are significant inversely correled to the french version of the pleasure scale: r = -.22, p = .0125 for the first, and r = -.26, p = .0027 for the second. The internal and concurrent validity of the french version of the revised social anhedonia scale should allow to improve our understanding of anhedonia in psychiatry and psychopathology.

IL-6 is a pleiotropic cytokine with still controversial role in tumorigenesis of different cancer types. Its promoter SNP-174 C/G is associated with increased IL-6 transcription and in some tumor types with elevated IL-6 serum levels. The role of IL-6 polymorphisms and IL-6 serum values and their correlation in the gastroenteropancreatic neuroendocrine tumors is lacking. We investigated for the first time frequencies of IL-6-174 genotypes in 80 GEP-NET patients and 162 age- and sex-matched healthy controls, serum values of IL-6 in GEP-NET patients and their correlation with IL-6-174 genotypes. To analyze IL6-174 C/G polymorphism PCR-NlaIII RFLP method was used, and serum levels were measured on Immulite analyzer by enzymatic solid-phase chemiluminescent immunometric method. Serum IL-6 values were elevated (>5.9 pg/ml) in 36.8% GEP-NET patients. Differences in genotypes distribution between patients and healthy controls as well as between patients with gastrointestinal and pancreatic neuroendocrine tumors (PETs) and functioning and nonfunctioning PETs were tested by chi(2) test and Fisher's Exact test. Analysis of variance (ANOVA with proc GLM in SAS/Stat) was performed for the group comparison. Level of significance was alpha=0.05. Patients with nonfunctioning PETs had only high expression IL-6-174 CG and GG genotypes and according to genotypes differed significantly (p=0.0289) from functioning PETs. High serum IL-6 values in all GEP-NET patients correlated significantly with GG IL-6-174 genotype (p=0.0139). Nonfunctioning PET patients had significantly (p=0.000777) higher IL-6 serum values in comparison to patients with functioning PETs and gastrointestinal NETs. Serum IL-6 values correlated significantly with IL-6-174 genotypes in nonfunctioning PETs and gastrointestinal NETs (p<0.05), but not in functioning PETs.

Intracellular redistribution of beta-catenin through mutation of the adenomatous polyposis coli (APC) gene has been proposed as an early tumorigenic event in most colorectal tumours. In serrated adenoma (SA), a newly recognised subtype of colorectal adenoma, APC mutations are uncommon, and the contribution of beta-catenin to tumorigenesis remains unclear. We compared intracellular localisation of beta-catenin and presence of mutations in exon 3 of beta-catenin between 45 SAs, with 71 conventional adenomas (CADs), and eight carcinomas invading the submucosa (SCAs). Widespread or focal nuclear beta-catenin expression was demonstrated in 7% of SAs (three out of 45), 61% of CADs (43 out of 71), and 88% of SCAs (seven out of eight). Cytoplasmic immunostaining for beta-catenin was demonstrated in 16% of SAs (seven out of 45), 77% of CADs (55 out of 71), and 88% of SCAs (seven out of eight). No mutation in exon 3 of beta-catenin was found in SAs or SCAs, while 7% of CADs (five out of 71) had beta-catenin mutations. No nuclear or cytoplasmic expression of beta-catenin was observed in the hyperplastic or conventionally adenomatous epithelium of mixed-type SAs. These findings suggest that beta-catenin mutation is unlikely to contribute to the tumorigenesis in SA, and that intracellular localisation of beta-catenin may not be associated with an early event of the tumour progression in most SAs.

The changes in serum concentrations of cytokines such as interleukin-1 (IL-1) beta, interleukin-6 (IL-6), tumor necrosis factor (TNF) alpha and a soluble-intercellular adhesion molecule (sICAM-1) has been investigated in patients with stable angina and acute myocardial infarction. Thirty-four patients with stable angina (SA), 15 with acute myocardial infarction (AMI), and 20 subjects in the control (C) group were included in the study. The mean serum concentrations of sICAM-1, IL-1-beta, IL-6, and TNF-alpha differed significantly among the three groups. Serum concentrations of IL-1 beta, sICAM-1, and TNF-alpha were comparable in the AMI and SA groups and higher than those found in the C group (p < 0.001). The serum concentration of IL-6 was more than twice as high in the AMI group as compared to the other two groups (p < 0.001). The mean serum concentrations of IL-1 beta, TNF-alpha, and IL-6 were comparable in the AMI and SA groups and higher than in the C group.

Sarcoidosis (SA) and diffuse interstitial fibrosis (DIF) are characterized by alveolitis, mast cell hyperplasia and increased fibroblast proliferation. Stem cell factor (SCF) stimulates proliferation of hematopoietic progenitor cells involved in mast and stromal cell interaction. We assessed the role of SCF secreted by alveolar fibroblasts (AFb) in the development of fibrosis of DIF and SA in six patients with SA and six patients with DIF. Bronchoalveolar lavage (BAL) was performed by conventional methods. A total of 500 cells were differentially counted from Giemsa-stained cytopreps. AFb and supernatants were recovered from long-term cultures of BAL cells and from 24 h cultures of confluent AFb. Levels of SCF were measured by ELISA. Alpha actin content of AFb was characterized by immunohistochemistry. The expression of AFb mRNA for IL1-alpha and beta, TGF-beta, IFN-gamma, IL-2, IL-4, IL-5 and IL-6 was determined by RT-PCR. There was a lymphocytic predominance in the SA patients and an increase in neutrophils and eosinophils in DIF. SCF secreted by AFb from DIF was significantly higher than in SA. TNF + IL-1 significantly decreased the secretion of SCF by AFb. There was a positive correlation between SCF levels and the percentage eosinophils but not for metachromatic cells. Alpha-actin expression of AFb in DIF was significantly higher than in SA. Cytokine mRNA was extracted from AFb of two SA and two DIF patients. The profile showed that only in stimulated AFb isolated from the DIF patients can IL-5 transcripts be visualized. In conclusion, AFb can contribute to the onset of fibrosis by secreting SCF and IL-5 which, in turn, may recruit eosinophils.

The objectives of this study were to evaluate the in vitro acaricidal effects of lyophilized extracts of four tannin rich plants (Acacia pennatula, Piscidia piscipula, Leucaena leucocephala and Lysiloma latisiliquum) against diverse stages of Rhipicephalus (Boophilus) microplus, and to asses whether tannins were involved in the acaricidal effect using polyethylene glycol (PEG) to block tannins. Larval immersion (LIT) and adult immersion (AIT) tests were used to evaluate the acaricidal effect of each of the lyophilized extracts against larval and adult stages of R. microplus respectively. Larvae and adult ticks were exposed to increasing concentrations of each plant extract (0, 1200, 2400, 4800, 9600 and 19,200 μg ml(-1)) for 10 min. Larval mortality was recorded at 48 h post-incubation. Adult mortality was recorded daily over 14 days, at which point their reproductive efficiency was evaluated. PEG was added to the extracts to verify whether tannins were involved in the acaricidal effect. The effect on egg laying inhibition and larval mortality was analyzed using the GLM procedure in SAS. A Kruskal-Wallis test was used to assess the effect of PEG on LIT results. Calculation of the lethal concentration 50 (LC50) was performed using a probit analysis. All extracts reduced the viability of R. microplus larval stages (P<0.001), and viability was restored with the addition of PEG suggesting an important role of tannins in the acaricidal effect (P<0.001). The LC50 values of L. latisiliquum and P. piscipula plant extracts were 6.402 and 2.466 μg ml(-1). None of the tannin-rich plant extracts affected adult mortality (P>0.05). Lysiloma latisiliquum extract inhibited egg hatching of R. microplus (P<0.01). Tannin-rich plant extracts from A. pennatula, P. piscipula, L. leucocephala and L. latisiliquum showed potential acaricidal activity. Further in vivo studies are needed to confirm this finding.

OBJECTIVE

To investigate hepatic stellate cells (HSC) responses at different differentiation stages on transforming growth factor-beta 1, and to elucidate the mechanisms of salvianolic acid - B (SA-B), a water soluble compound from Salvia miltiorrhiza, against hepatic fibrosis, relating to interference with TGF-beta 1 stimulated HSC activation and intracellular signal transduction via Smads.

METHODS

HSC was isolated from rat by in situ perfusion of liver and 8.2% nycondenz gradient centrifugation, and primarily cultured on uncoated plastic for 1 d, 4 d and 7 d respectively, which represented quiescent, intermediate and activated phenotypes. The cells were stimulated with 100 pmol/L TGF-beta 1, cell phenotypes were observed under inverted microscope, alpha-actin expression was checked by Western blot, and collagen secretion was measured with [(3)H] proline incorporation and collangenase digestion, then HSC at one definite differentiation stage that responded most sensitively to TGF-beta 1 was selected as the cell model for the following study. 0.1 micromol/L - 1 mmol/L SA-B was incubated with HSC and the cell proliferation was measured by intracellular [(3)H] thymidine pulse. SA-B was also incubated with TGF-beta 1 stimulated HSC, the collagen secretion was measured as above, alpha-actin and plasmin activator inhibitor-1 (PAI-1) were checked with Western blot, and alpha1 (I) procollagen mRNA levels were analyzed with Northern blot. The cytoplasmic and nuclear proteins were extracted, and cytoplasmic and nuclear Smad2, 3 expression and phosphorylation levels were measured with Western blot.

RESULTS

As culture duration prolonged, HSC phenotypes underwent activation gradually, accompanied by the increase of alpha-actin expression and collagen secretion. TGF-beta 1 increased the basal collagen levels at d1, d4 and d7 by 128.6%, 207.7% and 188.2% of the control respectively, while d4 HSC had the most sensitive response, and this intermediate HSC was used as cell model for the following study. Except 0.1 mmol/L-1 mmol/L SA-B caused parts of HSC death, 0.1 micromol/L-10 micromol/L SA-B had no influence on cell shape, but decreased HSC proliferation in a dose depend manner, by 76%, 60.1% and 47.8% of the control respectively. 1 micromol/L-10 micromol/L SA-B remarkably inhibited the collagen secretion of TGF-beta 1 stimulated HSC by 68.6% and 56.1% of the control, PAI-1 and alpha-actin expression, and down-regulated alpha 1 (I) pro-collagen gene expression. 0.1 micro mol/L approximately 10 micro mol/L SA-B decreased the cytoplasmic and nuclear Smad2, 3 protein expression, especially inhibited Smad2 phosphorylation and nuclear translocation.

CONCLUSIONS

SA-B obviously inhibits intermediate HSC proliferation, decreases TGF-beta 1 stimulated HSC activation and matrix protein and gene expression, and inhibited stimulated HSC Smad2, 3 protein expression, phosphorylation and nuclear translocation. The inhibition of TGF-beta 1 signaling in HSC and its biological responses is the important mechanism of SA-B against hepatic fibrosis.

Inter-alpha-inhibitor (IalphaI) is a serine proteinase inhibitor found in high concentrations in human plasma. The protein is composed of a light inhibitory chain called bikunin and two heavy chains of unknown function. The three polypeptide chains are covalently assembled via a carbohydrate cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherfurd, S., & Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751]. The aim of this study was to complete the primary structure by characterizing additional covalent posttranslational modifications of the heavy chains. Analysis revealed three N-linked oligosaccharides located on Asn251 and Asn554 of heavy chain 1 and on Asn64 of heavy chain 2: all these were complex biantennary structures composed of (Asn)-GlcNAc2-Man-(Man-GlcNAc-Gal-SA)2. In addition, the IalphaI heavy chains carried several O-linked glycans located on Thr619 of heavy chain 1 and a cluster of four O-linked oligosaccharides on Thr612, Ser619, Thr621, and Thr637 of heavy chain 2. The oligosaccharides were short (Ser/Thr)-GalNAc-Gal-SA trisaccharides. The IalphaI heavy chains contain nine Cys residues, of which eight are involved in disulfide bridges. The unpaired Cys residue residing on heavy chain 1, Cys26, appears to be modified by dihexosylation. The other Cys residues exclusively form intrachain disulfide bridges. In heavy chain 1 the two disulfide bonds are formed between Cys210 and Cys213 and between Cys234 and Cys506, and in heavy chain 2, between Cys207 and Cys210 and between Cys596 and Cys597. Interestingly, three of these four disulfides are formed between Cys residues that are either adjacent or only two amino acid residues apart.

The precentral extension of area 3 as well as the transition between the frontal operculum and insula (area G) comprises the primary gustatory cortex in the subhuman primate, receiving projections from the thalamic taste relay. However, in contrast to the extensive studies that have been carried out on the latter area, only a few taste units in the former area have been recorded. To clarify gustatory coding in area 3, we investigated the taste response properties of neurons in area 3 compared with those in area G in alert monkeys by infiltrating into their mouths seven taste stimuli [0.3 M sucrose (S), 0.1 M NaCl (N), 0.01 N HCl (H), 0.003 M quinine-HCl (Q), 0.1 M monosodium glutamate (MSG), distilled water (W), and orange juice (OR)] and artificial saliva (SA). A larger number of HCl-best units and a smaller number of quinine-best units were found in area 3 than in area G. The onset latency and response duration were significantly shorter in area 3 than in area G. Weighted multi-dimensional scaling showed that area G divided eight stimulants into four classes, i.e. two groups (H-Q-W and S-MSG-OR), N and SA, whereas area 3 divided them into three classes (N-MSG-W-OR, S-Q, and H-SA). This suggested that tastants not separated in area G were separated in area 3, and vice versa. This indicates that both areas complement each other in the representation of taste stimuli, each contributing to taste information processing in a different manner.

We describe the application of the molecular dynamics (MD) and molecular mechanics-generalized Born/surface area (MM-GB/SA) approaches to the simulation of the different biological activity of diethylstilbestrol (DES) on two highly homologous nuclear receptors-estrogen receptor alpha (ER-alpha) and estrogen-related receptor gamma (ERR-gamma). DES exerts an agonistic effect against ER-alpha and an antagonistic effect against ERR-gamma. Using the x-ray crystal structures of ER-alpha in the canonical agonist bound form (PDB code: 3ERD) and antagonist bound form (PDB code: 3ERT), ERR-gamma homology models have been constructed for the receptor in two different conformations. MM-GB/SA binding free energy calculations of DES in the ER-alpha and ERR-gamma structures suggest that DES exhibits a greater free energy of binding in the agonist bound conformation of ER-alpha, while the antagonist bound conformation is preferred for ERR-gamma. Further dissection of the free energy contributions coupled with calculation of the ligand binding pocket volume suggests that the van der Waals interactions for DES within the smaller binding pocket volume of ERR-gamma are less favorable and this is the main factor for DES antagonism in ERR-gamma. This approach has potential general applicability to the prediction of the biological activity of nuclear receptor ligands.

Cryptococcal meningitis is an important fungal infection among systemic lupus erythematosus patients. We conducted a pooled analysis and systematic review to describe the epidemiological and clinical profile of cryptococcal meningitis in systemic lupus erythematosus patients. From two hospitals in China and nine literature databases, cases and prevalence data were collected for pooled analysis and meta-analysis, respectively. Categorical variables of cases were compared using a χ(2)-test on the statistical program of SAS. A multiple regression analysis was performed to ascertain independent predictors significantly correlated with prognosis. Meta-analysis was conducted by the statistical program of R. The prevalence of cryptococcal meningitis in systemic lupus erythematosus patients was 0.5%. Patients were predominantly females and adults. A prednisone equivalent of more than 30 mg/day before infection was associated with higher mortality (odds ratio (OR)=9.69 (1.54, 60.73)). In all, 36.8-38.9% patients showed low lupus activity when they developed the crytococcal infection. Moreover, 38.2% of the patients were misdiagnosed. The estimated case-fatality rate was 23.6%. Our results suggest that more emphasis should be placed to further understand lupus-related cryptococcal meningitis and to develop better prophylaxis and management strategies to combat this condition.

When CHO cells are arrested in S-phase, they undergo repeated rounds of centrosome duplication without cell-cycle progression. While the increase is slow and asynchronous, the number of centrosomes in these cells does rise with time. To investigate mechanisms controlling this duplication, we have arrested CHO cells in S-phase for up to 72 h, and coordinately inhibited new centriole formation by treatment with the microtubule poison colcemid. We find that in such cells, the pre-existing centrosomes remain, and a variable number of foci--containing alpha/gamma-tubulin and centrin 2--assemble at the nuclear periphery. When the colcemid is washed out, the nuclear-associated foci disappear, and cells assemble new centriole-containing centrosomes, which accumulate the centriole scaffold protein SAS-6, nucleate microtubule asters, and form functional mitotic spindle poles. The number of centrosomes that assemble following colcemid washout increases with duration of S-phase arrest, even though the number of nuclear-associated foci or pre-existing centrosomes does not increase. This suggests that during S-phase, a cryptic generative event occurs repeatedly, even in the absence of new triplet microtubule assembly. When triplet microtubule assembly is restored, these cryptic generative events become realized, and multiple centriole-containing centrosomes assemble.

Antimutagens from gaiyou (Artemisia argyi Levl. et Vant., Compositae) were examined. The methanol extract prepared from aerial parts of this plant strongly reduced the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), when Salmonella typhimurium TA98 was used in the presence of the rat liver microsomal fraction. The antimutagens were purified chromatographically while monitoring the antimutagenic activity against Trp-P-2 with a modified Ames test employing a plate method. This purification resulted in the isolation of four strong antimutagens, 5,7-dihydroxy-6,3',4'-trimethoxyflavone (eupatilin), 5, 7,4'-trihydroxy-6,3'-dimethoxyflavone (jaceosidin), 5,7, 4'-trihydroxyflavone (apigenin) and 5,7, 4'-trihydroxy-3'-methoxyflavone (chrysoeriol) from the methanol extract. These antimutagenic flavones exhibited strong antimutagenic activity against not only Trp-P-2 but also against other heterocyclic amines, such as 3-amino-1,4-dimethyl-5H-pyrido[4, 3-b]indole (Trp-P-1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA(alpha)C) in S. typhimurium TA98. In contrast, they did not exhibit antimutagenic activity against benzo[a]pyrene (B[a]P), 4-nitroquinoline-1-oxide (4-NQO), 2-aminofluorene (2-AF), 2-nitrofluorene (2-NF) or furylfuramide (AF-2) in S. typhimurium TA98, or B[a]P, 4-NQO, 2-NF, AF-2, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium azide (SA) in Salmonella typhimurium TA100, whereas they decreased the mutagenicity caused by aflatoxin B(1) (AFB(1)) and 2-aminoanthracene (2-AA) in both of these tester strains. Regarding the structure-activity relationship, the tested flavones had distinct differences in the intensities of their antimutagenic activities according to the differences of their substitution patterns. Namely, the intensity of antimutagenic activities against Trp-P-2 decreased in the order of: 5,7,3',4'-tetrasubstituted flavones (IC(50): <0.1 mmol/plate), 5,7,4'-trisubstituted flavones (IC(50): 0.120-0.260 mmol/plate), 5,6,7,3',4'-pentasubstituted flavones (IC(50): 0.440-0. 772 mmol/plate). The four isolated flavones were also studied regarding their antimutagenic mechanisms with preincubation methods of the modified Ames test and emission spectroscopic analysis. The results suggested that all isolated flavones were desmutagens which directly inactivated Trp-P-2 or inhibited its metabolic activation.