IL-4 mediated by HSV vector suppresses morphine withdrawal response and decreases TNFα, NR2B, and pC/EBPß in the periaqueductal gray in rats.

Chronic opiates induce the development of physical dependence. Opioid physical dependence characterized by withdrawal symptoms, may have very long-lasting effects on the motivation for reward, including the incubation of cue-induced drug-seeking behavior. Elucidation of the mechanisms involved in physical dependence is crucial to developing more effective treatment strategies for opioid dependence. Chronic morphine induces production of proinflammatory cytokines in regional-specific sites of the brain. Interleukin-4 (IL-4) is a prototypical anti-inflammatory cytokine that globally suppresses proinflammatory cytokines. Here, we used recombinant herpes simplex virus vector S4IL4 that encode mouse il4 gene to evaluate the therapeutic potential of IL-4 in naloxone-precipitation morphine withdrawal (MW). One week after microinjection of the vector S4IL4 into the PAG LacZ or mouse IL-4 immunoreactivity in the vlPAG was visualized. ELISA assay showed that vector S4IL4 into the PAG induced the expression of IL-4. S4IL4 blunted the morphine withdrawal syndrome. S4IL4 suppressed the upregulated TNFα, NR2B and pC/EBPß in the PAG induced by MW. These results show that inhibition of proinflammatory factor in the PAG suppressed MW. This study may provide a novel therapeutic approach to morphine physical withdrawal symptoms.

Evaluation of skin cancer chemoprevention potential of sunscreen agents using the Epstein-Barr virus early antigen activation in vitro assay.

In our continuing search for novel cancer chemopreventive compounds of natural and synthetic origin, we have evaluated 14 commonly used ultraviolet (UV) sunscreen agents (designated UV-1 to UV-14) for their skin cancer chemoprevention potential. They belong to 8 different chemical categories: aminobenzoate (UV-5, UV-7, UV-8 and UV-14), benzophenone (UV-1, UV-2, UV-3 and UV-13), benzotriazole (UV-10), benzyloxyphenol (UV-9), cinnamate (UV-6), quinolone (UV-4), salicylate (UV-11) and xanthone (UV-12). In the in vitro assay employed, the sunscreens were assessed by their inhibition of the Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in human lymphoblastoid Raji cells. All sunscreens tested were found to exhibit anti-tumour promoting activity: listed in decreasing order, moderate (UV-11, UV-2, UV-7, UV-12, UV-3, UV-9 and UV-14) to weak (UV-1, UV-6, UV-8, UV-16, UV-5, UV-4 and UV-10) with octyl salicylate (UV-11) as the most potent and drometrizole (UV-10) as the least potent among the compounds evaluated. A plausible relationship between the antioxidant property of sunscreens and their ability to promote anti-tumour activity was noted. The results call for a comprehensive analysis of skin cancer chemoprevention potential of currently used UV sunscreen agents around the globe to identify those with the best clinical profile.

X-ray waveguide mode in resonance with a periodic structure.

We present a novel concept for x-ray waveguiding based on electromagnetism in photonic crystals, using a waveguide consisting of a pair of claddings sandwiching a core with a periodic structure. By confining the x rays undergoing multiple interference in the core by total reflection, a characteristic waveguide mode whose field distribution matches the periodicity of the core is formed. The distinctively low propagation loss enables the single-mode propagation of x rays. This concept opens broad application possibilities in x-ray physics from coherent imaging to x-ray quantum optics.

Effects of transglucosidase on diabetes, cardiovascular risk factors and hepatic biomarkers in patients with type 2 diabetes: a 12-week, randomized, double-blind, placebo-controlled trial.

In this 12-week, randomized, double-blind, placebo-controlled trial, the efficacy and safety of transglucosidase (TGD) were compared with placebo in patients with type 2 diabetes mellitus (T2DM). At 12 weeks, TGD 300 mg/day and TGD 900 mg/day significantly reduced HbA1c (0.18 and 0.21%) and insulin concentration (19.4 and 25.0 pmol/l), respectively, vs. placebo. TGD 300 mg/day and TGD 900 mg/day also significantly reduced low-density lipoprotein cholesterol (0.22 and 0.17 mmol/l, respectively). TGD 900 mg/day significantly reduced triglyceride by 0.24 mmol/l and diastolic blood pressure by 8 mmHg. Placebo was associated with a significant increase from baseline in body mass index, alanine aminotransferase and aspartate aminotransferase (0.17 kg/m(2) , 3 and 2 U/l, respectively), whereas TGD was not. TGD 300 mg/day significantly increased high-molecular-weight adiponectin by 0.6 µg/ml. Adverse events did not differ significantly between the groups. TGD resulted in lowering of HbA1c and blood insulin level and improvements in metabolic and cardiovascular risk factors in T2DM.

Cephalometric evaluation after two-stage palatoplasty combined with a Hotz plate: a comparative study between the modified Furlow and Widmaier-Perko methods.

The effects on craniofacial growth of two different soft palate repair techniques in two-stage palatoplasty were investigated. This was a retrospective, cross-sectional cohort study of 68 children with non-syndromic, complete unilateral cleft lip and palate. Thirty-four patients were treated with the modified Furlow method (F-group) and the remaining 34 with the Widmaier-Perko method (P-group). Craniofacial growth was assessed by analyzing 12 angular and 12 linear measurements on lateral cephalograms. Composite facial diagrams from the two groups were compared with those of a control non-cleft group. Angular and linear measurements did not differ significantly between the two groups, implying that the craniofacial morphology was not affected by the difference in soft palate repair technique. However, small differences in anterior nasal spine and posterior nasal spine were found in cleft patients compared with controls. These findings suggest that the modified Furlow and Widmaier-Perko methods have a similar impact on craniofacial growth. Considering speech function, the modified Furlow method provides better craniofacial growth and speech function. However, the long-term effects of both methods on craniofacial growth after growth cessation remain to be determined.

Role of the Gln/Glu residues of trichocellins A-II/B-II in ion-channel formation in lipid membranes and catecholamine secretion from chromaffin cells.

Trichocellins (TC) A-II and B-II, 20-residue peptaibols isolated from conidia of the fungus Trichoderma viride, have the same sequence except for the residue at position 18. Both TCs were found to form voltage-dependent ion-channels in bilayer lipid membranes (BLM) and to induce catecholamine secretion from bovine adrenal chromaffin cells through Ca2+ influx. TC-A-II (Gln18, neutral) was more effective than TC-B-II (Glu18, charged) for macroscopic current induction in BLMs and for catecholamine secretion from chromaffin cells, suggesting that Glu18 is unfavorable for the ion-channel formation in BLMs and chromaffin cell membranes. Nevertheless, single-channel recordings indicated that TC-B-II forms larger pores with longer open lifetimes than those of TC-A-II. This indicates that the negatively charged carboxyl group of Glu at position 18 stabilizes larger pores. The effects of the negative charge of Glu18 on the activities were confirmed by the use of a TC-B-II analog containing the methyl ester of Glu18.

Asymmetrical membrane fluidity of bovine adrenal chromaffin cells and granules and effect of trichosporin-B-VIa.

We examined membrane fluidity of bovine adrenal chromaffin cells and chromaffin granules using cationic trimethylammonium derivative of diphenylhexatriene (TMA-DPH) as a fluorescence probe. After adding TMA-DPH to the suspension of chromaffin cells and that of granules, it first bound to the outer layer of the plasma membrane of the cells and that of the granule membrane, then gradually penetrated the inner layer of each membrane and distributed to both leaflets of the respective membranes. Accompanying increases in the ratio of incorporated probe on the cytoplasmic side of the chromaffin cell membrane, its fluorescence anisotropy gradually decreased. However, in chromaffin granules, the fluorescence anisotropy gradually increased with increases in the ratio of incorporated probe. These findings suggest that the inner layer of the plasma membrane and outer layer of the granular membrane are more fluid than the corresponding side of each membrane, which is suitable for the fusion between both membranes. We also examined the effect of trichosporin-B-VIa, a fungal ion channel forming alpha-aminoisobutyric acid-containing peptide, on the fluidity of chromaffin cells using TMA-DPH. The peptide decreased the fluorescence anisotropy and increased the fluorescence intensity in the concentration range that induced Ca2+ dependent catecholamine secretion, suggesting that a change in lipid dynamics of the lipid bilayer of the plasma membrane was induced by this peptide.

Role of proline residue in the channel-forming and catecholamine-releasing activities of the peptaibol, trichosporin-B-VIa.

Trichosporin-B-VIa (TS-B-VIa) has a Pro14-kinked helical structure which is considered to be important for the formation of peptaibol-type ion-channels in lipid bilayer membranes. TS-B-VIa and its analog [Aib14]TS-B-VIa with Pro-->Aib substitution at position 14, resulting in a straight helical structure, were tested for ion-channel-forming activity in planar lipid bilayer membranes and for ability to induce catecholamine secretion from cultured bovine adrenal chromaffin cells. Voltage-dependent multi-channel conductance, which is characteristic of TS-B-VIa, was also observed for [Aib14]TS-B-VIa. In single-channel measurements, current fluctuations induced by [Aib14]TS-B-VIa had a shorter life-time and showed fewer substates than those induced by TS-B-VIa. Catecholamine secretion induced by these peptides at low concentrations is completely Ca(2+)-dependent. At high concentrations, TS-B-VIa-induced secretion was partly independent of external Ca2+, but this was not the case for the analog. The differences of behavior can be explained in terms of the differences of hydrophobicity, and magnitude of dipole moment due to the conformational changes around position 14 and the C-terminal domain caused by the Pro-->Aib substitution.

Pathway for Ca2+ influx into cells by trichosporin-B-VIa, an alpha-aminoisobutyric acid-containing peptide, from the fungus Trichoderma polysporum.

Trichosporin (TS) -B-VIa, a fungal alpha-aminoisobutyric acid (Aib) -containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both 45Ca2+ influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 microM) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 microM) was partly independent of the Ca2+. The concentration-response curves (2-5 microM) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 microM) -induced secretion was not antagonized by diltiazem, a blocker of L-type voltage-sensitive Ca2+ channels. The treatment of fura-2-loaded C6 glioma cells with TS-B-VIa (2-5 microM) led to an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+]i were only slightly observed in Ca(2+)-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C6 cells. The TS-B-VIa-induced increase in [Ca2+]i in the C6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+]1 in the C6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chrornaffin cells) and non-excitable cells (the C6 cells and the platelets) and that TS-B-VIa per se may form Ca(2+)-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes.

Responses of Xenopus laevis water nose to water-soluble and volatile odorants.

Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents, and inward currents in response to water-soluble and volatile odorants from receptor neurons in the lateral diverticulum (water nose) of the olfactory sensory epithelium of Xenopus laevis. The resting membrane potential was -46.5 +/- 1.2 mV (mean +/- SEM, n = 68), and a current injection of 1-3 pA induced overshooting action potentials. Under voltage-clamp conditions, a voltage-dependent Na+ inward current, a sustained outward K+ current, and a Ca2+-activated K+ current were identified. Application of an amino acid cocktail induced inward currents in 32 of 238 olfactory neurons in the lateral diverticulum under voltage-clamp conditions. Application of volatile odorant cocktails also induced current responses in 23 of 238 olfactory neurons. These results suggest that the olfactory neurons respond to both water-soluble and volatile odorants. The application of alanine or arginine induced inward currents in a dose-dependent manner. More than 50% of the single olfactory neurons responded to multiple types of amino acids, including acidic, neutral, and basic amino acids applied at 100 microM or 1 mM. These results suggest that olfactory neurons in the lateral diverticulum have receptors for amino acids and volatile odorants.

Structural homology between DNA binding sites of DNA polymerase beta and DNA topoisomerase II.

Unsaturated long-chain fatty acids selectively bind to the DNA binding sites of DNA polymerase beta and DNA topoisomerase II, and inhibit their activities, although the amino acid sequences of these enzymes are markedly different from each other. Computer modeling analysis revealed that the fatty acid interaction interface in both enzymes has a group of four amino acid residues in common, forming a pocket which binds to the fatty acid molecule. The four amino acid residues were Thr596, His735, Leu741 and Lys983 for yeast DNA topoisomerase II, corresponding to Thr79, His51, Leu11 and Lys35 for rat DNA polymerase beta. Using three-dimensional structure model analysis, we determined the spatial positioning of specific amino acid residues binding to the fatty acids in DNA topoisomerase II, and subsequently obtained supplementary information to build the structural model.

Gene transfer into intact plant cells by electroinjection through cell walls and membranes.

Tobacco mosaic virus (TMV) RNA was introduced directly into mesophyll cells of Nicotiana tabacum var. Samsun using electric-field pulses (electroinjection). The injected gene was successfully expressed in the recipient cells as judged by the assay for the virus coat protein using immunofluorescence and by the virus infectivity assay of the homogenate of the electroinjected cells for local lesions on tobacco leaves. As much as 50% of the cells that survived 24 days after electroinjection showed immunofluorescent specks.

Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein.

We previously established a novel mouse model for human aging and identified the genetic foundation responsible for it. A defect in expression of a novel gene, termed klotho (kl), leads to a syndrome resembling human aging in mice. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to beta-glucosidases. In this report, we present the entire mouse kl gene organization. The mouse kl gene spans about 50 kilobases and consists of five exons. The promoter region lacks a TATA-box and contains four potential binding sites for SP1. We further show that two kl gene transcripts encoding membrane or secreted protein are generated through alternative transcriptional termination. These findings provide fundamental information for further study of the kl gene which may regulate aging in vivo.

Homologous cysteine proteinase genes located on two different chromosomes from Trypanosoma rangeli.

DNA fragments were obtained by the polymerase chain reaction (PCR) using genomic DNA from T. rangeli as template and oligonucleotide primers encoding the active site amino acids of cysteine proteinase. After amplification by PCR, several DNA products were observed. These were purified and used as templates for a second round of PCR. This resulted in two DNA products of 475 and 498 bp. The 498 bp DNA (Tr-CP) contained both the sense and antisense primer sequences, and encoded a polypeptide having substantial homology with eukaryotic cysteine proteinases. The other product (Tr-DMR), which lacked the antisense primer sequence, encoded a polypeptide having homology with the DNA mismatch repair gene from Saccharomyces cerevisiae. The overall homology of the Tr-CP-encoded polypeptide to the cysteine proteinases from other trypanosome species was 69% identity (T. cruzi) and 73% identity (T. brucei), respectively. Northern blot analysis revealed that the Tr-CP gene was specifically expressed in T. rangeli as a 1.7 kb mRNA. A karyotype map of the chromosomes was also performed using pulsed-field gradient gel electrophoresis and Southern blot hybridization with these genes. T. rangeli has 14 chromosome bands ranging from 350 kb to 1.6 Mb, which were fewer in number and smaller in size compared with those from T. cruzi. The Tr-CP fragment and the Tr-DMR fragment hybridized with equal intensity on chromosome 1 (350 kb) and chromosome 2 (470 kb), respectively. These results suggest that non-pathogenic T. rangeli contains a conserved gene corresponding to the cysteine proteinase or a closely related enzyme, and that there is more than one copy of this gene, each found on different chromosomes.

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.

Gastroesophageal disease and nausea: does fundoplication help or hurt?

HYPOTHESIS: Nausea associated with gastroesophageal reflux disease is cured by laparoscopic Nissen fundoplication (LNF). DESIGN: Prospective cohort study of unselected patients who underwent LNF from January 1, 1995, through March 31, 1999. Patients were followed up by a physician for 6 to 36 months. SETTING: A large community teaching hospital. PATIENTS: One hundred consecutive patients with gastroesophageal reflux disease who underwent LNF; all patients were followed up. Patients were grouped according to the presence (group A, n = 33) or absence (group B, n = 67) of preoperative nausea. Interventions were LNF, esophageal manometry, 24-hour pH monitoring, and nuclear gastric emptying studies. MAIN OUTCOME MEASURES: Resolution of symptoms after LNF. RESULTS: Nausea was the most common atypical symptom of gastroesophageal reflux disease, occurring in 33 patients (33%). There were no differences in esophageal manometry or 24-hour pH results between groups. There was a female preponderance in group A (55% vs 33%; P = .003). Patients in group A had a higher prevalence of preoperative dysphagia (P = .02). Patients with persistent postoperative nausea had a higher prevalence of cough (P = .003) and dysphagia (P = .009). The LNF was more effective in reducing heartburn (95% reduction) and regurgitation (95% reduction) than cough and dysphagia (60% reduction). There was a 79% reduction in the number of patients with nausea (33 to 7; P<.001). CONCLUSION: Laparoscopic Nissen fundoplication is effective in eliminating nausea associated with gastroesophageal reflux disease and is not contraindicated in these patients.

Laparoscopic common bile duct exploration: long-term outcome.

HYPOTHESIS: Transcystic laparoscopic common bile duct exploration (LCBDE) with biliary endoscopy results in excellent long-term clinical outcome and patient satisfaction. DESIGN: Prospective cohort study of unselected patients found to have common bile duct stones during laparoscopic cholecystectomy between October 1989 and April 1998. A mailed survey assessed symptoms, outcome, and satisfaction. SETTING: A large community teaching hospital. PATIENTS: Two hundred seventeen patients with common bile duct stones. INTERVENTION: Transcystic LCBDE with choledochoscopy. MAIN OUTCOME MEASURES: Success of LCBDE, morbidity, postoperative symptoms, and satisfaction. RESULTS: One hundred sixteen surveys (54%) were returned. Mean follow-up was 60 months. The LCBDE procedure failed in 6 patients and endoscopic retrograde cholangiopancreatography was performed in 4 patients (3%). One patient had unsuspected retained stones. No patient had late recognition of retained stones or a bile duct stricture. Abdominal pain was present in 90 patients (89%) preoperatively and in 29 patients (26%) postoperatively (P = .001). The LCBDE procedure reduced 3 specific pain profiles: epigastric, from 47% (n = 54) to 7% (n = 8); back, from 31% (n = 36) to 6% (n = 7); and shoulder, from 18% (n = 21) to 2% (n = 2). When pain persisted, it was different in character in 15%. All nonpain symptoms (such as nausea, bloating, indigestion, and gas) were reduced from 78% (n = 91) to 34% (n = 39) (P = .001) except diarrhea. Diarrhea was present in 24 patients (22%) preoperatively and postoperatively, though it was a new postoperative symptom in 11 patients (11%). One hundred two patients (95%) were satisfied or mostly satisfied with LCBDE. CONCLUSIONS: Pain and nonpain symptoms, while reduced significantly after LCBDE, may persist. The LCBDE procedure does not result in common bile duct strictures or a significant rate of retained stones. This relatively new treatment for common bile duct stones is safe and effective.

Fungal metabolites. XXI. Characteristics of low energy collision induced dissociation of [M + 2H]2+, [M + H + Na]2+ and [M + 2Na]2+ of peptaibols using electrospray ionization mass spectrometry.

The mass spectral characteristics of selected peptaibols were investigated using electrospray ionization in conjunction with low-energy collision-induced dissociation (CID). Protonated and sodiated molecular species were selected as precursor ions and the resulting CID spectra are discussed with respect to fragmentation characteristics related to the structures of peptaibols. The CID spectra of peptaibols with acetylated N-terminus and with the sub-sequences of Aib-Pro were similar. The CID spectra of [M + 2Na]2+ provided structural information more than those of [M + 2H]2+ and [M + H + Na]2+. The CID of [M + 2Na]2+ can be used to deduce complete sequence information for peptaibols.

Transient expression of the beta-glucuronidase gene in tissues of Arabidopsis thaliana by bombardment-mediated transformation.

Particle bombardment has proved to be useful for the transformation of plants. We have previously reported successful transient expression of the beta-glucuronidase (GUS) gene in cultured plant cells and tissues and the stable transformation of various plants using a pneumatic particle gun. In this chapter, we describe transient expression of the GUS gene in Arabidopsis thaliana leaves and roots using the pneumatic particle gun.

Physiological mechanism for enhancement of paracellular drug transport.

We examined the action mechanisms of enhancers that improve paracellular drug transport. For sodium caprate (C10), the increase in the intracellular calcium level was considered to induce the contraction of calmodulin-dependent actin filaments, followed by dilation of the paracellular pathway. Although decanoylcarnitine (DC) also increased the intracellular calcium level, the action was independent of calmodulin and thus, the action mechanism of acylcarnitines was considered to differ from that of C10. Other acylcarnitines, lauroylcarnitine (LC) and palmitoylcarnitine (PC) and organic acids, tartaric acid (TA) and citric acid (CA) decreased the intracellular ATP level and the intracellular pH. From these results, it was considered that one of the action mechanism of acylcarnitines and organic acids is that the intracellular acidosis increases the calcium level through the decrease in ATP levels, followed by opening the tight junction. Membrane dysfunction which was expected from the above mechanism was assessed by the transport function of electrolytes. Membrane conductance, which was increased by C10, LC and PC, returned to the control value during a 3- to 6-h recovery period. On the other hand, Cl(-) ion secretion, which was obtained from short-circuit current (I(sc)), was decreased by these enhancers, but was normalized by C10 but not by LC and PC. Accordingly, C10 can be considered a safer enhancer than acylcarnitines.