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Objective: To describe the effects of hysterectomy on the anterior and posterior vaginal compartments and specific pelvic organ prolapsed quantification (POP-Q) data points., Methods: In a retrospective case-control study, data were analyzed from patients presenting at a urogynecologic center in Manhasset, USA, with urogenital prolapse between January 1, 2008, and December 31, 2010. Patients who underwent hysterectomy for indications not including prolapse were compared with age- and parity-matched women without prior hysterectomy. POP-Q points were measured and converted to stages for each vaginal compartment. The t test, Wilcoxon rank sum, and Fisher exact tests were used to compare normally distributed variables, nonparametric data, and categoric variables, respectively., Results: Seventy-one patients who underwent prior hysterectomy were matched to 71 patients with intact uteri. Women with prior hysterectomy had significantly greater prolapse at POP-Q point Bp and worse overall posterior compartment prolapse compared with those without prior hysterectomy. Women who underwent hysterectomy were more likely to have stage 2 or greater prolapse (OR 2.6, 95% CI, 1.3-5.2; P=0.01)., Conclusion: When matched for age and parity, women with prior hysterectomy had significantly greater posterior compartment prolapse compared with those without. Prior hysterectomy had no significant effect on the anterior vaginal compartment., (Copyright © 2012 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.)

INTRODUCTION: Lynch syndrome was first described in the 1950s however until recently it was rarely included in medical school curricula. As a result, many practicing physicians have limited exposure, potentially contributing to significant under diagnosis. As identification of Lynch syndrome prior to malignancy allows for intensified screening, prophylactic surgery and improved patient outcomes, all physicians should be aware of the characteristics of affected families. We aim to determine the overall level of awareness of Lynch syndrome among medical students at an American medical school. METHODS: A voluntary and anonymous questionnaire was delivered to students at an American medical school. The survey instrument assessed the respondent's perceived knowledge regarding the genetics and recommended screening for carriers of Lynch syndrome mutations. RESULTS: The questionnaire was distributed to the entire student body (405 students) with a response rate of 50%. Fifty-nine percent of students reported that they had learned about Lynch syndrome; 27% of first year students, 44% of second year students; 90% of third year students and 100% of fourth year students. Of the students familiar with Lynch syndrome, the reported knowledge of the underlying genetics was 46%, available genetic screening, 18%, criteria used to screen for the syndrome, 24%, recommendations for colon screening, 31% and recommendations for endometrial cancer screening, 17%. CONCLUSION: The majority of medical students surveyed had been exposed to Lynch syndrome and awareness increased over each year of education. Significantly more students were aware of recommendations for colon cancer screening than endometrial cancer screening (32% versus 17%, p = 0.01). Studies of the natural history of Lynch syndrome indicate that affected women are more likely to present with endometrial cancer than colon cancer and while there are no prospective data proving the efficacy of endometrial cancer screening in this high-risk population, the endometrium is easily accessible and can be sampled using simple office techniques. In addition, prophylactic hysterectomy and bilateral salpingo-oophorectomy are reasonable risk reducing interventions for the prevention of both uterine and ovarian cancer. Our findings suggest that increased emphasis must be placed on teaching the gynecologic manifestations of Lynch Syndrome in order to avoid the misconception that it is simply a colon cancer syndrome.

Aim: To evaluate the effect of age, body mass index (BMI), and parity on Pelvic Organ Prolapse Quantification (POP-Q) system measurements in women with symptomatic prolapse., Material and Methods: The charts of 603 women with symptomatic prolapse were reviewed. Patients with prior prolapse surgery or hysterectomy were excluded. POP-Q measurements were recorded by a single examiner. Data were analyzed using descriptive statistics, the Pearson's product-moment correlation, and Wilcoxon signed rank test., Results: Three hundred and eleven patients met inclusion criteria. Mean age was 63.4 years, mean parity was 2.6, and mean body mass index (BMI) was 26.6 kg/m(2) . Increasing age was correlated with worsening scores on Ba (r = 0.33, P < 0.0001), C (r = 0.14, P < 0.02), D (r = 0.14, P = 0.02) and Bp (r = 0.13, P = 0.02), while parity was only correlated with worsening scores on Aa (r = 0.12, P = 0.04). There was no correlation between BMI and any POP-Q data point., Conclusion: The effect of increasing parity was seen solely on point Aa in women with symptomatic prolapse. Age affected all vaginal compartments, while BMI had no impact on POP-Q data points., (© 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.)

Aims: To compare quality of life in patients with mixed urinary incontinence (MUI) to stress urinary incontinence (SUI) after treatment with a retropubic midurethral sling and concomitant prolapse repair., Methods: Sixty-three patients met inclusion criteria. Patients completed the short form of the urogenital distress inventory (UDI-6) questionnaire pre- and postoperatively. Concomitant procedures included anterior/posterior colporrhaphy, paravaginal repair, LeForte, vaginal hysterectomy, and sacrospinous ligament fixation., Results: Preoperatively, those with MUI had significantly higher UDI-6 scores (59.8 ± 16.6 vs. 42.3 ± 14.5, respectively, P < 0.0001). Postoperatively, there was no difference in UDI-6 scores (P = 0.87). Subjective cure rate for SUI was the same in patients with MUI versus SUI (73%). Patients with MUI demonstrated a significantly greater improvement in UDI-6 scores as compared to patients with SUI., Conclusions: Patients with MUI compared with SUI demonstrated a greater improvement in quality of life after midurethral sling placement and concomitant pelvic organ prolapse repair as assessed by a disease-specific quality of life questionnaire (UDI-6)., (Copyright © 2011 Wiley Periodicals, Inc.)

Objective: : The objective of the study was to determine the prevalence of microscopic hematuria (MH) in women with pelvic organ prolapse., Study Design: : Data of all patients presenting to a urogynecologic center with pelvic organ prolapse between 2008 and 2010 were reviewed. Microscopic hematuria was defined as 3 or more red blood cells per high-power field on at least 2 urinalysis specimens with negative cultures. Exclusion criteria included age younger than 40 years, presence of gross hematuria, menses, or urologic disease. Statistical analysis was performed using JMP 4.0.4 (SAS Institute, Cary, NC)., Results: : The population consisted of 572 women aged 40 to 96 years. Fifty-five patients (9.6%) met the criteria for MH, and 126 (22%) had 3 or more red blood cells per high-power field on 1 urinalysis. No cases of urologic malignancy were diagnosed on further workup. All patients with microhematuria had cystoceles on examination., Conclusions: : Our findings suggest that the prevalence of MH is increased in women with pelvic organ prolapse.

Objective: To compare changes in quality of life for women undergoing either suprapubic or transvaginal midurethral sling placement for stress urinary incontinence (SUI)., Methods: A retrospective cohort study was conducted involving 113 patients who underwent transvaginal or suprapubic midurethral sling placement for SUI over 2 years at a US institution. All patients completed a short form of the urogenital distress inventory (UDI-6) questionnaire preoperatively and postoperatively. Preoperative and postoperative UDI-6 scores were compared between the 2 groups. Data were analyzed using the Mann-Whitney U test and the Fisher exact test., Results: A midurethral sling was placed using the suprapubic approach in 70 patients, and 43 patients underwent the procedure using the transvaginal approach. Preoperatively, there was no statistical difference between mean UDI-6 scores for the suprapubic versus the transvaginal group (48.5 versus 51.0 P = 0.9). Postoperatively, patients who underwent a suprapubic procedure had significantly lower mean UDI-6 scores than those who underwent a transvaginal procedure (10.6 versus 17.0; P < 0.005)., Conclusion: Suprapubic midurethral sling placement was associated with a significantly greater quality of life postoperatively compared with the transvaginal procedure., (Copyright © 2011 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.)

Objective: The purpose of this study was to evaluate the significance of a thickened endometrial echo in an asymptomatic, postmenopausal patient., Study Design: A retrospective review was conducted of all women who underwent transvaginal ultrasonography between January 2003 and August 2008, were found to have an endometrial thickness of at least 5mm and were subjected to endometrial sampling., Results: Sixty-five postmenopausal women, without vaginal bleeding underwent ultrasonographic evaluation with subsequent endometrial sampling. The mean endometrial stripe thickness was 9.7 mm (range: 5.4-22). Four (6.2%) cases of simple/complex hyperplasia were identified and two (3.1%) cases of atypical hyperplasia were diagnosed. Zero (0%) specimens were identified as adenocarcinoma. Twenty-eight (43.1%) polyps and eleven (16.9%) leiomyomata were identified., Conclusion: The use of transvaginal sonography as a screening tool in this population is not validated and need not trigger routine evaluation., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

In vitro metabolism of methadone was investigated in cytochrome P450 (CYP) supersomes and phenotyped human liver microsomes (HLMs) to reconcile past findings on CYP involvement in stereo-selective metabolism of methadone. Racaemic methadone was used for incubations; (R)- and (S)-methadone turnover and (R)- and (S)-EDDP formation were determined using chiral liquid chromatography-tandem mass spectrometry. CYP supersome activity for methadone use and EDDP formation ranked CYP2B6 > 3A4 > 2C19 > 2D6 > 2C18, 3A7 > 2C8, 2C9, 3A5. After abundance scaling, CYP3A4, 2B6 and 2C19 accounted for 63-74, 12-32 and 1. 4-14% of respective activity. CYP2B6, 2D6 and 2C18 demonstrated a preference for (S)-EDDP formation; CYP2C19, 3A7 and 2C8 for (R)-EDDP; 3A4 none. Correlation analysis with 15 HLMs supported the involvement of CYP2B6 and 3A. The significant correlation of S/R ratio with CYP2B6 activity confirmed its stereo-selectivity. CYP2C19 and 2D6 inhibitors and monoclonal antibody (mAb) did not inhibit EDDP formation in HLM. Chemical and mAb inhibition of CYP3A in high 3A activity HLM reduced EDDP formation by 60-85%; inhibition of CYP2B6 in 2B6 high-activity HLM reduced (S)-EDDP formation by 80% and (R)-EDDP formation by 55%. Inhibition changed methadone metabolism in a stereo-selective manner. When CYP3A was inhibited, 2B6 mediated (S)-EDDP formation predominated; S/R stereo-selectivity increased. When 2B6 was inhibited (S)-EDDP formation fell and stereo-selectivity decreased. The results confirmed the primary roles of CYPs 3A4 and 2B6 in methadone metabolism; CYP2C8 and 2C9 did not appear involved; 2C19 and 2D6 have minimal roles. CYP2B6 is the primary determinant of stereo-selective metabolism; stereo-selective inhibition might play a role in varied plasma concentrations of the two enantiomers., (© 2010 The Authors. Basic & Clinical Pharmacology & Toxicology © 2010 Nordic Pharmacological Society.)

Background: This study was conducted to examine the pharmacokinetic interactions between buprenorphine/naloxone (BUP/NLX) and lopinavir/ritonavir (LPV/r) in HIV-seronegative subjects chronically maintained on BUP/NLX., Methods: This study was an open labeled pharmacokinetic study in twelve HIV-seronegative subjects stabilized on at least 3 weeks of BUP/NLX therapy. Subjects sequentially underwent baseline and steady-state pharmacokinetic evaluation of once-daily LPV/r (800/200 mg)., Results: Compared to baseline values, BUP AUC0-24h (46.8 vs. 46.2 ng*hr/mL) and Cmax (6.54 vs. 5.88 ng/mL) did not differ significantly after achieving steady-state LPV/r. Similar analyses of norBUP, the primary metabolite of BUP, demonstrated no significant difference in norBUP AUC0-24 hours (73.7 vs. 52.7 ng x h/mL); however, Cmax (5.29 vs. 3.11 ng/mL) levels were statistically different (P < 0.05) after LPV/r administration. Naloxone concentrations were similarly unchanged for AUC0-24 hours (0.421 vs. 0.374 ng x hr/mL) and Cmax (0.186 vs. 0.186 ng/mL). Using standardized measures, no objective opioid withdrawal was observed. The AUC0-24 hours and Cmin of LPV in this study did not significantly differ from historical controls (159.6 vs. 171.3 microg x hr/mL) and (2.3 vs. 1.3 microg/mL)., Conclusions: The addition of LPV/r to stabilized patients receiving BUP/NLX did not affect buprenorphine pharmacokinetics but did increase the clearance of norbuprenorphine. Pharmacodynamic responses indicate that the altered norbuprenorphine clearance did not lead to opioid withdrawal. Buprenorphine/naloxone and LPV/r can be safely coadministered without need for dosage modification.

HIV-infected patients with opioid dependence often require opioid replacement therapy. Pharmacokinetic interactions between HIV therapy and opioid dependence treatment medications can occur. HIV-seronegative subjects stabilized on at least 3 weeks of buprenorphine/naloxone (BUP/NLX) therapy sequentially underwent baseline and steady-state pharmacokinetic evaluation of open-label, twice daily tipranavir 500 mg co-administered with ritonavir 200 mg (TPV/r). Twelve subjects were enrolled and 10 completed the study. Prior to starting TPV/r, the geometric mean BUP AUC(0-24h) and C(max) were 43.9 ng h/mL and 5.61 ng/mL, respectively. After achieving steady-state with TPV/r (> or = 7 days), these values were similar at 43.7 ng h/mL and 4.84 ng/mL, respectively. Similar analyses for norBUP, the primary metabolite of BUP, demonstrated a reduction in geometric mean for AUC(0-24h) [68.7-14.7 ng h/mL; ratio=0.21 (90% CI 0.19-0.25)] and C(max) [4.75-0.94 ng/mL; ratio=0.20 (90% CI 0.17-0.23)]. The last measurable NLX concentration (C(last)) in the concentration-time profile, never measured in previous BUP/NLX interaction studies with antiretroviral medications, was decreased by 20%. Despite these pharmacokinetic effects on BUP metabolites and NLX, no clinical opioid withdrawal symptoms were noted. TPV steady-state AUC(0-12h) and C(max) decreased 19% and 25%, respectively, and C(min) was relatively unchanged when compared to historical control subjects receiving TPV/r alone. No dosage modification of BUP/NLX is required when co-administered with TPV/r. Though mechanistically unclear, it is likely that decreased plasma RTV levels while on BUP/NLX contributed substantially to the decrease in TPV levels. BUP/NLX and TPV/r should therefore be used cautiously to avoid decreased efficacy of TPV in patients taking these agents concomitantly.

We tested the hypothesis that primary cultures of human hepatocytes could predict potential drug interactions with methadone and buprenorphine. Hepatocytes (five donors) were preincubated with dimethyl sulfoxide (DMSO) (vehicle), rifampin, or nelfinavir before incubation with methadone or buprenorphine. Culture media (0-60 min) was analyzed by liquid chromatography-tandem mass spectrometry for R- and S-methadone and R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) or for buprenorphine, norbuprenorphine, and their glucuronides [buprenorphine-3-glucuronide (B-3-G) and norbuprenorphine-3-glucuronide (N-3-G)]. R- and S-EDDP were detected in three of five, four of five, and five of five media from cells pretreated with DMSO, nelfinavir, and rifampin. R-EDDP increased 3.1- and 26.5-fold, and S-EDDP increased 2.5- and 21.3-fold after nelfinavir and rifampin. The rifampin effect was significant. B-3-G production was detected in media of all cells incubated with buprenorphine and accounted for most of the buprenorphine loss from culture media; it was not significantly affected by either pretreatment. Norbuprenorphine and N-3-G together were detected in three of five, four of five, and five of five donors pretreated with DMSO, nelfinavir and rifampin, and norbuprenorphine in one of five, one of five, and two of five donors. Although there was a trend for norbuprenorphine (2.8- and 4.9-fold) and N-3-G (1.7- and 1.9-fold) to increase after nelfinavir and rifampin, none of the changes were significant. To investigate low norbuprenorphine production, buprenorphine was incubated with human liver and small intestine microsomes fortified to support both N-dealkylation and glucuronidation; N-dealkylation predominated in small intestine and glucuronidation in liver microsomes. These studies support the hypothesis that methadone metabolism and its potential for drug interactions can be predicted with cultured human hepatocytes, but for buprenorphine the combined effects of hepatic and small intestinal metabolism are probably involved.

The prevalence of allergic disorders is increasing around the world. This study used the standardized written questionnaire from International Study of Asthma and Allergies in Childhood (ISAAC, phase III) to collect two cross-sectional appraisals of schoolchildren 5 years apart-one performed 2002 and another on 2007, to focus on the secular trends in prevalence of allergies in central Taiwan. This questionnaire was administered to schoolchildren from over 46 schools across Changhwa County in 2002, and again in 2007. The present study is a follow-up study and uses data collected from a previously reported study conducted in the year 2002 of the same location using the same methodology. Although the repeated cross-sectional survey after a 5-year lapse suggests a mere increase of diagnosed asthma cases from 7.1% to 7.4%, the prevalence of its related symptoms--nocturnal cough, rhinitis, and eczema--was found to increase significantly by 2.6% (p < 0.0001), 6.8% (p < 0.001), and 5.9% (p < 0.001), respectively. Additionally, comparing the secular trends in asthma prevalence, rhinitis, and eczema between several polluted areas in Changhwa County with its less polluted neighbors in 2002 and 2007 indicates that schoolchildren in TaiHao area, which is noted to be polluted by a petrochemical company, has a significantly increased prevalence of wheezing (3.7%, p < 0.05) and nocturnal cough (3.6%, p < 0.05). For schoolchildren living in the coastal areas, a 6% (p < 0.05) increase in the prevalence of rhinitis is noted. The prevalence of eczema uniformly increased for all areas of Changhwa County (p < 0.01). In the downtown areas, besides eczema, the prevalence of asthma and rhinitis also increased significantly (p < 0.05). Of note is the consistent finding in these two periods of the higher prevalence of childhood asthma, rhinitis, and eczema in the downtown areas compared with that from both the polluted and coastal areas. The secular trends in prevalence of asthma, rhinitis, and eczema in these two periods seem to correlate to environmental factors. While the symptoms of asthma, rhinitis, and eczema in schoolchildren affected boys more than girls, it is observed that affected girls have more marked manifestations, although it did not reach statistical significance.

The reason for the increased risk for development of osteoarthritis (OA) after acute joint trauma is not well understood, but the mechanically injured cartilage may be more susceptible to degradative mediators secreted by other tissues in the joint. To establish a model for such interactions, we coincubated bovine cartilage tissue explants together with normal joint capsule and found a profound ( approximately 70%) reduction in cartilage proteoglycan biosynthesis. This reduction is due to release by the joint capsule of a heat-labile and non-toxic factor. Surprisingly, while cultured synovium is a canonical source of interleukin-1 (IL-1), blockade either by soluble IL-1 type II receptor (sIL-1r) or IL-1 receptor antagonist (IL-1RA) had no effect. Combined blockade of IL-1 and tumor necrosis factor alpha (TNF-alpha) also had no effect. To support the clinical relevance of the findings, we harvested joint capsule from post-mortem human knees. Human joint capsule from a normal adult knee also released a substance that caused an approximately 40% decrease in cartilage proteoglycan biosynthesis. Furthermore, this inhibition was not affected by IL-1 blockade with either sIL-1r or IL-1RA. These results suggest that joint capsule tissue from a normal knee joint can release an uncharacterized cytokine that potently inhibits cartilage biosynthetic activity by an IL-1- and TNF-independent pathway.

Cocaine withdrawal symptoms are thought to play a role in relapse; studies characterizing the symptomatology have yielded mixed findings. This study sought to examine the pharmacodynamic/pharmacokinetic profile of repeated high dose exposure to oral cocaine and characterize acute and protracted withdrawal in cocaine abusers. This study employed a repeated-dosing, single-blind design in which subjects (n = 9), resided for 40 days on a closed ward. They were maintained for two 4-day cocaine exposure periods (Days 1-4 & Days 9-12, cocaine 175 mg, p.o.; 5 hourly doses; 875 mg/day) separated by a 4-day matched placebo exposure period (Days 5-8). After these 12 days, an additional period of 28 days of placebo maintenance followed (Days 13-40). Test sessions were conducted during each phase; measures of mood, drug effects, sleep, pharmacokinetics, and prolactin were collected throughout the study. The dosing regimen produced cocaine plasma concentrations (Cmax of 680 ng/mL) two to threefold higher than typically seen in acute dose studies. Prototypic psychostimulant effects, including subjective ratings of euphoric effects (liking, high, good effects) and significant cardiopressor effects, were sustained during the active dosing periods, corresponding to the rise and fall of plasma cocaine. Withdrawal-like symptoms (i.e., disruptions of sleep, increased ratings of anxiety, irritability, crashing) were observed within 24-hr after cessation of dosing. Cocaine reduced prolactin acutely, but no sustained alterations were observed for this measure or for other signs or symptoms during the 28-day abstinence period. These findings indicate that exposure to controlled high doses of cocaine produces modest symptoms consistent with cocaine withdrawal within hours of cessation of dosing but provide no evidence of symptoms persisting beyond 24 hours., (2009 APA, all rights reserved.)

A rugged liquid chromatographic-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method was developed and validated for accurate monitoring of steady-state plasma aripiprazole. Haloperidol-d(4) was chosen as the internal standard. ESI of aripiprazole and haloperidol-d(4) yielded abundant MH(+) ions, m/z 448 and 379, respectively. These ions were collision-dissociated to respective product ions of m/z 285 and 168. Ion-suppression experiments with blank plasma extracts showed substantial depressions of the product ions at retention times between 0.5 to 2 min, prohibiting development of a high-throughput LC-MS-MS method. A steep-gradient elution LC permitted a robust LC-ESI-MS-MS method with a 12-min analysis time. Aripiprazole was quantified from 0.2-mL aliquots of human plasma with acceptable precision and accuracy down to a lower limit of quantitation of 2 ng/mL. Aripiprazole was stable in plasma samples stored at room temperature for 24 h or exposed to three freeze-thaw cycles and in processed extracts stored at -20 degrees C for six days or on the autosampler at 10 degrees C for four days. The method has been successfully used for determinations of steady-state concentrations of aripiprazole in human subjects given daily oral doses of 15 mg. All measured concentrations were well within the quantitative range of 2 to 400 ng/mL.

A liquid chromatography-electrospray ionization-tandem mass spectrometry method has been developed and validated to detect (R)- and (S)-methadone and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in human plasma with cross-validation to urine and liver microsomes. Use of deuterated internal standards and liquid-liquid extraction coupled with chiral separation provided baseline separation with a lower limit of quantitation (LLOQ) of 2.5 ng/mL. The LLOQ was established from comparison of signal in blanks from six different sources per matrix with the same sources fortified at the LLOQ (none exceeded 19% of LLOQ) and precision and accuracy at the LLOQ determined in the same six sources per matrix. The assay was precise (% coefficients of variation within 13.8%) and accurate (% targets within 15%) in all three matrices. No interference was seen from addition of other psychoactive drugs. Stability was determined in plasma (24 h at room temperature, 321 days at -20 degrees C, 3 freeze-thaw cycles); processed plasma samples (5 days at -20 degrees C, 12 days on autosampler); urine (24 h at room temperature); and stock solutions (20 h at room temperature, 61 days at -20 degrees C). Applications of varying degree are presented for each matrix. Plasma from five subjects maintained on 100 mg oral methadone per day permitted comparison of the pharmacokinetics of the enantiomers. The t(1/2) of (R)-methadone was significantly longer than for (S)-methadone, and (S)-methadone was more tightly protein bound. The C(max), AUC, C(min), and % protein bound of (S)-EDDP were significantly greater than (R)-EDDP, while the t(1/2) of (R)-EDDP was significantly greater than (S)-EDDP. In spot urines, (R)- was higher than (S)-methadone, and (S)- was generally higher than (R)-EDDP. (R)- and (S)-EDDP production was detected after incubation of therapeutic concentrations of racemic methadone with human liver microsomes, and (S)-EDDP production was twofold greater than (R)-EDDP in three human placental microsomes incubated with racemic methadone.

Poly(acrylic acid) (PAA) was attached on both termini of Pluronic P85 copolymer (EO27PO39EO27) via atom transfer radical polymerization (ATRP) to produce a novel block copolymer, PAA-b-P85-b-PAA (P85PAA). The P85PAA-DOX complex formation and drug loading were strongly dependent on the PAA segment length and pH, where the protonation of carboxyl groups in the PAA segment at pH < 7.2 reduced the binding sites of DOX onto P85PAA chains, resulting in a diminished DOX uptake at low pH. The composition of copolymer-DOX complexes at pH 7.2 was close to the stoichiometric 1:1 DOX:carboxyl molar ratio, confirming the dominance of electrostatic interactions between cationic DOX molecules and carboxyl groups. The stability study of the copolymer-DOX complex suggested that non-polyelectrolyte interactions may also participate in the complexation of drug and P85PAA block copolymer. DOX loading at pH 5.0 decreased to 60% of the total binding capacity, indicating that protonation of carboxyl groups reduced the DOX binding to P85PAA block copolymer. DOX release from the complex is a pH-responsive process, where the protonation of carboxyl groups at mildly acidic condition resulted in a faster dissociation of copolymer-DOX complex, leading to an accelerated release of DOX at pH 5.0. Thus, complexation of DOX with P85PAA yielded a drug delivery system affording a pH-triggered release of DOX in an acidic environment of pH 5.0.

Dermatofibrosarcoma protuberans (DFSP) is an uncommon, slow growing, sarcoma of dermal and subcutaneous tissue with an infiltrative growth pattern. Although DFSP has a high rate of local recurrence, it rarely metastasizes. DFSP is characterized by a chromosomal translocation involving the collagen type I a 1 (COL1A1) gene on chromosome 17 and the platelet-derived growth factor B-chain (PDGFB) gene on chromosome 22. Various exons of the COL1A1 gene have been reported to be involved in the fusion with exon 2 of the PDGFB gene. In this study, we examined the COL1A1-PDGFB fusion transcripts using frozen specimens from three patients with DFSP. The molecular biology study with reverse transcriptase polymerase chain reaction (RT- PCR) and sequencing showed that the ends of exons 25, 31, and 45 in the COL1A1 gene were fused with PDGFB. The exon 2 of the PDGFB gene fused with exon 31 of the COL1A1 gene was a novel fusion gene.

Several studies have reported sex differences in behavioral responses to cocaine whereby females display a greater degree of locomotor activity. Fluctuations in estrogen and progesterone during the estrous cycle have been postulated to underlie these behavioral differences. In this study, we tested the hypothesis that hormonal replacement (estrogen or progesterone) in ovariectomized rats affects cocaine pharmacokinetics. We found that estrogen replacement did not affect cocaine-induced locomotor activity, but progesterone attenuated locomotor counts in comparison with control groups receiving only sesame oil. Estrogen, however, decreased brain levels of cocaine and norcocaine 30 min after cocaine administration in comparison to the group-receiving vehicle at that time point. In addition, in progesterone-treated rats, levels of benzoylecgonine and ecgonine methylester were higher at 30 min post-administration than at 15 min. No changes were found in blood levels of the metabolites. These findings suggest that while progesterone has an impact on locomotor behavior, pharmacokinetic effects may have a limited role in mediating behavioral responses to cocaine.

Plasma levels of cocaine (COC) and two of its principle metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME) were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS) in samples collected up to 3 h after a subcutaneous injection of cocaine (10 mg/kg) on six different days between days 4 and 24 postpartum, a period of dramatic change in the endocrine state of the female rat. Locomotor activity was measured in the same animals during this period using automated animal activity monitors. Additional measures in males provide a link to existing literature. We found that plasma levels of cocaine and its metabolites, as well as their respective time courses, are remarkably uniform across the postpartum period in female rats, as are the effects of cocaine on locomotor activity. Data from males show accord with prior published values. COC and BE, but not EME levels, were higher in males, and the time courses of COC and BE levels after injection varied somewhat between postpartum females and males; however, neither baseline nor cocaine-induced locomotor activity differed between postpartum females and males. We conclude that in the postpartum rat, there are no significant differences in the peripheral processing or general accessibility of cocaine to the brain to activate motor systems across the postpartum period. These data are critical to our understanding of differences in the reward salience of cocaine across the postpartum period and in other adult rat models [Mattson BJ, Williams S, Rosenblatt JS, Morrell JI. Comparison of two positive reinforcing stimuli: pups and cocaine throughout the postpartum period. Behav Neurosci 2001;115:683-94, Mattson BJ, Williams SE, Rosenblatt JS, Morrell JI. Preferences for cocaine- or pup-associated chambers differentiate otherwise behaviorally identical postpartum maternal rats. Psychopharmacology (Berl) 2003;167:1-8].

Interleukin 8 (IL8) is usually produced in both epithelial and monocytic cells during bacterial infections, causing inflammation. Helicobacter pylori induces production of IL8 from gastric epithelial cells via its cag pathogenicity island (cag PAI) system, LPS and outer-membrane protein. In some bacteria, heat-shock protein 60 (HSP60) also elicits a strong pro-inflammatory response in cells of the innate immune system. Three recombinant H. pylori HSP60 (rHSP60) proteins of different sizes were produced and one of these was used to raise two monoclonal antibodies (2E7 and 7B5). IL8 production was found to be induced in cultured monocytic cells treated with H. pylori cells or rHSP60 proteins, as measured by ELISA, and the amount of IL8 produced was dose-dependent. Pre-incubation of H. pylori cells or rHSP60 preparations with the antibody 2E7 significantly inhibited IL8 production from monocytic cells. These results indicated that HSP60 is closely associated with IL8 production in monocytic cells.

Contact between Helicobacter pylori and gastric epithelial cells results in activation of NF-kappaB followed by secretion of interleukin (IL)-8. However, host-cell receptor(s) and their ligands involved in H. pylori-related IL-8 production have yet to be fully defined. In this study, the interaction between Toll-like receptors (TLRs), which are host receptors for pathogens involved in the innate immune response, and heat-shock protein (HSP) 60, an immune-potent antigen of H. pylori, was examined during H. pylori-induced IL-8 secretion in vitro. Recombinant H. pylori HSP60 (rHpHSP60) was prepared and added to cultured KATO III human gastric epithelial cells with or without pre-incubation with mouse monoclonal anti-TLR2 or anti-TLR4 antibodies. IL-8 mRNA expression and IL-8 protein release were analysed by Northern blotting and immunoassay. Involvement of NF-kappaB activation was analysed immunocytochemically by anti-NF-kappaB p65 antibody and ammonium pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-kappaB-mediated transcriptional activation. rHpHSP60 induced IL-8 mRNA expression and IL-8 secretion in a dose-dependent manner in KATO III cells. Anti-TLR2 antibody inhibited rHpHSP60-induced IL-8 secretion by 75 %, and anti-TLR4 antibody inhibited it by 30 %. rHpHSP60 induced nuclear translocation of NF-kappaB p65, which was inhibited by pretreatment with anti-TLR2 antibody. Treatment with PDTC significantly decreased the secretion of IL-8 induced by rHpHSP60. These findings suggest that H. pylori HSP60 activates NF-kappaB and induces IL-8 production through TLR-triggered pathways in gastric epithelial cells. Thus, it is possible that H. pylori HSP60 and TLR interaction in host cells contributes to the development of gastric inflammation caused by H. pylori infection.

Quetiapine is an atypical antipsychotic agent for the treatment of schizophrenia. After an oral dose it is absorbed rapidly and extensively metabolized in the liver, resulting in low plasma concentrations of the parent drug. A sensitive analytical method is needed. A liquid chromatographic-electrospray-tandem mass spectrometric (LC-ESI-MS-MS) method combined with a simple liquid-liquid extraction has been developed for the measurement of quetiapine in human plasma and in human liver microsomes (HLM). Clozapine is used as internal standard. Plasma samples or microsomes quenched with methanol (100 microL) were made basic and extracted with 3 mL n-butyl chloride. The reconstituted extracts were analyzed by LC-ESI-MS-MS. Selective reaction monitoring of MH(+) at m/z 384 and 327 resulted in strong fragment ions at m/z 253 and 192 for quetiapine and clozapine, respectively. Recovery of quetiapine and clozapine ranged from 62 to 73%. Intrarun accuracy and precision determined at 1.0 (lower limit of quantitation), 2.5, 200, and 400 ng/mL did not exceed 7% deviation from target and the %CV did not exceed 5.5%. The % target +/- %CV for interrun accuracy and precision were at least 95% +/- 7.4% at concentrations of 2.5, 200, and 400 ng/mL. Plasma samples (2.5 and 400 ng/mL) stored at room temperature for 24 h or after 3 cycles of freeze/thaw were all stable (maximum % deviation < or = 11.0%). Processed extracts (2.5 and 400 ng/mL) stored for 7 days at -20 degrees C or 6 days on the autosampler were all stable (maximum % deviation < or = 11.5%). The method has been used to study quetiapine utilization during incubation with HLM or with cDNA-expressed human cytochrom P450s (CYP). Quetiapine is extensively metabolized by CYP 3A4 and CYP 2D6 and to a lesser extent by CYP 3A7, CYP 3A5, and CYP 2C19.

Mice and ducks were subcutaneously immunized with recombinant whole heavy (H) chains of Clostridium botulinum type C and D neurotoxins, which were expressed as glutathione S-transferase fusion proteins. In the case of mice, it was confirmed that two immunizations with type C- and D-H chains, 10 microg each time, significantly increased the specific antibodies against 100-kDa H chains of type C and D neurotoxins in an immunoblot analysis and an enzyme-linked immunosorbent assay, respectively. The mice immunized with type C- and D-H chains showed no symptoms of botulism when they were challenged with C- and D-16 S toxins at doses, given intraperitoneally, of up to 10(5) and 10(6) minmum lethal doses (MLD), respectively, per mouse. Ducks were immunized with a total of 100 microg of type C-H chain. The ducks also developed specific antibodies to the type C-H chain and showed significant protection against a challenge with 10(3) duck MLD of C-16 S toxin given intravenously. These results indicate that recombinant whole H chains can be used as an effective and safe vaccine for type C and D botulism in domestic animals.

Female rats display a more robust behavioral response to acute cocaine administration than do male rats. However, a clear understanding of the biological mechanisms underlying these differences remains elusive. The present study investigated whether sexual dimorphisms in cocaine-induced motor behavior might be based on monoaminergic levels and/or cocaine pharmacokinetics. An acute injection of cocaine (5, 15, 20 or 30 mg/kg) or saline was administered to male and female rats, and behavioral activity was monitored for 3 h. Following acute cocaine or saline administration motor behavior varied according to dose and sex; overall, female rats displayed greater rearing counts and stereotypic scores, greater total locomotor counts at 15, 20, and 30 mg/kg of cocaine, and greater ambulatory counts at 20 and 30 mg/kg of cocaine than did male rats. Neurochemical determinations in post-mortem tissue showed that both male and female rats had increases in total dopamine (DA) in the caudate putamen (CPu) 15 min following cocaine administration. Additionally, male rats had a decrease in dihydroxyphenylacetic acid (DOPAC)/DA turnover. Female rats showed significant reductions in total levels of DA, DOPAC, HVA, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), and DOPAC/DA turnover in the nucleus accumbens (NAc). Male rats displayed a reduction only in DOPAC/DA turnover and increases in 5-HT in the NAc following cocaine administration. Furthermore, sex differences in cocaine metabolism were observed where females had greater brain/blood levels of norcocaine and ecgonine methyl ester while male rats had higher blood levels of benzoylecgonine. These results suggest that sex differences in the behavioral responses to cocaine administration could be explained in part by intrinsic differences in both monoaminergic levels and metabolic processes.

Gas chromatography/mass spectrometry (GC/MS) is often used for detection and measurement of cocaine metabolites in biological specimens. However, cocaine N-oxide, a recently identified metabolite of cocaine, is thermally degraded when introduced into a GC/MS. The major degradation products are cocaine and norcocaine. When cocaine N-oxide was measured in rat plasma using liquid chromatography in combination with electrospray ionization-mass spectrometry (LC/ESI-MS), the cocaine N-oxide concentrations in the rat plasma were reported to be as high as 30% of the cocaine concentrations. However, in our study involving LC/ESI-MS/MS analysis of plasma collected from human subjects following administration of oral cocaine, we determined that the concentrations of cocaine N-oxide relative to the cocaine concentrations never exceeded 3%. This suggests that determination of cocaine concentration in human plasma by GC/MS analysis will not significantly distort the actual cocaine concentrations due to thermal conversion of cocaine N-oxide to cocaine. In the work reported here, we compared results obtained using GC/MS, LC/ESI-MS/MS, and liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) to determine thermal degradation of cocaine N-oxide. LC/ ESI-MS/MS was selected to determine cocaine, benzoylecgonine, and cocaine N-oxide, and LC/APCI-MS/MS was selected to determine ecgonine methyl ester and norcocaine in plasma collected from three human subjects participating in a clinical study. The resulting time course data provide additional information into kinetic interrelationships between cocaine N-oxidation and cocaine hydrolysis.

The efficacy of using additives to improve the shelf life of low-fat (approximately 18%) Chinese-style sausages was demonstrated. Sausages containing 3% sodium lactate (SL), 0.2% trisodium phosphate (TSP) or 0.2% potassium sorbate (KS) compared with the control (CONT) were manufactured and stored at 4°C for 12 weeks. Treatment TSP was the highest in pH during any storage intervals. Generally speaking, all microbial counts were lower for SL but higher for TSP; nonetheless, all treatments had bacterial counts less than 10(7) CFU/g. Low-fat Chinese-style sausage containing 3% sodium lactate resulted in better quality regarding physicochemical and microbiological characteristics. Results also suggested a potential utilization of sodium lactate as an antimicrobial agent.

In order to support studies on various medication protocols for the treatment of cocaine abuse, an accurate, precise, and sensitive (2.5 to 750 ng/mL) liquid chromatography-tandem mass spectrometry assay was developed to determine cocaine and benzoylecgonine in human plasma. Cocaine-d3 and benzoylecgonine-d3 were added as internal standards and samples were subjected to solid-phase extraction. Cocaine recovery was 94.4% and benzoylecgonine was 80.3% at 2.5 ng/mL. The selected reaction monitoring of parent ions at m/z 304 and 290 resulted in strong fragments at m/z 182 and 168 for cocaine and benzoylecgonine, respectively. The method was fully validated. The mean measured concentration at the 2.5 ng/mL, the lower limit of quantitation, was within 10.8% of the target and the precision determined at the low (5 ng/mL), medium (50 ng/mL), and high (650 ng/mL) quality controls ranged from 0.9 to 6.2 %CV. Cocaine and benzoylecgonine concentrations in plasma treated with 1% NaF showed changes of less than 10% when maintained at room temperature for up to 7 h and no significant changes when subjected to three freeze-thaw cycles. The concentrations of cocaine and benzoylecgonine remained stable in plasma samples stored at -20 degrees C for up to 11 months. Methanolic stock solutions of both analytes are stable, staying within 2% of the freshly prepared stock solutions, when stored at -20 degrees C for up to 235 days. Both extracted analytes reconstituted in methanolic solutions are stable for up to seven days whether stored at -20 degrees C or at room temperature on the autosampler. The method is rugged, rapid, and robust and has been applied to the batch analysis of more than 700 samples during pharmacokinetic profiling to assess potential interactions between intravenous (i.v.) cocaine challenge and treament medications. Results from three of these subjects receiving 40 mg (i.v.) cocaine demonstrate the utility of the method.

The present study assessed the roles of Adenomatous Polyposis Coli (APC) tumor suppressor gene during oral carcinogenesis. Reduction of APC transcript levels and APC loss of heterozygosity (LOH) were found in 39% (7/18) and 29% (10/34) cases of oral squamous cell carcinoma (OSCC), respectively. The apparent APC heterozygosity (27%) in non-cancerous matched oral tissue (NCMOT) adjacent to OSCC at an exon 11 locus was significantly lower than normally found in the Taiwanese population (49%). These findings suggest that the allelic status of APC could indicate a cancer risk. No polymorphism of 11307 allele of APC was identified in NCMOT or OSCC. Our data indicated that alterations of APC are frequent molecular changes of OSCC. Advances in understanding of the APC alterations that accompany OSCC development might provide a means for early diagnosis and possibly new therapeutic strategies.

Purpose: To assess the pharmacokinetics of Ftorafur (tegafur, FT), 5-fluorouracil (5-FU), and uracil in 31 cancer patients who were enrolled in phase I studies of oral uracil and FT (UFT). The correlation between pharmacokinetic parameters and toxic effects of UFT was evaluated., Methods: Uracil and FT were orally administered in a 4:1 molar ratio at FT doses of 200-400 mg/m2 per day. Patients also received leucovorin at 150 mg/day. Daily doses were divided into three doses and administered at 8-h intervals for 28 consecutive days. Plasma FT concentrations were measured by high-performance liquid chromatography, and plasma 5-FU and uracil concentrations were determined using gas chromatography-mass spectrometry. National Institutes of Health Common Toxicity Criteria were used for assessment of toxicity., Results: The concentrations of FT, 5-FU, and uracil showed wide interpatient variations. Maximum plasma concentrations (Cp(max)) of all three compounds were achieved in 0.3 to 4.0 h. At the various study doses, the terminal half-life (t 1/2beta) of FT ranged from 3.9 to 5.9 h, the area under the concentration-versus-time curve (AUC0-6h) ranged from 16,220 to 52,446 (ng/ml)h, the total clearance (ClT) ranged from 100 to 175 ml/min, and the steady-state volume of distribution (Vd(ss)) ranged from 18.3 to 28.7 l. The 5-FU generated from FT had an apparent distribution half-life (t 1/2alpha) and an apparent elimination half-life (t 1/2beta) of 0.3-1.3 h and 4.9-7.0 h, respectively. The AUC0-6h of 5-FU ranged from 120 to 325 (ng/ml)h. Uracil had a t 1/2alpha of 0.2-0.5 h and the level quickly returned to the endogenous level. The AUC0-6h for uracil ranged from 605 to 3764 (ng/ml)h, the ClT ranged from 3225 to 7748 ml/min, and the Vd(ss) ranged from 341 to 1354 l. The Cp(max) and AUC0-6h of both FT and uracil were significantly correlated with FT doses (P-values of 0.0244 and 0.0112) and with uracil doses (P-values of 0.0346 and 0.0083), respectively. In addition to interpatient variations, intrapatient variations were also observed in six patients who had pharmacology studies done on days 1 and 26+/-2 at the same study dose. We found that the repeated treatment with UFT caused cumulative increases in the values of Cp(max), Ctrough, and AUC0-6h of FT and 5-FU. The major toxic effects observed were diarrhea and nausea and vomiting. The occurrence of these toxic effects correlated significantly with the Cp(max) and AUC0-6h of 5-FU., Conclusions: The pharmacology studies showed that FT and uracil were readily absorbed orally and that FT was rapidly converted to 5-FU. The preliminary findings suggest that determination of plasma levels of 5-FU after oral administration of UFT may help predict subsequent toxic effects.

Oxidative modifications of low-density lipoproteins (LDL) may contribute to the pathogenesis of atherosclerosis. Although the oxidation products of the lipid components of LDL have been studied extensively, less is known about the oxidation products of the apoprotein, apolipoprotein B-100. To identify the specific oxidative modifications, we oxidized LDL in the presence of Cu(2+), treated with DNPH, precipitated and delipidated the protein, digested the protein with trypsin, and analyzed the peptides by high-performance liquid chromatography. We isolated nine peptides that exhibited measurable absorbance at 365 nm, which is characteristic of hydrazones derived from DNPH and is not observed in peptides derived from unoxidized LDL. Unexpectedly, we obtained the same peptides with absorbance at 365 nm in Cu(2+)-oxidized LDL not treated with DNPH. N-terminal sequence analyses and mass spectrometry indicated that the peptides isolated from the Cu(2+)-oxidized LDL all contained kynurenine residues in place of Trp residues found in the native apoprotein. The product profile we observed in Cu(2+)-oxidized LDL was remarkably different from the profiles observed in LDL oxidized by HOCl or myeloperoxidase in vitro, and the preferential oxidation of Trp to kynurenine in Cu(2+)-catalyzed oxidation of LDL contrasts with the products observed following oxidation of LDL with HOCl or myeloperoxidase. Our studies to date support the working hypothesis that the specific products of protein oxidation are sufficiently distinct to be developed as biomarkers of proposed mechanisms of oxidation of LDL and biological molecules in other toxicities and diseases.

Therapeutic doses of Ritalin, a racemic mixture of d- and l-threo-methyphenidate, result in low plasma concentrations of methylphenidate. In order to assess the safety and efficacy of methylphenidate, a sensitive analytical method is needed. A gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS) assay capable of measuring both d- and l-enantiomers in human plasma was developed and validated to support clinical studies involving administration of d,l-methylphenidate. d,l-Methylphenidate-d3 is added to 1-mL plasma samples. The plasma samples are made basic, mixed with isopropanol and extracted with hexane. The hexane extracts are then back-extracted into 0.1 N HCl. The acidified aqueous extract is made basic, cooled to ice temperature, and the methylphenidate derivatized with heptafluorobutyryl-l-prolyl chloride. The two diastereomeric derivatives are then extracted into hexane. The hexane extract is evaporated to dryness, reconstituted in ethyl acetate, and analyzed by GC-NCI-MS. This method can accurately (+/- 5% target) and precisely (< 11.1% coefficient of variation) quantitate enantiomers of threo-methylphenidate in human plasma and in the whole blood at concentrations ranging from 0.75 to 100 ng/mL. Plasma samples are stable for up to five freeze-thaw cycles when the duration of each cycle did not exceed 0.5 h. The drug degraded gradually when plasma samples were left at room temperature; a 6% loss at 3 h progressed to 17% at 12 h and to 35% at 24 h. Therefore, it is important that extraction of plasma samples begins within 0.5 h after samples are removed from the freezer. Whole blood stability results show that concentrations of methylphenidate in whole blood, with or without NaF, stored for up to 6 h at room temperature did not deviate from the target concentration by more than 13%. The derivatized methylphenidate in extract is stable at 4 degrees C for up to 10 days.

Oxidative modification of LDL may be important in the initiation and/or progression of atherosclerosis, but the precise mechanisms through which low density lipoprotein (LDL) is oxidized are unknown. Recently, evidence for the existence of HOCl-oxidized LDL in human atherosclerotic lesions has been reported, and myeloperoxidase (MPO), which is thought to act through production of HOCl, has been identified in human atherosclerotic lesions. In the present report we describe the formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive modifications in the apolipoprotein (apo) by exposure of LDL to myeloperoxidase in vitro. In contrast with the complex mixture of peptides from oxidation of LDL with reagent HOCl, oxidation with MPO in vitro produced a major tryptic peptide showing absorbance at 365 nm. This peptide was isolated and characterized as VELEVPQL(*C)SFILK..., corresponding to amino acid residues 53-66...on apoB-100. Mass spectrometric analyses of two tryptic peptides from oxidation of LDL by HOCl indicated formation of the corresponding methionine sulfoxide (M=O), cysteinyl azo (*C), RS -N= N-DNP, derivatives of EEL(*C)T(M=O)FIR and LNDLNS VLV(M=O)PTFHVPFTDLQVPS(*C)K, which suggest oxidation to the corresponding sulfinic acids (RSO2H) by HOCl. The present results demonstrate that DNPH-reactive modifications other than aldehydes and ketones can be formed in the oxidation of proteins and illustrate how characterization of specific products of protein oxidation can be useful in assessing the relative contributions of different and unexpected mechanisms to the oxidation of LDL and other target substrates. The data also suggest a direct interaction of the LDL particle with the active site on myeloperoxidase and indicate that effects of the protein microenvironment can greatly influence product formation and stability.

1. Contractile responses of smooth muscle from the Wistar rat urinary bladder were studied with the use of muscarinic agonists and antagonists. 2. McN-A-343 induced only weak contractile responses of the bladder muscle. In contrast, oxotremorine showed higher potency than either acetylcholine or bethanechol in inducing a contractile response (the respective pD2 values were 6.38 +/- 0.25, 4.82 +/- 0.24 and 4.42 +/- 0.14). 3. The M2 antagonists, methoctramine (10(-9) M to 10(-5) M) and gallamine (10(-9) M to 10(-5) M), did not reduce acetylcholine-induced (10(-5) M) contractions of the bladder muscle strip. On the other hand, 4-diphenyl-acetoxy-N-methyl piperidine methiodide (4-DAMP, 10(-10) M to 10(-7) M), an M3 receptor blocker, effectively antagonized the acetylcholine-induced contractions in a concentration-dependent manner. 4-DAMP had a similar pA2 value to those of the non-selective antagonists, atropine and scopolamine (pA2 values were 8.26 +/- 0.05, 8.36 +/- 0.05 and 8.41 +/- 0.11, respectively). Pirenzepine, and M1 blocker, antagonized the contractions at higher concentrations (10(-8) M to 10(-5) M, pA2 = 6.23 +/- 0.04). 4. It is concluded that (1) the dominant muscarinic receptor subtype responsible for smooth muscle contraction in the rat urinary bladder is M3; and (2) the muscarinic agonist oxotremorine was more potent than acetylcholine and bethanechol in inducing a contractile response.

Although there is growing evidence that neurotoxic molecules produced by HIV-1-infected mononuclear phagocytes damage neurons, the precise mechanisms of neuronal attack remain uncertain. One class of cytotoxin involves neuronal injury mediated via the NMDA receptor. We examined blood monocytes and brain mononuclear cells isolated at autopsy from HIV-1-infected individuals for the ability to release NMDA-like neuron-killing factors. We found that a neurotoxic amine, NTox, was produced by blood monocytes and by brain mononuclear phagocytes infected with retrovirus. In vivo injections of minute quantities of NTox produced selective damage to hippocampal pyramidal neurons. NTox can be extracted directly from brain tissues infected with HIV-1 and showed structural features similar to wasp and spider venoms. In contrast to NTox, HIV-1 infection did not increase the release of the NMDA excitotoxin quinolinic acid (QUIN) from mononuclear cells. Although we found modest elevations of QUIN in the CSF of HIV-1-infected individuals, the increases were likely attributable to entry through damaged blood-brain barrier. Taken together, our data pinpoint NTox, rather than QUIN, as a major NMDA receptor-directed toxin associated with neuro-AIDS.

Synaptosomes are nerve-end particles (NEP) isolated by using the technique of differential centrifugation. The synaptosome offers a good model for biochemical and pharmacological studies of the nerve endings. No report has been made on synaptosome isolation from the urinary bladder. The purpose of our work was to develop the use of synaptosome in the research of neurophysiology and neuropharmacology of the urinary bladder. Synaptosome-rich fraction was prepared from tissue homogenate of male Wistar rat urinary bladder by differential centrifugation (1000, 17,000 and 100,000 g) with discontinuous sucrose gradient. Electron microscopy showed synaptosomes as thin-walled bags containing a large number of synaptic vesicles. Two types of synaptosomes were easily discerned: those containing small agranular vesicles, and those containing dense-cored vesicles. The acetylcholine, norepinephrine, epinephrine and dopamine contents in the preparation were measured by the method of high-performance liquid chromatography. The respective concentrations were 300.4 +/- 30.1, 962.8 +/- 58.5, 617.3 +/- 59.8 and 1354.8 +/- 144.2 pmol/mg synaptosomal protein. In conclusion, it has been demonstrated that synaptosome-rich fractions can be prepared from the rat urinary bladder. Thus it is possible to apply this methodology for the investigation of the neurobiology of urinary bladders.

Three-month-old male Wistar rats were rendered diabetic with a single intravenous injection of streptozotocin (60 mg/kg body weight). Two weeks after induction of diabetes, synaptosome-rich fractions were prepared from urinary bladder tissue homogenate of the diabetic rats and control rats by differential centrifugation (1000 x g, 17,000 x g and 100,000 x g) with discontinuous sucrose gradient. Synaptosomal acetylcholine, norepinephrine, epinephrine and dopamine were measured by the method of high-performance liquid chromatography. The respective neurotransmitter concentrations for the diabetic rats were 1537.8 +/- 65.3, 4757.7 +/- 361.9, 3720.7 +/- 276.1, and 2447.8 +/- 196.8 pmol/mg synaptosomal protein, respectively; those for the control rats were 338.1 +/- 25.0, 1009.0 +/- 54.6, 645.3 +/- 52.2, and 1426.1 +/- 123.9 pmol/mg protein, respectively. Thus, the synaptosomal concentrations for all the measured neurotransmitters were significantly higher in the diabetic rats (P < 0.05 for each comparison). In conclusion, it has been demonstrated that the vesicle-bound acetylcholine and catecholamines in the synaptosome-rich fraction of the urinary bladder were significantly increased in 2-week diabetic rats. This finding would suggest impaired neurotransmitter release from both the bladder sympathetic and parasympathetic efferent nerve endings in early streptozotocin-induced diabetes.

Synaptosome-rich fractions were prepared from tissue homogenate of the urinary bladder of the spontaneously hypertensive rat and normotensive Wistar-Kyoto rat by differential centrifugation (1000 x g, 17 000 x g and 100 000 x g) with discontinuous sucrose gradient. Synaptosomal acetylcholine, norepinephrine, epinephrine and dopamine were measured by the method of high-performance liquid chromatography. The respective neurotransmitter concentrations for the normotensive rats were 300.4 +/- 30.1, 962.8 +/- 58.5, 617.3 +/- 59.8, and 1354.8 +/- 144.2 pmol/mg synaptosomal protein. For the hypertensive rats, the acetylcholine concentration (203.8 +/- 23.0 pmol/mg protein) was significantly lower (P < 0.05), while the norepinephrine, epinephrine and dopamine concentrations (1459.0 +/- 180.3, 971.3 +/- 62.2, and 2161.0 +/- 243.4 pmol/mg protein, respectively) were significantly higher (P < 0.05 for all) than those of the normotensive rats. In conclusion, it has been demonstrated that the vesicle-bound catecholamines in the synaptosome-rich fraction of the urinary bladder were significantly increased in hypertensive rats. On the contrary, the synaptosomal acetylcholine concentration was significantly decreased. These findings are suggestive of increased sympathetic innervation and decreased parasympathetic innervation in the urinary bladder of the spontaneously hypertensive rat.

Tissue concentration of norepinephrine and neuropeptide-Y immunoreactivity (NPY-IR) were measured in the urinary bladder, urethra, prostate and corpus cavernosum of the spontaneously hypertensive rat, as well as the normotensive Wistar-Kyoto rat. The results showed significantly increased tissue norepinephrine concentrations in the urinary bladder, urethra and prostate of the spontaneously hypertensive rat when compared to those of the normotensive rat (hypertensive, n = 18: 18.3 +/- 2.1, 14.9 +/- 1.7, 22.6 +/- 2.3 vs. normotensive, n = 18: 11.2 +/- 1.9, 10.4 +/- 1.3, 16.7 +/- 2.4 nmol/g tissue, respectively, P < 0.05 in each case). No difference was noted in the cavernosal tissue (hypertensive, n = 18: 11.3 +/- 1.6 vs. normotensive, n = 18: 10.1 +/- 1.8 nmol/g tissue, P > 0.01). Correspondingly, tissue NPY-IR was significantly increased in the bladder, urethra and prostate tissue of the spontaneously hypertensive rat (hypertensive, n = 18: 39.7 +/- 5.6, 25.3 +/- 3.4, 31.5 +/- 2.8 vs. normotensive, n = 18: 27.4 +/- 3.1, 18.6 +/- 2.7, 24.2 +/- 3.2 pmol/g tissue, respectively, P < 0.05 in each case). Again, no significant difference was observed in the cavernosal tissue (hypertensive, n = 18: 15.9 +/- 2.2 vs. normotensive, n = 18: 14.8 +/- 2.6 pmol/g tissue, P > 0.01). It is therefore concluded that increased tissue concentration of norepinephrine and NPY-IR were present in the urinary bladder, urethra and prostate of the spontaneously hypertensive rat. The significance of such biochemical findings needs further investigation but may suggest increased sympathetic innervation or activity. On the contrary, no corresponding changes were observed in the corpus cavernosum of the hypertensive rat.

Clinically, a 59% prevalence of impotence was reported among diabetic male patients. Neurological, vascular, endocrinologic and psychological factors are probably involved. Previous reports with the rat model found deterioration of sexual behavior and reproductive function caused by streptozotocin (STZ)-induced diabetes. The purpose of the present study was to develop the dark-cycle video recording methodology for the observation of rat sexual activities and to study the effect of STZ-induced diabetes on sexual performances of the rat. Adult male Wistar rats were rendered diabetic with intraperitoneal injection of STZ (60 mg/kg body weight). In the 4th week a diabetic rat or a control rat was caged with an adult ovariectomized female rat during the dark cycle. The female had been brought into behavioral estrus with intramuscular injection of 0.1 mg estradiol benzoate 3 days before and 1.0 mg progesterone 3 h before testing. Infrared-light-illuminated video recording was performed to evaluate the sexual performances. The mounting latency and frequency, intromission latency and frequency, the hit rate as well as the post-ejaculatory period of the diabetic rats were significantly deteriorated when compared with the controls (p < 0.05). However, the ejaculatory latency showed no significant difference between the two groups (p > 0.05). In conclusion, it has been demonstrated that with this methodology, behavioral studies on nocturnal animals like the rat can be carried out conveniently. It was shown that the sexual arousal mechanism and copulation-ejaculatory mechanism were both depressed in STZ-induced diabetic rats. The same study model can be used for further pathophysiological and pharmacological researches on the sexual behaviors of diabetic rats.

Soybean consumption may be protective for breast cancer, possibly due in part to the presence of the isoflavones daidzein and genistein, which are weakly estrogenic. The metabolism and disposition of these phytoestrogens during chronic soya exposure were studied on a metabolic unit. Six healthy 22- to 29-year-old women consumed an unrestricted hospital diet for most of the study and ingested 12 oz of soymilk with each meal for one month. At two-week intervals, excretion of isoflavones in urine was studied, during which time the subjects consumed a constant basal diet for three to four days, ingested the full daily 36-oz portion of soymilk within 30 minutes each day for one to two days, and collected urine continuously. Urinary recovery of genistein [initially 23.9 +/- 17.3% (SD) of ingested genistin + genistein], daidzein (initially 66.2 +/- 23.5% of ingested daidzin + daidzein), and equol (initially 28% of the ingested precursors daidzin + daidzein in 1 subject and < 1% in 5 subjects) decreased progressively over four weeks of daily soya ingestion by 42% for genistein (p < 0.05) and 31% for daidzein (p < 0.01) but increased by 3- to 100-fold for equol (4 subjects, p < 0.05). Total amounts excreted and peak levels were similarly affected. The absorption half-lives (t 1/2) for genistein and daidzein were initially 2.7 +/- 0.8 and 1.6 +/- 0.5 hours, respectively, and during four weeks of soymilk ingestion decreased to 2.0 +/- 0.6 (p = 0.04) and 1.4 +/- 0.2 hours (p = 0.06), respectively, suggesting more rapid absorption. The appearance t 1/2 of equol can be estimated for only one subject initially (2.9 hrs), but during four weeks of soya ingestion it could be estimated for three more subjects (4.7 +/- 2.3 hrs). The excretion t 1/2 values for genistein and daidzein were initially 6.7 +/- 0.8 and 4.4 +/- 0.7 hours, respectively, and during four weeks of soymilk ingestion decreased to 4.2 +/- 1.2 (p = 0.005) and 3.2 +/- 1.1 hours (p = 0.005), respectively, suggesting more rapid excretion. For equol, the excretion t 1/2 was initially 9.1 hours (1 subject), and after two and four weeks of soymilk ingestion it was 13.4 +/- 9.7 and 5.5 +/- 1.6 hours (4 subjects, p = 0.046, 2 wks vs. 4 wks), respectively. These results indicate that metabolism and disposition of ingested isoflavones are altered during chronic soya ingestion in women, perhaps from increased metabolic degradation to formation of nonisoflavone metabolites. Increased production of the longer- and stronger-acting estrogenic equol in some women during chronic soymilk ingestion may alter the estrogenic potency of dietary soya isoflavones.

The treatment effect of "ryu-wei-ti-huang-wan', a Chinese prescription composed of extracts from six plants, on diabetic impotence was evaluated in the present study. Adult male Wistar rats were divided into three groups: (1) rats rendered diabetic with a single intraperitoneal injection of streptozotocin (60 mg/kg body weight); (2) rats with streptozotocin-induced diabetes treated with a ryu-wei-ti-huang-wan powder preparation at a dose of 30 mg powder/kg body weight twice a day, and (3) control rats. A male rat was caged with an adult ovariectomized female rat during the dark cycle. Infra-red-light-illuminated video recording was utilized to evaluate the sexual performance. The diabetic rats exhibited depressed mounting activity and no intromission or ejaculation. After ryu-wei-ti-huang-wan treatment either for 1 day or 2 weeks, the diabetic rats showed significant improvement in mounting performance with preservation of intromission and ejaculation. No significant difference in the blood sugar level was noted between the treatment and non-treatment groups. In conclusion, the diabetic rats showed impairment in sexual arousal, erectile as well as ejaculatory functions. Ryu-wei-ti-huang-wan was effective in preserving these functions with a rapid onset. It is evident that the extracts exert the therapeutic effects not through a lowering of blood sugar. These substances are potentially useful in the clinical treatment of male diabetic impotence.

Microdialysis and gas chromatography/mass spectrometry was used for the measurement of extracellular N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) in rat hypothalamus. The sensitivity of the method for each of these compounds was approximately 5 pmol/30 microliters of dialysate. Baseline NAA concentrations in dialysate were estimated to be approximately 25 pmol/36 microliters, while that for NAAG was at or below the detection limit of 5 pmol/ 36 microliters. In vivo and in vitro calibrations of microdialysis probes showed that the recovery for NAA was approximately 10 percent. For NAAG, the in vitro recovery was 6.3%, and in vivo recovery, 11%. Depolarization stimulation using 100 mM KCl in the microdialysis perfusate was employed to measure extracellular NAA and NAAG concentrations. Extracellular NAA was elevated to approximately 70 pmol/36 microliters dialysate following depolarization. No significant elevation of NAAG was observed. By infusing known amounts of stable isotopically labeled NAAG-d3 via the microdialysis probe and measuring the isotopically labeled catabolic product, NAA-d3, in collected microdialysate, we were able to confirm the existence of one or more hydrolytic enzymes active towards NAAG in the hypothalamus. This finding suggest the possible involvement of active metabolic processes in the relationship between NAAG and NAA releases.

The role of peptides in mediating the nonadrenergic, noncholinergic (NANC) response of the rat urinary bladder was studied. Electrical stimulation of muscle strips from 3-month-old female Wistar rat urinary bladders in the presence of autonomic blockers (atropine 10(-6) mol/l, propanolol 10(-6) mol/l, phentolamine 10(-6) mol/l, and guanethidine 10(-6) mol/l) showed NANC contraction accounting for 60% of the maximum contractile responses at 40 Hz. Frequency-response studies showed that in the presence of alpha-chymotrypsin (2 U/ml, 30-min incubation), the NANC contractile responses to electrical stimulation at lower frequencies (3-10 Hz) were enhanced (p < 0.05; n = 9). However, no significant differences were observed at higher frequencies (20-40 Hz). With repetitive 4-Hz stimulation, alpha chymotrypsin caused a 19% increase in the NANC contractile response (p < 0.05; n = 8). It is postulated that the NANC response of the rat bladder smooth muscle is composed of an excitatory (contractile) and an inhibitory (relaxant) component. Some peptide(s) is/are responsible for mediating the inhibitory response.

Tissue polypeptide specific antigen (TPS) is the M3 epitope of the tissue polypeptide antigen, and a specific epithelial proliferation marker. To examine the benefit of urine TPS (UTPS) measurement in the diagnosis and classification of biological properties of transitional cell carcinoma (TCC), a radioimmunoassay of U-TPS was measured in patients with active TCC (n = 56), at tumor-free status (n = 36), with inflammatory urological disease (n = 44), and age-sex adjusted normal subjects (n = 75). Both neoplastic and inflammatory urological diseases had an increase in U-TPS levels (U/gm creatinine) compared to normal individuals (p = 0.0005), while it normalized in tumor-free condition (p = 0.007). For patients with active TCC, a strong positive association was observed between U-TPS values and both histological grading (p = 0.05) and positive cytology (p = 0.05). U-TPS levels were significantly higher in the presence of nodal or systemic metastasis (p = 0.008 by ANOVA test). Measurement of U-TPS appeared to be an indicator of poor outcome for patients with bladder cancer (p = 0.05 by t test) for a mean follow-up of 26 months. The results indicate that determination of U-TPS can be a supplement in assessing the biological properties of TCC, and may be helpful in identifying patients who need meticulous peri-operative staging survey.

Radiation cystitis with macroscopic hematuria has been a frustrating clinical problem for urologists. Since 1985 hyperbaric oxygen (HBO) has been used to treat this disease, showing favorable results. Between November 1989 and October 1992, 20 female patients with hemorrhagic radiation cystitis were treated with HBO at a pressure of 2.5 atm abs, breathing 100% O2 for 100 min in our multiplace hyperbaric chamber. After an average of 44 HBO sessions, macroscopic hematuria was completely halted in 16 patients (80%) and markedly decreased in 2 patients (10%). Comparison of the cystoscopic findings before and after HBO showed a significant decrease in hemorrhagic sites and telangiectasis of the bladder mucosa. One patient had urinary frequency and urgency without hematuria during her hospital stay. After 30 sessions of HBO therapy, her symptoms subsided, and the cystoscopic findings were much improved. Only one patient failed to respond to HBO and underwent ileal conduit diversion. The mean follow-up period was 14 mo. (5-41 mo.). From our clinical results and cystoscopic findings, we suggest that HBO is an effective and safe treatment for hemorrhagic radiation cystitis.

Reduced interarch distance, resulted from extrusion of antagonist teeth and/or down growth of alveolar bone due to long-time absence of natural teeth without adequate restoration in this area, is found in many cases. In such condition, it is difficult to fabricate a distal free-end removable partial denture with conventional resin base because of insufficient space for denture base resin, metal framework and artificial teeth, and the long-term prognosis of the denture is questionable due to minimized strength of the denture base. In this article, the authors demonstrate the clinical and laboratory methods and procedures for distal extension removable partial denture construction in reduced interach distance condition. Partial metal base in the insufficient vertical space area of the ordinary denture base is altered, the metal occlusal scheme and buccal resin facing are designed, instead of resin denture teeth. These designs provide strength, esthetic and masticatory function to the denture, and the denture base is convenient for future denture repair or relining.

The clinical significance of c-erb B-2 expression in urinary bladder cancer remains controversial. We performed an immunohistochemical study to examine the expression of c-erb B-2 in non-neoplastic urothelium (n = 12) and transitional cell carcinoma of the urinary bladder (n = 82). c-erb B-2 protein was localized in superficial and some intermediate cells of non-neoplastic urothelium. A total of 29 out of 82 (35%) tumors were positive for c-erb B-2 over-expression. There was no significant association of c-erbB-2 expression with tumor grade (p = 0.12), stage (p = 0.93), DNA ploidy status (p = 0.56) and the sex of patients (p = 0.5). Expression of epidermal growth factor receptor and Ki-67 index was available in 33 cases. Both parameters showed no apparent association with c-erbB-2 expression (p = 0.53 and 0.58 respectively). Factors correlated with poor patient survival by univariate analysis were tumor stage (p = 0.0001), tumor grade (p = 0.001), development of second recurrence (p = 0.002) and negative expression of c-erbB-2 (p = 0.017). Important indicators associated with first recurrence were tumor stage (p = 0.028), and c-erbB-2 expression with the risk of second recurrence (p = 0.031). Multivariate survival analysis revealed that tumor stage was among the most important prognostic factors (p = 0.029), followed by tumors without c-erbB-2 expression (p = 0.031) with a median follow-up at 46 months. The age of patients and c-erbB-2 expression were significantly associated with developing second recurrence (p = 0.031 and 0.046 respectively). The results indicate that expression of c-erbB-2 is independent of the stage and grade of bladder cancer. Although c-erbB-2 status can discriminate subpopulations with a high risk of recurrence, evaluation of its expression in paraffin-embedded tumors does not indicate poor prognosis for patients with urinary bladder cancer. To address this discrepancy a better understanding of the regulatory mechanism and physiological properties of c-erbB-2 protein in urothelium is required.

DNA ploidy analysis has been accepted as an important prognostic factor for patients with transitional cell carcinoma (TCC). However, there was few information dealing with the clinical relevance of slightly aberrant DNA content by flow cytometry (FCM). Here we present five cases of near-diploid (ND) tumours, with DNA index (DI) varying from 0.92 to 1.14, obtained from a prospective study of fifty-one cases (9.8%). The frequency of ND tumours showed a tendency to decrease with increasing tumour stage. Higher fraction of tumour proliferation, defined by Ki-67 index, showed a steady increment from 3.4 to 23.5% with occurrence of gross chromosomal changes. In contrast, the expression of epidermal growth factor receptor (EGFR) decreased from 48.3 to 35.3% for diploid (n = 29) through aneuploid (n = 17) tumours. All three ND bladder cancers had recurrence of one to three times with median follow-up of 36 months. The incidences of tumour recurrence (60%) and cancer death (20%) in ND tumours were intermediate between the aneuploid and diploid TCCs. But, flow DNA analysis of paraffin-embedded ND tumours revealed wide and symmetrical G0/G1 peak with DI varying from 5.6 to 13.0. Our limited experience suggests the necessity of special treatment for G0/G1 peaks having CV values greater than 5.5% from paraffin-embedded urothelial carcinomas.