Validation of cytochrome P450 time-dependent inhibition assays: a two-time point IC50 shift approach facilitates kinact assay design.

1. Recent guidance from the US Food and Drug Administration (USFDA) has advocated testing of time-dependent inhibition of cytochrome P450 (CYP), which can be addressed by performing IC(50) shift as well as K(I)/k(inact) determinations. 2. Direct (IC(50), K(i)) and time-dependent inhibition (IC(50) shift, K(I)/k(inact)) assays were validated in human liver microsomes with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis for the following enzyme/substrate/inhibitor combinations: CYP1A2/phenacetin/alpha-naphthoflavone/furafylline, CYP2C8/amodiaquine/montelukast/gemfibrozil-1-O-beta-glucuronide, CYP2C9/diclofenac/sulfaphenazole/tienilic acid, CYP2C19/S-mephenytoin/S-benzylnirvanol/S-fluoxetine, CYP2D6/dextromethorphan/quinidine/paroxetine, and CYP3A4/midazolam/testosterone/ketoconazole/azamulin/verapamil/diltiazem. IC(50) shift assays were performed with two pre-incubation time points (10 and 30 min) to facilitate k(inact) assay design. 3. Data obtained show good agreement with literature values. For rapid acting inhibitors, such as azamulin/CYP3A4 and tienilic acid/CYP2C9, the IC(50) shifts were similar at both time points suggesting a short maximum pre-incubation time with closely spaced time points for the K(I)/k(inact) assay. Slow acting inhibitors (such as verapamil/CYP3A4 or S-fluoxetine/CYP2C19) showed an increase in IC(50) shift between 10 and 30 min suggesting a longer maximum pre-incubation time with wider spacing of time points for K(I)/k(inact). 4. The two-time point IC(50) shift experiment proved to be an excellent method for the selection of appropriate K(I)/k(inact) assay parameters and is suitable for the routine analysis of P450 inhibition by drug candidates.

Transpupillary thermotherapy for the treatment of choroidal neovascularization in age-related macular degeneration in Taiwan.

PURPOSE: To evaluate the therapeutic outcome and the recurrence of choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) after transpupillary thermotherapy (TTT) in light-brown retinas. METHODS: A retrospective, non-randomized study of 58 eyes in 55 patients with subfoveal CNV treated with TTT was conducted. Power settings were set about half the value for Caucasian eyes. The outcome was assessed with best-corrected visual acuity, fluorescein angiography, indocyanine green angiography, and fundoscopic examination. RESULTS: Forty-four membranes were occult, six classic, and eight mixed. Mean follow-up was 16.6+/-10.7 months (range: 6-48 months). Membranes closed in 46 eyes. Iatrogenic complications included three subretinal haemorrhage, two retinal pigment epithelium tears, and two macular area cystic changes. In eyes with occult CNV, visual acuity improved in six (13.6%), 14 (31.8%) remained unchanged, and 24 (54.6%) deteriorated. For various CNV, average logMAR changes from baseline at last follow-up were 0.30 in occult, -0.08 in classic, and 0.59 in mixed (P<0.01). Thirty eyes experienced recurrence within 9.2+/-6.2 months (range: 2-22 months). Cumulative recurrence rate was 45% at 12 months and 71% at 22 months, with no significant difference between occult and non-occult type CNV. CONCLUSIONS: Transpupillary thermotherapy does not cure CNV secondary to AMD. High recurrence was found independent of CNV type. Most improved vision was found mostly in classic CNV. Complications associated with high energy level should be considered in light-brown retinas.

Potential routes of acquisition of Arcobacter species by piglets.

The aim of this study was to determine the prevalence and the transmission routes of Arcobacter spp. in sows and their offspring on a breeding farm. Twelve Arcobacter-positive sows and their litters were studied for this purpose. Analysis of rectal samples showed a high prevalence of Arcobacter spp. among the sows (approximately 42% of the sows carried one or more Arcobacter species). Intermittent excretion of one particular species and shifts in excretion from one species to another were observed in individual animals over time. The detection of Arcobacter spp. in amniotic fluid of the sows and in rectal samples from newborn piglets (ranging from 38.5-83.3% per litter), as well as the high similarity between PFGE profiles of Arcobacter isolates from sows and their respective newborns indicated the existence of an intra-uterine transmission route for Arcobacter spp. Specific antibodies against Arcobacter spp. were detected in colostrum by Western blot. At 2 weeks of age, only a few piglets were positive for Arcobacter. The reappearance of Arcobacter in these piglets at Week 3 and the shift in the Arcobacter species detected (from a prominent presence of A. cryaerophilus at birth to the presence of A. skirrowii and A. butzleri at 3 weeks after birth) showed that a post-natal infection route from their mothers, newcomers or the environment to the piglets existed. Thus, in this manuscript the transmission of Arcobacter spp. (both vertical and horizontal) from carrying sows to their offspring is demonstrated.

Ethyl acetate extract of Patrinia scabiosaefolia downregulates anti-apoptotic Bcl-2/Bcl-X(L) expression, and induces apoptosis in human breast carcinoma MCF-7 cells independent of caspase-9 activation.

Patrinia scabiosaefolia Fisch. is a Chinese medicinal herb used traditionally for treating intestinal carbuncle. Although Patrinia scabiosaefolia has also been suggested for cancer therapy, there has not been any scientific evidence supporting this application. In this study, a panel of human cancer cells, including breast carcinoma MCF-7; hepatocellular carcinoma HepG2; skin melanoma A375; lung carcinoma A549 and prostate adenocarcinoma PC-3, were treated in vitro with ethyl acetate extract of Patrinia scabiosaefolia (EAE-PS) for 48 h. Results from MTT study showed that MCF-7 was the most responsive (IC50 = 112.3 microg/ml) while PC-3 was the most resistant (IC50 = 348.7 microg/ml) one to cell growth inhibition. DNA flow cytometry demonstrated that EAE-PS induced apoptosis in the resistant MCF-7 cells by 14.5-fold of the control level after 36 h of treatment. Immunoblot studies further illustrated that although EAE-PS downregulated the anti-apoptotic Bcl-2/Bcl-X(L) expression in breast cancer cells, the induced apoptosis could not be prevented by the caspase-9 inhibitor (Z-LEHD-FMK). All these results suggest that EAE-PS retards MCF-7 cell growth by activating the caspase-independent mitochondrial cell death pathway. Results from this study support future research and development of the bioactive ingredients from Patrinia scabiosaefolia as anticancer agents, especially against those apoptosis-resistant cancers with deregulated Bcl-2/Bcl-X(L) expression.

Preclinical evaluation of a prostate-targeted gene-directed enzyme prodrug therapy delivered by ovine atadenovirus.

Gene-directed enzyme prodrug therapy (GDEPT) based on the Escherichia coli enzyme, purine nucleoside phosphorylase (PNP), provides a novel strategy for treating slowly growing tumors like prostate cancer (CaP). PNP converts systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine, that kills PNP-expressing and nearby cells by inhibiting DNA, RNA and protein synthesis. Reporter gene expression directed by a hybrid prostate-directed promoter and enhancer, PSMEPb, was assayed after plasmid transfection or viral transduction of prostate and non-CaP cell lines. Androgen-sensitive (AS) LNCaP-LN3 and androgen-independent (AI) PC3 human CaP xenografts in nude mice were injected intratumorally with an ovine atadenovirus vector, OAdV623, that carries the PNP gene under PSMEPb, formulated with cationic lipid for enhanced infectivity. Fludarabine phosphate was then given intraperitoneally for 5 days at 75 mg/m2/day. PNP expression was evaluated by enzymic conversion of its substrate using reverse phase HPLC. OAdV623 showed excellent in vitro transcriptional specificity for CaP cells. In vivo, expression of PNP persisted for > 6 days after OAdV623 injection and a single treatment provided 100% increase in tumor doubling time and > 50% inhibition of tumor growth for both LNCaP-LN3 and PC3 lines, with increased tumor necrosis and apoptosis and decreased tumor cell proliferation. OAdV623 significantly suppressed the growth of AS and AI human CaP xenografts in mice.

The effectiveness of Tsu-San-Li (St-36) and Tai-Chung (Li-3) acupoints for treatment of acute liver damage in rats.

This study investigated the effects of acupuncture on carbon tetrachloride (CCl4) induced acute liver injury in male rats (n=36). The experimental groups were injected with CCl4 before, during, or after acupuncture therapy. Acupoints similar to the human Tsu-San-Li (St-36) and Tai-Chung (Li-3) were needled bilaterally. Rats treated with CCl4 had higher levels of serum glutamate-oxalate-transaminase (sGOT) and serum glutamate-pyruvate-transaminase (sGPT). Comparing the experimental groups, biochemical and pathological parameters of liver injury were significantly reduced when rats were acupunctured after, not before, CCl4-induced hepatotoxicity. Acupuncture at the Tsu-San-Li and Tai-Chung acupoints cannot prevent acute liver injury but may be effective in treating liver injury induced by carbon tetrachloride in rats.

The effect of quadriceps femoris muscle strengthening exercises on spasticity in children with cerebral palsy.

BACKGROUND AND PURPOSE: The Bobath neurodevelopmental treatment approach advised against the use of resistive exercise, as proponents felt that increased effort would increase spasticity. The purpose of this study was to test the premise that the performance of exercises with maximum efforts will increase spasticity in people with cerebral palsy (CP). Spasticity, in the present study, was defined as a velocity-dependent hyperexcitability of the muscle stretch reflex. SUBJECTS: Twenty-four subjects with the spastic diplegic form of CP (mean age=11.4 years, SD=3.0, range=7-17) and 12 subjects without known neurological impairments (mean age=11.6 years, SD=3.5, range=7-17) were assessed. METHODS: Knee muscle spasticity was assessed bilaterally using the pendulum test to elicit a stretch reflex immediately before and after 3 different forms of right quadriceps femoris muscle exercise (isometric, isotonic, and isokinetic) during a single bout of exercise training. Pendulum test outcome measures were: (1) first swing excursion, (2) number of lower leg oscillations, and (3) duration of the oscillations. RESULTS: There were no changes in spasticity following exercise between the 2 groups of subjects. DISCUSSION AND CONCLUSION: These results do not support the premise that exercises with maximum efforts increase spasticity in people with CP.

Simultaneous capture of metal, sulfur and chlorine by sorbents during fluidized bed incineration.

Metal capture experiments were carried out in an atmospheric fluidized bed incinerator to investigate the effect of sulfur and chlorine on metal capture efficiency and the potential for simultaneous capture of metal, sulfur and chlorine by sorbents. In addition to experimental investigation, the effect of sulfur and chlorine on the metal capture process was also theoretically investigated through performing equilibrium calculations based on the minimization of system free energy. The observed results have indicated that, in general, the existence of sulfur and chlorine enhances the efficiency of metal capture especially at low to medium combustion temperatures. The capture mechanisms appear to include particulate scrubbing and chemisorption depending on the type of sorbents. Among the three sorbents tested, calcined limestone is capable of capturing all the three air pollutants simultaneously. The results also indicate that a mixture of the three sorbents, in general, captures more metals than a single sorbent during the process. In addition, the existence of sulfur and chlorine apparently enhances the metal capture process.

MDM2 expression in areca quid chewing-associated oral squamous cell carcinomas in Taiwan.

MDM2 (murine double minute gene 2) overexpression has been implicated in the pathogenesis of human tumors via inhibition of the p53 tumor suppressor protein. To investigate the potential involvement of MDM2 overexpression in the pathogenesis of oral squamous cell carcinomas (SCCs) in Taiwan, we examined the expression of MDM2 protein and its relationship to p53 protein levels in 52 oral SCCs using antibodies to MDM2 and p53. Of the 52 patients, 36 (69 %) had tumors with positive MDM2 nuclear staining and 32 (61%) had tumors with p53 nuclear staining. Co-expression of MDM2 protein and p53 was detected in 25 (48%) cases; and 9 (17%) tumors showed neither MDM2 protein nor p53 staining. A significant correlation was observed between MDM2 protein and p53 expression in 38 cases with an areca quid (AQ) chewing habit (P=0.032). No significant correlation was found between the degree of MDM2 protein staining and the patients' ages, sex, cancer location, clinical staging, primary tumor TNM status or histological differentiation of SCC at the time of initial presentation. Kaplan-Meier analysis showed that either MDM2 protein expression or co-expression of p53 and MDM2 protein did not relate significantly to patient overall survival. Nevertheless, the high prevalence of MDM2 protein overexpression found in this study suggest that MDM2 may also participate in the carcinogenesis of AQ chewing-associated oral SCCs in Taiwan.

Inhibitory effect of anti-pyretic and anti-inflammatory herbs on herpes simplex virus replication.

The increasing clinical use of acyclovir, ganciclovir, and foscarnet against herpes simplex virus (HSV), varicella-zoster virus, and cytomegalovirus has been associated with the emergence of drug-resistant herpesvirus strains. To develop anti-HSV compounds from plants, 31 herbs used as antipyretic and anti-inflammatory agents in Chinese medicine were screened. Five different preparations (cold aqueous, hot aqueous, ethanolic, acid ethanolic, and methanolic) from 31 herbs were analyzed by plaque reduction assay, and 7 extracts. which showed significant antiviral activities, were further elucidated for their antiviral mechanisms. Our results showed that ethanolic extract of Rheum officinale and methanolic extract of Paeonia suffruticosa prevented the process of virus attachment and penetration. Aqueous extract of P. suffruticosa and ethanolic extract of Melia toosendan inhibited virus attachment to cell surface. Aqueous extract of Sophora flavescens and methanolic extract of M. toosendan showed no effect on virus attachment and penetration. These data indicated that these 4 herbs have a potential value as a source of new powerful anti-HSV compounds.

Induction of the c-jun protooncogene expression by areca nut extract and arecoline on oral mucosal fibroblasts.

To investigate the mechanisms of areca quid (AQ)-induced carcinogenesis, expression of c-fos and c-jun protooncogenes was examined in human oral mucosal fibroblasts after exposure to areca nut extracts (ANE) or arecoline. We found that treatment of cells with 200 microg/ml ANE or 10 microg/ml arecoline for 1 h induced about three-fold increase in c-jun mRNA levels. This increase was transient and the level of c-jun mRNAs returned rapidly to control levels thereafter. However, ANE and arecoline did not induce c-fos mRNA expression at detectable levels. During AQ chewing, oral mucosal cells are continuously stimulated by ANE and arecoline. Persistent induction of the c-jun protooncogene by ANE and arecoline may be one of the mechanisms in the carcinogenesis of oral squamous cell carcinoma in Taiwan. Furthermore, we observed that pre-incubation of cells with either N-acetyl-cysteine [a glutathione (GSH) precursor] or L-buthionine-S,R-sulfoximine (a specific inhibitor of GSH biosynthesis) had a minimal effect on arecoline-induced c-jun expression. Therefore, arecoline-induced c-jun expression is independent of GSH depletion.

HIV-associated eosinophilic folliculitis in a Chinese woman: a case report and a survey in Hong Kong.

This is the first survey of eosinophilic folliculitis (EF) in patients infected with the human immunodeficiency virus (HIV) in Hong Kong. The present report provides the local data on HIV-associated eosinophilic folliculitis (HIV-EF) and includes the first Chinese, heterosexual female patient with this condition. This is a retrospective study on all HIV-positive patients (n = 451) attending the outpatient clinic of the AIDS Unit in Hong Kong. Patients diagnosed as having EF with histological support were included for analysis. The data were presented by descriptive method. Three patients were identified, all of them had skin biopsies done which confirmed the diagnosis; including the female case. Recognition of HIV-EF is important because it is indicative of significant immunosuppression with risk of opportunistic infection. We concluded that HIV-EF is no longer an exclusive male disease in homosexual patients only. We expect more female patients or heterosexual subjects who are HIV positive developing this disease in the future.

PIP: A retrospective record review of HIV-associated eosinophilic pustular folliculitis (EF) cases in 451 HIV-positive patients attending an outpatient AIDS clinic in Hong Kong revealed the first female case of HIV-EF in a Chinese heterosexual woman. A 45-year-old HIV-positive woman presented with a 2-week history of pruritic papulopustular eruption on the face. Clinical examination revealed multiple erythematous follicular papules and pustules on her face and neck. Histologic examination of a skin biopsy indicated spongiosis and exocytosis in the epidermis involving infundibulum and sebaceous glands with micropustule formation. This patient's EF responded well to treatment with ultraviolet B phototherapy. This record review also revealed 2 HIV-EF cases in Chinese men (1 homosexual and 1 heterosexual). In previous series, HIV-EF has been found only in homosexual men.

Anti-inflammatory effect of magnolol, isolated from Magnolia officinalis, on A23187-induced pleurisy in mice.

In the present study, A23187-induced pleurisy in mice was used to investigate the anti-inflammatory effect of magnolol, a phenolic compound isolated from Chinese medicine Hou p'u (cortex of Magnolia officinalis). A23187-induced protein leakage was reduced by magnolol (10 mg kg-1, i.p.), indomethacin (10 mg kg-1, i.p.) and BW755C (30 mg kg-1, i.p.). A23187-induced polymorphonuclear (PMN) leucocyte infiltration in the pleural cavity was suppressed by magnolol and BW755C, while enhanced by indomethacin. Like BW755C, magnolol reduced both prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels in the pleural fluid of A23187-induced pleurisy, while indomethacin reduced PGE2 but increased LTB4 formation. In the rat isolated peripheral neutrophil suspension, magnolol (3.7 microM) and BW755C (10 microM) also suppressed the A23187-induced thromboxane B2 (TXB2) and LTB4 formation. These results suggest that magnolol, like BW755C, might be a dual cyclo-oxygenase and lipoxygenase inhibitor. The inhibitory effect of magnolol on the A23187-induced pleurisy is proposed to be, at least partly, dependent on the reduction of the formation of eicosanoids mediators in the inflammatory site.

Seasonal prevalence of air-borne pollen and spores in Kuala Lumpur, Malaysia.

Aerosampling using Rotorod samplers was conducted in the Institute for Medical Research, Kuala Lumpur, Malaysia, from December 1991 to November 1993. Samples were collected twice a week between 10.00 hours to 12.00 hours. Rods were stained and examined microscopically. A total of 8 and 20 types of pollens and mold spores were collected, respectively. More mold spores were collected than pollens. Grass pollen constituted more than 40 percent of total pollen counts. Gramineae pollen counts peaked in March and September. The most abundant mold spore was Cladosporium followed by Rust, Nigrospora, Curvularia and Smut. Cladosporium counts peaked in February and August. Rust counts peaked in June and December whereas counts for Nigrospora peaked in February and October. Highest counts of Smut were recorded in March and October. Curvularia counts peaked in January, June and September.

Production of selenomethionine-labeled recombinant human neutrophil collagenase in Escherichia coli.

Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca2+ and Zn2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.

Laboratory evaluation of two commercial repellants against Leptotrombidium fletcheri (Acari: Trombiculidae).

Two commercial repellants were evaluated in the laboratory against Leptotrombidium fletcheri chiggers. The active ingredient in one was DEET and in the other was citrus oil. Excito-toxicity effect was studied and it was determined by the time ("escape time") chiggers took to move off filter papers treated with the repellants. All chiggers exposed on filter papers treated with DEET died and did not move off the treated papers. None of the chiggers that were placed on papers treated with citrus oil were killed. Escape times on papers treated with a 2-sec spray of citrus oil were longer than those for the 4- and 8-sec sprays. The weights of citrus oil deposited increased with increasing spray times. Electron microscopy showed that the repellants had no effect on the texture of the filter papers. It was concluded that the spray containing DEET was more effective; however, both repellants should be further evaluated under field conditions for protection against chigger bites.

Effects of a long-chain fatty amine on mammary carcinogenesis induced in female Sprague-Dawley rats by DMBA.

Reports that protein kinase C is inhibited by sphingosine and other long-chain amines and the suggestion that promotion of mammary carcinogenesis by dietary fat is mediated by protein kinase C prompted us to investigate the effects of a long-chain amine, 1-octadecylamine, on mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthracene in rats fed a high-fat diet. Rats fed the amine sulfate at a level of 0.01% in a semipurified diet containing 20% corn oil developed more tumors than those fed the high-fat diet alone, although body weight gain was inhibited slightly. Rats fed the amine sulfate at 0.1% of the diet developed very few tumors compared with those fed either the high-fat diet or a low-fat diet containing 5% corn oil. At the higher level, the C18 amine also caused a marked inhibition of body weight gain.

Thermoradiotherapy of choroidal melanoma. Clinical experience.

Thermoradiotherapy (TRT) was used to treat 18 patients with choroidal melanoma. Techniques and clinical observations using a plaque-type device capable of delivering microwave hyperthermia to intraocular tumors are described. Iodine-125 plaque irradiation (48-88 Gy to apex), together with microwave hyperthermia (46 degrees-52.5 degrees C to base), were given to patients during one brachytherapy session. Since October 1985, 15 medium and 3 large-sized tumors were treated. Clinical observations include partial clearing of six vitreous opacities as well as three retinal detachments noted before treatment. Objective measurements of improved visual acuity were noted in seven of the nine cases. All tumors responded to treatment, but one tumor had regrowth and the eye was enucleated. These data suggest that a microwave plaque can be used to deliver hyperthermia to human choroidal melanomas. Within the range of the follow-up period, no side effects that might preclude the use of this hyperthermia system for choroidal melanoma treatment were noted.