The measurement of nicotine in human plasma by high-performance liquid chromatography-electrospray-tandem mass spectrometry

The authors describe a reverse-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry method for the measurement of nicotine in human plasma. Samples (500 muL) with added deuterium-labeled d(3)-nicotine as an internal standard (IS) were treated with a 2-step process of ether extraction (6 mL) followed by back-extraction into 0.1% formic acid (50 muL). Chromatography was performed on a phenyl Novapak column with a mobile phase consisting of 50% 10 mM ammonium fortriate (pH 3.3) and acetonitrile (50:50, vol/vol). A flow rate of 0.2 mL/min resulted in a total analysis time of 5 minutes per sample. Mass spectrometric detection was by selected reactant monitoring (nicotine m/z 163.2 --> 130.2; IS m/z 166.2 --> 87.2). The assay was linear from 0.5 to 100 mug/L (r > 0.993, n = 9). The accuracy and imprecision of the method for quality control sampleswere 87.5% to 113% and < 10.2%, respectively. Interday accuracy and imprecision at the limit of quantification (0.5 mug/L) was 113% and 7.2% (n = 4). The process efficiency for nicotine in plasma was > 75%. The method described has good process efficiency, stabilized nicotine, avoided concentration steps, and most importantly minimized potential contamination. Further, we have established that water-based standards and controls are interchangeable with plasma-based samples. This method was used successfully to measure the pharmacokinetic profiles of subjects involved in the development of an aerosol inhalation drug delivery system.

Rapid determination of the active leflunomide metabolite A77 1726 in human plasma by high-performance liquid chromatography

A simple method for the measurement of the active leflunomide metabolite A77 1726 in human plasma by HPLC is presented. The sample workup was simple, using acetonitrile for protein precipitation. Chromatographic separation of A77 1726 and the internal standard, alpha-phenylcinnamic acid, was achieved using a C-18 column with UV detection at 305 nm. The assay displayed reproducible linearity for A77 1726 with determination coefficients (r(2)) > 0.997 over the concentration range 0.5-60.0 mug/ml. The reproducibility (%CV) for intra- and inter-day assays of spiked controls was

Modulation of proliferation-specific and differentiation-specific markers in human keratinocytes by SMAD7

We examined the potential role of SMAD7 in human epidermal keratinocyte differentiation. Overexpression of SMAD7 inhibited the activity of the proliferation-specific promoters for the keratin 14 and cdc2 genes and reduced the expression of the mRNA for the proliferation-specific genes cdc2 and E2F1. The ability of SMAD7 to suppress cdc2 promoter activity was lost in transformed keratinocyte cell lines and was mediated by a domain(s) located between aa 195-395 of SMAD7. This domain lies outside the domain required to inhibit TGFbeta1 signaling, suggesting that this activity is mediated by a novel functional domain(s). Examination of AP1, NFkappaB, serum response element, Gli, wnt, and E2F responsive reporters indicated that SMAD7 significantly suppressed the E2F responsive reporter and modestly increased AP1 activity in proliferating keratinocytes. These data Suggest that SMAD7 may have a role in TGFbeta-independent signaling events in proliferating/undifferentiated keratinocytes. The effects of SMAD7 in differentiated keratinocytes indicated a more traditional role for SMAD7 as an inhibitor of TGFbeta action. SMAD7 was unable to initiate the expression of differentiation markers but was able to superinduce/derepress differentiation-specific markers and genes in differentiated keratinocytes. This latter role is consistent with the ability of SMAD7 to inhibit TGFbeta-mediated suppression of keratinocyte differentiation and suggest that the opposing actions of SMAD7 and TGFbeta may serve to modulate squamous differentiation. (C) 2004 Elsevier Inc. All rights reserved.

Secretoneurin is released into human airways by topical histamine but not capsaicin

Background: The neuropeptide secretoneurin, with potential relevance to leukocyte trafficking, is present in nerves of the nasal mucosa in allergic rhinitis and may be released in response to allergen and histamine exposure. There is no information on the occurrence and mechanisms of release of secretoneurin in healthy human airways. Methods: The presence of secretoneurin in nasal biopsies and its release in response to nasal capsaicin and histamine challenges were examined. Symptoms and lavage fluid levels of fucose were recorded as markers of effects in part produced by neural activity. Bronchial histamine challenges followed by sputum induction and analysis of secretoneurin were also carried out. Results: Nerves displaying secretoneurin immunoreactivity abounded in the nasal mucosa. Nasal capsaicin challenge produced local pain (P < 0.05) and increased the levels of fucose (P < 0.05), but failed to affect the levels of secretoneurin. Nasal histamine challenge produced symptoms (P < 0.05) and increased the mucosal output of secretoneurin (P < 0.05) and fucose (P < 0.05). Bronchial histamine challenge increased the sputum levels of secretoneurin (P < 0.05). Conclusions: We conclude that secretoneurin is present in healthy human airways and that histamine evokes its release in both nasal and bronchial mucosae. The present observations support the possibility that secretoneurin is involved in histamine-dependent responses of the human airway mucosa.

Genetic study of alcoholism and novel gene expression in the alcoholic brain

Alcohol dependence may result from neuroadaptation involving alteration of gene expression after long-term alcohol exposure. The systematic study of gene expression profiles of the human alcoholic brain was initiated using the method of polymerase chain reaction (PCR)-differential display and was followed by DNA microarray. To date, more than 100 alcohol-responsive genes have been identified from the frontal cortex, motor cortex and nucleus accumbens of the human brain. These genes have a wide range of functions in the brain and indicate diverse actions of alcohol on neuronal function. This review discusses the current information on the genetic basis of alcoholism and the induction and characterization of these alcohol-responsive genes.

Case report: unusual presentation of Fasciolopsis buski in a Viet Namese child

A Viet Namese child presented with a history of abdominal pain. Shortly afterwards, he vomited eight live trematode flukes that were collected and morphologically identified as Fasciolopsis buski. The identification was confirmed by DNA analysis. Adult worms of F buski from humans are very rarely seen except at autopsy, and this is the first such report from Viet Nam. (C) 2003 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Hypermethylation of the 5 ' CpG island of the gene encoding the serine protease Testisin promotes its loss in testicular tumorigenesis

The Testisin gene (PRSS21) encodes a glycosylphosphatidylinositol (GPI)-linked serine protease that exhibits testis tissue-specific expression. Loss of Testisin has been implicated in testicular tumorigenesis, but its role in testis biology and tumorigenesis is not known. Here we have investigated the role of CpG methylation in Testisin gene inactivation and tested the hypothesis that Testisin may act as a tumour suppressor for testicular tumorigenesis. Using sequence analysis of bisulphite-treated genomic DNA, we find a strong relationship between hypermethylation of a 385 bp 50 CpG rich island of the Testisin gene, and silencing of the Testisin gene in a range of human tumour cell lines and in 100% (eight/eight) of testicular germ cell tumours. We show that treatment of Testisin-negative cell lines with demethylating agents and/or a histone deacetylase inhibitor results in reactivation of Testisin gene expression, implicating hypermethylation in Testisin gene silencing. Stable expression of Testisin in the Testisin-negative Tera-2 testicular cancer line suppressed tumorigenicity as revealed by inhibition of both anchorage-dependent cell growth and tumour formation in an SCID mouse model of testicular tumorigenesis. Together, these data show that loss of Testisin is caused, at least in part, by DNA hypermethylation and histone deacetylation, and suggest a tumour suppressor role for Testisin in testicular tumorigenesis.

Novel variants in growth differentiation factor 9 in mothers of dizygotic twins

Context: Genes from the ovarian bone morphogenetic signaling pathway (GDF9 and BMP15) are critical for normal human fertility. We previously identified a deletion mutation in GDF9 in sisters with spontaneous dizygotic (DZ) twins, but the prevalence of rare GDF9 variants in twinning families is unknown. Objective: The objective was to evaluate the frequency of rare variants in GDF9 in families with a history of DZ twinning. Design and Subjects: We recruited 3450 individuals from 915 DZ twinning families (1693 mothers of twins) and 1512 controls of Caucasian origin. One mother of DZ twins was selected from 279 of the 915 families, and a DNA sample was screened for rare variants in GDF9 using denaturant HPLC. Variants were confirmed by DNA sequencing and genotyped in the entire sample by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Results: We found two novel insertion/deletions (c.392-393insT, c.1268-1269delAA) and four missense alterations in the GDF9 sequence in mothers of twins. Two of the missense variants (c.307C > T, p.Pro103Ser and c.362C > T, p.Thr121Leu) were located in the proregion of GDF9 and two (c.1121C > T, p.Pro374Leu and c.1360C > T, p.Arg454Cys) in the mature protein region. For each variant, the frequencies were higher in cases compared with controls. The proportion of mothers of DZ twins carrying any variant (4.12%) was significantly higher (P < 0.0001) than the proportion of carriers in controls (2.29%). Conclusion: We describe new variants in the GDF9 gene that are significantly more common in mothers of DZ twins than controls, suggesting that rare GDF9 variants contribute to the likelihood of DZ twinning.