N-in-one determination of retention factors for drugs by immobilized artificial membrane chromatography coupled to atmospheric pressure chemical ionization mass spectrometry.

Immobilized artificial membrane (IAM) chromatography is widely used in drug discovery for ranking the absorption properties of drug candidates. In this work an IAM chromatography method using atmospheric pressure chemical ionization mass spectrometric detection (IAM/APCI-MS) was developed for the determination of log k(IAM) values for a mixture of compounds (9-in-one). Values were calculated from isocratic runs (0, 10, 20, 30, 35% acetonitrile) in both positive and negative modes. Good correlation (r(2) = 0.97) was achieved for n-in-one results obtained with ammonium acetate buffer and mass spectrometry, compared with the traditional method involving single compound analysis with phosphate buffered saline and an ultraviolet detector. A gradient elution method providing fast determination of relative log k(IAM) values in a single IAM/APCI-MS run was demonstrated for the same compounds.

The autoimmune regulator: a key toward understanding the molecular pathogenesis of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is an autoimmune disease with autosomal recessive inheritance. APECED is characterized by the breakdown of tolerance to several organ-specific selfantigens. The symptoms of APECED fall into three main categories: autoimmune polyendocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The gene defective in APECED, AIRE, has been cloned and numerous mutations in this gene have been found in patients with APECED. AIRE is predicted to encode a 545-amino-acid protein containing structural domains characteristic for transcription regulators. The protein has been shown to act as a transcriptional activator in vitro. The AIRE protein is mainly localized to the nucleus, where it can be detected as speckles resembling nuclear bodies. In humans, the expression of AIRE has been observed predominantly in immunologically relevant tissues, especially the thymus. Recently, we have shown in the mouse that Aire is also expressed in various tissues and cell types outside the immune system.

Effect of nitro groups and alkyl chain length on the negative ion tandem mass spectra of alkyl 3-hydroxy-5-(4'-nitrophenoxy) and alkyl 3-hydroxy-5-(2', 4'-dinitrophenoxy) benzoates

Tandem mass spectrometric behaviour was studied for a small combinatorial library of alkyl 3-hydroxy-5-(4'-nitrophenoxy) benzoates (A1-A5) and alkyl 3-hydroxy-5-(2', 4'-dinitrophenoxy) benzoates (B1-B5). The spectra were recorded by negative ion electrospray low-energy collision induced dissociation (CID) tandem mass spectrometry. The product ion spectra of [M - H](-) of the benzoates A1-A5 are similar, as are those of benzoates B1-B5. However, the spectra of the B series compounds differ significantly from those of the A series owing to the second electron-withdrawing nitro substituent in the B compounds. In addition, the length of the alkyl chain has an effect on the fragmentation. However, both series of compounds exhibit an abundant nitrophenoxy ion formed by the loss of 3-hydroxybenzoate. This is at m/z 138 in A1-A5 and at m/z 183 in B1-B5. A precursor ion scan of the nitrophenoxy ion provides a rapid method to identify the synthesised compounds in this type of combinatorial mixture. Copyright 1999 John Wiley & Sons, Ltd.

Functional consequences of amyloidosis mutation for gelsolin polypeptide -- analysis of gelsolin-actin interaction and gelsolin processing in gelsolin knock-out fibroblasts.

Gelsolin, an actin-modulating protein, derived from a single gene exists in intracellular and secreted forms. A point mutation at position 187 of both forms of gelsolin causes familial amyloidosis of the Finnish type (FAF). Here, we expressed both isoforms of the wild-type and FAF mutant gelsolin in mouse embryonic gelsolin-null fibroblasts. We demonstrate that the FAF mutation does not interfere with the normal actin-modulating function of intracellular gelsolin, and that aberrant processing of secreted FAF gelsolin to FAF amyloid precursor takes place in the gelsolin-negative background. These results suggest that, in patients with FAF, symptoms are caused by the accumulation in their tissues of amyloid derived from plasma gelsolin and are not due to functional differences in cytoplasmic gelsolin.

Enzyme-assisted synthesis and structural characterization of nitrocatechol glucuronides.

Enzyme-assisted synthesis and characterization are described for 3-O-beta-D-glucuronides 1b-4b of the aglycons E- and Z-2-cyano-N, N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide (entacapone), 1a and 2a, respectively, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2, 4-pentanedione (nitecapone) 3a and 4'-methyl-3, 4-dihydroxy-5-nitrobenzophenone (tolcapone) 4a, and 1-o- and 2-o-glucuronides 5b and 6b of the aglycon 1, 2-dihydroxy-4-nitrobenzene 5a. Liver microsomes from rats pretreated with Aroclor 1254 were used as catalyst in the synthesis. Glucuronidation was regio- and stereoselective in the case of 1a-4a; only one product was observed by HPLC, HPTLC, and NMR. The glucuronidation of 1,2-dihydroxy-4-nitrobenzene 5a resulted in equal amounts of 1-O-beta-D- and 2-O-beta-D-glucuronides. Purification of the crude products by C18 solid-phase extraction and/or flash chromatography gave compounds 1b-6b in 38-98% yields (50-84 mg). The structures of the glucuronides were characterized on the basis of UV and IR spectra and confirmed with FAB-MS and NMR spectroscopy.

Cells of the neuronal lineage play a major role in the generation of amyloid precursor fragments in gelsolin-related amyloidosis.

Gelsolin-related amyloidosis or familial amyloidosis, Finnish type (FAF) (OMIM No105120) is a hereditary amyloid disease caused by a mutation in a precursor protein for amyloid (gelsolin) and characterized by corneal dystrophy and polyneuropathy. In vitro expression of the FAF-mutant (Asp187 --> Asn/Tyr) secretory gelsolin in COS cells leads to generation of an aberrant polypeptide presumably representing the precursor for tissue amyloid. Here, we provide evidence that this abnormal processing results from defective initial folding of the secreted FAF gelsolin due to the lack of the Cys188-Cys201 disulfide bond, normally formed next to the FAF mutation site. We compared cells of different tissue origin and discovered a dramatic difference between the amount of cleavage of FAF gelsolin to the amyloid precursor in neuronal and non-neuronal cells. More than half of the mutant gelsolin was cleaved in PC12 and in vitro differentiated human neuronal progenitor cells. In contrast, human fibroblasts and Schwannoma cell cultures showed only a limited capacity to cleave FAF gelsolin, although the cleavage mechanism per se seems to be similar in the various cell types. The present findings of processing and distribution of secreted FAF gelsolin in the neuronal cells emphasize the role of neurons in the tissue pathogenesis of this amyloid polyneuropathy.

Identification of the circulating amyloid precursor and other gelsolin metabolites in patients with G654A mutation in the gelsolin gene (Finnish familial amyloidosis): pathogenetic and diagnostic implications.

Familial amyloidosis of the Finnish type (FAF) is an autosomal dominant type of systemic amyloidosis caused by a G654A (Asn-187) or G654T (Tyr-187) mutation in the gelsolin gene. Herein we show that patients with the Asn-187 gelsolin mutation have, in addition to full-sized gelsolin, a series of lower-Mr C-terminal fragments of gelsolin (Mr of 70,000-45,000) in the circulation, and that a 50 to 55-kd fragment of gelsolin is excreted in the urine. In homozygous FAF (Asn-187), the 65-kd fragment, which contains the amyloid-forming region (Ala173-Met243), and the 55-kd fragment, which is devoid of that region, are the major gelsolin species in plasma; whereas normal gelsolin, as well as a 70-kd fragment identified as the C-terminal portion of gelsolin starting at Glu122, and a 45-kd fragment starting at Ser384, are minor components. In patients heterozygous for the Asn-187 mutation--the usual form of the expression of the dominant disease--normal-sized gelsolin is the major circulating form; the 65- and 55-kd fragments represent minor components. Immunodetection of the plasma 65-kd gelsolin fragment, which is disease-specific, and measurement of the urinary gelsolin fragment provide useful means for diagnosing FAF.

Tissue distribution and levels of gelsolin mRNA in normal individuals and patients with gelsolin-related amyloidosis.

We measured quantitatively the mRNA levels of intracellular and secretory forms of gelsolin, an actin-modulating protein, in human tissues from subjects of different ages. The intracellular gelsolin mRNA constituted the major type of gelsolin steady-state mRNA in all tissues analyzed. Both forms of gelsolin were expressed in most adult tissues, with particularly high mRNA levels in all types of muscle and interestingly in skin. Between the adult and infantile tissues the most striking difference in expression levels was found in liver, as the adult liver contained only a subtle amount of gelsolin mRNA. Skin and muscle samples from patients with gelsolin-related amyloidosis (FAF), with significantly increased concentrations of serum gelsolin, did not reveal an increased expression of the gene, and both mutant and wild-type alleles were expressed in equal amounts. The high level of expression of the gelsolin gene in the skin in general could locally contribute to the characteristic skin amyloidosis in FAF patients.

In vitro expression analysis shows that the secretory form of gelsolin is the sole source of amyloid in gelsolin-related amyloidosis.

Amyloidoses are a group of diseases where abnormal fibrillar protein deposits accumulate in patients' tissues. In familial amyloidosis of the Finnish type (FAF), or gelsolin-related amyloidosis, the amyloid subunit protein consists of gelsolin peptides of amino acids 173-243 with the disease causing substitution at Asp187. Gelsolin is an actin-modulating protein and exists in both secretory and intracellular forms both encoded by a single gene in chromosome 9. We have previously shown that the FAF-associated forms of secretory gelsolin carrying the Asp187Asn or Asp187Tyr mutations are abnormally processed in cells, resulting in the secretion of an aberrant 68 kDa carboxyterminal fragment. Here we demonstrate by N-terminal sequencing that the amino terminus of this abnormal fragment is the amino acid 173 and thus represents the N-terminus of the FAF amyloid. We also provide evidence that the same truncated gelsolin can be found among the aberrant gelsolin fragments detected in patients' CSF. Finally, we also expressed the FAF-associated forms of intracellular gelsolin in COS-1 cells, and found no abnormality in their processing opposite to secretory form. Our results provide strong evidence that the secretory gelsolin is solely responsible for the amyloid formation in FAF.

Toward understanding the pathogenic mechanisms in gelsolin-related amyloidosis: in vitro expression reveals an abnormal gelsolin fragment.

Gelsolin-related amyloidosis, also called familial amyloidosis, Finnish type (FAF) is an autosomal dominantly inherited disorder characterized by progressive polyneuropathy and corneal lattice dystrophy. All the analyzed patients are found to carry a nucleotide substitution of A or T for G654 in their gelsolin gene, which at the protein level results in the conversion of the 187 amino acid residue, aspartic acid, to asparagine or tyrosine, respectively. In this study, we transfected mammalian mesenchymal COS-1 cells with a derivative of the expression vector pCD-X containing cDNA coding for the wild-type (D187) and mutant forms (N187 and Y187) of plasma gelsolin. Both disease-associated mutant forms of gelsolin were found to be abnormally processed, which led to the secretion of an aberrant 68 kDa gelsolin fragment into the culture media. This fragment most probably represents a carboxy-terminal part of the protein and contains the suggested amyloid-forming sequence. Initial data were also obtained for involvement of a metalloendoprotease in the pathologic processing. This aberrant proteolysis is likely to represent a crucial initiator step in the cascade resulting in amyloid accumulation in patients' tissues.