Studies on the metabolism and toxicological detection of the Eschscholtzia californica alkaloids californine and protopine in urine using gas chromatography-mass spectrometry

Eschscholtzia californica preparations are in use as phytopharmaceuticals and as herbal drugs. Studies are described on the metabolism and the toxicological analysis of the Eschscholtzia californica alkaloids californine and protopine in rat urine using gas chromatography-mass spectrometry. The identified metabolites indicated that californine is extensively metabolized by N-demethylation and/or single or double demethylenation with consecutive catechol-O-methylation of one of the hydroxy groups. Protopine, however, only undergoes extensive demethylenation of the 2,3-methylenedioxy group followed by catechol-O-methylation. All phenolic hydroxy metabolites were found to be partly conjugated. The authors' systematic toxicological analysis procedure using full-scan gas chromatography-mass spectrometry after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of the main metabolites of californine and protopine in rat urine after a dose which should correspond to that of drug users. Therefore, use of Eschscholtzia californica preparations should also be detectable in human urine by the authors' systematic toxicological analysis procedure.     

Structure-activity relationship studies on 2-heteroaryl-4-arylimidazoles NPY5 receptor antagonists

A series of 2-heteroaryl-4-arylimidazoles with potent in vitro activity at the NPY5 receptor was developed. Introduction of electron-withdrawing groups on the 4-aryl ring led to a significant improvement of in vitro potency. Several analogues from this series had anorectic activity in rodent feeding models, but were also found to have undesired behavioral effects in spontaneous locomotor activity.     

Two supernumerary marker chromosomes, originating from chromosomes 6 and 11, in a child with developmental delay and craniofacial dysmorphism

The interpretation of the significance of marker chromosomes, which can be encountered at prenatal diagnosis, is extremely problematic. Various factors contribute to the difficulty of clarifying the phenotypic risks of supernumerary marker chromosomes, including differences in the size, structure, and origin of marker chromosomes, as well as the occurrence of multiple marker chromosomes of different origin in the same proband. Research on marker chromosomes is currently in a data-accumulation phase. We report the presence of two marker chromosomes, originating from chromosomes 6 and 11, in a child with developmental delay and craniofacial dysmorphism and discuss the related literature.     

Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha

Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and importin beta. Srp1p, the Saccharomyces cerevisiae homologue of importin alpha, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state. We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p. Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC). Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain. We show that Nup2p effectively releases the NLS protein from importin alpha-importin and beta and strongly binds to the importin heterodimer via Srp1p. Kap95p (importin beta) is released from this complex by a direct interaction with Gsp1p-GTP. These data suggest that besides Gsp1p, which disassembles the NLS-importin alpha-importin beta complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p. We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import. Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC. The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus.     

Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL). New gain of function mutations in the loop region

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence. Not all of the resulting M. tuberculosis mutants displayed a gain of function phenotype; for instance, normal phenotypes were noted for mutations at Ala(20), the analogue of the highly sensitive Gly(22) site in E. coli. A previously unnoticed intersubunit hydrogen bond in the extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking studies indicated that these sites are spatially close under physiological conditions. In general, mutation at these positions results in a gain of function phenotype, which provides strong evidence for the importance of the loop region in MscL channel function. No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment. Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL.     

Molecular cloning, chromosome mapping and characterization of UBQLN3 a testis-specific gene that contains an ubiquitin-like domain

The sequence of the ubiquitin protein is highly conserved between species and has facilitated the cloning of numerous ubiquitin-like proteins. In the present study, we report the cloning of the cDNA for human ubiquilin 3 (UBQLN3). The deduced amino acid sequence of UBQLN3 contains a UBQ domain (ubiquitin-like) in the amino terminus as well as two highly conserved domains found in several recently cloned ubiquitin-like proteins. One of these domains, termed the NP domain, is a highly conserved 93 amino acid region present in UBQLN3 and several ubiquitin-like proteins. The last conserved domain is the UBA domain (ubiquitin-associated) found in a variety of proteins of the ubiquination pathway. The human UBQLN3 gene was mapped to the 11p15 region of chromosome 11. Northern blot analysis of multiple human and mouse tissues demonstrated UBQLN3 mRNA expression specifically in testis.