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PubMed1997

Identification of complex genomic breakpoint junctions in the t(9;11) MLL-AF9 fusion gene in acute leukemia.

The MLL gene at chromosome 11, band q23, is involved in translocations with as many as 40 different chromosomal bands. Virtually all breakpoints occur within an 8.3 kb BamHI fragment and result in 5' MLL fused to partner genes in a 5'-3' orientation. The translocation t(9;11)(p22;q23), which results in the fusion of MLL to AF9, is the most common of the 11q23 chromosomal abnormalities observed in de novo acute myeloid leukemia (AML), in therapy related leukemia (t-AML), and rarely in acute lymphoblastic leukemia (ALL). We have studied 24 patients with a t(9;11) and an MLL rearrangement, including 19 patients with AML, four with t-AML, and one with ALL. To understand the mechanisms of this illegitimate recombination, we cloned and sequenced the t(9;11) translocation breakpoint junctions on both derivative chromosomes from one AML patient and from the Mono Mac 6 (MM6) cell line, which was derived from a patient with AML. Two different complex junctions were noted. In the AML patient, both chromosome 11 and 9 breaks were staggered, occurred in Alu DNA sequences, and resulted in a 331 bp duplication. In the MM6 cell line, breaks in chromosomes 11 and 9 were also staggered, but, in contrast to the finding in the AML patient, the breaks did not involve Alu DNA sequences and resulted in a 664 bp deletion at the breakpoints. Using reverse transcriptase (RT-) PCR, we analyzed 11 patient samples, including the two just described, for MML-AF9 fusions. The fusion occurred in six of seven AML patients, two of two t-AML patients, one patient with ALL, and in the MM6 cell line. Interestingly, all of the breaks within the AF9 gene in AML patients occurred in the central AF9 exon, called Site A by others, whereas in the single ALL patient the breakpoint mapped to a more 3' region of the AF9 gene. Our data, when combined with those of others, suggest that the fusion point within the AF9 gene, and thus the amount of AF9 material included in the MLL-AF9 fusion gene product, may influence the phenotype of the resulting leukemia. This further supports the proposal that the MML translocation partner genes play a critical role in the leukemogenic process.

Acute Disease↗
PubMed2001

Amniotomy plus intravenous oxytocin for induction of labour.

BACKGROUND: Induction of labour is a common obstetric intervention. Amniotomy alone for induction of labour is reviewed separately and oxytocin alone for induction of labour is being prepared for inclusion in The Cochrane Library. This review will address the use of the combination of these two methods for induction of labour in the third trimester. This is one of a series of reviews of methods of cervical ripening and labour induction using standardised methodology. OBJECTIVES: To determine, from the best available evidence, the efficacy and safety of amniotomy and intravenous oxytocin for third trimester induction of labour. SEARCH STRATEGY: The Cochrane Pregnancy and Childbirth Group Trials Register, the Cochrane Controlled Trials Register and reference lists of articles were searched. Date of last search: May 2001. SELECTION CRITERIA: The criteria for inclusion included the following: (1) clinical trials comparing amniotomy plus intravenous oxytocin used for third trimester cervical ripening or labour induction with placebo/no treatment or other methods listed above it on a predefined list of labour induction methods; (2) random allocation to the treatment or control group; (3) adequate allocation concealment; (4) violations of allocated management not sufficient to materially affect conclusions; (5) clinically meaningful outcome measures reported; (6) data available for analysis according to the random allocation; (7) missing data insufficient to materially affect the conclusions. DATA COLLECTION AND ANALYSIS: Trial quality assessment and data extraction were done by both reviewers. A strategy was developed to deal with the large volume and complexity of trial data relating to labour induction. This involved a two-stage method of data extraction. The initial data extraction was done centrally, and incorporated into a series of primary reviews arranged by methods of induction of labour, following a standardised methodology. The data is to be extracted from the primary reviews into a series of secondary reviews, arranged by category of woman. MAIN RESULTS: Seventeen trials involving 2566 women were included. Amniotomy and intravenous oxytocin were found to result in fewer women being undelivered vaginally at 24 hours than amniotomy alone (relative risk (RR) 0.03, 95% confidence intervals (CI) 0.001-0.49). This finding was based on the results of a single study of 100 women. As regards secondary results amniotomy and intravenous oxytocin resulted in significantly fewer instrumental vaginal deliveries than placebo (RR 0.18, CI 0.05-0.58). Amniotomy and intravenous oxytocin resulted in more postpartum haemorrhage than vaginal prostaglandins (RR 5.5, CI 1.26-24.07). Significantly more women were also dissatisfied with amniotomy and intravenous oxytocin when compared with vaginal prostaglandins, RR 53, CI 3.32-846.51. REVIEWER'S CONCLUSIONS: Data on the effectiveness and safety of amniotomy and intravenous oxytocin are lacking. No recommendations for clinical practice can be made on the basis of this review. Amniotomy and intravenous oxytocin is a combination of two methods of induction of labour and both methods are utilised in clinical practice. If their use is to be continued it is important to compare the effectiveness and safety of these methods, and to define under which clinical circumstances one may be preferable to another.

Amnion↗
PubMed1987

Interdisciplinary andrology. STDs. AIDS research.

Multidisciplinary andrology deals with clinical application and modern technology for the evaluation and differential diagnosis of male infertility with emphasis on morphological, anatomical, biochemical, immunological, hereditary, and microbiological parameters. Little is known about the effects of diet, disease, stress, environmental, and drugs on male-related unexplained infertility of couples. Regional, national, and international centers of multidisciplinary andrology should provide (1) extensive and unique clinical services; (2) a computerized "patient referral center," (3) self-learning packages (slides/tape programs) for patients; and (4) a computer link to the National Library of Medicine and the Drug Information Center. Specialized laboratories and clinics can be served by expert consultants, visiting professors, bilingual and well-trained clinicians, nurses, laboratory technologists, computer operators, and related allied health personnel. Patient education pamphlets, updated every few years, can be distributed during training workshops when an extensive network of remote teleprinters can be utilized. Qualified client location may install a printer to allow on-site printing of reports in the shortest possible time. Special mailing containers are provided to clients who wish to mail their laboratory specimens. Other clinical services may include the following: 1. Central source of communication and information in andrology; 2. International roster of multidisciplinary andrology centers; 3. Patient referral to centers and consultations for developing countries; 4. Screening of husbands and wives for in vitro fertilization/embryo transfer (IVF/ET); 5. Screening of couples with unexplained infertility for sexually transmitted diseases (STDs) including AIDS; 6. Exchange of research material and methodology; 7. Coordination of multicenter research; 8. Organizing training workshops for clinicians, nurses, and laboratory technicians; 9. Establishing a repository of films, video tapes, slides, catalogs, instrumentation, books, SEM photos, and atlases; 10. Publication and editorial assistance; 11. Consultation for the appropriate selection, purchase, and quality control of instrumentation (all on one computer system); 12. Evaluation of new diagnostic tools for idiopathic infertility and fertility regulation; 13.(ABSTRACT TRUNCATED AT 400 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed2006

Home Study Course: summer 2006.

OBJECTIVE: The Home Study Course is intended for the practicing colposcopist or practitioner who is seeking to develop or enhance his or her colposcopic skills. The goal of the course is to present colposcopic cases that are unusual or instructive in terms of appearance, presentation, or management, or that demonstrate new and important knowledge in the area of colposcopy or pathology. Participants may benefit from reading and studying the material or from testing their knowledge by answering the questions. ACCME ACCREDITATION: The American Society for Colposcopy and Cervical Pathology (ASCCP) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. The American Society for Colposcopy and Cervical Pathology designates this educational activity for a maximum of 1 AMA PRA Categorys I Creditt. Physicians should only claim credit commensurate with the extent of their participation in the activity. ASCCP also designates their educational activity for 1 Category 1 credit hour of the ASCCP's Program for Continuing Professional Development. Credit is available for those who choose to apply. The Home Study Course is planned and produced in accordance with the ACCME's Essential Areas and Elements. DISCLOSURES: The clinical history and images in the Home Study Course may represent an actual case, but not always. To improve educational quality, some gross, cytological, or histological images may come from photographic libraries. Good teaching cases are often difficult to obtain, and we encourage our readers to submit cases with high-quality images to the Home Study Course editor or executive editor to consider for publication. Lastly, faculty must disclose any significant financial interest or relationship with proprietary entities that may have a direct relationship to the subject matter. For this course, the authors had no such relationship to report.

Adult↗
PubMed2006

Home study course: fall 2006.

OBJECTIVE: The Home Study Course is intended for the practicing colposcopist or practitioner who is seeking to develop or enhance his/her colposcopic skills. The goal of the course is to present colposcopic cases that are unusual or instructive in terms of appearance, presentation, or management, or that demonstrate new and important knowledge in the area of colposcopy or pathology. Participants may benefit from reading and studying the material or from testing their knowledge by answering the questions. ACCME ACCREDITATION: The American Society for Colposcopy and Cervical Pathology (ASCCP) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. The ASCCP designates this education activity for a maximum of 1 American Medical Association Physician's Recognition Award Category I Credit. Physicians should only claim credit commensurate with the extent of their participation in the activity. The ASCCP also designates their educational activity for 1 Category 1 credit hour of the ASCCP's Program for Continuing Professional Development. Credit is available for those who choose to apply. The Home Study Course is planned and produced in accordance with the ACCME's Essential Areas and Elements. DISCLOSURES: The clinical history and images in the Home Study Course may represent an actual case, but not always. To improve educational quality, some gross, cytological, or histological images may come from photographic libraries. Good teaching cases are often difficult to obtain, and we encourage our readers to submit cases with high-quality images to the Home Study Course editor or executive editor to consider for publication.

Adult↗
PubMed2003

Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus-insect cell expression system.

We report gene cloning, plasmid construction, baculovirus expression, purification, and biological activity testing of the human hematopoietic cytokine interleukin-3. cDNA was constructed from extracted total RNA of Jurkat cells. Both signal and structural fragment of interleukin-3 were cloned from this cDNA library, modified by adding a hexahistidine-tag at the C-terminus, and introduced into the pBacPAK9 transfer vector to generate recombinant baculoviruses. For protein expression High Five cells were infected either in spinner flasks or 2.5-L bioreactors in batch culture yielding levels of 1.5-3 mg L(-1) interleukin-3 in the cell culture supernatant. Interleukin-3 was purified by a single step chromatography using cobalt metal affinity resins, which yielded a highly stable and soluble protein. N-terminal amino acid sequencing of the purified interleukin-3 showed correct cleavage of the signal peptide during protein processing. The two N-glycosylation sites were found to be occupied by 100 and 35%, respectively, with an N-glycan pattern of paucimannosidic structures, which are typical for recombinant glycoproteins produced by High Five lepidopteran cells. The specific biological activity of purified interleukin-3 was several times higher when compared with different lots of commercially available material from Escherichia coli. The results indicate that the strategy we used in this experiment is a straightforward and convenient way for recombinant protein preparation and can be adapted to produce other recombinant cytokines.

Amino Acid Sequence↗
PubMed1998

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly.

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 'invades' the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

Amino Acid Sequence↗
PubMed2003

Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis (para rubber tree).

Latex of Hevea brasiliensis (Willd. ex A, Juss.) Mull. Arg. (Brazilian rubber tree) contains 30-50% (w/w) of natural rubber (cis-1,4-polyisoprene), which is an important raw material for many industrial uses. In order to gain insights into the molecular events occurring in latex, we analyzed more than 20,000 cDNA-AFLP-based TDFs (transcription-derived fragments) and 1176 ESTs. The results revealed several novel features of the latex transcriptome. First, the repertoire of the genes expressed in latex is unique. Only seven gene families accounted for more than 51% of the latex transcriptome. Among them, two of the most abundant ESTs were the genes encoding rubber particle proteins REF (rubber elongation factor) and SRPP (small rubber particle protein), comprising 29% of the total ESTs. Unexpectedly, several genes involved in the rubber biosynthesis were expressed at low levels in the latex. In fact, genes encoding cis -prenyltransferase (CPT), a potential candidate for rubber polymerase, were not present in the EST pool because of their low expression level. However, we were able to clone four full-length cDNAs by screening the same latex cDNA library used in the EST analysis and confirmed their enzyme activity in vitro. The second most abundant transcripts were defense- or stress-related genes, suggesting that defense is one of the functions of laticifers. Finally, the presence of the non-mevalonate DXP/MEP pathway for IPP synthesis in latex was noted by up-regulation of the 1-deoxy-D-xylulose 5-phosphate synthase gene.

Amino Acid Sequence↗
PubMed1998

Who needs evidence-based health care?

The vast amount of published material in clinical and biomedical sciences, and conflicting results on diagnostic and therapeutic procedures may introduce doubts in decision-making for patient care. Information retrieving skills and the critical appraisal of published literature, together with elaboration of practice guidelines based on epidemiological methodology, form the basis of the trend towards evidence-based health care, which aims to overcome these problems. A survey conducted by questionnaire at the Chaim Sheba Medical Center analyzed which types of information sources are considered most relevant and useful for patient care by a cross-section of physicians with varying degrees of experience. They considered review articles and meta-analyses extremely reliable for information purposes, while for practical patient-care purposes they tended to rely more on the opinions of peers and experts. As the requirements of evidence-based health care may influence the attitudes of clinicians to the published literature and its evaluation, they have implications for medical libraries and information centers. Specifically, information specialists will be called upon more and more to impart information-retrieval and critical appraisal skills to clinicians. The involvement of information specialists in information gathering and selection will provide added value to the expertise and knowledge of in-house experts for decision-making.

Attitude of Health Personnel↗
PubMed1996

Cytogenetic and molecular analysis in trisomy 12p.

We studied a male patient with de novo pure trisomy 12p syndrome by molecular analysis and fluorescence in situ hybridization (FISH) with markers from chromosome 12. G-banding studies demonstrated a 46,XY, 22p+ karyotype and the banding pattern and clinical findings suggested that the extra chromosomal material was derived from 12p. Trisomy 12p was confirmed by dosage analysis with chromosome 12p markers and FISH analysis with a whole chromosome 12 paint. The de novo re-arranged chromosome was of paternal origin. A comparison of the clinical and cytogenetic findings in this patient was made with previously described cases of trisomy 12p. We propose a classification system for 12p trisomy in order to better characterize the correlative relationships between specific cytogenetic constitution and phenotype.

Abnormalities, Multiple↗
PubMed2003

A reference ontology for biomedical informatics: the Foundational Model of Anatomy.

The Foundational Model of Anatomy (FMA), initially developed as an enhancement of the anatomical content of UMLS, is a domain ontology of the concepts and relationships that pertain to the structural organization of the human body. It encompasses the material objects from the molecular to the macroscopic levels that constitute the body and associates with them non-material entities (spaces, surfaces, lines, and points) required for describing structural relationships. The disciplined modeling approach employed for the development of the FMA relies on a set of declared principles, high level schemes, Aristotelian definitions and a frame-based authoring environment. We propose the FMA as a reference ontology in biomedical informatics for correlating different views of anatomy, aligning existing and emerging ontologies in bioinformatics ontologies and providing a structure-based template for representing biological functions.

Abstracting and Indexing↗
PubMed2006

Microbial community analysis of a biogas-producing completely stirred tank reactor fed continuously with fodder beet silage as mono-substrate.

The bioconversion of renewable raw material to biogas by anaerobic microbial fermentation processes in completely stirred tank reactors (CSTR) is a valuable alternative resource of energy especially for rural areas. However, knowledge about the microorganisms involved in the degradation of plant biomass is still poor. In this study, a first analysis of the biogas-forming process within a CSTR fed continuously with fodder beet silage as mono-substrate is presented in the context of molecular data on the microbial community composition. As indicated by the conventional process parameters like pH value, content of volatile fatty acids, N:P ratio and the biogas yield, the biogas-forming process within the CSTR occurred with a stable and efficient performance. The average biogas yield based on volatile solids was 0.87m(3)kg(-1) at an organic loading rate of 1.2-2.3kgm(-3)d(-1). This amounts to 94% of the theoretical maximum. In order to identify microorganisms within the CSTR, a 16S rDNA clone library was constructed by PCR amplification applying a prokaryote-specific primer set. One hundred and forty seven clones were obtained and subsequently characterized by amplified rDNA restriction analysis (ARDRA). The sequences of 60 unique ARDRA patterns were estimated in a length of approximately 800-900bp each. Four of them were assigned to the domain Archaea and 56 to the domain Bacteria. Within the domain Archaea, all clones showed a close relationship to methanogenic species. Major bacterial groups represented in the clone library were the class Clostridia of the phylum Firmicutes (22% of all 16S rDNA clones), the class Deltaproteobacteria of the phylum Proteobacteria (24%), the class Bacilli of the phylum Firmicutes (22%) and members of the phylum Bacteroidetes (21%). Within these major groups, the highest biodiversity was found within the class Clostridia (35% of all operational taxonomic units). Members of the phyla Actinobacteria and Spirochaetes were represented only by 5 and 2 clonal sequences, respectively.

Archaea↗
PubMed2004

Molecular cloning and characterization of a cytochrome P450 taxoid 2alpha-hydroxylase involved in Taxol biosynthesis.

The Taxol biosynthetic pathway, arising from the primary isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate in yew (Taxus), consists of approximately twenty steps, at least nine of which are thought to be cytochrome P450-mediated oxygenations. Several oxygenases involved in the early hydroxylation steps of the pathway have been identified and the corresponding genes have been cloned; however, defining the enzymes and their genes responsible for oxygenations in the central portion of the pathway is more difficult because neither the exact sequence of reactions nor the relevant intermediates are known. A surrogate substrate, (+)-taxusin (taxa-4(20),11(12)-dien-5alpha,9alpha,10beta,13alpha-tetraol tetraacetate), that was previously employed in the isolation of a taxoid 7beta-hydroxylase, was used here to functionally screen a family of cytochrome P450 oxygenases originating from a Taxus cell EST library. This in vivo screen in yeast led to the identification of a 1488bp cDNA clone (encoding a 495 residue protein) that was capable of producing 2alpha-hydroxytaxusin from taxusin with a K(m) value of 10.5 +/- 2.7 microM and k(cat) of about 0.05 s(-1) for the surrogate substrate. This structurally typical cytochrome P450 resembles most closely the previously isolated taxoid 7beta-hydroxylase, which also uses taxusin as a substrate, and both 2alpha- and 7beta-hydroxylases are capable of the reciprocal conversion of their respective pentaol tetraacetate products to the common hexaol tetraacetate. This C2-hydroxylase would appear to mediate the mid-pathway functionalization of the C2-position of the taxane core that ultimately bears a benzoyl group as an important Taxol pharmacophore. Overexpression of this cytochrome P450 taxoid 2alpha-hydroxylase in Taxus cells may improve Taxol yields and could prove useful in the production of other 2alpha-hydroxy taxoids as starting materials for subsequent acylation at this position.

Amino Acid Sequence↗
PubMed2004

Pyridoxine biosynthesis in yeast: participation of ribose 5-phosphate ketol-isomerase.

To identify the genes involved in pyridoxine synthesis in yeast, auxotrophic mutants were prepared. After transformation with a yeast genomic library, a transformant (A22t1) was obtained from one of the auxotrophs, A22, which lost the pyridoxine auxotrophy. From an analysis of the plasmid harboured in A22t1, the RKI1 gene coding for ribose 5-phosphate ketol-isomerase and residing on chromosome no. 15 was identified as the responsible gene. This notion was confirmed by gene disruption and tetrad analysis on a diploid prepared from the wild-type and the auxotroph. The site of mutation on the RKI1 gene was identified as position 566 with a transition from guanine to adenine, resulting in amino acid substitution of Arg-189 with lysine. The enzymic activity of the Arg189-->Lys (R189K) mutant of ribose 5-phosphate ketolisomerase was 0.6% when compared with the wild-type enzyme. Loss of the structural integrity of the protein seems to be responsible for the greatly diminished activity, which eventually leads to a shortage of either ribose 5-phosphate or ribulose 5-phosphate as the starting or intermediary material for pyridoxine synthesis.

Aldose-Ketose Isomerases↗
PubMed1996

Representation of unique sequences in libraries of randomized nucleic acids.

From a library of nucleic acid molecules, which are randomized in parts of their sequence, unique sequence variants can be selected for specific properties. The planning of such an in vitro selection experiment requires some consideration regarding how much DNA template or RNA transcript should be used initially. The amount applied depends on the number of randomized nucleotides and on the expectations of how often each conceivable and unique sequence combination should be represented in the experimental pool. We display graphs describing the probability for the representation of unique nucleic acid molecules in a randomized pool as a function of the mean representation k, defined by the ratio of sampled nucleic acid molecules to conceivable sequence combinations and we summarize the amounts required to represent unique sequences with 99% likelihood. The probability of representation, P = 1-e-k, can be applied also to 'sub-saturated' pools (k < 1) of nucleic acids with long randomized domains, where it is impossible to provide sufficient material for full sequence representation.

Forecasting↗
PubMed2005

Radiation shielding and dosimetry experiments updates in the SINBAD database.

The Shielding Integral Benchmark Archive Database (SINBAD) is an internationally established set of radiation shielding and dosimetry data related to experiments relevant in reactor shielding, fusion blanket neutronics and accelerator shielding. In addition to the characterisation of the radiation source, it describes shielding materials and instrumentation and the relevant detectors. The experimental results, be it dose or reaction rates, or unfolded spectra, are presented in tabular ASCII form that can easily be exported to different computer environments for further use. Most sets in SINBAD also contain the computer model used for the interpretation of the experiment and, where available, results from uncertainty analysis. This is an international effort between the Organization for Economic Cooperation and Development, Nuclear Energy Agency Data Bank (http://www.nea.fr/html/databank/) (OECD/NEA Data Bank) and Oak Ridge National Laboratory, Radiation Safety Information Computational Center (http://www-rsicc.ornl.gov/rsic.html) (ORNL/RSICC). Cooperation from many organisations, authors and benchmark analysts have helped SINBAD become a 'living database'--one which involves continuous information updates, preservation and additions of nuclear benchmarks in the areas of fusion, fission and accelerator science and engineering. This paper focuses on the increased comprehensiveness of experiments that have been carried out in recent years and the validation of computer code and cross section library using these experiments.

Database Management Systems↗
PubMed2004

HPLC determination of mycophenolic acid and mycophenolic acid glucuronide in human plasma with hybrid material.

Mycophenolic acid (MPA), the active metabolite of the prodrug mycophenolate mofetil is an immunosuppressive agent which inhibits inosine monophosphate dehydrogenase. MPA is metabolised to phenolic glucuronide (MPAG) that may be hydrolysed in vivo to form free MPA. Drug monitoring is required in patients with multi-organ failure. Here, we report a HPLC method with organic/inorganic hybrid material for the simultaneous analysis of MPA and MPAG in human plasma. MPA and MPAG and carboxy butoxy ether mycophenolic acid (MPAC) used as internal standard were analysed on a bonded X-Terra column with a linear gradient elution mode using orthophosphoric acid and acetonitrile as eluents. Sample treatment procedure consists of deproteinisation with acetonitrile. Analytical recoveries were higher than 98 and 89% at concentrations ranging from 1 to 25 and 20 to 200mg/L for MPA and MPAG, respectively. Calibration curves fitted by plotting the peak area ratio (compound of interest/internal standard) versus concentration were linear in the range 0.2-50mg/L for MPA and in the range 1-500mg/L for MPAG. The quantification limit was 0.2mg/L for MPA and 1mg/L for MPAG with a coefficient of variation less than 20% for a 500microL sample volume. Intra- and inter-assay coefficient of variation was lower than 7% for all compounds. Detection was performed at 215nm. Peak identity was confirmed through library matching by comparison with reference spectra. The X-Terra column provides good peak shape and may be used at low pH with a long life-time column. This HPLC method using a simple sample treatment procedure appears suitable for therapeutic drug monitoring in organ-transplant patients. The method is sensitive enough for monitoring MPA and MPAG during pharmacokinetic studies.

Chromatography, High Pressure Liquid↗
PubMed2003

Discovery of a new phosphotyrosine mimetic for PTP1B using breakaway tethering.

Protein tyrosine phosphatases play important roles in many signaling cascades involved in human disease. The identification of druglike inhibitors for these targets is a major challenge, and the discovery of suitable phosphotyrosine (pY) mimetics remains one of the key difficulties. Here we describe an extension of tethering technology, "breakaway tethering", which is ideally suited for discovering such new chemical entities. The approach involves first irreversibly modifying a protein with an extender that contains both a masked thiol and a known pY mimetic. The extender is then cleaved to release the pY mimetic, unmasking the thiol. The resulting protein is screened against a library of disulfide-containing small molecule fragments; any molecules with inherent affinity for the pY binding site will preferentially form disulfides with the extender, allowing for their identification by mass spectrometry. The ability to start from a known substrate mimimizes perturbation of protein structure and increases the opportunity to probe the active site using tethering. We applied this approach to the anti-diabetic protein PTP1B to discover a pY mimetic which belongs to a new molecular class and which binds in a novel fashion.

Binding Sites↗