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Biomedical subjects

M Lui

Publications and source records attributed to M Lui.

At least 19 recordsLinked to original sources
PubMed2001

Apocrine adenoma of the breast: diagnosis on large core needle biopsy.

Apocrine adenoma is a rare benign epithelial tumour of the breast that can radiologically present as a well-defined opacity. The clinical, radiological and pathological findings of apocrine adenoma of the breast in a 72-year-old woman are described. Histologically, the tumour is characterised by a circumscribed proliferation of metaplastic apocrine cells which may contain calcifications. Pathological assessment of the lesion is essential in making the diagnosis.

Adenoma↗
PubMed1999

Identification of a putative effector protein for rab11 that participates in transferrin recycling.

We have identified and cloned the cDNA for a 912-aa protein, rab11BP, that interacts with the GTP-containing active form of rab11, a GTP-binding protein that plays a critical role in receptor recycling. Although rab11BP is primarily cytosolic, a significant fraction colocalizes with rab11 in endosomal membranes of both the sorting and recycling subcompartments. In vitro binding of rab11 to native rab11BP requires partial denaturation of the latter to expose an internal binding site located between residues 334 and 504 that is apparently masked by the C-terminal portion of the protein, which includes six repeats known as WD40 domains. Within the cell, rab11BP must undergo a conformational change in which the rab11-binding site becomes exposed, because when coexpressed with rab11 in transfected cells the two proteins formed abundant complexes in association with membranes. Furthermore, although overexpression of rab11BP did not affect transferrin recycling, overexpression of a truncated form of the protein, rab11BP(1-504), that includes the rab11-binding site but lacks the WD40 domains inhibited recycling as strongly as does a dominant negative rab11 mutant protein that does not bind GTP. Strikingly, the inhibition caused by the truncated rab11BP was prevented completely when the cells also expressed a C-terminally deleted, nonprenylatable form of rab11 that, by itself, has no effect on recycling. We propose that rab11BP is an effector for rab11, whose association with this GTP-binding protein is dependent on the action of another membrane-associated factor that promotes the unmasking of the rab11-binding site in rab11BP.

3T3 Cells↗
PubMed1998

Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis.

Mass spectrometry occupies a central position in most current protein identification schemes. So-called 'mass fingerprinting' techniques rely on composite mass patterns of proteolytic fragments, or dissociation products thereof, to query databases. Keys to successful analysis of ever smaller amounts are sensitivity and complete spectral information, both of which depend for a large part on proper sample preparation. Clean-up and concentration of peptide mixtures over eppendorf gel loading tips filled with chromatographic media (i.e. 'micro-tips') are believed to be quite useful in this regard. We have studied quantitative and qualitative aspects of polypeptide extraction using these small manual devices. Optimization of sample volume and additives, micro-tip bed volume, and eluent composition and volume, all contribute to effective recovery (approximately 65-70%, on average). Improper digest conditions can, in fact, lead to far bigger losses, suggesting the need for at least trace amounts of Zwittergent 3-16. Of particular interest is our finding that partial fractionation, obtained by two-step micro-tip elution, generally results in more and better signals during subsequent mass analysis. Thus, by using optimized micro-tips, in combination with adequate sample handling and instrumentation, direct mass spectrometric identification can be routinely and successfully done in any resource facility type setting.

Amino Acid Sequence↗
PubMed1998

Identification of new mediator subunits in the RNA polymerase II holoenzyme from Saccharomyces cerevisiae.

Mediator was isolated from yeast on the basis of its requirement for transcriptional activation in a fully defined system. We have now identified three new members of mediator in the low molecular mass range by peptide sequence determination. These are the products of the NUT2, CSE2, and MED11 genes. The product of the NUT1 gene is evidently a component of mediator as well. NUT1 and NUT2 were earlier identified as negative regulators of the HO promoter, whereas mutations in CSE2 affect chromosome segregation. MED11 is a previously uncharacterized gene. The existence of these proteins in the mediator complex was verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.

Amino Acid Sequence↗
PubMed1998

The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain.

Mediator was resolved from yeast as a multiprotein complex on the basis of its requirement for transcriptional activation in a fully defined system. Three groups of mediator polypeptides could be distinguished: the products of five SRB genes, identified as suppressors of carboxy-terminal domain (CTD)-truncation mutants; products of four genes identified as global repressors; and six members of a new protein family, termed Med, thought to be primarily responsible for transcriptional activation. Notably absent from the purified mediator were Srbs 8, 9, 10, and 11, as well as members of the SWI/SNF complex. The CTD was required for function of mediator in vitro, in keeping with previous indications of involvement of the CTD in transcriptional activation in vivo. Evidence for human homologs of several mediator proteins, including Med7, points to similar mechanisms in higher cells.

Amino Acid Sequence↗
PubMed1997

The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101.

Two highly similar, PtdIns(4,5)P2-selective, G beta gamma-activated PI3Ks were purified from pig neutrophil cytosol. Both were heterodimers, were composed of a 101 kDa protein and either a 120 kDa or a 117 kDa catalytic subunit, and were activated greater than 100-fold by G beta gammas. Peptide sequence-based oligonucleotide probes were used to clone cDNAs for the p120 and p101 species. The cDNA of p120 is highly related to p110 gamma, while the cDNA of p101 is not substantially related to anything in current databases. The proteins were expressed in and purified from insect and mammalian cells. They bound tightly to one another, both in vivo and in vitro, and in so doing, p101 amplified the effect of G beta gammas on the PI3K activity of p120 from less than 2-fold to greater than 100-fold.

Amino Acid Sequence↗
PubMed1996

Methodical analysis of protein-nitrocellulose interactions to design a refined digestion protocol.

We have analyzed the efficacies of seven different organic solvents, 13 organic bases, and 17 detergents to dissociate electroblotted proteins from nitrocellulose. Most efficient were a 1% piperidine-40% acetonitrile mixture and 1% concentrations of either cetyltrimethylammonium bromide or several polyoxyethylene and zwitterionic detergents at pH > or = 8.5. In general, detergent-promoted elution varied with pH (8.5-->12.0) and temperature (37-->65 degrees C) in a detergent-dependent and protein-dependent manner, making for unpredictable results of any combination. However, Zwittergent 3-16, as the major exception, eluted proteins of different sizes and properties almost equally well at pH 8.5. This preferred effect is not enhanced by addition of organic solvent. In fact, detergent/acetonitrile mixtures were generally less efficient for protein elution. Zwittergent 3-16 (1% in 100 mM NH4HCO3) proved to be the most powerful additive for one-step enzymatic digestion of nitrocellulose-bound proteins, both in terms of highest recoveries (including endoproteinase Lys-C digestions) and general applicability, a major improvement over the use of Tween 80 and hydrogenated Triton X-100. The new digest procedure takes just 2 h to complete and requires 5 ng protease per 1 microliter buffer per 1 mm2 of nitrocellulose; relatively large pieces of membrane (> or = 100 mm2) can be successfully processed. The same detergent is a satisfactory additive for digestion of polyvinylidene difluoridebound proteins as well. Moreover, Zwittergent 3-16 is fully compatible with the usual downstream procedures. It is uv (OD214) transparent and does not interfere with high-performance liquid chromatography in any other way. Apparent incompatibility with direct (i.e., "prechromatography") matrix-assisted laser-desorption ionization time-of-flight mass spectrometry can be either reduced by methanol washing of the dried sample and activation of a low-mass gate or eliminated by a simple microliter cleanup procedure.

Chromatography, High Pressure Liquid↗
PubMed1994

Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR gamma and RXR alpha.

Previously, we identified a novel transcription factor, ARF6, as a key regulator of the tissue-specific adipocyte P2 (aP2) enhancer. In order to identify the proteins which comprise the adipocyte ARF6 complex, we have purified this DNA binding activity from a cultured adipocyte cell line. We have developed a system for growth and differentiation of HIB-1B brown adipocytes in suspension culture that facilitates the production of large quantities of adipocyte nuclear extract. ARF6 was purified from HIB-1B nuclear extract by a combination of conventional and sequence-specific DNA affinity chromotography. Chemical sequencing and mass spectral analysis of tryptic peptides derived from the purified polypeptides identifies the ARF6 complex as a heterodimer of the retinoid X receptor alpha (RXR alpha) and the murine peroxisome proliferator activated receptor gamma (PPAR gamma). Of the known PPAR gamma isoforms, PPAR gamma is the predominant form expressed in adipose tissue. These results suggest that PPAR gamma 2 serves a unique function among PPAR family members as an important regulator of adipocyte-specific gene expression.

Adipocytes↗
PubMed1994

Metal chelates as reversible stains for detection of electroblotted proteins: application to protein microsequencing and immunoblotting.

Coomassie brilliant blue and Ponceau red have traditionally been used to stain electroblotted proteins, since they are compatible with existing N-terminal and internal protein microsequencing as well as with immunoblotting procedures. With recent improvements in sequencing and immunoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alternatives to conventional stains. Electroblotted proteins were detected by blocking nonspecific sites with polyvinylpyrrolidone-40 followed by incubation in metal chelate solutions at acidic pH values. Two of the most promising metal chelate stains were the Ferrozine/ferrous complex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electroblots were successfully stained using iron chelates. When these two stains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stains was substantiated by incubating stained membranes at neutral to basic pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapidly elute the complexes from the bound proteins. The chelate stains were determined to be fully compatible with immunoblotting, N-terminal, and in situ internal protein microsequencing.

Amino Acid Sequence↗
PubMed1994

Microbore reversed-phase high-performance liquid chromatographic purification of peptides for combined chemical sequencing-laser-desorption mass spectrometric analysis.

An optimized microbore RP-HPLC system (1.0 mm I.D. columns) for the purification of low picomole amounts (< 5 pmol) of peptides is described. It is comprised of commercially available columns, instrument components and parts. These were selected on the basis of a comparative evaluation and to yield the highest resolution and most efficient peak collection. The sensitivity of this system equals, probably surpasses, that of advanced chemical microsequencing for which 2-4 pmol of peptide are minimally required. As an automated sequencer cannot be "on-line" connected with a micro-preparative HPLC system, fractions must be collected and transferred. With a typical flow of 30 microliters, efficient manual collection is possible and fractions (about 20 microliters in volume) can still be handled without unacceptable losses, albeit with great precaution. Furthermore, major difficulties were encountered to efficiently and quantitatively load low- or sub-picomole amounts of peptide mixtures onto the RP-HPLC column for separation. Discipline and rigorous adherence to sample handling protocols are thus on order when working at those levels of sensitivity. With adequate instrumentation and handling procedures in place, we demonstrate that low picomole amounts of peptides can now be routinely prepared for analysis by combined Edman-chemical sequencing-matrix-assisted laser-desorption mass spectrometry (MALDI-MS). The integrated method was applied to covalent structural characterization of minute quantities of a gel-purified protein of known biological function but unknown identity. The results allowed unambiguous identification and illustrated the power of MALDI-MS-aided interpretation of chemical sequencing data: accurate peptide masses were crucial for (i) confirmation of the results, (ii) deconvolution of mixed sequences, (iii) proposal of complete structures on the basis of partial sequences, and (iv) confirmation of protein identification (obtained by database search with a single, small stretch of peptide sequence) by "mass matching" of several more peptides with predicted proteolytic fragments.

Amino Acid Sequence↗
PubMed1994

RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs.

The immunosuppressants rapamycin and FK506 bind to the same intracellular protein, the immunophilin FKBP12. The FKB12-FK506 complex interacts with and inhibits the Ca(2+)-activated protein phosphatase calcineurin. The target of the FKBP12-rapamycin complex has not yet been identified. We report that a protein complex containing 245 kDa and 35 kDa components, designated rapamycin and FKBP12 targets 1 and 2 (RAFT1 and RAFT2), interacts with FKBP12 in a rapamycin-dependent manner. Sequences (330 amino acids total) of tryptic peptides derived from the 245 kDa RAFT1 reveal striking homologies to the yeast TOR gene products, which were originally identified by mutations that confer rapamycin resistance in yeast. A RAFT1 cDNA was obtained and found to encode a 289 kDa protein (2549 amino acids) that is 43% and 39% identical to TOR2 and TOR1, respectively. We propose that RAFT1 is the direct target of FKBP12-rapamycin and a mammalian homolog of the TOR proteins.

Amino Acid Sequence↗
PubMed1994

High-sensitivity sequencing of large proteins: partial structure of the rapamycin-FKBP12 target.

We report on studies leading to refinements of various steps of the protein internal sequencing process. Specifically, the developments comprise (1) higher-sensitivity chemical sequencing through background reduction; (2) improved peptide recovery from rapid in situ digests of nanogram amount, nitrocellulose-bound proteins; and (3) accurate UV spectroscopic identification of Trp- and Cys-containing peptides. In addition, we describe strategies for 2-dimensional liquid chromatographic peptide isolation from complex mixtures and a multi-analytical approach to peptide sequence analysis (Edman sequencing, matrix-assisted laser desorption mass spectrometry, and UV spectroscopy). Both strategies were applied in tandem to the primary structural analysis of a gel-purified, 250-kDa protein (mammalian target of rapamycin-FKBP12 complex), available in low picomolar quantities only. More than 300-amino acids worth of sequence was obtained in mostly uninterrupted stretches, several containing Trp, Cys, His, and Ser. That information has allowed the matching of a biological function of a mammalian protein to a yeast gene product with a well-characterized mutant phenotype. The results also demonstrate that extended chemical sequencing analysis (e.g., 26 successive amino acids) is now feasible, starting with initial yields well below 1 pmol.

Amino Acid Sequence↗
PubMed1990

Expression of key enzymes of purine and pyrimidine metabolism in a hepatocyte-derived cell line at different phases of the growth cycle.

The effect of growth phase on enzymatic activities of the de novo and salvage pathways for purine and pyrimidine nucleotide synthesis was studied in a hepatocyte-derived cell line from the rat. The cells were in lag phase after plating for 36 h; log phase started at 48 h and persisted up to 120 h of culture. Then the cells stopped growing and entered into plateau phase (144 h). In non-proliferating cells (144 h of culture) the basal activities of the enzymes of purine de novo biosynthesis were 1.7- to 6.8-fold higher than in normal rat liver, those of pyrimidine de novo synthesis showed 0.6- to 30-fold increase in activity. The purine salvage enzymes were unchanged, and the pyrimidine salvage enzymes were 3.1- to 7.4-fold higher compared to normal liver. During the growth cycle all enzymes except the purine salvage enzymes, which did not change, showed a peak in activity at 72 h of culture (log phase). The increase in activity in log phase compared to plateau phase was 1.3- to 2.4-fold for purine de novo synthetic enzymes, 1.1- to 2.4-fold for pyrimidine de novo enzymes, and 1.4- to 4.7-fold for pyrimidine salvage enzymes. The specific activities of the enzymes in exponentially growing cells were comparable either to that in 24-h regenerating liver, or to that in hepatomas of low or medium growth rate. It was concluded that the enzymatic pattern and metabolic state of the cells shared some features with regenerating liver, others with tumors, although they were not tumorigenic after transplantation into athymic nude mice.

Animals↗