ATPase activity of purified and reconstituted multidrug resistance protein MRP1 from drug-selected H69AR cells.

The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which express high levels of this protein. A purification procedure comprised of a differential two-step solubilization of MRP1 from plasma membranes with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate followed by immunoaffinity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 microgram of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gels. After reconstitution of purified MRP1 into proteoliposomes, kinetic analyses indicated that its K(m) for ATP hydrolysis was 104+/-22 microM with maximal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was further characterized with various inhibitors and exhibited an inhibition profile that distinguishes it from P-glycoprotein and other ATPases. The ATPase activity of reconstituted MRP1 was stimulated by the conjugated organic anion substrates leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 microM, respectively. MRP1 ATPase was also stimulated by glutathione disulfide but not by reduced glutathione or unconjugated chemotherapeutic agents. This purification and reconstitution procedure is the first to be described in which the ATPase activity of the reconstituted MRP1 retains kinetic characteristics with respect to ATP-dependence and substrate stimulation that are very similar to those deduced from transport studies using MRP1-enriched plasma membrane vesicles.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.     

High-resolution comparative physical mapping of mouse Chromosome 10 in the region of homology with human Chromosome 21

Comparative mapping of human and mouse chromosomes can be used to predict locations of homologous loci between the species, provides the substrate to examine the process of chromosomal evolution, and facilitates the continuing development of mouse genetic models for human disorders. A YAC contig of the region of mouse Chromosome (Chr) 10 (MMU10) that demonstrates conserved linkage with the distal portion of human Chr 21 (HSA21) has been constructed. The contig contains all known genes mapped in both species, defines the proximal region of homology between MMU10 and HSA22, and contains the evolutionary junction between HSA21 and HSA22 on MMU10. It consists of 23 YACs and 2 PACs, and covers 3.2 Mb of MMU10. The average marker density for this region is 1 marker/69 kb. Nine of 22 expressed sequences are mapped here for the first time in mouse, and two are newly characterized expressed sequences. The contig also contains 12 simple sequence repeats (SSRs) and 16 YAC and PAC endclone markers. YAC fragmentation analysis was used to create a physical map for the proximal 2.2 Mb of the contig. Cloning of the corresponding region of HSA21 has proven difficult, and the mouse contig includes segments absent from previously described sequence ready maps of HSA21. Received: 22 July 1998 / Accepted: 13 November 1998     

The type I and type II 11beta-hydroxysteroid dehydrogenase enzymes.

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11β-hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11β-HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11β-HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11β-HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11β-HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11β-HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11β-HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11β-HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20–30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of >60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11β-HSD domains A–D to a cleft in the protein structure (cofactor binding domain), two parallel β-sheets, and an -helix (active site), respectively.     

Sensitive method for the quantitation of droloxifene in plasma and serum by high-performance liquid chromatography employing fluorimetric detection

A simple and highly sensitive reversed-phase fluorimetric HPLC method for the quantitation of droloxifene from rat, monkey, and human plasma as well as human serum is described. This assay employs solid-phase extraction and has a dynamic range of 25 to 10 000 pg/ml. Sample extraction (efficiencies >86%) was accomplished using a benzenesulfonic acid (SCX) column with water and methanol rinses. Droloxifene and internal standard were eluted with 1 ml of 3.5% (v/v) ammonium hydroxide (30%) in methanol. Samples were quantited using post-column UV-photochemical cyclization coupled with fluoremetric detection with excitation and emission wavelengths of 260 nm and 375 nm, respectively. Relative ease of sample extraction and short run times allow for the analysis of approximately 100 samples per day.     

Structural analysis of a highly acetylated protein using a curved-field reflectron mass spectrometer

Matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry (MS/MS) were used to determine the multiple acetylation sites in the histone acetyltransferase (HAT): p300-HAT. Partial cleavage of the peptides containing acetylated lysine residues by trypsin provided a set of nested sequences that enabled us to determine that multiple acetylation occurs on the same molecule. At the same time, cleavages resulting in a terminal unacetylated lysine suggested that not all of these sites are fully modified. Using MS and MS/MS, we were able to characterize both the unmodified and acetylated tryptic peptides covering more than 82% of the protein.     

Activity of alpha- and theta-defensins against primary isolates of HIV-1

Theta-defensins are lectin-like, cyclic octadecapeptides found in the leukocytes of nonhuman primates. They are also homologues of the more familiar alpha-defensins expressed by humans and certain other mammals. This study compares the ability of six theta-defensins (hominid retrocyclins 1-3 and rhesus theta-defensins 1-3) and four human alpha-defensins (human neutrophil peptides (HNPs) 1-4) to bind gp120 and CD4. In addition, we compared the ability of these theta-defensins and HNP-1 to protect J53-BL cells (an indicator cell line) from primary HIV-1 isolates that varied in subtype and coreceptor usage. The most potent theta-defensin, retrocyclin-2, bound with exceptionally high affinity to gp120 (K(D), 9.4 nM) and CD4 (K(D), 6.87 nM), and its effectiveness against subtype B isolates (IC(50), 1.05 +/- 0.28 microg/ml; 520 +/- 139 nM) was approximately twice as great as that of HNP-1 on a molar basis. We also show, for the first time, that human alpha-defensins, HNPs 1-3, are lectins that bind with relatively high affinity to gp120 (K(D) range, 15.8-52.8 nM) and CD4 (K(D) range, 8.0-34.9 nM). Proteins found in human and FBS bound exogenous HNP-2 and retrocyclin-1, and competed with their ability to bind gp120. However, even the low concentrations of alpha-defensins found in normal human serum suffice to bind over half of the gp120 spikes on HIV-1 and a higher percentage of cell surface CD4 molecules. Although this report principally concerns the relationship between carbohydrate-binding and the antiviral properties of alpha- and theta-defensins, the lectin-like behavior of defensins may contribute to many other activities of these multifunctional peptides.     

Rapid annexin-V labeling in synaptosomes

Distal neuronal terminals may be the site of apoptotic events and early synapse loss in neurodegenerative disease. To examine apoptosis in synaptic regions, we established a cell-free assay using a rat brain crude synaptosomal preparation (P-2 fraction) as a model system. The apoptosis marker annexin-V was used to measure phosphatidylserine (PS) exposure, and to ensure that only intact terminals were assayed, synaptosomes were dual labeled with a viability marker (calcein AM). Fluorescence was quantified by flow cytometry analysis. Annexin-V labeling increased rapidly in synaptosomes, following a 1 min incubation with staurosporine. However, increased caspase-3-like activity was not measured until 30 min with a fluorometric assay. The addition of a peptide inhibitor of caspase-3-like activity (Ac-DEVD-CHO) during homogenization was not able to block the initial increase in annexin labeling, but resulted in a partial blockade of annexin labeling after 30 min. These data demonstrate that PS externalization and caspase activation occur rapidly in this widely used neurochemical preparation.