Accuracy of prediction equations to estimate submaximal VO2 during cycle ergometry: the HERITAGE Family Study

Calcium-binding S100 proteins are thought to play a central role in calcium-mediated signal transduction pathways. They consist of two helix-loop-helix, calcium-binding EF-hand domains. A characteristic feature is their tendency to form homo- and/or heterodimeric complexes. This report presents for the first time a functional "in vivo" approach to the analysis of S100 protein dimerization. Using the two-hybrid system we analyzed the dimerization of MRP8 (S100A8) and MRP14 (S100A9), two S100 proteins expressed in myeloid cells. It is reported that the MRP8-MRP14 heteromer is the clearly preferred complex in both man and mouse. The ability to homodimerize, however, appears to be restricted to the murine MRPs. Interaction analysis of chimeric murine/human MRP14 proteins indicates, that the C-terminal EF-hand domain plays a prominent role in MRP8-MRP14 interaction and determines the specificity of dimerization. Site-directed mutagenesis of four evolutionary conserved hydrophobic amino acids, which have been recently supposed to be essential for S100 protein dimerization, suggests that at least one of these, namely the most N-terminal located residue, is not critical for dimerization.     

Epitope insertion favors a six transmembrane domain model for the carboxy-terminal portion of the multidrug resistance-associated protein

The overexpression of the multidrug resistance protein, MRP, in mammalian cells is associated with pleiotropic resistance to cytotoxic drugs. MRP is an integral membrane protein which belongs to the family of ATP-binding cassette transporters. Secondary structure predictions combined with biochemical analyses suggest that MRP encodes 11 transmembrane (TM) domains in the amino-terminal half of the protein and four or six transmembrane domains in the carboxy-terminal half of the protein. To gain insight into the membrane topology of the carboxy-terminal half of MRP, small, antigenic hemagglutinin (HA) epitopes (YPYDVPDYAS) were inserted within six predicted hydrophilic subfragments of this region (938, 1001, 1084, 1175, 1222, 1295). These epitope-tagged MRP variants were expressed in HeLa cells to evaluate their ability to confer resistance to the drug etoposide (VP-16). Insertion of the HA epitopes at positions 938, 1001, and 1222 resulted in functional proteins, while epitope insertion at positions 1084, 1175, and 1295 abrogated MRP function. The intracellular versus extracellular location of the HA epitopes present in biologically active MRP variants was then established in intact and permeabilized cells by immunofluorescence using an anti-HA antibody. Epitopes inserted at positions 1001 and 1222 were located on the extracellular side of the plasma membrane, while the epitope inserted at position 938 was located intracellularly. These results are consistent with a six TM rather than a four TM domain model for the membrane portion of the carboxy-terminal half of MRP.     

Multidrug resistance-associated protein in acute myeloid leukemia: No impact on treatment outcome

Drug resistance remains a major problem in the treatment of patients with acute myeloid leukemia (AML). Expression of the MDR1 gene in leukemic cells was shown previously to be associated with worse clinical outcome of the patients. The multidrug resistance-associated protein (MRP) has been shown recently to be another protein causing the multidrug resistance phenotype in cell lines, but its impact on clinical outcome in patients with AML remains to be proven. To determine the clinical significance of MRP in patients with de novo AML, we have studied the MRP expression in leukemic cells and its association with both response to induction chemotherapy and survival of the patients. MRP gene expression was determined by immuno-cytochemistry (n = 80) by means of the monoclonal antibodies QCRL-1 and QCRL-3. MRP expression was low, intermediate, and high in 19, 55, and 26% of the patients, respectively. High MRP expression was independent of age and sex of the patients, WBC count, and percentage of blasts. However, high MRP expression was more frequent in the FAB M5 subtype as compared to the other subtypes. MRP expression had no impact on clinical outcome. The complete remission rates were 65, 68, and 63% for patients with low, intermediate, and high expression, respectively. Overall survival was also independent of MRP expression. In contrast, patients with P-glycoprotein-positive AML had lower complete remission rates and shorter durations of survival. These data indicate that MRP is expressed in patients with de novo AML but, in contrast to P-glycoprotein, does not predict for outcome of induction chemotherapy or survival.     

Placentas and foetal health

The placenta provides many critical services to the developing foetus. Proper placental implantation, growth and function are necessary for normal foetal growth and development. Placental structure varies widely among species but all mammalian placentas have a convoluted materno-foetal interface that may be quite simple or highly complex; the more complicated interdigitations tend to characterise smaller placentas that have limited areas of contact between the placenta and the endometrium. The intimacy of the contact between maternal and foetal tissue varies from apposition only, as in the equine placenta, to invasion of maternal vessels by foetal cells, as in man. Abnormalities identified in the human placenta provide insight into maternal and foetal diseases that affect pregnancy outcome.     

Expression of the multidrug resistance-associated protein (MRP) gene in urothelial carcinomas

The intrinsic or acquired resistance of urothelial cancer to chemotherapy is one major obstacle to successful treatment. Generally, the expression level of P-glycoprotein in urothelial cancer is low, so we accordingly investigated the expression of multidrug resistance-associated protein (MRP). We examined the expression of MRP mRNA by means of slot-blotting samples of 11 renal pelvic and/or ureteral tumors, 33 bladder tumors, one lung metastasis from a ureter tumor, 7 non-cancerous urothelia from patients with transitional-cell carcinoma (TCC) and one urothelium from a patient with renal-cell carcinoma (RCC). We also estimated, by Southern blotting, whether or not the MRP gene was amplified in clinical specimens that overexpressed MRP mRNA. MRP was detected immunohistochemically using a polyclonal antibody against MRP. In all, 5 of 11 renal pelvic and/or ureter tumors (45.5%), 17 of 33 bladder tumors (51.5%) and 4 of 7 non-cancerous urothelia of TCC patients (57.1%) expressed more than 2-fold the MRP mRNA levels of drug-sensitive human KB cells. There was no significant difference in the MRP mRNA level between primary and recurrent tumors. Low-grade urothelial carcinomas (G1 and G2 TCCs) expressed significantly higher levels of MRP mRNA than the high-grade G3 TCC. The MRP gene was not amplified in urothelial carcinomas, irrespective of their expression levels of MRP mRNA. Immunohistochemically, MRP was located mainly on the plasma membrane, but also detected on the cytoplasm of cancer cells. MRP may be one mechanism responsible for intrinsic drug resistance in low-grade urothelial cancer.     

Nortriptyline and cognitive-behavioral therapy in the treatment of cigarette smoking

During the process of evolution, ancestral lysozymes evolved into calcium-binding lysozymes by acquiring three critical aspartate residues at positions 86, 91 and 92. To investigate the process of the acquisition of calcium-binding ability, two of the aspartates were partially introduced into human lysozyme at positions 86, 91 and 92. These mutants (HLQ86D, HLA92D and HLQ86D/D91Q/A92D), having two critical aspartates in calcium-binding sites, were expressed in Escherichia coli as non-active inclusion bodies. For the preparation of lysozyme samples, a refolding system using thioredoxin was established. This system allowed for effective refolding of wild-type and mutant lysozymes, and 100% of activity was recovered within 4 days. The calcium ion dependence of the melting temperature (Tm) of wild-type and mutant lysozymes was investigated by differential scanning calorimetry at pH 4.5. The Tm values of wild-type, HLQ86D and HLA92D mutants were not dependent on calcium ion concentration. However, the Tm of HLQ86D/D91Q/A92D was 4 degrees higher in the presence of 50 mM CaCl2 than in its absence, and the calcium-binding constant of this mutant was estimated to be 2.25(+/-0.25)x10(2) M(-1) at pH 4.5. Moreover, the calcium-binding ability of this mutant was confirmed by the result using Sephadex G-25 gel chromatography. These results indicate that it is indispensable to have at least two aspartates at positions 86 and 92 for acquisition of calcium-binding ability. The process of the acquisition of calcium-binding site during evolution of calcium-binding lysozyme is discussed.     

High level expression and dimer characterization of the S100 EF-hand proteins, migration inhibitory factor-related proteins 8 and 14

The phenotypical and functional heterogeneity of different macrophage subpopulations are defined by discrete changes in the expression of two S100 calcium-binding proteins, migration inhibitory factor-related proteins (MRPs) 8 and 14. To further our understanding of MRP8 and MRP14 in the developmental stages of inflammatory responses, overexpression of the MRPs was obtained through a combination of a T7-based expression vector and the Escherichia coli BL21 (DE3) cell line. An efficient, two-step chromatographic protocol was then developed for rapid, facile purification. Extensive biophysical characterization and chemical cross-linking experiments show that MRP8 and MRP14 form oligomers with a strong preference to associate as a heterodimer. Heteronuclear NMR experiments indicate that a specific well packed dimer is formed only in equimolar mixtures of the two proteins. Our results suggest that there is a unique complementarity in the interface of the MRP8/MRP14 complex that cannot be fully reproduced in the MRP8 and MRP14 homodimers.     

Difloxacin reverses multidrug resistance in HL-60/AR cells that overexpress the multidrug resistance-related protein (MRP) gene

In this study, we have examined the in vitro chemosensitizing activity of difloxacin, a quinolone antimicrobial agent, in the multidrug-resistant human myeloid leukemia HL-60/AR cell line. HL-60/AR cells were found to overexpress multidrug resistance-associated protein (MRP) mRNA as compared to HL-60 cells. Difloxacin, in a concentration-dependent manner, increased the sensitivity of HL-60/AR cells to daunorubicin, adriamycin, and vincristine, and partially corrected the altered drug transport. In addition, difloxacin corrected subcellular distribution of adriamycin by inducing redistribution of the drug from the perinuclear region to the nucleus in HL-60/AR cells. The chemosensitizing effect of difloxacin was observed at clinically achievable concentrations. We conclude that difloxacin is an effective chemosensitizer of MRP-associated multidrug-resistant tumor cells and is a potential candidate for clinical use to reverse multidrug resistance.     

Membrane topology and glycosylation of the human multidrug resistance-associated protein

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.     

A novel surface marker (B203.13) of human haemopoietic progenitors, preferentially expressed along the B and myeloid lineages

We used a monoclonal antibody (mAb) (B203.13, IgM) generated from a mouse immunized with the human B/myeloid bi-phenotypic B1b cell line, to analyse haemopoietic cells. The antigen recognized by this mAb is expressed on most adult and umbilical cord blood CD21+ B cells, at minimal density on mature monocytes, and is undetectable on granulocytes, T, natural killer (NK) cells, and erythrocytes. Within umbilical cord blood and adult bone marrow haemopoietic progenitor cells, the B203.13 mAb recognized a surface marker, present on progenitor cells of several haemopoietic lineages, that was transiently expressed on early erythroid and T/NK progenitors, and was preferentially maintained on cells of the B and myeloid lineages. Within the CD34+ cells, B203.13 was expressed on early committed myeloid (CD33+) and erythroid (CD71dim) progenitor cells, as confirmed in colony formation assays. The mAb also reacted with cells of B and myeloid chronic leukaemias and cell lines. These data define B203.13 mAb as a novel reagent useful for the characterization of haemopoietic progenitors and leukaemias.     

Autacoid interactions in the regulation of blood flow in the human placenta

1. Interactions between autacoids may play important roles in the regulation of blood flow in the foetal placenta. In order to investigate this aspect of placental haemodynamics, human normal-term placentae were perfused in vitro and the responses of the foetal vessels to various combinations of vasoactive agents were determined. 2. Vasoconstriction responses to 5-hydroxytryptamine (5-HT) were potentiated in the presence of endothelin-1 (ET-1), the thromboxane A2-mimetic U46619 and a nitric oxide synthase inhibitor, N-nitro-L-arginine (NOLA), but not in the presence of angiotensin II. 3. N-Nitro-L-arginine caused vasoconstriction of the perfused placenta and indomethacin attenuated this effect and blocked the potentiation of the 5-HT response by NOLA. 4. Indomethacin did not affect ET-1-induced pressure increases and infusion of U46619 had no effect on release of ET-like immunoreactivity into the foetal placental circulation. 5. The present study provides evidence of interactions between several autacoids in human perfused placentae in vitro. These interactions may play important roles in foetal placental haemodynamics in normal or pathological situations.     

Morphologic findings in fetuses and placentas of late abortion in the 2nd trimester

To investigate possible causes of abortion (and intrauterine foetal death) we reviewed clinical and morphological data of foetuses and placentas morphologically from 830 spontaneous abortions seen during a 12 years period (1978-1989) at the Institute of Pathology, University of Leipzig, and the Pathological Institute of Hoyerswerda. Our review showed that definite and possible causes of foetal death and abortion could be classified as placental, foetal maternal, and clinical. Placental changes, which included infection of the foetal membranes, disturbances of the uteroplacental circulation (abruptio placentae with bleeding) and placental dysmaturity, were the most important causes and accounted for 73.8% of the cases. Foetal causes mainly comprised multiple twin pregnancies and foetal malformations. In 20 cases (2.4%) we found malformations as a cause of foetal death and consecutive abortion. Overall, malformations were found in 7.5% of the cases examined. Maternal and obstetric complications of pregnancy were less frequent. In 16.5% of our cases, the cause of the abortion or intrauterine death remained obscure. However, since 1989, genetic analysis and prenatal diagnostic procedures have improved, bringing a greater knowledge on the spectrum and aetiology of possible developmental disorders in the foetus. This should reduce the number of unexplained abortions.     

Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.     

Multidrug resistance-related transport proteins in isolated human brain microvessels and in cells cultured from these isolates

The multidrug transporter, P-glycoprotein (Pgp), at the blood-brain barrier is thought to be important for limiting access of toxic agents to the brain, but controversy surrounds its cellular location, whether on endothelium or on adjacent astrocyte foot processes. In the present study, the distribution of protein and mRNA for Pgp and for another transporter, multidrug resistance-associated protein (MRP), is compared with that for the endothelial marker, platelet-endothelial cell adhesion molecule-1 (PECAM-1) and for the astrocyte-derived glial fibrillary acidic protein (GFAP) in microvessels isolated from human brain and in cells grown from these microvessels. Activities of the multidrug transporters are assessed in the cultured cells from the effects of transport inhibitors on intracellular [3H]vincristine accumulation. The isolated microvessels show strong immunocytochemical staining for Pgp and PECAM-1 and little or no staining for GFAP and MRP, and they contain mRNAs detectable by RT-PCR encoding only Pgp and PECAM-1, but not GFAP or MRP. Thus, Pgp may well be synthesised and expressed on cells within the microvessels rather than on adherent astrocyte foot processes. In cells grown from the microvessels, although PECAM-1 remains, Pgp expression decreases and MRP appears. Evidence suggests these multidrug transporters are functionally active in the cultured cells.     

Simultaneous detection of beta-galactosidase activity and surface antigen expression in viable haematopoietic cells

The quantitation of intracellular beta-galactosidase activity has been described for viable cells. By using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG) in conjunction with flow cytometry, the proportion of positive cells as well as the level of expression can be determined. In this paper we describe beta-galactosidase expression in lymphoid and myeloid cells from transgenic mice that widely express beta-galactosidase from an inserted lacZ transgene. Both foetal and adult haematopoietic tissues are able to express beta-galactosidase. The intracellular fluorescence reflecting beta-galactosidase activity can be readily combined with fluorescently labelled antibodies against cell surface antigens. Thus, beta-galactosidase can be used as a marker in transplantation experiments to study the development of lymphoid and myeloid precursor cells.     

ATP-dependent glutathione conjugate export pump

KCP4 cells are resistant to cisplatin and have a GS-X pump different from MRP. The GS-X pump was suggested to be involved in reducing the accumulation of cisplatin in KCP-4 cells. The expression of cMOAT was 4-to 6-fold higher in KCP-4 cells and two other cisplatin-resistant human cell lines. It is still not clear whether the cisplatin resistance in KCP-4 cells are attributed to cMOAT. Other members of the MRP/GS-X pump family have been reported. Amino acid sequence of EBCR of rabbit is 91% identical to that of human cMOAT. On the view of cancer chemotherapy, it is very important to understand the structure and function of GS-X pumps since they may be involved in not only drug resistance but also drug metabolism and side effects.