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Context: Products of at least five specific steroidogenic genes, including steroidogenic acute regulatory protein (StAR), which facilitates the entry of cytosolic cholesterol into the mitochondrion, side chain cleavage P450 enzyme, 3beta-hydroxysteroid-dehydrogenase-2, 17-hydroxylase/17-20-lyase, and aromatase, which catalyzes the final step, are necessary for the conversion of cholesterol to estrogen. Expression and biological activity of StAR and aromatase were previously demonstrated in endometriosis but not in normal endometrium. Prostaglandin E2 (PGE2) induces aromatase expression via the transcriptional factor steroidogenic factor-1 (SF1) in endometriosis, which is opposed by chicken-ovalbumin upstream-transcription factor (COUP-TF) and Wilms' tumor-1 (WT1) in endometrium., Objective: The aim of the study was to demonstrate a complete steroidogenic pathway leading to estrogen biosynthesis in endometriotic cells and the transcriptional mechanisms that regulate basal and PGE2-stimulated estrogen production in endometriotic cells and endometrium., Results: Compared with normal endometrial tissues, mRNA levels of StAR, side chain cleavage P450, 3beta-hydroxysteroid-dehydrogenase-2, 17-hydroxylase/17-20-lyase, aromatase, and SF1 were significantly higher in endometriotic tissues. PGE2 induced the expression of all steroidogenic genes; production of progesterone, estrone, and estradiol; and StAR promoter activity in endometriotic cells. Overexpression of SF1 induced, whereas COUP-TFII or WT1 suppressed, StAR promoter activity. PGE2 induced coordinate binding of SF1 to StAR and aromatase promoters but decreased COUP-TFII binding in endometriotic cells. COUP-TFII or WT1 binding to both promoters was significantly higher in endometrial compared with endometriotic cells., Conclusion: Endometriotic cells contain the full complement of steroidogenic genes for de novo synthesis of estradiol from cholesterol, which is stimulated by PGE2 via enhanced binding of SF1 to promoters of StAR and aromatase genes in a synchronous fashion.

Background: The benefits of estrogen plus progestin in healthy post-menopausal women remain uncertain. Tibolone, with its in vitro documented inhibitory effects on estrogens metabolism and its selective action on breast, may be an alternative that could favorably influence the health benefit of hormone replacement therapy., Methods: We studied the effect of tibolone on the tumor growth of MCF-7 cells implanted in 40 ovariectomized nude mice, receiving subcutaneous pellets of 17beta-estradiol, estrone, estrone-sulfate or vehicle, and daily gavages of tibolone or placebo., Results: Tibolone, although used at high dose, did not stimulate nor inhibit the estrogen-induced tumors, nor the tumors in estrogen-deprived mice. Measurements of plasma levels of estrogens indicated that tibolone potently stimulated sulfotransferase activity, but intra-tumor levels of estrogens were not significantly modified by tibolone., Conclusions: This in vivo study performed with high dose of orally administered tibolone that allowed its hepatic conversion into active metabolites has shown no significant effect on breast tumors growth. Tibolone increased the circulating sulfated estrogens by its activity on the hepatic sulfation but not the intra-tumor levels of estrogens (free or sulfated). However, further studies of dose-response curve and molecular markers are needed to exclude definitely a stimulatory effect of tibolone on tumor growth.

Bacterial colony counting is a significant technical hurdle for vaccine studies as well as various microbiological studies. We now show that an automated colony counter can process images obtained with a digital camera or document scanner and that any laboratory can efficiently have bacterial colonies enumerated by sending the images to a laboratory with a colony counter via internet.

Multidrug resistance presents a major obstacle to the treatment of infectious diseases and cancer. LmrA, a bacterial ATP-dependent multidrug transporter, mediates efflux of hydrophobic cationic substrates, including antibiotics. The substrate-binding domain of LmrA was identified by using photo-affinity ligands, proteolytic degradation of LmrA, and identification of ligand-modified peptide fragments with matrix-assisted laser desorption ionization/time of flight mass spectrometry. In the nonenergized state, labeling occurred in the alpha-helical transmembrane segments (TM) 3, 5 and 6 of the membrane-spanning domain. Upon nucleotide binding, the accessibility of TM5 for substrates increased, whereas that of TM6 decreased. Inverse changes were observed upon ATP-hydrolysis. An atomic-detail model of dimeric LmrA was generated based on the template structure of the homologous transporter MsbA from Vibrio cholerae, allowing a three-dimensional visualization of the substrate-binding domain. Labeling of TM3 of one monomer occurred in a predicted area of contact with TM5 or TM6 of the opposite monomer, indicating substrate-binding at the monomer/monomer interface. Inverse changes in the reactivity of TM segments 5 and 6 suggest that substrate binding and release involves a repositioning of these helices during the catalytic cycle.

The active efflux of toxic compounds by (multi)drug transporters is one of the mechanisms that bacteria have developed to resist cytotoxic drugs. The authors describe the role of the lactococcal secondary multidrug transporter LmrP in the resistance to a broad range of clinically important antibiotics. Cells expressing LmrP display an increased resistance to the lincosamide, streptogramin, tetracycline and 14- and 15-membered macrolide antibiotics. The streptogramin antibiotic quinupristin, present in the fourth-generation antibiotic RP 59500, can inhibit LmrP-mediated Hoechst 33342 transport, but is not transported by LmrP, indicating that quinupristin acts as a modulator of LmrP activity. LmrP-expressing Lactococcus lactis cells in which a proton-motive force is generated accumulate significantly less tetracycline than control cells without LmrP expression. In contrast, LmrP-expressing and control cells accumulate equal amounts of tetracycline in the absence of metabolic energy. These findings demonstrate that the increased antibiotic resistance in LmrP-expressing cells is a result of the active extrusion of antibiotics from the cell.

LmrA is a 590-amino acid membrane protein which confers multidrug resistance on Lactococcus lactis cells by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane at the expense of ATP hydrolysis. Its structural and functional characteristics place it in the P-glycoprotein cluster of the ATP-binding cassette transporter superfamily, making it the first prokaryotic multidrug transporter of this cluster. The number of compounds recognized and transported by LmrA is remarkably vast and includes many lipophilic cations as well as a record of eight classes of clinically relevant broad-spectrum antibiotics. Homologs of LmrA have been found in pathogenic bacteria, suggesting that these putative efflux pumps may play a crucial role in antibiotic resistance of human pathogens. Recent evidence indicates that LmrA is functional as a homodimer, consistent with the overall structure of P-glycoprotein, and mediates drug transport by an alternating two-site transport mechanism. Copyright 2000 Harcourt Publishers Ltd.

One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in Lactococcus lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

Lactococcus lactis possesses an ATP-binding cassette transporter, LmrA, which is a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein, and is able to transport a broad range of structurally unrelated amphiphilic drugs. A histidine tag was introduced at the N-terminus of LmrA to facilitate purification by nickel affinity chromatography. The histidine-tagged protein was overexpressed in L. lactis using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. This system allowed us to get functional overexpression of LmrA up to a level of 30% of total membrane protein. For reconstitution, LmrA was solubilized with dodecylmaltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecylmaltoside-destabilized, preformed liposomes prepared from L. lactis phospholipids. The detergent was removed by adsorption onto polystyrene beads. The LmrA protein was reconstituted in a functional form, and mediated the ATP-dependent transport of the fluorescent substrate Hoechst-33342 into the proteoliposomes. Interestingly, reconstituted LmrA also catalyzed the ATP-dependent transport of fluorescent phosphatidylethanolamine, but not of fluorescent phosphatidylcholine. These data demonstrate that LmrA activity is independent of accessory proteins and support the notion that LmrA may be involved in the transport of specific lipids or lipid-linked precursors in L. lactis.

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette (ABC) superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in L. lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

The secondary multidrug transporter LmrP of Lactococcus lactis mediates the efflux of Hoechst 33342 from the cytoplasmic leaflet of the membrane. Kinetic analysis of Hoechst 33342 transport in inside-out membrane vesicles of L. lactis showed that the LmrP-mediated H(+)/Hoechst 33342 antiport reaction obeyed Michaelis-Menten kinetics, with a low apparent affinity constant of 0.63 microM Hoechst 33342 (= 0.5 mmol Hoechst 33342/mol phospholipid). Several drugs significantly inhibited LmrP-mediated Hoechst 33342 transport through a direct interaction with the protein rather than through dissipation of the proton motive force or reduction of the membrane partitioning of Hoechst 33342. The characterization of the mechanism of inhibition of LmrP-mediated Hoechst 33342 transport indicated competitive inhibition by quinine and verapamil, noncompetitive inhibition by nicardipin and vinblastin, and uncompetitive inhibition by TPP(+). The three types of inhibition of LmrP-mediated Hoechst 33342 transport in inside-out membrane vesicles indicate for the first time the presence of multiple drug interaction sites in a secondary multidrug transporter.

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. The secondary multidrug transporter LmrP and the ATP-binding cassette (ABC) type multidrug transporter LmrA in Lactococcus lactis are representatives of the two major classes of multidrug transporters found in pro- and eukaryotic organisms. Therefore, knowledge of the molecular properties of LmrP and LmrA will have a wide significance for multidrug transporters in all living cells, and may enable the development of specific inhibitors and of new drugs which circumvent the action of multidrug transporters. Interestingly, LmrP and LmrA are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA will represent an intriguing new area of research.

The histidine-tagged secondary multidrug transporter LmrP was overexpressed in Lactococcus lactis, using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. LmrP-mediated H+/drug antiport activity in inside-out membrane vesicles was inhibited by detergents, such as Triton X-100, Triton X-114, and Tween 80, at low concentrations that did not affect the magnitude or composition of the proton motive force. The inhibition of the activity of LmrP by detergents restricted the range of compounds that could be used for the solubilization and reconstitution of the protein because low concentrations of detergent are retained in proteoliposomes. Surprisingly, dodecyl maltoside did not modulate the activity of LmrP. Therefore, LmrP was solubilized with dodecyl maltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecyl maltoside-destabilized, preformed liposomes prepared from Escherichia coli phospholipids and egg phosphatidylcholine. Reconstituted LmrP mediated the transport of multiple drugs in response to an artificially imposed pH gradient, demonstrating that the protein functions as a proton motive force-dependent multidrug transporter, independent of accessory proteins. These observations are relevant for the effective solubilization and reconstitution of multidrug transporters belonging to the major facilitator superfamily, which, in view of their broad drug specificity, may strongly interact with detergents.

Aberrant aromatase expression in stromal cells of endometriosis gives rise to conversion of circulating androstenedione to estrone in this tissue, whereas aromatase expression is absent in the eutopic endometrium. In this study, we initially demonstrated by Northern blotting transcripts of the reductive 17beta-hydroxysteroid dehydrogenase (17betaHSD) type 1, which catalyzes the conversion of estrone to 17beta-estradiol, in both eutopic endometrium and endometriosis. Thus, it follows that the product of the aromatase reaction, namely estrone, that is weakly estrogenic can be converted to the potent estrogen, 17beta-estradiol, in endometriotic tissues. It was previously demonstrated that progesterone stimulates the inactivation of 17beta-estradiol through conversion to estrone in eutopic endometrial epithelial cells. Subsequently, 17betaHSD type 2 was shown to catalyze this reaction, and its transcripts were detected in the epithelial cell component of the eutopic endometrium in secretory phase. Because 17beta-estradiol plays a critical role in the development and growth of endometriosis, we studied 17betaHSD-2 expression in endometriotic tissues and eutopic endometrium. We demonstrated, by Northern blotting, 17betaHSD-2 messenger ribonucleic acid (RNA) in all RNA samples of secretory eutopic endometrium (n=12) but not in secretory samples of endometriotic lesions (n=10), including paired samples of endometrium and endometriosis obtained simultaneously from eight patients. This messenger RNA was not detectable in any samples of proliferative eutopic endometrium or endometriosis (n=4) as expected. Next, we confirmed these findings by demonstration of immunoreactive 17betaHSD-2 in epithelial cells of secretory eutopic endometrium in 11 of 13 samples employing a monoclonal antibody against 17betaHSD-2, whereas 17betaHSD-2 was absent in paired secretory endometriotic tissues (n=4). Proliferative eutopic endometrial (n=8) and endometriotic (n=4) tissues were both negative for immunoreactive 17betaHSD-2, except for barely detectable levels in 1 eutopic endometrial sample. Finally, we sought to determine whether deficient 17betaHSD-2 expression in endometriotic tissues is due to impaired progesterone action in endometriosis. We determined by immunohistochemistry the expression of progesterone and estrogen receptors in these paired samples of secretory (n=4) and proliferative (n=4) eutopic endometrium and endometriosis, and no differences could be demonstrated. In conclusion, inactivation of 17beta-estradiol is impaired in endometriotic tissues due to deficient expression of 17betaHSD-2, which is normally expressed in eutopic endometrium in response to progesterone. The lack of 17betaHSD-2 expression in endometriosis is not due to alterations in the levels of immunoreactive progesterone or estrogen receptors in this tissue and may be related to an inhibitory aberration in the signaling pathway that regulates 17betaHSD-2 expression.

The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli. The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli. LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Deltapsi) and the transmembrane proton gradient (DeltapH), pointing to an electrogenic drug/proton antiport mechanism. Ethidium bromide, a substrate for LmrP, inhibited the LmrP-mediated TPP+ extrusion from right-side-out membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs. Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relation between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer. This observation indicates that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium. This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transporter occurs by a "hydrophobic vacuum cleaner" mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA.

Respiratory impairment in myasthenia gravis is usually attributed to weakness of the diaphragm and thoracic chest wall muscles, and is rarely attributed to upper airway obstruction. Myasthenia gravis is characterized by weakness of the striated muscles and usually affects those innervated by the bulbar cranial nerves. Weakness of these bulbar and upper airway muscles can lead to upper airway obstruction. To our knowledge, there are only five case reports in the literature associating upper airway obstruction with myasthenia gravis. Therefore, we attempted to further define its occurrence in myasthenia gravis patients by reviewing their flow volume loops. We present a case of upper airway obstruction causing respiratory symptoms in a myasthenia gravis patient. We then surveyed a total of 61 patients with myasthenia gravis who were tested in our pulmonary function laboratory between February 1990 and August 1993. Of these 61 patients, 12 had flow volume loops and 7 of these 12 disclosed a pattern of extrathoracic upper airway obstruction. The FVC was 80% or more in five of seven patients. Our data suggest that upper airway obstruction is much more common in patients with myasthenia gravis than previously recognized. In conclusion, we recommend the performance of flow volume loops in patients with myasthenia gravis to evaluate their respiratory impairment.

A description and demonstration of a fiber interferometer that uses a short segment of silica hollow-core fiber spliced between two sections of single-mode fiber to form a mechanically robust in-line cavity are presented. The hollow-core fiber is specifically manufactured to have an outer diameter that is equal to the outer diameter of the single-mode lead fibers, thereby combining the best qualities of existing intrinsic and extrinsic Fabry-Perot sensors. A dynamic strain resolution of ~22 nepsilon/ radicalHz at frequencies of >5 Hz with a sensor gauge length of 137 microm is demonstrated.

Antibiotics which affect protein synthesis have been reported to alter in vitro function of polymorphonuclear neutrophil leukocytes (PMNs). We studied chemiluminescence of PMNs from 30 healthy adults during phagocytosis of opsonized zymosan. Similar chemiluminescence values were obtained before, during, and after oral therapy with erythromycin, rosaramicin, or placebo. While macrolide antibiotic therapy did not alter this PMN function, we conclude that chemiluminescence is a reproducible technique which is well suited for serial evaluation of PMN activity.

A luminol-enhanced chemiluminescence assay was used to measure light elaborated by peripheral blood polymorphonuclear leukocytes (PMNs) and by pulmonary alveolar macrophages (PAMs) from 14 healthy, normal subjects and 16 patients with sarcoidosis. Resting peripheral blood PMNs (incubated only in medium) from patients with sarcoidosis generated substantially more chemiluminescence than PMNs from normal control subjects (p = 0.002). With zymosan stimulation, greater chemiluminescence was produced by PMNs from untreated patients with sarcoidosis than from normal subjects (p less than 0.05), whereas no differences were noted with latex stimulation. Chemiluminescence for resting PAMs was not different between normal subjects and patients with sarcoidosis. However, PAMs from untreated patients with sarcoidosis had higher chemiluminescence with latex particle ingestion than PAMs from normal subjects (p less than 0.05), but no differences in PAM chemiluminescence were found when zymosan was the phagocytic particle. Increased chemiluminescence by PMNs and PAMs from patients with sarcoidosis may reflect phagocyte activation in the disease process.

In a search for dietary factors that might stimulate enterocyte proliferation, we developed an assay for thymidine incorporation into DNA using harvested crypt cells from mature rat small intestine. Human colostrum stimulated a significant increase in thymidine incorporation into rat crypt cell DNA during a 60-min period of incubation. When the protein with biological activity was purified to a single peak by sequential ion exchange and gel filtration chromatography, it was found to have the characteristics of lactoferrin. The protein was identical to lactoferrin standards by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, and double-diffusion immunologic precipitation. All available human lactoferrins stimulated thymidine uptake and all reacted with a lactoferrin polyclonal antibody. Human lactoferrin appears to be a potent activator of thymidine incorporation into DNA in incubated rat crypt cells, a nutritional function not previously reported.

Serial quantitative nasal cultures were performed on 87 healthy nasal carriers of Staphylococcus aureus, who were randomly assigned to 7 days of oral therapy with erythromycin, rosamicin (an investigational macrolide antibiotic), or placebo. Staphylococcal carrier rates decreased during therapy with both antibiotics; however, erythromycin was significantly more effective in lowering carrier rates than was rosamicin. The anti-staphylococcal effects of both antibiotics were similar when the mean numbers of S. aureus isolated from positive cultures during therapy were compared. Side effects to each regimen were minimal.

Healthy nasal carriers of Staphylococcus aureus were randomly assigned to one of three treatment regimens: josamycin (1.5 g/day), erythromycin stearate (1.0 g/day), or placebo, each administered orally for 7 days. Quantitative nasal cultures were obtained from each subject at intervals before, during, and after treatment. All 25 placebo-treated subjects had positive nasal cultures for S. aureus at all culture intervals. Both josamycin and erythromycin were equally effective in reducing the carrier rates and in decreasing the total numbers of S. aureus isolated from subjects with positive cultures during treatment. No increase in in vitro antibiotic resistance was detected in isolates obtained after therapy. Both antibiotics were well tolerated, and toxicity was not encountered with either drug.