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Biomedical subjects

R Liu

Publications and source records attributed to R Liu.

At least 19 recordsLinked to original sources
PubMed2000

Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor.

Histone acetylation alters chromatin state by modifying lysines on histone and plays an important role in modulating gene transcription. A dynamic balance of histone acetylation/deacetylation is maintained by histone acetyltransferases and histone deacetylases. Emerging evidence suggests that a family of histone deacetylases may exist to regulate diverse cellular functions, including chromatin structure, gene expression, cell cycle progression, and oncogenesis. We describe here a novel human histone deacetylase, named HDAC8, cloned from human kidney. HDAC8 encodes 377 amino acid residues and shares extensive homology to several known HDACs, in particular a histone deacetylase from Arabidopsis thaliana. Northern blot analyses revealed that HDAC8 expression pattern for HDAC8 is distinct from that for HDAC1 and HDAC3, and expression of HDAC8 mRNA occurs in multiple organs including heart, lung, kidney, and pancreas. HDAC8 mRNA was also observed in several cell lines derived from cancerous tissues. When expressed in HEK293 cells, HDAC8 exhibited deacetylase activity toward acetylated histone, indicating that this protein is a bona fide histone deacetylase. Its histone deacetylase activity was inhibited by trichostatin and other known histone deacetylase inhibitors. Furthermore, active recombinant HDAC8 was expressed and purified from Escherichia coli. When ectopically expressed in cells, HDAC8 was found to be localized to the nucleus. Co-transfection experiments demonstrated that expression of HDAC8 repressed a viral SV40 early promoter activity. These results indicate that HDAC8 is a novel member of the histone deacetylase family, which may play a role in the development of a broad range of tissues and potentially in the etiology of cancer.

3T3 Cells↗
PubMed2000

Inducible chemoresistance to 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothe cin (CPT-11) in colorectal cancer cells and a xenograft model is overcome by inhibition of nuclear factor-kappaB activation.

Limited studies have indicated that some chemotherapy agents activate the transcription factor nuclear factor-kappaB (NF-kappaB), and that this leads to suppression of the apoptotic potential of the chemotherapy. In contrast, it was reported recently that stable inhibition of NF-kappaB in four different cancer cell lines did not lead to augmentation of the chemotherapy-induced apoptosis. In this study, we have focused on colorectal cancer, which is known to be highly resistant to genotoxic chemotherapy and gamma irradiation. We show that the topoisomerase I inhibitor 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) activates NF-kappaB in most colorectal cancer cell lines. We then examine a therapeutic strategy that uses adenovirus-mediated transfer of the super-repressor IkappaBalpha to inhibit NF-kappaB activation as an adjuvant approach to promote chemosensitivity in colorectal tumor cells to treatment with CPT-11. These data demonstrate that the protection from apoptosis induced in response to CPT-11 treatment is effectively inhibited by the transient inhibition of NF-kappaB in a variety of human colon cancer cell lines and in a tumor xenograft model, resulting in a significantly enhanced tumoricidal response to CPT-11 via increased induction of apoptosis. These findings indicate that the activation of NF-kappaB by chemotherapy is an important underlying mechanism of inducible chemoresistance.

Adenoviridae↗
PubMed2000

Constructing high complexity synthetic libraries of long ORFs using in vitro selection.

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel.

Amino Acid Sequence↗
PubMed2000

Ribozyme ablation demonstrates that the cardiac subtype of the voltage-sensitive calcium channel is the molecular transducer of 1, 25-dihydroxyvitamin D(3)-stimulated calcium influx in osteoblastic cells.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) stimulates transmembrane influx of Ca(2+) through L-type voltage-sensitive Ca(2+) channels (VSCCs) in ROS 17/2.8 osteoblastic cells. Ca(2+) influx modulates osteoblastic activities including matrix deposition, hormone responsiveness, and Ca(2+)-dependent signaling. 1, 25(OH)(2)D(3) also regulates transcript levels encoding VSCCs. L-type VSCCs are multisubunit complexes composed of a central pore-forming alpha(1) subunit and four additional subunits. The alpha(1) subunit is encoded by one gene in a multimember family, defining tissue-specific subtypes. Osteoblasts synthesize two splice variants of the alpha(1C) cardiac VSCC subtype; however, the molecular identity of the 1,25(OH)(2)D(3)-regulated VSCC remained unknown. We created a ribozyme specifically cleaving alpha(1C) mRNA. To increase target ablation efficiency, the ribozyme was inserted into U1 small nuclear RNA (snRNA) by engineering the U1 snRNA expression cassette, conferring the ribozyme transcript with stabilizing stem-loops at both sides and the Sm binding site that facilitates localization into nucleoplasm. After transfection of ROS 17/2.8 cells with U1 ribozyme-encoding vector, stable clonal cells were selected in which the expression of alpha(1C) transcript and protein were strikingly reduced. Ca(2+) influx assays in ribozyme transfectants showed selective attenuation of depolarization and 1, 25(OH)(2)D(3)-regulated Ca(2+) responses. We conclude that the cardiac subtype of the L-type VSCC is the transducer of stimulated Ca(2+) influx in ROS 17/2.8 osteoblastic cells.

Alternative Splicing↗
PubMed2000

Age-associated decline in gamma-glutamylcysteine synthetase gene expression in rats.

Although glutathione (GSH) concentration has been reported to diminish with age, the mechanism underlying such age-associated decline in the GSH content is not well understood. In this study, we compared the gene expression of both subunits of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, in young, adult, and old Fisher 344 rats. It was found that GCS activity was significantly decreased with increased age in liver, kidney, lung, and red blood cells (RBC). Parallel with the decreased enzyme activity, the protein and mRNA contents of both GCS subunits also changed inversely with age in liver, kidney, and lung, implying a decreased GCS gene expression during aging. Such a reduced GCS gene expression was accompanied by a decline in total GSH content without any change in cysteine concentration. Furthermore, the decreased GCS gene expression in old rats was not associated with a decline in the plasma insulin or cortisol level. This study showed, for the first time, that the expression of both GCS subunit genes was decreased in some organs of old rats, which would result in a reduced rate of GSH biosynthesis. Such decline in GSH synthetic capacity may underlie the observed decrease in GSH content during aging.

Aging↗
PubMed2000

Analysis of mechanisms involved in the prevention of gamma irradiation-induced apoptosis by hGM-CSF.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) induces proliferation and sustains viability of the mouse interleukin (IL)-3 dependent lymphoid cell line BA/F3 expressing the hGM-CSF receptor. Caspase-3 like enzyme activity and DNA fragmentation were augmented by depletion of this factor from the cell, and exposure to gamma irradiation accelerated kinetics of these events. Anti gamma irradiation-induced apoptosis occurred through various mutant GM-CSF receptors and only the box1 region was essential while the C terminal region, including tyrosine residues which are required for MAPK cascade activation, was dispensable. Consistent with this notion, the addition of PD98059 had no effect on this activity thereby indicating that activation of MAPK is not essential for the activity. As expected, gamma irradiation increased p53 protein and bax mRNA levels and the presence of hGM-CSF dramatically modulated bax/bcl-X(L) ratio. The PI-3K specific inhibitor wortmannin did not affect hGM-CSF dependent anti gamma irradiation induced apoptosis nor bcl-X(L) induction, thus bcl-X(L) but not PI-3K pathway seems to be involved in hGM-CSF dependent anti gamma irradiation-induced apoptosis. It is well documented that the boxl region is essential for GM-CSF dependent activation of JAK2 and JAK2 specific inhibitor AG490 suppressed anti gamma, irradiation-induced apoptosis by hGM-CSF. An artificial JAK2 activating molecule in which extracellular and the transmembrane of beta(c) fused with whole JAK2 can sustain BA/F3 cells survival and proliferation mIL-3 independently, but these cells are susceptible to gamma irradiation. Furthermore GyrB/Jak2, which can activate STAT5 but not the MAPK cascade nor survival of BA/F3 cells, also could not prevent gamma irradiation-induced apoptosis. Although JAK2 is essential for hGM-CSF dependent anti gamma irradiation-induced apoptosis, it appeared that JAK2 does not seem sufficient for the activity.

Animals↗
PubMed2000

Double-stranded RNA injection produces null phenotypes in zebrafish.

Zebrafish is a simple vertebrate that has many attributes that make it ideal for the study of developmental genetics. One feature that has been lacking in this model system is the ability to disable specifically targeted genes. Recently, double-stranded RNA has been used to silence gene expression in the nematode Caenorhabditis elegans. We have found that expression of the green fluorescent protein (GFP) from a microinjected plasmid vector can be suppressed in zebrafish embryos by the coinjection of a double-stranded RNA that is specifically targeted to GFP. To determine that double-stranded RNA can attenuate endogenous gene expression, single-cell zebrafish embryos were injected with double-stranded RNA specifically targeted to Zf-T and Pax6.1. We found that microinjection of double-stranded Zf-T RNA resulted in a high incidence of a phenotype similar to that of ntl. Furthermore, Zf-T gene expression could not be detected by in situ hybridization and the message was decreased by 75% by semiquantitative RT-PCR in 12-h embryos that had been injected with the double-stranded RNA. Expression of the zebrafish genes sonic hedgehog and floating head was altered in the embryos microinjected with the Zf-T double-stranded RNA in a manner that is remarkably similar to the zebrafish no-tail mutant. Microinjection of double-stranded RNA targeted to Pax6.1 was associated with depressed expression of Pax6. 1 and resulted in absent or greatly reduced eye and forebrain development, similar to the phenotype seen in mouse mutants. Simultaneous injection of Pax6.1 and Zf-T resulted in embryos lacking notochords, eyes, and brain structures.

Animals↗
PubMed2000

Pharmacophore/receptor models for GABA(A)/BzR subtypes (alpha1beta3gamma2, alpha5beta3gamma2, and alpha6beta3gamma2) via a comprehensive ligand-mapping approach.

Pharmacophore/receptor models for three recombinant GABA(A)/BzR subtypes (alpha1beta3gamma2, alpha5beta3gamma2, and alpha6beta3gamma2) have been established via an SAR ligand-mapping approach. This study was based on the affinities of 151 BzR ligands at five distinct (alpha1-3,5,6beta3gamma2) recombinant GABA(A)/BzR receptor subtypes from at least nine different structural families. Examination of the included volumes of the alpha1-, alpha5-, and alpha6-containing subtypes indicated that region L(2) for the alpha5-containing subtype appeared to be larger in size than the analogous region of the other receptor subtypes. Region L(Di), in contrast, appeared to be larger in the alpha1 subtype than in the other two subtypes. Moreover, region L(3) in the alpha6 subtype is either very small or nonexistent in this diazepam-insensitive subtype (see Figure 16 for details) as compared to the other subtypes. Use of the pharmacophore/receptor models for these subtypes has resulted in the design of novel BzR ligands (see 27) selective for the alpha5beta3gamma2 receptor subtype. alpha5-Selective ligand 27 when injected directly into the hippocampus did enhance memory in one paradigm (Bailey et al., unpublished observations); however, systemic administration of either 9 or 27 into animals did not provide an observable enhancement. This result is in complete agreement with the observation of Liu (1996). It has been shown (Liu, 1996; Wisden et al., 1992) that in the central nervous system of the rat (as well as monkeys and pigeons) there are several native subtypes of the GABA(A) receptor which exhibit different functions, regional distributions, and neuronal locations. Although 27 binds more potently at alpha5beta3gamma2 receptor subtypes and is clearly an inverse agonist (Liu et al., 1996; Liu, 1996), it is possible that this ligand acts as an agonist at one or more subtypes. Liu (1996) clearly showed that a number of imidazobenzodiazepines were negative modulators at one subtype and agonists at another. Therefore, selectivity for a particular subtype at this point is not sufficient to rule out some physiological effect at other GABA(A)/BzR subtypes. The inability of 27 to potentiate memory when given systemically is again in support of this hypothesis, especially since alpha1beta2gamma2 subtypes are distributed throughout the brain (Wisden et al., 1992). A drug delivered systemically is far more likely to interact with all subtypes than one delivered to a specific brain region. This observation (systemic vs intrahippocampal) provides further support for the design of more subtype-specific ligands at the BzR to accurately define their pharmacology, one key to the design of new drugs with fewer side effects.

Animals↗
PubMed2000

A specific inhibitor of janus kinase-3 increases survival in a transgenic mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disorder involving the motor neurons of cortex, brain stem, and spinal cord. About 10% of all ALS patients are familial cases (FALS), of which 20% have mutations in the Cu, Zn-superoxide dismutase (SOD1) gene. The murine model for FALS, which overexpresses a FALS variant of the SOD1 gene, exhibits progressive limbic paralysis followed by death. Treatment of FALS mice with WHI-P131, a specific inhibitor of Janus kinase 3 (JAK3), increased survival by more than two months, suggesting that specific inhibitors of JAK3 may be useful in the treatment of human ALS. These results uniquely establish JAK3 as a novel molecular target for the treatment of FALS.

Animals↗
PubMed2000

The introduction of strain and its effects on the structure and stability of T4 lysozyme.

In order to try to better understand the role played by strain in the structure and stability of a protein a series of "small-to-large" mutations was made within the core of T4 lysozyme. Three different alanine residues, one involved in backbone contacts, one in side-chain contacts, and the third adjacent to a small cavity, were each replaced with subsets of the larger residues, Val, Leu, Ile, Met, Phe and Trp. As expected, the protein is progressively destabilized as the size of the introduced side-chain becomes larger. There does, however, seem to be a limit to the destabilization, suggesting that a protein of a given size may be capable of maintaining only a certain amount of strain. The changes in stability vary greatly from site to site. Substitution of larger residues for both Ala42 and Ala98 substantially destabilize the protein, even though the primary contacts in one case are predominantly with side-chain atoms and in the other with backbone. The results suggest that it is neither practical nor meaningful to try to separate the effects of introduced strain on side-chains from the effects on the backbone. Substitutions at Ala129 are much less destabilizing than at sites 42 or 98. This is most easily understood in terms of the pre-existing cavity, which provides partial space to accommodate the introduced side-chains. Crystal structures were obtained for a number of the mutants. These show that the changes in structure to accommodate the introduced side-chains usually consist of essentially rigid-body displacements of groups of linked atoms, achieved through relatively small changes in torsion angles. On rare occasions, a side-chain close to the site of substitution may change to a different rotamer. When such rotomer changes occur, they permit the structure to dissipate strain by a response that is plastic rather than elastic. In one case, a surface loop moves 1.2 A, not in direct response to a mutation, but in an interaction mediated via an intermolecular contact. It illustrates how the structure of a protein can be modified by crystal contacts.

Alanine↗
PubMed2000

Extracellular signal-regulated kinase 1/extracellular signal-regulated kinase 2 mitogen-activated protein kinase signaling and activation of activator protein 1 and nuclear factor kappaB transcription factors play central roles in interleukin-8 expression stimulated by monosodium urate monohydrate and calcium pyrophosphate crystals in monocytic cells.

OBJECTIVE: Monosodium urate monohydrate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals cause acute gout and pseudogout, respectively. Because acute gout and pseudogout appear to be dependent on interleukin-8 (IL-8)-induced neutrophil ingress, this study was undertaken to define and compare how MSU and CPPD crystals stimulate IL-8 messenger RNA (mRNA) expression in mononuclear phagocytes. METHODS: MSU and CPPD crystal-induced mitogen-activated protein kinase (MAPK) signal transduction and IL-8 transcriptional activation were studied in human monocytic cells, using the THP-1 cell line. RESULTS: MSU and CPPD crystals (0.5 mg/ml) induced activation of c-Jun N-terminal kinase, extracellular signal-regulated kinase 1 (ERK-1)/ERK-2, and p38 MAPK pathways in THP-1 cells. Activation of the ERK-1/ERK-2 pathway was essential for MSU and CPPD crystal-induced IL-8 mRNA expression, whereas the p38 pathway played a greater role in IL-8 mRNA expression in response to CPPD crystals in comparison with MSU crystals. Both crystals induced the binding of nuclear factor kappaB (NF-kappaB), including the NF-kappaB complex c-Rel/RelA, and activator protein 1 (AP-1, including N-terminal phosphorylated c-Jun) to the IL-8 promoter. Both crystals induced transcriptional activation of the IL-8 promoter, which was dependent on activation of c-Rel/RelA and AP-1. Activation of the NF-IL-6 transcription factor played a lesser role. Finally, crystal-induced IL-8 promoter activation was mediated by activation of the ERK-1/ERK-2 pathway, as demonstrated by transfection of dominant-negative raf-1. CONCLUSION: These results indicate that ERK-1/ ERK-2 signaling and transcriptional activation through AP-1 and NF-kappaB are essential for the induction of IL-8 expression in mononuclear phagocytes in response to CPPD and MSU crystals.

Calcium Pyrophosphate↗
PubMed2000

Evaluation of the Effect of Radiation Therapy to Nasopharyngeal Carcinoma by Positron Emission Tomography with 2-

Purpose: This investigation evaluated the effectiveness of positron emission tomography with 2-[F-18]fluoro-2-deoxy-D-glucose (PET-FDG) in assessing residual tumor or tumor recurrence in postradiation nasopharyngeal carcinoma (NPC) patients.Procedures: Forty-six patients with histologically proven NPC who received radiotherapy were included. PET-FDG images were analyzed by a semiquantitative method, metabolic ratio (tumor to cerebellum ratio).Results: The overall sensitivity and specificity of PET-FDG to detect residual tumors and recurrent lesions in the postradiation patients were 80% (12/15) and 87% (27/31), respectively. In patients with PET-FDG 6 months after radiation therapy, the sensitivity and specificity raised to 92% (11/12) and 100% (20/20), respectively.Conclusions: PET-FDG is effective in the evaluation of NPC treated with radiation. The optimal timing in assessing residual tumor or tumor recurrence in postradiation patients should be 6 months or later.

Journal Article↗
PubMed2000

Reassignment of fundamental vibrational modes of cyclic S4N3 cation.

MP2/6 31G* calculations were carried out to investigate the vibrational spectrum of cyclic S4N3+. The results indicate that previous assignments of several fundamental vibrational modes are in error. On the basis of the calculated results, reassignments of these modes are proposed.

Cations↗
PubMed2000

Leukocyte functions in 2 cases of Papillon-Lefèvre syndrome.

AIM: To investigate the rôle of leukocytes in the pathogenesis of Papillon-Lefevre syndrome (PLS). METHODS: Peripheral blood polymorphonuclear neutrophils (PMNs), monocytes (MNs) and gingival crevicular fluid (GCF) were obtained from 2 cases of PLS with typical features. The chemotaxis of PMNs and MNs were evaluated using a modified Boyden chamber. The adherence of PMNs was determined by adherence of PMNs to petri dishes. Interleukin-8 (IL-8) in GCF was detected by sandwich ELISA. Elastase activity in GCF was measured with a low molecular weight substrate (S-2484) specific for granulocyte elastase. RESULTS: PMNs from both patients showed depressed chemotactic response to FMLP and IL-8. Total amounts of IL-8 in GCF from the 2 patients were much higher than those of the normal controls. Elastase activity was not significantly different from that of the controls. The adherence of PMN and the chemotaxis of MN in the 2 patients were normal. CONCLUSION: The depressed chemotactic response of PMN leads to decreased recruitment of PMN and/or release of lysozyme from PMN in the diseased gingival tissue, increasing the susceptibility of PLS patients to periodontal infection.

Adult↗
PubMed2000

Renal NO production and the development of hypertension.

The juxtaglomerular apparatus (JGA) has the very important functions of detecting the fluid flow rate to the distal tubule and thus controlling the glomerular filtration rate (GFR) (tubuloglomerular feedback mechanism [TGF]) and renin release from the afferent arteriole. In studies of the TGF it has been evident that the sensitivity of this mechanism can be reset. Volume expansion will reset it to a low sensitivity leading to a high GFR and urine excretion rate, while dehydration will sensitize the TGF mechanism, giving rise to a low GFR and low urine excretion rate. Furthermore, we have found that in animals that spontaneously develop hypertension there is initially a sensitization of the TGF, leading to a reduced GFR and urine excretion rate, with fluid volume retention in the body and a consequent rise in blood pressure. When the pressure is raised, the TGF characteristics are normalized. In the macula densa (MD) cells in the JGA, there is a large production of NO from neuronal NOS. This production continuously reduces TGF sensitivity and is apparently impaired in animals that spontaneously develop hypertension. When we added an nNOS inhibitor to the drinking water for several weeks while measuring blood pressure, we found an increase in blood pressure after 3-4 weeks of treatment. This effect was abolished by a high salt diet. From these investigations, it also appeared as if nNOS-derived NO inhibited renin release. Experiments have also indicated that NO may resensitize inhibited G-protein coupled purinergic receptors.

Animals↗
PubMed2000

Isoflurane-sevoflurane adminstration before ischemia attenuates ischemia-reperfusion-induced injury in isolated rat lungs.

BACKGROUND: The effects of volatile anesthetics on ischemia-reperfusion (IR)-induced lung injury are not clear. The authors investigated the effects of preadministration of isoflurane and sevoflurane on IR-induced lung injury in an isolated buffer-perfused rat lung model. METHODS: Isolated rat lungs were designated into four groups: control group (n = 6): perfusion for 120 min without ischemia; IR group (n = 6): interruption of perfusion and ventilation for 60 min followed by reperfusion for 60 min; sevoflurane (SEVO)-IR (n = 6) and isoflurane (ISO)-IR (n = 6) groups: 1 minimum alveolar concentration (MAC) isoflurane or sevoflurane was administered for 30 min, followed by 60 min ischemia, then 60 min reperfusion. The authors measured the coefficient of filtration (Kfc) of the lung, lactate dehydrogenase (LDH) activity, tumor necrosis factor alpha, and nitric oxide metabolites (nitrite + nitrate) in the perfusate and the wet-to-dry lung weight ratio. RESULTS: IR caused significant increases in the coefficient of filtration (approximately sevenfold at 60 min of reperfusion compared with baseline; P < 0.01), the wet-to-dry lung weight ratio, the rate of increase of lactate dehydrogenase activity, and tumor necrosis factor a in the perfusate, and caused a significant decrease in nitric oxide metabolites in the perfusate. Administration of 1 MAC isoflurane or sevoflurane before ischemia significantly attenuated IR-induced increases in the coefficient of filtration and the wet-to-dry lung weight ratio, inhibited increases in the rate of increase of lactate dehydrogenase activity and tumor necrosis factor alpha in the perfusate, and abrogated the decrease in nitric oxide metabolites in the perfusate. No difference was found between the SEVO-IR and ISO-IR groups. CONCLUSION: Isoflurane and sevoflurane administered before ischemia can attenuate IR-induced injury in isolated rat lungs.

Anesthetics, Inhalation↗
PubMed1999

Insights into the structure and substrate interactions of the P-glycoprotein multidrug transporter from spectroscopic studies.

The P-glycoprotein multidrug transporter is a 170-kDa efflux pump which exports a diverse group of natural products, chemotherapeutic drugs, and hydrophobic peptides across the plasma membrane, driven by ATP hydrolysis. The transporter has been proposed to interact with its drug substrates within the membrane environment; however, much remains to be learned about the nature and number of the drug binding site(s). The two nucleotide binding domains are responsible for ATP binding and hydrolysis, which is coupled to drug movement across the membrane. In recent years, P-glycoprotein has been purified and functionally reconstituted in amounts large enough to allow biophysical studies. The use of spectroscopic techniques has led to insights into both its secondary and tertiary structure, and its interaction with nucleotides and drugs. In this review, we will summarise what has been learned by application to purified P-glycoprotein of fluorescence spectroscopy, circular dichroism spectroscopy and infra-red spectroscopy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗