The effects of anthracycline drugs on the conformational distribution of mouse P-glycoprotein explains their transport rate differences.

P-glycoprotein (Pgp) is an ATP-dependent efflux transporter and plays a major role in anti-cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti-cancer drug efflux are not well understood. Even subtle differences in the anti-cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, this study focused on two closely-related anthracycline drugs, daunorubicin (DNR), and doxorubicin (DOX), with mouse Pgp. While only differing by a single hydroxyl functional group, DNR has a 4 to 5-fold higher transport rate than DOX. They both non-competitively inhibited Pgp-mediated ATP hydrolysis below basal levels. The Km of Pgp-mediated ATP hydrolysis extracted from the kinetics curves was lower for DOX than DNR. However, the dissociation constants (KDs) for these drugs determined by fluorescence quenching were virtually identical. Acrylamide quenching of Pgp tryptophan fluorescence to probe the tertiary structure of Pgp suggested that DNR shifts Pgp to a "closed" conformation, while DOX shifts Pgp to an "intermediate" conformation. The effects of these drugs on the Pgp conformational distributions in a lipid bilayer were also examined by atomic force microscopy (AFM). Analysis of AFM images revealed that DNR and DOX cause distinct and significant shifts in the conformational distribution of Pgp. The results were combined to build a conformational distribution model for anthracycline transport by Pgp.

The conformation and dynamics of P-glycoprotein in a lipid bilayer investigated by atomic force microscopy.

The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range of drugs through ATP hydrolysis and ligand-induced conformational changes. Deciphering these structural changes is key to understanding the molecular basis of transport and to developing molecules that can modulate efflux. Here, atomic force microscopy (AFM) is used to directly image individual Pgp transporter molecules in a lipid bilayer under physiological pH and ambient temperature. Analysis of the Pgp AFM images revealed "small" and "large" protrusions from the lipid bilayer with significant differences in protrusion height and volume. The geometry of these "small" and "large" protrusions correlated to the predicted extracellular (EC) and cytosolic (C) domains of the Pgp X-ray crystal structure, respectively. To assign these protrusions, simulated AFM images were produced from the Pgp X-ray crystal structures with membrane planes defined by three computational approaches, and a simulated 80 ŠAFM cantilever tip. The theoretical AFM images of the EC and C domains had similar heights and volumes to the "small" and "large" protrusions in the experimental AFM images, respectively. The assignment of the protrusions in the AFM images to the EC and C domains was confirmed by changes in protrusion volume by Pgp-specific antibodies. The Pgp domains showed a considerable degree of conformational dynamics in time resolved AFM images. With this information, a model of Pgp conformational dynamics in a lipid bilayer is proposed within the context of the known Pgp X-ray crystal structures.

Characterization of a large genomic deletion in four Irish families with C7 deficiency.

Inherited deficiency of the seventh complement component (C7) is associated with increased susceptibility to Neisseria meningitidis infections. The disease is rare in most Western countries. Here we report new investigations of a large, but incompletely characterized genomic deletion of exons 8 and 9 [c.739-?_1093+?del], previously identified in three unrelated Irish families with C7 deficiency. We have analysed DNA from one individual, who is homozygous for the deletion, by PCR using primers progressively proximal to the deleted exons. Thus we were able to map the deletion boundaries. Amplification across the breakpoint and sequencing revealed an indel mutation that included a 6.4kb deletion together with an insertion of a novel 8bp sequence [c.739+1262_1270-2387delinsGCAGGCCA]. We demonstrated the same defect in the C7 deficient patients from each family and developed a duplex PCR method to enable the detection of alleles containing the deletion in heterozygotes. A member of a fourth family was found to be homozygous for the deletion defect. Thus, the deletion defect may be a more commonly distributed cause of C7 deficiency in Ireland.

Plasmodesmal targeting and intercellular movement of potato mop-top pomovirus is mediated by a membrane anchored tyrosine-based motif on the lumenal side of the endoplasmic reticulum and the C-terminal transmembrane domain in the TGB3 movement protein.

Live-cell fluorescence microscopy was used to investigate the third triple gene block protein (TGB3) of potato mop-top pomovirus and its role in assisted targeting of TGB2 to plasmodesmata (PD). Wild-type and mutant TGB3 proteins were expressed under the control of the 35S promoter or from a virus reporter clone. Assisted targeting of TGB2 to PD was optimal when the proteins were expressed from a bicistronic plasmid in the relative ratios expected in a virus infection, suggesting that excess TGB3 inhibited PD localisation. Contrary to the generally accepted view, bimolecular fluorescence complementation showed that the TGB3 N terminus is located in the cytosol. Mutational analysis to dissect TGB3 sub domain functions showed that PD targeting was mediated by a composite signal comprising an ER-lumenal tyrosine-based motif and the C-terminal transmembrane domain. Mutation of either of these domains also abolished cell-to-cell movement of the virus. The results are discussed in the context of TGB3 membrane topology.

A novel mutation in a patient with a deficiency of the eighth component of complement associated with recurrent meningococcal meningitis.

INTRODUCTION: Complement component C8 is one of the five terminal complement components required for the formation of the membrane attack complex. Complete absence of C8 results in increased susceptibility to gram-negative bacteria such as Neisseria species. MATERIALS AND METHODS: Two functionally distinct C8 deficiency states have been described: C8 alpha-gamma deficiency has been predominantly reported amongst Afro-Caribbeans, Hispanics, and Japanese and C8beta mainly in Caucasians. RESULTS: We report a case of functional and immunochemical deficiency of the complement component C8, diagnosed in a Caucasian adult following three episodes of meningitis. Western blotting and hemolytic assay demonstrated absence of C8beta. In genetic studies, the common exon 9 C > T transition responsible for 85% of C8beta deficiencies was not found. Two mutations were identified: a novel duplication mutation, c.1047_1053 dupGGCTGTG in exon 7 that introduces a frame shift, resulting in the addition of seven novel amino acid residues and a premature stop codon, and a previously reported mutation, c.271C > T in exon 3. The parents each expressed one of these mutations, confirming compound heterozygosity. DISCUSSION: This is the first report of a duplication mutation in C8beta deficiency and extends the molecular heterogeneity of the disorder.

Systemic lactose intolerance: a new perspective on an old problem.

Intolerance to certain foods can cause a range of gut and systemic symptoms. The possibility that these can be caused by lactose has been missed because of "hidden" lactose added to many foods and drinks inadequately labelled, confusing diagnosis based on dietary removal of dairy foods. Two polymorphisms, C/T13910 and G/A22018, linked to hypolactasia, correlate with breath hydrogen and symptoms after lactose. This, with a 48 hour record of gut and systemic symptoms and a six hour breath hydrogen test, provides a new approach to the clinical management of lactose intolerance. The key is the prolonged effect of dietary removal of lactose. Patients diagnosed as lactose intolerant must be advised of "risk" foods, inadequately labelled, including processed meats, bread, cake mixes, soft drinks, and lagers. This review highlights the wide range of systemic symptoms caused by lactose intolerance. This has important implications for the management of irritable bowel syndrome, and for doctors of many specialties.

Hepatoerythropoietic porphyria: a missense mutation in the UROD gene is associated with mild disease and an unusual porphyrin excretion pattern.

Hepatoerythropoietic porphyria (HEP) is an uncommon inherited cutaneous porphyria, related to porphyria cutanea tarda, that results from severe uroporphyrinogen decarboxylase (UROD) deficiency. It is characterized clinically by the onset in early childhood of severe lesions on sun-exposed skin. We describe a man aged 38 years with an unusually mild form of the disease that started in his early teens. Our data confirm that homozygosity for the F46L mutation in the UROD gene causes a mild form of HEP and show that this genotype may be associated with a unique urinary porphyrin excretion pattern in which pentacarboxylic porphyrin predominates.

The inhibitor DBMIB provides insight into the functional architecture of the Qo site in the cytochrome b6f complex.

Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-13412], we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q(o) site of the cytochrome (cyt) b(6)f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b(6)f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q(o) site. With a single DBMIB at the Q(o) site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b(6)f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc(1) complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt b(L) heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q(o) pocket. With >or=2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISP(B) position of the chicken mitochondrial cyt bc(1) complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b(6)f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc(1) complex suggest that the soluble ISP domain of the spinach cyt b(6)f complex can rotate by at least 53 degrees, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc(1) complex and the M. laminosus and C. reinhardtii cyt b(6)f complexes.

The 16189 variant of mitochondrial DNA occurs more frequently in C282Y homozygotes with haemochromatosis than those without iron loading.

BACKGROUND: Patients with hereditary haemochromatosis (HH) are usually homozygous for the C282Y mutation in the HFE gene. They have variable expression of iron overload and present with a variety of complications, including liver disease, diabetes, arthropathy, fatigue, and cardiomyopathy. The mitochondrial 16189 variant is associated with diabetes, dilated cardiomyopathy, and low body fat at birth, and might contribute to genetic predisposition in further multifactorial disorders. The objective of this study was to determine the frequency of the 16189 variant in a range of patients with haemochromatosis, who had mutations in the HFE gene. METHODS: Blood DNA was analysed for the presence of the 16189 variant in British, French, and Australian C282Y homozygotes and controls, with known iron status, and in birth cohorts. RESULTS: The frequency of the mitochondrial 16189 variant was found to be elevated in individuals with haemochromatosis who were homozygous for the C282Y allele, compared with population controls and with C282Y homozygotes who were asymptomatic (42/292 (14.4%); 102/1186 (8.6%) (p = 0.003); and 2/64 (3.1%) (p = 0.023), respectively). CONCLUSIONS: Iron loading in C282Y homozygotes with HH was exacerbated by the presence of the mitochondrial 16189 variant.

Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves.

The sink-source transition in tobacco leaves was studied noninvasively using transgenic plants expressing the green-fluorescent protein (GFP) under control of the Arabidopsis thaliana SUC2 promoter, and also by imaging transgenic plants that constitutively expressed a tobacco mosaic virus movement protein (MP) fused to GFP (MP-GFP). The sink-source transition was measured on intact leaves and progressed basipetally at rates of up to 600 microns/h. The transition was most rapid on the largest sink leaves. However, leaf size was a poor indicator of the current position of the sink-source transition. A quantitative study of plasmodesmatal frequencies revealed the loss of enormous numbers of simple plasmodemata during the sink-source transition. In contrast, branched plasmodesmata increased in frequency during the sink-source transition, particularly between periclinal cell walls of the spongy mesophyll. The progression of plasmodesmal branching, as mapped by the labelling of plasmodesmata with MP-GFP fusion, occurred asynchronously in different cell layers, commencing in trichomes and appearing lastly in periclinal cell walls of the palisade layer. It appears that dividing cells retain simple plasmodesmata for longer periods than nondividing cells. The rapid conversion of simple to branched plasmodesmata is discussed in relation to the capacity for macromolecular trafficking in developing leaf tissues.

Inhibitor "double occupancy" in the Q(o) pocket of the chloroplast cytochrome b6f complex.

Electron paramagnetic resonance (EPR) spectra of the "Rieske" 2Fe-2S cluster revealed that two molecules of the inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) can bind to each monomer of the spinach cytochrome (cyt) b6f complex, both in isolated form and in intact thylakoid membranes. Binding to the high-affinity site, which accounts for the observed inhibitory effects, caused small shifts in the g(x) transition of the 2Fe-2S cluster EPR spectrum, similar to those induced by stigmatellin or 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether (DNP-INT). Occupancy of the low-affinity site was only observed after addition of superstoichiometric amounts of the inhibitor and was accompanied by the appearance of a g = 1.94 EPR signal. The shape of the equilibrium binding titration curve, the effects on the 2Fe-2S EPR spectrum, and the ability of the DBMIB binding to displace DNP-INT were consistent with two molecules of DBMIB binding at the Q(o) pocket, with the strongly binding species binding close to the 2Fe-2S cluster. Possible implications of these findings for so-called "double-occupancy" models for Q(o) site catalysis are discussed.

Alternative splicing and tissue-specific transcription of human and rodent ubiquitous 5-aminolevulinate synthase (ALAS1) genes.

The rate of haem synthesis in non-erythroid mammalian tissues is controlled by the ubiquitous isoform of 5-aminolevulinate synthase (ALAS1). In order to explore the regulation of mammalian ALAS1 genes, we have investigated the transcription of the human and rat genes. The 17 kb human gene differs from the rat gene in containing an additional untranslated exon that is alternatively spliced to produce a longer, minor mRNA transcript. Relative amounts of the two transcripts were similar in all tissues examined. Analysis of mRNA transcripts in human and rat tissues revealed tissue-specific differences in the use of transcription start sites by closely similar core promoters. In brain, initiation was from sites within and upstream from the TATA box, including an initiator-like element. In liver, initiation was TATA-driven from a single downstream site that appeared to be used exclusively for induction by drugs. Intermediate patterns were observed in other tissues and cell lines. Mutation of the TATA box did not impair transcription in transfected HeLa cells but activated upstream start sites, recapitulating the brain pattern. Our findings indicate that the conformation of the core ALAS1 promoter that directs assembly of the transcription pre-initiation complex may vary between tissues and have implications for understanding the tissue-specific regulated expression of this gene.

Tuberization in potato involves a switch from apoplastic to symplastic phloem unloading.

Phloem unloading was studied in potato plants in real time during the early stages of tuberization using carboxyfluorescein (CF) as a phloem-mobile tracer, and the unloading pattern was compared with autoradiography of tubers that had transported (14)C assimilates. In stolons undergoing extension growth, apoplastic phloem unloading predominated. However, during the first visible signs of tuberization, a transition occurred from apoplastic to symplastic transport, and both CF and (14)C assimilates subsequently followed identical patterns of phloem unloading. It is suggested that the switch to symplastic sucrose unloading may be responsible for the upregulation of several genes involved in sucrose metabolism. A detailed analysis of sugar levels and (14)C sugar partitioning in tuberizing stolons revealed a distinct difference between the apical region of the tuber and the subapical region. Analysis of invertase activity in nontuberizing and tuberizing stolons revealed a marked decline in soluble invertase in the subapical region of swelling stolons, consistent with the switch from apoplastic to symplastic unloading. However, cell wall-bound invertase activity remained high in the apical 1 to 2 mm of tuberizing stolons. Histochemical analysis of potato lines transformed with the promoter of an apoplastic invertase gene (invGE) linked to a reporter gene also revealed discrete gene expression in the apical bud region. Evidence is presented that the apical and lateral tuber buds function as isolated domains with respect to sucrose unloading and metabolism.

Co-inheritance of mutations in the uroporphyrinogen decarboxylase and hemochromatosis genes accelerates the onset of porphyria cutanea tarda.

Porphyria cutanea tarda is a skin disease caused by photosensitization by porphyrins whose accumulation is caused by deficiency of hepatic uroporphyrin- ogen decarboxylase activity. Mutations in the uroporphyrinogen decarboxylase gene are present in the low-penetrant, autosomal dominant familial form but not in the commoner sporadic form of porphyria cutanea tarda. We have investigated the relationship between age of onset of skin lesions and mutations (C282Y, H63D) in the hemochromatosis gene in familial (19 patients) and sporadic porphyria cutanea tarda (65 patients). Familial porphyria cutanea tarda was identified by mutational analysis of the uroporphyrinogen decarboxylase gene. Five previously described and eight novel mutations (A80S, R144P, L216Q, E218K, L282R, G303S, 402-403delGT, IVS2 + 2 delTAA) were identified. Homozygosity for the C282Y hemochromatosis mutation was associated with an earlier onset of skin lesions in both familial and sporadic porphyria cutanea tarda, the effect being more marked in familial porphyria cutanea tarda where anticipation was demonstrated in family studies. Analysis of the frequencies of hemochromatosis genotypes in each type of porphyria cutanea tarda indicated that C282Y homozygosity is an important susceptibility factor in both types but suggested that heterozygosity for this mutation has much less effect on the development of the disease.

Non-erythroid form of acute intermittent porphyria caused by promoter and frameshift mutations distant from the coding sequence of exon 1 of the HMBS gene.

Acute intermittent porphyria (AIP) is a low-penetrant, autosomal dominant disorder caused by mutations in the HMBS gene. The gene is transcribed from two promoters to produce ubiquitous and erythroid isoforms of porphobilinogen deaminase, which differ only at their NH2 ends. In the classical form of AIP, both isoforms are deficient, but about 5% of families have the non-erythroid variant in which only the ubiquitous isoform is affected. Previously identified mutations in this variant have been within or close to the coding region of exon 1 of the HMBS gene, the only exon that is expressed solely in the ubiquitous isoform. Here, we describe mutations in the ubiquitous promoter (-154delG) and in exon 3 (41delA) that cause the non-erythroid variant. Reporter gene and electrophoretic mobility shift assays show that the G nucleotide at position -154, the most 5' of several transcription-initiation sites in the ubiquitous HMBS promoter, which lies immediately 3' to a transcription-factor IIB binding motif, is essential for normal transcription. The frameshift mutation in exon 3 introduces a stop codon into mRNA for the ubiquitous isoform only. Our investigations identify two new mechanisms for production of the non-erythroid variant of AIP and demonstrate that mutational analysis for diagnosis of this variant needs to include wider regions of the HMBS gene than indicated by previous reports. Furthermore, they show that deletion of one of several transcription initiation sites in the promoter of a housekeeping gene that lacks both TATA and initiator elements can produce disease.

Inhibitory copper binding site on the spinach cytochrome b6f complex: implications for Qo site catalysis.

The isolated cytochrome (cyt) b(6)f complex from spinach is inhibited by Cu(2+) with a K(D) of about 1 microM at pH 7.6 in the presence of 1.6 microM decyl-plastoquinol (C(10)-PQH(2)) as a substrate. Inhibition was competitive with respect to C(10)-PQH(2) but noncompetitive with respect to horse heart cyt c or plastocyanin (PC). Inhibition was also pH-sensitive, with an apparent pK at about 7, above which inhibition was stronger, suggesting that binding occurred at or near a protonatable amino acid residue. Equilibrium binding titrations revealed ca. 1.4 tight Cu(2+) binding sites with a K(D) of about 0.5 microM and multiple (>8) weak (K(D) > 50 microM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu(2+). A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu(2+) was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu(2+) and the cyt f (Fe(3+)) heme at a distance of between 30 and 54 A, depending upon the relative orientations of Cu(2+) and cyt f heme g-tensors. Two-pulse electron spin-echo envelope modulation (ESEEM) and 4-pulse 2-dimensional hyperfine sublevel correlation (2D HYSCORE) measurements of Cu(2+) bound to isolated cyt b(6)f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu(2+) ligand as an imidazole nitrogen of a His residue, and electron-nuclear double resonance (ENDOR) confirmed the presence of a directly coordinated nitrogen. A model of the 3-dimensional structure of the cytochrome b(6)f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc(1) complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu(2+). Two of these are located on the "Rieske" iron-sulfur protein protein (ISP) while the third is found on the cyt f protein. None of these potential ligands appear to interact directly with the quinol oxidase (Q(o)) binding pocket. A model is thus proposed wherein Cu(2+) interferes with the interaction of the ISP protein with the Q(o) site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP "domain movement" in Q(o) site catalysis are discussed.

Variegate porphyria in Western Europe: identification of PPOX gene mutations in 104 families, extent of allelic heterogeneity, and absence of correlation between phenotype and type of mutation.

Variegate porphyria (VP) is a low-penetrance, autosomal dominant disorder characterized clinically by skin lesions and acute neurovisceral attacks that occur separately or together. It results from partial deficiency of protoporphyrinogen oxidase encoded by the PPOX gene. VP is relatively common in South Africa, where most patients have inherited the same mutation in the PPOX gene from a common ancestor, but few families from elsewhere have been studied. Here we describe the molecular basis and clinical features of 108 unrelated patients from France and the United Kingdom. Mutations in the PPOX gene were identified by a combination of screening (denaturing gradient gel electrophoresis, heteroduplex analysis, or denaturing high-performance liquid chromatography) and direct automated sequencing of amplified genomic DNA. A total of 60 novel and 6 previously reported mutations (25 missense, 24 frameshift, 10 splice site, and 7 nonsense) were identified in 104 (96%) of these unrelated patients, together with 3 previously unrecognized single-nucleotide polymorphisms. VP is less heterogeneous than other acute porphyrias; 5 mutations were present in 28 (26%) of the families, whereas 47 mutations were restricted to 1 family; only 2 mutations were found in both countries. The pattern of clinical presentation was identical to that reported from South Africa and was not influenced by type of mutation. Our results define the molecular genetics of VP in western Europe, demonstrate its allelic heterogeneity outside South Africa, and show that genotype is not a significant determinant of mode of presentation.

Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.

Leaves undergo a sink-source transition during which a physiological change occurs from carbon import to export. In sink leaves, biolistic bombardment of plasmids encoding GFP-fusion proteins demonstrated that proteins with an Mr up to 50 kDa could move freely through plasmodesmata. During the sink-source transition, the capacity to traffic proteins decreased substantially and was accompanied by a developmental switch from simple to branched forms of plasmodesmata. Inoculation of sink leaves with a movement protein-defective virus showed that virally expressed GFP, but not viral RNA, was capable of trafficking between sink cells during infection. Contrary to dogma that plasmodesmata have a size exclusion limit below 1 kDa, the data demonstrate that nonspecific "macromolecular trafficking" is a general feature of simple plasmodesmata in sink leaves.