A cyclodextrin dimer with a photocleavable linker as a possible carrier for the photosensitizer in photodynamic tumor therapy

A cyclodextrin dimer has been synthesized with two β-cyclodextrins linked by a flexible chain containing a carbon–carbon double bond. This dimer binds and solubilizes a phthalocyanine-based photosensitizer that generates singlet oxygen on irradiation. When the complex is irradiated, the singlet oxygen cleaves the carbon–carbon link, and the cyclodextrins are released, liberating the photosensitizer into the light path. Ideas about how this phenomenon could be used to make photodynamic tumor therapy into a more selective process are described.

Specific molecular recognition of mixed nucleic acid sequences: An aromatic dication that binds in the DNA minor groove as a dimer

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.

d-dimer testing as an adjunct to ultrasonography in patients with clinically suspected deep vein thrombosis: prospective cohort study

Objective To investigate the efficacy of using a rapid plasma d-dimer test as an adjunct to compression ultrasound for diagnosing clinically suspected deep vein thrombosis.

Human fatty acid synthase: Role of interdomain in the formation of catalytically active synthase dimer

The human and animal fatty acid synthases are dimers of two identical multifunctional proteins (Mr 272,000) arranged in an antiparallel configuration. This arrangement generates two active centers for fatty acid synthesis separated by interdomain (ID) regions and predicts that two appropriate halves of the monomer should be able to reconstitute an active fatty acid synthesizing center. This prediction was confirmed by the reconstitution of the synthase active center by using two heterologously expressed halves of the monomer protein. Each of these recombinant halves of synthase monomer contains half of the ID regions. We show here that the fatty acid synthase activity could not be reconstituted when the ID sequences present in the two recombinant halves are deleted, suggesting that these ID sequences are essential for fatty acid synthase dimer formation. Further, we confirm that the ID sequences are the only regions of fatty acid synthase monomers that showed significant dimer formation, by using the yeast two-hybrid system. These results are consistent with the proposal that the ID region, which has no known catalytic activity, associates readily and holds together the two dynamic active centers of the fatty acid synthase dimer, therefore playing an important role in the architecture of catalytically active fatty acid synthase.

Effects of mutations involving cell division, recombination, and chromosome dimer resolution on a priA2∷kan mutant

Recombinational repair of replication forks can occur either to a crossover (XO) or noncrossover (non-XO) depending on Holliday junction resolution. Once the fork is repaired by recombination, PriA is important for restarting these forks in Escherichia coli. PriA mutants are Rec− and UV sensitive and have poor viability and 10-fold elevated basal levels of SOS expression. PriA sulB mutant cells and their nucleoids were studied by differential interference contrast and fluorescence microscopy of 4′,6-diamidino-2-phenylindole-stained log phase cells. Two populations of cells were seen. Eighty four percent appeared like wild type, and 16% of the cells were filamented and had poorly partitioned chromosomes (Par−). To probe potential mechanisms leading to the two populations of cells, mutations were added to the priA sulB mutant. Mutating sulA or introducing lexA3 decreased, but did not eliminate filamentation or defects in partitioning. Mutating either recA or recB virtually eliminated the Par− phenotype. Filamentation in the recB mutant decreased to 3%, but increased to 28% in the recA mutant. The ability to resolve and/or branch migrate Holliday junctions also appeared crucial in the priA mutant because removing either recG or ruvC was lethal. Lastly, it was tested whether the ability to resolve chromosome dimers caused by XOs was important in a priA mutant by mutating dif and the C-terminal portion of ftsK. Mutation of dif showed no change in phenotype whereas ftsK1∷cat was lethal with priA2∷kan. A model is proposed where the PriA-independent pathway of replication restart functions at forks that have been repaired to non-XOs.

A new isoleucine substitution of Val-20 in transthyretin tetramers selectively impairs dimer-dimer contacts and causes systemic amyloidosis.

The most frequent form of inherited amyloidoses is associated with mutations in the transthyretin (TTR) gene coding for 127-amino acid residues of four identical, noncovalently linked subunits that form a pair of dimers in the plasma protein complex. Amyloid fibrils containing the variant and to a lesser extent the wild-type form of the TTR molecule are deposited in various organs, including peripheral nerves and the myocardium, with polyneuropathy and cardiomyopathy as major clinical manifestations. So far, more than 40 distinct amino acid substitutions distributed throughout the TTR sequence over 30 positions have been found to be correlated with an increased amyloidogenicity of TTR. Most of these amyloidogenic amino acid substitutions are suspected to alter the conformation and stability of the monomer. Here we identify and characterize by protein and DNA analysis a novel amyloidogenic Val-20 to Ile mutation in a German three-generation family. The index patient suffered from severe amyloid cardiomyopathy at the age of 60. Conformational stability and unfolding behavior of the Ile-20 monomer in urea gradients was found to be almost indistinguishable from that of wild-type TTR. In contrast, tetramer stability was significantly reduced in agreement with the expected change in the interactions between the two opposing dimers via the side chain of Ile-20. Our observations provide strong evidence for the view that amyloidogenic amino acid substitutions in TTR facilitate the conversion of tetrameric TTR complexes into those conformational intermediates of the TTR folding pathway that have an intrinsic amyloidogenic potential.

The rat extracellular superoxide dismutase dimer is converted to a tetramer by the exchange of a single amino acid.

Extracellular superoxide dismutase (EC-SOD) is a secreted Cu and Zn-containing glycoprotein. While EC-SOD from most mammals is tetrameric and has a high affinity for heparin and heparan sulfate, rat EC-SOD has a low affinity for heparin, does not bind to heparan sulfate in vivo, and is apparently dimeric. To examine the molecular basis of the deviant physical properties of rat EC-SOD, the cDNAs of the rat and mouse EC-SODs were isolated and the deduced amino acid sequences were compared with that of human EC-SOD. Comparison of the sequences offered no obvious explanation of the differences. Analysis of a series of chimeric and point mutated EC-SODs showed that the N-terminal region contributes to the oligomeric state of the EC-SODs, and that a single amino acid, a valine (human amino acid position 24), is essential for the tetramerization. This residue is replaced by an aspartate in the rat. Rat EC-SOD carrying an Asp --> Val mutation is tetrameric and has a high heparin affinity, while mouse EC-SOD with a Val --> Asp mutation is dimeric and has lost its high heparin affinity. Thus, the rat EC-SOD dimer is converted to a tetramer by the exchange of a single amino acid. Furthermore, the cooperative action of four heparin-binding domains is necessary for high heparin affinity. These results also suggest that tetrameric EC-SODs are not symmetrical tetrahedrons, but composed of two interacting dimers, further supporting an evolutionary relationship with the dimeric cytosolic Cu and Zn-containing SODs.

A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

The dimer-dimer interaction surface of the replication terminator protein of Bacillus subtilis and termination of DNA replication.

The replication terminator protein (RTP) of Bacillus subtilis causes polar fork arrest at replication termini by sequence-specific interaction of two dimeric proteins with the terminus sequence. The crystal structure of the RTP protein has been solved, and the structure has already provide valuable clues regarding the structural basis of its function. However, it provides little information as to the surface of the protein involved in dimer-dimer interaction. Using site-directed mutagenesis, we have identified three sites on the protein that appear to mediate the dimer-dimer interaction. Crystallographic analysis of one of the mutant proteins (Y88F) showed that its structure is unaltered when compared to the wild-type protein. The locations of the three sites suggested a model for the dimer-dimer interaction that involves an association between two beta-ribbon motifs. This model is supported by a fourth mutation that was predicted to disrupt the interaction and was shown to do so. Biochemical analyses of these mutants provide compelling evidence that cooperative protein-protein interaction between two dimers of RTP is essential to impose polar blocks to the elongation of both DNA and RNA chains.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

Human white blood cells contain cyclobutyl pyrimidine dimer photolyase.

Although enzymatic photoreactivation of cyclobutyl pyrimidine dimers in DNA is present in almost all organisms, its presence in placental mammals is controversial. We tested human white blood cells for photolyase by using three defined DNAs (supercoiled pET-2, nonsupercoiled bacteriophage lambda, and a defined-sequence 287-bp oligonucleotide), two dimer-specific endonucleases (T4 endonuclease V and UV endonuclease from Micrococcus luteus), and three assay methods. We show that human white blood cells contain photolyase that can photorepair pyrimidine dimers in defined supercoiled and linear DNAs and in a 287-bp oligonucleotide and that human photolyase is active on genomic DNA in intact human cells.

Specificity of dimer formation in tropomyosins: influence of alternatively spliced exons on homodimer and heterodimer assembly.

Tropomyosins consist of nearly 100% alpha-helix and assemble into parallel and in-register coiled-coil dimers. In vitro it has been established that nonmuscle as well as native muscle tropomyosins can form homodimers. However, a mixture of muscle alpha and beta tropomyosin subunits results in the formation of the thermodynamically more stable alpha/beta heterodimer. Although the assembly preference of the muscle tropomyosin heterodimer can be understood thermodynamically, the presence of multiple tropomyosin isoforms expressed in nonmuscle cells points toward a more complex principle for determining dimer formation. We have investigated the dimerization of rat tropomyosins in living cells by the use of epitope tagging with a 16-aa sequence of the influenza hemagglutinin. Employing transfection and immunoprecipitation techniques, we have analyzed the dimers formed by muscle and nonmuscle tropomyosins in rat fibroblasts. We demonstrate that the information for homo- versus heterodimerization is contained within the tropomyosin molecule itself and that the information for the selectivity is conferred by the alternatively spliced exons. These results have important implications for models of the regulation of cytoskeletal dynamics.

Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium.

Mutations in Cu/Zn superoxide dismutase (SOD), a hallmark of familial amyotrophic lateral sclerosis (FALS) in humans, are shown here to confer striking neuropathology in Drosophila. Heterozygotes with one wild-type and one deleted SOD allele retain the expected 50% of normal activity for this dimeric enzyme. However, heterozygotes with one wild-type and one missense SOD allele show lesser SOD activities, ranging from 37% for a heterozygote carrying a missense mutation predicted from structural models to destabilize the dimer interface, to an average of 13% for several heterozygotes carrying missense mutations predicted to destabilize the subunit fold. Genetic and biochemical evidence suggests a model of dimer dysequilibrium whereby SOD activity in missense heterozygotes is reduced through entrapment of wild-type subunits into unstable or enzymatically inactive heterodimers. This dramatic impairment of the activity of wild-type subunits in vivo has implications for our understanding of FALS and for possible therapeutic strategies.

A single GAL4 dimer can maximally activate transcription under physiological conditions.

Most eukaryotic promoters contain multiple binding sites for one or more transcriptional activators that interact in a synergistic manner. A common view is that synergism is a manifestation of the need for many contacts between activators and the general transcription machinery that a single activator presumably cannot fulfill. In this model, various combinations of protein-protein interactions control the level of gene expression. However, we show here that under physiological conditions, a single binding site and presumably GAL4 can activate transcription to the maximum possible level in vivo. Synergistic effects in this natural system are shown to be consistent with cooperative DNA binding. These results point to DNA occupancy as the major element in fine tuning gene expression in the galactose regulon.