Dynamic interplay between adhesive and lateral E-cadherin dimers

E-cadherin, an adhesive transmembrane protein of epithelial adherens junctions, forms two types of detergent-resistant dimers: adhesive dimers consisting of cadherin molecules derived from two neighboring cells and lateral dimers incorporating cadherins of the same cell. Both dimers depend on the integrity of the same residue, Trp156. While the relative amounts of these complexes are not certain, we show here that in epithelial A-431 cells, adhesive dimers may be a prevalent form. Inactivation of the calcium-binding sites, located between successive cadherin ectodomains, drastically reduced the amount of adhesive dimers and concomitantly increased the amount of lateral dimers. A similar interdependence of adhesive and lateral dimers was observed in digitonin-permeabilized cells. In these cells, adhesive dimers immediately disassembled after lowering the Ca2+ concentration below 0.1 mM. The disappearance of adhesive dimers was counterbalanced by an increase in Trp156-dependent lateral dimers. Increasing the calcium concentration to a normal level rapidly restored the original balance between adhesive and lateral dimers. We also present evidence that E-cadherin dimers in vivo have a short lifetime. These observations suggest that cadherin-mediated adhesion is based on the dynamic cycling of E-cadherin between monomeric and adhesive dimer states.     

Pyrimidine Dimers in the DNA of Paramecium aurelia

The production and fate of thymine-containing pyrimidine dimers in Paramecium aurelia DNA was investigated in three experimental series: production of dimers by UV irradiation, fate of dimers in the dark, and “loss of photoreactivability of dimers.” It is shown that cyclobutyl dimers are made by UV irradiation of Paramecium DNA in vivo, that because of cytoplasmic absorption the number of dimers made in DNA irradiated in vivo is much lower than in DNA irradiated in vitro, that dimers are lost from animals incubated in the dark after irradiation, and that all the dimers that remain in the animals can be destroyed by photoreactivating illumination. Since mutation induction is photoreactivable, these and previous photoreactivation data suggest that pyrimidine dimers are important in mutation induction in P. aurelia.     

New chemical and biological aspects of artemisinin-derived trioxane dimers.

Joining two 10-deoxoartemisinin trioxane units via a p-diacetylbenzene linker produces new C-10 non-acetal dimers and. 1H NMR spectroscopy allows unambiguous assignment of the stereochemistry at C-10 in these dimers. Successful replacement of both carbonyl oxygen atoms in these diketone dimers by fluorine atoms produces new tetrafluorinated dimers and. Each dimer was evaluated in vitro for antimalarial, antiproliferative, and antitumor activities; ketone dimers and, more than fluorinated dimers and, are promising for chemotherapy of both malaria and cancer.     

Roles of PsbI and PsbM in photosystem II dimer formation and stability studied by deletion mutagenesis and X-ray crystallography

PsbM and PsbI are two low molecular weight subunits of photosystem II (PSII), with PsbM being located in the center, and PsbI in the periphery, of the PSII dimer. In order to study the functions of these two subunits from a structural point of view, we crystallized and analyzed the crystal structure of PSII dimers from two mutants lacking either PsbM or PsbI. Our results confirmed the location of these two subunits in the current crystal structure, as well as their absence in the respective mutants. The relative contents of PSII dimers were found to be decreased in both mutants, with a concomitant increase in the amount of PSII monomers, suggesting a destabilization of PSII dimers in both of the mutants. On the other hand, the accumulation level of the overall PSII complexes in the two mutants was similar to that in the wild-type strain. Treatment of purified PSII dimers with lauryldimethylamine N-oxide at an elevated temperature preferentially disintegrated the dimers from the PsbM deletion mutant into monomers and CP43-less monomers, whereas no significant degradation of the dimers was observed from the PsbI deletion mutant. These results indicate that although both PsbM and PsbI are required for the efficient formation and stability of PSII dimers in vivo, they have different roles, namely, PsbM is required directly for the formation of dimers and its absence led to the instability of the dimers accumulated. On the other hand, PsbI is required in the assembly process of PSII dimers in vivo; once the dimers are formed, PsbI was no longer required for its stability.     

Targeting the insulin receptor with hormone and peptide dimers

Insulin is a key hormone involved in the regulation of overall energetic homeostasis of the organism. The dimeric character of the receptor for insulin evokes ideas about its activation or inhibition with peptide dimers that could either trigger or block the structural transition of the insulin receptor, leading to its activation. Herewith, we present the chemical engineering and biological characterization of several series of insulin dimers or dimers of specific peptides that should be able to bind receptors for insulin or insulin growth factor 1. The hormones or peptides in the dimers were interconnected with different linkers, consisting of triazole moieties and 3, 6, 8, 11, or 23 polyethylene glycol units. The prepared dimers were weaker in binding to insulin receptors than human insulin. However, some of the insulin dimers showed preferential binding specificity toward the isoform A of the insulin receptor, and the insulin dimers also stimulated the insulin receptor more strongly than would be consistent with their binding affinities. Our results suggest that designing insulin dimers may be a promising strategy for modulating the ability of the hormone to activate the receptor or to alter its specificity toward insulin receptor isoforms.     

Ultraviolet radiation-induced lethality and repair of pyrimidine dimers in fish embryos

Pimephales promelas (fathead minnow) embryos were used to show a correlation between induction of pyrimidine dimers in DNA and embryo death. Embryo killing was measured by a lack of heart-beat and blood circulation at 48 h post-ultraviolet radiation (UVR). When the embryos were exposed to various doses of UVR from a FS-40 sunlamp followed by exposure to photoreactivating light (PRL) (320-400 nm), the number of pyrimidine dimers decreased significantly. The photorepair of dimers was accompanied by a substantial increase in embryo survival. When embryo killing was examined as a function of the number of dimers present, dimers were identified as a major lesion involved in UVR-induced killing in these fish embryos. This in vivo study on photoreactivation treatment of fish embryos shows a direct association between UVR-induced pyrimidine dimers and embryo killing. In addition, when embryos were held in the dark for 9 h after UVR, 50% of the dimers were removed by excision repair.     

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.     

Multicopy plasmid instability: the dimer catastrophe hypothesis

Multimer formation reduces plasmid copy number and is an established cause of segregational instability. Nevertheless, it is difficult to rationalize observations that low levels of dimers can cause severe instability, if we assume they are distributed evenly in cell populations. We report here that dimer distribution is in fact heterogeneous in recombination-proficient strains. Most cells in the population contain only monomers; dimers are confined to a small sub-population from which plasmid-free daughters arise at high frequency. In a rec⁺ culture where 4% of pBR322 molecules are dimers, more than half are in dimer-only cells. We show that this situation is inevitable because dimers replicate at twice the rate of monomers. Runaway multimerization is avoided because dimer-containing cells grow more slowly than their monomer-containing counterparts. A computer simulation is used to show how dimers proliferate after formation by homologous recombination. The equilibrium concentration of dimers is proportional to the inter-plasmid recombination rate and is essentially independent of the rate at which homologous recombination converts dimers to monomers.     

DNA Repair in Potorous tridactylus

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times (~ 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure ~ 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though ~ 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.     

Photoreactivation and dark repair of ultraviolet light-induced pyrimidine dimers in chloroplast DNA.

A UV-specific endonuclease was used to detect ultraviolet light-induced pyrimidine dimers in chloroplast DNA of Chlamydomonas reinhardi that was specifically labeled with tritiated thymidine. All of the dimers induced by 100 J/m2 of 254 nm light are removed by photoreaction. Wild-type cells exposed to 50 J/m2 of UF light removed over 80% of the dimers from chloroplast DNA after 24 h of incubation in growth medium in the dark. A UV- sensitive mutant, UVS1, defective in the excision of pyrimidine dimers from nuclear DNA is capable of removing pyrimidine dimers from chloroplast DNA nearly as well as wild-type, suggesting that nuclear and chloroplast DNA dark-repair systems are under separate genetic control.     

Efficiency of formation of pyrimidine dimers in SV40 chromatin in vitro

The efficiency of formation of pyrimidine dimers by 254-nm light was studied in mixtures of SV40 chromatin and DNA extracted from that chromatin. At high doses (beyond 380 J/m2), fewer dimers are formed in chromatin than in DNA for a given dose of radiation. This difference is about 10% as saturation with pyrimidine dimers is approached at 6840 J/m2. Conversely, at biologically repairable doses (up to 40 J/m2, less than 2 dimers/genome), significantly more dimers are produced in the chromatin than in the DNA. A maximum increase of about 50% occurs at doses producing 0.5--20 dimers/genome. With isolated nucleosomes from this chromatin, a maximum increase in dimer formation of 77% was observed. Therefore, the increased dimer formation in the whole chromatin can be wholly accounted for in the nucleosome portion.     

Beta gamma dimers of G proteins inhibit atrial muscarinic K+ channels

It has been proposed that beta gamma dimers of signal-transducing G proteins mediate muscarinic activation of atrial K+ channels. We examined this hypothesis by testing the effects of beta gamma dimers from four sources (human erythrocytes, human placenta, bovine brain, and bovine retina) on single channel muscarinic K+ (K+[acetylcholine (ACh)]) currents in inside-out membrane patches of adult guinea pig atria. None of the four beta gamma dimer preparations stimulated K+[ACh] currents; on the contrary, each inhibited the currents whether the currents were activated with GTP alone (agonist-independent activity) or with GTP plus a muscarinic agonist (agonist-dependent activity). Detergents at concentrations used to suspend erythrocyte, brain, and placental beta gamma dimers had no effect by themselves, and detergents were not used with the retinal beta gamma dimers. We conclude that beta gamma dimers do not mediate stimulatory effects of the endogenous G protein that regulates the K+ channels. In fact beta gamma dimers appear to inhibit activation by the endogenous G alpha subunits. Further insight into the role of beta gamma dimers came from the observation that agonist-independent GTP-activated K+[ACh] currents were inhibited by beta gamma dimers at about one-tenth the concentration required to inhibit agonist-dependent activation. One possibility is that dimeric beta gamma may have a higher affinity for free alpha subunits than for alpha subunits associated with agonist-occupied receptors. Thus, in addition to the known requirement of beta gamma dimers for the interaction of alpha subunits with receptors, beta gamma dimers may also improve the signal-to-noise ratio for agonists by reducing agonist-independent background activities.     

Chemotaxis kinase CheA is activated by three neighbouring chemoreceptor dimers as effectively as by receptor clusters

Chemoreceptors are central to bacterial chemotaxis. These transmembrane homodimers form trimers of dimers. Trimers form clusters of a few to thousands of receptors. A crucial receptor function is 100‐fold activation, in signalling complexes, of sensory histidine kinase CheA. Significant activation has been shown to require more than one receptor dimer but the number required for full activation was unknown. We investigated this issue using Nanodiscs, soluble, nanoscale (～10 nm diameter) plugs of lipid bilayer, to limit the number of neighbouring receptors contributing to activation. Utilizing size‐exclusion chromatography, we separated primary preparations of receptor‐containing Nanodiscs, otherwise heterogeneous for number and orientation of inserted receptors, into fractions enriched for specific numbers of dimers per disc. Fractionated, clarified Nanodiscs carrying approximately five dimers per disc were as effective in activating kinase as native membrane vesicles containing many neighbouring dimers. At five independently inserted dimers per disc, every disc would have at least three dimers oriented in parallel and thus able act together as they would in native membrane. We conclude full kinase activation involves interaction of CheA with groups of three receptor dimers, presumably as a trimer of dimers, and that more extensive interactions among receptors are not necessary for full kinase activation.     

In vitro multimerization of von Willebrand factor is triggered by low pH. Importance of the propolypeptide and free sulfhydryls

Large von Willebrand factor (vWf) multimers are assembled by the formation of disulfide bonds between dimers in trans Golgi and post-Golgi compartments. We were able to reproduce this process in a cell-free system using purified vWf dimers. The multimers formed in vitro extended to 5 x 10(6) Da and were similar in size distribution to those secreted constitutively by endothelial cells in culture. Multimerization occurred only at acidic pH with an optimum at pH 5.8 and needed the continued presence of an acidic pH for it to proceed. Pro-vWf dimers multimerized, whereas mature dimers failed to assemble into large multimers. Multimerization required the propolypeptide to be a contiguous part of pro-vWf subunits since free propolypeptide did not promote multimerization of mature dimers. In addition, multimers formed in the presence of both pro-vWf and mature vWf dimers incorporated only pro-vWf dimers. Two out of six available monoclonal antibodies to the prosequence inhibited multimerization. Multimerization was also inhibited by chemical blocking of free sulfhydryl(s). The free sulfhydryl(s) were localized to the mature region of the pro-vWf by examination of the mature subunit and the propolypeptide derived by proteolytic cleavage of pro-vWf subunits.     

Potential of mean force calculations of the stacking-unstacking process in single-stranded deoxyribodinucleoside monophosphates.

The free energy of the stacking-unstacking process of deoxyribodinucleoside monophosphates in aqueous solution has been investigated by potential of mean force calculations along a reaction coordinate, defined by the distance between the glycosidic nitrogen atoms of the bases. The stacking-unstacking process of a ribodinucleoside monophosphate was observed to be well characterized by this coordinate, which has the advantage that it allows for a dynamical backbone and flexible bases. All 16 naturally occurring DNA dimers composed of the adenine, cytosine, guanine, or thymine bases in both the 5' and the 3' positions were studied. From the free-energy profiles we observed the deepest minima for the stacked states of the purine-purine dimers, but good stacking was also observed for the purine-pyrimidine and pyrimidine-purine dimers. Substantial stacking ability was found for the dimers composed of a thymine base and a purine base and also for the deoxythymidylyl-3',5'-deoxythymidine dimer. Very poor stacking was observed for the dCpdC dimer. Conformational properties and solvent accessibility are discussed for the stacked and unstacked dimers. The potential of mean force profiles of the stacking-unstacking process for the DNA dimers are compared with the RNA dimers.     

The influence of the wavelength of ultraviolet radiation on survival,mutation induction and DNA repair in irradiated chinese hamster cells

Chinese hamster ovary cells were used to compare the cytotoxicity and mutagenicity of far-UV radiation emitted by a low-pressure mercury, germicidal lamp (wavelength predominantly 254 nm) with that of near-UV radiation emitted by a fluorescent lamp with a continuous spectrum (Westinghouse “Sun Lamp”), of which only the radiation with wavelengths greater than 290 nm or greater than 310 nm was transmitted to the cells. The radiation effects were compared on the basis of an equal number of pyrimidine dimers, the predominant lesion induced in DNA by far-UV, for the induction of which much more energy is needed with near-UV than with 254-nm radiation.The numbers of dimers induced were determined by a biochemical method detecting UV-endonuclease-susceptible sites. The equivalence of these sites with pyrimidine dimers was established, qualitatively and quantitatively, in studies with enzymic photoreactivation in vitro and chromatographic analysis of dimers.On the basis of induced dimers, more cells were killed by >310-nm UV than by >290-nm UV; both forms of radiation were more cytotoxic than 254-nm UV when equal numbers of dimers were induced. Moreover, 5–6 times as many mutants were induced per dimer by >310-nm UV than by >290-nm UV; the latter appeared approximately as mutagenic as 254-nm UV. The differences in lethality and mutagenicity were not caused by differences in repair of dimers: cells with an equal number of dimers induced by either 254-nm or near-UV showed the same removal of sites susceptible to a UV endonuclease specific for dimers, as well as an identical amount of repair replication.The results indicate that near-UV induces, besides pyrimidine dimers, other lesions that appear to be of high biological significance.     

The ecto-5'-nucleotidase subunits in dimers are not linked by disulfide bridges but by non-covalent bonds

It has long been considered that ecto-5'-nucleotidase (eNT) dimers consist of subunits linked by disulfide bonds. Hydrophilic (6.7S) and amphiphilic (4.0S) dimers were separated by sedimentation analysis of eNT purified from bull seminal plasma. Hydrophilic (4. 2S) and amphiphilic (2.6S) eNT monomers were obtained after reduction of disulfide bonds in dimers. The amphiphilic eNT dimers or monomers were converted into their hydrophilic variants with phosphatidylinositol-specific phospholipase C. SDS-PAGE plus Western blot showed 68 kDa subunits, regardless of the addition of beta-mercaptoethanol to the SDS mixture. Active eNT monomers were obtained by addition of 1 M guanidinium chloride (Gdn) to dimers, and unfolded subunits by addition of 4 M Gdn. The results unambiguously demonstrate that the subunits in eNT dimers are not linked by disulfide bridges, but by non-covalent bonds, and that dissociation precedes inactivation and unfolding.     

Proximity of repair patches to persistent pyrimidine dimers in DNA of normal human and xeroderma pigmentosum cells

The proximity of repair patches to persistent pyrimidine dimers in normal human cells and xeroderma pigmentosum group C and D cells was analyzed by sequential digestion of repaired DNA with Micrococcus luteus UV-endonuclease and Escherichia coli DNA polymerase I. Although this enzymatic digestion removed one-third of the pyrimidine dimers, less than 3% of the label associated with repair patches and a similar amount of uniformly labeled DNA were removed. The repair patches therefore appear to be similarly distant from persistent dimers in all cell types, and, in particular, are not adjacent to unexcised dimers in xeroderma pigmentosum group D cells. A previous model that suggested that patches are inserted adjacent to dimers in xeroderma pigmentosum group D cells receives no support from these results.