Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control.

We have demonstrated that controlled electric fields can be used to regulate transport, concentration, hybridization, and denaturation of single- and double-stranded oligonucleotides. Discrimination among oligonucleotide hybrids with widely varying binding strengths may be attained by simple adjustment of the electric field strength. When this approach is used, electric field denaturation control allows single base pair mismatch discrimination to be carried out rapidly (<15 sec) and with high resolution. Electric field denaturation takes place at temperatures well below the melting point of the hybrids, and it may constitute a novel mechanism of DNA denaturation.

Electric field directed nucleic acid hybridization on microchips.

Selection and adjustment of proper physical parameters enables rapid DNA transport, site selective concentration, and accelerated hybridization reactions to be carried out on active microelectronic arrays. These physical parameters include DC current, voltage, solution conductivity and buffer species. Generally, at any given current and voltage level, the transport or mobility of DNA is inversely proportional to electrolyte or buffer conductivity. However, only a subset of buffer species produce both rapid transport, site specific concentration and accelerated hybridization. These buffers include zwitterionic and low conductivity species such as: d- and l-histidine; 1- and 3-methylhistidines; carnosine; imidazole; pyridine; and collidine. In contrast, buffers such as glycine, beta-alanine and gamma-amino-butyric acid (GABA) produce rapid transport and site selective concentration but do not facilitate hybridization. Our results suggest that the ability of these buffers (histidine, etc.) to facilitate hybridization appears linked to their ability to provide electric field concentration of DNA; to buffer acidic conditions present at the anode; and in this process acquire a net positive charge which then shields or diminishes repulsion between the DNA strands, thus promoting hybridization.

Biodistribution of monoclonal antibodies: scale-up from mouse to human using a physiologically based pharmacokinetic model.

The efficacy of a novel diagnostic or therapeutic agent depends on its selective localization in a target tissue. Biodistribution studies are expensive and difficult to carry out in humans, but such data can be obtained easily in rodents. We have developed a physiologically based pharmacokinetic model for scaling up data from mice to humans, the first such model for genetically engineered macromolecules that bind to their targets in vivo, such as mAbs. The mathematical model uses physiological parameters including organ volumes, blood flow rates, and vascular permeabilities; the compartments (organs) are connected anatomically. This allows the use of scale-up techniques to predict antibody distribution in humans. The model was tested with data obtained in human patients for the biodistribution of a mAb against carcinoembryonic antigen. The model was further tested for a two-step protocol: bifunctional antibodies and radiolabeled hapten, which compared favorably with data in both mice and humans. The model was useful for optimization of treatment parameters, such as dose and time interval of injections, binding affinities, and choice of molecular carrier. This framework may be applicable to other genetically engineered molecules (e.g., growth factors, antisense oligonucleotides, and gene-carrying vectors).

How the anti-(metal chelate) antibody CHA255 is specific for the metal ion of its antigen: X-ray structures for two Fab'/hapten complexes with different metals in the chelate.

Antibodies with bound metal-chelate haptens provide new means for exploiting the diverse properties of metallic elements. The murine monoclonal antibody CHA255 (IgG1 lambda) binds the metal-chelate hapten indium (III)-4-[N'-(2-hydroxyethyl)thioureido]-L-benzyl-EDTA (designated In-EOTUBE) with high affinity (K(a) = 1.1 x 10(10) M-1). Antibody binding is highly specific for the indium chelate; the affinity decreases as much as 10(4) with other metals, even those having ionic radii close to indium. To better understand this selectivity, the crystal structure of the antigen-binding fragment (Fab') of CHA255 complexed with its hapten, In(III)-EOTUBE, was determined by molecular replacement and refined at 2.2-A resolution. The structure of CHA255 Fab' complexed with Fe(III)-EOTUBE was also determined and refined at 2.8-A resolution. In both structures, the hapten's EDTA moiety is half-buried near the center of the complementarity-determining regions (CDR's). Five of the six CDR's on the Fab' interact with the hapten through protein side-chain atoms (but not main-chain atoms). A novel feature of the In-EOTUBE/Fab' complex is coordination of the indium by N epsilon of one histidine from the heavy chain's third CDR (distance = 2.4 A). The histidine coordination is not observed in the Fe-EOTUBE/Fab' complex, due mainly to a slightly different hapten conformation that reduces metal accessibility; this may partially explain the 20-fold lower affinity of CHA255 for iron hapten. An unexpected feature of the Fab' overall is an elbow angle of 193 degrees (the angle between the pseudodyad axes of the Fab's constant and variable domains).

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. III. EPR measurements of the reduced acceptor complex.

Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.

An ultrastructural study of glycosaminoglycans associated with collagen and other constituents of the cat anulus fibrosus.

It has been confirmed that glycosaminoglycan is associated with collagen and other constituents of the anulus fibrosus. In cells, it appears to be present on the rough endoplasmic reticulum and is removed by hyaluronidase digestion and thus may consist of chondroitin sulphate. In pericellular material it appears as fibrils and granules, is unaffected by hyaluronidase and may consist of keratan sulphate or dermatan sulphate. Collagen fibrils contain hyaluronidase-digestable glycosaminoglycan (such as chondroitin sulphate) in the form of peripheral rings that appear as bands in longitudinal sections of the fibrils.

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. I. Static magnetization measurements.

We have measured the static magnetization of unreduced and reduced reaction centers that vary in their quinone content. Measurements were performed in the temperature range 0.7 degrees K less than T less than 200 degrees K and magnetic fields of up to 10 kG. The electronic g-value, crystal field parameters D, E, and the exchange interaction, J, between the quinone spin and Fe2+ were determined using the spin Hamiltonian formalism. The effective moment mu eff/Fe2+ of both reduced and unreduced samples were determined to be 5.35 +/- 0.15 Bohr magnetons. This shows, in agreement with previous findings, that Fe2+ does not change its valence state when the reaction centers are reduced. Typical values of D congruent to +5 cm-1 and E/D congruent to 0.27 are consistent with Fe being in an octahedral environment with rhombic distortion. The values of D and E were approximately the same for reaction centers having one and two quinones. These findings imply that quinone is most likely not a ligand of Fe. The Fe2+ and the spin on the quinone in reduced reaction centers were found to be coupled with an exchange interaction 0 less than /J/ less than 1 cm-1. The validity of the spin Hamiltonian was checked by using an orbital Hamiltonian to calculate energy levels of the 25 states of the S = 2, L = 2 manifold and comparing the magnetization of the lowest five states with those obtained from the spin Hamiltonian. Using the orbital Hamiltonian, we calculated the position of the first excited quintet state to be 340 cm-1 above the ground state quintet. This is in good agreement with the temperature dependence of the quadrupole splitting as determined by Mossbauer spectroscopy.

New host record and notes on mallophaga from the white-necked raven (Corvus cryptoleucus Couch).

Fifty-eight white-necked ravens (Corvus cryptoleucus) were examined for ectoparasites. Brueelia afzali and Philopterus ocellatus osborni were the most abundant mallophagans while Colpocephalum fregili and Myrsidea interruptus were the least common. The white-necked raven is listed as a new host for P. ocellatus osborni. Host sexual selection is indicated by statistical treatment of data obtained from ecologically distinct host populations.

Glycosaminoglycans in the touch corpuscle of the dog.

Alcian Blue staining of the touch corpuscle of dog skin indicates that glycosaminoglycans are present as globular masses in some of the basal cells and are also seen in high concentration in the connective tissue core. The function of these basal cells is not clear but they may be responsible for the production of the large amount of glycosaminoglycan present in the underlying dermis.

Correlation between Alcian Blue stainig of glycosaminoglycans of cat nucleus pulposus and TEM x-ray probe microanalysis.

The nucleus pulposus of cat intervertebral disc was examined after staining glycosaminoglycans with Alcian Blue and the results correlated with TEM X-ray probe microanalysis. In unstained sections a difference in copper levels between tissues and resin was detected. In tissue stained with Alcian blue before embedding, the copper levels were slightly increased and the morphological appearance of the intercellular material was amorphous. In sections restained after cutting, the relative levels of copper in the resin were considerably increased and tissue levels were significantly higher than in the resin. Moreover, the morphology of the intercellular material was altered from a rather amorphous material to a network. Sulphur levels behaved in similar manner to copper levels but any correlation between the elements was due to factors unrelated to glycosaminoglycan staining and probably resulted from contaminating sulphur.

Staining of glycosaminoglycans of intervertebral disc tissue.

Intervertebral discs of six species were stained with Alcian blue and PAS methods to identify glycosaminoglycans. The polyanions are most concentrated in 'ring-like' pericellular areas and in material lying between individual lamellae and bundles of collagen. A slight increase occurs early in life and towards the disc centre. The concentration of glycosaminoglycans cannot be correlated with species which are known to suffer from clinical disc disease, though there appears to be a higher concentration in the discs of the cat and dog than in other species studied.

Leaching of glycosaminoglycan from cow uterus and vagina into fixative solution.

Samples from the genital tract of two cows in metoestrus and two in dioestrus were fixed with formalin-cetyltrimethylammonium bromide and the amount of glycosaminoglycan leached into this histological fixative was estimated using an Alcian Blue method. Uronic acid content was estimated by a carbazole method. Total glycosaminoglycan was estimated after digestion with papain. No more than 10% of the glycosaminoglycan was soluble in the fixative and in comsequence 90% is potentially stainable by Alcian Blue in tissue sections. Total glycosaminoglycan in uterus and vagina is higher in metoestrus than in dioestrus. Estimation of uronic acid in the leached material confirmed the results and indicated that little of it consisted of mucin or keratan sulphate.

Staining of glycosaminoglycans in intervertebral disc cells.

Disc material from horse, ox, sheep, pig, dog and cat was stained by the Alcian-blue-critical electrolyte concentration technique and with the standard and two-step periodic acid Schiff methods. The effects of pretreatment with hyaluronidase and with chondroitinase was also evaluated. There appears to be a small increase in total cellular glycosaminoglycan content with age in all species: cellular material of high molecular weight however only increases in aged animals. The degree of sulphation of cellular glycosaminoglycans does not vary with age or with position in the disc.

Age changes in Alcian blue staining of glycosaminoglycans in sheep articular cartilage.

Alcian blue staining of glycosaminoglycans in sheep articular cartilage is described and discussed. Chodrocytes contain mainly low molecular weight chondroitin sulphate in the outer layers but keratan sulphate increases with depth. Protein-polysaccharide interaction increases with age but the cellular staining decreases in intensity with age. Lacunar capsules contain material of higher molecular weight, much of which is chondroitin sulphate, but no protein-polysaccharide interaction occurs. The matrix of the outer layers is deeply stained while that of the inner layer is pale and consists mainly of keratan sulphate. When stained in high salt concentrations (greater than 0-7 M) the matrix of the middle layer shows two zones; hyaluronidase treatment allows these zones to be differentiated at lower salt concentrations.

Uridine diphosphoglucose dehydrogenase in the intervertebral disc.

Intervertebral discs of an old sheep and a young pig were examined for the presence of cells containing the enzyme uridine diphosphoglucose dehydrogenase. In the sheep, the inner anulus had a higher proportion of active cells than the outer anulus; in the pig, there was no difference. From a consideration of cell numbers, it is suggested that there is an accumulation of glycosaminoglycans in the centre of the disc rather than an increased production rate. Notochordal cells in the pig disc contain uridine diphosphoglucose dehydrogenase and are capable of producing glycosaminoglycans.

Glycosaminoglycan staining in diseasesed dog skin.

Diseased skin of dogs was stained using the critical electrolyte concentration-Alcian Blue method, PAS methods, and the high iron diamine technique. Digestion with testicular hyaluronidase and chondroitinase was also used to evaluate the staining results. Diseased skin exhibits a tendency for the glycosaminoglycans to revert to the condition seen in juvenile normal skin: epidermal glycoprotein content falls, total glycosaminoglycan content and the proportion undigested by hyaluronidase rises, and sulphation falls. In collagen, both hyaluronidase-stable material and sulphation increase, but follicle basement membrane does not show this trend towards the juvenile state.

Fragility of the skin in a cat.

Skin that showed abnormal fragility was investigated by histochemical and other methods. There was a reduction in the amount of acid glycosaminoglycan but collagen cross-linkage and electron microscopic appearance of the fibres was normal. This differs from the condition of dermatosparaxis noted in other species.