Evidence for glucose-mediated covalent cross-linking of collagen after glycosylation in vitro.

Rabbit forelimb tendons incubated for 15 or 21 days at 35 degrees C in the presence of 8 or 24 mg of glucose/ml were shown to change their chemical, biochemical and mechanical characteristics. The tendons treated with glucose contained up to three times as much hexosyl-lysine and hexosylhydroxylysine as did control tendons as judged by assay of NaB3H4-reduced samples. Measurement of the force generated on thermal contraction showed significant increases in glycosylated tendons compared with controls, indicating the formation of new covalent stabilizing bonds. This conclusion was supported by the decreased solubility of intact tendons and re-formed fibres glycosylated in vitro, and by the evidence from peptide maps of CNBr-digested glucose-incubated tendons. The latter, when compared with peptide maps of control tendons, revealed the presence of additional high-Mr peptide material. These peptides appear to be cross-linked by a new type of covalent bond stable to mild thermal and chemical treatment. This system in vitro provides a readily controlled model for the study of the chemistry of changes brought about in collagen by non-enzymic glycosylation in diabetes.     

Orbitally magnetized organic solids

The preparation is discussed of polymeric organic solids in which large ring-like delocalized electronic orbitals can be made to enclose or trap magnetic flux. The situation is analogous to snapping shut a loop of superconductor while it is in a magnetic field. The operation of the Meissner effect acts to retain the trapped flux and create remant magnetization.     

Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus

Hepatitis B virus (HBV) has not yet been propagated in vitro, and knowledge concerning its reaction with receptors on target cells remained scant. We have located within the HBV envelope proteins a sequence mediating the attachment of HBV to human hepatoma HepG2 cells. A synthetic peptide analog (PLGFFPDHQLDPAFGANSNNPDWDFNP) is recognized by both cell receptors and anti-HBV antibodies and elicits antibodies reacting with native HBV. The synthetic peptide is a promising immunogen expected to elicit protective antibodies based on the concept of the attachment blockade pathway of virus neutralization. The approach described here, based on anti-peptide antisera and the binding of peptide analogs to cell receptors is generally applicable for the delineation of cell receptor binding sites on viruses with known gene sequences.     

Heat modifiability and detergent solubility of outer membrane proteins of Rhodopseudomonas sphaeroides

The outer membrane fraction from Rhodopseudomonas sphaeroides was isolated by isopycnic density centrifugation. The purity of this fraction was assayed by several methods. When the outer membrane fraction obtained after French press lysis of cells was compared with the outer membrane fragments released during spheroplast formation, the polypeptide profiles were identical. Detergent solubilization of membrane fractions showed that Triton X-100 nonselectively solubilizes both the cytoplasmic membrane and the outer membrane, whereas Deriphat 160 selectively solubilizes the cytoplasmic membrane. Several outer membrane polypeptides, including the major outer membrane protein, exhibited changes in electrophoretic mobility that depended upon the temperature of solubilization in sodium dodecyl sulfate. Solubilization at room temperature in the presence of ions reproduced the effect of thermal denaturation on the major outer membrane polypeptide.     

EPR and M?ssbauer studies of protocatechuate 4,5-dioxygenase. Characterization of a new Fe2+ environment

Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni has been purified to homogeneity and crystallized. The iron containing, extradiol dioxygenase is shown to be composed of two subunit types (alpha, Mr = 17,700 and beta, Mr = 33,800) in a 1:1 ratio; such a composition has not been observed for other extradiol dioxygenases. The 4.2 K M?ssbauer spectrum of native protocatechuate 4,5-dioxygenase prepared from cells grown in 57Fe-enriched media consists of a doublet with quadrupole splitting, delta EQ = 2.22 mm/s, and isomer shift delta Fe = 1.28 mm/s, demonstrating a high spin Fe2+ site. These parameters, and the temperature dependence of delta EQ, are unique among enzymes but are strikingly similar to those reported for the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, suggesting very similar ligand environments. The Fe2+ of protocatechuate 4,5-dioxygenase can be oxidized, for instance by H2O2, to yield high spin Fe3+ with EPR g values around g = 6 (and g = 4.3). In the oxidized state, protocatechuate 4,5-dioxygenase is inactive; the iron, however, can be rereduced by ascorbate to yield active enzyme. Our data suggest that protocatechuate binds to Fe2+; the spectra indicate that the ligand binding is heterogenous. The M?ssbauer spectra observed here are fundamentally different from those reported earlier (Zabinski, R., Münck, E., Champion, P., and Wood, J. M. (1972) Biochemistry 11, 3212-3219). The spectra of the earlier (reconstituted) preparations, which had substantially lower specific activities, probably reflect adventitiously bound Fe3+. We discuss here how adventitiously bound iron can be identified and removed. The Fe2+ which is present in native protocatechuate 4,5-dioxygenase and its complexes with substrates and inhibitors reacts quantitatively with nitric oxide to produce a species with electronic spin S = 3/2. The EPR and M?ssbauer spectra of these complexes compare favorably with EDTA . Fe(II) . NO. We have studied the latter complex extensively and have analyzed the M?ssbauer spectra with an S = 3/2 spin Hamiltonian. EPR spectra show that protocatechuate 4,5-dioxygenase-NO complexes with substrates or inhibitors are heterogeneous and consist of several well defined subspecies. The data show that NO, and presumably also O2, has access to the active site Fe2+ in the enzyme-substrate complex. The use of EPR-detectable NO complexes as a rapid and sensitive tool for the study of the EPR silent active site iron of extradiol dioxygenases is discussed.     

Studies of the ferredoxin from Thermus thermophilus

The soluble ferredoxin from Thermus thermophilus was examined by M?ssbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.     

Linkage Modification with Mixed Random Mating and Selfing: A Numerical Study

Although recombination cannot increase under conditions of random mating or complete selfing in regimes of constant selection, with mixed random mating and selfing, selection for increased recombination can occur. For some fitness regimes there may be selection for reduced recombination with both low and high degrees of selfing but selection for increased recombination with moderate degrees of selfing. With some fitness regimes there is a historical effect: depending on which equilibrium a population starts from, there may be selection for either increased or decreased recombination. In other cases the direction of selection may be determined by the present state of individuals within the population. If recombination is already fairly limited, there may be selection for further reduction. If recombination is already fairly frequent, there may be selection for increased recombination. For certain symmetric viability systems there may be an intermediate value of the recombination fraction between 0 and 0.5 toward which the population will evolve. Although it is not yet possible to classify precisely those fitness matrices that can exhibit selection for increased recombination, it does appear that selection for increased recombination can occur only if at least two of the double homozygotes are less fit than would be expected on the basis of a comparison of the fitnesses of the single and double heterozygotes on an additive scale.     

The periodate oxidation of bovine bone sialoprotein, and some observations on its structure

1. Bovine bone sialoprotein (mol.wt. 23000) contains N-acetylneuraminic acid and N-glycollylneuraminic acid, fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine residues in the form of a very small number, perhaps one, of highly branched oligosaccharide structures linked covalently to peptide. 2. Periodate oxidation of the sialoprotein results in quantitative destruction only of the sialic acid and fucose residue consistent with the earlier findings of their positions as terminal groups. 3. Terminal sialic acid residues are attached to galactopyranose residues by 2,3-linkages, and to some N-acetylgalactosamine residues (at C-6). 4. Sequential Smith degradation indicates that N-acetylgalactosamine residues may be present as points of branching (linked in C-1, C-3 and C-6) and N-acetylglucosamine residues are located in the inner part of the structure, adjacent to the carbohydrate–peptide bond(s). 5. Mannose residues appear to be linked in the 1,3-positions.     

Modification of the zona-free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility

These studies were designed to evaluate the ability of the zona-free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona-free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P < .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona-free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona-free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona-free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona-free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.