Production of antibody to lipopolysaccharide (LPS) after immunization with a LPS-polymyxin B-agarose immunogen

A method was devised to produce antibodies to lipopolysaccharide (LPS) in guineapigs following a single immunization, The antigen was prepared by mixing polymyxin B-agarose with LPS from Escherichia coli O55:B5. Use of the agarose support allowed purification of the complex by simple washing procedures. Twenty-nine days after a single injection of the immunogen mixed with Freund complete adjuvant all animals demonstrated antibody to the LPS portion of the complex. No antibodies were detected to the polymyxin B component. Typical titres of LPS as measured by ELISA were 2¹¹. After, a booster immunization, titres of LPS antibody were further inceased and a greater avidity was noted. In contrast to other methods which have been employed for production of antibody to LPS, use of the polymyxin B-agarose complex has the following advantages: ease of antigen preparation, ready purification of the complex, potent immunostimulation, and under the conditions employed here, LPS-specific antibody production, without accompanying antibody to polymyxin B.     

Immunocytochemical localization of callose in root cortical cells parasitized by the ring nematodeCriconemella xenoplax

Summary Under hypoxia (10 and 5% partial oxygen tension) meristematic cells ofAllium cepa L. roots acquired new cycle kinetics, characterized by reduced but constant rates of root growth. Under these conditions, there was preferential lengthening of G₁ and of the last third of the S period, S₃. Since hyperoxygenation shortened S₃ but not G₁ in these cells, the high sensitivity of late replication to environmental oxygen is demonstrated. The preferential depression of the replication rate when those cells replicated the last third of their DNA was not associated with diminished cell size. Rather, the lower the oxygen level the larger the mean size of the cycling cells. Under anoxia (0% oxygen tension) the rate of growth slowed, accompanied by preferential accumulation of cells in G₁. However, steady state kinetics of root growth was not achieved under these extreme conditions.Abbreviations Mean cell length - LI labelling index or frequency of cells with labelled nuclei after [³H]thymidine - G₁, S, G₂ pre-replicative, replicative, and post-replicative periods of the interphase of cycling cells - M mitosis     

Ultrastructure of appressorium development by basidiospore germlings of the rust fungusGymnosporangium juniperi-virginianae

Summary Basidiospore germlings ofG. juniperi-virginianae readily formed appressoria (infection structures) on dialysis membranes. These specimens could be effectively freeze-substituted and processed for study with transmission electron microscopy. Appressorium formation on these membranes appeared to be very similar to that occurring on host leaves up to the point of penetration peg formation. A germ tube emerged laterally from each spore, grew until it contacted the membrane, and then differentiated into a swollen appressorium whose end was flattened against the membrane. The fungal wall in contact with the membrane became very thin. A region devoid of most organelles developed in the appressorium tip. Numerous filasomes and microvesicles accumulated in this region. Eventually, a structure known as the appressorial cone formed at the end of the appressorium. This structure was deposited outside the plasma membrane in direct contact with the dialysis membrane. Basidiospores and appressoria appeared to be effectively stuck to the dialysis membrane by a fibrillar, extracellular matrix. This substance appeared as a diffuse network on young germ tubes, but subsequently assumed the appearance of an electron-dense layer or coating on appressoria and basidiospores.     

Oxidation state dependence of proton exchange near the iron-sulfur centers in ferredoxins and high-potential iron-sulfur proteins

For both the [2Fe-2S] and the [4Fe-4S] ferredoxins, dialysis against 2H2O prior to single electron reduction leads to the appearance of a deuterium modulation pattern in the electron spin echo decay envelope indicative of deuteron-proton exchange very near the paramagnetic center. In contrast, if the ferredoxin is exposed to 2H2O after its reduction in H2O, far less deuterium exchange near the metal center takes place. Thus, proton exchange with solvent is in part dependent on the redox state of the protein. For high potential iron-sulfur proteins, this type of proton-deuteron exchange near the metal center does not occur unless the protein is partially unfolded in dimethylsulfoxide in 2H2O.     

Electron spin echo studies of cytochrome c oxidase

We have studied the linear electric field effect in pulsed EPR of the "EPR-detectable copper" signal of beef heart cytochrome c oxidase and have compared our results with those for a variety of square planar and tetrahedral Cu(II) model compounds and with Cu(II) proteins containing either type 1 or type 2 copper. The electric field induced g shifts (linear electric field effect) for cytochrome oxidase are comparable in magnitude to those for simple Cu(II) complexes and for some copper proteins containing type 2 sites. The shifts are smaller than those for tetrahedral copper complexes and for type 1 copper sites. However, the magnetic field dependence of the linear electric field effect does not resemble that observed for any Cu(II) complex studied nor for type 1 copper. These findings cannot be reconciled with the tetrahedral Cu(II) model proposed by Greenaway, Chan, and Vincow ((1977) Biochim. Biophys. Acta 490, 62-78, 1977) to explain the unusual EPR spectrum of cytochrome oxidase.     

Demonstration of nitrogen coordination in metal--bleomycin complexes by electron spin--echo envelope spectroscopy

We have studied the Cu(II), Co(II), and Fe(III) complexes of the antineoplastic drug bleomycin by using electron spin--echo envelope spectroscopy. For all three complexes, nitrogen coordination of the metal ions is demonstrated. For the Cu(II)-- and Co(II)--drug complexes, we have been able to identify imidazole as a metal ligand.     

Nucleotide substitution in the amino acid acceptor stem of lysine transfer RNA causes missense suppression

Previous results from this laboratory indicated that, in Escherichia coli K12, a new class of missense suppressors, which read the lysine codons AAA and AAG, may be misacylated lysine transfer RNAs. We therefore isolated and determined the nucleotide sequence of the lysine tRNA from two of the suppressor strains. In each case, we found both wild-type and mutant species of lysine tRNA, a result consistent with evidence that there are two genes for lysine tRNA in the E coli genome. The wild-type sequence was essentially identical to that reported for lysine tRNA from E. coli B. The mutant species isolated from each suppressor strain had a U for C70 nucleotide substitution, demonstrating that the AAG suppressor is a mutant lysine tRNA. The nucleotide substitution in the amino acid acceptor stem is consistent with the in vivo evidence that the suppressor corrects AAA and AAG missense mutations by inserting an amino acid other than lysine during polypeptide synthesis. This report represents the first verification of missense suppression caused by misacylation of a mutant tRNA.