The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.     

Effect of light intensity on macromolecular synthesis in cyanobacteria

The light-dependent incorporation of NaH¹⁴CO₃ into low molecular weight compounds, polysaccharide, or protein was determined in cultures of the cyanobacteriumMerismopedia tenuissima incubated at a series of light intensities. There was an inverse relationship between incorporation into polysaccharide and protein. At light intensities of 90 E/m²/sec or above, relative incorporation of radioisotope into polysaccharide was greatest and relative incorporation into protein was lowest. Optimal relative protein accumulation occurred in samples incubated at 20 E/m²/sec. A broader optimum of light intensity for maximal protein accumulation was found if ammonia rather than nitrate was the nitrogen source. Physiological adaptation of cultures to growth at a particular light intensity did not alter the pattern of macromolecular incorporation when those cultures were tested over the series of light intensities. The response of cultures ofOscillatoria rubescens to light intensity was similar to that ofM. tenuissima, although incorporation into low molecular weight compounds was significantly greater.The effect of light intensity on macromolecular synthesis in a natural population ofOscillatoria rubescens was also determined. A pattern similar to that observed in batch cultures ofO. rubescens was occasionally found, but in other experiments there was no increase in relative protein incorporation when light intensity was decreased.     

Replication through an abasic DNA lesion: structural basis for adenine selectivity

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A‐rule. In this study, we present X‐ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non‐instructive lesions by DNA polymerases at least from this sequence family.