Detection of plaque-forming cells in the peripheral blood of actively immunized humans.

Ten adult human volunteers were immunized with Salmonella typhi and their peripheral blood leukocytes were collected for 14 days after immunization. These peripheral blood leukocyted, rich in lymphocytes, were plaqued in a modified Jerne assay against sheep erythrocytes coated with either Salmonella or Escherichia lipopolysaccharide. A specific direct and indirect PFC response developed in immunized individuals by day 7 and peaked at day 10. This vigorous PFC response rapidly declined to normal levels by day 14. This marked and specific PFC response of human peripheral blood leukocytes may be developed as a useful tool for monitoring the humoral immune response of patients with Gram-negative bacterial infections.     

Methods for evaluating the morphological and immunohistochemical properties of human tumor colonies grown in soft agar

Clonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson's Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best's carmine, Page-Green method for inclusion bodies, and Snook's reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis.     

Mutagenic activity of Fusarium moniliforme isolates in the Salmonella typhimurium assay.

A total of 33 isolates of Fusarium moniliforme from several food or feed crops were grown on sterile cracked corn, and chloroform-isopropanol extracts were assayed for mutagenic activity in the Salmonella typhimurium-microsome system by using tester strain TA98 or TA100 or both. Extracts of 21 (64%) of the isolates assayed against TA100 were mutagenic. Activities of seven of these extracts were increased markedly with incorporation of the liver homogenate (S-9) into the assay. Seven (33%) of the isolates assayed against TA98 were weakly active, with the liver homogenate having little effect on reversion rates.     

The effects of modification with N-bromosuccinimide on the binding of ligands to dihydrofolate reductase.

The binding constants of substrate, inhibitors and coenzymes to native Lactobacillus casei dihydrofolate reductase and to the enzyme modified (at Trp-21) by N-bromosuccinimide have been determined using fluorimetric and spectrophotometric methods. The modification leads to only modest decreases (factors of 2-4) in the binding of substrate or substrate analogues, but the effects of coenzyme binding are much larger. The binding of NADPH is decreased by a factor of 200, but that of NADP+ by only a factor of 4, indicating a clear difference in their mode of interaction with the enzyme. The nature of this difference is discussed in the light of crystallographic and n.m.r. studies of the enzyme.     

Osmotic regulation: physiological significance of proteolytic and nonproteolytic activation of isofloridoside-phosphate synthase

When cells of Poterioochromonas malhamensis Peterfi are exposed to media of increased osmotic strength, both the internal pool of isofloridoside, and activity in homogenates of isofloridoside-phosphate synthase increase, proportional to the degree of osmotic stress. During the first few minutes of exposure of cells to higher osmolalities, an early relatively small increase in enzyme activity was observed. At the same time a progressive activation of the enzyme in homogenates was noted, providing bovine serum albumin had been omitted from the homogenizing buffer. This in vitro activation was also proportional to the degree of prior osmotic stress, was more pronounced in the presence of fluoride, and was inhibited strongly by adding bovine serum albumin or other proteins. Since earlier work had demonstrated activation of the synthase by adding exogenous proteases, it is likely that this in vitro activation was due to protease activity in the homogenate. The presumed protease must have acquired activity in the cells in response to osmotic stress, and is likely to be responsible for the observed in vivo activation of this biosynthetic enzyme.     

Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy.

Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)--CN in the partially reduced state. Hence as haem a3+(3)--CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)--CN . Cu+a3.     

Hydrocarbon production in Anacystis montana and Botryococcus braunii

The production of labelled aliphatic hydrocarbons in Anacystis montana and Botryococcus braunii has been studied using Na₂CO₃ [¹⁴C] as a carbon source. The major hydrocarbon produced by A. montana is pentadecane (ca 93%) accompanied by a pentadecene (ca 4%) and other hydrocarbons in the range C₁₃-C₁₇. Long chain (C₂₁-C ₃₃) hydrocarbons could not be detected in this organism. The variety of unsaturated hydrocarbons (C₂₅-C₃₁) previously reported in Botryococcus braunii is confirmed and contrasts with the synthesis of unsaturated C₁₇ hydrocarbons only, in axenic cultures prepared from single cell isolates of this colonial alga.