Regulation of natural killer cell activity by macrophages in the rheumatoid joint and peripheral blood

Recently, in another study, we observed that indomethacin, a prostaglandin synthetase inhibitor, significantly increased NK activity in both normal and rheumatoid arthritis (RA) peripheral blood (PB) but not in RA synovial fluid (SF). Because macrophages are a major source of prostaglandins, we examined the effect of macrophage-enriched adherent cells (AC) on NK activity as measured by a 3-hr Cr-release assay with K 562 cells. The removal of AC resulted in increased (p less than 0.01) NK activity in both normal and RA PB. In contrast, the removal of AC from RA SF resulted in a significant decrease (p less than 0.001) of NK activity. By using only nonadherent cells (NAC), NK activity in RA SF and synovial tissue (ST) was significantly reduced when compared to autologous RA PB (p less than 0.001). Enhancement of NK activity of SF NAC by both poly I:C and IL 2 was not dependent on AC. Mixing experiments demonstrated that the addition of synovial AC for 16 hr increased NK activity of synovial NAC to a level similar to that of unseparated mononuclear cells, whereas autologous PB AC suppressed NK activity of PB NAC. PB AC, when added to SF NAC, also increased NK activity. Supernatants from synovial mononuclear cells were stimulatory of synovial NAC NK activity, whereas normal PB mononuclear supernatants were suppressive. These observations document 1) a significant reduction of NAC-mediated NK activity in the rheumatoid joint as compared to PB from the same patient, and 2) that AC modulate NK activity differently in the rheumatoid joint as compared to RA or normal PB.     

Comparison of the data, analysis, and results of X-ray absorption studies of cytochrome c oxidase

Differences in the methods of analysis of X-ray absorption data used by Powers et al. [Powers, L., Blumberg, W. E., Chance, B., Barlow, C., Leigh, J., Jr., Smith, J., Yonetani, T., Vik, S., & Peisach, J. (1979) Biochim. Biophys. Acta 547, 520-538; Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] and Scott et al. [Scott, R., Schwartz, J., & Cramer S. (1986) Biochemistry 25, 5546-5555] are clarified. In addition, we compare the X-ray absorption data and results for resting cytochrome c oxidase reported by both groups using the same analysis method and conclude apart from any assumptions that the data are not identical.     

A rapid non-radioactive procedure for plaque hybridization using biotinylated probes prepared by random primed labeling

A simple, non-radioactive method has been developed for the rapid screening of phage libraries. In the present study, nanogram amounts of a small restriction fragment (135 bp) were biotinylated via random primed labeling and used to probe cDNA libraries using a modified plaque hybridization protocol. The high backgrounds that are seen typically with avidin/biotin-based methods for plaque hybridization were eliminated by incubation of filters with one of several different proteases prior to hybridization. A comparison of several detection systems indicated that streptavidin conjugated to calf intestinal alkaline phosphatase (AP) was the most sensitive, yielding signals comparable to those obtained with 32P-labeled probes. The times required for phage growth and pre-hybridization were reduced substantially, permitting a convenient one-day screening protocol. Nitrocellulose filters gave the best signal to noise ratio, although "streaking" of plaque DNA was observed occasionally; this problem can be overcome by using nylon-based membranes, which allows exact visualization of the positive plaques. The method was highly reliable; 29 out of 33 putative clones retested positive and the authenticity of these was confirmed by DNA sequence analysis. The random primed biotinylation procedure has been utilized successfully with several different cDNA fragments and has proven useful for other hybridization-based methods (Northern and Southern blots), without the problems associated with the use of radiolabeled probes.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Rapid purification of calmodulin and S-100 protein by affinity chromatography with melittin immobilized to sepharose

Melittin-Sepharose was prepared for Ca2+-dependent affinity chromatography of calmodulin and S-100 protein. This matrix exhibits extremely high capacity (approximately 10 mg calmodulin/ml gel), low nonspecific binding, and excellent recovery (greater than 90%) under optimal conditions. Recovery of calmodulin from melittin-Sepharose was related to the degree of saturation of column capacity with lower yields when only partial saturation was achieved. Large-scale, simultaneous purification of calmodulin and S-100 protein from brain was carried out using selective adsorption to organomercurial agarose followed by melittin-Sepharose chromatography; yields were 250-300 mg of calmodulin and 200-300 mg of S-100 per kg tissue. Calmodulin also was purified in a single step from bovine testis supernatant using melittin-Sepharose in yields comparable to those from brain.     

Polymorphism in a casein fraction of sows' milk

Polyacrylamide vertical gel electrophoresis was used to separate the caseins of sows' milk. Polymorphism was found in a region of the gels designated Cn₃. The Cn₃ polymorphism consists of two bands which appear to be controlled by two codominant alleles designated Cn ₃ ^A and Cn ₃ ^B . Homozygotes possess one band and heterozygotes posses both bands of equal intensity giving the following phenotypes: Cn₃A, Cn₃AB, and Cn₃B. A chi-square test for goodness of fit of the observed phenotypes to that expected by the Hardy-Weinberg equilibrium formula and a segregation analysis of eight matings involving 19 female progeny conform to the hypothesis that the Cn₃ polymorphism is controlled by two codominant autosomal alleles. Further family studies will be necessary to confirm the genetic control of the Cn₃ polymorphism.Deceased.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A.nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.     

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A. nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.