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.     

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.     

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.     

Calcineurin is associated with the cytoskeleton of cultured neurons and has a role in the acquisition of polarity.

Calcineurin is a calmodulin-dependent serine-threonine phosphatase found in many cell types but most abundant in neurons. To determine its localization in developing neurons, dissociated cultures from embryonic day 15 rat cerebellum were analyzed immunocytochemically after treatment with cytoskeletal-disrupting drugs. During the initial outgrowth of neurites, calcineurin is enriched in growth cones where its localization depends upon the integrity of both microtubules and actin filaments. Treatment with cytochalasin shifts calcineurin from the growth cone to the neurite shaft, and with nocadozole calcineurin translocates to the cell body. Therefore calcineurin is well positioned to mediate interactions between cytoskeletal systems during neurite elongation. By 14 d in culture, when the neurons have developed extensive neuronal contacts and synapses are present, calcineurin is predominantly in the neurite shaft. Incubation of cultured cells with Cyclosporin A or a specific peptide, both of which selectively inhibit calcineurin's phosphatase activity, prevented axonal elongation. Because the microtubule-associated protein tau appears to play a key role in asymmetric neurite elongation, we examined modifications in its phosphorylation state resulting from calcineurin inhibition. In contrast to the normal development of cerebellar macroneurons in which reactivity with the phosphorylation-dependent antibody, tau-1, progressively increases, there was a persistent inhibition of tau-1 reactivity in cells exposed to Cyclosporin A. These findings suggest a role for calcineurin in regulating tau phosphorylation and possibly modulating other steps required for the determination of polarity.     

PATTERNS IN LEAF MORPHOLOGY AND PHOTOSYNTHESIS IN SHOOTS OF SASSAFRAS ALBIDUM (LAURACEAE)

Trees of Sassafras albidum (Nuttall) Nees. display leaves that fall into discrete categories of form. Unlobed leaves, with undissected margins, predominate at proximal and distal nodes of shoots, while lobed leaves are most common at intermediate nodes. In this study we investigated the hypothesis that shoots of sassafras respond to environmental changes over the growing season by generating predictable nodal patterns for leaf morphology and physiology. We recorded leaf shape category and measured leaf surface area, node number, chlorophyll content, nitrogen content, and photosynthesis in four trees. Over 1,000 measurements of photosynthesis were made in situ, using the LI-Cor LI 6200 portable photosynthesis system. The two trees growing under heavy shade had few lobed leaves (2.0% and 18.8%) and often had negative carbon balance, with positive net photosynthesis occurring during sun flecks. The two trees growing in more open conditions had many more lobed leaves (56.3% and 61.0%) and higher, less variable net photosynthetic rates. As indicated by chlorophyll and nitrogen contents, the highest leaf photosynthetic rates occurred at intermediate nodes (nodes six to nine), and this was shifted distally along the shoot during the growing season. Leaves at intermediate nodes also had the largest surface areas and the greatest frequency of lobing. In comparative experiments with models, lobing was shown to enhance free-convectional heat loss relative to unlobed leaves of the same surface area. Due to their large surface area, these leaves also have the highest rates of whole leaf photosynthesis. Under conditions of equivalent photosynthetically active radiation, lobed leaves had higher rates of net photosynthesis than did unlobed leaves. We conclude that shoots of sassafras produce groups of leaves with predictably different morphological and physiological specializations.