Glucocorticoid modulation of lymphokine-induced macrophage proliferation

The ability of glucocorticoids to modify lymphokine-induced macrophage proliferation, an in vitro correlate of cellular immunity in the guinea pig, was investigated. Lymphocyte production of macrophage mitogenic factor (MMF) was decreased in the presence of physiological concentrations of glucocorticoids. Inhibition was concentration dependent (IC₅₀ of triamcinolone acetonide (TA): 2 × 10^?9M), glucocorticoid specific, and reversed by cortexolone. In contrast, pharmacological concentrations of glucocorticoids were necessary to inhibit macrophage proliferation induced by suboptimal dilutions of MMF. This inhibition was concentration dependent (IC₅₀ of TA: 4 × 10^?7M), glucocorticoid specific, and reversed by cortexolone. At supraoptimal dilutions of MMF, glucocorticoids caused a twofold potentiation of MMF-induced macrophage proliferation. Potentiation was concentration dependent (EC₅₀ of TA: 3 × 10^?8M), glucocorticoid specific, reversed by glucocorticoid antagonists, and occurred in the presence of indomethacin. Thus, glucocorticoids regulate both the initiation and effector phases of this in vitro model of delayed hypersensitivity. However, the results indicate that the major mechanism of glucocorticoid-mediated anti-inflammatory action occurs at the level of the MMF-producing lymphocyte rather than at the effector macrophage, as MMF-induced proliferation is likely controlled by opposing glucocorticoid-sensitive mechanisms.     

An activating amino acid substitution in the c-abl oncogene protein fails to produce a local conformational change

Thebcr-abl chimeric gene of Philadelphia chromosome positive chronic myelogenous leukemias is only weakly transforming. This transformation activity is greatly enhanced by a Lys-for-Glu substitution at position 832 in the c-abl gene, as occurs in the highly transforming v-abl genes. It has been suggested that this mutation results in a significant structural change in the encoded protein product. Using conformational energy analysis, we have determined the allowed low-energy conformations for residues 828–836 of this protein with Lys and Glu at position 832. In both cases, the overwhelmingly preferred conformation for this region is a bend-helix motif. The helix terminates at residue 836, and there are no discernible differences in conformation between the Lys- and Glu-containing sequences. These results suggest that the activating amino acid substitution at position 832 in the c-abl protein product does not produce its effect via a local conformational change.     

Use of Ultrafiltration To Isolate Viruses from Seawater Which Are Pathogens of Marine Phytoplankton

Viruses may be major structuring elements of phytoplankton communities and hence important regulators of nutrient and energy fluxes in aquatic environments. In order to ascertain whether viruses are potentially important in dictating phytoplankton community structure, it is essential to determine the extent to which representative phytoplankton taxa are susceptible to viral infection. We used a spiral ultrafiltration cartridge (30,000-molecular-weight cutoff) to concentrate viruses from seawater at efficiencies approaching 100%. Natural virus communities were concentrated from stations in the Gulf of Mexico, a barrier island pass, and a hypersaline lagoon (Laguna Madre) and added to cultures of potential phytoplankton hosts. By following changes in in vivo fluorescence over time, it was possible to isolate several viruses that were pathogens to a variety of marine phytoplankton, including a prasinophyte (Micromonas pusilla), a pennate diatom (likely a Navicula sp.), a centric diatom (of unknown taxa), and a chroococcoid cyanobacterium (a Synechococcus sp.). As well, we observed changes in fluorescence in cultures of a cryptophyte (a Rhodomonas sp.) and a chlorophyte (Nannochloropsis oculata) which were consistent with the presence of viral pathogens. Although pathogens were isolated from all stations, all the pathogens were not isolated from every station. Filterability studies on the viruses infecting M. pusilla and the Navicula sp. showed that the viruses were consistently infective after filtration through polycarbonate and glass-fiber filters but were affected by most other filter types. Establishment of phytoplankton-pathogen systems will be important in elucidating the effect that viruses have on primary producers in aquatic systems.     

Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen

A rapid, inexpensive and sensitive high-performance liquid chromatographic method for the quantitation of ibuprofen enantiomers from a variety of biological fluids is reported. This method uses a commercially available internal standard and has significantly less interference from endogenous co-extracted solutes than do previously reported methods. The method involves the acid extraction of drug and internal standard [(±)-fenoprofen] from the biological fluid with isooctane—isopropanol (95:5) followed by evaporation and derivatization with enthylchloroformate and R-(+)-α-phenylethylamine. Excellent linearity was observed between the peak-area ratio and enantiomer concentration (r > 0.99) over a concentration range of 0.25–50 μg/ml. This method is suitable for the quantitation of ibuprofen from single-dose pharmacokinetic studies involving either rats or humans.     

Reversed-phase high-performance liquid chromatography analysis of 6-thioguanine applicable to pharmacologic studies in humans

6-Thioguanine (6TG) and its metabolites were analyzed in human plasma with a reversed-phase high-performance liquid chromatographic method. 6TG and related compounds were extracted from plasma with an equal volume of 2 N perchloric acid at a 50–100% recovery efficiency. The neutralized extracts were chromatographed on a μBondapak C₁₈ column by two separate isocratic conditions. 6TG, 6-thiouric acid, 6-thioxanthine, 6-thioguanosine, and 6-methylthiouric acid were analyzed with 0.01 M sodium acetate, pH 3.5–10% methanol as the mobile phase and 340 nm for detection. 6-Methylthioguanine and three unknown metabolites were separated with acetate—25% methanol and 310 nm detection. One of the unknowns was identified as 6-methylthioguanosine. External standard calibration was used for quantitation. The 6TG detection limit was 0.8 nmol/ml in plasma.     

Extensive homology between poly(A)-containing mRNA and purified nominal poly(A)-lacking mRNA in mouse kidney

To investigate poly(A)-lacking mRNA in mouse kidney, we studied a fraction of renal mRNA that does not bind to oligo(dT)-cellulose but can be purified by benzoylated cellulose chromatography. Nominal poly(A)-lacking mRNA and poly(A)-containing mRNA have complete nucleotide sequence homology, suggesting that kidney does not contain mRNAs that are not represented in the polyadenylated RNA fraction. Translation products directed by nominal poly(A)-lacking mRNA and poly(A)-containing mRNA are qualitatively and quantitatively similar in one-dimensional polyacrylamide gels. [3H]cDNA transcribed from poly(A)-containing mRNA hybridizes with its template and with nominal poly(A)-lacking mRNA to the same extent (95%) and with the same kinetics; reaction of [3H]cDNA to nominal poly(A)-lacking mRNA with the two mRNA populations gives the same result. The extensive homology these two mRNA populations share is important to the interpretation of mRNA lifetime and to the analysis of authentic poly(A)-lacking mRNAs.     

Association of poly(adenylate)-deficient messenger ribonucleic acid with membranes in mouse kidney

To describe further the metabolism of messenger ribonucleic acid (mRNA) in mouse kidney, we examined newly synthesized mRNA deficient in poly(adenylate) [poly(A)]. Approximately 50% of renal polysomal mRNA that labeled selectively in the presence of the pyrimidine analogue 5-fluoroorotic acid lacks or is deficient in poly(A) as defined by its ability to bind to poly(A) affinity columns. Nearly one-half of this poly(A)-deficient mRNA is associated uniquely with a cellular membrane fraction detected by sedimentation of renal cytoplasm in sucrose density gradients containing EDTA and nonionic detergents. Poly(A+) mRNA and poly(A)-deficient mRNA [poly(A-) mRNA] have similar modal sedimentation coefficients (20-22 S) and similar cytoplasmic distribution. Although 95% of newly synthesized poly(A+) mRNA is released in 10 mM EDTA as 20-90 S ribonucleoproteins from polysomes greater than 80 S, only 55% of poly(A)-deficient mRNA is released under the same conditions. Poly(A)-deficient mRNA recovered from greater than 80 S ribonucleoproteins resistant to EDTA treatment lacks ribosomal RNA, is similar in size to poly(A+) mRNA, and is associated with membranous structures, since 70% of poly(A)-deficient mRNA in EDTA-resistant ribonucleoproteins is released into the 20-80 S region by solubilizing membranes with 1% Triton X-100. These membrane-associated renal poly(A-) mRNAs could have unique coding or regulatory functions.     

An improved spectrophotometric assay for human plasma carboxypeptidase N

A continuous spectrophotometric assay for human plasma carboxypeptidase N utilizing furylacryloyl-alanyl-lysine is described. Synthesis was made by use of 9-(2-sulfo)fluorenylmethyloxycarbonyl (Sulfmoc) chloride as the N-?-amino-blocking group for lysine. The substrate has the advantage of containing a chromophore which allows difference measurements above 324 nm. The kinetic parameters K_m and $\text{K}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ have been determined for furylacryloyl-alanyl-lysine and -arginine. Difference measurements were related to micromoles of lysine or arginine released and were expressed as units.