S.M. Pemrick

The retinoid receptors belong to a large superfamily of ligand-inducible transcription factors that include the steroid, vitamin D and thyroid hormone receptors, the peroxisome proliferator-activated receptor, the insect edysteroid receptor, and a number of orphan receptors whose ligands are unknown. All nuclear receptors have several well-characterized structural domains, including a conserved DNA-binding domain, and a ligand binding domain at the carboxyl terminus of the receptor. The RAR and RXR classes of nuclear retinoic acid receptors are each composed of alpha, beta and gamma subtypes with more than one isoform for each receptor subtype. Data from many investigators suggest there are RAR- and RXR-dependent gene pathways, and that the individual receptor subtypes may control distinct gene expression patterns. In addition, RXR has been found to heterodimerize with other nuclear receptors to form active transcriptional complexes, which influence the activity of a variety of gene pathways important in growth and differentiation. As a result, retinoids have been useful clinical agents in Dermatology and Oncology. However, upon prolonged exposure to retinoic acid, resistance to retinoids has often been encountered both in the clinical setting and in long-term cell culture (HL60R and RAC65 cells). In the latter case, retinoid resistance has been associated with a mutation in the RAR gene which transcribes a RAR receptor truncated at the C-terminal end. These mutated RAR receptors exhibit a reduced affinity for retinoic acid while retaining the ability to bind to a retinoic acid response element on DNA. As a result, these mutant receptors exhibit dominant-negative activity by binding to the DNA without activating transcription and by competing with other receptors for sites on the response element. In fact, dominant-negative activity may be very important in the development of many neoplastic diseases, including acute promyelocytic leukemia (APL), where a t(15;17) chromosomal translocation fuses the PML gene to the RAR gene, to produce a PML-RAR fusion protein in large excess in the cell. However, retinoid resistance in the patient is most probably the result of pharmacokinetic problems, whereby, with continuous retinoid treatment, the plasma levels of retinoic acid gradually decrease to below that required to maintain differentiation of leukemic cells in vivo. A major challenge for drug discovery is to design a drug which circumvents these pharmacokinetic problems either by designing novel drug delivery systems or by employing retinoids which do not bind to CRABP, such as 9-c-RA.(ABSTRACT TRUNCATED AT 400 WORDS)

Incubation of rabbit skeletal myosin with an extract of light chain kinase plus ATP phosphorylated the L2 light chain and modified the steady state kinetics of the actomyosin ATPase. With regulated actin, the ATPase activity of phosphorylated myosin (P-myosin) was 35 to 181% greater than that of unphosphorylated myosin when assayed with 0.05 to 5 micro M Ca2+. Phosphorylation had no effect on the Ca2+ concentration required for half-maximal activity, but it did increase the ATPase activity at low Ca2+. With pure actin, the percentage of increase in the actomyosin ATPase activity correlated with the percentage of phosphorylation of myosin. Steady state kinetic analyses of the actomyosin system indicated that 50 to 82% phosphorylation of myosin decreased significantly the Kapp of actin for myosin with no significant effect on the Vmax. Phosphorylaton of heavy meromyosin similarly modified the steady state kinetics of the acto-heavy meromyosin system. Both the K+/EDTA- and Mg-ATPase activities of P-myosin and phosphorylated heavy meromyosin were within normal limits indicating that phosphorylaiion had not altered significantly the hydrolytic site. Phosphatase treatment of P-myosin decreased both the level of phosphorylation of L2 and the actomyosin ATPase activity to control levels for unphosphorylated myosin. It is concluded levels for unphosphorylated myosin. It is concluded from these results that the ability of P-myosin to modify the steady state kinetics of the actomyosin ATPase was: 1) specific for phosphorylation; 2) independent of the thin filament regulatory proteins.

SUMMARY Daily patterns of incorporation of carbon dioxide and inorganic phosphate were measured in phytoplankton from Lake George, New York, in January and February 1972. Rates of photosynthesis oscillated in phase for the entire assemblage and for individual cells of the diatoms Asterionella, Tabellaria , and Fragilaria. The photosynthetic capacity was maximal in the early afternoon. Daily patterns of phosphate uptake were also rhythmic. At ambient concentrations the assemblage takes up phosphate maximally in the morning while individual cells of the large diatom take it up maximally in the evening. A kinetic analysis of phosphate uptake indicated 2 velocities of uptake for cells of Tabellaria and Fragilaria: a hyperbolic function at small and an apparently linear relationship at relatively large concentrations. The large diatoms, in contrast to the total assemblage, functioned maximally at 2 separate times of the day: in the evening at ambient levels and in the morning at 0.4 μM and larger concentrations. Temporal stratification of the nutrient niche may be achieved by several uptake mechanisms in the algal cell that function at different times of the day or with a variable uptake velocity.

Ca2+ -activated neutral protease (CAF) was capable of degrading myosin over a 200-fold range of protease concentrations. CAF selected the heavy chain of myosin, although either prolonged exposure to or high concentrations of the protease degraded the L1, but not the L2 or L3, light chains of myosin. The following results indicated that during the first hour of digestion, under conditions where native myosin was the substrate, CAF selected for the "head" region of the myosin heavy chain: (a) large heavy chain fragments of identical molecular weight were produced from filamentous and from soluble myosin; (b) light meromyosin was not a substrate; (c) agents known to bind to the head of myosin (actin, MgATP, and L2) had both a qualitative and quantitative effect on degradation; and (d) similar cleavage sites could be demonstrated for myosin and for heavy meromyosin (HMM) despite the fact that HMM was a much poorer substrate than myosin. This observation is interpreted as an indication that the conformation of myosin heavy chain is altered in the preparation of HMM. The principal cleavage sites on the heavy chain of myosin were 20,000, 35,000 and 50,000 D from the N-terminus, producing large fragments with molecular weights of 180,000, 165,000, and 150,000 which comprised a "nicked" species of myosin. This nicked species retained both normal solubility properties and normal hydrolytic activities. For this reason, it is concluded that "nicked myosin" is an important pathophysiological species.

Abstract The rate of hydrogen ion liberation during the hydrolysis of ATP by Subfragment I was followed in a stopped flow spectrophotometer. At pH 8, 20°, and in the presence of MgCl2 the initial rate of hydrogen ion liberation was greater than the steady state rate and the approach to a steady state rate could be described by a single exponential term. The rate constant characterizing the exponential term increased with MgATP concentration from 0.01 to 0.1 mm above which an average limiting value of 128 s-1 was reached. It was confirmed that two exponential terms are required to describe the course of the reaction for myosin. These results can be interpreted as indicating interaction between the two ATPase sites on intact myosin.