The measurement of protein synthesis in biological systems

Attempts to quantitate metabolism in the lung and other tissues using radioactive precursors may be subject to significant errors arising from inappropriate assumptions regarding precursor metabolism, compartmentation and specific radioactivity. This article reviews the type and magnitude of error which may complicate such measurements by presenting specific data from experiments using radioactive amino acids to estimate the rate of protein synthesis. The applicability of these observations to other metabolic systems is discussed briefly in order to develop a more general awareness of the errors which may result from incomplete validation of experimental measurements using radioisotopes.     

Predation by squirrel monkeys and double-toothed kites on tent-making bats

Central American squirrel monkeys (Saimiri oerstedi) appear to recognize the modified leaves that phyllostomid bats utilize for diurnal roost sites. The monkeys visually and manually search these bat tents for both bats and insects. Adult males are the most successful at capturing bats. Nonvolant juvenile bats are more vulnerable to monkey predation than are adults. Bats that escape monkey predation frequently are captured by doubletoothed kites (Harpagus bidentatus) that tend foraging troops of monkeys. Predation by squirrel monkeys, coupled with that of double-toothed kites, may be a significant source of mortality for tent-making bats.     

Activation of the acetyl-coenzyme A:lysoplatelet-activating factor acetyltransferase regulates platelet-activating factor synthesis in human endothelial cells.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a phospholipid with many physiological actions. It is synthesized by endothelial cells and a variety of others in response to stimulation with receptor-mediated agonists. In endothelial cells it remains associated with the surface of the cell and serves as a signal for adhesive interactions with leukocytes. Thus, its synthesis must be precisely regulated. In previous work we have shown that PAF synthesis is regulated at the initiating step, a phospholipase A2. Here we demonstrate that the subsequent step of PAF synthesis, the acetyl-CoA:lyso-PAF acetyltransferase, is rapidly activated when cells are exposed to thrombin or other agonists. We found that the activity increased from basal values (5 nmol/mg/min) to approximately 3-fold higher within 1 min following the addition of agonists. The enzyme activity returned to basal levels within 10 min. The pattern of activation and inactivation suggested covalent modification of the enzyme. This was supported in experiments in which we showed that homogenates had stable enhanced activity and that there was no evidence for an activator or inhibitor. Pretreatment of the cells with vanadate, an inhibitor of protein phosphatases, markedly prolonged the activation state. In subsequent studies we pretreated intact cells with vanadate to block inactivation of the enzyme and then measured the accumulation of PAF in response to thrombin. We found that it was markedly augmented and prolonged. From this we conclude that the synthesis of PAF in intact cells is regulated by the activity of the acetyltransferase. We characterized requirements for activation of acetyltransferase and found that it was not dependent on the influx of intracellular calcium but that calcium entry did influence the length of time for which the enzyme was activated. The acetyltransferase in endothelial cells was shown to be a specific enzyme that did not catalyze the transfer of long chain acyl groups from acyl-CoA to lysophospholipids and demonstrated modest specificity for the acceptor lysophospholipids. These results suggest that activation of the acetyltransferase is a crucial determinant of the amount of PAF synthesized in activated endothelial cells.     

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

We examined the effects of peroxide on the sarco(endo)plasmicreticulum Ca²⁺ (SERCA) pump in pigcoronary artery endothelium and smooth muscle at three organizationallevels: Ca²⁺ transport inpermeabilized cells, cytosolicCa²⁺ concentration in intactcells, and contractile function of artery rings. We monitored theATP-dependent, azide-insensitive, oxalate-stimulated ⁴⁵Ca²⁺uptake by saponin-permeabilized cultured cells. Low concentrations ofperoxide inhibited the uptake less effectively in endothelium than insmooth muscle whether we added the peroxide directly to theCa²⁺ uptake solution or treatedintact cells with peroxide and washed them before the permeabilization.An acylphosphate formation assay confirmed the greater resistance ofthe SERCA pump in endothelial cells than in smooth muscle cells.Pretreating smooth muscle cells with 300 µM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolicCa²⁺ concentration in aCa²⁺-free solution, but it did notaffect the endothelial cells. Peroxide pretreatment inhibited theCPA-induced contraction in deendothelialized arteries with a 50%inhibitory concentration of 97 ± 13 µM, but up to 500 µMperoxide did not affect the endothelium-dependent, CPA-inducedrelaxation. Similarly, 500 µM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced,endothelium-dependent relaxation by only 40 ± 13%. The greaterresistance of the endothelium to reactive oxygen may be importantduring ischemia-reperfusion or in the postinfection immune response.

     

Development of the retinal tapetum lucidum of the walleye (Stizostedion vitreum vitreum)

The development of the retinal tapetum lucidum within the cells of the retinal pigment epithelium (RPE) has been investigated by both light and electron microscopy in the walleye (Stizostedion vitreum vitreum) in specimens ranging in total length from 25-140 mm. In addition changes in the arrangement of the photoreceptors (both rods and cones) in both light and dark-adaptation have also been studied. At 25 mm no evidence of a tapetum is present. At about 30 mm it makes its initial appearance as granular bodies formed within the apical smooth endoplasmic reticulum (SER) cisternae of the RPE cells in the superior temporal fundus. The developing tapetum then spreads peripherally and continues to thicken in existing areas. By 90 mm it is well established throughout the fundus but always appears better developed in the superior fundus. By 125-140 mm it is essentially adult in appearance. At 60-70 mm the rods and cones begin to form bundles producing macroreceptors of 20-30 photoreceptors. In dark-adaptation the rod bundles are retracted and have one or more cone cells centrally located in each bundle, with the bundles separated from one another by melanosomes. Initially when no tapetal material is present, post-larval walleye are positively phototactic and feed on zooplankton. In the adult condition when a tapetum lucidum and large macroreceptors are present, the walleye is negatively phototactic and feeds almost exclusively on larger organisms such as other fish.     

Co-segregation of a gene encoding a deletion ligand for Tcrb-V3+ T cells with Mtv-3

A gene encoding the endogenous superantigen Mls^c, which deletes Tcrb-V3⁺ T cells in the NOD inbred mouse strain, was found to co-segregate with Mtv-3 on chromosome 11. This identifies a fourth gene encoding a deletion ligand for Tcrb-V3⁺ T cells and extends recently published observations in support of the hypothesis that a number of endogenous superantigens are the products of Mtv proviruses. Address correspondence and offprint requests to : K. Tomonari.     

RAC1 induces nuclear alterations through the LINC complex to enhance melanoma invasiveness

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.     

Liver cells secrete the plasma form of platelet-activating factor acetylhydrolase.

Platelet-activating factor (PAF) is a phospholipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) with diverse physiological effects. It has been implicated as a mediator of inflammation, allergy, shock, and thrombosis. Plasma contains an enzyme, PAF acetylhydrolase, that catalyzes the degradation of PAF, and the level of this enzyme may regulate the concentration of PAF in the blood and extracellular spaces under some conditions. Thus, the cellular source(s) of this enzyme and the factors that regulate its synthesis and secretion are issues that may have important physiological and pathological implications. We found that cultures of Hep G2, a human hepatocarcinoma line, secreted PAF acetylhydrolase activity. Optimal secretion occurred in medium that contained serum, and the newly secreted PAF acetylhydrolase was associated with high density and low density lipoproteins (LDL and HDL, respectively), just as the enzyme is in plasma. In the absence of serum. PAF acetylhydrolase was secreted with a particle that had a density similar to HDL. Apolipoproteins B and E were found in the same fractions. We tested the effects of a variety of hormones on the secretion of PAF acetylhydrolase and found that secretion was inhibited by 17 alpha-ethynylestradiol with a maximal effect at 30 microM. This may account for the observation of others that estrogens reduce the activity of PAF acetylhydrolase in the plasma. The PAF acetylhydrolase secreted by Hep G2 cells appeared to be identical to the enzyme in human plasma based on substrate specificity, association with LDL and HDL, response to inhibitors, and reactivity with antibodies against the plasma PAF acetylhydrolase. In conclusion, we have demonstrated that hepatocytes in culture secrete a PAF acetylhydrolase that is apparently identical to the plasma form. The secretion is constitutive but may also be regulated in response to hormonal stimulation.