Have the bloody cells gone to our heads?

Recent studies have shown that cells expressing neuronal antigens can be derived from a bone marrow transplant. A new report lends support to and extends these previous results by presenting compelling morphological evidence for the generation and integration of highly differentiated bone marrow-derived neurons.     

The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.     

Identification and design of a novel series of MGAT2 inhibitors

[Acyl CoA]monoacylglycerol acyltransferase 2 (MGAT2) is of interest as a target for therapeutic treatment of diabetes, obesity and other diseases which together constitute the metabolic syndrome. In this Letter we report our discovery and optimisation of a novel series of MGAT2 inhibitors. The development of the SAR of the series and a detailed discussion around some key parameters monitored and addressed during the lead generation phase will be given. The in vivo results from an oral lipid tolerance test (OLTT) using the MGAT2 inhibitor (S)-10, shows a significant reduction (68% inhibition relative to na?ve, p <0.01) in plasma triacylglycerol (TAG) concentration.     

Thoracoabdominal restriction in supine men: CT and lung function measurements

Thoracoabdominal restriction was brought on by means of a corset, and the subsequent effects on thoracic dimensions and lung tissue were studied by computerized tomography (CT) and by various lung function tests in supine healthy volunteers (mean age 30 yr). Restriction caused reductions in total lung capacity (helium equilibration) from mean 6.84 to 4.80 liters, in functional residual capacity (FRC) from 2.65 to 2.08 liters, and in vital capacity from 5.16 to 3.45 liters. Closing capacity (single-breath N2 washout) fell from 2.42 to 1.88 liters, thus matching the reduction in FRC. The static pressure-lung volume curve was shifted to the right by 1.5 cmH2O at 50% of total lung capacity. However, no change in the slope of the curve was observed. The diaphragm was moved cranially by 1.2 cm, and the thoracic cross-sectional area was reduced by a mean 32 cm2 at a level just above the diaphragm. No changes in the lung tissue were seen on CT scanning. Gas exchange, as assessed by multiple inert gas elimination technique and arterial blood gas analysis, was unaffected by restriction. It is concluded that in supine subjects, thoracoabdominal restriction that reduces FRC by 0.6 liter is not accompanied by atelectasis (normal CT scan). In this respect the result differs from that found in anesthetized supine subjects who show the same fall in FRC and atelectasis in dependent lung regions.     

Toxic lead exposure in the urban rock dove

Thirteen adult urban rock doves (Columba livia), 12 captured alive and one found dead, were studied from the Baltimore zoo. The mean concentration of lead in the blood for the 12 live birds was 184.5 +/- 531.2 (range 10.5-1,870 micrograms/dl). Three of the 13 birds with high measured blood and tissue lead concentrations were found at necropsy with lead shot pellets in their gizzards. Correlations were not found between concentrations of lead in the blood and body weight or hematocrit. Conversely, high correlations were noted between concentrations of lead in the blood and measured liver and kidney concentrations (r = 0.946, P less than 0.01; r = 0.993, P less than 0.01, respectively). Numbers of intranuclear acid-fast inclusions per 10 consecutive fields (100x oil immersion lens) correlated well with measured kidney lead concentrations (r = 0.990, P less than 0.001).     

Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells

Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1. In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E. coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAI-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin).     

Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli

Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.     

Anion effects on the reaction of lecithin-cholesterol acyltransferase with discoidal complexes of phosphatidylcholines . apolipoprotein A-I . cholesterol

Discoidal complexes of phosphatidylcholine (PC) . apolipoprotein A-I . cholesterol were prepared with egg PC, palmitoyloleoylPC, dipalmitoylPC, or dimyristoylPC, and were used as substrates of purified lecithin-cholesterol acyltransferase to investigate the effects of neutral salts on the enzymatic reaction. Sodium fluoride, chloride and bromide concentrations up to 1 M, did not affect the properties of the substrate particles, but caused marked and distinct changes in the activity of the enzyme with the various PC particles. The effects of salts were largely due to the anions, which followed the order of the lyotropic series in their inactivating capacity: F- less than Cl- less than Br- less than NO3- less than I- less than SCN-. Sodium salts (F-, Cl-, and Br-) produced a very large increase in the pH optimum of the enzymatic reaction (7.4 to at least 8.5) essentially obliterating the ionization of a functional group with pK of 8.1. The kinetics of the enzymatic reaction revealed major differences among the PC particles, and different responses of their kinetic parameters with increasing salt concentrations. The conclusions reached in this work are the following: (1) The relative reactivity of PC substrates, in discoidal particles, with lecithin-cholesterol acyltransferase depends strongly on the concentration and type of salts in the medium. (2) Anions (in lyotropic series) rather than cations affect the enzymatic reaction. (3) There are functional groups with pK of 8.1 which are affected markedly in their ionization behavior by anion binding. (4) The active site of lecithin-cholesterol acyltransferase and its interaction with anions are affected by the exact nature of the PC-apolipoprotein interface.