Spontaneous Aggregation and Cytotoxicity of the β-Amyloid Aβ1–40: A Kinetic Model

The time dependency of the spontaneous aggregation of the fibrillogenic β-Amyloid peptide, Aβ^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Aβ antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 μM amyloid peptide. The anti–Aβ antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length Aβ was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Aβ^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Aβ^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Aβ^1–40 in vitro and possibly in vivo.     

Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell

One of the major properties of the semi-synthetic minimal cell, as a model for early living cells, is the ability to self-reproduce itself, and the reproduction of the boundary layer or vesicle compartment is part of this process. A minimal bio-molecular mechanism based on the activity of one single enzyme, the FAS-B (Fatty Acid Synthase) Type I enzyme from Brevibacterium ammoniagenes, is encapsulated in 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes to control lipid synthesis. Consequently molecules of palmitic acid released from the FAS catalysis, within the internal lumen, move toward the membrane compartment and become incorporated into the phospholipid bilayer. As a result the vesicle membranes change in lipid composition and liposome growth can be monitored. Here we report the first experiments showing vesicles growth by catalysis of one enzyme only that produces cell boundary from within. This is the prototype of the simplest autopoietic minimal cell.     

Identifying essential genes in bacterial metabolic networks with machine learning methods

Background  

Identifying essential genes in bacteria supports to identify potential drug targets and an understanding of minimal requirements for a synthetic cell. However, experimentally assaying the essentiality of their coding genes is resource intensive and not feasible for all bacterial organisms, in particular if they are infective.     

Metabolism of carbon tetrachloride to phosgene

Aerobic incubation of hepatic microsomal fractions in the presence of carbon tetrachloride, NADPH and cysteine resulted in the formation of phosgene which was identified by gas chromatography/mass spectrometry as the adduct, 2-oxothiazolidine-4-carboxylic acid, formed by its reaction with cysteine. [¹³C]-Carbon tetrachloride was metabolized to 2-[¹³C]-oxothiazolidine-4-carboxylic acid the , when carbon tetrachloride was incubated in the presence of [¹⁸O]-O₂, 2- [¹⁸O]-oxothiazolidine-4-carboxylic acid was formed. The reaction was inhibited by carbon monoxide showing the involvement of the cytochrome P-450-dependent mixed function oxidase system. The metabolism of carbon tetrachloride to phosgene may play a role in the production of hepatotoxicity by this compound.     

Trees on networks: resolving statistical patterns of phylogenetic similarities among interacting proteins

Background  

Phylogenies capture the evolutionary ancestry linking extant species. Correlations and similarities among a set of species are mediated by and need to be understood in terms of the phylogenic tree. In a similar way it has been argued that biological networks also induce correlations among sets of interacting genes or their protein products.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

Effect of tranquilizers on the total acetylcholine content and acetylcholinesterase activity in the brain tissue of Arvicanthis niloticus

The effect of reserpine and meprobamate on the total acetylcholine content and acetylcholinesterase activity in the brain tissue of the kusu rat, Arvicanthis niloticus, was studied. The total acetylcholine content and acetylcholinesterase activity were determined 1 hr after i.p. injection of different doses of reserpine (0.25, 0.5 and 1 mg/ml/100 g body wt) and meprobamate (6.25, 12.5 and 25 mg/ml/100 g body wt). The effect of different time intervals (1, 10, 30 min, 1, 2.5, 5, 8, 12, 24 and 48 hr) on the total acetylcholine content and acetylcholinesterase activity was investigated after i.p. injection of 0.5 mg of reserpine and 12.5 mg of meprobamate/ml/100 g body wt. Both reserpine and meprobamate caused an increase in the total ACh content in the brain tissue of Arvicanthis niloticus which was suggested to be due to a decrease in the release of ACh, since both reserpine and meprobamate inhibited AChE activity after some tested periods. The effect of meprobamate was observed to be stronger than that of reserpine.     

Fluorescent analysis of alpha-keto acids in serum and urine by high-performance liquid chromatography

A selective procedure using synthetic substrates for determination of exo-1,4,-beta-glucanases in a mixture of exoglucanases , endoglucanases , and beta-glucosidases is formulated. The heterobiosides , p- nithrophenyl -beta-D- cellobioside ( pNPC ) or p-nitrophenyl-beta-D-lactoside ( pNPL ), were used as selective substrates for the measurement of exoglucanase activity. The exoglucanases (especially cellobiohydrolases , which split off cellobiose units from the nonreducing end of the cellulose chain) specifically act on the agluconic bond (between p-nitrophenyl and the disaccharide moiety) and not on the holosidic bond (between the two glucose units of cellobiose). The interfering effect of beta-glucosidase, which acts on both agluconic and holosidic bonds, is overcome by the addition of D-glucono-1,5-delta-lactone, a specific inhibitor of beta-glucosidases. The interference of endoglucanases , which also act on both agluconic and holosidic bonds, can be compensated for by prior standardization of the assay procedure with a purified endoglucanase from the studied mixture of cellulases.