Amyloid-like properties of bacterial inclusion bodies

Bacterial inclusion bodies are major bottlenecks in protein production, narrowing the spectrum of relevant polypeptides obtained by recombinant DNA. While regarded as amorphous deposits formed by passive and rather unspecific precipitation of unfolded chains, we prove here that they are instead organized aggregates sharing important structural and biological features with amyloids. By using an Escherichia coli beta-galactosidase variant, we show that aggregation does not necessarily require unfolded polypeptide chains but rather depends on specific interactions between solvent-exposed hydrophobic stretches in partially structured species. In addition, purified inclusion bodies are efficient and highly selective nucleation seeds, promoting deposition of soluble homologous but not heterologous polypeptides in a dose-dependent manner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which, jointly with Fourier transform infra red spectroscopy data, indicates a high level of organized intermolecular beta-sheet structure. The evidences of amyloid-like structure of bacterial inclusion bodies, irrespective of potential applications in bioprocess engineering, prompts the use of bacterial models to explore the molecular determinants of protein aggregation by means of simple biological systems.     

Metabolic and Regulatory Roles of Leucine in Neural Cells

Dietary leucine transported into the brain parenchyma serves several functions. Most prominent is the role of leucine as a metabolic precursor of fuel molecules, α-ketoisocaproate and ketone bodies. As alternatives to glucose, these compounds are forwarded by the producing astrocytes to the adjacent neural cells. Leucine furthermore participates in the maintenance of the nitrogen balance in the glutamate/glutamine cycle pertinent to the neurotransmitter glutamate. Leucine also serves as a regulator of the activity of some enzymes important for brain energy metabolism. Another role of leucine as an informational molecule is in mTOR signaling that participates in the regulation of food ingestion. The importance of leucine for brain function is stressed by the fact that inborn errors in its metabolism cause metabolic diseases often associated with neuropathological symptoms. In this overview, the current knowledge on the metabolic and regulatory roles of this essential amino acid in neural cells are briefly summarized. Special issue dedicated to Dr. Frode Fonnum.     

植物残体与泥炭和泥炭矿体分类的初步探讨

泥炭植物残体是泥炭和泥炭矿体的主要组分。泥炭的植物组成与泥炭的分解度和营养状况密切相关。在进行泥炭分类时,植物残体组成是其重要依据。各泥炭矿体特征间“质”的差异也反映在泥炭植物残体组成成分的不同上。因此,在进行泥炭矿体分类时,可将矿层的植物残体组分作其依据。本文拟将我国的泥炭矿体分为富营养、中营养、贫营养和混合四个泥炭矿体型;并续分出十一个泥炭矿体组。     

Cellular stress induces cytoplasmic RNA granules in fission yeast

Severe stress causes plant and animal cells to form large cytoplasmic granules containing RNA and proteins. Here, we demonstrate the existence of stress-induced cytoplasmic RNA granules in Schizosaccharomyces pombe. Homologs to several known protein components of mammalian processing bodies and stress granules are found in fission yeast RNA granules. In contrast to mammalian cells, poly(A)-binding protein (Pabp) colocalizes in stress-induced granules with decapping protein. After glucose deprivation, protein kinase A (PKA) is required for accumulation of Pabp-positive granules and translational down-regulation. This is the first demonstration of a role for PKA in RNA granule formation. In mammals, the translation initiation protein eIF2α is a key regulator of formation of granules containing poly(A)-binding protein. In S. pombe, nonphosphorylatable eIF2α does not block but delays granule formation and subsequent clearance after exposure to hyperosmosis. At least two separate pathways in S. pombe appear to regulate stress-induced granules: pka1 mutants are fully proficient to form granules after hyperosmotic shock; conversely, eIF2α does not affect granule formation in glucose starvation. Further, we demonstrate a Pka1-dependent link between calcium perturbation and RNA granules, which has not been described earlier in any organism.     

High‐level production of human interleukin‐10 fusions in tobacco cell suspension cultures

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale‐up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin‐10 (IL‐10) in tobacco BY‐2 cell suspension and evaluated the effect of an elastin‐like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL‐10 accumulation. We report the highest accumulation levels of hIL‐10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL‐10‐ELP has cytokine activity, its activity is reduced compared to unfused IL‐10, likely caused by interference of ELP with folding of IL‐10. Green fluorescent protein has no effect on IL‐10 accumulation, but examining the trafficking of IL‐10‐GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full‐size fusion remains in the whole‐cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL‐10‐GFP‐ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER.     

Drosophila social clustering is disrupted by anesthetics and in narrow abdomen ion channel mutants

Members of many species tend to congregate, a behavioral strategy known as local enhancement. Selective advantages of local enhancement range from efficient use of resources to defense from predators. While previous studies have examined many types of social behavior in fruit flies, few have specifically investigated local enhancement. Resource‐independent local enhancement (RILE) has recently been described in the fruit fly using a measure called social space index (SSI), although the neural mechanisms remain unknown. Here, we analyze RILE of Drosophila under conditions that allow us to elucidate its neural mechanisms. We have investigated the effects of general volatile anesthetics, compounds that compromise higher order functioning of the type typically required for responding to social cues. We exposed Canton‐S flies to non‐immobilizing concentrations of halothane and found that flies had a significantly decreased SSI compared with flies tested in air. Narrow abdomen (na) mutants, which display altered responses to anesthetics in numerous behavioral assays, also have a significantly reduced SSI, an effect that was fully reversed by restoring expression of na by driving a UAS‐NA rescue construct with NA‐GAL4. We found that na expression in cholinergic neurons fully rescued the behavioral defect, whereas expression of na in glutamatergic neurons did so only partially. Our results also suggest a role for na expression in the mushroom bodies (MBs), as suppressing na expression in the MBs of NA‐GAL4 rescue flies diminishes SSI. Our data indicate that RILE, a simple behavioral strategy, requires complex neural processing .     

Inhibition of spontaneous network activity in neonatal hippocampal slices by energy substrates is not correlated with intracellular acidification

Several energy substrates complementary to glucose, including lactate, pyruvate and β-hydroxybutyrate, serve as a fuel for neurons. It was reported recently that these substrates can substantially modulate cortical excitability in neonatal slices. However, complementary energy substrates (CES) can also induce an intracellular acidification when added exogenously. Therefore, action of CES on the neuronal properties governing excitability in neonatal brain slices may be underlain by a change in the cell energy status or by intracellular acidification, or both. Here, we attempt to elucidate these possibilities in neonatal hippocampus by recording neuronal population activity and monitoring intracellular pH. We show that a spontaneous network activity pattern, giant depolarizing potentials (GDPs), characteristic for the neonatal hippocampal slices exposed to artificial cerebrospinal fluid, is strongly inhibited by CES and this effect is unlikely to be caused by a subtle intracellular acidification induced by these compounds. Indeed, a much stronger intracellular acidification in the HCO(3) -free solution inhibited neither the GDP frequency nor the GDP amplitude. Therefore, modulation of neuronal energy homeostasis is the most likely factor underlying the effect of lactate, pyruvate and β-hydroxybutyrate on network excitability in neonatal brain slices.     

Biologically active constituents from the fruiting body of Taiwanofungus camphoratus

Five new benzenoids, benzocamphorins A-E (1-5), and 10 recently isolated triterpenoids, camphoratins A-J (16-25), together with 23 known compounds including seven benzenoids (6-12), three lignans (13-15), and 13 triterpenoids (26-38) were isolated from the fruiting body of Taiwanofungus camphoratus. Their structures were established by spectroscopic analysis. Selected compounds were examined for cytotoxic and anti-inflammatory activities. Compounds 9 and 21 showed moderate cytotoxicity against MCF-7 and Hep2 cell lines with ED₅₀ values of 3.4 and 3.0 ??g/mL, respectively. Compounds 21, 25, 26, 29-31, 33, and 36 demonstrated potent anti-inflammatory activity by inhibiting lipopolysaccharide (LPS)-induced nitric oxide (NO) production with IC₅₀ values of 2.5, 1.6, 3.6, 0.6, 4.1, 4.2, 2.5, and 1.5 ??M, respectively, which were better than those of the nonspecific nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine methyl ester (l-NAME) (IC₅₀: 25.8 ??M). These results may substantiate the use of T. camphoratus in traditional Chinese medicine (TCM) for the treatment of inflammation and cancer-related diseases. The newly discovered compounds deserve further development as anti-inflammatory candidates.     

Computational analysis of residue contributions to coiled-coil topology

A variety of features are thought to contribute to the oligomeric and topological specificity of coiled coils. In previous work, we examined the determinants of oligomeric state. Here, we examine the energetic basis for the tendency of six coiled-coil peptides to align their α-helices in antiparallel orientation using molecular dynamics simulations with implicit solvation (EEF1.1). We also examine the effect of mutations known to disrupt the topology of these peptides. In agreement with experiment, ARG or LYS at a or d positions were found to stabilize the antiparallel configuration. The modeling suggests that this is not due to a-a' or d-d' repulsions but due to interactions with e' and g' residues. TRP at core positions also favors the antiparallel configuration. Residues that disfavor parallel dimers, such as ILE at d, are better tolerated in, and thus favor the antiparallel configuration. Salt bridge networks were found to be more stabilizing in the antiparallel configuration for geometric reasons: antiparallel helices point amino acid side chains in opposite directions. However, the structure with the largest number of salt bridges was not always the most stable, due to desolvation and configurational entropy contributions. In tetramers, the extent of stabilization of the antiparallel topology by core residues is influenced by the e' residue on a neighboring helix. Residues at b and c positions in some cases also contribute to stabilization of antiparallel tetramers. This work provides useful rules toward the goal of designing coiled coils with a well-defined and predictable three-dimensional structure.