Self-assembly and Self-replication of Short Amphiphilic β-sheet Peptides

Most self-replicating peptide systems are made of α-helix forming sequences. However, it has been postulated that shorter and simpler peptides may also serve as templates for replication when arranged into well-defined structures. We describe here the design and characterization of new peptides that form soluble β-sheet aggregates that serve to significantly accelerate their ligation and self-replication. We then discuss the relevance of these phenomena to early molecular evolution, in light of additional functionality associated with β-sheet assemblies.     

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

The synergistic anti-inflammatory effects of lycopene, lutein, β-carotene, and carnosic acid combinations via redox-based inhibition of NF-κB signaling

Inflammatory mediators and cytokines play important roles in the pathogenesis of a vast number of human diseases; therefore much attention is focused on blunting their proinflammatory modes of action. The aims of the present research were to assess the effectiveness of combinations of carotenoids and phenolics, at concentrations that can be achieved in blood, to inhibit the release of inflammatory mediators from macrophages exposed to lipopolysaccharide (LPS) and to determine what the anti-inflammatory effect of the phytonutrient combinations was in an in vivo mouse model of peritonitis. Preincubation of mouse peritoneal macrophages with lycopene (1μM) or Lyc-O-Mato (1μM) and carnosic acid (2μM), lutein (1μM), and/or β-carotene (2μM) 1h before addition of LPS for 24h caused a synergistic inhibition of NO, prostaglandin E(2), and superoxide production derived from downregulation of iNOS, COX-2, and NADPH oxidase protein and mRNA expression and synergistic inhibition of TNFα secretion. We surmise that the anti-inflammatory action of the phytonutrient combinations used probably resides in their antioxidant properties, because they caused an immediate, efficient, and synergistic inhibition of LPS-induced internal superoxide production leading to a marked decrease in ERK and NF-κB activation. The anti-inflammatory effects of the selected phytonutrient combinations were also demonstrated in a mouse model of peritonitis: their supplementation in drinking water resulted in attenuation of neutrophil recruitment to the peritoneal cavity and in inhibition of inflammatory mediator production by peritoneal neutrophils and macrophages.     

How persons become things: economic and epistemological changes among Nayaka hunter‐gatherers

The ontologies and epistemologies of hunter‐gatherers have attracted growing attention in recent years as these people are undergoing changes. We examine these changes, focusing on one particular case based on our studies of the South Indian Nayaka; they have recently added cultivation and animal husbandry to their partially ongoing hunting and gathering life‐style. Resisting analysis based on an assumed forest/domesticated dichotomy, we show that forest and domesticated animals and plants are both regarded as sentient co‐dwellers in some cases, and as objects in others, depending not on what they are in essence, or where they are, but on when, by whom, and for what purpose they are approached. We argue that pockets of utilitarian framing emerge within the continuing relational epistemology of the Nayaka along with a growing departure from immediacy in the production‐consumption nexus. In these pockets, the vivid presence of animals and plants is concealed, and they no longer appear as persons but as things.     

Further support for the involvement of phytoehrome in stomatal movement

The involvement of phytochrome in stomatal movement in Commelina communis L. is indicated by the following observations: 1) Short irradiation with red or blue light causes opening, of isolated stomata and swelling of guard cell protoplasts. This is reversed by subsequent far red irradiation. 2) In a similar way, stomatal response to prolonged irradiation with red or blue light is decreased by concomitant far red irradiation. 3) Pretreatment with filipin, which interferes with phytochrome binding to membranes, decreases stomatal opening in red and blue light. The stomatal responses to blue and red light are modified by DCMU, N₂, CO_2-enriched atmosphere, and CO_2-free air, which are known to affect, among other processes, chlorophyll fluorescence. Increased chlorophyll fluorescence by DCMU, N₂ and CO₂-enriched atmosphere enhanced stomatal opening in blue light and inhibited it in red light. CO₂-free air, which decreases chlorophyll fluorescence, had the opposite effect.     

The nuclear location signal

A short sequence of predominantly basic amino acids Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val from SV40 Large T is responsible for the normal nuclear location of the protein. Alteration of Lys-128 to each of six different residues other than Arg renders Large T cytoplasmic, whereas single amino acid changes in the surrounding region impair but do not prevent nuclear accumulation. When transposed to the amino terminus of cytoplasmic Large T species, or Escherichia coli beta-galactosidase or of chicken muscle pyruvate kinase, the sequence around Lys-128 of Large T is able to direct the recipient protein to the nucleus. This demonstrates that these amino acids can be sufficient for nuclear location and can act as a nuclear location signal. A computer search of over 2500 proteins reveals that some other nuclear proteins (for example, BK virus Large T, SV40 VP2 and adenovirus 72kDa DNA binding protein) contain very similar basic tracts, but so too do some presumed non-nuclear proteins (for example, poliovirus VP3). We suggest that the related sequence acts as the nuclear location signal in the other nuclear proteins but that the sequence does not function in all cases, perhaps because it is not accessible. A similar, but shorter or less basic sequence, was detected in a number of other nuclear proteins, for example, polyoma virus Large T, SV40 VP1 and several histones. However, such sequences were also found in many other proteins. Perhaps the shorter basic sequences can also act as nuclear location signals, but to be functional they need to be exposed (for example, at the amino terminus of the protein as in SV40 VP1) or to be present in multiple copies.     

The abnormal location of cytoplasmic SV40 large T is not caused by failure to bind to DNA or to p53.

We have examined the large T encoded by an SV40 mutant, d10, which fails to localize to the nucleus. The DNA sequence of the mutant predicts the alteration of Lys 128----Thr within the sequence 127 Lys Lys Lys Arg Lys 131 of large T. The results show that d10 large T is capable of binding to SV40 DNA, to cellular DNA and to the cellular phosphoprotein p53 as well as wild-type large T. These data suggest that the cytoplasmic location of d10 large T is not due to an inability of the protein to be retained within the nucleus, but argues instead that the protein fails to reach the nucleus because it contains a defective nuclear location signal.     

Nucleotide sequence of the dihydrofolate reductase gene of methotrexate-resistant Lactobacillus casei

The nucleotide sequence of the dihydrofolate reductase (DHFR) gene of a methotrexate-resistant strain of Lactobacillus casei, which is the source of DHFR for nuclear magnetic resonance (NMR) studies, has been determined. The derived amino acid sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at position 8 and proline instead of leucine at position 90. The nucleotide sequences of 320-bp 5' and 335-bp 3' flanking regions of this gene have also been determined.     

The effect of abscisic acid on epidermal cells: protoplast swelling and ATPase activity

Isolated epidermal protoplasts of Commelina communis L. increase in volume in the presence of KCl. Since this swelling is an osmotic phenomenon it reflects K⁺ influx. ATP slightly decreased the volume of the protoplasts, pointing towards the possibility that K⁺ uptake is passive. On the other hand abscisic acid (ABA) and sodium orthovanadate increased the swelling, and their effect was reversed by ATP. This may support the suggestion that ABA inhibits the active and ATPase-mediated relase of K⁺ from epidermal cells. Mg²⁺-dependent, K⁺-stimulated ATPase activity was found in the microsomal fraction from epidermal cells. This activity was vandadate sensitive. ABA increased the basal activity in the presence of Mg²⁺ but inhibited the K⁺ stimulation.     

K, Mg-ATPase activity in guard cells of Commelina communis

Siliqua development was studied in the wild type line Landsberg erecta and the GA-sensitive mutant ga-1 of Arabidopsis thaliana (L.) Heynh. Reciprocal crosses between wild type and ga-l mutant, and self pollinations of either parent have shown that siliqua growth is determined by endogenous GAs originating from maternal tissues and embryo. The ga-1 mutant either self pollinated or cross-pollinated with wild type pollen showed reduced siliqua growth, which to a large extent could be overcome by exogenously applied GAs. The siliquae of the ga-1 mutant possessed very reduced ent -kaurene synthesizing capacity and no detectable endogenous GA-activity indicating an early block in the GA-biosynthetic pathway. Seed weight is not affected by GA-deficiency during development.