This chapter presents a protocol for studying the effects of curcumin in a colorectal cell line and a mouse model of colitis-associated colon carcinogenesis. The protocol using the CT26 cell line incorporates cell proliferation, migration, invasion, spheroid formation, cell cycle, polymerase chain reaction (PCR), and western blot analyses. For the mouse model, this involved a macroscopic and histological examination of the colon and assays for oxidative damage markers.

Intracellular platelet activating-factor acetylhydrolase type II (PAF-AH II) is a 40-kDa monomeric enzyme. It was originally identified as an enzyme that hydrolyzes the acetyl group of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). As a member of phospholipase A2 super family, PAF-AH II has broad substrate specificity. It can hydrolyze phospholipids with relatively short-length or oxidatively modified sn-2 chains which endows it with various functions such as protection against oxidative stress, transacetylase activity and producing lipid mediators. PAF-AH II has been proven to be involved in several diseases such as allergic diseases, oxidative stress-induced injury and ischemia injury, thus it has drawn more attention from researchers. In this paper, we outline an entire summary of PAF-AH II, including its structure, substrate specificity, activity assay, inhibitors and biological activities.

Naturally occurring constrained peptides are frequently used as scaffolds for bioactive peptide grating due to their high stability. Here, we used in silico methods to design several constrained peptides comprising a scorpion toxin scaffold, a MDM2 binding epitope, and a cluster of positively charged residues. The designed peptides displayed varied binding affinity to MDM2 despite differing by only one or two residues. One of the peptides, SC426, had nanomolar binding affinity (KD =6.6±2.6 nm) to MDM2, and exhibited stronger inhibitory activity on the proliferation of HCT116 cells (p53-wild type) and SW480 cells (p53-mutant) than that of nutlin-3a. Binding mode analysis of the designed peptide at MDM2 suggests that the conserved \"FWL\" epitope was buried in the hydrophobic binding pocket, and the residues located at the periphery of the binding site contributed to the high binding affinity of SC426. Overall, in silico design of miniproteins with therapeutic potential through epitope grafting to the naturally occurring constrained peptide is an effective strategy.

Muraymycins are a subclass of antimicrobially active uridine-derived natural products. Biological data on several muraymycin analogues have been reported, including some inhibitory in vitro activities toward their target protein, the bacterial membrane enzyme MraY. However, a structure-activity relationship (SAR) study on naturally occurring muraymycins based on such in vitro data has been missing so far. In this work, we report a detailed SAR investigation on representatives of the four muraymycin subgroups A-D using a fluorescence-based in vitro MraY assay. For some muraymycins, inhibition of MraY with IC50 values in the low-picomolar range was observed. These inhibitory potencies were compared with antibacterial activities and were correlated to modelling data derived from a previously reported X-ray crystal structure of MraY in complex with a muraymycin inhibitor. Overall, these results will pave the way for the development of muraymycin analogues with optimized properties as antibacterial drug candidates.

Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

Lactoferrin is a glycoprotein with two globular lobes, each having two domains. Since the discovery of its antimicrobial properties, efforts have been made to find peptides derived from this protein showing antimicrobial properties. Most peptides initially studied were derived from Lactoferricin B, obtained from the protein by digestion with pepsin. More recently, a new family of antimicrobial peptides (AMPs) derived from Lactoferrin was discovered by Bolcher et al, and named Lactoferrampin (LFampin). The original sequence of LFampin contained residues 268 - 284 from the N1 domain of Lactoferrin. From this peptide, the Bolscher\'s group synthesized a collection of peptides obtained by extension and / or truncation at the C or N-terminal sides, in order to unravel the main structural features responsible for antimicrobial action. Here, we present results for three of these peptides, namely LFampin 265 - 284, LFampin 265 - 280, and LFampin 270 - 284. The peptides were tested against bacteria (E. coli and S. sanguinis), fungi (C. albicans), and model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG), and their mixtures at a ratio of 3 : 1 (DMPC : DMPG (3 : 1)). The ability to adopt a helical conformation was followed by a circular dichroism (CD), and the perturbation of the gel to the liquid-crystalline phase transition of the membrane was characterized by differential scanning calorimetry (DSC). Distinct behavior was observed in the three peptides, both from the microbiology and model membrane studies, with the biophysical results showing excellent correlation with the microbiology activity studies. LFampin 265 - 284 was the most active peptide toward the tested microorganisms, and in the biophysical studies it showed the highest ability to form an α-helix and the strongest interaction with model membranes, followed by LFampin 265 - 280. LFampin 270 - 284 was inactive, showing marginal secondary structure and no interaction with the pathogen model membranes.