pVEC hydrophobic N‐terminus is critical for antibacterial activity

Cell‐penetrating peptides (CPPs) are commonly defined by their shared ability to be internalized into eukaryotic cells, without inducing permanent membrane damage, and to improve cargo delivery. Many CPPs also possess antimicrobial action strong enough to selectively lyse microbes in infected mammalian cultures. pVEC, a CPP derived from cadherin, is able to translocate into mammalian cells, and it is also antimicrobial. Structure‐activity relationship and sequence alignment studies have suggested that the hydrophobic N‐terminus (LLIIL) of pVEC is essential for this peptide's uptake into eukaryotic cells. In this study, our aim was to examine the contribution of these residues to the antimicrobial action and the translocation mechanism of pVEC. We performed antimicrobial activity and microscopy experiments with pVEC and with del5 pVEC (N‐terminal truncated variant of pVEC) and showed that pVEC loses its antimicrobial effect upon deletion of the LLIIL residues, even though both peptides induce membrane permeability. We also calculated the free energy of the transport process using steered molecular dynamic simulations and replica exchange umbrella sampling simulations to compare the difference in uptake mechanism of the 2 peptides in atomistic detail. Despite the difference in experimentally observed antimicrobial activity, the simulations on the 2 peptides showed similar characteristics and the energetic cost of translocation of pVEC was higher than that of del5 pVEC, suggesting that pVEC uptake mechanism cannot be explained by simple passive transport. Our results suggest that LLIIL residues are key contributors to pVEC antibacterial activity because of irreversible membrane disruption.     

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%–75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.     

Quantitative comparison of cancer and normal cell adhesion using organosilane monolayer templates: an experimental study on the anti-adhesion effect of green-tea catechins

The main constituent of green tea, (?)-Epigallocatechin-3-O-gallate (EGCG), is known to have cancer-specific chemopreventive effects. In the present work, we investigated how EGCG suppresses cell adhesion by comparing the adhesion of human pancreatic cancer cells (AsPC-1 and BxPC-3) and their counterpart, normal human embryonic pancreas-derived cells (1C3D3), in catechin-containing media using organosilane monolayer templates (OMTs). The purpose of this work is (1) to evaluate the quantitativeness in the measurement of cell adhesion with the OMT and (2) to show how green-tea catechins suppress cell adhesion in a cancer-specific manner. For the first purpose, the adhesion of cancer and normal cells was compared using the OMT. The cell adhesion in different type of catechins such as EGCG, (?)-Epicatechin-3-O-gallate (ECG) and (?)-Epicatechin (EC) was also evaluated. The measurements revealed that the anti-adhesion effect of green-tea catechins is cancer-specific, and the order is EGCG?ECG>EC. The results agree well with the data reported to date, showing the quantitativeness of the new method. For the second purpose, the contact area of cells on the OMT was measured by reflection interference contrast microscopy. The cell-OMT contact area of cancer cells decreases with increasing EGCG concentration, whereas that of normal cells remains constant. The results reveal a twofold action of EGCG on cancer cell adhesion—suppressing cell attachment to a candidate adhesion site and decreasing the contact area of the cells—and validates the use of OMT as a tool for screening cancer cell adhesion.     

Positive and negative cis-acting DNA domains are required for spatial and temporal regulation of gene expression by a seed storage protein promoter.

Mutations affecting spatial and temporal regulation of a beta-phaseolin gene encoding the major storage protein of bean (Phaseolus vulgaris) were analyzed by stable and transient transformation approaches. The results substantiate the value of transient assays for rapid determination of the functionality of cis-acting sequences and the importance of stable transformation to identify tissue-specific determinants. Spatial information is specified primarily by two upstream activating sequences (UAS). UAS1 (-295 to -109) was sufficient for seed-specific expression from both homologous and heterologous (CaMV 35S) promoters. In situ localization of GUS expression in tobacco embryos demonstrated that UAS1 activity was restricted to the cotyledons and shoot meristem. A second positive domain, UAS2 (-468 to -391), extended gene activity to the hypocotyl. Temporal control of GUS expression was found to involve two negative regulatory sequences, NRS1 (-391 to -295) and NRS2 (-518 to -418), as well as the positive domain UAS1. The deletion of either negative element caused premature onset of GUS expression. These findings indicate combinatorial interactions between multiple sequence motifs specifying spatial information, and provide the first example of the involvement of negative elements in the temporal control of gene expression in higher plants.     

Xylanase and itaconic acid production by Aspergillus terreus NRRL 1960 within a biorefinery concept

Production of two industrially important products, xylanase and itaconic acid (IA), by Aspergillus terreus NRRL 1960 from agricultural residues was investigated within a biorefinery concept. Biological pretreatment was applied to lignocellulosic materials by using A. terreus, which produced xylanase while growing on agricultural residues. For IA production, already grown cells were transferred into a new medium. The first step provided not only the pretreatment of lignocellulosic material in order to be used as feedstock but also production of xylanase. For this purpose, cotton stalk, sunflower stalk and corn cob were used as carbon sources as lignocellulosic material. Among them, the highest xylanase production was obtained on corn cob. By application of two-step fermentation, about 70 IU/mL xylanase and 18 g/L IA production levels were achieved. This study shows the stepwise usage potential of the microorganism as a tool in a biorefinery concept.