Contents of Volume 53

Plant Molecular Biology -     

An Insertion Peptide in Yeast Glycyl-tRNA Synthetase Facilitates both Productive Docking and Catalysis of Cognate tRNAs

The yeast Saccharomyces cerevisiae possesses two distinct glycyl-tRNA synthetase (GlyRS) genes: GRS1 and GRS2. GRS1 is dually functional, encoding both cytoplasmic and mitochondrial activities, while GRS2 is dysfunctional and not required for growth. The protein products of these two genes, GlyRS1 and GlyRS2, are much alike but are distinguished by an insertion peptide of GlyRS1, which is absent from GlyRS2 and other eukaryotic homologues. We show that deletion or mutation of the insertion peptide modestly impaired the enzyme''s catalytic efficiency in vitro (with a 2- to 3-fold increase in K_m and a 5- to 8-fold decrease in k_cat). Consistently, GRS2 can be conveniently converted to a functional gene via codon optimization, and the insertion peptide is dispensable for protein stability and the rescue activity of GRS1 at 30°C in vivo. A phylogenetic analysis further showed that GRS1 and GRS2 are paralogues that arose from a gene duplication event relatively recently, with GRS1 being the predecessor. These results indicate that GlyRS2 is an active enzyme essentially resembling the insertion peptide-deleted form of GlyRS1. Our study suggests that the insertion peptide represents a novel auxiliary domain, which facilitates both productive docking and catalysis of cognate tRNAs.     

Kinetic Process of β-Amyloid Formation via Membrane Binding

Recently we have studied thermodynamics of membrane-mediated β-amyloid formation in equilibrium experiments using penetratin-lipid mixtures. The results showed that penetratin bound to the membrane interface in the α-helical conformation when the peptide/lipid (P/L) ratios were below a lipid-dependent critical value P/L^∗. When P/L reached P/L^∗, small β-aggregates emerged, which served as the nuclei for large β-aggregates. Here we studied the corresponding kinetic process to understand the potential barriers for the membrane-mediated β-amyloid formation. We performed kinetic experiments using giant unilamellar vesicles made of 7:3 DOPC/DOPG. The observed time behavior of individual giant unilamellar vesicles, although complex, exhibited the physical effects seen in equilibrium experiments. Most interestingly, a potential barrier appeared to block penetratin from translocating across the bilayer. As a result, the kinetic value for the critical threshold P/L^∗ is roughly one-half of the value measured in equilibrium where peptides bind symmetrically on both sides of lipid bilayers. We also investigated the similarity and differences between the charged and neutral lipids in their interactions with penetratin. We reached an important conclusion that the bound states of peptides in lipid bilayers are largely independent of the charge on the lipid headgroups.     

Pattern analysis of stem cell differentiation during <Emphasis Type="Italic">in vitro Arabidopsis</Emphasis> organogenesis

Plant somatic cells have the capability to switch their cell fates from differentiated to undifferentiated status under proper culture conditions, which is designated as totipotency. As a result, plant cells can easily regenerate new tissues or organs from a wide variety of explants. However, the mechanism by which plant cells have such remarkable regeneration ability is still largely unknown. In this study, we used a set of meristem-specific marker genes to analyze the patterns of stem cell differentiation in the processes of somatic embryogenesis as well as shoot or root organogenesis in vitro. Our studies furnish preliminary and important information on the patterns of the de novo stem cell differentiation during various types of in vitro organogenesis.     

ACE/ACE2 Ratio and MMP-9 Activity as Potential Biomarkers in Tuberculous Pleural Effusions

Objective: Pleural effusion is common problem, but the rapid and reliable diagnosis for specific pathogenic effusions are lacking. This study aimed to identify the diagnosis based on clinical variables to differentiate pleural tuberculous exudates from other pleural effusions. We also investigated the role of renin-angiotensin system (RAS) and matrix metalloproteinase (MMPs) in the pathogenesis of pleural exudates.Experimental design: The major components in RAS and extracellular matrix metabolism, including angiotensin converting enzyme (ACE), ACE2, MMP-2 and MMP-9 activities, were measured and compared in the patients with transudative (n = 45) and exudative (n = 80) effusions. The exudative effusions were come from the patients with tuberculosis (n = 20), pneumonia (n = 32), and adenocarcinoma (n = 28).Results: Increased ACE and equivalent ACE2 activities, resulting in a significantly increased ACE/ACE2 ratio in exudates, were detected compared to these values in transudates. MMP-9 activity in exudates was significantly higher than that in transudates. The significant correlation between ACE and ACE2 activity that was found in transudates was not found in exudates. Advanced analyses showed significantly increased ACE and MMP-9 activities, and decreased ACE2 activity in tuberculous pleural effusions compared with those in pneumonia and adenocarcinoma effusions. The results indicate that increased ACE and MMP-9 activities found in the exudates were mainly contributed from a higher level of both enzyme activities in the tuberculous pleural effusions.Conclusion: Interplay between ACE and ACE2, essential functions in the RAS, and abnormal regulation of MMP-9 probably play a pivotal role in the development of exudative effusions. Moreover, the ACE/ACE2 ratio combined with MMP-9 activity in pleural fluid may be potential biomarkers for diagnosing tuberculous pleurisy.     

Mitochondrial glutathione modulates TNF-alpha-induced endothelial cell dysfunction.

The effect of glutathione (GSH) depletion by L-buthionine-[S,R]-sulphoximine (BSO) on tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression and mononuclear leukocyte adhesion to human umbilical vein endothelial cells (HUVECs) was investigated. Cells with marked depletion of cytoplasmic GSH, but with an intact pool of mitochondrial GSH, only slightly enhanced TNF-alpha-induced E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression, compared with the control. However, TNF-a-induced expression of both molecules was markedly enhanced when the mitochondrial GSH pool was diminished to <15% of the control. In contrast, TNF-alpha-induced intercellular adhesion molecule-1 (ICAM-1) expression was not affected by the depletion of either cytoplasmic or mitochondrial GSH. Marked enhancement of TNF-alpha-induced adhesion molecule expression by the depletion of mitochondrial GSH resulted in increased in mononuclear leukocyte adhesion to treated HUVECs, compared with the control. These effects parallel reactive oxygen species (ROS) formation by the depletion of mitochondrial but not cytoplasmic GSH. Our findings demonstrate that depletion of mitochondrial GSH renders more ROS generation in HUVECs, and mitochondrial GSH modulates TNF-alpha-induced adhesion molecule expression and mononuclear leukocyte adhesion in HUVECs.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.