Spatial alterations of De Novo purine biosynthetic enzymes by Akt-independent PDK1 signaling pathways.

A macromolecular complex of the enzymes involved in human de novo purine biosynthesis, the purinosome, has been shown to consist of a core assembly to regulate the metabolic activity of the pathway. However, it remains elusive whether the core assembly itself can be selectively controlled in the cytoplasm without promoting the purinosome. Here, we reveal that pharmacological inhibition of the cytoplasmic activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1) selectively promotes the formation of the core assembly, but not the purinosome, in cancer cells. However, alternative signaling cascades that are associated with the plasma membrane-bound PDK1 activity, including Akt-mediated cascades, regulate neither the core assembly nor the purinosome in our conditions. Along with immunofluorescence microscopy and a knock-down study against PDK1 using small interfering RNAs, we reveal that cytoplasmic PDK1-associated signaling pathways regulate subcellular colocalization of three enzymes that form the core assembly of the purinosome in an Akt-independent manner. Collectively, this study reveals a new mode of compartmentalization of purine biosynthetic enzymes controlled by spatially resolved signaling pathways.

Subcellular functions of proteins under fluorescence single-cell microscopy.

A cell is a highly organized, dynamic, and intricate biological entity orchestrated by a myriad of proteins and their self-assemblies. Because a protein's actions depend on its coordination in both space and time, our curiosity about protein functions has extended from the test tube into the intracellular space of the cell. Accordingly, modern technological developments and advances in enzymology have been geared towards analyzing protein functions within intact single cells. We discuss here how fluorescence single-cell microscopy has been employed to identify subcellular locations of proteins, detect reversible protein-protein interactions, and measure protein activity and kinetics in living cells. Considering that fluorescence single-cell microscopy has been only recently recognized as a primary technique in enzymology, its potentials and outcomes in studying intracellular protein functions are projected to be immensely useful and enlightening. We anticipate that this review would inspire many investigators to study their proteins of interest beyond the conventional boundary of specific disciplines. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides.

All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.

An interactive RADIANCE toolkit for customizable CT dose monitoring and reporting.

The need for tools to monitor imaging-related radiation has grown dramatically in recent years. RADIANCE, a freely available open-source dose-monitoring tool, was developed in response to the need for an informatics solution in this realm. A number of open-source as well as commercial solutions have since been developed to enable radiology practices to monitor radiation dose parameters for modalities ranging from computed tomography to radiography to fluoroscopy. However, it is not sufficient to simply collect this data; it is equally important to be able to review it in the appropriate context. Most of the currently available dose-monitoring solutions have some type of reporting capability, such as a real-time dashboard or a static report. Previous versions of RADIANCE have included a real-time dashboard with pre-set screens that plot effective dose estimates according to different criteria, as well as monthly scorecards to summarize dose estimates for individuals within a radiology practice. In this work, we present the RADIANCE toolkit, a customizable reporting solution that allows users to generate reports of interest to them, summarizing a variety of metrics that can be grouped according to useful parameters. The output of the toolkit can be used for real-time dose monitoring or scheduled reporting, such as to a quality assurance committee. Making dose parameter data more accessible and more meaningful to the user promotes dose reduction efforts such as regular protocol review and optimization, and ultimately improves patient care by decreasing unnecessary radiation exposure.

A novel adipose-specific gene deletion model demonstrates potential pitfalls of existing methods.

Adipose-specific gene deletion in mice is crucial in determining gene function in adipocyte homeostasis and the development of obesity. We noted 100% mortality when the Hdac3 gene was conditionally deleted using Fabp4-Cre mice, the most commonly used model of adipose-targeted Cre recombinase. However, this surprising result was not reproduced using other models of adipose targeting of Cre, including a novel Retn-Cre mouse. These findings underscore the need for caution when interpreting data obtained using Fabp4-Cre mice and should encourage the use of additional or alternative adipose-targeting Cre mouse models before drawing conclusions about in vivo adipocyte-specific functions.