Determination of pyrrolidone carboxylate and gamma-glutamyl amino acids by gas chromatography.

Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and γ-glutamyl amino acids are described. These intermediates of the γ-glutamyl cycle were separated by ion exchange chromatography and converted to their N-acyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-1-propanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several γ-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.     

Comparison of pre-treatment and in-vivo dosimetry for advanced radiotherapy of prostate cancer

BackgroundThe usage of advanced radiotherapy techniques requires validation of a previously calculated dose with the precise delivery with a linear accelerator. This study aimed to review and evaluate new verification methods of dose distribution. Moreover, our purpose was to define an internal protocol of acceptance for in-vivo measurements of dose distribution.Materials and methodsThis study included 43 treatment plans of prostate cancer calculated using the Monte Carlo algorithm. All plans were delivered using the Volumetric Modulated Arc Therapy (VMAT) technique of advanced radiotherapy by the linear accelerator Elekta VersaHD. The dose distribution was verified using: MatriXX, iViewDose, and in-vivo measurements. The verification also included recalculation of fluence maps of quality assurance plans in another independent algorithm.ResultsThe acceptance criterion of 95% points of dose in agreement was found for pre-treatment verification using MatriXX; the average γ value was 99.09 ± 0.93 (SD) and 99.64 ± 0.35 (SD) for recalculation in the Collapse Cone algorithm. Moreover, using the second algorithm in the verification process showed a positive correlation ρ = 0.58, p < 0.001. However, verification using iViewDose in a phantom and in-vivo did not meet this γ-pass rate.ConclusionsEvaluation of gamma values for in-vivo measurements utilizing iViewDose software was helpful to establish an internal dosimetry protocol for prostate cancer treatments. We assumed value at a minimum of 50% points of the dose in agreement with the 3%/3 mm criterion as an acceptable compliance level. The recalculated dose distribution of QA plans in regard to the Collapse Cone algorithm in the other treatment planning system can be used as a pre-treatment verification method used by a medical physicist in their daily work. The effectiveness of use in iViewDose software, as a pre-treatment tool, is still debatable, unlike the MatriXX device.     

Subcellular Distribution of Prolyl Endopeptidase and Cation-Sensitive Neutral Endopeptidase in Rabbit Brain

Abstract: The subcellular distribution of prolyl endopeptidase, and of cationsensitive neutral endopeptidase, two enzymes actively metabolizing many neuropeptides, was determined in homogenates of rabbit brain. The subcellular distribution of both enzymes was more similar to lactate dehydrogenase, a cytoplasmic enzyme marker, than to choline acetyltransferase, a synaptosomal marker. Only 35% of the activity of these two neutral endopeptidases was found in the crude mitochondrial fraction (P₂), the bulk of the remaining activity being associated with the high-speed supernatant. Prolyl endopeptidase and cation-sensitive neutral endopeptidase thus can be regarded as mainly cytoplasmic enzymes in the rabbit brain.     

Phosphorylation of the multicatalytic proteinase complex from bovine pituitaries by a copurifying cAMP-dependent protein kinase

The multicatalytic proteinase complex (MPC) constitutes a major nonlysosomal proteolytic system that may play an important role in the processing of biologically active peptides and enzymes, as well as in intracellular metabolism. We report that at least two of its subunits of MW 28,800 (S2) and 27,000 (S3) are phosphorylated by a cAMP-dependent protein kinase (PK-A) that copurifies with the complex isolated from bovine pituitaries. The cAMP-induced phosphorylation was time dependent and inhibited by a PK-A inhibitor. Although not an integral part of the complex, PK-A activity was still present even in 1700-fold-purified and apparently homogeneous preparations by criteria of nondissociating polyacrylamide gel electrophoresis. Furthermore, we present evidence that the copurification of the two enzymes is not species or tissue specific, or dependent on a single method of purification. The copurifying kinase was stimulated 10-fold by cAMP (10 microM) and 2- to 3-fold by a peptide substrate of the MPC, but was unaffected by protein kinase C activators (calcium and a phospholipid mixture). These findings suggest that protein phosphorylation may represent a mechanism for regulating the activity of the multicatalytic proteinase complex.     

Innate immunity orchestrates the mobilization and homing of hematopoietic stem/progenitor cells by engaging purinergic signaling—an update

Purinergic Signalling - Bone marrow (BM) as an active hematopoietic organ is highly sensitive to changes in body microenvironments and responds to external physical stimuli from the surrounding...     

Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L.

Hemp (Cannabis sativa L.) is an emerging dioecious crop grown primarily for grain, fiber, and cannabinoids. There is good evidence for medicinal benefits of the most abundant cannabinoid in hemp, cannabidiol (CBD). For CBD production, female plants producing CBD but not tetrahydrocannabinol (THC) are desired. We developed and validated high‐throughput PACE (PCR Allele Competitive Extension) assays for C. sativa plant sex and cannabinoid chemotype. The sex assay was validated across a wide range of germplasm and resolved male plants from female and monoecious plants. The cannabinoid chemotype assay revealed segregation in hemp populations, and resolved plants producing predominantly THC, predominantly CBD, and roughly equal amounts of THC and CBD. Cultivar populations that were thought to be stabilized for CBD production were found to be segregating phenotypically and genotypically. Many plants predominantly producing CBD accumulated more than the current US legal limit of 0.3% THC by dry weight. These assays and data provide potentially useful tools for breeding and early selection of hemp.     

Flexible docking of peptides to proteins using CABS‐dock

Molecular docking of peptides to proteins can be a useful tool in the exploration of the possible peptide binding sites and poses. CABS‐dock is a method for protein–peptide docking that features significant conformational flexibility of both the peptide and the protein molecules during the peptide search for a binding site. The CABS‐dock has been made available as a web server and a standalone package. The web server is an easy to use tool with a simple web interface. The standalone package is a command‐line program dedicated to professional users. It offers a number of advanced features, analysis tools and support for large‐sized systems. In this article, we outline the current status of the CABS‐dock method, its recent developments, applications, and challenges ahead.     

Crystal structure of Nsp15 endoribonuclease NendoU from SARS‐CoV‐2

Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS‐CoVs and Middle East Respiratory Syndrome coronavirus (MERS‐CoVs), the detailed information about SARS‐CoV‐2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high‐throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS‐CoV‐2 proteins and structures. Here we report two high‐resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.