25-hydroxycholesterol is produced by testicular macrophages during the early postnatal period and influences differentiation of Leydig cells in vitro

Leydig cells develop inappropriately in animals lacking testicular macrophages. We have recently found that macrophages from adult animals produce 25-hydroxycholesterol, an oxysterol involved in the differentiation of hepatocytes and keratinocytes. Therefore, we hypothesized that testicular macrophages also produce 25-hydroxycholesterol during the early postnatal period and that this oxysterol plays a role in the differentiation of Leydig cells. We assessed the production of 25-hydroxycholesterol and 25-hydroxylase mRNA by cultured testicular macrophages from rats at 10, 20, and 40 days of age. We also tested the long-term effects of 25-hydroxycholesterol on basal and LH-stimulated testosterone production, and 3beta-hydroxysteroid dehydrogenase activity as end points of Leydig cell differentiation in vitro. We found that testicular macrophages from animals at all ages produced both 25-hydroxycholesterol and 25-hydroxylase mRNA, with macrophages from 10-day-old animals having the highest steady-state levels of message. We also found that chronic exposure of Leydig cells to 25-hydroxycholesterol increased basal production of testosterone but decreased LH-stimulated steroidogenesis at all ages. Finally, 25-hydroxycholesterol increased 3beta-hydroxysteroid dehydrogenase activity in both progenitor and immature Leydig cells. These findings support the hypothesis that testicular macrophages play an important role in the differentiation of Leydig cells through the secretion of 25-hydroxycholesterol.     

Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.     

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties

Background

Small molecule inhibitors of biologically important protein–glycosaminoglycan (GAG) interactions have yet to be identified.

Methods

Compound libraries were screened in an assay of L-selectin–IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models.

Results

Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity.

Conclusions

A new class of compounds, SMIGs, inhibits protein–GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events.

General significance

SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action — inhibiting protein–GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.     

Purification of valyl-tRNA synthetase high-molecular-mass complex from rabbit liver

A high-molecular-mass complex containing valyl-tRNA synthetase has been purified to homogeneity from rabbit liver. The molecular mass of the complex is about 800 kDa. The complex consists of four polypeptides of 130, 50, 40 and 30 kDa.     

Relative contribution of specific sources of systematic errors and analytical imprecision to metabolite analysis by HPLC–ECD

Objective interpretation of metabolomics data requires understanding both analytical and biological measurement errors. Here we address analytical measurement errors, the sources of these errors, and how this variability can impact metabolomic profiles. Sources considered include room temperature exposure (which could affect sample stability), spiking with authentic standards, the number of study replicates, the overall temporal design of the experimental series, and the complexity of the biological matrix of the samples (individual or pooled sera). The study focused on the analysis of 80 rat sera metabolites by HPLC coupled with coulometric array detectors. Time delay and room temperature exposure had minimal effects on the total relative metabolite concentrations and variability (mean: 94–98% of control, CV_median: ±5–7%), but the concentrations of some specific metabolites were significantly altered. Changes observed in the concentrations of specific metabolites ranged as high as ±7-fold, with changes in variability ranging from 0.3% to 68%. Spiked samples demonstrated more complex behavior when allowed to decay over time than did control samples. The spiking of sera and standards with 43 known metabolites increased variability of the apparent concentrations of metabolites up to 24% as opposed to 3% in pure sera. Increased variability was metabolite-specific. In both pure and spiked sera, 80–95% of metabolites had CVs equivalent to standard analytical CVs for these metabolites. Experimental design, number of replicates, and complexity of the biological matrix had comparable effects. These results suggest that, under carefully controlled conditions, these analytical issues are not significant sources of variability relative to biological variation for most metabolites.This work was supported by NIA-AG15354.     

Simultaneous determination of tricarboxylic acid cycle metabolites by high-performance liquid chromatography with ultraviolet detection

Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.     

G-protein-coupled receptor 1, G-protein Galpha-subunit 1, and prephenate dehydratase 1 are required for blue light-induced production of phenylalanine in etiolated Arabidopsis

Different classes of plant hormones and different wavelengths of light act through specific signal transduction mechanisms to coordinate higher plant development. A specific prephenate dehydratase protein (PD1) was discovered to have a strong interaction with the sole canonical G-protein Galpha-subunit (GPA1) in Arabidopsis (Arabidopsis thaliana). PD1 is a protein located in the cytosol, present in etiolated seedlings, with a specific role in blue light-mediated synthesis of phenylpyruvate and subsequently of phenylalanine (Phe). Insertion mutagenesis confirms that GPA1 and the sole canonical G-protein-coupled receptor (GCR1) in Arabidopsis also have a role in this blue light-mediated event. In vitro analyses indicate that the increase in PD1 activity is the direct and specific consequence of its interaction with activated GPA1. Because of their shared role in the light-mediated synthesis of phenylpyruvate and Phe, because they are iteratively interactive, and because activated GPA1 is directly responsible for the activation of PD1; GCR1, GPA1, and PD1 form all of or part of a signal transduction mechanism responsible for the light-mediated synthesis of phenylpyruvate, Phe, and those metabolites that derive from that Phe. Data are also presented to confirm that abscisic acid can act through the same pathway. An additional outcome of the work is the confirmation that phenylpyruvate acts as the intermediate in the synthesis of Phe in etiolated plants, as it commonly does in bacteria and fungi.     

Dissociation of neuronal and psychophysical responses to local and global motion

Most neurons in cortical area MT (V5) are strongly direction selective, and their activity is closely associated with the perception of visual motion. These neurons have large receptive fields built by combining inputs with smaller receptive fields that respond to local motion. Humans integrate motion over large areas and can perceive what has been referred to as global motion. The large size and direction selectivity of MT receptive fields suggests that MT neurons may represent global motion. We have explored this possibility by measuring responses to a stimulus in which the directions of simultaneously presented local and global motion are independently controlled. Surprisingly, MT responses depended only on the local motion and were unaffected by the global motion. Yet, under similar conditions, human observers perceive global motion and are impaired in discriminating local motion. Although local motion perception might depend on MT signals, global motion perception depends on mechanisms qualitatively different from those in MT. Motion perception therefore does not depend on a single cortical area but reflects the action and interaction of multiple brain systems.