High-content analysis of CCR2 antagonists on human primary monocytes

The monocyte chemoattractant protein 1 (MCP-1)-driven activation of CC-type chemokine receptor 2 (CCR2) is one of the early key events to induce monocyte migration toward centers of inflammation. In this work, the authors analyzed MCP-1 internalization into primary human monocytes using partially automated liquid handling, automated fluorescence microscopic imaging, and a specific image analysis algorithm. A fluorophore-conjugated form of MCP-1 was rapidly endocytosed and retained by the monocytes. The CCR2 dependency of the MCP-1 internalization was demonstrated by the use of BMS CCR2 22, a CCR2-specific antagonist. The apparent inhibitory potencies of a series of small-molecule CCR2 antagonists were determined and compared in five assay formats, including the high-content analysis assay described in this work. Interestingly, some but not all antagonists showed markedly different inhibitory behaviors in the five readout systems, with an up to more than 100-fold difference between the highest and the lowest apparent inhibitory potencies. These findings raise the distinct possibility that some CCR2 antagonists are capable of discriminating between different functional states of the CCR2 receptor(s) and suggest strategies for the identification of functionally selective CCR2 antagonists with increased therapeutic advantage over nonselective antagonists.     

Effects of Piper hispidinervum on spermatogenesis and histochemistry of ovarioles of Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), not only damages crops, but controlling its population also requires synthetic insecticides, which leads to selection of resistant populations and environmental contamination. Essential oils are an alternative for controlling this insect. There are few studies of the effects of these oils on the insect's reproductive system. We evaluated the effects of the long pepper, Piper hispidinervum, essential oil on the gonads of the armyworm and tested its possible influence on the fertility of this insect. Dosages of 30 and 50 mg/ml were tested in 3^rd instar caterpillars using the leaf immersion method. Testes and ovarioles were collected, fixed with 10% formalin and embedded in Historesin. The sections were stained with toluidine blue and Mallory trichrome to detect connective tissue, periodic acid-Schiff to detect neutral carbohydrates, and bromophenol blue to detect proteins. We found that the long pepper essential oil affected negatively the spermatogenesis and altered the histochemistry of the ovarioles of S. frugiperda. The effects of long pepper oil suggest that it is a promising tool for controlling the armyworm pest.     

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50–60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.     

Maturation of human tripeptidyl-peptidase I in vitro

Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal aminopeptidase that cleaves off tripeptides from the free N termini of oligopeptides and also shows minor endopeptidase activity. TPP I is synthesized as a preproenzyme. Its proenzyme autoactivates under acidic conditions in vitro, resulting in a rapid conversion into the mature form. In this study, we examined the process of maturation in vitro of recombinant latent human TPP I purified to homogeneity from secretions of Chinese hamster ovary cells overexpressing TPP I cDNA. Autoprocessing of TPP I proenzyme was carried out at a wide pH range, from approximately 2.0 to 6.0, albeit with different efficiencies depending on the pH and the type of buffer. However, the acquisition of enzymatic activity in the same buffer took place in a narrower pH "window," usually in the range of 3.6-4.2. N-terminal sequencing revealed that mature, inactive enzyme generated during autoactivation at higher pH contained N-terminal extensions (starting at 6 and 14 amino acid residues upstream of the prosegment/mature enzyme junction), which could contribute to the lack of activity of TPP I generated in this manner. Autoprocessing was not associated with any major changes of the secondary structure of the proenzyme, as revealed by CD spectroscopy. Both the activation and proteolytic processing of the recombinant TPP I precursor were primarily concentration-independent. The addition of the mature enzyme did not accelerate the processing of the proenzyme. In addition, the maturation of the proenzyme was not affected by the presence of glycerol. Finally, the proenzyme with the active site mutated (S475L) was not processed in the presence of the wild-type enzyme. All of these findings indicate a primarily intramolecular (unimolecular) mechanism of TPP I activation and autoprocessing and suggest that in vivo mature enzyme does not significantly participate in its own generation from the precursor.     

Whole-Embryo Modeling of Early Segmentation in Drosophila Identifies Robust and Fragile Expression Domains

Segmentation of the Drosophila melanogaster embryo results from the dynamic establishment of spatial mRNA and protein patterns. Here, we exploit recent temporal mRNA and protein expression measurements on the full surface of the blastoderm to calibrate a dynamical model of the gap gene network on the entire embryo cortex. We model the early mRNA and protein dynamics of the gap genes hunchback, Kruppel, giant, and knirps, taking as regulatory inputs the maternal Bicoid and Caudal gradients, plus the zygotic Tailless and Huckebein proteins. The model captures the expression patterns faithfully, and its predictions are assessed from gap gene mutants. The inferred network shows an architecture based on reciprocal repression between gap genes that can stably pattern the embryo on a realistic geometry but requires complex regulations such as those involving the Hunchback monomer and dimers. Sensitivity analysis identifies the posterior domain of giant as among the most fragile features of an otherwise robust network, and hints at redundant regulations by Bicoid and Hunchback, possibly reflecting recent evolutionary changes in the gap-gene network in insects.