Quantification of Beta Adrenergic Receptor Subtypes in Beta-Arrestin Knockout Mouse Airways

In allergic asthma Beta 2 adrenergic receptors (β₂ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β₂AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β₂AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β₂AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β₂ARs predominate over β₁ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β₂AR expression. These data provide further evidence that β₂ARs are expressed in greater abundance than β₁ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β₂AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference point for future studies exploiting these knockout mice in various disease models including asthma.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Semi-synthetic aristolactams—inhibitors of CDK2 enzyme

Several analogs of aristolochic acids were isolated and derivatized into their lactam derivatives to study their inhibition in CDK2 assay. The study helped to derive some conclusions about the structure–activity relation around the phenanthrin moiety. Semi-synthetic aristolactam 21 showed good activity with inhibition IC₅₀ of 35 nM in CDK2 assay. The activity of this compound was comparable to some of the most potent synthetic compounds reported in the literature.     

Identification of a targeting factor for posttranslational membrane protein insertion into the ER

Hundreds of proteins are anchored in intracellular membranes by a single transmembrane domain (TMD) close to the C terminus. Although these tail-anchored (TA) proteins serve numerous essential roles in cells, components of their targeting and insertion pathways have long remained elusive. Here we reveal a cytosolic TMD recognition complex (TRC) that targets TA proteins for insertion into the ER membrane. The highly conserved, 40 kDa ATPase subunit of TRC (which we termed TRC40) was identified as Asna-1. TRC40/Asna-1 interacts posttranslationally with TA proteins in a TMD-dependent manner for delivery to a proteinaceous receptor at the ER membrane. Subsequent release from TRC40/Asna-1 and insertion into the membrane depends on ATP hydrolysis. Consequently, an ATPase-deficient mutant of TRC40/Asna-1 dominantly inhibited TA protein insertion selectively without influencing other translocation pathways. Thus, TRC40/Asna-1 represents an integral component of a posttranslational pathway of membrane protein insertion whose targeting is mediated by TRC.     

Effect of different light regimes on fitness of two species of Drosophila montium subgroup

Most organisms possess “biological chronometers” in the form of circadian clocks. Organism possessing circadian clock gains fitness advantage in two ways, by synchronizing its behavior through physiological process and secondly by coordinating its internal metabolic process. Environmental manipulations of circadian clocks have been shown to affect many life-history-related traits. Life-history traits are important components of fitness. To enhance individual fitness, organism has to synchronize the physiology with the surrounding environment. The present investigations were made to understand whether rhythm changes affect fitness of two co-existing species of montium a subgroup of Drosophila. The stocks were maintained at 20 ± 1 °C with 75% RH. Fitness such as fecundity, male lifetime fertility, female lifetime fertility, and longevity was assessed in LD (light/dark), LL (continuous light), and DD (continuous dark) for 15 and 30th generations. Fecundity was assessed in 25 pairs of flies for 20 days, and fertility and longevity was assessed in 10 pairs of flies until lifetime. The result revealed differential effect of light regimes on the two different species of Drosophila. Although the two species are related, effect of the three light regimes, LD, LL, and DD on them was different. It is evident that these two species although genetically related exhibit different responses to different light regimes.     

Fen1 mutations that specifically disrupt its interaction with PCNA cause aneuploidy-associated cancer

DNA replication and repair are critical processes for all living organisms to ensure faithful duplication and transmission of genetic information. Flap endonuclease 1 (Fen1), a structure-specific nuclease, plays an important role in multiple DNA metabolic pathways and maintenance of genome stability. Human FEN1 mutations that impair its exonuclease activity have been linked to cancer development. FEN1 interacts with multiple proteins, including proliferation cell nuclear antigen (PCNA), to form various functional complexes. Interactions with these proteins are considered to be the key molecular mechanisms mediating FEN1's key biological functions. The current challenge is to experimentally demonstrate the biological consequence of a specific interaction without compromising other functions of a desired protein. To address this issue, we established a mutant mouse model harboring a FEN1 point mutation (F343A/F344A, FFAA), which specifically abolishes the FEN1/PCNA interaction. We show that the FFAA mutation causes defects in RNA primer removal and long-patch base excision repair, even in the heterozygous state, resulting in numerous DNA breaks. These breaks activate the G2/M checkpoint protein, Chk1, and induce near-tetraploid aneuploidy, commonly observed in human cancer, consequently elevating the transformation frequency. Consistent with this, inhibition of aneuploidy formation by a Chk1 inhibitor significantly suppressed the cellular transformation. WT/FFAA FEN1 mutant mice develop aneuploidy-associated cancer at a high frequency. Thus, this study establishes an exemplary case for investigating the biological significance of protein-protein interactions by knock-in of a point mutation rather than knock-out of a whole gene.