VU6005806/AZN-00016130, an advanced M4 positive allosteric modulator (PAM) profiled as a potential preclinical development candidate

This letter describes progress towards an M₄ PAM preclinical candidate that resulted in the discovery of VU6005806/AZN-00016130. While the thieno[2,3-c]pyridazine core has been a consistent feature of key M₄ PAMs, no work had previously been reported with respect to alternate functionality at the C3 position of the pyridazine ring. Here, we detail new chemistry and analogs that explored this region, and quickly led to VU6005806/AZN-00016130, which was profiled as a putative candidate. While, the β-amino carboxamide moiety engendered solubility limited absorption in higher species precluding advancement (or requiring extensive pharmaceutical sciences formulation), VU6005806/AZN-00016130 represents a new, high quality preclinical in vivo probe.     

A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes

Aims: Despite the fact that the entire genome sequence of probiotic Lactobacillus casei has recently been available, their mechanisms of beneficial effects are poorly clarified, probably because of the lack of an efficient mutagenesis system. The aim of this study was to establish a practical random mutagenesis system of L. casei using the Tn5 transposome complexes. Methods and Results: We optimized the conditions for transformation using a plasmid pUCYIT356‐1‐Not2 and then transposition reaction using Tn5 transposome system for L. casei ATCC 27139. Tn5 insertion library of this strain being consisted of 9408 mutants was constructed by repeating the mutagenesis procedure. To examine the utility of this mutagenesis system, we screened a panel of insertion mutants for nutrient requirements. Six auxotrophic mutants were isolated and their Tn5 insertion sites were determined by inverse PCR, which demonstrated that insertions occur randomly throughout the whole bacterial genome. Conclusions: Tn5 transposome system functioned efficiently to generate transposon insertion mutants of L. casei and enabled to construct useful L. casei Tn5 insertion library at optimized conditions for transformation and transposition. Significance and Impact of the Study: The availability of this system facilitates the study of the mechanisms of beneficial effects of L. casei for human health.     

Modulation of surface proteins by calcium in Chinese hamster lung cells

Investigations on the role of calcium in regulation of cell morphology of Chinese hamster lung cells (V79) revealed that cells grown with additional calcium (5 mM) in the growth medium (Ham's F12) adhere more tightly to the substratum than those grown in F12 alone. Additional calcium in the medium did not cause any changes in the structural membrane proteins or glycoproteins. Radioiodination of the surface membrane proteins of cells grown with or without additional calcium showed distinct differences in the labeling profile. The most striking change observed in cells grown with additional calcium was a very heavily labeled protein band at 70 K molecular weight. Two bands at approx. 100 K and 42 K were also heavily labeled. In contrast, the amount of radioactivity of a protein band at 52 K decreased in the cells grown in additional calcium. In general, cells grown with additional CaCl² were better iodinated than those grown in growth medium alone. The results demonstrate that calcium modulates surface proteins of V79 cells and this modulation may account for the changes observed in the cell morphology.     

The effects of alcohol on rat placenta

In this study, daily food and water consumption and body weights, histopathology of placenta, tenascin (TN), type IV collagen and EGF and its receptor immunolocalization in the placenta of albino rats treated with two doses of alcohol (1 and 5 g kg(-1) day(-1)) were determined. Alcohol was administered in three different periods i.e. the whole 4 weeks before the pregnancy, during the pregnancy, and during the 4 weeks before the pregnancy plus pregnancy itself. The samples of placenta obtained from control and treated rats on days 10, 12, 14, 16, 18, 20 and 21 of gestation were evaluated morphologically and fixed for histology and immunohistochemistry. Some differences in food and water consumption between the groups were determined. The placental weight, especially in the groups receiving 1 and 5 g kg(-1) day(-1) alcohol during the pregnancy, showed increases. The changes in placental histology such as increases in the number and the size of trophoblastic giant cells, cytoplasmic dissolution and nuclear polymorphism, degenerations in spongiotrophoblasts, hyperemia at the basal zone and labyrinth, hyperplasia at the labyrinth and irregular vascularization were seen particularly in the groups receiving alcohol during the pregnancy, and during the 4 weeks before the pregnancy plus pregnancy itself. Increases in the immunolocalization of TN and type IV collagen and decreases in the immunolocalization of EGF and EGFR in the placentas of alcohol-receiving rats were found. In conclusion, ethanol treatment during pregnancy in rats affected placentation and the immunolocalization of TN, type IV collagen, EGF and EGFR in the placentas.     

Hypertrophic cardiomyopathy-causing Asp175asn and Glu180gly Tpm1 mutations shift tropomyosin strands further towards the open position during the ATPase cycle

To understand the molecular mechanism by which the hypertrophic cardiomyopathy-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin alter contractile regulation, we labeled recombinant wild type and mutant α-tropomyosins with 5-iodoacetamide-fluorescein and incorporated them into the ghost muscle fibers. The orientation and mobility of the probe were studied by polarized fluorimetry at different stages of the ATPase cycle. Multistep alterations in the position and mobility of wild type tropomyosin on the thin filaments during the ATP cycle were observed. Both mutations were found to shift tropomyosin strands further towards the open position and to change the affinity of tropomyosin for actin, with the effect of the Glu180Gly mutation being greater than Asp175Asn, showing an increase in the binding strong cross-bridges to actin during the ATPase cycle. These structural changes to the thin filament are likely to underlie the observed increased Ca²⁺-sensitivity caused by these mutations which initiates the disease remodeling.     

ER stress impairs MHC Class I surface expression and increases susceptibility of thyroid cells to NK-mediated cytotoxicity

We recently reported that, in thyroid cells, ER stress triggered by thapsigargin or tunicamycin, two well known ER stressing agents, induced dedifferentiation and loss of the epithelial phenotype in rat thyroid cells. In this study, we sought to evaluate if, in thyroid cells, ER stress could affect MHC class I expression and the possible implications of this effect in the alteration of function of natural killer cells, suggesting a role in thyroid pathology. In both, a human line of fetal thyroid cells (TAD-2 cells) and primary cultures of human thyroid cells, thapsigargin and tunicamicin triggered ER stress evaluated by BiP mRNA levels and XBP-1 splicing. In both cell types, TAD-2 cell line and primary cultures, major histocompatibility complex class I (MHC-I) plasmamembrane expression was significantly reduced by ER stress. This effect was accompanied by signs of natural killer activation. Thus, natural killer cells dramatically increased IFN-γ production and markedly increased their cytotoxicity against thyroid cells. Together, these data indicate that ER stress induces a decrease of MHC class I surface expression in thyroid cells, resulting in reduced natural killer-cell self-tolerance.     

Water quality and diversity of the Recent ostracod fauna in lowland springs from Lombardy (northern Italy)

Sedimentary records provide important information for understanding changes in the history of eutrophication in Lake Taihu. In addition, the catchment nutrient model SWAT provides a powerful tool to examine eutrophic changes in a long-term context. Since it is difficult to evaluate impacts of natural eutrophic development and anthropogenic changes in catchment discharge and land use, simulation of past changes provides a mirror on processes and dynamics. Boundaries in the simulations are set to a pre-industrial time to evaluate natural-agricultural nutrient changes in Taihu Basin a 100 years ago. Total nitrogen (TN) and total phosphorus (TP) in the main channel flowing into the lake are simulated in four sub-basins for 200 model years. Results show that modeling can capture basic features of basin nutrient development, where mean TN concentration (0.12 mg l⁻¹) can be compared in broad scale to mean TN concentration (0.17 mg kg⁻¹) from Lake Taihu sedimentary cores dating back about 100 years. Spatial nutrient simulations suggest that the two major nutrient sources are from the southwestern sub-basin (48% TN and 68% TP of the basin total) and the northwestern sub-basin (18% TN and 17% TP). There are differences of +7.3 × 10⁴ kg TN and +2.0 × 10⁵ kg TP between total input and output values, simulating mean annual amounts of nutrient deposited into the lake. TN and TP concentration differences between input and output sub-basins become smaller in the second 100 years than the first 100 years, suggesting a 100 year period to reach a balance of net nutrients. Catchment nutrient modeling provides a basis to evaluate how nutrient production and balance responded to environmental changes over 200 years in Taihu Basin.     

富营养水体中总氮与总磷比对苦草生长的影响

在室外受控实验条件下,研究了富营养水体中不同氮磷比(TN/TP)(12.5∶1、25∶1、50∶1)对苦草(Vallisneria spiralis)生长的影响.结果表明:当TN/TP为25∶1时,苦草的生长状态最好,苦草生物量的增长率最大,分别高于TN/TP为12.5∶1和50∶1时的54%和31%;当TN/TP为25∶1时,附着生物尤其是附着藻类的生长状态最差,对苦草生长的不利影响最小;可见,富营养水体中适宜于苦草生长的TN/TP为25∶1;TN/TP为25∶1这一比例的应用具有相对性,当水体营养浓度低于限制水平时,该比例适用,如果水体中营养盐浓度超过限制水平时,该比例则不适用.