A high-throughput method for enzyme kinetic studies

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 degrees C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery.     

Differential cell surface properties of vegetative Bacillus

Aims:  The genus Bacillus encompasses a wide range of species which display varying pathogenic abilities. The hydrophobicity of a range of Bacillus species was determined to evaluate the correlation between bacterial hydrophobicity and pathogenicity.
Methods and Results:  Bacterial adhesion to hydrocarbon assays were used to determine the hydrophobicity of various Bacillus species. Significant differences in the hydrophobicity of vegetative Bacilli were found. Specifically, vegetative Bacillus anthracis or Bacillus thuringiensis cells were highly hydrophobic whereas Bacillus cereus or Bacillus subtilis were only slightly hydrophobic using this test. Cell adhesion assays using A549 or J774 cells were used to demonstrate a correlation between the bacterial hydrophobicity profiles with the ability to adhere to the mammalian cell lines.
Conclusions:  The ability of Bacillus species to adhere to mammalian cell lines correlates with the hydrophobicity of the bacteria and also correlates with the relative pathogenicity of some of the Bacillus species tested.
Significance and Impact of the Study:  This work suggests that study of the physical-chemical properties of vegetative cells could inform future approaches for the rapid identification and discrimination of potentially pathogenic Bacilli .     

Environmental controls over carbon exchange of three forest ecosystems in eastern China

Net ecosystem productivity (NEP) was continuously measured using the eddy covariance (EC) technique from 2003 to 2005 at three forest sites of ChinaFLUX. The forests include Changbaishan temperate mixed forest (CBS), Qianyanzhou subtropical coniferous plantation (QYZ), and Dinghushan subtropical evergreen broad‐leaved forest (DHS). They span wide ranges of temperature and precipitation and are influenced by the eastern Asian monsoon climate to varying extent. In this study, we estimated ecosystem respiration (RE) and gross ecosystem productivity (GEP). Comparison of ecosystem carbon exchange among the three forests shows that RE was mainly determined by temperature, with the forest at CBS exhibiting the highest temperature sensitivity among the three ecosystems. The RE was highly dependent on GEP across the three forests, and the ratio of RE to GEP decreased along the North–South Transect of Eastern China (NSTEC) (i.e. from the CBS to the DHS), with an average of 0.77 ± 0.06. Daily GEP was mainly influenced by temperature at CBS, whereas photosynthetic photon flux density was the dominant factor affecting the daily GEP at both QYZ and DHS. Temperature mainly determined the pattern of the interannual variations of ecosystem carbon exchange at CBS. However, water availability primarily controlled the interannual variations of ecosystem carbon exchange at QYZ. At DHS, NEP attained the highest values at the beginning of the dry seasons (autumn) rather than the rainy seasons (summer), probably because insufficient radiation and frequent fog during the rainy seasons hindered canopy photosynthesis. All the three forest ecosystems acted as a carbon sink from 2003 to 2005. The annual average values of NEP at CBS, QYZ, and DHS were 259 ± 19, 354 ± 34, and 434 ± 66 g C m⁻² yr⁻¹, respectively. The slope of NEP that decreased with increasing latitude along the NSTEC was markedly different from that observed on the forest transect in the European continent. Long‐term flux measurements over more forest ecosystems along the NSTEC will further help verify such a difference between the European forest transect and the NSTEC and provide insights into the responses of ecosystem carbon exchange to climate change in China.     

Rac1 Regulates Endometrial Secretory Function to Control Placental Development

During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.     

Amyloid-Related Memory Decline in Preclinical Alzheimer’s Disease Is Dependent on APOE ε4 and Is Detectable over 18-Months

High levels of β-amyloid (Aβ) in the brain and carriage of the APOE ε4 allele have each been linked to cognitive impairment in cognitively normal (CN) older adults. However, the relationship between these two biomarkers and cognitive decline is unclear. The aim of this study was to investigate the relationship between cerebral Aβ level, APOE ε4 carrier status, and cognitive decline over 18 months, in 317 cognitively healthy (CN) older adults (47.6% males, 52.4% females) aged between 60 and 89 years (Mean = 69.9, SD = 6.8). Cognition was assessed using the Cogstate Brief Battery (CBB) and the California Verbal Learning Test, Second Edition (CVLT-II). Planned comparisons indicated that CN older adults with high Aβ who were also APOE ε4 carriers demonstrated the most pronounced decline in learning and working memory. In CN older adults who were APOE ε4 non-carriers, high Aβ was unrelated to cognitive decline in learning and working memory. Carriage of APOE ε4 in CN older adults with low Aβ was associated with a significantly increased rate of decline in learning and unexpectedly, improved cognitive performance on measures of verbal episodic memory over 18 months. These results suggest that Aβ and APOE ε4 interact to increase the rate of cognitive decline in CN older adults and provide further support for the use of Aβ and APOE ε4 as biomarkers of early Alzheimer’s disease.     

Geographical variation in size in the European rabbit Oryctolagus cuniculus (Lagomorpha: Leporidae) in western Europe and North Africa

Craniometric data of nine characters from 260 skulls of Oryctolagus cuniculus from western Europe and northern Africa were gathered to examine phenotypic variation in size, using both univariate and multivariate analyses. Although animals from the northern part of Europe were found to be larger than those from southern Europe, variation in size occurred in a continuous cline. Skull size was positively correlated with latitude and negatively correlated with ambient temperature. On the basis of skull morphology, there is no evidence for separation of O. cuniculus into the two or more subspecies commonly described. Further data, particularly from genetic material, are required to clarify the taxonomic status of the rabbit in Europe.     

Inhibition of Francisella tularensis LVS infection of macrophages results in a reduced inflammatory response: evaluation of a therapeutic strategy for intracellular bacteria

Francisella tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. A flow cytometric assay was developed to measure bacterial uptake, using a fluorescein isothiocyanate-labelled anti-F. tularensis lipopolysaccharide antibody in conjunction with antibodies to cell surface markers, in order to determine the specific cell phenotypes that were positive for the bacteria. Several phagocytic inhibitors were evaluated in macrophage cell lines and a lung homogenate assay to determine whether the uptake of F. tularensis strain LVS could be altered. Our data show that cytochalasin B, LY294002, wortmannin, nocodazole, MG132 and XVA143 inhibitors reduced LVS uptake by >50% in these assays without having significant cytotoxic effects. Furthermore, a reduction in the inflammatory cytokines monocyte chemoattractant protein-1, interleukin-6 and tumour necrosis factor-α was found in the supernatant of lung tissue infected with LVS when the inhibitory compounds were present. Similarly, there was an alteration in bacterial uptake and a reduction in the inflammatory cytokine response following the administration of wortmannin to LVS-infected mice. Although wortmannin treatment alone did not correlate with the enhanced survival of LVS-infected mice, these inhibitors may have utility in combination therapeutic approaches or against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.