UCN-01 alters phosphorylation of Akt and GSK3beta and induces apoptosis in six independent human neuroblastoma cell lines

In this study we evaluated UCN-01, a small molecule that inhibits protein kinases by interacting with the ATP-binding site, as a potential anti-cancer agent for neuroblastoma. UCN-01 was effective at inducing apoptosis in six neuroblastoma cell lines with diverse cellular and genetic phenotypes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assays, detection of active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) confirmed that UCN-01 induced apoptosis. Cell cycle analysis determined that the UCN-01 treated cells accumulated in S phase by 16 h. Unlike vinblastine and docetaxel that increased survivin expression, UCN-01 treatment did not increase X-linked inhibitor of apoptosis protein (XIAP) and survivin levels. Analysis of specific phosphoepitopes on chk1/2, Akt, and GSK3beta following UCN-01 treatment determined that there was no significant change in phospho-chk1/2. However, there was decreased immunoreactivity at Ser473 and Thr308 of Akt and Ser9 of GSK3beta by 4 h indicating that the Akt survival pathway and downstream signalling was compromised. Thus, UCN-01 was effective at inducing apoptosis in neuroblastoma cell lines.     

Molecular cloning and expression analysis of the mevalonate kinase gene from Arabidopsis thaliana

Mevalonate kinase (MVK), the enzyme that catalyzes the phosphorylation of mevalonate to produce mevalonate 5-phosphate, is considered as a potential regulatory enzyme of the isoprenoid biosynthetic pathway. The Arabidopsis thaliana MVK gene corresponding to the MVK cDNA previously isolated has been cloned and characterized. RNAse protection analysis indicated that the expression of the MVK gene generates three mRNA populations with 5 ends mapping 203, 254 and 355 nt upstream of the MVK ATG start codon. Northern blot analysis showed that the MVK mRNA accumulates preferentially in roots and inflorescences. Histochemical analysis, with transgenic A. thaliana plants containing a translational fusion of a 1.8 kb fragment of the 5 region of the MVK gene to the -glucuronidase (GUS) reporter gene, indicated that the MVK 5-flanking region directs widespread expression of the GUS gene throughout development, although the highest levels of GUS activity are detected in roots (meristematic region) and flowers (sepals, petals, anthers, style and stigmatic papillae). The expression pattern of the MVK gene suggests that the role of the encoded MVK is the production of a general pool of mevalonate-5-phosphate for the synthesis of different classes of isoprenoids involved in both basic and specialized plant cell functions. Functional promoter deletion analysis in transfected A. thaliana protoplasts indicated that regulatory elements between positions –295 and –194 of the MVK 5-flanking region are crucial for high-level MVK gene expression.     

Production of C2 toxin by Alexandrium tamarense CI01 using different culture methods

The dinoflagellate Alexandrium tamarense CI01 wasgrown in three types of cultures: batch culture, semi-continuous culture andtemporary culture, to investigate the effects of different culture methods oncell growth and the productivity of C2 toxin (C2, a paralytic shellfish toxin).In the batch cultures, cells grew in three phases: a short lag phase, anexponential phase with a specific growth rate () of 0.78day^–1 and a relatively long stationary phase. Themaximum toxin productivity was 1.2 mol L^–1 or77 fmol cell^–1 in 9 days. In the semi-continuouscultures, cells grewin response to the dilution cycles, with values being 0.64, 0.32 and 0.35day^–1 for one-day, two-day and three-day cycles,respectively. The toxin yield was about one half of that of the batch cultures.A "temporary" culture method was used to maintain the metabolically activecellsremoved from the semi-continuous cultures, in a nutrient-depleted condition, toachieve a high toxin productivity of 1.0 molL^–1 in 4 days. Thus,the semi-continuous culture method provided an efficient means to generateamounts of metabolically active algal cells. The temporary culture offered aneffective way to produce C2. The highest yields of C2 were obtained in3–4days when the temporary cultures were aerated.     

Separation of epithelial cells from suspensions of cells from the hamster parotid gland in an isokinetic density gradient of Ficoll in tissue culture medium.

Epithelial cells were separated from suspensions of hamster parotid cells by velocity sedimentation in an isokinetic gradient and by isopycnic sedimentation. Epithelial cells were 48.1 ± 18.0% of the cells in the starting sample suspensions of cells from the disaggregated hamster parotid glands. The purest gradient fractions following velocity sedimentation in a previously described isokinetic gradient contained 98.8 ± 1.8% epithelial cells. The purest fractions obtained from isopycnic sedimentation contained 99.9 ± 0.2% epithelial cells. Purification of parotid epithelial cells by velocity sedimentation in the isokinetic gradient seems preferable to purification using isopycnic centrifugation because a larger proportion of the epithelial cells are obtained in the zone of the gradient which contains highly purified epithelial cells and because velocity sedimentation requires lower centrifugal forces for a shorter period of time.     

The Genetic Variant of SARS-CoV-2: would It Matter for Controlling the Devastating Pandemic?

The pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is far from being controlled despite the great effort that have been taken throughout the world. Herd immunity through vaccination is our major expectation to rein the virus. However, the emergence of widespread genetic variants could potentially undermine the vaccines. The evidence that some variants could evade immune responses elicited by vaccines and previous infection is growing. In this review, we summarized the current understanding on five notable genetic variants, i.e., D614G, Cluster 5, VOC 202012/01, 501Y.V2 and P.1, and discussed the potential impact of these variants on the virus transmission, pathogenesis and vaccine efficacy. We also highlight that mutations in the N-terminal domain of spike protein should be considered when evaluating the antibody neutralization abilities. Among these genetic variants, a concern of genetic variant 501Y.V2 to escape the protection by vaccines was raised. We therefore call for new vaccines targeting this variant to be developed.     

SRA的RNA和蛋白质均有转录辅激活功能

类固醇受体激活物（steroid receptor activator, SRA）是一种 类固醇受体辅激活物.最初的研究认为,SRA只存在RNA形式,不存在蛋白质 形式.但是后来的研究发现,SRA是在RNA和蛋白质两个水平上发挥功能的分 子,其cDNA序列存在687 bp保守的核心区域,该核心区域对其发挥转录共激 活活性是必需的.SRA的RNA形式主要参与核受体的转录共激活作用,其表达 与乳腺癌的发生有很大关系,SRA的蛋白质形式（steroid receptor activator protein ,SRAP）也具有类似的功能.但是不同于RNA形式, SRAP可结合到特定基因的启动子区域,并起到阻遏物的作用.本文对SRA的 特点、表达及功能等方面的最新研究进展及其可能的作用机制与作用形式 进行概述.     

Thermal niche for in situ seed germination by Mediterranean mountain streams: model prediction and validation for Rhamnus persicifolia seeds

Background and Aims

Mediterranean mountain species face exacting ecological conditions of rainy, cold winters and arid, hot summers, which affect seed germination phenology. In this study, a soil heat sum model was used to predict field emergence of Rhamnus persicifolia, an endemic tree species living at the edge of mountain streams of central eastern Sardinia.

Methods

Seeds were incubated in the light at a range of temperatures (10–25 and 25/10 °C) after different periods (up to 3 months) of cold stratification at 5 °C. Base temperatures (T_b), and thermal times for 50 % germination (θ₅₀) were calculated. Seeds were also buried in the soil in two natural populations (Rio Correboi and Rio Olai), both underneath and outside the tree canopy, and exhumed at regular intervals. Soil temperatures were recorded using data loggers and soil heat sum (°Cd) was calculated on the basis of the estimated T_b and soil temperatures.

Key Results

Cold stratification released physiological dormancy (PD), increasing final germination and widening the range of germination temperatures, indicative of a Type 2 non-deep PD. T_b was reduced from 10·5 °C for non-stratified seeds to 2·7 °C for seeds cold stratified for 3 months. The best thermal time model was obtained by fitting probit germination against log °Cd. θ₅₀ was 2·6 log °Cd for untreated seeds and 2·17–2·19 log °Cd for stratified seeds. When θ₅₀ values were integrated with soil heat sum estimates, field emergence was predicted from March to April and confirmed through field observations.

Conclusions

T_b and θ₅₀ values facilitated model development of the thermal niche for in situ germination of R. persicifolia. These experimental approaches may be applied to model the natural regeneration patterns of other species growing on Mediterranean mountain waterways and of physiologically dormant species, with overwintering cold stratification requirement and spring germination.     

Pharmacological properties of native CaCCs and TMEM16A

We previously reported that TREK-1 gating by internal pH and pressure occurs close to or within the selectivity filter. These conclusions were based upon kinetic measurements of high-affinity block by quaternary ammonium (QA) ions that appeared to exhibit state-independent accessibility to their binding site within the pore. Intriguingly, recent crystal structures of two related K2P potassium channels were also both found to be open at the helix bundle crossing. However, this did not exclude the possibility of gating at the bundle crossing and it was suggested that side-fenestrations within these structures might allow state-independent access of QA ions to their binding site. In this addendum to our original study we demonstrate that even hydrophobic QA ions do not access the TREK-1 pore via these fenestrations. Furthermore, by using a chemically reactive QA ion immobilized within the pore via covalent cysteine modification we provide additional evidence that the QA binding site remains accessible to the cytoplasm in the closed state. These results support models of K2P channel gating which occur close to or within the selectivity filter and do not involve closure at the helix bundle crossing.