Phosphorylation of 338SSYY341 regulates specific interaction between Raf-1 and MEK1

The present study characterizes the interaction between the Raf-1 kinase domain and MEK1 and examines whether the magnitude of their interaction correlates to the ability of Raf to phosphorylate MEK1. Here we show that the minimal domain required for the Raf kinase activity starts from tryptophan 342. Maximal binding of the Raf kinase domain to MEK1 and its kinase activity are achieved upon phosphorylation of the region (338)SSYY(341) in response to 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA), or mutation of Y340Y341 to aspartic acids. Conversely, the TPA-stimulated MEK binding and kinase activity are diminished when this region is deleted or Ser(338) and Ser(339) are mutated to alanines. We also show that the integrity of the Raf ATP-binding site is necessary for the interaction between Raf-1 and MEK1. Furthermore, two MEK-binding sites are identified; the first is localized between amino acids 325 and 349, and the second is within the region between amino acids 350 and 648. Separately, the binding of each site to MEK1 is weak, but in a cis context, they give rise to a much stronger association, which can be further stimulated by TPA. Finally, we find that tryptophan 342, which is conserved among the Raf family and other protein kinases, is essential for the Ser(338) phosphorylation of the full-length Raf and its binding to MEK1. Taken together, our results indicate that the phosphorylation of Ser(338) and Tyr(341) on Raf exerts an important effect on reconfiguring the two MEK-binding sites. As a result, these two sites coordinate to form a high affinity MEK-binding epitope, leading to a marked increase in Raf kinase activity.     

心肌细胞和血管平滑肌细胞收缩调控机制的研究进展

心脏和血管构成体内的心血管系统,两者都具有收缩性。心脏收缩要求在很短的时间内升高室内压,因此要求细胞收缩快速和有力,这就需要细胞的收缩结构和钙调控过程能满足其要求。血管收缩缓慢而持久,其收缩结构及机制也正好与之功能相适应。本文从细胞水平讨论了心脏和血管的收缩结构和收缩机制,以及钙调控机制,并分别对两者之间的异同点作了介绍。     

Insertional mutagenesis of a fungal biocontrol agent led to discovery of a rare cellobiose lipid with antifungal activity

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.     

Structural and functional studies with antibodies to the integrin beta 2 subunit. A model for the I-like domain

To establish a structure and function map of the beta2 integrin subunit, we mapped the epitopes of a panel of beta2 monoclonal antibodies including function-blocking, nonblocking, and activating antibodies using human/mouse beta2 subunit chimeras. Activating antibodies recognize the C-terminal half of the cysteine-rich region, residues 522-612. Antibodies that do not affect ligand binding map to residues 1-98 and residues 344-521. Monoclonal antibodies to epitopes within a predicted I-like domain (residues 104-341) strongly inhibit LFA-1-dependent adhesion. These function-blocking monoclonal antibodies were mapped to specific residues with human --> mouse knock-out or mouse --> human knock-in mutations. Combinatorial epitopes involving residues distant in the sequence provide support for a specific alignment between the beta-subunit and I domains that was used to construct a three-dimensional model. Antigenic residues 133, 332, and 339 are on the first and last predicted alpha-helices of the I-like domain, which are adjacent on its "front." Other antigenic residues in beta2 and in other integrin beta subunits are present on the front. No antigenic residues are present on the "back" of the domain, which is predicted to be in an interface with other domains, such as the alpha subunit beta-propeller domain. Most mutations in the beta2 subunit in leukocyte adhesion deficiency are predicted to be buried in the beta2 subunit I-like domain. Two long insertions are present relative to alpha-subunit I-domains. One is tied down to the back of the I-like domain by a disulfide bond. The other corresponds to the "specificity-determining loop" defined in beta1 and beta3 integrins and contains the antigenic residue Glu(175) in a disulfide-bonded loop located near the "top" of the domain.     

林隙（gap）更新动态研究进展

林隙（ｇａｐ）更新动态研究进展臧润国（中国林业科学研究院生态环境研究所,北京１０００９３）ＲｅｓｅａｒｃｈＡｄｖａｎｃｅｓｏｆＧａｐＲｅｇｅｎｅｒａｔｉｏｎＤｙｎａｍｉｃｓ．ＺａｎｇＲｕｎｇｕｏ（ＩｎｓｔｉｔｕｔｅｏｆＥｃｏｌｏｇｙａｎｄＥｎｖｉｒ...     

Combining Serum Cystatin C with Total Bilirubin Improves Short-Term Mortality Prediction in Patients with HBV-Related Acute-On-Chronic Liver Failure

Background & Aims

HBV-related acute-on-chronic liver failure (HBV-ACLF) is a severe liver disease which results in a high mortality in China. To early predict the prognosis of the patients may prevent the complications and improve the survival. This study was aimed to develop a new prognostic index to estimate the survival related to HBV-ACLF.

Methods

Consecutive patients with HBV-ACLF were included in a prospective observational study. Serum Cystatin C concentrations were measured by using the particle-enhanced immunonephelometry assay. All of the patients were followed for at least 3 months. Cox regression analysis was carried out to identify which factors were predictive of mortality. The area under the receiver operating characteristic curve (AUC) was used to evaluate the efficacy of the variates for early predicting mortality.

Results

Seventy-two patients with HBV-ACLF were recruited between January 2012 and January 2013. Thirty patients died (41.7%) during 3-months followed up. Cox multivariate regression analysis identified serum cystatin C (CysC) and total bilirubin (TBil) were independent factors significantly (P < 0.01) associated with survival. Our results further showed that new prognostic index (PI) combining serum CysC with TBil was a good indicator for predicting the mortality of patients with HBV-ACLF. Specifically, the PI had a higher accuracy than the CTP, MELD, or MELD-Na scoring for early prediction short-term survival of HBV-ACLF patients with normal levels of serum creatinine (Cr). The survival rate in low risk group (PI < 3.91) was 94.3%, which was markedly higher than those in the high-risk group (PI ≥ 3.91) (17.4%, P < 0.001).

Conclusion

We developed a new prognostic index combining serum CysC with TBil which early predicted the short-term mortality of HBV-ACLF patients.     

A New Approach to Segment Both Main and Peripheral Retinal Vessels Based on Gray-Voting and Gaussian Mixture Model

Vessel segmentation in retinal fundus images is a preliminary step to clinical diagnosis for some systemic diseases and some eye diseases. The performances of existing methods for segmenting small vessels which are usually of more importance than the main vessels in a clinical diagnosis are not satisfactory in clinical use. In this paper, we present a method for both main and peripheral vessel segmentation. A local gray-level change enhancement algorithm called gray-voting is used to enhance the small vessels, while a two-dimensional Gabor wavelet is used to extract the main vessels. We fuse the gray-voting results with the 2D-Gabor filter results as pre-processing outcome. A Gaussian mixture model is then used to extract vessel clusters from the pre-processing outcome, while small vessels fragments are obtained using another gray-voting process, which complements the vessel cluster extraction already performed. At the last step, we eliminate the fragments that do not belong to the vessels based on the shape of the fragments. We evaluated the approach with two publicly available DRIVE (Staal et al., 2004) and STARE (Hoover et at., 2000) datasets with manually segmented results. For the STARE dataset, when using the second manually segmented results which include much more small vessels than the first manually segmented results as the “gold standard,” this approach achieved an average sensitivity, accuracy and specificity of 65.0%, 92.1% and 97.0%, respectively. The sensitivities of this approach were much higher than those of the other existing methods, with comparable specificities; these results thus demonstrated that this approach was sensitive to detection of small vessels.     

Deletion of aquaporin-4 in APP/PS1 mice exacerbates brain Aβ accumulation and memory deficits

Background

Preventing or reducing amyloid-beta (Aβ) accumulation in the brain is an important therapeutic strategy for Alzheimer’s disease (AD). Recent studies showed that the water channel aquaporin-4 (AQP4) mediates soluble Aβ clearance from the brain parenchyma along the paravascular pathway. However the direct evidence for roles of AQP4 in the pathophysiology of AD remains absent.

Results

Here, we reported that the deletion of AQP4 exacerbated cognitive deficits of 12-moth old APP/PS1 mice, with increases in Aβ accumulation, cerebral amyloid angiopathy and loss of synaptic protein and brain-derived neurotrophic factor in the hippocampus and cortex. Furthermore, AQP4 deficiency increased atrophy of astrocytes with significant decreases in interleukin-1 beta and nonsignficant decreases in interleukin-6 and tumor necrosis factor-alpha in hippocampal and cerebral samples.

Conclusions

These results suggest that AQP4 attenuates Aβ pathogenesis despite its potentially inflammatory side-effects, thus serving as a promising target for treating AD.