Substrate Compliance versus Ligand Density in Cell on Gel Responses

Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on “rigid” glass or “stiff” gels than on “soft” gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.     

靴隼雕繁殖习性初报

国内关于靴隼雕(Hieraaetus pennata)资料缺乏,仅有几例新纪录报道,无繁殖信息。本文报道2010~2016年间靴隼雕在新疆南部和北部的繁殖与分布状况。靴隼雕巢址选择开阔的地带,营巢于大树上(n=7),巢距地面高度在7~12 m,巢直径约74~102 cm。窝卵数2~3枚,孵卵期37~40 d,育雏期48~58 d,繁殖期持续4~5个月。育雏前期与后期亲鸟的行为变化较大。其巢区附近野生动物资源丰富,食物以鸟类为主,主要是水鸟的幼鸟(体重小于300 g);哺乳类包括草兔(Lepus capensis)、大耳猬(Hemiechinus auritus)、鼹形田鼠(Ellobius talpinus)等。在新疆和硕利用红外相机监测32 d,共收获77 894张图片。育雏期人工观察16 d,约248 h,同时拍下行为照片及录像作为辅助记录。育雏前期,雌鸟的陪护时间占88.43%,雄鸟只有3.26%。亲鸟育雏期的活动节律、投食次数呈现单峰型;续巢频次则为双峰型。在中亚,靴隼雕有明显"东扩"之趋势。通过野外观察靴隼雕的行为,了解繁殖状况,积累基础资料,对其种群保护和管理具有意义。     

Phospholipid diversity: Correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called “fusion proteins”. This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

Differential PKC-dependent and -independent PKD activation by G protein α subunits of the Gq family: selective stimulation of PKD Ser⁷⁴⁸ autophosphorylation by Gαq

Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gαq-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family Gα and stimulated with aluminum fluoride (AlF4⁻). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser⁷⁴⁴, a prominent and rapid PKC transphosphorylation site, and Ser⁷⁴⁸, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4⁻ potently induced PKD activation and Ser⁷⁴⁴ and Ser⁷⁴⁸ phosphorylation, in the presence of cotransfected Gαq, Gα11, Gα14 or Gα15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked Gα11-, Gα14-, and Gα15-mediated enhancement of Ser⁷⁴⁸ phosphorylation induced by AlF4⁻, and largely abolished Ser⁷⁴⁴ phosphorylation. In contrast, Ser⁷⁴⁸ phosphorylation was almost completely intact, and Ser⁷⁴⁴ phosphorylation was significantly activated in cells cotransfected with Gαq. Importantly, the differential Ser⁷⁴⁸ phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gαq but not Gα11. Taken together, our results suggest that Gαq, but not the closely related Gα11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser⁷⁴⁸.     

Staurosporine-induced apoptosis presents with unexpected cholinergic effects in a differentiated neuroblastoma cell line

Apoptosis of cholinergic neurons is one of the core hallmarks of Alzheimer’s disease. SH-SY5Y neuroblastoma cells differentiated to the cholinergic phenotype were exposed to 100 nM staurosporine. Over a treatment period of 24 h, the pro- and anti-apoptotic factors, caspase-3 and Bcl-2, as well as LDH release as a measure of cell viability, were assessed in conjunction with the number of apoptotic cells by means of fluorescence-activated cell sorting. Caspase-3 activity and LDH release increased by 30% and 20% over controls, respectively, while Bcl-2 levels rose by 200% over controls. Furthermore, staurosporine treatment resulted in decreased acetylcholinesterase (AChE) enzymatic activity and decreased protein levels of the AChE splice variant tailed AChE (AChE-T). Only a slight increase in levels of readthrough AChE (AChE-R) was observed. Likewise, staurosporine reduced levels and activity of the cholinergic players choline acetyltransferase and high affinity choline uptake. The present study demonstrates that treatment with staurosporine leads to apoptotic events, which, however, are not reflected in the increased AChE activity and the alterations of AChE isoforms expression that are usually seen in apoptotic conditions. The effects of various additional phosphorylation inhibitors on AChE activity suggest that these unexpected cholinergic effects, firstly, are linked to the impact of staurosporine on phosphorylation and, secondly, reveal themselves in a first phase of cellular adaption that precedes neurotoxicity and subsequent cell death.     

The neural adhesion molecule L1CAM confers chemoresistance in human glioblastomas

Glioblastoma multiforme (GBM) represents the most common and malignant brain tumor. GBM tissues exhibit elevated expression of the transforming growth factor-beta1 (TGF-β1) and the adhesion molecule L1CAM. This study investigated the mechanism of L1CAM regulation in GBM cells and its role in the mediation of chemoresistance. L1CAM expression levels varied in GBM cells being highest in A172 cells and low in T98G cells. Inhibition of TGF-β1 signaling in A172 cells reduced L1CAM expression and vice versa stimulation with exogenous TGF-β1 led to upregulation of L1CAM in T98G cells. Additionally, TGF-β1 and L1CAM expression increased during differentiation of glioma stem-like cells. L1CAM expressing GBM cells and differentiated glioma stem-like cells showed a reduced apoptotic response after treatment with the chemotherapeutic drug temozolomide. Accordingly, siRNA-mediated knock-down of L1CAM in A172 cells and differentiated glioma stem-like cells increased chemosensitivity, whereas overexpression of L1CAM in T98G cells and glioma spheroids diminished the apoptotic response. Elevated L1CAM expression caused a diminished expression of caspase-8 in GBM and differentiated glioma stem-like cells. These data show that TGF-β1 dependent upregulation of L1CAM expression in GBM cells leads to the downregulation of caspase-8 and apoptosis resistance pointing to L1CAM as potential target for improved therapy of GBM patients.     

Killing of raptors on grouse moors: evidence and effects

Owing to the intensity of game management in Britain, managers of grouse moors have illegally killed raptors to increase the numbers of Red Grouse Lagopus l. scotica available for shooting. This paper summarizes evidence for the recent scale of illegal raptor killing on grouse moors and its effects on populations. It provides insights into how raptors themselves respond demographically to different levels of killing. Over Britain as a whole, most raptors have increased and expanded considerably since the 1970s, in response to reduced killing and nest destruction, and the diminished impacts of organo‐chlorine pesticides; however, in recent decades the populations of some species have declined on and around grouse moors. This is widely evident in Hen Harrier Circus cyaneus, Peregrine Falcon Falco peregrinus and Golden Eagle Aquila chrysaetos populations and in more restricted areas also in Northern Goshawk Accipiter gentilis and Red Kite Milvus milvus populations, in all of which illegal killing has been sufficient to affect numbers over wider areas. The evidence consists mainly of: (1) greater disappearance of nesting pairs, lower breeding densities or reduced occupancy of apparently suitable traditional territories on grouse moors compared with other areas; (2) reduced nest success compared with other areas; (3) reduced adult survival compared with other areas; (4) reduced age of first breeding, reflecting the removal of adults from nesting territories and their replacement by birds in immature plumage; (5) greater levels of disappearance of satellite‐tracked birds on grouse moors than elsewhere; and (6) the finding of poisoned baits and traps, and shot or poisoned carcasses of raptors. Not all these types of evidence are available for every species, and other types of evidence are available for some. The Common Buzzard Buteo buteo is currently the most numerous raptor in Britain and also seems to be killed in the greatest numbers. Other raptor species, including Merlin Falco columbarius, Common Kestrel Falco tinnunculus and Eurasian Sparrowhawk Accipiter nisus which nest on or near grouse moors, have little or no significant impact on grouse and are killed less often or not at all. In the absence of illegal killing, some raptor species breed as well or better on grouse moors than in other habitats. Merlins, in particular, seem to thrive on grouse moors, benefiting from the management involved (including predator control). Other aspects of illegal raptor killing are discussed, including suggestions for ways in which it might be reduced.     

Reduced cellular Ca availability enhances TDP-43 cleavage by apoptotic caspases

Accumulation of transactive response DNA binding protein (TDP-43) fragments in motor neurons is a post mortem hallmark of different neurodegenerative diseases. TDP-43 fragments are the products of the apoptotic caspases-3 and -7. Either excessive or insufficient cellular Ca²⁺ availability is associated with activation of apoptotic caspases. However, as far as we know, it is not described whether activation of caspases, due to restricted intracellular Ca²⁺, affects TDP-43 cleavage. Here we show that in various cell lineages with restricted Ca²⁺ availability, TDP-43 is initially cleaved by caspases-3 and -7 and then, also by caspases-6 and -8 once activated by caspase-3. Furthermore, we disclose the existence of a TDP-43 caspase-mediated fragment of 15 kDa, in addition to the well-known fragments of 35 and 25 kDa. Interestingly, with respect to the other two fragments this novel fragment is the major product of caspase activity on murine TDP-43 whereas in human cell lines the opposite occurs. This outcome should be considered when murine models are used to investigate TDP-43 proteinopathies.