RETRACTED ARTICLE: Age-dependent Increase in Desmosterol Restores DRM Formation and Membrane-related Functions in Cholesterol-free DHCR24−/− Mice

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24^−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24^−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24^−/− mouse. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Structure and electrostatic property of cytoplasmic domain of ZntB transporter

ZntB is the distant homolog of CorA Mg²⁺ transporter within the metal ion transporter superfamily. It was early reported that the ZntB from Salmonella typhimurium facilitated efflux of Zn²⁺ and Cd²⁺, but not Mg²⁺. Here, we report the 1.90 Å crystal structure of the intracellular domain of ZntB from Vibrio parahemolyticus. The domain forms a funnel-shaped homopentamer that is similar to the full-length CorA from Thermatoga maritima, but differs from two previously reported dimeric structures of truncated CorA intracellular domains. However, no Zn²⁺ or Cd²⁺ binding sites were identified in the high-resolution structure. Instead, 25 well-defined Cl⁻ ions were observed and some of these binding sites are highly conserved within the ZntB family. Continuum electrostatics calculations suggest that the central pore of the funnel is highly attractive for cations, especially divalents. The presence of the bound Cl⁻ ions increases the stability of cations along the pore suggesting they could be important in enhancing cation transport.     

Neuropeptide glutamic-isoleucine (NEI) specifically stimulates the secretory activity of gonadotrophs in primary cultures of female rat pituitary cells

The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1–400 × 10⁻⁸ M) and time periods (1–5 h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100 × 10⁻⁸ M) and GnRH (0.1 and 1 × 10⁻⁹ M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4 h of NEI incubation. NEI (100 × 10⁻⁸ M) induced a significant LH secretion after 2 h of stimulus, reaching a maximum response 4 h later. A rapid and remarkable LH release was induced by NEI (400 × 10⁻⁸ M) 1 h after stimulus, attaining its highest level at 2 h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10 × 10⁻⁸ M), caused no action when used alone, its co-incubation with GnRH (1 × 10⁻⁹ M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.     

A versatile chiral selector for determination of enantiomeric composition of fluorescent and nonfluorescent chiral molecules using steady-state fluorescence spectroscopy

A fluorescent chiral molecular micelle (FCMM), poly (sodium N-undecanoyl-L-phenylalaninate) (poly-L-SUF), was developed as a chiral selector for enantiomeric recognition and determination of enantiomeric composition of four fluorescent and four nonfluorescent chiral molecules by use of steady-state fluorescence spectroscopy. The influence of FCMM concentration, buffer pH and complexation medium on FCMM-analyte host-guest complexation, and the emission spectral properties of the resulting complexes were investigated. The chiral interactions of the analytes,1,1'-binaphthyl-2,2'-diamine, 1-(9-anthryl)-2,2,2-trifluoroethanol, propranolol, naproxen, chloromethyl menthyl ether (CME), citramalic acid, tartaric acid, and limonene (LIM), in the presence of poly-L-SUF were based on diastereomeric complex formation. The figures of merit obtained from the partial-least-squares regression modeling of the calibration samples suggested good prediction ability for the validation of six of the eight chiral analytes. Better host-guest complexation of the more hydrophobic molecules, CME and LIM, were obtained in methanol/water mixtures, resulting in better predictability of the regression models. Prediction ability of the models was evaluated by use of the root-mean-square percent relative error (RMS%RE) and was found to range from 1.77 to 15.80% (buffer), 1.26 to 7.95% (25:75 methanol/water), and 1.21 to 4.28% (75:25 methanol/water).     

The Flowering Unit in the Synflorescences of Amaranthaceae

The structure of the synflorescence and the flowering units in Amaranthaceae are characterized. The synflorescence is polytelic. In the flowering unit we recognize the main florescence and the enrichment zone. The florescences may consist of: (1) Fully developed partial florescences bearing three or more flowers; (2) Partial florescences reduced to one or a few fertile flowers having prophylls with more or less modified axillary productions; or (3) No partial florescences but solitary flowers having prophylls with no axillary productions. We described the flowering unit in species with florescences bearing a solitary flower and the flowering unit in species with florescences bearing partial florescences. Hypothesized developmental processes are described, with a view to finding relationships among different models characterized in the family as well as defining characters for cladistic studies, which may be useful to depict all the variations observed.     

Cytometric analysis for drug-induced steatosis in HepG2 cells

Drugs are capable of inducing hepatic lipid accumulation. When fat accumulates, lipids are primarily stored as triglycerides which results in steatosis and provides substrates for lipid peroxidation. An in vitro multiparametric flow cytometry assay was performed in HepG2 cells by using fluorescent probes to analyze cell viability (propidium iodide, PI), lipid accumulation (BODIPY493/503), mitochondrial membrane potential (tetramethyl rhodamine methyl ester, TMRM) and reactive oxygen species generation (ROS) (2′,7′-dihydrochlorofluorescein diacetate, DHCF-DA) as functional markers. All the measurements were restricted to live cells by gating the cells that excluded PI or those that exhibited the typical forward and side scatter features of live cells. The assay was qualified by analyzing a number of selected model drugs with a well documented induction of steatosis in vivo using different mechanisms as positive controls and several non-steatosic compounds as negative controls. For the cytometric screening assay, the concentrations tested were up to the corresponding IC₁₀ value determined by the MTT assay. Among the parameters analyzed, increased BODIPY fluorescence was the most sensitive and selective marker of drug-induced steatosis. However, a more consistent predictive approach was the combination of two endpoints: lipid accumulation and ROS generation. The assay correctly identified 100% of steatosis-positive and steatosis-negative compounds, and a high steatosis risk was predicted for amiodarone, doxycycline, tetracycline and valproate treatments at therapeutic doses. The results suggest that this cell-based assay may be a useful approach to identify the potential of drug candidates to induce steatosis.     

Loss of ��-Dystroglycan Laminin Binding in Epithelium-derived Cancers Is Caused by Silencing of LARGE

The interaction between epithelial cells and the extracellular matrix is crucial for tissue architecture and function and is compromised during cancer progression. Dystroglycan is a membrane receptor that mediates interactions between cells and basement membranes in various epithelia. In many epithelium-derived cancers, β-dystroglycan is expressed, but α-dystroglycan is not detected. Here we report that α-dystroglycan is correctly expressed and trafficked to the cell membrane but lacks laminin binding as a result of the silencing of the like-acetylglucosaminyltransferase (LARGE) gene in a cohort of highly metastatic epithelial cell lines derived from breast, cervical, and lung cancers. Exogenous expression of LARGE in these cancer cells restores the normal glycosylation and laminin binding of α-dystroglycan, leading to enhanced cell adhesion and reduced cell migration in vitro. Our findings demonstrate that LARGE repression is responsible for the defects in dystroglycan-mediated cell adhesion that are observed in epithelium-derived cancer cells and point to a defect of dystroglycan glycosylation as a factor in cancer progression.Normal epithelial cells are tightly associated with one another and with the underlying basement membrane to maintain tissue architecture and function. During cancer progression, primitive cancer cells escape from this control by modifying the binding affinities of their cell membrane receptors. Several receptors have been described as important for this process. Of these, the integrins are the best studied (1). The receptor dystroglycan has been reported to be required for the development and maintenance of epithelial tissues (2, 3). A direct requirement for dystroglycan in epithelia is further demonstrated by the profound effect that loss of dystroglycan expression has on cell polarity and laminin binding in cultured mammary epithelial cells (4, 5). However, dystroglycan is not only important in the establishment and maintenance of epithelial structure. Associations have also been made between the loss of α-dystroglycan immunoreactivity and cancer progression in tumors of epithelial origin, including breast, colon, cervix, and prostate cancers (4, 6–9). The dystroglycan loss of function could thus serve as an effective means by which cancerous cells modify their adhesion to the extracellular matrix (ECM).²Dystroglycan is a ubiquitously expressed cell membrane protein that plays a key function in cellular integrity, linking the intracellular cytoskeleton to the extracellular matrix. The dystroglycan gene encodes a preprotein that is cleaved into two peptides (10). The C-terminal component, known as β-dystroglycan, is embedded within the cell membrane, whereas the N-terminal component, α-dystroglycan, is present within the extracellular periphery but remains associated with β-dystroglycan through non-covalent bonds. β-Dystroglycan binds to actin (11), dystrophin (11), utrophin (11), and Grb2 (12) through its C-terminal intracellular domain. α-Dystroglycan, on the other hand, binds to ECM proteins that contain laminin globular domains including laminins (13, 14), agrin (15), and perlecan (16), as well as to the transmembrane protein neurexin (17). α-Dystroglycan is extensively decorated by three different types of glycan modifications: mucin type O-glycosylation, O-mannosylation, and N-glycosylation. The state of α-dystroglycan glycosylation has been shown to be critical for the ability of the protein to bind to laminin globular domain-containing proteins of the ECM (18).Previous studies of epithelium-derived cancers (4, 9) demonstrated that the loss of immunoreactivity of α-dystroglycan antibodies correlates with tumor grade and poor prognosis. This reduced detection of α-dystroglycan, however, is based on a loss of α-dystroglycan reactivity to antibodies (known as IIH6 and VIA4-1) that recognize the laminin-binding glyco-epitope of α-dystroglycan, i.e. the protein is only functional when it is glycosylated in such a way (henceforth, referred to as functional glycosylation). However, in most of the cancer samples that have been studied to date, β-dystroglycan is expressed at normal levels at the cell membrane. Thus, the aforementioned cancer-associated loss of α-dystroglycan expression may reflect a failure in the post-translational processing of dystroglycan rather than in the synthesis of α-dystroglycan itself.A similar defect in dystroglycan has been reported in a group of congenital muscular dystrophies (19). This spectrum of human developmental syndromes involves the brain, eye, and skeletal muscle and shows a dramatic gradient of phenotypic severity that ranges from the most devastating in Walker-Warburg syndrome to the least severe in limb-girdle muscular dystrophy. Six distinct known and putative glycosyltransferases have been shown to underlie these syndromes: protein O-mannosyltransferase 1 (POMT1), protein O-mannosyltransferase 2 (POMT2), protein O-mannose β-1,2-acetylglucosaminyltransferase 1 (POMGnT1), like acetylglucosaminyltransferase (LARGE), Fukutin, and Fukutin-related protein (FKRP) (20–25). Indeed, all muscular dystrophy patients with mutations in any of these genes fail to express the functionally glycosylated α-dystroglycan epitope that is recognized by the IIH6 and VIA4-1 antibodies.To investigate the molecular mechanism responsible for the loss of α-dystroglycan in epithelium-derived cancers and its role in metastatic progression, we examined the expression and glycosylation status of α-dystroglycan in a group of breast, cervical, and lung cancer cell lines. Here we report that although α-dystroglycan is expressed in the metastatic cell lines MDA-MB-231, HeLa, H1299, and H2030, it is not functionally glycosylated. In screening these cell lines for expression of the six known α-dystroglycan-modifying proteins, we observed that only one, LARGE, was extensively down-regulated. We also report that the ectopic restoration of LARGE expression in these cell lines led not only to the production of a functional dystroglycan but also to the reversion of certain characteristics associated with invasiveness, namely cell attachment to ECM proteins and cell migration.     

Calpain Expression and Activity during Lens Fiber Cell Differentiation

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was αII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.Calpains comprise a family of cysteine proteases named for the calcium dependence of the founder members of the family, the ubiquitously expressed enzymes, calpain 1 (μ-calpain) and calpain 2 (m-calpain). The calpain family includes more than a dozen members with sequence relatedness to the catalytic subunits of calpain 1 and 2. Calpains have a modular domain architecture. By convention, the family is subdivided into classical and nonclassical calpains, according to the presence or absence, respectively, of a calcium-binding penta-EF-hand module in domain IV of the protein (1). Classical calpains include calpain 1, 2, 3, 8, 9, and 11. Nonclassical calpains include calpain 5, 6, 7, 10, 12, 13, and 14.Transgenic and gene knock-out approaches in mice have demonstrated an essential role for calpains during embryonic development. Knock-out of the small regulatory subunit (Capn4) results in embryonic lethality (2, 3). Similarly, inactivation of the Capn2 gene blocks development between the morula and blastocyst stage (4). In humans, mutations in CAPN3 underlie limb-girdle muscular dystrophy-2A, and polymorphisms in CAPN10 may predispose to type 2 diabetes mellitus (5, 6).Even under conditions of calcium overload, where calpains are presumably activated maximally, only a subset (<5%) of cellular proteins are hydrolyzed (7). Calpains typically cleave their substrates at a limited number of sites to generate large polypeptide fragments that, in many cases, retain bioactivity. Thus, under physiological conditions, calpains probably participate in the regulation of protein function rather than in non-specific protein degradation.More than 100 proteins have been shown to serve as calpain substrates in vitro, including cytoskeletal proteins (8), signal transduction molecules (9), ion channels (10), and receptors (11). In vivo, calpains are believed to function in myoblast fusion (12), long term potentiation (13), and cellular mobility (14). Unregulated calpain activity, secondary to intracellular calcium overload, is associated with several pathological conditions, including Alzheimer disease (15), animal models of cataract (16), myocardial (17), and cerebral ischemia (18).In addition to their domain structure, calpains are often classified according to their tissue expression patterns. Calpain 1, 2, and 10 are widely expressed in mammalian tissues, but other members of the calpain family show tissue-specific expression patterns. Calpain 8, for example, is a stomach-specific calpain (19), whereas expression of calpain 9 is restricted to tissues of the digestive tract (20). The expression of calpain 3 was originally thought to be limited to skeletal muscle (21), but splice variants of calpain 3 have since been detected in a range of tissues. At least 12 isoforms of calpain 3 have been described in rodents (22), of which several are expressed in the mammalian eye, including Lp82 (lens), Cn94 (cornea), and Rt88 (retina) (23).Calpains have been studied intensively in the ocular lens because of their suspected involvement in lens opacification (cataract). Calpain-mediated proteolysis of lens crystallin proteins causes increased light scatter (24). Unregulated activation of calpains is observed in rodent cataract models (25), where calpain-mediated degradation of crystallin proteins (26) and cytoskeletal elements (27) is commonly observed. Calpain inhibitors are effective in delaying or preventing cataract in vitro (28, 29) and in vivo (30, 31).It is likely, however, that calpains have important physiological roles in the lens beyond their involvement in tissue pathology. Terminal differentiation of lens fiber cells involves a series of profound morphological and biochemical transformations. For example, differentiating lens fiber cells undergo an enormous (>100-fold) increase in cell length, accompanied by extensive remodeling of the plasma membrane system (32). Early in the differentiation process, fusion pores are established between cells, as neighboring fibers are incorporated into the lens syncytium (33). A later stage of fiber cell differentiation involves the dissolution of all intracellular organelles, a process that is thought to eliminate light-scattering particles from the light path and contribute to the transparency of the tissue (34). Any or all of these phenomena might require the developmentally regulated activation of calpains. This is consistent with our previous observation that in calpain 3 knock-out mice the transition zone is altered, suggesting a change in the differentiation program (35).In the current study, therefore, we examined the depth-dependent expression pattern and activity of calpains in the mouse lens. Fluorogenic substrates were microinjected into the intact lens to visualize calpain activity directly, and proteomic approaches were used to identify endogenous calpain substrates. The cleavage pattern of one of these, αII-spectrin, was examined in detail. Immunocytochemical and immunoblot analysis with wild type and calpain 3-null lenses indicated that αII-spectrin is a specific calpain 3 substrate in maturing lens fiber cells. Together, the data suggest that calpains are activated relatively late in fiber cell differentiation and may contribute to the remodeling of the membrane cytoskeleton that accompanies fiber cell maturation.     

Structural aspects of the distinct biochemical properties of glutaredoxin 1 and glutaredoxin 2 from Saccharomyces cerevisiae

Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower K_M for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK_a of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK_a values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 Å, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity.