Motor-free mitochondrial presequence translocase drives membrane integration of preproteins

The mitochondrial inner membrane is the central energy-converting membrane of eukaryotic cells. The electrochemical proton gradient generated by the respiratory chain drives the ATP synthase. To maintain this proton-motive force, the inner membrane forms a tight barrier and strictly controls the translocation of ions. However, the major preprotein transport machinery of the inner membrane, termed the presequence translocase, translocates polypeptide chains into or across the membrane. Different views exist of the molecular mechanism of the translocase, in particular of the coupling with the import motor of the matrix. We have reconstituted preprotein transport into the mitochondrial inner membrane by incorporating the purified presequence translocase into cardiolipin-containing liposomes. We show that the motor-free form of the presequence translocase integrates preproteins into the membrane. The reconstituted presequence translocase responds to targeting peptides and mediates voltage-driven preprotein translocation, lateral release and insertion into the lipid phase. Thus, the minimal system for preprotein integration into the mitochondrial inner membrane is the presequence translocase, a cardiolipin-rich membrane and a membrane potential.     

Modified Ceria for “Low‐Temperature” CO2 Utilization: A Chemical Looping Route to Exploit Industrial Waste Heat

Efficient CO₂ utilization is key to limit global climate change. Carbon monoxide, which is a crucial feedstock for chemical synthesis, can be produced by splitting CO₂. However, existing thermochemical routes are energy intensive requiring high operating temperatures. A hybrid redox process (HRP) involving CO₂‐to‐CO conversion using a lattice oxygen‐deprived redox catalyst at relatively low temperatures (<700 °C) is reported. The lattice oxygen of the redox catalyst, restored during CO₂‐splitting, is subsequently used to convert methane to syngas. Operated at temperatures significantly lower than a number of industrial waste heat sources, this cyclic redox process allows for efficient waste heat‐utilization to convert CO₂. To enable the low temperature operation, lanthanum modified ceria (1:1 Ce:La) promoted by rhodium (0.5 wt%) is reported as an effective redox catalyst. Near‐complete CO₂ conversion with a syngas yield of up to 83% at low temperatures is achieved using Rh‐promoted LaCeO_4?x. While La improves low‐temperature bulk redox properties of ceria, Rh considerably enhances the surface catalytic properties for methane activation. Density functional theory calculations further illustrate the underlying functions of La‐substitution. The highly effective redox catalyst and HRP scheme provide a potentially attractive route for chemical production using CO₂, industrial waste heat, and methane, with appreciably lowered CO₂ emissions.     

Ral Overactivation in Malignant Peripheral Nerve Sheath Tumors

Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.The Ras family of guanine-nucleotide bound proteins exerts a fundamental role in cell biology and constitutes an important area of cancer research due to its significant involvement in the development and progression of malignancies (8, 10, 17, 18, 32). Ras-like (Ral) proteins are crucial members of this family and have been shown to play a pivotal role in human tumors (7, 28, 41, 66, 70). Because Ral guanine nucleotide exchange factors (Ral-GEFs) are direct effectors of Ras, the Ral signaling pathway has been traditionally considered a Ras-effector pathway. Activation of Ras (in resemblance to Ral) is regulated by two classes of proteins: Ras-GEFs (e.g., SOS) and Ras- GTPase activating proteins (Ras-GAPs such as neurofibromin). The latter induces hydrolysis of Ras from the active (GTP) form to the inactive (GDP) form (13). Ral-GEFs include two main groups: the proteins that are stimulated by Ras because of their carboxy-terminal Ras binding domain (RalGDS, RGL1, and RGL2) and the proteins that are activated by substrates of PI3K through a pleckstrin homology domain on their C-terminal (RALGPS1 and RALGPS2) (19). Although highly similar to Ras, Ral proteins (RalA and RalB) involve a series of distinctly different effectors that influence gene expression and translation through interaction with ZO-1-associated nucleic acid binding protein (ZONAB) and RalA binding protein 1 (RalBP1) (11, 23, 33). RalB directly interacts with the SEC5 subunit of exocyst to facilitate the host defense response (48, 58).In addition to overactivation of GEFs, inactivation of GAPs is another mechanism for overactivation of GTP-bound proteins. The lack of neurofibromin (encoded by NF1 on human chromosome 17q11.2), a Ras-GAP protein, is the main molecular event in neurofibromatosis type 1 (NF-1), an autosomal-dominant human genetic disease occurring in approximately 1 in 2,500 to 3,500 births (22, 27, 42). One of the main tumor-causing effects of inactivating mutations in the tumor suppressor NF1 gene is postulated to be the subsequent activation of Ras (3, 29, 53, 57, 69). With two main functional domains, SEC14 and Ras-GAP, neurofibromin is best known for its Ras-GAP function. Although the yeast SEC14p is shown to be involved in regulating intracellular proteins and lipid trafficking, the function of its homologous domain in neurofibromin is unknown (49, 62). Although neurofibromas are the most common tumors in NF-1, 10% of patients with plexiform develop malignant peripheral nerve sheath tumors (MPNSTs), which are typically high grade and often fatal (21, 34, 65).The molecular events involved in the malignant transformation of benign neurofibromas to MPNST are poorly defined. Usually arising in the third through sixth decades of life, these tumors are composed of tightly packed hyperchromatic spindle-shaped cells with frequent mitotic figures. Inactivation of both copies of the NF1 gene has been demonstrated in benign human neurofibromas and shown to cause tumors in murine models (56). Loss of heterozygosity of NF1 and p53 has frequently been observed in human MPNST (35, 47, 54). Recombinant mouse strains (NP mice), which harbor inactivated Nf1 and p53 alleles (cis-Nf1^+/−:p53^+/−), demonstrate the cumulative effects of loss of both Nf1 and p53 genes in the etiology of MPNST (14, 68).In the present study, we show that while both Ras activation and activation of a series of its downstream effector pathways are observed in a fraction of MPNST cells, RalA is activated globally in all studied mouse and human MPNST cells and tumor samples. Our results also explain the involvement of this signaling molecule in a series of key biological functions of MPNST cells, as shown in a variety of in vitro assays and an in vivo model of MPNST. Such information may play a role in designing novel therapies for treatment of MPNST or other tumors with overactivation of the Ral pathway.     

Vitamin C and vitamin E restore the resistance of GSH-depleted lens cells to H2O2

A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether other antioxidants can compensate for GSH depletion in protection against oxidative insults. Rabbit lens epithelial cells were depleted of > 75% of intracellular GSH by 25-200 microM buthionine sulfoximine (BSO). Depletion of GSH by BSO alone had little direct effect on cell viability, but resulted in an approximately 30-fold increase in susceptibility to H(2)O(2)-induced cell death. Experimentally enhanced levels of nonprotein sulfhydryls other than GSH (i.e., N-acetylcysteine) did not protect GSH-depleted cells from H(2)O(2)-induced cell death. In contrast, pretreatment of cells with vitamin C (25-50 microM) or vitamin E (5-40 microM), restored the resistance of GSH-depleted cells to H(2)O(2). However, concentrations of vitamin C > 400 microM and vitamin E > 80 microM enhanced the toxic effect of H(2)O(2). Although levels of GSH actually decreased by 10-20% in cells supplemented with vitamin C or vitamin E, the protective effects of vitamin C and vitamin E on BSO-treated cells were associated with significant ( approximately 70%) decreases in oxidized glutathione (GSSG) and concomitant restoration of the cellular redox status (as indicated by GSH:GSSG ratio) to levels detected in cells not treated with BSO. These results demonstrate a role for vitamin C and vitamin E in maintaining glutathione in its reduced form. The ability of vitamin C and vitamin E in compensations for GSH depletion to protect against H(2)O(2)-induced cell death suggests that GSH, vitamin C, and vitamin E have common targets in their actions against oxidative damage, and supports the preventive or therapeutic use of vitamin C and E to combat age- and pathology-associated declines in GSH. Moreover, levels of these nutrients must be optimized to achieve the maximal benefit.     

On the Mathematical Reconstruction of Two Dimensional Plants

A set of 10, chosen medicinal plants (some of them with a reputation as remedies for tuberculosis) has been investigated through Partitioned Iterated Function Systems-Semi Fractals with Angle (PIFS-SFA) coding, Lempel, Ziv, Welch with quantization and noise (LZW-QN) compression, and surface density statistics (f(α)-SDS) discrimination techniques. The final outcomes of this quantitative analysis were, firstly: the linear ordering of the plants in question accompanied by the hope that it reflects their medical significance, secondly: the mathematical representation of each of the plants, and thirdly: the impressive compression achieved, leading to remarkable computer memory saving, and still permitting successful pattern recognition i.e., proper identification of the plant from the compressed image.     

An excursion through the ultrastructural world of quick-frozen pancreatic islets

In this article we have presented a philosophical and historical perspective on quick freezing, freeze-drying, freeze-substitution, and immunocytochemical localization of pancreatic islet hormones. A compilation of our findings indicates that quick-freezing does not produce any gross distortion of islet tissue; the amount of usable islet tissue for ultrastructural analysis is approximately 13 micron deep from the frozen edge; three different cell types can be identified in quick-frozen tissue based on general morphological characteristics; freeze-substitution with tetrahydrofuran produces a unique ultrastructural appearance in which ribosomes are particularly striking; with the use of protein A-gold, insulin and glucagon can be localized immunocytochemically on silver-gray (50-nm-thick) sections treated with 1% ovalbumin at room temperature overnight; secretory granules of quick-frozen alpha and beta cells may exist in either a swollen or condensed state; swollen beta cell secretory granules contain a filamentous material that demonstrates immunogold labeling for insulin; insulin and glucagon can be localized within the cisternae of endoplasmic reticulum; our methods provide not only discrete immunocytochemical localization of hormone, but also well-preserved cellular compartments; energy electron loss spectroscopy (EELS) has shown that quantifiable nitrogen maps can be used as an index of hormone packaging in secretory granules; and the sectioning properties of secretory granules at the ultramicrotome change when islet tissue is unosmicated and sectioned on glycerol.     

Intracellular electrophysiology of mammalian peptidergic neurons in rat hypothalamic slices

The magnocellular neuropeptidergic cells (MNCs) of the paraventricular and supraoptic nuclei have been a model for biochemical and physiological studies of peptidergic neurons in the mammalian brain, but nearly all the electrophysiological studies of these vasopressinergic and oxytocinergic neuroendocrine cells are based on extracellular recordings. This paper reviews recent literature on electrophysiological properties of neurons in the magnocellular nuclei in which the rat in vitro slice preparation and intracellular recording were used. Spontaneously occurring action potentials and synaptic potentials (excitatory and inhibitory) have been observed in hypothalamic slices. The spike patterns have included slow and irregular firing, short rapid bursts of inactivating spikes, and slow phasic discharge with prolonged active and silent periods. Some studies have shown that increased osmolality causes neuronal firing, but this area is controversial. Intracellular injections of lucifer yellow have shown that some MNCs are dye-coupled and electron microscopic observations with the freeze-fracture technique have revealed occasional gap junctions, thus suggesting that some MNCs are electrotonically coupled. Both excitatory and inhibitory postsynaptic potentials have been evoked with extracellular stimulation. Therefore, action potentials, synaptic potentials, burst discharges, and probably electrotonic coupling have been found with intracellular recording in mammalian neuroendocrine cells. Future studies with intracellular recording and staining followed by immunohistochemical identification of cells should provide significant new information on the membrane physiology and synaptic pharmacology of vasopressinergic and oxytocinergic cells.     

Tubuläre intranukleäre Einschlußkörper beiLinaria vulgaris,L. alpina (Scrophulariaceae) undIncarvillea variabilis (Bignoniaceae)

The epidermal nuclei of the ovary inLinaria vulgaris andL. alpina, and the nuclei in leaves ofIncarvillea variabilis often contain crystalloid, tubular inclusions. The tubuli in bothLinaria species have an average diameter of 200 Å with a width of 70 Å in the clear and are arranged in a higly organized manner: Parallel tubuli form layers which build up a crystal lattice. There are three directions of tubuli in each inclusion: In each layer they are aligned in an angle of 60° to those of the next, so that the tubuli of the fourth layer are in the same direction as those of the first one. InIncarvillea the tubuli (each with an average diameter of 220 Å, and 70 Å width in the clear) also exhibit compact packing, but their arrangement is not so highly ordered as inLinaria.