Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Endovascular hypothermia improves post-resuscitation myocardial dysfunction by increasing mitochondrial biogenesis in a pig model of cardiac arrest

The aim of the study was to investigate the effects of endovascular hypothermia on mitochondrial biogenesis in a pig model of prolonged cardiac arrest (CA). Ventricular fibrillation was electrically induced, and animals were left untreated for 10 min; then after 6min of cardiopulmonary resuscitation (CPR), defibrillation was attempted. 25 animals that were successfully resuscitated were randomized into three groups: Sham group (SG, 5, no CA), normal temperature group (NTG, 5 for 12 h observation and 5 for 24 h observation), and endovascular hypothermia group (EHG, 5 for 12 h observation and 5 for 24 h observation). The core temperatures (T_c) in the EHG were maintained at 34 ± 0.5 °C for 6 h by an endovascular hypothermia device (Coolgard 3000), then actively increased at the speed of 0.5 °C per hour during the next 6 h to achieve a normal body temperature, while T_c were maintained at 37.5 ± 0.5 °C in the NTG. Cardiac and mitochondrial functions, the quantification of myocardial mitochondrial DNA (mtDNA), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF)-1, and NRF-2 were examined. Results showed that myocardial and mitochondrial injury and dysfunction increased significantly at 12 h and 24 h after CA. Endovascular hypothermia offered a method to rapidly achieve the target temperature and provide stable target temperature management (TTM). Cardiac outcomes were improved and myocardial injuries were alleviated with endovascular hypothermia. Compared with NTG, endovascular hypothermia significantly increased mitochondrial activity and biogenesis by amplifying mitochondrial biogenesis factors’ expressions, including PGC-1α, NRF-1, and NRF-2. In conclusions, endovascular hypothermia after CA alleviated myocardial and mitochondrial dysfunction, and was associated with increasing mitochondrial biogenesis.     

Mössbauer spectroscopy of iron-containing dermal granules from Molpadia intermedia

Dermal granules containing hydrous ferric oxide cores from Molpadia intermedia were studied by Mössbauer spectroscopy from 1.5 t0 300 K and in magnetic fields up to 80 kOersted at 4.2 K. A magnetic phase transition to an antiferromagnetically ordered state is observed at 10 K. The results are compared with the magnetic behavior of micellar cores of ferritin from eukaryotes and iron-storage materials from prokaryotes.     

A transient voltage-dependent outward current in cultured cerebellar granules

Granule cells were dissociated from rat cerebella with a procedure that yields a 98% pure cell population. Potassium currents in these cells were studied using the patch-clamp technique. Depolarizing pulses of 10 mV step and 100 ms duration from a holding potential of –80 mV elicited two different potassium outward currents: a transient, low-voltage activated component and a long lasting, high-voltage activated component. At +30 mV, the total current reached an amplitude of 2 nA (mean value of 15 experiments). The reversal potential of the transient current, estimated by measuring tail currents, was –77 mV, close to that predicted by the Nernst equation. The transient current was half inactivated with a holding potential of –78 mV and completely inactivated with –50 mV or more positive holding potentials. Finally, the current decay could be fitted by the sum of two exponentials with time constants of about 20 and 250 ms.     

Translocation and proteolytic processing of nascent secretory polypeptide chains: two functions associated with the ribosomal domain of the endoplasmic reticulum

Rat liver microsomes were subfractionated by isopycnic centrifugation in sucrose gradient. The subfractions were assayed for translocation and proteolytic processing of nascent polypeptides in a rabbit reticulocyte lysate programmed with total RNA from human term placenta. The distribution of the translocation and processing of prelactogen through the gradient correlated with that of the microsomal RNA (ribosomes). Microsomes became inactive upon incubation with elastase, but the proteolyzed membranes recovered their activity by recombination with the soluble and active fragment of the docking protein (SRP-receptor) from dog pancreas. When this fragment was combined with the gradient subfractions, or with the subfractions inactivated by incubation with elastase, the density profile of the translocation activity remained similar to that of RNA. Thus, its distribution cannot be accounted for merely by that of the docking protein; another membrane constituent, still unidentified, is both necessary for translocation of polypeptides and restricted to the rough portions of the endosplamic reticulum. Signal peptidase was assayed in the absence of protein synthesis, by use of preformed prelactogen and detergent-disrupted microsomes. Its density distribution was also similar to that of RNA. Several components of the endosplamic reticulum now appear to be segregated within restricted areas on either side of the membrane, and to make up a biochemically distinct domain. We propose to call it the ribosomal domain in consideration of its contribution to protein biosynthesis by bound ribosomes. This domain probably accounts for a greater part of the membrane area at the cytoplasmic than at the luminal surface, as postulated earlier to explain how enzymes of the cytoplasmic surface are relatively less abundant in the rough microsomes than those of the luminal surface [Amar-Costesec A. & Beaufay H. (1981) J. Theor. Biol. 89, 217-230].     

Tridimensional analysis of the formation of secretory vesicles in the Golgi apparatus of absorptive columnar cells of the mouse colon

The tridimensional structure of the Golgi apparatus has been studied in the absorptive cells of the mouse colon by means of reduced osmium postfixation and phosphatase cytochemistry. In thick sections of tissue impregnated with osmium tetroxide or treated with a technique to demonstrate TPPase activity, the Golgi formed a continuous ribbon-like structure capping the upper pole of the nucleus. Along the longitudinal axis of this ribbon, compact zones made up of superposed flattened saccules alternated with less compact zones which consisted of highly perforated saccules or bridging anastomosed tubules. In the cis-trans axis, the following elements were observed: (1) a cis element consisting of a continuous osmiophilic tubular network; (2) two or three subjacent elements selectively perforated by wells; (3) a trans compartment made up of two or three TPPase-reactive sacculotubular elements, some showing a "peeling-off" configuration. In some regions, the first flattened saccule of this trans compartment displayed discrete ovoid dilatations, located in compact zones and containing a dense granulofibrillar material; in the subjacent elements this material was seen concentrated in nodular swellings, at the intersection of the meshes of anastomosed membranous tubules. 100-300 nm vesicles containing a similar dense granulofilamentous material were observed in the trans Golgi zone and interspersed in the supranuclear cytoplasm between the Golgi zone and the apical surface of the cell. Smaller vesicles 80-100 nm in diameter containing a fine dusty material were also seen in proximity. These morphological observations suggested that at least two kinds of material were segregated in the saccules of the trans compartment and packaged in vesicles of two class sizes that detached from the Golgi stack on its trans aspect.     

Electron-microscopic studies on the threshold value of calcium concentration for the release of storage granules and the acceleration of their degradation in the rat parathyroid gland

Summary To determine both a threshold value of calcium concentration (CC) for the release of storage granules and that for the acceleration of degradation of these granules, the rat parathyroid glands were perfused in situ with HEPES-Ringer solutions containing different concentration of Ca²⁺ for 10 min. With perfusates containing 0.83–1.21 mM Ca²⁺ (equivalent to 8–11 mg/dl serum calcium), the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged. With perfusates containing 0.83 mM Ca²⁺ (7.5 mg/dl) or less, however, both NSG-I and NSG-II decreased remarkably and the former was larger than the latter. On the contrary, with perfusates containing 1.27 mM Ca²⁺ (11.5 mg/dl) or more, NSG-II increased and the ratio of NSG-I to NSG-II was changed reversely. We concluded that a thereshold value of CC required for the release of storage granules may be present between 0.88 and 0.83 mM Ca²⁺ (8 and 7.5 mg/dl) and that a threshold value of CC for accelerating the transformation of type-I granules into type-II, the degradation of storage granules, may be situated at about 1.27 mM Ca²⁺ (11.5 mg/dl). Additionally, it was suggested that both prosecretory and storage granules are not only formed at the innermost Golgi cisterna but also at the trans-Golgi network.     

Recent developments in chloroplast protein transport

Most proteins located in chloroplasts are encoded by nuclear genes, synthesized in the cytoplasm, and transported into the organelle. The study of protein uptake by chloroplasts has greatly expanded over the past few years. The increased activity in this field is due, in part, to the application of recombinant DNA methodology to the analysis of protein translocation. Added interest has also been gained by the realization that the transport mechanisms that mediate protein uptake by chloroplasts, mitochondria and the endoplasmic reticulum display certain characteristics in common. These include amino terminal sequences that target proteins to particular organelles, a transport process that is mechanistically independent from the events of translation, and an ATP-requiring transport step that is thought to involve partial unfolding of the protein to be translocated. In this review we examine recent studies on the binding of precursors to the chloroplast surface, the energy-dependent uptake of proteins into the stroma, and the targeting of proteins to the thylakoid lumen. These aspects of protein transport into chloroplasts are discussed in the context of recent studies on protein uptake by mitochondria.Abbrevlations CAT chloramphenicol acetyl transferase - CCCP carbonylcyanide m-chlorophenylhydrazone - DHFR dihydrofolate reductase - EPSP 5-enol-pyruvylshikimate-3-phosphate - ER endoplasmic reticulum - LHCP light harvesting chlorophyll a/b apoprotein - NPT neomycin phosphotransferase - oATP adenosine-2,3-dialdehyde-5-triphosphate - P-inorganic phosphate Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - SRP signal recognition particle     

飞蝗复眼生理和结构上的节律变化

采用细胞内记录和光镜方法研究了飞蝗(Locusta migratoria)夜间和日间在暗适应和明适应状态下小网膜细胞角敏感度以及晶锥和小网膜细胞之间区域结构上的变化.结果表明小网膜细胞角敏感度的变化不仅仅由于晶锥周围主色素细胞色素颗粒的移动,而且也由于小眼感杆束结构上的节律变化.