Intracellular Localization of Jasmonate-Induced Proteins in Barley Leaves

The plant growth substance jasmonic acid and its methyl ester (JA-Me) induce a set of proteins (jasmonate-induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one-dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno-gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP-23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP-37 was detected in vacuoles and in the nucleoplasm; JIP-66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP-100 was found within the stromal fraction of chloroplasts; JIP-70 is present in the peroxisome and the nucleus; JIP-50 and JIP-6 accumulate in vacuoles. The location of JIP-66 and JIP-6 confirms their possible physiological role deduced from molecular analysis of their cDNA.     

Glutamine synthetase in alder (Alnus glutinosa) root nodules. Purification, properties and cytoimmunochemical localization

Chromatographic, kinetic and regulatory properties of glutamine synthetase (GS) were investigated in root nodules of Alnus glutinosa L. (Gaertn) grown in a culture chamber. By DEAE Sephacel column chromatography and polyacrylamide gel electrophoresis, a major form of the enzyme was identified. Molecular weight was about 360 000 for the native protein and 43 000 for the subunits. Optimum pH was about 7.3 and Km for glutamate and ATP were 0.9 m M and 0.5 m M , respectively. By immunofluorescent techniques GS was localized in the inner cortical cells but not in vesicles of alder root nodules.     

Chemiluminescence energy transfer: a new technique applicable to the study of ligand--ligand interactions in living systems

Chemiluminescent molecules can be readily detected in the range 10(-15) to 10(-18) mol, and potentially at least down to 10(-20) mol reacting/s. The chemiluminescent compound aminobutylethylisoluminol (ABEI) and its isothiocyanate derivative have been synthesized. The ABEI was coupled to rabbit immunoglobulin and cyclic AMP. These labeled antigens were stable for at least 9 months and were used to establish chemiluminescent immunoassays. When these chemiluminescent-labeled antigens bound to their respective fluorescein-labeled antibodies, a wavelength shift towards the green was detected in the chemiluminescence. This was due to chemiluminescence energy transfer and used to establish an homogenous immunoassay which could measure these antigens in biological samples at least as sensitively as conventional radioimmunoassays.     

A comparative study of the effect of modification of the surface of human platelets on the receptors for aggregated immunoglobulins and for ristocetin-von Willebrand factor

The receptors for aggregated immunoglobulin G (IgG) (an Fc receptor) and for ristocetin-von Willebrand factor on human platelets were studied by means of various modifications of the platelet surface. The expression of these receptors was measured by the agglutination of platelets to ristocetin in the presence of von Willebrand factor, which is part of the factor VIII complex, and by the binding of aggregated IgG coupled to ³H-labelled diazobenzen. Treatment of platelets with chymotrypsin, trypsin, papain and pronase which removed protein and glycoprotein from the platelet under conditions where the release reaction was inhibited caused loss of the expression of the receptor for ristocetin-von Willebrand factor and an enhancement of that for aggregated IgG. Induction of membrane changes with ADP and of the release reaction with the ionophore A23187 abolished agglutination to ristocetin-von Willebrand factor but did not alter the receptor for aggregated IgG. Possible contributions of unspecific membrane changes, produced by protease treatment of platelets, to the modification of receptor expression were eliminated by the use of formaldehyde-treated platelets. Trypsin, papain and pronase destroyed the ability of these platelets to agglutinate to ristocetin-von Willebrand factor but produced no change in the binding of aggregated IgG. Therefore, the receptor for ristocetin-von Willebrand factor is truly sensitive to proteolysis while the Fc receptor is not, but is partially masked by protease-sensitive material.     

Intracellular localization of sirtuin and cell length analysis of Lactobacillus paracasei suggest possible role of sirtuin in cell division and cell shape regulation

Sirtuin has been associated in prolonging lifespan of different model organisms. It has been shown to have an enzymatic activity of NAD⁺-dependent protein deacetylation targeting acetylated proteins. To determine targets and possible roles of sirtuin (LpSirA) in the Lactobacillus paracasei BL23, deletion (ΔsirA), sirtuin overexpressor (highsirA) and GFP fusion (highsirA-Venus) strains were generated, and microscopic localization and cell length analysis were done. Microscopic analysis revealed localization of LpSirA at cell division plates, at cell poles and all throughout the cell length in a spiral manner. Cell length analysis revealed that 46.9% of the ΔsirA cells were observed to be shorter (<2 μm), whereas 12.6% of the highsirA cells were observed to be longer (>4 μm) in comparison with the wild-type with only 17.1% short cells and 5.3% long cells. Our results suggest that sirtuin may have an essential role in cell division and cell shape regulation.     

Small GTP-binding proteins in Plasmodium falciparum

Summary— During its erythrocytic life cycle Plasmodium falciparum exchanges compounds with host cells through phagocytosis and exocytosis. In eucaryotic cells, small GTP-binding proteins of the Ras superfamily appear to be involved in different steps of membrane trafficking and in intracellular signals. In this paper, we investigate the Rab4, Rab6 and Ras-related proteins in P falciparum infected red cells. We report that P falciparum Rab and Ras-related proteins could be distinguished from their counterparts by iso-electrofocusing and immunoblotting. The localization of P falciparum Rab 4 and Rab 6 was studied by immunogold electron microscopy on ultrathin frozen sections of infected red blood cells. Rab4 parasite-relate protein was found associated with the membranes of early endosome-like structures near the parasite plasma membrane. Rab6-related protein was associated with the Golgi/trans Golgi network, as already suggested by immunofluorescence microscopy studies and Ras-related protein was cytoplasmic and plasma membrane-associated. These results are in accordance with their mammalian counterparts and support the implication of Rab-related proteins in vesicular trafficking in Plasmodium.     

Replication of bacteriophage f1: A complex containing gene II protein in gene V mutant-infected bacteria

A dense complex has been isolated from bacteria infected with gene V amber mutant f 1 bacteriophage. The major protein in this complex is the f 1 bacteriophage-specific gene II protein. Other proteins in the complex include the f 1 bacteriophage coat protein and proteins which migrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with the f1 bacteriophage-specific gene III, gene IV and X protein. A protein of approximately 20,000 M_r is also present in the complex. Examination of bacteria infected with gene V mutant f1 bacteriophage revealed the complex as a densely staining amorphous body which appears to be associated with the cytoplasmic membrane. Bacteria infected with f1 bacteriophage that contain amber mutations in genes other than gene V do not contain this complex.     

Impact of heterodimerization on intracellular localization of the ecdysteroid receptor (EcR)

Initially, nuclear import of the ecdysteroid receptor (EcR) in vertebrate cells (CHO-K1 and COS-7) does not afford a heterodimerization partner. Later on, EcR is retained in the nucleus only in the presence of a heterodimerization partner. Ultraspiracle (Usp) is more efficient compared to its vertebrate orthologue RXR and leads to an exclusively nuclear localization of EcR even in the absence of ligand. The DNA binding domain of the heterodimerization partner is important for retainment of EcR in the nucleus as shown by Usp4 (Usp(R130C)), which has lost its DNA binding capability. The C-terminal end of Usp (Usp(Delta205-508)) encompassing the C-terminal part of the D-domain and the E- and F-domains are essential for retainment of EcR in the nucleus. Nuclear localization is further influenced by cell-specific factors, since hormone and heterodimerization stabilizes the EcR protein in a cell-specific way.     

Potassium transport and intracellular potassium activities in rabbit gallbladder

Summary Intracellular K activities, (K) _c , in rabbit gallbladder were determined using conventional and ion-selective microelectrodes. (K) _c averaged 73mm and was 1.5 times that predicted for an equilibrium distribution of the ion across both apical and basolateral membranes. Thus, K must be actively transported into the cell, and the responsible mechanism is almost certainly the Na–K exchange pump in the basolateral membrane.Measurements of the bidirectional transepithelial fluxes of⁴²K indicate that K is secreted into the mucosal solution at a rate of 0.8 eq/cm² hr; this value is only 6% of the rate of transcellular Na absorption by this epithelium.Calculation of the conductance of the basolateral membrane,G ^s, reveals that it is too low to account for the maintenance of the steady-state (K) _c by a 3 Na2 K pump mechanism at the basolateral membrane if K exit across that barrier is entirely electrodiffusional.Our results together with those of others strongly suggest that a significant fraction of downhill K exit from the cell across the basolateral membrane is nonconductive and coupled to the movement of some other ion, perhaps Cl.     

Detection of disease-specific augmentation of abnormal immunoglobulin G in sera of patients with rheumatoid arthritis

Galactose-free immunoglobulin G (IgG), which is known to be higher in the sera of patients with rheumatoid arthritis, was prepared from IgG of healthy volunteers using enzymes. Its reactivity to lectins was analyzed. The galactose-free IgG showed no reactivity to Ricinus communis agglutinin 120 but displayed greater reactivity to concanavalin A and Lens culinaris lectin than did intact human IgG. Then, IgG in serum samples was bound to protein A immobilized on a nitrocellulose membrane, and its reactivity to biotinylated concanavalin A was measured with streptavidin-conjugated horseradish peroxidase. When the reactivity to concanavalin A of IgG in sera from healthy individuals and patients with rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, or hepatic disease was compared, higher levels were shown in patients with RA, notably in 60% of the seronegative patients and 80% of the early phase patients. Therefore, it was suggested that augmentation of the abnormal IgG in sera was highly specific to patients with RA and that this novel serum test could be very useful for an accurate diagnosis of this disease.