Assocation of the 33 kDa extrinsic polypeptide (water-splitting) with PS II particles: immunochemical quantification of residual polypeptide after membrane extraction

Various washing procedures were tested on Triton-prepared PS II particles for their ability to remove the 33 kDa extrinsic polypeptide (33 kDa EP) associated with the water-splitting complex. Residual 33 kDa EP was evaluated by Coomassie blue staining of SDS gels of washed particles and by Western blotting with an antibody specific for the 33 kDa EP. A wash with 16 mM Tris buffer, pH 8.3, inhibited water-splitting activity but did not remove all the 33 kDa EP. Sequential washes with 30 mM octyl glucoside (pH 8.0 and 6.8), and a single wash with 0.8 M Tris were also ineffective in removing all the 33 kDa EP. Washing with 1 M CaCl₂ was more effective in removing 33 kDa EP; while only a faint trace of protein was detectable by Coomassie-staining, immunoblotting revealed a considerable remainder. The treated particles retained some water-splitting activity. The two step procedure of Miyao and Murata (1984) involving 1 M NaCl and 2.3 M urea was most effective, removing all but a trace of antibody positive protein. Our finding suggests that (1) the degree of depletion of the 33 kDa EP cannot be judged on the basis of Coomassie stain alone, and (2) this extrinsic protein is very tightly associated with the membrane, perhaps via a hydrophilic portion of this otherwise hydrophilic protein. The results also suggest that the presence or absence of the 33 kDa protein per se is not the primary determinant of residual water splitting activity.Abbreviations Chl chlorophyll - DCPIP dichlorophenolindophenol - DPC diphenolcarbazide - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2(N-morpholino)ethanesulfonic acid - SDS sodium dodecyl sulfate - Tris Tris(hydroxymethyl)aminomethane     

Characterization of cDNA clones encoding the extrinsic 23 kDa polypeptide of the oxygen-evolving complex of photosystem II in pea

The 23 kDa polypeptide of the oxygen-evolving complex of photosystem II has been extracted from pea photosystem II particles by washing with 1 M NaCl and purified by anion-exchange chromatography. The N-terminal amino acid sequence has been determined and specific antisera have been raised in rabbits and used to screen a pea-leaf cDNA library in gt11. Determination of the nucleotide sequence of two clones provided the nucleotide sequence for the full 23 kDa polypeptide. The deduced amino acid sequence showed it to code for a mature protein of 186 amino acid residues with an N-terminal presequence of 73 amino acid residues showing a high degree of conservation with previously reported 23 kDa sequences from spinach and Chlamydomonas. Southern blots of genomic DNA from pea probed with the labelled cDNA gave rise to only one band suggesting that the protein is encoded by a single gene. Northern blots of RNA extracted from various organs indicated a message of approximately 1.1 kb, in good agreement with the size of the cDNA, in all chlorophyll-containing tissues. Western blots of protein extracted from the same organs indicated that the 23 kDa polypeptide was present in all major organs of the plant except the roots.Abbreviations bis-Tris bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - pfu plaque-forming units     

Differential expression of 240 kDa ConA-binding glycoprotein in vitro and in vivo detected by immunochemical methods

The expression of the 240 ConA-binding glycoprotein (240 kDa), a marker of synaptic junctions isolated from the rat cerebellum, was studied by immunocytochemical techniques in forebrain and cerebellum from rat and chicken, and in chick dorsal root ganglia. Parallel studies were carried out either on tissue sections or in dissociated cell cultures. In all cases non neuronal cells were not immunostained. The tissue sections of cerebellum from rat and chick exhibited 240 kDa glycoprotein immunoreactivity, especially in the molecular layer, while the forebrain sections from rat and chick did not show any significant immunostaining. In contrast, in dissociated forebrain cell cultures, all neuronal cells expressed 240 kDa glycoprotein immunoreactivity, while glial cells remained totally unlabelled. In tissue sections of dorsal root ganglion (DRG), sensory neurons expressed the 240 kDa only after the embryonic day (E 10). A large number of small neurons in the dorsomedial part of DRG were immunostained with 240 kDa glycoprotein antiserum, whereas only a small number of neurons in the ventrolateral part of the ganglia displayed 240 kDa immunoreactivity. In dissociated DRG cells cultures (mixed or neuron-enriched DRG cell cultures) all the neuronal perikarya but not their processes were stained. These studies indicate that 240 kDa glycoprotein expression is completely modified in cultures of neurons of CNS or PNS since the antigen becomes synthetized in high amount by all cells independent of synapse formation. This demonstrates that the expression of 240 kDa is controlled by the cell environment.     

3-[18F]Acetylcyclofoxy: a useful probe for the visualization of opiate receptors in living animals

A fluoro-analogue of the potent narcotic antagonist, naltrexone, was synthesized and shown to bind with high affinity to opiate receptors in vitro. 3-[18F]acetylcyclofoxy was prepared via a one-step triflate displacement reaction with the positron emitting 18F ion from tetraethylammonium [18F] fluoride. 3-[18F]acetylcyclofoxy accumulation in opiate receptor rich brain regions of both rat and baboon is shown to be completely displaced by the active enantiomer of naloxone [-)-naloxone) while the identical dose of the pharmacologically inert (+)-naloxone has no detectable effect. Moreover, both rat and baboon brain showed the well documented, typical opiate receptor distribution so that basal ganglia and thalamus are clearly visible in the living baboon brain up to 95 min after intravenous injection of 3-[18F] acetylcyclofoxy. We expect that 3-[18F )acetylcyclofoxy will be a useful probe for visualizing opiate receptors in living humans.     

Expression profiles of interferon‐stimulated gene 15 and prostaglandin synthases in the ovine lymph nodes during early pregnancy

Lymph nodes are distributed all over the body and are part of the lymphatic system. The interferon‐stimulated gene 15 kDa protein (ISG15) and prostaglandins (PGs) are involved in the establishment of pregnancy and are expressed in the uterus during early pregnancy in sheep. In this study, the ovine lymph nodes were obtained on Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX‐1), COX‐2, prostaglandin E (PGE) synthase (PTGES), and a PGF synthase (aldo‐keto reductase family 1, member B1, AKR1B1) were detected by quantitative real‐time polymerase chain reaction, western blot analysis, and immunohistochemistry analysis. Our results showed that there were peaks in the expression of mRNAs and the proteins of ISG15, COX‐1, COX‐2, PTGES, and AKR1B1 in the lymph nodes during early pregnancy and that the COX‐2 and AKR1B1 proteins were limited to the subcapsular sinus and lymph sinuses. In conclusion, the ISG15, COX‐1, COX‐2, PTGES, and AKR1B1 were upregulated in the maternal lymph nodes, which may be beneficial for the development of conceptus, maternal systemic immunoregulation, and anti‐luteolysis during early pregnancy in sheep.     

The FKBP12 subunit modifies the long-range allosterism of the ryanodine receptor

Ryanodine receptors (RyRs) are large conductance intracellular channels controlling intracellular calcium homeostasis in myocytes, neurons, and other cell types. Loss of RyR’s constitutive cytoplasmic partner FKBP results in channel sensitization, dominant subconductance states, and increased cytoplasmic Ca²⁺. FKBP12 binds to RyR1’s cytoplasmic assembly 130?Å away from the ion gate at four equivalent sites in the RyR1 tetramer. To understand how FKBP12 binding alters RyR1’s channel properties, we studied the 3D structure of RyR1 alone in the closed conformation in the context of the open and closed conformations of FKBP12-bound RyR1. We analyzed the metrics of conformational changes of existing structures, the structure of the ion gate, and carried out multivariate statistical analysis of thousands of individual cryoEM RyR1 particles. We find that under closed state conditions, in the presence of FKBP12, the cytoplasmic domain of RyR1 adopts an upward conformation, whereas absence of FKBP12 results in a relaxed conformation, while the ion gate remains closed. The relaxed conformation is intermediate between the RyR1-FKBP12 complex closed (upward) and open (downward) conformations. The closed-relaxed conformation of RyR1 appears to be consistent with a lower energy barrier separating the closed and open states of RyR1-FKBP12, and suggests that FKBP12 plays an important role by restricting conformations within RyR1’s conformational landscape.     

The renaissance of Ca2+-binding proteins in the nervous system: secretagogin takes center stage

Effective control of the Ca²⁺ homeostasis in any living cell is paramount to coordinate some of the most essential physiological processes, including cell division, morphological differentiation, and intercellular communication. Therefore, effective homeostatic mechanisms have evolved to maintain the intracellular Ca²⁺ concentration at physiologically adequate levels, as well as to regulate the spatial and temporal dynamics of Ca²⁺signaling at subcellular resolution. Members of the superfamily of EF-hand Ca²⁺-binding proteins are effective to either attenuate intracellular Ca²⁺ transients as stochiometric buffers or function as Ca²⁺ sensors whose conformational change upon Ca²⁺ binding triggers protein-protein interactions, leading to cell state-specific intracellular signaling events. In the central nervous system, some EF-hand Ca²⁺-binding proteins are restricted to specific subtypes of neurons or glia, with their expression under developmental and/or metabolic control. Therefore, Ca²⁺-binding proteins are widely used as molecular markers of cell identity whilst also predicting excitability and neurotransmitter release profiles in response to electrical stimuli. Secretagogin is a novel member of the group of EF-hand Ca²⁺-binding proteins whose expression precedes that of many other Ca²⁺-binding proteins in postmitotic, migratory neurons in the embryonic nervous system. Secretagogin expression persists during neurogenesis in the adult brain, yet becomes confined to regionalized subsets of differentiated neurons in the adult central and peripheral nervous and neuroendocrine systems. Secretagogin may be implicated in the control of neuronal turnover and differentiation, particularly since it is re-expressed in neoplastic brain and endocrine tumors and modulates cell proliferation in vitro. Alternatively, and since secretagogin can bind to SNARE proteins, it might function as a Ca²⁺ sensor/coincidence detector modulating vesicular exocytosis of neurotransmitters, neuropeptides or hormones. Thus, secretagogin emerges as a functionally multifaceted Ca²⁺-binding protein whose molecular characterization can unravel a new and fundamental dimension of Ca²⁺signaling under physiological and disease conditions in the nervous system and beyond.     

Si RNA inhibition of GRP58 associated with decrease in mitomycin C-induced DNA cross-linking and cytotoxicity

The anti-cancer drug mitomycin C is metabolically activated to bind and cross-link DNA. The cross-linking contributes significantly to the cytotoxicity. The complex chemical structure of mitomycin C allows its metabolism by several known (cytosolic NAD(P)H:quinone oxidoreductase and microsomal NADPH:cytochrome P450 reductase) and unknown enzymes. The identification of new enzymes/proteins that metabolize mitomycin C and like drugs is an area of significant research interest since these studies have direct implications in drug development and clinical usage. In the present studies, we have investigated a role of cytosolic glucose regulatory protein GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. The control and GRP58 siRNA were transfected in human colon carcinoma HCT116 cells in culture. The transfection of GRP58 siRNA but not control siRNA significantly inhibited GRP58 in human colon carcinoma HCT116 cells. The inhibition of GRP58 led to decrease in mitomycin C-induced DNA cross-linking and cytotoxicity. These results establish a role of GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. Site-directed mutagenesis of cysteines to serines in thioredoxin domains of GRP58 and cross-linking assays revealed that both N- and C-terminal thioredoxin domains are required for GRP58-mediated mitomycin C-induced DNA cross-linking. These results suggest that GRP58 might be an important target enzyme for further studies on mitomycin C and similar drug therapy.     

Plasmodium yoelii: axenic development of the parasite mosquito stages

Study of the parasite mosquito stages of Plasmodium and its use in the production of sporozoite vaccines against malaria has been hampered by the technical difficulties of in vitro development. Here, we show the complete axenic development of the parasite mosquito stages of Plasmodium yoelii. While we demonstrate that matrigel is not required for parasite development, soluble factors produced and secreted by Drosophila melanogaster S2 cells appear to be crucial for the ookinete to oocyst transition. Parasites cultured axenically are both morphologically and biologically similar to mosquito-derived ookinetes, oocysts, and sporozoites. Axenically derived sporozoites were capable of producing an infection in mice as determined by RT-PCR; however, the parasitemia was significantly much less than that produced by mosquito-derived sporozoites. Our cell free system for development of the mosquito stages of P. yoelii provides a simplified approach to generate sporozoites that may be for biological assays and genetic manipulations.     

The involvement of the 67 kDa laminin receptor-mediated modulation of cytoskeleton in the degranulation inhibition induced by epigallocatechin-3-O-gallate

Recently, we have reported that (-)-epigallocatechin-3-O-gallate (EGCG) acts as an inhibitor of degranulation. However, the inhibitory mechanism for degranulation is still poorly understood. Here we show that suppression of exocytosis-related myosin II regulatory light chain phosphorylation and alteration of actin remodeling are involved in the inhibitory effect of EGCG on the calcium ionophore-induced degranulation from human basophilic KU812 cells. Surface plasmon resonance assay also revealed that EGCG binds to the cell surface, and the disruption of lipid rafts resulted in reduction of EGCG's ability. We have previously identified the raft-associated 67kDa laminin receptor (67LR) as an EGCG receptor on the cell surface. Treatment of the cells with anti-67LR antibody or RNA interference-mediated downregulation of 67LR expression abolished the effects of EGCG. These findings suggest that EGCG-induced inhibition of the degranulation includes the primary binding of EGCG to the cell surface 67LR and subsequent modulation of cytoskeleton.