Suppression of lymphocyte spontaneous proliferative response by proteolipid protein peptide in patients with HAM/TSP

To understand the immune mechanism suggested in HTLV-I-associated myelopathy (HAM/TSP), we investigated T cell response to proteolipid protein (PLP). Because of high autologous proliferative response (APR) of peripheral blood mononuclear cells (PBMC) in culture, the lymphocyte proliferation assay was not useful in this disease. Unexpectedly, however, APR was profoundly (70–98%) suppressed in 6 of 9 cases when PLP peptide 105-124 was added in the culture. PLP peptide 85-104 or 145-159 also suppressed APR in a few cases. Time course study showed that the peptide-mediated suppression became apparent after day 4 in culture. The results can be interpreted as that suppressor cells recognizing the PLP peptides were present in the PBMC of HAM/TSP patients and suppressed the APR as the consequence of antigen specific response. This may indicate that a T cell response to certain PLP determinants is involved in the pathomechanism of HAM/TSP at least in part. Molecular mimicry between PLP and HTLV-I mayaccount for the T cell sensitization to PLP in HAM/TSP.Special issue dedicated to Dr. Marjorie B. Lees.     

Evaluation of the role of State transitions in determining the efficiency of light utilisation for CO2 assimilation in leaves

Wheat leaves were exposed to light treatments that excite preferentially Photosystem I (PS I) or Photosystem II (PS II) and induce State 1 or State 2, respectively. Simultaneous measurements of CO₂ assimilation, chlorophyll fluorescence and absorbance at 820 nm were used to estimate the quantum efficiencies of CO₂ assimilation and PS II and PS I photochemistry during State transitions. State transitions were found to be associated with changes in the efficiency with which an absorbed photon is transferred to an open PS II reaction centre, but did not correlate with changes in the quantum efficiencies of PS II photochemistry or CO₂ assimilation. Studies of the phosphorylation status of the light harvesting chlorophyll protein complex associated with PS II (LHC II) in wheat leaves and using chlorina mutants of barley which are deficient in this complex demonstrate that the changes in the effective antennae size of Photosystem II occurring during State transitions require LHC II and correlate with the phosphorylation status of LHC II. However, such correlations were not found in maize leaves. It is concluded that State transitions in C₃ leaves are associated with phosphorylation-induced modifications of the PS II antennae, but these changes do not serve to optimise the use of light absorbed by the leaf for CO₂ assimilation.Abbreviations Fm, Fo, Fv maximal, minimal and variable fluorescence yields - Fm, Fv maximal and variable fluorescence yields in a light adapted state - LHC II light harvesting chlorophyll a/b protein complex associated with PS II - q_P photochemical quenching - A₈₂₀ light-induced absorbance change at 820 nm - _{PS I}, _{PS II} relative quantum efficiencies of PS I and PS II photochemistry - _{CO 2} quantum yield of CO₂ assimilation     

Response ofBrassica napus L. Microspore-derived embryos to exogenous abscisic acid and desiccation

Summary Microspore-derived embryos fromBrassica napus cv. Topas (low erucic acid) and Reston (high erucic acid) were subjected to treatment with abscisic acid (ABA) during late-stage embryo development and then dried under controlled relative humidities to mature dry seed levels of moisture. Exogenously medium-supplied ABA arrested growth and development, reduced moisture content, increased total fatty acids on a dry weight basis, and stimulated systhesis of proteins in microspore-derived embryos. ABA also resulted in a higher proportion of 22∶1 in cv. Reston (high 22∶1) and increased the level of fatty acid unsaturation in cv. Topas (low 22∶1). The accumulation of two proteins that co-migrated with cruciferin and napin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gels were also promoted by exposure to ABA, and the degree of accumulation was dependent on the concentration and time of application of ABA. Controlled desiccation of microspore embryos, used to simulate normal maturation and dehydration of zygotic embryos during seed development, did not seem to cause an increase of either storage proteins, total fatty acids, or 22∶1 (in cv. Reston), suggesting that dehydration is not a prerequisite for these processes, at least in culturedBrassica embryos.     

Regional Distribution and Quantitative Measurement of the Phosphoinositidase C-Linked Guanine Nucleotide Binding Proteins G1α and Gqα in Rat Brain

Abstract: Levels of the guanine nucleotide binding proteins G₁₁α and G_qα, which produce receptor regulation of phosphoinositidase C., were measured immunologically in 13 regions of rat central nervous system. This was achieved by immunoblotting membranes from these regions with antisera (CQ series) that identify these two polypeptides equally, following separation of the membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis conditions that can resolve G_qα and G₁₁α. In all regions examined, G_qα was more highly expressed than G₁₁α. Ratios of levels of G_qα to G₁₁α varied between the regions from 5:1 to 2:1. Quantitative measurements of the levels of G_qα and G₁₁α in each region were obtained by comparison with known amounts of purified liver G_qα and G₁₁α and with E. coli expressed recombinant G_qα. Areas that expressed G_qα highly included olfactory bulb (930 ng/ mg of membrane protein), frontal cortex (700 ng/mg of membrane protein), parietal occipital cortex (670 ng/mg of membrane protein), caudate putamen (1,003 ng/mg of membrane protein), hippocampus (1,045 ng/mg of membrane protein), hypothalamus (790 ng/mg of membrane protein), and cerebellum (950 ng/mg of membrane protein). More modest levels were observed in thalamus (450 ng/mg of membrane protein), pituitary (480 ng/mg of membrane protein), optic chiasma (330 ng/mg of membrane protein), and spinal cord (350 ng/mg of membrane protein). Gna was more evenly expressed with values ranging from about 170 ng/mg of membrane protein in spinal cord and optic chiasma to close to 300 ng/mg of membrane protein in regions expressing high levels of G_qα. A third polypeptide could be identified by the CQ antisera in all brain regions. The possibility that this polypeptide is the α subunit of G₁₄ is discussed.     

Fed-batch cultivation of recombinant escherichia coli JM103 and production of the fusion protein SPA::EcoRI in a 60-L working volume airlift tower loop reactor

SPA::EcoRI fusion protein was produced by Escherichia coli JM103 carrying the multicopy expression plasmid pMTC48, the multicopy repressor plasmid pRK248, and the multicopy protection plasmid pEcoR4 in a 60-L working volume airlift tower loop reactor on M9 minimal medium with glucose. Cell mass concentration, total cell count, number of colony-forming units, specific growth rate, yield coefficient, and metabolite (acetate, pyruvate, succinate, lactate, ethanol) concentrations were monitored during the growth phase and gene expression. Gene expression was induced by temperature shift or chemically by isopropyl-thiogalactosidase in the airlift tower loop reactor (ALTR) at constant cultivation time and in a small stirred tank reactor at different cultivation times. During induction, the cultivation medium was supplemented with concentrated Luria-Bertani (LB) medium. The intracellular enzyme activity was evaluated as a function of the time after the start of the induction. It was found that the reduction of the glucose concentration and increase of the dissolved oxygen concentration reduced the acetate produced and increased the intracellular enzyme activity. (c) 1993 John Wiley & Sons, Inc.     

Biochemical characterization and crystallization of porin from Rhodopseudomonas blastica

Oligomeric porin of the phototrophic bacterium Rhodopseudomonas blastica DSM 2131 was obtained from cell envelopes by differential temperature extraction in the presence of detergent and salt. The isolated porin exhibited strong porin activity after reconstitution into lipid bilayer membranes. The effective channel diameter for the trimer was estimated as 1.5 nm from single channel conductance measurements in the presence of 1 M KCl. Moderate cation-selectivity was observed. Oligomeric porin migrated as a single band (apparent molecular weight 81 kDa) on sodium dodecyl sulfate polyacrylamide gelelectrophoresis when solubilized below 70 °C. The oligomers were converted into monomers on heating to 70 °C or above forming two bands with apparent molecular weight of 36 kDa and 35 kDa. The oligomer was not sensitive to EDTA. Its molecular weight was determined to be 119.3 kDa by analytical ultracentrifugation. The isoelectric point was 5.7. Circular dichroism data indicated a high content of -sheet structure. Gasphase sequencing of the N-terminal residues revealed the sequence: NH₂-Glu-Ile-Ser-Leu-Asn-Gly-Tyr-Gly-Arg-Phe. Crystals with a maximal side length of 300 m and diffracting to 0.32 nm resolution were obtained with the porin oligomer in the presence of C8E4 and 1,2,3-heptanetriol by using the vapor phase equilibration technique.Abbreviations C8E4 n-octyl tetraoxyethylene - M_r apparent molecular weight - Octyl-POE n-octyl polyoxyethylene - LDAO N,N-dimethyl dodecyl aminoxide - LPS lipopolysaccharide - PAGE polyacrylamide gel-electrophoresis - PEG polyethylene glycol     

Chemistry and biology of enzymes in protein glutathionylation

Protein S-glutathionylation is emerging as a central oxidation that regulates redox signaling and biological processes linked to diseases. In recent years, the field of protein S-glutathionylation has expanded by developing biochemical tools for the identification and functional analyses of S-glutathionylation, investigating knockout mouse models, and developing and evaluating chemical inhibitors for enzymes involved in glutathionylation. This review will highlight recent studies of two enzymes, glutathione transferase omega 1 (GSTO1) and glutaredoxin 1 (Grx1), especially introducing their glutathionylation substrates associated with inflammation, cancer, and neurodegeneration and showcasing the advancement of their chemical inhibitors. Lastly, we will feature protein substrates and chemical inducers of LanC-like protein (LanCL), the first enzyme in protein C-glutathionylation.     

The MitoLuc Assay System for Accurate Real-Time Monitoring of Mitochondrial Protein Import Within Mammalian Cells

Mitochondrial protein import is critical for organelle biogenesis, bioenergetic function, and health. The mechanism of which is poorly understood, particularly of the mammalian system. To address this problem we have established an assay to quantitatively monitor mitochondrial import inside mammalian cells. The reporter is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a purified recombinant precursor protein destined for import. Following import the complementary fragments combine to form an active luciferase–providing a sensitive, accurate and continuous measure of protein import. This advance allows detailed mechanistic examination of the transport process in live cells, including the analysis of import breakdown associated with disease, and high-throughput drug screening. Furthermore, the set-up has the potential to be adapted for the analysis of alternative protein transport systems within different cell types, and multicellular model organisms.     

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.