RasG protein accumulation occurs just prior to amoebae emergence during spore germination in Dictyostelium discoideum

Abstract RasG protein levels in dormant and germinating spores of Dictyostelium discoideum strains JC1 and SG1 were estimated by Western blotting. Ras Glevels were very low in dormant spores and remained low during the lag period, regardless of whether spores were heat activated or treated with autoactivator during the early stages of spore germination. RasG levels increased late during spore swelling just prior to the emergence stage of germination. These data are consistent with a requirement for RasG during vegetative growth.     

Chaetomella acutiseta produces chaetomellic acids A and B which are reversible inhibitors of farnesyl-protien transferase

Chaetomellic acids A and B, isolated from Chaetomella acutiseta, are specific inhibitors of farnesyl-protein transferase that do not inhibit geranylgeranyl transferase type 1 or squalene synthase. Chaetomellic acids A and B are reversible inhibitors, resemble farnesyl diphosphate and probably inhibit FPTase by substituting for farnesyl diphosphate. Chaetomellic acid production appears to be widespread within the genus Chaetomella. Correspondence to: R. B. Lingham     

Primary renal hemangiosarcoma in a moustached tamarin

A wild-caught adult female Saguinus mystax died after 54 months in captivity. At necropsy, a small reddish zone in the renal cortex of one kidney was shown histologically to be a hemangiosarcoma.     

Polymorphism of the tumor necrosis factor beta gene in systemic lupus erythematosus: TNFB-MHC haplotypes

We investigated the Nco I restriction fragment length polymorphism (RFLP) of the tumor necrosis factor beta (TNFB) gene in 173 patients with systemic lupus erythematosus (SLE), 192 unrelated healthy controls, and eleven panel families, all of German origin. The phenotype frequency of the TNFB*1 allele was significantly increased in patients compared to controls (63.6% vs 47.1%, RR = 1.96, p <0.002). The results of a two-point haplotype statistical analysis between TNFB and HLA alleles show that there is linkage disequilibrium between TNFB*1 and HLA-A1, Cw7, B8, DR3, DQ2, and C4A DE. The frequency of TNFB*1 was compared in SLE patients and controls in the presence or absence of each of these alleles. TNFB*1 is increased in patients over controls only in the presence of the mentioned alleles. Therefore, the whole haplotype A1, Cw7, B8, TNFB*1, C4A DE, DR3, DQ2 is increased in patients and it cannot be determined which of the genes carried by this haplotype is responsible for the susceptibility to SLE. In addition, two-locus associations were analyzed in 192 unrelated healthy controls for TNFB and class I alleles typed by serology, and for TNFB and class II alleles typed by polymerase chain reaction/oligonucleotide probes. We found positive linkage disequilibrium between TNFB*1 and the following alleles: HLA-A24, HLA-B8, DRB1*0301, DRB1*1104, DRB1*1302, DQA1*0501, DQB1*0201, DQB1*0604, and DPB1*0101. TNFB*2 is associated with HLA-B7, DRB1*1501, and DQB1*0602.This study was supported by grants from the Federal Ministry of Research and Technology (BMFT/DFVLR, 01 VM 8608/9), the German Academic Exchange Service (DAAD, 322/501/014/0), and SFB (217).This work is part of the doctoral thesis of M. P. Bettinotti.     

Discrete steps in dioxygen activation—The cytochrome oxidase/O2 reaction

The kinetic constraints that are imposed on cytochrome oxidase in its dual function as the terminal oxidant in the respiratory process and as a redox-linked proton pump provide a unique opportunity to investigate the molecular details of biological O₂ activation. By using flow/flash techniques, it is possible to visualize individual steps in the O₂-binding and reduction process, and results from a number of spectroscopic investigations on the oxidation of reduced cytochrome oxidase by O₂ are now available. In this article, we use these results to synthesize a reaction mechanism for O₂ activation in the enzyme and to simulate time-concentration profiles for a number of intemediates that have been observed experimentally. Kinetic manifestation of the consequences of coupling exergonic electron transfer to endergonic proton translocation emerge from this analysis. Energetic efficiency in this process apparently requires that potentially toxic intermediate oxidation states of dioxygen accumulate to substantial concentration during the reduction reaction.     

Extracellular Neuroactive Amino Acids in the Rat Striatum During Ischaemia: Comparison Between Penumbral Conditions and Ischaemia with Sustained Anoxic Depolarisation

Abstract: Changes in the extracellular levels of excitatory and inhibitory amino acid transmitters were studied in the rat striatum during penumbral ischaemia using intracerebral microdialysis. Effects of penumbral forebrain ischaemia were compared with those of ischaemia with sustained anoxic depolarisation and K⁺ (100 m M ). Comparisons were also made between different groups of animals at 2 and 24 h after dialysis probe implantation. The K⁺ stimulus did not provoke any release of excitatory amino acids in the 24-h group, probably reflecting a decrease of functional synapses adjacent to the probe. During 30 min of penumbral ischaemia, excitatory amino acids did not reach critical concentrations in the extracellular fluid, and increases in levels of inhibitory/modulatory amino acids were similar. On the other hand, severe transient ischaemia resulted in massive synchronous release of many neuroactive excitatory and inhibitory compounds, in both the 2- and 24-h groups. These and other data suggest that changes during severe ischaemia may arise from both neurotransmitter and metabolic pools. It is concluded that is- chaemic damage in the penumbra may not be related to extracellular neuroactive amino acid changes generated within this region.     

Adoptive immunotherapy with peripheral blood lymphocytes cocultured in vitro with autologous tumor cells and interleukin-2

A clinical trial of adoptive immunotherapy was carried out with peripheral blood lymphocytes (PBL), cocultured in vitro with autologous tumor cells and interieukin-2 (IL-2), in 14 patients with advanced melanoma. PBL from these patients were cocultured with irradiated autologous tumor cells for 7 days, which was followed by expansion in IL-2-containing medium. These lymphocytes were returned to the patient along with intravenous IL-2 at doses up to 2×10⁶ IU m^–2 day^–1. A dose of 300 mg/m² cyclophosphamide was administered to each patient intravenously 4 days prior to each treatment. Following coculture, the lymphocytes were primarily CD3⁺ T cells and they expressed varied degrees of cytotoxicity against autologous melanoma cells. In 9 patients the activated cells were al least 80% CD4⁺ and in 2 cases they were mostly CD8+. Some of the activated cells exhibited suppressor or helper activity in a functional regulatory coculture assay. No major therapeutic response was observed in this study. Minor responses were observed in 2 patients. Toxicities were those expected from the IL-2 dose administered.This work has been supported by an American Cancer Society Institutional Research Grant (ACS-IRG 91-230), by the University of Connecticut Clinical Research Center (grant 0021), and by the Hartford Hospital Research Fund (grant 1017-20-018). Dr. Sporn is a recipient of American Cancer Society Clinical Oncology Career Development Award 90-230     

An SAR sequence containing 395 bp DNA fragment mediates enhanced,gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants

A 395 bp fragment located downstream from the soybean heat shock geneGmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybeanin vitro. Chimaeric genes containing the SAR_L fragment either at one side (5 or 3) or at both sides of a heat shock promoter-regulated -glucuronidase reporter gene were constructed. A five-to nine-fold increase of heat-inducible -glucuronidase activity was observed in transgenic tobacco plants containing constructs with SAR_L fragments either at both sides or with at least one SAR_L copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these. plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies in transgenic plants.     

A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The K_D value observed is in accordance with the IC₅₀ determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.