Postsurgical adjuvant chemoimmunotherapy with recombinant interleukin-2 and 1,3-bis-(2-chloroethyl)-1-nitrosurea on spontaneous metastases of a non-immunogenic murine tumour

Summary The efficacy of the association of recombinant interleukin-2 (rIL-2) with chemotherapy has been investigated on an experimental model representative of clinical tumours, i.e. on post-surgical spontaneous metastases of a non-immunogenic tumour. We used the M5076 ovarian reticulum cell sarcoma, which metastatizes to the liver after intra-footpad implantation. Such a tumour appeared to be non-immunogenic by a variety of commonly used in vivo assays. Four clinically widely employed drugs, i.e. doxorubicin,cis-diamminedichloroplatinum II, cyclophosphamide and 1,3-bis-(2-chloroethyl)-1-nitrosurea (BCNU), were tested and BCNU proved to be the most effective one when administered as single injection at the maximum tolerated dose (33 mg/kg i.p.) 1 day after tumour excision. When moderate doses of rIL-2 (6 × 10⁵ IU in three injections per day for 5 days) were administered at three different intervals after BCNU, namely before the nadir of white blood cells (1 day after BCNU), at the nadir (3 days after BCNU) or at recovery (6 days after BCNU), no increase in BCNU antitumour activity was observed. The same results were obtained by administering rIL-2 for 5 days before BCNU. Higher doses of rIL-2 (1.2 × 10⁶ IU in three injections per day for 5 days), which were always well tolerated in sham-excised non-tumour-bearing mice, proved lethal in two out of four experiments in tumour-bearing animals. In the two experiments in which no lethality was observed, the administration of high doses of rIL-2 1 or 6 days after BCNU significantly increased the antitumour activity of BCNU alone. rIL-2 alone was not active even when administered at high doses. These results indicate that high but not moderate doses of rIL-2 may increase the activity of BCNU against a non-immunogenic tumour. Moreover, they suggest that rIL-2 tolerability is reduced in tumour-bearing mice.     

Detection,characterization, and phytotoxic activity of the nucleoside antibiotics,Blasticidin S and 5-Hydroxylmethyl-Blasticidin S

Screening for herbicidal compounds carried out on culture broths of Streptomyces strains isolated from soil resulted in the detection of potent phytotoxic activity. The active principles were isolated, and identified as the nucleoside antibiotics Blasticidin S (Bl-S) and 5-Hydroxymethyl-Blasticidin S (H-M-Bl-S). Bl-S was more active than H-M-Bl-S on seedling germination in petri dishes and in postemergence greenhouse tests. Moreover both antibiotics were more phytotoxic to dicotyledonous than to monocotyledonous plants. The increased sensitivity of dicots was confirmed in carrot and rice cell cultures. Both compounds also inhibited [¹⁴C]amino acid incorporation into proteins of rice and carrot cell cultures. Protein synthesis was more affected in carrot than rice.     

Interaction of hemin with placental glutathione transferase

To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport.     

Spermine-binding protein and polyamine metabolism in duodenal mucosa of chick embryo

The spermine-binding activity of a cytosolic protein from chick intestine increases during embryogenesis and in the first week of life. Ornithine and S-adenosylmethionine decarboxylase activities assayed under the same experimental conditions increase showing a maximum at day 18 and 20 respectively. The behaviour of either enzyme activity is reflected in the pattern of duodenal polyamine concentration measured during the same period. The possibility that duodenal spermine-binding protein may be correlated with spermine accumulation in the tissue is discussed.     

Book Reviews

Book Reviewed in this article:
Serotonin Receptor Subtypes: Pharmacological Significance and Clinical Implications (International Academy for Biomedical and Drug Research, Vol.1) edited by S. Z. Langer, N. Brunello, G. Racagni, and J. Mendlewicz.
Protocols for Neural Cell Culture edited by S. Fedoroff and A. Richardson.
Molecular Genetic Approaches to Neuropsychiatric Disease edited by J. Brosius and R. T. Fremeau.
Insulin: Molecular Biology to Pathology edited by F. M. Ashcroft and S. J. H. Ashcroft.
Adenosine in the Nervous System edited by T. W. Stone.     

Synonymous and Nonsynonymous Substitutions in Genes from Gramineae: Intragenic Correlations

In this work, we have investigated the relationships between synonymous and nonsynonymous rates and base composition in coding sequences from Gramineae to analyze the factors underlying the variation in substitutional rates. We have shown that in these genes the rates of nucleotide divergence, both synonymous and nonsynonymous, are, to some extent, dependent on each other and on the base composition. In the first place, the variation in nonsynonymous rate is related to the GC level at the second codon position (the higher the GC₂ level, the higher the amino acid replacement rate). The correlation is especially strong with T₂, the coefficients being significant in the three data sets analyzed. This correlation between nonsynonymous rate and base composition at the second codon position is also detectable at the intragenic level, which implies that the factors that tend to increase the intergenic variance in nonsynonymous rates also affect the intragenic variance. On the other hand, we have shown that the synonymous rate is strongly correlated with the GC₃ level. This correlation is observed both across genes and at the intragenic level. Similarly, the nonsynonymous rate is also affected at the intragenic level by GC₃ level, like the silent rate. In fact, synonymous and nonsynonymous rates exhibit a parallel behavior in relation to GC₃ level, indicating that the intragenic patterns of both silent and amino acid divergence rates are influenced in a similar way by the intragenic variation of GC₃. This result, taken together with the fact that the number of genes displaying intragenic correlation coefficients between synonymous and nonsynonymous rates is not very high, but higher than random expectation (in the three data sets analyzed), strongly suggests that the processes of silent and amino acid replacement divergence are, at least in part, driven by common evolutionary forces in genes from Gramineae. Received: 2 July 1998 / Accepted: 18 April 1999     

Rejuvenated Vintage Tissue Sections Highlight Individual Antigen Fate During Processing and Long-term Storage

Antigen-bearing proteins become progressively unavailable to immunodetection after prolonged storage of routine sections, exposed to a variety of agents, such as moisture, oxygen, and temperature. By proteomic analysis, the antigens are retained in the sections and definitely in the tissue block, pointing to fixation-independent, storage time–dependent protein modifications. Based on previous experience, we hypothesized that a combined exposure to a reducing agent and to chemicals favoring protein conformation changes would reverse the masking in aged sections. Disaccharides, lactose and sucrose, and a surfactant, added to a standard antigen retrieval buffer, reverse the negative changes in aged sections. Furthermore, they provide enhanced access to antigens in freshly cut sections, but not universally, revealing additional factors, besides heat and calcium chelation, required for antigen retrieval of individual proteins:     

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role

The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe²⁺-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.     

The influence of aluminium availability on phosphate uptake in Phaseolus vulgaris L. and Phaseolus lunatus L.

Aluminium toxicity is one of the major limiting factors of crop productivity on acid soils. High levels of available aluminium in soil may induce phosphorus deficiency in plants. This study investigates the influence of Aluminium (Al) on the phosphate (P_i) uptake of two Phaseolus species, Phaseolus vulgaris L. var. Red Kidney and Phaseolus lunatus L. The two bean species were treated first with solutions of Al at different concentrations (0, 25, 50 and 100 μM, pH 4.50) and second with solutions of P_i (150 μM) at pH 4.50. The higher the Al concentration the higher the Al concentration sorbed but P. vulgaris L var. Red Kidney adsorbed significantly more Al than P. lunatus L. Both species released organic acids: P. vulgaris L var. Red Kidney released fumaric acid and P. lunatus L. fumaric and oxalic acids which could have hindered further Al uptake.The two bean species showed a sigmoid P_i uptake trend but with two different mechanisms. P. vulgaris L var. Red Kidney showed a starting point of 3 h whereas P. lunatus L. adsorbed P_i immediately within the first minutes. In addition, P. vulgaris L var. Red Kidney presented significantly higher P_i uptake (higher uptake rate ‘k’ and higher maximum adsorption ‘a’ of the kinetic uptake model). The Al treatments did not significantly influence P_i uptake. Results suggest that P. lunatus L. might adopt an external Al detoxification mechanism by the release of oxalic acid. P. vulgaris L var. Red Kidney on the other hand seemed to adopt an internal detoxification mechanism even if the Al sorbed is poorly translocated into the shoots. More detailed studies will be necessary to better define Al tolerance and/or resistance of Phaseolus spp.     

Proteomic changes in rat serum, polymorphonuclear and mononuclear leukocytes after chronic nicotine administration

In order to gain information about the effect triggered at the molecular level by nicotine, its neuroimmunomodulatory properties and its impact on the pathogenesis of inflammatory diseases, peripheral blood serum and leukocytes of rat submitted to passive nicotine administration were subjected to proteomic investigation. Serum, polymorphonuclear (PMN) and mononuclear (MN) leukocytes from chronically treated animals and from control animals were analysed by a two-dimensional (2-D) gel electrophoresis/mass spectrometry approach to detect differentially expressed proteins. The nicotine regimen selected is known to have a stimulatory effect on locomotor activity and to produce a sensitisation of the mesolimbic dopamine system mechanism involved in addiction development. After 2-D gel analysis and matching, 36 spots in serum, seven in PMN and five in MN were found to display a statistical difference in their expression and were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry peptide fingerprinting for protein identification. Fifteen different proteins were identified. The results indicate an overall impact of nicotine on proteins involved in a variety of cellular and metabolic pathways, including acute phase response (suggesting the effect on inflammatory cascades and more in general on the immune system), oxidative stress metabolism and assembly and regulation of cytoskeleton. In particular, the observed changes imply a general reduction in the inflammatory response with a concomitant increased unbalance of the oxidative stress metabolism in the periphery and point to a number of potential noninvasive markers for the central nervous system (CNS) and non-CNS mediated activities of nicotine.