Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

Effect of murine interferon α/β on tumor-induced suppressor function

T-lymphocyte-mediated immunosuppression has been described in several animal models and in man. In animal models, T-cell-mediated immunosuppression can hasten the development of cancers, permit the growth of tumors in immunocompetent hosts, and inhibit otherwise effective antitumor immunotherapy. Cyclophosphamide can abrogate the T-cell-mediated immunosuppression. However, inappropriately administered cyclophosphamide can adversely affect antitumor immunity. On the basis of data showing that interferon / (IFN/) and IFN selectively abrogate the T-cell-mediated dinitrofluorobenzene-specific suppressor function, we investigated the efficacy of purified murine IFN/ in manipulating tumorinduced T-cell-mediated immunosuppression in the wellcharacterized P815 mastocytoma model. In this model, generation of cytotoxicity in vitro and its inhibition by T cells correlates with antitumor immunity in vivo. We report that IFN/ selectively diminishes the generation of tumor-induced suppressor activity.     

A new approach to predicting protein folding types

A new method is proposed for predicting the folding type of a protein according to its amino acid composition based on the following physical picture: (1) a protein is characterized as a vector of 20-dimensional space, in which its 20 components are defined by the compositions of its 20 amino acids; and (2) the similarity of two proteins is proportional to the mutual projection of their characterized vectors, and hence inversely proportional to the size of their correlation angle. Thus, the prediction is performed by calculating the correlation angles of the vector for the predicted protein with a set of standard vectors representing the norms of four protein folding types (i.e., alla, all ,a+, anda/). In comparison with the existing methods, the new method has the merits of yielding a higher rate of correct prediction, displaying a more intuitive physical picture, and being convenient in application. For instance, in predicting the 64 proteins in the development set based on which the standard vectors are derived, the average accuracy rate is 83.6%, which is higher than that obtained for the same set of proteins by any of the existing methods. The average accuracy predicted for an independent set of 35 proteins of known X-ray structure is 91.4%, which is significantly higher than any of the reported accuracies so far, implying that the new method is of great value in practical application. All of these have demonstrated that the new method as proposed in this paper is characterized by an improved feature in both self-consistency and extrapolating-effectiveness.On sabbatical leave from Department of Physics, Tianjin University, Tianjin, China.     

Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat

Summary. We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.     

Energy Metabolism of Synaptosomal Subpopulations from Different Neuronal Systems of Rat Hippocampus: Effect of L-Acetylcarnitine Administration In Vivo

The maximum rate (V_max) of some enzyme activities related to glycolysis, Krebs' cycle, acetylcholine catabolism and amino acid metabolism were evaluated in different types of synaptosomes obtained from rat hippocampus. The enzyme characterization was performed on two synaptosomal populations defined as large and small synaptosomes, supposed to originate mainly from the granule cell glutamatergic mossy fiber endings and small cholinergic nerve endings mainly arising from septohippocampal fiber synapses, involved with cognitive processes. Thus, this is an unique model of pharmacological significance to study the selective action of drugs on energy metabolism of hippocampus and the sub-chronic i.p. treatement with L-acetylcarnitine at two different dose levels (30 and 60 mg · kg^–1, 5 day a week, for 4 weeks) was performed. In control animals, the results indicate that these two hippocampal synaptosomal populations differ for the potential catalytic activities of enzymes of the main metabolic pathways related to energy metabolism. This energetic micro-heterogeneity may cause their different behaviour during both physiopathological events and pharmacological treatment, because of different sensitivity of neurons. Therefore, the micro-heterogeneity of brain synaptosomes must be considered when the effect of a pharmacological treatment is to be evaluated. In fact, the in vivo administration of L-acetylcarnitine affects some specific enzyme activities, suggesting a specific molecular trigger mode of action on citrate synthase (Krebs' cycle) and glutamate-pyruvate-transaminase (glutamate metabolism), but mainly of small synaptosomal populations, suggesting a specific synaptic trigger site of action. These observations on various types of hippocampal synaptosomes confirm their different metabolic machinery and their different sensitivity to pharmacological treatment.     

Models for glycosidic juvenogens: Enzymic formation of selected alkyl-β-D-glucopyranosides and alkyl-β-D-galactopyranosides under microwave irradiation

2-(4-Methoxybenzyl)-1-cyclohexyl--d-glucopyra nosides (1b and 2b) and 2-(4-methoxybenzyl)-1-cyclohexyl--d-galactopy ranosides (1c and 2c), models for glycosidic juvenogens, were synthesized using either D-glucose or D-galactose [in their natural form (3 and 5) or activated form (4 and 6)], and the respective racemic cis or trans isomers of 2-(4-methoxybenzyl)-1-cyclohexanol (1a and 2a) by either enzymic reverse hydrolysis or transglycosylation under both standard heating and microwave irradiation. Commercially available almond -glucosidase (EC 3.2.1.21) or -galactosidase (EC 3.2.1.23) from Escherichia coli were employed using different acetonitrile/water mixtures [9/1 (v/v) for the reverse hydrolysis, and 4/1 (v/v) for the transglycosylation].     

Melatonin enhances Th2 cell mediated immune responses: Lack of sensitivity to reversal by naltrexone or benzodiazepine receptor antagonists

Chronic administration of melatonin for 5 days to antigen-primed mice increased the production of pro-inflammatory cytokine IL10 but decreased the secretion of antiinflammatory cytokine TNF-. These results further confirm that melatonin activates Th2like immune response. Whether melatoninmediated Th2 response is dependent on opioid or central and peripheral benzodiazepine receptors was also examined. Hence, melatonin was administered to antigen-sensitised mice with either naltrexone (a opioid receptor antagonist) or flumazenil (a central benzodiazepine receptor antagonist) or PK11195 (a peripheral benzoidiazepine receptor antagonist). No significant difference in melatonin-induced Th2 cell response was observed by naltrexone, flumazenil or PK11195 treatment. These findings suggest that the Th2 cell response induced by melatonin in antigen sensitised mice neither dependent on endogenous opioid system nor is modulated through the central or peripheral benzodiazepine receptors.     

Tnf-α and IL-1α inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: Evidence for primary impairment of mitochondrial function

Cytokines such as tumor necrosis factor (TNF) and Interleukin-1 (IL1) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNF and IL1. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNF (1, 10, 100, 1000 I.U./ml) and IL1 (0.1, 1, 10, 100 I.U./ ml).TNF- and IL1- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNF and IL1 and a subsequent down regulation of PDH activity.     

Promotion of murine antitumour activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α

Summary The effect of prothymosin (ProT) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10⁵ syngeneic leukaemic L1210 cells developed ascites within 8–12 days and died 10–14 days later. Treatment with ProT consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%–60% of the animals up to 70 days. The most effective treatment schedule of ProT was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor (TNF) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF and tumoricidal activity peaked 7 days after the last injection of ProT and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT and a wide range of thymosin 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF and exhibited negligible tumoricidal activity. Our data demonstrate that ProT has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProT-activated PE cells. These cells were demonstrated to produce high levels of TNF in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.Supported by a CEC grant to Dr. M. Papamichail     

“Gomori-positive” neurosecretion in the rat after deafferentation of the medial basal hypothalamus

A portion of the "Gomori-positive" peptidergic neurosecretory (NS) cells in the paraventricular and especially in the supraoptic and postoptic nuclei degenerate three weeks after deafferentation of the medial basal hypothalamus. Most of the remaining NS cells show signs of high activity. Regenerating NS fibres form "muffs" around the blood vessels laterally from the lesion; some of them enter the "isolated" area or persist there if a thin layer of the brain tissue is left somewhere untouched under the basal end of the cut. The regenerating NS fibres are also found outside the nervous tissue: within the scar tissue, in the proliferating connective tissue of the brain sheet below the basal end of the cut and in the mantel plexus area. The NS fibres make close contact with blood vessels invading or penetrating the vascular wall. It is suggested that peptide neurohormones discharged from the "Gomori-positive" NS terminals enter the general blood circulation as well as the portal blood at the site of these newly formed axovasal contacts. It is supposed that under these conditions monoaminergic terminals do not discharge monoamines because no stimulation of monoamine-producing NS cells occurs with deafferentation.     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

An efficient synthesis of 3′-amino-3′-deoxythymidine derivatives

An efficient method of reduction of 3-azido-3-deoxythymidine and its 5-protected derivatives to 3-aminothymidine derivatives on a palladium catalyst using ammonium formate as a source of hydrogen was suggested.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 147–150.Original Russian Text Copyright © 2005 by Seregin, Chudinov, Yurkevich, Shvets.     

Genomic DNA can be used with cationic methods for highly efficient transformation of maize protoplasts

Summary Efficient delivery of genomic DNA fragments to maize protoplasts was obtained by new methods using the polycation Polybrene or Lipofectin cationic liposomes. Stable kanamycin-resistant secondary transformants were recovered after transfection with genomic DNA from a maize cell line that had previously been tagged with the bacterial gene neomycin phosphotransferase (nptII) in a first-round transformation. The frequency of secondary transformants with nptII-homologous DNA sequences was 3% or 6% of all randomly picked microcalli after Polybrene or Lipofectin-mediated transfection, respectively. Transformation with genomic DNA by these methods may allow easy transfer of uncloned genes encoding desirable characteristics to crop species that can be regenerated from protoplasts.     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

Novel plasmid marker rescue transformation system for molecular cloning in Bacillus subtilis enabling direct selection of recombinants

Summary A versatile plasmid marker rescue transformation system was developed for homology-facilitated cloning in Bacillus subtilis. It is based on the highly efficient host-vector system 6GM15-pHPS9, which allows the direct selection of recombinants by means of -galactosidase -complementation. The system offers several advantages over previously described cloning systems: (1) the convenient direct selection of recombinants; (2) the ability to effectively transform B. subtilis competent cells with plasmid monomers, which allows the forced cloning of DNA fragments with high efficiency; (3) the availability of 6 unique target sites, which can be used for direct clone selection, SphI, NdeI, NheI, BamHI, SmaI and EcoRI; and (4) the rapid segregational loss of the helper plasmid from the transformed cells.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Chromosome and isoenzyme studies on cells derived from protoplast fusion of Nicotiana glauca with glycine max-Nicotiana glauca cell hybrids

Summary The somatic hybrids of Glycine max (L)Merr.-Nicotiana glauca Grah. exhibited a preferential loss of N. glauca chromosomes. When protoplasts from such hybrid cells were back fused twice to N. glauca protoplasts, a considerable increase in stability of the N. glauca chromosomes was observed. Gel electrophoresis studies of aspartate aminotransferase showed that the chromosome(s) responsible for this enzyme was stabilized in the back fused hybrid cell lines. The data suggest that the back fusion technique described in this study might aid in stabilizing somatic hybrids.NRCC No. 18040     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.