Complete resistance to pepper mild mottle tobamovirus mediated by viral replicase sequences partially depends on transgene homozygosity and is based on a gene silencing mechanism

We have previously distinguished two different types of resistance response, complete and delayed, to the pepper mild mottle tobamovirus (PMMoV) in Nicotiana benthamiana plants transformed with the 54kDa region of the viral replicase gene. The present study was undertaken to assess a potential relationship between transgene dose and the type of resistance observed in the 54kDa transgenic plants. By genetic and molecular analysis, we demonstrated that complete resistance against PMMoV infection was strictly correlated with homozygosity of the transgene in the R1 and R2 generation plants. Progeny hemizygous for the TDNA locus showed delayed resistance, but none of the progeny exhibited the complete resistance response. Thus, the resistance to PMMoV varied in a gene dosagedependent manner. However, a limited number of individuals of the R2 progeny from homozygous plants displayed complete resistance, while the remaining plants showed delayed resistanc; the ratio between the two resistance responses was roughly similar to the ratio observed in either the R2 progeny from hemizygous plants or the original R1 generation plants. R2 homozygous plants displaying complete resistance contained undetectable levels of 54kDa mRNA, although the transgene was accurately transcribed in these plants. These results suggest that transgene homozygosity is a necessary but not a sufficient condition to confer complete resistance to PMMoV by a gene silencing mechanism.     

Interferons up-regulate with different potency HLA class I antigen expression in M14 human melanoma cell line. Possible interaction with glucocorticoid hormones

The relative potency of interferon (IFN), interferon (IFN), and interferon (IFN) in inducing the expression of HLA class I antigens, as well as their capacity to counteract the inhibition induced by glucocorticoid hormones on HLA class I antigen expression, were analysed in the human melanoma cell line M14, both at membrane and at mRNA level. The data obtained indicate that (a) IFN enhance with different potency (IFN>IFN>IFN) the expression of HLA class I antigens in M14 cells, (b) prednisone inhibits HLA class I antigen expresion, (c) glucocorticoid hormones, when associated with IFN or IFN, inhibit the HLA class I enhancement induced by IFN alone, and ffinally, (c) the association between 1 M prednisone or 1 M deflazacort and IFN seems to potentiate the enhancing capacity of IFN on the expression of HLA class I molecules at the mRNA level. These findings, if confirmed, might indicate that IFN and glucocorticoid hormones are not mutually exclusive in the management of human melanoma.     

Low-dose melphalan-induced shift in the production of a Th2-type cytokine to a Th1-type cytokine in mice bearing a large MOPC-315 tumor

The current studies demonstrate that MOPC-315 tumor cells secrete large amounts of interleukin-10 (IL-10), which contributes to the inhibitory activity of MOPC-315 culture supernatants for the in vitro generation of antitumor cytotoxicity by MOPC-315-immune spleen cells. Moreover, addition of neutralizing monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of cells from the tumor infiltrated spleens of mice bearing a large MOPC-315 tumor resulted in the generation of enhanced anti-MOPC-315 cytotoxicity. In contrast, addition of monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of splenic cells from mice that are in the final stages of immune-mediated tumor eradication as a consequence of low-dose melphalan (l-phenylalanine mustard; L-PAM) therapy (and whose spleens no longer contain metastatic tumor cells) did not lead to enhancement in the in vitro generation of antitumor cytotoxicity. The cessation of IL-10 secretion as a consequence of low-dose L-PAM therapy of MOPC-315 tumor bearers was found to be accompanied by the acquisition of the ability to secrete interferon (IFN) by the splenic cells. In addition, by day 2 after low-dose L-PAM therapy a drastic decrease in the amount of IL-10 secreted by the s.c. tumor nodules was noted, which preceded the accumulation of tumor-infiltrating lymphocytes capable of secreting IFN. Thus, low-dose L-PAM therapy of mice bearing a large MOPC-315 tumor leads to a shift in cytokine production from a Th2-type cytokine to a Th1-type cytokine, and it is conceivable that this shift in cytokine production plays an important role in the low-dose L-PAM-induced acquisition of antitumor immunity by hitherto immunosuppressed mice bearing a large MOPC-315 tumor.Supported by research grant IM-435 from the American Cancer Society and CA54413 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of career development award CA-01350 from the National Cancer Institute     

Cytokine regulation of cell-to-cell interactions in lymphokine-activated killer cell cytotoxicity in vitro

The permanent pancreas carcinoma cell line, PCI-24, was developed in order to analyse cytokine regulation on pancreas carcinoma and lymphokine-activated killer (LAK) cell interaction. PCI cells expressed ICAM-1 and HLA-ABC, but not HLA-DR antigens. PCI cells showed augmented ICAM-1 and HLA-ABC expression when incubated with interferon (IFN) and tumour necrosis factor . A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN in close association with ICAM-1 up-regulation on PCI cells. In addition, natural attachment was significantly inhibited by anti-LFA-1 and anti-ICAM-1 antibody treatments. Cytotoxicity of the LAK cells against PCI cells was also significantly inhibited with the same treatment. Thus, the attachment of LAK cells to PCI cells through LFA-1/ICAM-1 molecules appeared to be essential for the cytotoxicity for PCI cells. Pretreatment of PCI cells, but not of LAK cells, with IFN or other cytokines resulted in a decrease of susceptibility for LAK cell cytotoxicity. The decreased susceptibility inversely correlated with HLA-ABC expression on the PCI cells. The collective evidence indicates that, although LAK cell attachment to pancreas carcinoma cells through the LFA-1/ICAM-1 molecule is augmented by IFN, IFN treatment of pancreas carcinoma cells reduces LAK cell cytotoxicity possibly through an increase in HLA-ABC or a regulation of molecules closely associated to HLA-ABC expression.     

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.     

The uptake and metabolism of fructose‐1,6‐diphosphate in rat cardiomyocytes

Fructose1,6diphosphate (FDP) is a glycolytic intermediate which has been theorized to increase the metabolic activity of ischemic tissues. Here we examine the effects of externally applied FDP on cardiomyocyte uptake and metabolism. Adult rat cardiomyocytes were isolated and exposed to varying concentrations (0, 5, 25 and 50 mM) of FDP for either 1, 16 or 24 h of hypoxia (95% N₂/5% CO₂), each time period followed by a 1 h reoxygenation (95% air/5% CO₂). The uptake of FDP by rat cardiomyocytes was more concentrationdependent than timedependent. Furthermore, the uptake of FDP by the cardiomyocytes was similar in the hypoxia and normoxia treated cells. Alamar Blue, a redox indicator that is sensitive to metabolic activity, was used to monitor the effects of the FDP on cardiomyocyte metabolism. In the 1 h hypoxia or normoxia group, the 5, 10 and 25 mM FDP showed a significant increase in metabolism compared to the control cells. When the length of hypoxia was extended to 16 h, all doses of FDP were greater than control. And at the 24 h hypoxia or normoxia time period, only the 10, 25 and 50 mM FDP groups were greater than control. The results indicate a non-linear trend between the external concentration of FDP and the changes noted in metabolism. The findings from this study indicate that a narrow concentration range between 5–10 mM augments cardiomyocyte metabolism, but higher or lower doses may have little additional affect.     

Role of ubiquitin‐proteasome‐dependent proteolytic process in degradation of muscle protein from diabetic rabbits

The activity of ATP, ubiquitin (Ub)dependent proteases partially purified from skeletal muscle (psoas) from alloxan diabetic rabbits was determined at different periods of insulin deficiency. Two days after alloxan injection, no change was observed in the activity of ATP, Ubdependent proteases, but this activity increased 3 and 5 days after diabetes induction, attaining 181% of control values on the 5th day. However, after this early rise, the activity of muscle ATP, Ubdependent proteases decreased, returning to values that did not differ significantly from controls 7 and 10 days after alloxan injection. After 15 days, the activity of these proteases was 57% lower than in muscle from control rabbits. Both the initial increase and the subsequent fall in the activity of the enzymes were prevented by insulin treatment of alloxan diabetic rabbits. The data suggest that Ubproteasomedependent proteolysis have an important role in the control of muscle protein degradation and may be regulated by insulin.     

Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis

The effect of intraperitoneal administration of tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the tocopherol treated group were not observed. The light emission was significantly higher in the control than in the tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbateFe²⁺ lipid peroxidation. The protector effect observed by tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

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.     

Exploration of RNA 3′ terminal locations in rat ribosome

The locations of the 3 ends of RNAs in rat ribosome were studied by a fluorescencelabeling method combined with high hydrostatic pressure and agarose electrophoresis. Under physiological conditions, only the 3 ends of 28 S and 5.8 S RNA in 80 S ribosome could be labeled with a high sensitive fluorescent probe – fluorescein 5thiosemicarbazide (FTSC), indicating that the 3 termini of 28 S and 5.8 S RNA were located on or near the surface of 80 S ribosome. The 3 terminus of 5 S RNA could be attacked by FTSC only in the case of the dissociation of the 80 S ribosome into two subunits induced by high salt concentration (1 M KCl) or at high hydrostatic pressure, showing that the 3 end of 5 S RNA was located on the interface of two subunits. However, no fluorescencelabeled 18 S RNA could be detected under all the conditions studied, suggesting that the 3 end of 18 S RNA was either located deeply inside ribosome or on the surface but protected by proteins. It was interesting to note that modifications of the 3 ends of ribosomal RNAs including oxidation with NaIO₄, reduction with KBH₄ and labeling with fluorescent probe did not destroy the translation activity of ribosome, indicating that the 3 ends of RNAs were not involved in the translation activity of ribosome.     

Differential molecular responses to abscisic acid and osmotic stress in viviparous maize embryos

Substantial quantities of mRNA encoding the abundant Em polypeptide accumulate, in planta, in developing embryos of maize (Zea mays L.). By contrast, accumulation of Em mRNA is only barely detectable in embryos with the vp-5/vp-5 genotype [an abscisic acid (ABA)-deficient viviparous phenotype]. Em mRNA is not detectable within viviparous embryos of the vp-1/vp-1 genotype that are non-responsive to ABA. Culture of immature wild-type and vp-5/vp-5 embryos in the presence of exogenous ABA or of an osmotically active agent prevents precocious germination and results in expression of the Em genes. When vp-1/vp-1 embryos are cultured under similar conditions, only the application of osmotic stress prevents precocious germination. However, Em mRNA does not accumulate either in ABA-treated or stressed, arrested embryos, indicating a requirement for ABA perception through a VP-1-mediated mechanism for Em gene expression. Nevertheless, vp-1/vp-1 embryos do show both ABA and stress responses at the molecular level. Treatment with ABA causes the accumulation of mRNA encoding a polypeptide of approx. 30 kDa, whilst osmotic stress induces the accumulation both of a 30-kDa polypeptide and a set of approx. 20-kDa polypeptides. This indicates the existence of discrete, parallel ABA and stress response pathways in developing maize embryos.Abbreviations ABA abscisic acid - cDNA copy-DNA - DAP days after pollination - kDa kilodaltons - MS Murashige and Skoog medium - LEA late embryogenesis abundant - NEpHGE non-equilibrium pH gradient gel electrophoresis - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Effects of D‐mannoheptulose upon D‐glucose metabolism in tumoral pancreatic islet cells

D[³H]mannoheptulose was recently reported to be poorly taken up by tumoral pancreatic islet cells of the RINm5F and INS1 lines. We have now investigated the effects of Dmannoheptulose upon Dglucose metabolism in these two cell lines. Dmannoheptulose (1.0–10.0 mM) only caused a minor decrease of Dglucose metabolism in RINm5F cells, whether at low (1.1 mM) or higher (8.3 mM) Dglucose concentration. A comparable situation was found in INS1 cells examined after more than 20 passages. In both cases, however, the hexaacetate ester of Dmannoheptulose (5.0 mM) efficiently inhibited Dglucose metabolism. In the INS1 cells, the relative extent of the inhibitory action of Dmannoheptulose upon Dglucose metabolism increased from 12.4 ± 2.6 to 38.3 ± 3.8% as the number of passages was decreased from more than 20 to 13–15 passages, the latter percentage remaining lower, however, than that recorded in INS1 cells also examined after 13–15 passages but exposed to Dmannoheptulose hexaacetate (66.9 ± 2.2%). These findings when compared to our recent measurements of D[³H]mannoheptulose uptake, reinforce the view that the entry of the heptose into cells and, hence, its inhibitory action on Dglucose metabolism are dictated by expression of the GLUT2 gene.     

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     

“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.     

Differential zinc and DNA binding by partial peptides of human protamine HP2

The Zn(II) binding by partial peptides of human protamine HP2: HP2_1–15; HP2_1–25, HP2_26–40, HP2_37–47, and HP2_43–57 was studied by circular dichroism (CD). Precipitation of a 20mer DNA by these partial peptides and the effects of Zn(II) thereon were investigated using polyacrylamide gel electrophoresis (GE). The results of this study suggest that reduced HP2 (thiol groups intact) can bind Zn(II) at various parts of the molecule. In the absence of DNA, the primary Zn(II) binding site in reduced HP2 is located in the 37–47 sequence (involving Cys37, His39, His43, and Cys47), while in the presence of DNA, the strongest Zn(II) binding is provided by sequences 12–22 (by His12, Cys13, His19, and His22) and 43–57 (His43, Cys47, Cys53, and His57). In its oxidized form, HP2 can bind zinc through His residues of the 7–22 sequence. Zn(II) markedly enhances DNA binding by all partial peptides. These findings suggest that Zn(II) ions may be a regulatory factor for sperm chromatin condensation processes.     

Natural-killer-stimulatory effect of combined low-dose interleukin-2 and interferon β in hairy-cell leukemia patients

The association of low doses of interleukin-2 (IL-2; 5 IU/ml) and interferon (IFN; 10 IU/ml) induced an additive or synergic stimulatory effect on natural killer (NK) activity (32%) in peripheral blood samples from hairy-cell leukemia patients, both those with active disease and those in remission. The synergic NK stimulatory effect was more commonly found in samples from patients with active disease, while the additive effect was more frequent in the patients in remission. The IL-2/IFN combination provoked a nonadditive nonsynergic NK-stimulatory effect in a further 19.8% samples. The targets of the IL-2/IFN combination were typical NK cells, as shown by the fact that there was increased cytotoxicity (synergic, additive or nonadditive nonsynergic) against the K562, but not the Daudi cell line in peripheral blood mononuclear cell samples treated with the combination of the two cytokines. When CD16⁺/CD56⁺ or CD57⁺/CD16⁺/CD56⁺ cells were removed, the NK-stimulatory effect was lost. The fact that the NK-cell-enhancing activity of the IL-2/IFN combination was reduced when Percoll fractions 2 and 3 were used, but still persisted in 66% of tests, may have been due to cytotoxicity being higher in the untreated fractions 2 and 3 than in the untreated unfractionated samples. One of the factors responsible for the NK-stimulatory effect appears to be the capacity of the IL-2/IFN combination to trigger an increase in IFN synthesis. If similar experiments give like results in samples from patients suffering from other B-cell lymphoproliferative, or HIV-associated disorders, all of which are characterized by a deficiency in NK activity, it should be possible to use low-dose IL-2/IFN to treat these disorders and, perhaps, residual neoplastic disease without exposing the patient to undue toxicity. Further, by testing other combinations one should be able to identify the lowest IL-2 and IFN doses that would effectively boost the additive or synergic effect in a greater number of cases.