Biological activities of human interferon and 2′–5′ oligoadenylates in plants

Exogenous human interferon 2 (IFN) and 2–5 oligoadenylates (2–5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1–1 u/ml and 2–5A at 1–10 nM.     

Structure–activity relationships in human RNA 3′-phosphate cyclase

RNA 3′-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2′,3′ cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP_i; transfer of AMP from Rtc1 to an RNA 3′-phosphate to form an RNA(3′)pp(5′)A intermediate; and attack by the terminal nucleoside O2′ on the 3′-phosphate to form an RNA 2′,3′ cyclic phosphate product and release AMP. Here we used the crystal structure of Escherichia coli RtcA to guide a mutational analysis of the human RNA cyclase Rtc1. An alanine scan defined seven conserved residues as essential for the Rtc1 RNA cyclization and autoadenylylation reactions. Structure–activity relationships were clarified by conservative substitutions. Our results are consistent with a mechanism of adenylate transfer in which attack of the Rtc1 His320 nucleophile on the ATP α phosphorus is facilitated by proper orientation of the PP_i leaving group via contacts to Arg21, Arg40, and Arg43. We invoke roles for Tyr294 in binding the adenine base and Glu14 in binding the divalent cation cofactor. We find that Rtc1 forms a stable binary complex with a 3′-phosphate terminated RNA, but not with an otherwise identical 3′-OH terminated RNA. Mutation of His320 had little impact on RNA 3′-phosphate binding, signifying that covalent adenylylation of Rtc1 is not a prerequisite for end recognition.     

Nuclease activity in human metaphase chromosomes substituted with 5′-bromodeoxyuridine

Human metaphase chromosomes, substituted with 5-bromodeoxyuridine (BrdUrd) for one, two or three rounds of replication, were briefly pretreated with ultraviolet light (UV), in the presence of 33258 Hoechst, and subsequently digested with either exonuclease III or S1 nuclease. Pretreatment alone was not sufficient to induce sister chromatid differential staining (SCD), but allowed subsequent digestion with exonuclease III or S1. Such enzymes were found to induce SCD with ethidium bromide, as unifilarly BrdUrd-substituted chromatids (TB) were more resistant than bifilarly substituted chromatids (BB). Other experiments with DNase I or the AluI and HaeIII restriction endonucleases showed that only HaeIII was capable of inducing SCD by attacking BB more than TB chromatids preincubated with UV in the presence of Hoechst. SCD with exonuclease III/S1 nuclease seems to be due to (1) UV-induced DNA debromination occurring twice in BB as opposed to TB chromatids, and (2) alteration of chromatin protein structure occurring to a different extent in differently BrdUrd-substituted chromatids. Our findings with endonucleases, on the contrary, may depend on the capacity of enzymatic cleavage to cancel the different protein alterations induced differentially by UV in TB as opposed to BB chromatids.     

Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.     

Identification of elements in human long 3′ UTRs that inhibit nonsense-mediated decay

The nonsense-mediated mRNA decay (NMD) pathway serves an important role in gene expression by targeting aberrant mRNAs that have acquired premature termination codons (PTCs) as well as a subset of normally processed endogenous mRNAs. One determinant for the targeting of mRNAs by NMD is the occurrence of translation termination distal to the poly(A) tail. Yet, a large subset of naturally occurring mRNAs contain long 3′ UTRs, many of which, according to global studies, are insensitive to NMD. This raises the possibility that such mRNAs have evolved mechanisms for NMD evasion. Here, we analyzed a set of human long 3′ UTR mRNAs and found that many are indeed resistant to NMD. By dissecting the 3′ UTR of one such mRNA, TRAM1 mRNA, we identified a cis element located within the first 200 nt that inhibits NMD when positioned in downstream proximity of the translation termination codon and is sufficient for repressing NMD of a heterologous reporter mRNA. Investigation of other NMD-evading long 3′ UTR mRNAs revealed a subset that, similar to TRAM1 mRNA, contains NMD-inhibiting cis elements in the first 200 nt. A smaller subset of long 3′ UTR mRNAs evades NMD by a different mechanism that appears to be independent of a termination-proximal cis element. Our study suggests that different mechanisms have evolved to ensure NMD evasion of human mRNAs with long 3′ UTRs.     

Electrophoretic and kinetic studies of human erythrocytes deficient in pyrimidine 5′-nucleotidase

Summary A new case of a defect in red cell pyrimidine 5-nucleotidase (P5N) activity was found in a large family from Guadeloupe in the West Indies. The propositus presented a characteristic hemolytic anemia with red cell basophilic stippling, an increased GSH level, and a shift of the peak in absorbance of nucleotide. The enzyme activity of the deficient red cells was about 14% that of normal. The electrophoretic pattern of P5N activity from the deficient red cells differed from that of the normal. The P5N activity of the deficient red cells was distinct from that of the control in terms of its Km and of the effects of pH on its maximum activity and heat stability. The significance of such differences is discussed.     

Kinetic and electrophoretic studies of human erythrocytes deficient in pyrimidine 5′-nucleotidase

Summary The mutant enzyme of a patient with hereditary pyrimidine 5-nucleotidase deficiency was analyzed biochemically. Partially purified by DEAE-Sephadex and concentrated by ultrafiltration, the enzyme had a high K_m for the substrate uridine monophosphate. Utilization of the substrate cytidine monophosphate was normal, but utilization of adenosine monophosphate was greatly increased. The enzyme was stable to heat; the pH optimum was acidic. Electrophoresis of the enzyme revealed a very faint, slower than normal band.     

Phosphatidylinositol 3′-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3′-kinase (PI 3′-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3′-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3′-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3′-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca²⁺ levels that can be effected by Ca²⁺ mobilized from intracellular stores in the absence of Ca²⁺ influx regulate PA-induced chemotaxis. Furthermore, PI 3′-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP₃), suggesting that PI 3′-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3′-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3′-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Molecular basis of 2′,3′-dideoxycytidine-induced drug resistance in human cells

Human monoblastoid cells (U937) grown in the presence of therapeutically relevant dideoxycytidine concentrations (0.1 M) become resistant to the drug thanks to an altered deoxycytidine kinase. In this paper we show that deoxycytidine kinase mRNA is significally reduced in drug-resistant U937 cells (U937-R) although the deoxycytidine kinase promoter is normal. A number of nucleotide deletions, insertions and substitutions was found in the coding region of deoxycytidine kinase gene. Several identified mutations result in truncated forms of the enzyme or in the introduction of stop codons: in one case a complete lack of exon 4 was found. Thus, the deoxycytidine kinase gene accumulates mutations at a very high rate, as already reported for other cytidine analogues (i.e. Ara C ) suggesting that the design of new antiviral or anticancer drugs of the cytidine family should take into account the potential development of cell resistance as a critical factor in drug failure.     

Inhibition of collagen synthesis by α, α′-dipyridyl in human skin fibroblasts in culture

Summary In human diploid skin fibroblasts in culture we have shown that nonhydroxylated collagen precursors remain in the cell when proline hydroxylation is inhibited by α, α′-dipyridyl, a chelator of ferrous ions. The inhibition of proline hydroxylation is reversed by addition of fresh medium containing 50 μg per ml of sodium ascorbate, whereupon nonhydroxylated collagen precursors are hydroxylated within the cell and extruded into the medium. Extrusion of collagen already formed within the cell is not appreciably affected by α, α′-dipyridyl inhibition. Under normal conditions collagen is released from the monolayer into the medium within 3 hr of a pulse ofL[¹⁴C]proline. In the presence of α, α′-dipyridyl, about 35% of theL[¹⁴C]proline incorporated into protein is released into the medium within 8 hr as a proline-rich, hydroxyproline-deficient protein; at the same time, approximately 15% of the protein-boundl-[¹⁴C]proline remains in the cell for as long as 12 hr. When proline hydroxylation is restored after 2 and 12 hr of α, α′-dipyridyl inhibition, approximately the same amount of hydroxyproline is formed after each time interval in the monolayer. Therefore, nonhydroxylated collagen precursors retained in the cell are not appreciably degraded during at least 12 hr of inhibition by α, α′-dipyridyl and are extruded into the medium only upon restoration of hydroxylation. This work was supported in part by a grant from the Easter Seal Research Foundation, and by Project 236, Health Services and Mental Health Administration, Department of Health, Education and Welfare, Grant HD-03110 from the National Institute of Child Health and Human Development, an American Cancer Society Institutional Grant (1N 15-J), a General Research Support Award (5-S01-FR-05406) from the National Institutes of Health, a University Research Council Grant, a National Science Foundation Equipment Grant (GB-4577), and a Research Career Development Award (5-K3-AM-5058) from the National Institute of Arthritis and Metabolic Disease (G.K.S.).     

The proposed 5′-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5′-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577–584) mentioned that this phenomenon resulted from the presence of a 5′-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5′-[³H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [³H]Adenosine derived from 5′-[³H]AMP hydrolysis was further transformed into [³H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [³H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5′-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5′-nucleotidase inhibitor. We did not observe 5′-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.     

Localization of adenylate cyclase and 5′-nucleotidase activities in human thyroid follicular cells

Summary The localization of adenylate cyclase and 5-nucleotidase activities in the follicular cells of adenomatous goiter and normal thyroid was studied by light and electron microscopy. Simultanous biochemical measurement for both activities was carried out to confirm the histochemical findings. Adenylyl-imidodiphosphate (AMP-PNP) was used as an effective substrate for adenylate cyclase. The specificity of the adenylate cyclase reaction was also examined by adding oxalacetic acid or PCMB as an adenylate cyclase inhibitor, and by adding sodium fluoride or TSH as an adenylate cyclase stimulator to the reaction mixture. In the case of tissue from adenomatous goiter, a large amount of the reaction product of the adenylate cyclase activity was found uniformly in the apical and lateral plasma membrane and not in the basal plasma membrane. In the cases of normal thyroid, a small amount of the reaction product of adenylate cyclase activity was demonstrated, and only in the lateral plasma membrane of the follicular cells. On the other hand, the histochemical localization of 5-nucleotidase activity was the same in adenomatous goiter and normal thyroid. The reaction product of 5-nucleotidase activity was found predominantly in the apical plasma membrane of the follicular cells. The biochemical findings indicated that the activity of adenylate cyclase per gram tissue was approximately 2 times higher in the case of adenomatous goiter than that in the case of normal thyroid, while the 5-nucleotidase activity in adenomatous goiter was in slightly higher level than in normal thyroid. Thus the histochemically demonstrable amount of adenylate cyclase and 5-nucleotidase reflected the activity levels measured biochemically. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that this structure may not play any important role in TSH reception.     

An EcoRI RFLP in the 5′ region of the human NF1 gene

Von Recklinghausen neurofibromatosis or type l neurofibromatosis (NF1), is one of the most common autosomal dominant disorders. NF1 is characterized by neurofibromas, café-au-lait spots and Lisch nodules of the iris. The NF1 gene is located in 17q11.2. The restriction fragment length polymorphism reported here will be useful in linkage analysis in NF1 families.     

Studies of effects of cyclic adenosine 3′,5′-monophosphate in regulation of human hemopoiesis in vitro

Summary Prostaglandin E₁ (PGE₁), high concentrations of dibutyryl cyclic AMP (dbcAMP), and theophylline were strikingly inhibitory both to tritiated thymidine ([³H]TdR) incorporation into bone marrow deoxyribonucleic acid (DNA) in vitro and to granulocytic colony growth. Autoradiography revealed that lower concentrations of dbcAMP were stimulatory to red blood cell precursors. This study was supported in part by United States Public Health Service Grant AM15163, by Health Research Council of the City of New York Career Scientist Award I-683, and by a Veterans Administration Medical Investigatorship to V. H.     

Static magnetic field inhibits 5′ nucleotidase activity in cancerous and non-cancerous human gastric tissues

Abstract

The aim of the present study is to investigate possible effects of static magnetic field (SMF) on 5′ nucleotidase (5′NT-CD73) and xanthine oxidase (XO) activities in cancerous and non-cancerous human gastric tissues in order to contribute to the elucidation of the anticancer activity of SMF. Cancerous and non-cancerous human gastric tissues removed from patients by surgical operations were used in the studies. SMF was created using two static magnets. Before and after treatment with SMF, 5′NT and XO activities in the tissue samples were measured. 5′NT activity was found to be lowered, but no significant change was observed in XO activity in the gastric tissues treated with the SMF. Our results suggest that SMF inhibits 5′NT enzyme in gastric tissues significantly. It is supposed that in addition to other proposed mechanisms, inhibition of purine catabolic activity due to inhibition of some key enzymes in the DNA turn-over like 5′NT might also play part in the anticancer activity of SMF.     

Intron 2 of human beta-globin in 3′-untranslated region enhances expression of chimeric genes

The possibility of enhancing heterologous gene expression in mammalian cells by the introduction of an intron in 3′ untranslated region (UTR) was investigated. To this end, a fragment of human betaglobin gene with intron 2 and flanked exon regions was introduced into the vector-encoding green fluorescent protein TagGFP2 after the TagGFP2 stop-codon (Int⁺). The distance between the stop-codon and the exon junction was 35 nucleotides. It ensured that Int+ mRNA was resistant to degradation by nonsense mediated decay (NMD) machinery. A control vector Intcontained corresponding intronless sequence of the beta-globin mRNA. On the same plasmid, the second gene encoded far-red fluorescent protein Katushka was used to normalize fluorescence for transfection efficiency and expression level in individual cells. Transiently transfected HEK293T cells were analysed by flow cytometry. It was shown that cells transfected with plasmid carrying the Int+ gene possess 1.8 ± 0.2 fold higher green fluorescence compared to Intcells. The observed effect was used to enhance expression of destabilized variants of yellow fluorescent protein TurboYFP-dest with high degradation rate in mammalian cells. We believe that introduction of beta-globin intron in the 3′-UTR of the chimeric gene can be used to enhance its expression and may be advantageous in some cases when usage of 5′ UTR intron is inappropriate.     

Cytoprotective effects of 6′-O-galloylpaeoniflorin against ultraviolet B radiation-induced cell damage in human keratinocytes

The cytoprotective effects of 6′-O-galloylpaeoniflorin against injury and death of human HaCaT keratinocytes resulting from ultraviolet B radiation were investigated. 6′-O-galloylpaeoniflorin exhibited the capacity to scavenge intracellular reactive oxygen species (ROS) generated by ultraviolet B radiation. 6′-O-galloylpaeoniflorin also attenuated ultraviolet B-induced oxidative macromolecular damage to DNA, lipids, and proteins, decreasing the number of DNA strand breaks, the level of 8-isoprostane (a biomarker of lipid peroxidation), and the level of protein carbonylation. Moreover, 6′-O-galloylpaeoniflorin rescued HaCaT cells from ultraviolet induced cell death, by downregulating the mitochondrial apoptotic pathway. Taken together, these results indicate that 6′-O-galloylpaeoniflorin has the potential to be developed as a medical agent against ROS-mediated skin diseases.