Induction of human high K(M) 5'-nucleotidase in cultured 293 cells

Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.     

Inhibition of phospholipid methylation in isolated rat hepatocytes by analogues of adenosine and S-adenosylhomocysteine

Phospholipid methylation in isolated hepatocytes was inhibited in the presence of 3-deazaadenosine (ID₅₀ = 1.7 μM) 9-β-d-arabinofuranosyladenine (ID₅₀ = 6.0 μM), S-tubercidinylhomocysteine (ID₅₀ = 30 μM), and 5′-deoxy-5′-isobutylthioadenosine (ID₅₀ = 177 μM). A transient inhibitory effect was observed with adenosine, whereas S-adenosyl-l-homocysteine and Sinefungin were essentially without effect. The inhibition of phospholipid methylation by S-tubercidinylhomocysteine and 9-β-d-arabinofuranosyladenine showed a lag-phase, whereas the effect of the other inhibitors was apparent within a few minutes. Cells exposed to 9-β-d-arabinofuranosyladenine or 3-deazaadenosine accumulated large amounts of AdoHcy, and adenosine induced a transient increase in the AdoHcy level. In addition, 3-deazaadenosine served as a precursor for the formation of S-3-deazaadenosylhomocysteine, which accumulated rapidly in cells exposed to this agent. The inhibitory effects of 3-deazaadenosine, 9-β-d-arabinofuranosyladenine and adenosine could be explained by the increase in total nucleosidylhomocysteine induced by these agents. In contrast, only a slight (less than 2-fold) increase in S-adenosyl-l-homocysteine content was observed in hepatocytes treated with 5′-deoxy-5′-isobutylthioadenosine, and this metabolic effect could not explain the inhibition of phospholipid methylation induced by this agent. None of the compounds tested reduced the amount nor the specific radioactivity of S-adenosylmethionine. Biological processes determining the inhibitory effects of adenosine, S-adenosyl-l-homocysteine and their analogues on phospholipid methylation in intact cells are discussed.     

Regulation of the CYP27B1 5′-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of λ DNA have been compared. A common ^5′GAGACC_3′CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5 ′ G G T C T C N ↓ 3 ′ C C A G A G N N N N N ↑     

Adenosine Deaminase and 5′Nucleotidase Activities in Peripheral Blood T Cells of Multiple Sclerosis Patients

Multiple sclerosis (MS) is one of the most common causes of neurological disability in young and middle-aged adults and is thought to be mediated by autoreactive T cells. Activities of adenosine deaminase (ADA) and 5′(nucleotidase (5′NT), which are involved in the differentiation and maturation of the lymphoid system, were measured in peripheral blood T cells from 21 MS patients and in 23 age and sex matched healthy controls to determine whether an association existed between these enzyme abnormalities and cellular immune functions. ADA and 5′NT activities were found significantly decreased in MS patients (P < .001 and P < .01 respectively) when compared with controls. Low levels of ADA and 5′NT activities were found irrespective of whether patients had relapsing–remitting or chronic progressive MS. These findings suggest that low levels of these enzyme activities in T cells may be related to the persistent abnormalities in T cell function in the clinical course of MS.     

Sequence analysis of rice rubi3 promoter gene expression cassettes for improved transgene expression

In a construct containing a GUS reporter gene driven by the 5′ regulatory elements from rubi3, expression was enhanced 4-fold when a 20-nucleotide (nt) GUS 5′ untranslated sequence was replaced with 9 nt sequences derived from rubi3′s second exon. The roles of the sequences immediately upstream from the GUS translation initiation codon, and their significance in gene expression, were investigated. Sequence analysis suggests that complementarity between sequences immediately 5′ of a translation initiation codon and the rice 17S rRNA may be responsible for the reduction in protein levels from constructs containing the GUS leader sequence. The results demonstrate an affect sequences immediately upstream from transgenic coding sequences have on expression, and when using the rubi3 5′ regulatory sequence in particular.     

Towards the development of a nuclear transformation system for <Emphasis Type="Italic">Dunaliella tertiolecta</Emphasis>

A nuclear transformation system for the microalga Dunaliella tertiolecta was explored using electroporation. Plasmids incorporating the D. tertiolecta RbcS1 5′ and 3′ untranslated regions flanking the Streptoalloteichus hindustanus gene encoding bleomycin resistance (ble) were introduced into D. tertiolecta cells both transiently and stably. Southern hybridisation was used to examine the fate of the plasmid following electroporation and revealed that the DNA was entering the cells but was quickly degraded. Using the same procedure one stably transformed line was recovered.     

Cytosolic acidification leads to Ca mobilization from intracellular stores in single and populational parietal cells and platelets

Regulatory relationship and gain control between cytosolic free Ca²⁺ concentration (Ca_i) and cytosolic pH (pH_i) were evaluated by two different cell types, gastric parietal cells, and blood platelets. Studies were carried out in both single cells and populations of cells, using Ca²⁺-indicative probe fura-2 (1-(2-(5′-carboxyoxazol-2′-yl)-6-aminobenzofuran-5-oxy)-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid) and pH-indicative probe BCECF (2′,7′-bis(carboxyethyl) carboxyfluorescein). Stimulation of single and populational parietal cells and platelets with gastrin and thrombin, respectively, resulted in an increase in Ca_i. In both populational cell types, an initial change in pH_i during agonist stimulation occurred almost simultaneously with the mobilization of Ca²⁺; an initial transient decrease in pH_i was followed by a slower increase in pH_i above the prestimulation level. When populational platelets were preloaded with the Ca²⁺ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′tetraacetic acid), the thrombin-induced initial large increase in Ca_i was apparently inhibited, whereas the pH_i decrease induced by thrombin was not altered. This suggests that the initial Ca_i change is not a prerequisite for the pH_i change. The effect of pH_i on Ca_i was examined next. In both single and populational cell types, application of the K⁺-H⁺ ionophore nigericin, which induced a transient decrease in pH_i, led to the release of Ca²⁺ from intracellular stores. In single parietal cells double-labeled with fura-2 and BCECF, a temporal decrease in pH_i preceded the rise in Ca_i after stimulation with nigericin. A decrease in pH_i, and an increase in Ca_i occurred at 1.5 and 4 s, respectively. In single parietal cells, replacement of medium Na⁺ with N-methyl- -glucamine (NMG⁺), which also induced a decrease in pH_i, resulted in repetitive Ca²⁺ spike oscillations. The source of Ca²⁺ utilized for the Ca²⁺ oscillation that was induced by NMG⁺ originated from the agonist-sensitive pool. Thus, several maneuvers, which were capable of decreasing pH_i, led to an increase in Ca_i. Cytosolic acidification may be a part of the trigger for Ca²⁺ mobilization from intracellular stores in both parietal cells and platelets.     

DNA sequence dependence of guanine-O alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea

After intracellular in vitro exposure to the mutagenic and carcinogenic N-nitroso compounds N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), respectively, the average relative amounts of the premutational lesion O⁶-alkylguanine represent about 6% and 8% of all alkylation products formed in genomic DNA. At the level of individual DNA molecules gunine-O⁶ alkylation does nor occur at random; rather, the probability of a substitution reaction at the nucleophilic O⁶ atom is influenced by nucleotide sequence, DNA conformation, and chromatin structure. In the present study, 5 different double-stranded polydeoxynucleotides and 15 double-stranded oligodeoxynucleotides (24-mers) were reacted with MeNU or EtNU in vitro under standardized conditions. Using a competitive radioimmunoassay in conjunction with an anti-(O⁶-2′-deoxyguanosine) monoclonal antibody, the frequency of guanine-O⁶ alkylation was found to be strongly dependent on the nature of the nucleotides flanking guanine on the 5t́ and 3′ sides. Thus, a 5′ neighboring guanine, followed by 5t́ adenine and 5′ cytosine, provided an up to 10-fold more ‘permissive’ condition for O⁶-alkylation of the central guanine than a 5′ thymine (with a 5-methylcytocine in the 5′ position being only slightly less inhibitory). Thymine and cytosine were more ‘permissive’ when placed 3′ in comparison with their affects in the 5′ flanking position.     

Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function

A novel mutant of the LLC-PK₁ renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N⁶-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V₂ receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK₁ cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5′-[β,γ-imino] triphosphate, 8-bromo adenosine-3′,5′-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK₁ and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V₂ receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V₂ receptor gene using DNA-transfer.     

Isolation and characterization of two asporogenous rifampicin resistant mutants of B. thuringiensis

Two rifampicin resistant mutants, Rif^r12 and Rif^r15, of B. thuringiensis Berliner have been isolated and characterized as true asporogenous mutants. Cells of Rif^r12 were blocked between stage I and stage II in the sporulation sequence; whereas cells of Rif^r15 mutant were blocked between stage II and stage III. It has been shown that two active forms of RNA-polymerase were present at t5 in Rif^r15 cells; as for wild type strain, the subunit composition of form I and form II were respectively ββ'_mα2 and β′βα2. In Rif^r12 cells, only one enzymatic form was found at t5 ; the subunit composition was determined as β′βσ_mα2 ; such a composition was characteristic for sporulation enzyme of wild type strain at t1,5. It is concluded that the sigma modification which occurs at about t1, is anterior to the β′ modification which is closely correlated with the forespore septum completion (t2). Thus, the timing of the modifications of B. thuringiensis RNA-polymerase previously suggested was clearly confirmed through the present study.In addition, both mutants present reduced levels of intracellular proteolytic activities, as compared with wild type strain, and the role of proteases is discussed.