Involvement of protein kinase C in fluoride-induced apoptosis in different types of lung cells.
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Apoptosis↗
Biomedical subjects
H Kersten
Publications and source records attributed to H Kersten.
Collaboration with school nurses: improving the effectiveness of tuberculosis screening.
OBJECTIVE: To compare tuberculosis skin test (TST) reading rates between children whose tests were read by school nurses following specific requests by physicians and those who relied on their parents to get their tests read, either at school or at the physician's office. DESIGN: A randomized controlled trial. SETTING: An urban hospital-based pediatric practice. PARTICIPANTS: Healthy low-income Hispanic and African American children aged 5 to 17 years whose physicians ordered TSTs at their routine physical examinations. Subjects attended 1 of 68 public schools. Nurses at these schools were willing to read student TSTs, and received instructions about how to read and report the results back to the physician's office. INTERVENTION: Subjects were randomized to a control group (routine TST placement, with no physician-to-school nurse communication) or to an intervention group (routine TST placement, with physician-to-school nurse communication). MAIN OUTCOME MEASURES: Tuberculosis skin test reading rates between the 2 groups were compared. Impediments to TST reading and reporting were investigated. RESULTS: One hundred thirty-four children were enrolled, 54 (40%) in the control group and 80 (60%) in the intervention group. More patients in the intervention group had their TSTs read by 72 hours compared with those in the control group (74 [92%] vs 30 [56%]; P<.001). The low reading rate in the control group was best attributed to communication failures. CONCLUSION: Systematic collaboration with school nurses can increase TST reading rates.
Adolescent↗
Serine 90 is required for enzymic activity by tRNA-guanine transglycosylase from Escherichia coli.
An Escherichia coli mutant described by Noguchi et al. [Noguchi, S., et al. (1982) J. Biol. Chem. 275, 6544-6550] contains tRNA lacking the hypermodified wobble nucleoside queuosine (Q) due to an inactive tRNA-guanine transglycosylase (TGT). TGT catalyzes the posttranscriptional base exchange of the Q precursor preQ1 with the genetically encoded guanine in tRNA(Asp,Asn,His,Tyr). The mutant tgt gene was cloned and sequenced; it contained a single point mutation resulting in the change of serine 90 to phenylalanine. Overexpression of the mutant gene yielded TGT(S90F) that showed a reduced solubility and did not purify in the same fashion as the wild-type enzyme. TGT(S90F) has no detectable enzymic activity. To determine whether serine 90 performs a catalytic role in the TGT reaction or whether the loss of activity was caused solely by a conformational change of the enzyme, we used site-specific mutagenesis to construct serine-to-alanine (S90A) and serine-to-cysteine (S90C) mutants. Both S90A and S90C mutants were purified in a manner identical to that used for the wild-type enzyme. SDS-PAGE of dimethyl suberimidate-cross-linked mutants showed a pattern identical to that of the wild-type TGT, indicative of a trimeric quaternary structure. Native PAGE of wild-type and mutant TGTs in the absence and presence of substrate tRNA exhibited band shifts indicating that both mutants retain the ability to bind tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
The nutrient factor queuine protects HeLa cells from hypoxic stress and improves metabolic adaptation to oxygen availability.
Queuine (q), a cyclopentendiol derivative of 7-aminomethyl-7-deazaguanine, is a nutrient factor for lower and higher eukaryotes, except yeast; it is synthesized in eubacteria partly at the level of tRNA. In eukaryotes q is preferentially inserted into the wobble position of specific tRNAs in differentiated and adult tissues, but occurs mainly free in embryonic and fast proliferating cells. HeLa cells grow to a higher cell density under aerobic than under hypoxic conditions only when supplemented with q. Here we show that in hypoxically grown HeLa cells, sufficiently supplied with q, free q accumulated when serum factors become limiting while the respective tRNAs remained completely q deficient. In these cells the levels of lactate dehydrogenase A (LDH A) mRNA and of LDH A protein were at least twofold higher than in aerobically grown cells, independent of the absence or presence of q. In response to q the LDH A4 isoenzyme was further activated by a post-translational mechanism. In q-deficient HeLa cells the activity of the major anoxic stress protein, LDHk, increased as a result of hypoxia; this increase was suppressed by q. In aerobically grown, q-deficient cells significant activities of LDH A4 and LDHk were present; both activities were markedly lowered by q, while the mitochondrial electron flow was improved. The results show that free q is essential for relieving hypoxic stress in HeLa cells that results from oxygen limitation.
Adaptation, Physiological↗
Genes, enzymes and coenzymes of queuosine biosynthesis in procaryotes.
In almost all known tRNAs that are specific for Asp, Asn, His or Tyr the wobble position of the anticodon is occupied by the hypermodified tRNA nucleoside queuosine. This unusual deazaguanine derivative is synthesised only in eubacteria. The biosynthesis, as investigated in Escherichia coli, is accomplished in four steps involving many unprecedented enzymatic reactions.
Bacteria↗
Transfer and isomerization of the ribose moiety of AdoMet during the biosynthesis of queuosine tRNAs, a new unique reaction catalyzed by the QueA protein from Escherichia coli.
The enzyme QueA of E coli is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. The enzyme catalyzes the synthesis of an epoxycyclopentane moiety and transfers this compound to specific tRNAs containing the queuosine precursor 7-(aminomethyl)-7-deazaguanine (preQ1). S-adenosylmethionine (AdoMet) is the sole cofactor that is required for this reaction (Slany et al, 1993, Biochemistry 32, 7811-7817). To proof that the ribose moiety of AdoMet is the precursor of the epoxycyclopentane moiety, labeled AdoMet, was generated from different types of 3H ATP and methionine by the AdoMet synthetase enzyme (MetK) from E coli. The resulting 3H labeled AdoMet was directly used as the cofactor for the QueA reaction. Using [2,5', 8-3H]ATP, containing tritium at C5' of the ribose ring, resulted in an incorporation of radioactivity into preQ1 tRNA, whereas this was not the case when [2,8-3H]ATP was applied. A model for the reaction catalyzed by the S-adenosylmethionine:tRNA ribosyltransferase-isomerase QueA is proposed.
Adenosine Triphosphate↗
Organization and functions of genes in the upstream region of tyrT of Escherichia coli: phenotypes of mutants with partial deletion of a new gene (tgs).
A delta tyrT::kan mutant from Escherichia coli K-12 (DTK-12) shows a transient growth lag that is caused by glycine starvation (U. Michelsen, M. Bösl, T. Dingermann, and H. Kersten, J. Bacteriol. 171:5987-5994, 1989). The same deletion, transduced into the relA1 spoT1 mutant CA274 to construct strain DTC274, caused complete growth inhibition in glucose minimal medium. Here, we show that the tyrT 5' region contains three new open reading frames in the order ORF37-->ORF34-->ORF32-->tyrT and that the delta tyrT::kan allele used previously deletes tyrT as well as a carboxy-terminal portion of ORF32. A plasmid encoding ORF32 totally complemented the inability of strain DTC274 to grow on glucose minimal medium as well as the transient glycine starvation phenomenon in DTK-12, and ORF32 was designated tgs. Partial deletion of tgs, cotransduced with the marker delta tyrT::kan, was responsible for the completely different phenotypes of the deletion mutants DTK-12 and DTC274. The deduced Tgs protein sequence showed significant homology to the PurN protein of E. coli and to enzymes with glycinamide ribonucleotide transformylase activity. We discuss whether growth inhibition in strain DTC274 may be caused by synergistic effects with the preexisting mutations relA1 and spoT1. The deduced protein sequence of ORF37 showed striking similarity to regulator response proteins and is probably a new member of this family. A spontaneous mutation in ORF37, caused by the integration of an insertion element, IS1, exhibited no phenotype.
Acyltransferases↗
Modulation of mammalian cell proliferation by a modified tRNA base of bacterial origin.
Addition of the q-base to q-deficient non-transformed mammalian cells stimulated their proliferation. The q-base also improved proliferation of some cancer-derived cell lines, but inhibited proliferation of others. The proliferation of HeLa-S3 carcinoma cells was stimulated by q under aerobic conditions, but was inhibited when the cells had shifted their energy metabolism towards glycolysis as the result of oxygen limitation. Q-deficient cells could not adapt their proliferation to the respective oxygen tension. The q-base stimulated the proliferation of non-transformed fibroblasts but inhibited proliferation of the same cell line, when aerobic glycolysis was increased after transformation with the ras gene. The results suggest that the q-base permits mammalian cells to adapt their proliferation to their specific metabolic state.
Animals↗
A new function of S-adenosylmethionine: the ribosyl moiety of AdoMet is the precursor of the cyclopentenediol moiety of the tRNA wobble base queuine.
Queuosine (Q) [7-(((4,5-cis-dihydroxy-2-cyclopenten-1-yl)amino)methyl)-7-deaz agu anosine] usually occurs in the first position of the anticodon of tRNAs specifying the amino acids asparagine, aspartate, histidine, and tyrosine. The hypermodified nucleoside is found in eubacteria and eucaryotes. Q is synthesized de novo exclusively in eubacteria; for eucaryotes the compound is a nutrient factor. In Escherichia coli the Q precursor (oQ), carrying a 2,3-epoxy-4,5-dihydroxycyclopentane ring, is formed from tRNA precursors containing 7-(aminomethyl)-7-deazaguanine (preQ1) by the queA gene product. A genomic queA mutant accumulating preQ1 tRNA was constructed. The QueA enzyme was overexpressed as a fusion protein with the glutathione S-transferase from Schistosoma japonicum and purified to homogeneity by affinity and anion-exchange chromatography. The enzyme QueA synthesizes oQ from preQ1 in a single S-adenosylmethionine- (AdoMet-) requiring step, indicating that the ribosyl moiety of AdoMet is transferred and isomerized to the epoxycyclopentane residue of oQ. The identity of oQ was verified by HPLC and directly combined HPLC/mass spectrometry. The formation of oQ was reconstituted in vitro, applying a synthetic RNA. A 17-nucleotide microhelix (corresponding to the anticodon stem and loop of tRNA(Tyr) from E. coli) is sufficient to act as the RNA substrate for oQ synthesis. We propose that QueA is an S-adenosylmethionine:tRNA ribosyltransferase-isomerase.
Amino Acid Sequence↗
Modulation of epidermal growth factor receptor activity and related responses by the 7-deazaguanine derivative, queuine.
Epidermal growth factor (EGF) induces autophosphorylation of its cognate receptor at tyrosine residues. Here we show that queuine (q), a widely distributed modified guanine analogue occurring free or as a tRNA wobble base, modulates this EGF receptor activity in vitro and in intact cells. Autophosphorylation of the immunopurified receptor from human A431 epidermoid carcinoma cells was enhanced three to fourfold in the presence of physiological concentrations of q. Using a membrane fraction of A431 cells, a twofold increase in autophosphorylation activity in the presence of q was observed, however, only when the receptor was activated by the ligand. In intact A431 cells, q enhanced the initial ligand-induced autophosphorylation of the EGF receptor three to fourfold. However, upon longer treatment of the cells with EGF in the presence of q, significantly less autophosphorylated receptor was detectable compared with stimulation of cells in the absence of q. A similar q-dependent modulation of EGF receptor autophosphorylation was observed also in human cervical carcinoma cells HeLa-S3. Treatment of q-deficient HeLa cells with EGF induced the c-fos gene expression, transiently increased the activity of the anoxic stress protein LDH k, and stimulated proliferation. Treatment of HeLa cells with EGF in the presence of q resulted in a delayed c-fos gene expression and an accelerated increase and decrease of LDH k activity. The stimulatory effect of low doses of EGF on HeLa cell proliferation was completely antagonized in the presence of q. The results suggest that the mitogenic signalling initiated by the EGF receptor is modulated by q.
Cell Line↗
The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site.
The tgt/sec operon in E. coli consists of five genes: queA, tgt, ORF12, secD, and secF. QueA and Tgt participate in the biosynthesis of the hypermodified t-RNA nucleoside Queuosine, whereas SecD and SecF are involved in protein secretion. Examination of the promoter region of the operon showed structural similarity to promoter regions of the rrn-operons. An upstream activation sequence (UAS) containing a potential binding site for the factor of inversion stimulation (FIS) was found. Gel retardation assays and DNaseI footprinting indicated, that FIS binds specifically and with high affinity to a site centred at position -58. Binding of FIS caused bending of the DNA, as deduced from circular permutation analysis. Various 5' deletion mutants of the promoter region were constructed and fused to a lacZ reporter gene to determine the influence of the UAS element on the promoter strength. An approximately two-fold activation of the promoter by the UAS element was observed.
Base Sequence↗
Cloning and characterization of a human angiotensin II type 1 receptor.
A human liver cDNA library was screened using a rat type 1 angiotensin II receptor cDNA coding sequence as a probe. cDNA clones were isolated which encoded a protein of 359 amino acids that shared 94.4% and 95.3% identify to rat and bovine type 1 angiotensin II receptors, respectively. Ligand binding studies of the cloned receptor expressed in COS cells suggested that it is pharmacologically a type 1 angiotensin II receptor subtype. Electrophysiological studies of the receptor expressed in Xenopus laevis oocytes revealed that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of calcium. Southern and Northern blot analyses indicated that the cloned receptor is represented as a single copy in the human genome and is expressed in many tissues of different histogenic origin with the exception of brain, where mRNA transcripts were barely detectable.
Amino Acid Sequence↗
The cyclophilin multigene family of peptidyl-prolyl isomerases. Characterization of three separate human isoforms.
Cyclophilin (CyP), a major cytosolic protein possessing peptidyl-prolyl cis-trans isomerase activity, has been implicated as the specific receptor of the immunosuppressive drug cyclosporin A (CsA). To identify other potential CsA receptors related to CyP, two human cDNA libraries were screened under low stringency conditions using human CyP cDNA (encoding hCyP1) as a probe. Two cDNAs were identified which encode distinct proteins related to human hCyP1. These two novel proteins, designated hCyP2 and hCyP3, share 65 and 76% amino acid sequence homology with hCyP1, respectively. Both hCyP2 and hCyP3 contain NH2-terminal hydrophobic extensions of 32 and 42 amino acids, respectively. Protein-specific antibodies revealed the predominant association of hCyP2 and hCyP3 with membranes and subcellular organelles, which suggests that the amino-terminal leader sequences of the two CyP isoforms may act as signal peptides. In contrast to the results with hCyP1, Southern blot analysis indicated that both hCyP2 and hCyP3 gene sequences are represented infrequently in the human genome. Northern and Western blot analysis showed that the distribution of mRNA and proteins of the three hCyPs in differing tissues and cell types was similar. Each hCyP protein was expressed in Escherichia coli, purified, and shown to be an active peptidyl-prolyl isomerase. Substrate specificity was examined with 11 synthetic peptides (Suc-Xaa-Yaa-Pro-Phe-4-nitroanilide), and inhibition of the peptidyl-prolyl isomerase activities associated with hCyP1, hCyP2, and hCyP3 was studied with CsA, MeAla6-CsA and MeBm2t1-CsA. From both equilibrium considerations and the results of kinetic characterizations it is proposed that of these three CyP proteins, hCyP1 is the most likely intracellular target for CsA.
Amino Acid Isomerases↗
A novel RNA product of the tyrT operon of Escherichia coli.
The tyrT operon of E. coli and several other tRNA operons of E. coli show striking structural features: they contain repeated sequence units including a 19bp motif resembling the 3' end of the corresponding mature tRNA. A novel RNA, encoded by the repeated sequence of the tyrT operon, was identified. The RNA, characterized by primer extension and Nuclease-S1 analysis, contained 171 nucleotides and terminated with the 19bp motif of the CCA-end of tRNA(1Tyr). The RNA, designated as rtT RNA, is probably released from the primary transcript of tyrT during tRNA processing, it includes the coding capacity for the arginine rich peptide Tpr. Predictions of secondary folding resulted a rather stable RNA structure with a free energy of -44.3kcal/mol. A weak ribosome binding site was found, preceding the second possible AUG initiator codon for Tpr. The comparison of rtT RNA with putative transcripts from the repeated sequences associated with related tRNA genes showed common features with respect to primary structure, arrangement and secondary folding. In E. coli cultures the lag-phase during growth, caused by transient glycine or by isoleucine limitation, was found to be overcome or markedly shortened in the presence of rtT RNA. These and previously reported results suggest a modulatory effect of rtT RNA on stringent response.
Amino Acid Sequence↗
Structure and organization of Escherichia coli genes involved in biosynthesis of the deazaguanine derivative queuine, a nutrient factor for eukaryotes.
The plasmid pPR20 contains the gene tgt, which encodes tRNA guanine transglycosylase (Tgt), on a 33-kbp DNA insert from a region around 9 min on the Escherichia coli linkage map. The plasmid was subcloned to determine the sequence and organization of the tgt gene. Tgt is a unique enzyme that exchanges the guanine residue with 7-aminomethyl-7-deazaguanine in tRNAs with GU(N) anticodons. After this exchange, a cyclopentendiol moiety is attached to the 7-aminomethyl group of 7-deazaguanine, resulting in the hypermodified nucleoside queuosine (Q). Here we give the complete sequence of a 3,545-bp StuI-BamHI DNA fragment where we found the tgt gene and three previously unknown genes encoding proteins with calculated molecular masses of 42.5 (Tgt), 14, 39, and 12 kDa. The gene products were characterized on sodium dodecyl sulfate gels after synthesis in a combined transcription-translation system. The mRNA start sites of the open reading frames (ORFs) were determined by primer extension analysis. Plasmids containing the ORF encoding the 39-kDa protein (ORF 39) complemented a mutation in Q biosynthesis after the Tgt step. This gene was designated queA. The genes are arranged in the following order: ORF 14 (transcribed in the counterclockwise direction), queA, tgt, and ORF 12 (all transcribed in the clockwise direction). The organization of the promoter sequences and the termination sites suggests that queA, tgt, and ORF 12 are localized on a putative operon together with the genes secD and secF.
Amino Acid Sequence↗
The deazaguanine-derivative, queuine, affects cell proliferation, protein phosphorylation and the expression of the proto oncogenes c-fos and c-myc in HeLa cells.
In higher eukaryotes the hypermodified guanine analogue, queuine (7-(5-[(1S,4S,5R)-4,5-dihydroxy-2-cyclopentene-1-yl) amino)-methyl)-7-deazaguanine), occurs free or as modified nucleoside (Q) in the anticodon of specific tRNAs. Fast proliferating tissues and tumors contain considerable amounts of free queuine and Q-deficient tRNAs. Here we show that HeLa cells can be grown in the absence or presence of queuine. In response to queuine, and under appropriate conditions, (i) the proliferation of HeLa cells was stimulated, (ii) the steady-state level of c-fos mRNA was reduced, contrary that of c-myc mRNA was elevated, and (iii) in cytosolic extracts protein phosphorylation especially for a 42 kDa protein was significantly increased. The results suggest that queuine substitutes for growth factors in a signal transduction pathway.
Blotting, Northern↗
Queuine modulates growth of HeLa cells depending on oxygen availability.
HeLa cells can be grown in media supplemented with horse serum that is lacking the nutrient factor queuine. The addition of 1 X 10(-8) M queuine to aerobically grown cells caused a slight, but significant, inhibition of growth, whereas cell proliferation was stimulated increasingly when the concentration of queuine was raised from 3 X 10(-8) M to 3 X 10(-7) M. This was also observed when the cells were transiently starved of serum factors. When the cells were grown under hypoxic stress, but otherwise identical conditions, they responded to queuine in an opposite manner. Under conditions of mitogenic stimulation, characteristic new proteins were found in cytosolic, nuclear and mitochondrial fractions of aerobically grown cells. The effects of queuine on cell proliferation at low concentrations are assumed to be mediated by the free base, whereas the effects at higher concentrations possibly involve both, queuine and Q-tRNAs. The 'Q system' appears to mediate growth control in dependence on oxygen availability.
Cell Division↗
Possible involvement of queuine in control mechanisms of protein synthesis and protein phosphorylation in eukaryotes.
The functional role of the deazaguanine-derivative queuine was investigated using virus-transformed erythroleukaemic cells of mice as a model. The two-dimensional patterns of [35S]methionine-labelled proteins on two-dimensional O'Farrell gels of queuine-deficient (Q-), compared with queuine-supplemented (Q+) growing cells, showed specific characteristic alterations in the synthesis of 36 and 42 kd basic proteins. According to pI values and immunoreactivity with anti-LDH antibodies, the 36 kd proteins represent various forms of LDH A subunits or closely related proteins. Cell-free systems of protein synthesis were established from growing (Q-) or (Q+) cells. Addition of 3 x 10(-8) M queuine to the (Q-) in vitro system inhibited the incorporation of [35S]methionine into total protein to approximately 20%; raising the concentration of queuine up to 1 x 10(-6) M did not increase the inhibitory effect appreciably. In the (Q-) system, a series of 36 kd proteins, with pI values corresponding to LDH A isoforms, were synthesized. The in vitro synthesis of these proteins was completely inhibited by addition of queuine at a concentration of 3 x 10(-8) M. Furthermore, the expression of certain other proteins was lower in the (Q+) than in the (Q-) in vitro system. Labelling of growing (Q+) or (Q-) cells with [32P]orthophosphate and subsequent analysis of phosphoproteins on two-dimensional O'Farrell gels showed that queuine inhibited the synthesis of distinct phosphoproteins. Protein synthesis performed in cell-free (Q-) or (Q+) systems in the presence of non-labelled amino acids and 32P-labelled gamma ATP also indicated that queuine interferes with the synthesis and/or phosphorylation of particular phosphoproteins.
Animals↗