Biomedical subjects

A A Thomas

Publications and source records attributed to A A Thomas.

At least 37 records · Page 2Linked to original sources

PubMed1998

Detection of HLA-specific antibodies by PRA-STAT and their association with transplant outcome.

OBJECTIVE: The aim was to investigate the correlation between renal transplant outcome and the presence of HLA-specific antibodies detected using the ELISA kit PRA-STAT as compared with complement-dependent cytotoxicity (CDC). METHOD: 295 sera from 95 renal transplant recipients (99 transplants) were investigated for the presence of HLA-specific antibodies using both PRA-STAT and CDC. The patients were divided into group I (49 transplants failed within 1 month) and group II (50 successful transplants). RESULTS: The concordance between PRA-STAT and CDC for the detection of HLA class I-specific antibodies was 87.8% (259 of 295). For 19 sera, antibodies were detected only by PRA-STAT; for 17 sera, antibodies were detected only by CDC. No donor-specific antibodies were detected by either technique for patients in group II. For four group I patients (six sera), donor-specific IgG antibodies were detected only by PRA-STAT (one before, three after transplant) and all four transplants failed. For five other group I patients (six sera), donor HLA-specific antibodies were detected only by CDC (one before, four after transplant) and all five transplants failed. The antibodies detected before transplant by CDC were shown to be IgM alloantibodies. CONCLUSION: This study showed that PRA-STAT could detect HLA-specific IgG antibodies relevant to transplant outcome that were not detected by CDC. However, it could not detect IgM alloantibodies that were also shown to be important. PRA-STAT is therefore a useful addition to a histocompatibility laboratory's screening repertoire only when used in conjunction with other techniques.

Adult↗

PubMed1997

Inactivation of eIF2B and phosphorylation of PHAS-I in heat-shocked rat hepatoma cells.

Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43 degrees C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.

Animals↗

PubMed1997

Translational properties of the untranslated regions of the p10 messenger RNA of Autographa californica multicapsid nucleopolyhedrovirus.

Protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mRNAs of Autographa californica multicapsid nucleopolyhedrovirus. The untranslated regions (UTRs) of the p10 mRNA of this baculovirus were studied by in vitro translation and after transfection into Spodoptera frugiperda insect cells. Lysates from insect cells were optimized for translation of in vitro transcripts containing p10 sequences. The lysates were used to measure the effects of various deletions in either the 5' or 3'UTR on protein synthesis. Transcripts containing the p10 5'UTR were translated efficiently. Large deletions in the 5'UTR severely decreased this efficiency. Deletions in the 3'UTR negatively affected expression of the reporter gene in vivo; however, no effect on translational efficiency in the insect-cell lysates was measured. The translational properties of the p10 transcripts were very similar in lysates made from either uninfected or baculovirus-infected insect cells. Determination of optimal salt conditions for either uncapped or capped transcripts showed that the p10 5'UTR was used very efficiently for translation initiation in vitro, even in the absence of a cap-structure at its 5' end. Addition of cap-analogue to the in vitro translation assays did not inhibit p10 5'UTR-driven translation, while translation of a cap-dependent mRNA was severely inhibited. These data suggest that the very late mRNAs of baculovirus are translated in a cap-independent manner.

Animals↗

PubMed1996

Phosphorylation of the eIF4E-binding protein PHAS-I after exposure of PC12 cells to EGF and NGF.

PHAS-I or the eIF4E-binding protein 1 regulates the cap-binding activity of eIF4E by sequestering eIF4E. Binding of elF4E to PHAS-I is regulated by phosphorylation of PHAS-I. PC12 cells were used to study the signal transduction pathway leading to phosphorylation of PHAS-I. Both EGF and NGF induced phosphorylation of PHAS-I. Wortmannin, a PI-3 kinase inhibitor, staurosporine, a PKC inhibitor, and rapamycin, a FRAP inhibitor all blocked the phosphorylation of PHAS-I. Of the three inhibitors, only wortmannin was able to inhibit MAPK phosphorylation. This excludes a role for MAPK in NGF- and EGF-induced PHAS-I phosphorylation in PC12 cells. Apparently, PHAS-I was phosphorylated in a PI-3 kinase-, PKC-, and FRAP-dependent manner after EGF or NGF stimulation. Only PI-3 kinase and FRAP are involved in the regulation of the basal level of PHAS-I phosphorylation.

Androstadienes↗

PubMed1996

Phosphorylation state of the cap-binding protein eIF4E during viral infection.

The eukaryotic translation initiation factor eIF4E, the cap-binding protein, seems to play an essential role in the establishment of the host shut-off after viral infection. Infection with adenovirus and influenza virus caused dephosphorylation of eIF4E and an involvement of a viral protein was suggested. In this report, we studied several other viruses for their ability to change the phosphorylation state of eIF4E, and we looked for the mechanism of eIF4E dephosphorylation. First, it was shown that after encephalomyocarditis virus (EMCV) and poliovirus infection, dephosphorylation of eIF4E occurred. Dephosphorylation of eIF4E was not observed after Semliki Forest virus and reovirus infection. An artificial increase of the level of phosphorylated eIF4E by treating the cells with the phosphatase inhibitor okadaic acid changed neither the kinetics of EMCV and poliovirus infection, nor that of host shut-off. Infections with the uv-treated EMCV showed that the virus binding or entry into the cell initiates eIF4E dephosphorylation. Besides this entry-induced eIF4E dephosphorylation, dephosphorylation was also induced by blocking protein synthesis with the initiation inhibitor pactamycin, or with the elongation inhibitor cycloheximide. We conclude that eIF4E is dephosphorylated by entry of EMCV, and the effect is strengthened by the decrease in cap-dependent translation.

Alphavirus Infections↗

PubMed1996

Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA.

Foot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5' untranslated region (5'UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5'UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products. After in vitro transcription with T7 RNA polymerase and translation in rabbit reticulocyte lysate, the two CAT products, resulting from initiation from the two initiation codons, were quantified. The downstream initiator AUG (AUGLb) was selected more efficiently in the wild-type 5'UTR. In truncated RNA, the upstream initiation site (AUGLab) was more efficiently utilized than in the wild-type 5'UTR. Protein synthesis initiation factors were added to translation assays to determine whether these factors influenced initiation site selection. Addition of eIF-2 and of eIF-2B changed the selection process for both types of RNA. These factors induced a 2.5-fold higher usage of the upstream AUGLab for wild-type and 5'UTR-truncated RNA. A change in mRNA concentration also induced a change in the usage of initiation codons; however, the effect of eIF-2 was measured over a broad range of mRNA concentrations. In conclusion, eIF-2 mediates the recognition of the initiation codon during both cap-dependent and internal ribosome entry site-dependent initiation.

Animals↗

PubMed1995

The human insulin-like growth factor II leader 1 contains an internal ribosomal entry site.

Insulin-like growth factor II is a small peptide growth hormone, encoded by four mRNAs with unique 5' untranslated regions and identical coding regions. The 5' untranslated region transcribed from promoter 1 is 598 nt (leader 1). The properties of this leader 1 suggest a strong regulation of translation; the high G + C-content, the presence of an upstream open reading frame, and the length of the 5' UTR are 3 elements which prohibit efficient translation and which may modulate expression. In this paper we show that the human IGFII leader 1 harbours sequence elements that allow translation initiation to occur by internal initiation on the IGF sequence. This mode of initiation was described first for picornaviral mRNAs, that are naturally uncapped. The IGFII leader 1-dependent expression in HeLa cells was resistant to infection with poliovirus; abrogation of cap-dependent initiation by poliovirus had apparently no effect on IGFII expression. Moreover, a downstream CAT-cistron in a bicistronic construct was translated upon insertion of the leader 1 sequence. The translational properties of the IGFII leader 1 suggest that internal initiation on this leader may be modulated during proliferation or differentiation, enabling cell-stage dependent expression of IGFII.

Base Sequence↗

PubMed1995

The amino acid sequence of eukaryotic translation initiation factor 1 and its similarity to yeast initiation factor SUI1.

Eukaryotic initiation factor eIF-1 was purified from rabbit reticulocytes. Amino acid sequence analysis revealed that the protein contained a blocked amino-terminus. After cleavage with the endoproteinase Asp-N, three peptides were sequenced. The obtained partial sequences were identical to sequences of SUI1ISO1, the human homologue of the yeast translation initiation factor SUI1. The SUI1 gene product was identified as a protein involved in the recognition of the protein synthesis initiation codon. A similar mode of action has been suggested for eIF-1.

Amino Acid Sequence↗

PubMed1995

Proteins binding to the leader of the 6.0 kb mRNA of human insulin-like growth factor 2 influence translation.

The leader of the 6.0 kb human insulin-like growth factor 2 (IGF-2) mRNA, leader 3, has been reported to partially repress translation. In the regulation of this phenomenon, RNA-binding proteins may play a role. Using UV-irradiation crosslinking, we found specific binding of four proteins (57, 43, 37 and 36 kDa) to this leader. Binding of these proteins to RNA proved to be highly sensitive to the potassium chloride concentration in the buffer solution, each protein having its own optimum. The 57 kDa protein was indistinguishable by size, binding properties and immunoprecipitation from the polypyrimidine tract binding protein (PTB), first described as a nuclear protein binding to the polypyrimidine tracts (PPTs) in introns. Cross-competition experiments showed that leader 3 has a much higher affinity for this 57 kDa protein than the PPT on which PTB was originally characterized. By competition with different fragments of leader 3, we were able to localize the binding of the 57 kDa protein to a 162 nt RNA fragment (AsnI-PvuII) in the 3'-part of the leader. When placed before a chloramphenicol acetyltransferase (CAT) open reading frame, this RNA fragment stimulated translation in reticulocyte lysate 3-fold, while other fragments of leader 3 repressed translation. The efficient translation directed by the 162 nt AsnI-PvuII fragment fused to CAT could be repressed by adding free AsnI-PvuII RNA fragment, indicating that the high translation efficiency of the AsnI-PvuII-CAT synthetic mRNA was due to the binding of protein and not to the structure of the RNA itself.

Base Sequence↗

PubMed1995

Phosphorylation of eIF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells.

Mitogenic stimulation of protein synthesis is accompanied by an increase in eIF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on eIF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than eIF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of eIF-4E is present in P19 cells; the EGF-induced change in phosphorylation of eIF-4E in these cells is likely to be regulated by a change in eIF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not eIF-4E phosphorylation.

Amino Acid Sequence↗

PubMed1994

Influence of the four leader sequences of the human insulin-like-growth-factor-2 mRNAs on the expression of reporter genes.

The human insulin-like-growth-factor-2 (IGF-2) gene generates mRNAs with four different leader sequences, but with identical coding and trailing regions. Previous research has revealed that the leader-2-containing and leader-4-containing mRNAs are completely polysomal, whereas mRNAs possessing leader-3 are predominantly present in the untranslated free messenger ribonucleoprotein particle (mRNP), both in cell lines and in foetal liver tissue. To investigate the influence of the IGF-2 leader sequences on expression of the gene, IGF-2 leader-luciferase and leader-chloramphenicol acetyltransferase fusion constructs were transfected transiently into different cell lines. In these experiments, the levels of expression obtained by constructs with leader-1, leader-2 and leader-4 were very similar, both at the level of mRNA and protein. Leader-3, however, strongly repressed the expression of the fusion mRNA via an unknown mechanism. This repression appeared to be confined to nucleotides at positions 328-906 of the leader sequence. The remaining 5' part of the leader sequence was efficient both in RNA expression and in translation, but the 3' part of the leader (nucleotides 906-1180) again moderately repressed luciferase expression, possibly due to endonucleolytic cleavage in this region of the RNA. To evaluate the effect of the IGF-2 leaders on in vitro translation, leader-chloramphenicol acetyltransferase fusion mRNAs were synthesized and translated in reticulocyte lysates. Compared to a chloramphenicol acetyltransferase control RNA, leader-1-chloramphenicol acetyltransferase mRNA translated over 20-fold less efficiently, whereas leader-2 repressed translation of its chloramphenicol acetyltransferase mRNA moderately (3-5 fold). Despite a general improvement of the translation efficiency upon translation in HeLa lysate, these discrepancies with the transfection data persisted. Translation of leader-3-containing mRNAs in reticulocyte lysates was barely detectable. The whole 5' region of leader-3, up to nucleotide 614, could be shown to be repressive. Only leader-4 directed translation of the chloramphenicol acetyltransferase open reading frame efficiently. As with leader-1 and leader-2, this L4-chloramphenicol acetyltransferase mRNA translated in a cap-dependent manner under the conditions of our experiments; translation of this mRNA was relatively resistant to addition of cap analogue. We conclude that all four IGF-2 leader sequences differ in their translational properties. This makes it likely that changes in the translational machinery will affect the expression of the various IGF-2 mRNAs differentially.

Base Sequence↗

PubMed1994

Translation initiation on the insulin-like growth factor II leader 1 is developmentally regulated.

The majority of cellular mRNAs have relatively short and unstructured 5' untranslated regions (UTRs) that allow efficient translation, such as the beta-globin mRNA. An exception to this rule is the group of growth factor mRNAs which, in general, have long 5' UTRs with a high G + C content. An example is insulin-like growth factor II (IGF-II), which is encoded by four mRNAs, arising from four different promoters. Transcripts having the human IGF-II leader 1 are only expressed in adult liver where IGF-II protein synthesis is solely under direction of this 5' UTR. We investigated the translational efficiency in vitro of this 5' UTR, linked to the chloramphenicol acetyltransferase (CAT) encoding region. As expected from the primary structure of IGF-II leader 1, translational efficiency was very low compared with beta-globin 5' UTR-CAT mRNA. Addition of cell extract from undifferentiated P19 embryonal carcinoma (EC) cells preferentially stimulated translation of an IGF-II 5' UTR RNA construct. No translational stimulation was found when cell extract from differentiated P19 EC cells was added. In contrast with the beta-globin 5' UTR, translation initiation on the IGF-II 5' UTR was not dependent on the presence of a cap structure. The results imply that only in undifferentiated P19 EC cells and not in their differentiated derivatives is a factor present that specifically stimulates IGF-II RNA translation, thereby suggesting translational regulation of IGF-II production during early embryonic development. A mechanism for translation initiation on the 5' UTR of IGF-II is discussed.

Adenoviridae↗

PubMed1994

Basepairing with 18S ribosomal RNA in internal initiation of translation.

In concert with the translation initiation factors 'trans-acting' factors function specifically during internal initiation on picornaviral mRNAs. Of these trans-acting factors, two have been identified as the La-protein and the polypyrimidine tract binding protein. Within the internal ribosomal entry site on the viral RNA, sequences are present that direct the ribosome to the initiation codon. We suggest that selection of the correct AUG initiation codon occurs through basepairing with a part of 18S ribosomal RNA.

Base Composition↗

PubMed1994

Characterization of the tobacco eIF-4A gene family.

Characterization of cDNAs encoding eukaryotic translation initiation factor 4A (eIF-4A) indicates the expression of a minimum of ten related genes in tobacco leaf cells. The ten groups fall into two gene families, NeIF-4A2 and NeIF-4A3. The majority of the cDNAs exhibit significant sequence similarity to the NeIF-4A2 family at both the DNA and deduced amino acid levels. Northern analysis using specific probes indicates variable expression of four family members in various tobacco organs. Western analysis, using an anti-tobacco eIF-4A polyclonal antibody, reveals a complex pattern of immunologically related polypeptides of approximately 46 kDa. Subcellular fractionation suggests that at least one eIF-4A-related polypeptide is located in the chloroplast where it is ribosome-associated.

Amino Acid Sequence↗

PubMed1994

Initiation of protein synthesis in eukaryotes.

The study of the regulation of initiation of protein synthesis has recently gained momentum because of the established relationship between translation initiation, cell growth and tumorigenesis. Therefore much effort is devoted to the role of protein kinases which are activated in signal transduction cascades and which are responsible for the phosphorylation of a number of initiation factors. These specific factors are mainly involved in the binding of messenger RNA to the 40S ribosome, a process that makes the unwinding of the 5' untranslated region necessary. It appears that the phosphorylation of these factors increases their ability for cap recognition and helicase activity. The enhanced phosphorylation of the messenger binding factors results not only in an overall stimulation of translation, but especially weak messengers are positively discriminated. The above mechanisms mainly deal with qualitative control of translation, i.e., messenger selection, but phosphorylation also plays a role in quantitative regulation of protein synthesis. The generation of active eIF-2, the initiation factor that binds the Met-tRNA(i) and GTP, is dependent on a factor involved in the GDP-GTP exchange. Phosphorylation of eIF-2 results in sequestration of the exchange factor and a slowing down of the rate of initiation.

Animals↗

PubMed1994

Binding of eukaryotic initiation factor-2 and trans-acting factors to the 5' untranslated region of encephalomyocarditis virus RNA.

The encephalomyocarditis virus 5' untranslated region (EMC 5' UTR) has a binding site for eukaryotic initiation factor eIF-2. Mutations in the 3' end or deletion of the 5' end of the internal ribosomal entry site had a negative effect on the binding of eIF-2 to the EMC 5' UTR. The binding of eIF-2 to the mutant 5' UTRs was completely inhibited by the addition of competitor tRNA. Cross-linking of the EMC 5' UTR with proteins from rabbit reticulocyte lysates showed binding of trans-acting factors p52 and p57. Deletions in the 5' end of the internal ribosomal entry site resulted in a loss of the ability to bind trans-acting factor p57, in accordance with literature data, while p52 binding to these deletion mutants was weak compared to the wildtype EMC 5' UTR. Mutations in the 3' part of the 5' UTR of EMC still resulted in binding of both trans-acting factors, as with wild type RNA, but binding was more sensitive to competitor tRNA when compared to the binding of p52/p57 to the wild type 5' UTR.

Base Sequence↗

PubMed1992

Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency.

Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in-vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV-infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV-directed shut off of host protein synthesis. The interaction of translation initiation factors with the 5' cap structure was studied. Transcripts prepared in vitro using T7 RNA polymerase were capped and methylated posttranscriptionally with [32P]-GTP and S-adenosyl-L-methionine to yield cap-labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the cap-specific cross-linking of eukaryotic initiation factors (eIF) eIF-4E and eIF-4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap-binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5' cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross-linking assay, it was demonstrated that eIF-4F, and also eIF-4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV-induced shut off of host cell protein synthesis.

Base Sequence↗

PubMed1992

Dependence of the adenovirus tripartite leader on the p220 subunit of eukaryotic initiation factor 4F during in vitro translation. Effect of p220 cleavage by foot-and-mouth-disease-virus L-protease on in vitro translation.

The adenovirus tripartite leader (TPT) 5' untranslated region (5'UTR) allows translation in poliovirus-infected cells, in which the p220 subunit of eukaryotic initiation factor 4F is degraded. This p220-independent translation was investigated by measuring in vitro translation in a reticulocyte lysate of a reporter gene, chloramphenicol acetyltransferase, coupled to the TPT 5'UTR. The p220 subunit was degraded by translation of a foot-and-mouth-disease L-protease construct. Surprisingly, the TPT 5'UTR was dependent on intact p220, as are other naturally capped mRNA species. Translation of encephalomyocarditis virus RNA was p220 independent, as expected from its ability to support internal, cap-independent initiation. In vitro protein-synthesis experiments with purified initiation factors confirmed the dependence of TPT mRNA translation on eukaryotic initiation factor 4F. The relationship between adenovirus TPT-5'UTR-directed translation and poliovirus-induced host cell shut-off is discussed.

Adenoviridae↗