Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) has an essential role in multiple signaling pathways, including cell proliferation and migration, through extracellular ligand binding and subsequent activation of its intracellular tyrosine kinase (TK) domain. The non-small cell lung cancer (NSCLC)-associated EGFR mutants, L858R and G719S, are constitutively active and oncogenic. They display sensitivity to TK inhibitors, including gefitinib and erlotinib. In contrast, the secondary mutation of the gatekeeper residue, T790M, reportedly confers inhibitor resistance on the oncogenic EGFR mutants. In this study, our biochemical analyses revealed that the introduction of the T790M mutation confers gefitinib resistance on the G719S mutant. The G719S/T790M double mutant has enhanced activity and retains high gefitinib-binding affinity. The T790M mutation increases the ATP affinity of the G719S mutant, explaining the acquired drug resistance of the double mutant. Structural analyses of the G719S/T790M double mutant, as well as the wild type and the G719S and L858R mutants, revealed that the T790M mutation stabilizes the hydrophobic spine of the active EGFR-TK conformation. The Met790 side chain of the G719S/T790M double mutant, in the apo form and gefitinib- and AMPPNP-bound forms, adopts different conformations that explain the accommodation of these ligands. In the L858R mutant structure, the active-site cleft is expanded by the repositioning of Phe723 within the P-loop. Notably, the introduction of the F723A mutation greatly enhanced the gefitinib sensitivity of the wild-type EGFR in vivo, supporting our hypothesis that the expansion of the active-site cleft results in enhanced gefitinib sensitivity. Taken together, our results provide a structural basis for the altered drug sensitivities caused by distinct NSCLC-associated EGFR mutations.

Interference with interaction between eukaryotic translation initiation factor 4G and poly(A)-binding protein in Xenopus oocytes leads to inhibition of polyadenylated mRNA translation and oocyte maturation.

The interaction between eukaryotic translation initiation factor 4G (eIF4G) and the poly(A)-binding protein (PABP) facilitates translational initiation of polyadenylated mRNAs. It was shown recently that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces polyadenylated mRNA translation and dramatically inhibits progesterone-induced oocyte maturation. These results strongly suggest that the eIF4G-PABP interaction plays a critical role in the translational control of maternal mRNAs during oocyte maturation. In the present work, we employed another strategy to interfere eIF4G-PABP interaction in Xenopus oocytes. The amino-terminal part of eIF4GI containing the PABP-binding site (4GNt-M1) was expressed in Xenopus oocytes. 4GNt-M1 could bind to PABP in oocytes, which suggests that 4GNt-M1 may evict PABP from the endogenous eIF4G. The expression of 4GNt-M1 resulted in reduction of polyadenylated mRNA translation. Furthermore, 4GNt-M1 inhibited progesterone-induced oocyte maturation. In contrast, 4GNt-M2, in which the PABP-binding sequences were mutated to abolish the PABP-binding activity, could not inhibit polyadenylated mRNA translation or oocyte maturation. These results further support the idea that the eIF4G-PABP interaction is critical for translational regulation of maternal mRNAs in oocytes.

[Clinical laboratory data and reference intervals standardized in Fukuoka].

In Fukuoka whose population is approximately five million inhabitants, surveys on the accuracy of laboratory data have been performed by the Fukuoka Prefecture Medical Association for the last 30 years. We have been attempting to evaluate the data for routine use since 1988, and it has become possible to share laboratory data between all institutions in Fukuoka prefectures. As a result, reference intervals for 23 clinical chemistry analytes were established in 1995, to which were added in 1996 five serum protein constituents that have been utilized for clinical examinations. Methods for documentations and monitorings the data obtained in the prefecture were also established, standardization of the above analytes extended to 97% of the institutions in the prefecture. Results for 14 of the 23 clinical chemistry analytes have become highly reliable and clinically useful as differences between institutions in terms of results have narrowed. Standardization of other analytes is now in progress.

Standardization of laboratory data and establishment of reference intervals in the Fukuoka Prefecture: a Japanese perspective.

Standardization of 22 clinical chemistry analytes and five serum protein constituents has been performed in the Fukuoka Prefecture, which has a population of approximately five million. The standardization project was established to determine reference intervals for these analytes by educating physicians, medical technologists and staff of medical institutions, and by daily or monthly monitoring the use of common control samples through e-mail. Standardization extended to 97% of the institutions in the prefecture. Results for 14 of the 22 clinical chemistry analytes have become highly reliable and differences between institutions decreased. Standardization of other analytes is now in progress. Regional collaboration based on international guidelines led to a significant improvement in interlaboratory comparability. Areas where further improvements are needed have been identified.

Failure in the detection of aberrant mRNA from the heterozygotic splice site mutant allele for protein S in a patient with protein S deficiency.

A 29-year-old male patient with acute arterial obstruction and a medical history including thrombosis in the deep veins and pulmonary infarction presented with a reduced level of both protein S (PS) activity and free PS. Sequencing of the genomic PS gene in this patient revealed that the patient was heterozygous for the mutant PS allele, in which a nucleotide substitution occurred at the donor splice site in intron 12 (GT to GA). The patient was heterozygous for PS genes having dimorphic codons for Pro626 (CCA/CCG) and the aberrant allele in this patient was associated with the CCA form. Allelic exclusion of PS expression was demonstrated by use of Pro626 (CCA/CCG) dimorphism and only a normal mRNA sequence derived from the CCG-allele was identified in the patient. These findings suggested that the mutation at the splice site in the PS gene caused either defective production of mRNA or the gene may have produced extremely unstable RNA products, leading to reduced levels of PS activity and free PS in this patient.

A novel splice acceptor site mutation of protein S gene in affected individuals with type I protein S deficiency: allelic exclusion of the mutant gene.

Sequencing studies of the protein S gene (PROS1) in a Japanese patient suffering from recurrent thrombosis revealed the following. The proband and his first daughter, but not the second daughter, were having the type I protein S (PS) deficiency due to a novel point mutation from A to G at the intronic acceptor splice site in intron 13 of the PROS1. In the affected daughter, exclusion of the aberrant allele was assessed by the BstX1 dimorphism of PROS1 at Pro626 (CCG/CCA). The reduced PS activities in the proband and his first daughter were apparently due to defective production of mRNA from the mutant allele.

Translational regulation of the mRNA encoding the eukaryotic translation initiation factor 4E in Xenopus.

We have cloned the cDNA for Xenopus eukaryotic translation initiation factor 4E (eIF4E). Here we show that translation of a luciferase mRNA that contains the 5' untranslated region derived from Xenopus eIF4E is active in fertilized eggs, but is repressed in oocytes. The results suggest that the expression of Xenopus eIF4E is regulated at the translation level.

Analysis of the isoform of Xenopus euakryotic translation initiation factor 4E.

We have found two isoforms of the eukaryotic translation initiation factor 4E (eIF4E) in Xenopus laevis. These proteins differ in length by 18 amino acids. Overexpression of either of the two eIF4E proteins modestly increase translation in Xenopus oocytes. The results suggest that both of these two isoforms function in translation.

Polysomes of eukaryotic cells observed by electron microscopy.

Shapes of polysomes of eukaryotic cells were determined by surface spreading of cells. We examined unicellular protozoan Tetrahymena cells, rabbit reticulocytes, and cultured MDCK, BHK and HeLa cells. Polysomes had ring-, 8- and caterpillar-shaped forms, indicating that eukaryotic cells contain fundamentally circular polysomes. Isolated and partially purified polysomes from Tetrahymena cells had features of polysomes similar to those of spread cells. These findings indicate that circular polysomes are not an effect of the spreading, but are actual features of the cells.

Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation.

The poly(A)-binding protein Pab1p interacts directly with the eukaryotic translation initiation factor 4G (eIF4G) to facilitate translation initiation of polyadenylated mRNAs in yeast [1,2]. Although the eIF4G-PABP interaction has also been demonstrated in a mammalian system [3,4], its biological significance in vertebrates is unknown. In Xenopus oocytes, cytoplasmic polyadenylation of several mRNAs coincides with their translational activation and is critical for maturation [5-7]. Because the amount of PABP is very low in oocytes [8], it has been argued that the eIF4G-PABP interaction does not play a major role in translational activation during oocyte maturation. Also, overexpression of PABP in Xenopus oocytes has only a modest stimulatory effect on translation of polyadenylated mRNA and does not alter either the efficiency or the kinetics of progesterone-induced maturation [9]. Here, we report that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces translation of polyadenylated mRNA and dramatically inhibits progesterone-induced maturation. Our results show that the eIF4G-PABP interaction is critical for translational control of maternal mRNAs during Xenopus development.

Isolation and characterization of Xenopus laevis aldolase B cDNA and expression patterns of aldolase A, B and C genes in adult tissues, oocytes and embryos of Xenopus laevis.

Following previous cloning and expression studies of Xenopus aldolase C (brain-type) and A (muscle-type) cDNAs, we cloned here two Xenopus aldolase B (liver-type) cDNAs (XALDB1 and XALDB2, 2447 and 1490 bp, respectively) using two different liver libraries. These cDNAs had very similar ORF with only one conservative amino acid substitution, but 3'-UTR of XALDB1 contained ca. 1 kb of unrelated reiterated sequence probably ligated during library construction as shown by genomic Southern blot analysis. In adult, aldolase B mRNA (ca. 1.8 kb) was expressed strongly in kidney, liver, stomach, intestine, moderately strongly in skin, and very weakly in all the other tissues including muscles and brain, which strongly express aldolase A and C mRNAs, respectively. In oocytes and early embryos, aldolase A and C mRNAs occurred abundantly as maternal mRNAs, but aldolase B mRNA occurred only at a residual level, and its strong expression started only after the late neurula stage, mainly in liver rudiment, pronephros, epidermis and proctodeum. Thus, active expression of the gene for aldolase B, involved in dietary fructose metabolism, starts only later during development (but before the feeding stage), albeit genes for aldolases A and C, involved in glycolysis, are expressed abundantly from early stages of embryogenesis, during which embryos develop depending on yolk as the only energy source.

A case of congenital afibrinogenemia: fibrinogen Hakata, a novel nonsense mutation of the fibrinogen gamma-chain gene.

Congenital afibrinogenemia due to a novel homozygous nonsense mutation of the fibrinogen gamma-chain gene, fibrinogen Hakata, was found in an 18-year-old Japanese girl who had received supplemental fibrinogen therapy since she was 4 months old. The plasma fibrinogen concentrations of the proband were measured as less than 10 mg/dl by a functional method and less than 17 mg/dl by an immunological method. Fibrinogen concentrations of her family were in the range of 94-164 mg/dl. The proband and her family had no other clinical symptoms. Genomic DNA of the proband and her family was isolated from leukocytes, and all exons of fibrinogen subunits and their intron/exon boundaries were analyzed. A genetic mutation, a guanine-to-thymine (G-to-T) transversion at the nucleotide position of 5860, was identified on exon 7 of the gamma-chain gene. This mutation changed the codon for the 231st residue of the gamma-chain from GAG (Glu) to TAG (stop). No other mutation was observed. Aalpha, Bbeta and gamma chains were observed in plasma of the heterozygous family members. However, only a trace amount of Aalpha chain and no gamma chain was detected in the plasma of the proband.

Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex formation.

A translation initiation factor, eIF4E, of Xenopus laevis was purified by affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the binding activity of eIF4E with 4E-BP2. Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex formation of 4E-BP2 with eIF4E; the dissociation constant of eIF4E for the cap-analogue was estimated to be 10(-2)-10(-4) M. These results suggest that the participation of another initiation factor is required for eIF4E to recognize the cap structure in vivo. The results reported in this paper support "the performed complex model" of Lee et al., in which eIF4E binds to the mRNA cap structure after the initiation factors have formed the initiation complex eIF4F.

New sensitive method for the detection of the A3243G mutation of human mitochondrial deoxyribonucleic acid in diabetes mellitus patients by ligation-mediated polymerase chain reaction.

An adenine-to-guanine mutation at nucleotide position (np) 3243 in the mitochondrial tRNALeu(UUR) gene is closely associated with various clinical phenotypes of diabetes mellitus. Because the mutation creates a new restriction site for the restriction enzyme ApaI, the mutation is usually detected and quantified by ApaI cleavage of the PCR products including np 3243. The sensitivity of the conventional method is, however, 5-10% heteroplasmy. The percentage of heteroplasmy of the mutation is usually highest in the affected tissues and is much lower in peripheral blood cells, which are used most frequently for the analysis. The sensitivity of the conventional method, however, is not sufficient to detect the mutation from peripheral blood cells. Utilizing ligation-mediated polymerase chain reaction, we have developed a feasible and sensitive method to detect 0.01% heteroplasmy of the 3243 mutation in peripheral leukocytes.

Disulfide bond formation is not involved in cap-binding activity of Xenopus translation initiation factor eIF-4E.

The eukaryotic initiation factor eIF-4E from Xenopus laevis was expressed in Escherichia coli and refolded in an active form. To define the cysteine residues forming a disulfide bond in Xenopus eIF-4E, each of the 3 cysteine residues was changed to serine by site-directed mutagenesis. Cap-binding activities of the mutant proteins were evaluated by 7-methyl-GTP(m7GTP)-affinity column chromatography. Even the mutant protein containing no cysteine showed an affinity for m7GTP. From the above results and the estimation of the sulfhydryl groups by Ellman's assay method, we concluded that a disulfide bond is not involved in the active Xenopus eIF-4E.

Expression of Xenopus laevis translation initiation factor 4E (eIF-4E) by baculovirus-insect cell system.

A gene encoding Xenopus laevis eIF-4E was cloned into a transfer vector, and its gene expression was attempted in cells of E. coli, yeast and insect. Effective expression of the active eIF-4E was achieved in the soluble fraction of the insect cell Sf9, which was infected with the recombinant baculovirus. Overexpression of the eIF-4E protein caused remarkable change in the shape of the cells.

Characteristic distribution of bases and codons around the initiation and termination codons in whole reading frames in bacteria and yeast genomes.

Recently the complete nucleotide sequence of the entire genome was determined for yeast and a few kinds of bacterium. To see characteristic features of base sequence in the cistron (actually the open reading frame, ORF) and in the regions around a cistron (ORF), the biases of appearance frequency of bases from the base ratio were studied statistically. In the regions before the base biases were observed. The characteristic base distribution patterns were similar to all the cases of bacteria, but different from yeast. The base biases are reflected on the appearance frequency of amino acids near the N-termini and C-termini of proteins. The characteristic biases found in the amino acid sequence of the N-terminal part of bacteria proteins are different from that in yeast proteins.

Transmission electron and atomic force microscopic observation of polysomes on carbon-coated grids prepared by surface spreading.

A simple modification of surface spreading that enables easy examination of subcellular structures by both transmission electron microscopy (TEM) and atomic force microscopy (AFM) was developed in the present study. Specimens were adsorbed onto carbon-coated electron microscopy (EM) grids with parlodion backing for comparison of images obtained using TEM and AFM. Fine structures of ribosomes and polysomes were observed in this study. Washing the mounted specimen with detergent such as 0.05% Photo-Flo 200 before conducting negative staining or metal shadowing brought clear visualization of ribosomes and polysomes. The same preparation could apply to AFM imaging without any additional treatment.