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Twenty patients with advanced gastric cancer were treated with FAP.MMC (5-FU 350 mg/m2 i.v. on days 1-3, ADM 40 mg/m2 i.v. on day 1, CDDP 20 mg/m2 i.v. on days 1-3, MMC 6 mg/m2 i.v. on day 1), administering 5-FU, ADM and CDDP every 4 weeks and MMC every 8 weeks. Fourteen patients were evaluable for responses. Four (29%) partial responses and two minor responses were observed. The median duration of partial response was 3.8 months (range 2.5-7 months). The median overall survival time was 5 months (range 1.5-15 months). Leukopenia was relatively severe, with a median WBC nadir of 1,300/mm3. Nausea and vomiting were frequent but moderate. However, these toxicities were clinically manageable. FAP.MMC was thus considered effective for advanced gastric cancer.

A phase II study of Prednimustine (PMN) was conducted for follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL). PMN at a dose of 40-60 mg/body, p.o. every day, was administered to 17 patients with FL and 4 with CLL. The dose and schedule of PMN was modified according to hematological toxicity. Among the 17 patients with FL, there were 6 (35.3%) CRs and 7 (41.2%) PRs, with a high response rate of 76.5%. Among the 4 patients with CLL, there were 2 PRs and one case which showed clinical improvement. PMN was effective for cases of FL and CLL refractory to alkylating agents, and therefore the result suggested a lack of clinical cross-resistance to these agents. As to the side effects observed in patients with FL, mild leukopenia (median of lowest count 3,150/mm3) occurred. One case experienced anorexia, while increased appetite was observed in 4 cases. We conclude that PMN is effective for FL and CLL, and that in addition, it has an advantage in that its mild side effects allow long-term administration through outpatient clinics, so that the quality of life for patients is not impaired.

From July 1983 to April 1986, four patients (three with ventricular septal perforations and one with left ventricular free-wall rupture) underwent replacement of the myocardium with a Dacron prosthesis for complications of acute myocardial infarction. There were three males and one female, ages ranging from 53 to 70 years (mean 63.3). Three of the four patients survived; the one with the ventricular septal perforation died of severe cardiac failure five days after operation. Replacement of the infarcted myocardium with a Dacron prosthesis seems to be an excellent operative technique for the treatment of complications of acute myocardial infarction when the left ventricular cavity is predicted to be small after resection of the myocardium.

Homology maps between bacteriophages phi81, phi80 and lambda were constructed on the basis of electron microscopy observation of DNA heteroduplexes. In phi81/phi80 heteroduplex, the left half and the right terminal region of 13% the total molecular length were highly homologous, while the remaining region covering the early gene cluster was entirely nonhomologous. In phi81/lambda heteroduplex, high-degree homologies were detected at the left 14% terminal region covering the head gene cluster, the central 3.8% region covering the att-int-xis region and the 1.3% Q homology region. Low-degree homologies of shorter length were scattered at the tail gene cluster, b2 region, cIII region, PQ region and SR region. Comparing our results with the homology maps of other lambdoid phages reported by Simon et al. (1971) and Fiandt et al. (1971), a phylogenic relation of phi81 to other lambdoid phages and the role of recombination in the course of divergence of lambdoid phages are discussed.

Eight patients with various hematological malignancies were treated with chlorambucil, an analogue of nitrogen mustard. Five patients with CLL, 2 patients with malignant lymphoma, and one each with pseudolymphoma of the lung and primary macroglobulinemia were given 4-6 mg of chlorambucil per day for 1-2 weeks at 2-4 week intervals or a daily dose of 6 mg continuously. Two cases of CLL and one each of follicular lymphoma and pseudolymphoma achieved partial remission. Side effects included myelosuppression, especially leukopenia. Chlorambucil can be used orally without severe toxicity for longer periods and the major indication for this drug seems to be CLL and favorable type of malignant lymphoma.

Combined valvular and coronary surgery were performed on 9 patients between 1973 and 1986. Valve replacements were consist of 5 AVR (including 1 translocated AVR and 1 Bentall operation), 2 MVR and 2 double valve replacement. Coronary surgery were of 6 aorto-coronary bypass using great saphenous veins, 1 innominate artery-coronary bypass using a Dacron prosthesis, 1 punch-out of coronary ostium and 1 dilatation of the left coronary ostium with a vein patch. Retrograde infusion of cardioplegic solution from the coronary sinus were done in patients with coronary ostial stenoses. One emergency case with acute cardiac failure died immediately after operation, and one with double valve and one with mitral valve replacement died of hepatic failure and multiple organ failure respectively within 1 year after surgery. Combined valvular and coronary surgery are necessary for patients with both diseases. Although the results of double valve replacement were not satisfactory, they would be improved using intra-aortic balloon pump and ventricular assist devices actively.

The nucleotide sequence of a region of Marchantia polymorpha chloroplast DNA was determined. On this DNA sequence (3.38kb), three open reading frames (ORFs) and three putative tRNA genes were detected in the following order: -ORF701-tRNASer(UGA)-ORF702-tRNAGly(GCC)-initiator tRNAMet(CAU)-ORF703-. The ORF703 is composed of 100 codons in which those for lysine (15%) and arginine (11%) are abundant, and could be accounted for as a counterpart of E. coli ribosomal protein S14 since they share 45% homology in the amino acid sequences. The ORF701 appears to code for a membrane protein, showing a periodic appearance of seven clusters of hydrophobic amino acids. Although the mechanisms remain unknown, the ORF701 causes a streptomycin-sensitive phenotype in resistant mutants of E. coli. The ORFs and tRNA genes are separated from each other by extremely AT-rich spacers containing sequences of dyad symmetry. The third letter positions of the codons in the ORFs are also rich in A and T residues.

SM-5887, a new totally synthetic anthracycline derivative, was studied in a phase I setting. Twenty-nine evaluable courses of treatment were conducted in groups at doses increasing from 10 to 130 mg/m2. At 130 mg/m2 the median lowest WBC count was 0.7 x 10(3)/mm3 (range 0.3-1.8) and the median lowest platelet count was 57 x 10(3)/mm3 (range 4-176). Nonhematological side effects were mild gastrointestinal symptoms and hair loss. The recommended dose and schedule for a phase II setting is 100 mg/m2 every 3 weeks.

The complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. Other genes involved in photosynthesis, respiratory electron transport, and membrane-associated transport in chloroplasts were predicted by the amino acid sequence homology and secondary structure of gene products. Thirty-three open reading frames in the liverwort chloroplast genome remain unidentified. However, most of these open reading frames are also conserved in the chloroplast genomes of two species, a liverwort, Marchantia polymorpha, and tobacco, Nicotiana tabacum, indicating their active functions in chloroplasts.

We have isolated mutations in the Escherichia coli glnS gene encoding glutaminyl-tRNA synthetase [GlnS; L-glutamine:tRNAGln ligase (AMP-forming), EC 6.1.1.18] that give rise to gene products with altered specificity for tRNA and are designated "mischarging" enzymes. These were produced by nitrosoguanine mutagenesis of the glnS gene carried on a transducing phage (lambda pglnS+). We then selected for mischarging of su+3 tRNATyr with glutamine by requiring suppression of a glutamine-requiring beta-galactosidase amber mutation (lacZ1000). Three independently isolated mutants (glnS7, glnS8, and glnS9) were characterized by genetic and biochemical means. The enzymes encoded by glnS7, glnS8, and glnS9 appear to be highly selective for su+3 tRNATyr, because in vivo mischarging of other amber suppressor tRNAs was not detected. The GlnS mutants described here retain their capacity to correctly aminoacylate tRNAGln. All three independently isolated mutant genes encode proteins with isoelectric points that differ from those of the wild-type enzyme but are identical to each other. This suggests that only a single site in the enzyme structure is altered to give the observed mischarging properties. In vitro aminoacylation reactions with purified GlnS7 protein show that this enzyme can also mischarge some tRNA species lacking the amber anticodon. This is an example of mischarging phenotype conferred by a mutation in an aminoacyl-tRNA synthetase gene; the results are discussed in the context of earlier genetic studies with mutant tRNAs.

The nucleotide sequence (25,320 base-pairs) of a part of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha was determined. This region encodes putative genes for four tRNAs, isoleucine tRNA(CAU), arginine tRNA(CCG), proline tRNA(UGG) and tryptophan tRNA(CCA); eight photosynthetic polypeptides, the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL), 51,000 Mr photosystem II chlorophyll alpha apoprotein (psbB), apocytochrome b-559 polypeptides (psbE and psbF), 10,000 Mr phosphoprotein (psbH), cytochrome f preprotein (petA), cytochrome b6 polypeptide (petB), and cytochrome b6/f complex subunit 4 polypeptide (petD); 13 ribosomal proteins (L2, L14, L16, L20, L22, L23, L33, S3, S8, S11, S12, S18 and S19); initiation factor 1 (infA); ribosome-associating polypeptide (secX); and alpha subunit of RNA polymerase (rpoA). Functionally related genes were located in several clusters in this region of the genome. There were two ribosomal protein gene clusters: rpl23-rpl2-rps19-rpl22-rps3-rpl16-+ ++rpl14-rps8-infA-secX-rps11-rpoA, with a gene arrangement similar to that of the Escherichia coli S10-spc-alpha operons, and the rps12'-rpl20-rps18-rpl33 cluster. There were gene clusters encoding photosynthesis components such as the psbB-psbH-petB-petD and the psbE-psbF clusters. Thirteen open reading frames, ranging in length from 31 to 434 amino acid residues, remain to be identified.

The nucleotide sequence (56,410 base-pairs) of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha has been determined. The sequence starts from one end (JLA) of the large single-copy region and encompasses genes for 21 tRNAs, six ATPase subunits (atpA, atpB, atpE, atpF, atpH and atpI), two photosystem I polypeptides (psaA and psaB), four photosystem II polypeptides (psbA, psbC, psbD and psbG), five ribosomal proteins (rps2, rps4, rps7, rps'12 and rps14), and three RNA polymerase subunits (rpoB, rpoC1 and rpoC2). In addition, we detected 18 open reading frames ranging from 29 to 2136 amino acid residues long, four of which share significant amino acid sequence homology to those of an Escherichia coli malK protein (designated mbpX), human mitochondrial ND2 (ndh2) and ND3 (ndh3) of a respiratory chain NADH dehydrogenase, or a bacterial antenna protein of a light-harvesting complex (lhcA). Sequence analysis suggests that four tRNA genes and six protein genes might be split by introns; they are trnG(UCC), trnK(UUU), trnL(UAA), trnV(UAC), atpF, ndh2, rpoC1, rps'12, ORF135 and ORF167. In the large single-copy region described here, the gene organization deduced is highly conserved with respect to that of higher plants, but an inversion of some 30,000 base-pairs flanked by trnL(CAA) and trnD(GUC) was seen between the liverwort and tobacco chloroplast genomes.

An efficient method was devised to isolate temperature sensitive mutants of E. coli defective in tRNA biosynthesis. Mutants were selected for their inability to express suppressor activity after su3(+)-transducing phage infection. In virtually all the mutants tested, temperature sensitive synthesis of tRNA(Tyr) was demonstrated. Electrophoretic fractionation of (32)P labeled RNA synthesized at high temperature showed in some mutants changes in mobility of the main tRNA band and the appearance of slow migrating new species of RNA. Temperature sensitive function of mutant cells was also evident in tRNA synthes: directed by virulent phage T4 and BF23. We conclude that although the mutants show individual differences, many are temperature sensitive in tRNA maturation functions. In spite of much information on the structure and function of transfer RNA (tRNA), our knowledge concerning the biosynthesis of tRNA is relatively poor. It is generally assumed that complete tRNA molecules are made via a series of processing steps from the original transcription products of tRNA genes which are presumably unmodified and longer than mature tRNA molecules. In the case of tyrosine suppressor tRNA of su3(+), an unmodified precursor RNA carrying additional residues at the 3' and 5' ends has been isolated (1,2), and an endonuclease cleaving at the 5' side of this precursor has been identified in E. coli (3). In the case of T4 encoded tRNA, a large precursor molecule for several tRNA's has been reported (4). Some enzymes that catalyze the modifications have also been described (5). However, the over-all picture and the precise mechanisms of tRNA maturation are as yet largely unkown. For study of tRNA biosynthesis in E. coli, a genetic approach may prove useful, as has been the case in other biosynthetic pathways. In order to obtain mutants blocked in any of the intermediary steps of tRNA synthesis, we have developed an efficient selection system that enriches these mutants. Since any mutational block in tRNA biosynthesis might well be lethal, we looked for conditional lethal mutants in which the defect in tRNA synthesis occurs only at high temperature. In this selection system, the su3 gene carried by a temperate phage was newly introduced into cells(su(-)) and those cells incapable of synthesizing su3(+) tRNA at high temperature were selected. Such mutants were easily enriched by using conditions in which cells expressing suppressor activity were killed by two virulent phages. In this communication, we report the method for isolation of mutants and some characterization of tRNA synthesis in these mutants. Recently, Schedl and Primakoff (6) have independently isolated thermosensitive mutants of E. coli defective in tRNA synthesis which may or may not be different types from ours.

SM-5887, a new totally synthetic anthracycline derivative was studied in a phase I setting. Twenty-nine evaluable courses of treatment were conducted in groups at doses increasing from 10 to 130 mg/m2. Myelosuppression was the dose-limiting toxicity and a MTD was 130 mg/m2. At 130 mg/m2 the median lowest white blood cell count was 0.7 x 10(3)/cmm (range: 0.3-1.8) and the median lowest granulocyte count was 0.1 x 10(3)/cmm (range: 0-0.7) and the median lowest platelet count was 57 x 10(3)/cmm (range: 4-176). Non-hematologic side effects were mild gastrointestinal symptoms and hair loss. The recommended dose and schedule for phase II setting is 100 mg/m2 every 3 weeks.

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.

One hundred and six patients with advanced breast cancer were treated with chemoendocrine therapy consisting of adriamycin (40 mg/m2) i.v. on day 1 and cyclophosphamide (130 mg/m2) i.v. daily for 5 days every 3 weeks, ftorafur (500 mg/m2) and tamoxifen (40 mg) orally daily. Of 82 evaluable patients, 16 showed complete response (20%), 32 partial response (39%), 32 no change (39%), and two progressive disease (2%). The overall response rate was 59%, and the median duration of response was 16.3 (3.5-67+) months with a median survival time from the start of chemoendocrine therapy of 25.5 (3.5-67+) months. The median survival time of responders (32.5 months) was significantly longer than that of non-responders (15.3 months). The major toxicities were hair loss, G1 symptoms, and hematological toxicity, but these were clinically well tolerated. No serious cardiac, renal or liver damage was seen. These results indicated that the addition of tamoxifen to the ACF regimen increased the number of complete responses and prolonged the survival time of responders.

An Escherichia coli DNA fragment containing an Su+6 amber suppressor gene (supP) was cloned into a lambda gt lambda Ch vector by the shotgun method, selecting a Su+6 transducing phage lambda pSu+6. Through prophage integration followed by induction occurring at the transducing region of the lambda pSu+6 in Su- E. coli, a counterpart transducing phage carrying the wild-type allele (Su degrees 6) was isolated (lambda pSu degrees 6). The fingerprint of a tRNA encoded by lambda pSu degrees 6 was identical to that of an unidentified tRNAE previously reported (Ikemura & Ozeki, 1977). The cloverleaf structure of this tRNA was determined by combining the results of tRNA analysis and DNA sequencing of the gene. Judging from the anticodon of 5'-CAA-3', Su degrees 6 tRNA was identified as a new type of leucine isoacceptor in E. coli. Unlike other suppressors analyzed, Su+6 tRNA differed by two nucleotides from Su degrees 6 tRNA; one at the anticodon (CAA to CUA) and the other at the junction of D- and anticodon-stem (A27 to G27). DNA sequence analysis revealed that a single stretch of tRNA is flanked by the putative sequences of promoter and terminator. Thus a single copy of the Su degrees 6 tRNA gene constitutes a solitary tRNA transcription unit. Southern blotting showed only one copy of Su degrees 6 tRNA gene per haploid genome of E. coli. Since this single gene can mutate to the Su+6 suppressor, the Su degrees 6 leucine tRNA may be accounted as a dispensable species among the leucine isoacceptor tRNAs. Two possible open reading frames are found immediately following the Su degrees 6 tRNA gene.

RNAs synthesized in Escherichia coli infected with virulent phages T4, T5, T7 and BF23 were labelled with 32PO4 3- after phage infection. [32P]RNAs of low molecular weight were separated by two-dimensional polyacrylamide gel electrophoresis, in which electrophoresis was carried out in two dimensions at different concentrations of acrylamide. The fractionated RNAs were characterized by RNA-fingerprint patterns made after T1 ribonuclease digestion. The two-dimensional gel of 10% yields 20% acrylamide was suitable for RNA of less than 200 nucleotides, while that of 5% yields 10% was preferred for RNAs of about 150--400 nucleotides. With T4 phage, 16 RNA species were separable on a single slab gel. Among those, 11 were identified as the known RNA species, including eight T4 tRNAs, one tRNA precursor and two non-tRNA molecules. In the case of T5 and BF23, more than 20 RNA species were separated on a slab gel; 15 or more RNAs were found in the 4-S RNA region, and several in 5-S and 6-S region. The RNA-fingerprint patterns of many BF23 RNAs were very similar to those of corresponding RNAs of T5. Pseudouridine and ribosylthymidine, minor nucleosides generally present in tRNA, were found in several BF23 4-S RNAs tested. Possibility of those BF23 4-S RNAs as tRNAs is discussed. With phage T7, three RNAs were detected, two of which were much smaller than tRNAs.

About fifty suppressor sensitive (sus) mutants of phage phi80 were isolated after hydroxylamine treatment, and these were classified into fourteen cistrons. In vitro and in vivo complementation experiments revealed that at least five cistrons were concerned with head formation and that at least six cistrons were concerned with tail formation in phi80. Some presumed "early" mutants were also found. Defective lysogens were isolated from a phi80-lysogenic nonpermissive strain using the colicin plate method (Gratia, 1966). In these strains deletions which affected the colicin B receptor gene extended for varying distances into prophage phi80. Marker rescue experiments were carried out with these deletion lysogens by infecting various sus mutants, and the gene order in prophage phi80 was determined. Clustering of all head genes and also of tail genes, as in phage lambda, were demonstrated by the results of the prophage deletion mapping as well as the two-factor crosses performed among the sus mutants. Moreover, the gross gene arrangement of phi80 was also similar to that of phage lambda: namely, the cluster of head genes was found to be located at one end of the vegetative map of phi80, being followed by that of tail genes, and presumed "early" genes are located at the other end of the map.

Growth of phage BF23 was restricted in Escherichia coli K-12 strains carrying a colicin I factor (ColIb); most infected cells lysed early without producing progeny phages. Either addition of chloramphenicol before phage infection or ultraviolet irradiation of phage prevented early abortive lysis, an indication that certain phage functions are required for this phenomenon. Very little or no phage-induced lysozyme was synthesized in the infected ColI(+) cells. This result suggests that early abortive lysis was not due to the lysozyme action. A small fraction (0.05) of BF23-infected ColI(+) cells showed normal phage growth. This "escaped growth" may reflect the physiological state of the host bacteria rather than the heterogeneity of the infecting phage. Host-controlled modification was not observed. A phage mutant, BF23hI, able to grow on ColI(+) cells, was isolated and was characterized to be recessive to the wild-type BF23 in its ability to undergo early abortive lysis. Among the T series phages, T5 induced early abortive lysis, and growth of T5 was restricted upon infection to ColI(+) cells. These results and the other observations, including the occurrence of phenotypic mixing between BF23 and T5, suggest that these two phages are related to each other even though the receptor sites for BF23 and T5 are apparently different.