Heterogeneity in the substitution process of amino acid sites of proteins coded for by mitochondrial DNA. Several forms of maximum likelihood models are applied to aligned amino acid sequence data coded for in the mitochondrial DNA of six species (chicken, frog, human, bovine, mouse, and rat). These models range in form from relatively simple models of the type currently used for inferring phylogenetic tree structure to models more complex than those that have been used previously. No major discrepancies between the optimal trees inferred by any of these methods are found, but there are huge differences in adequacy of fit. A very significant finding is that the fit of any of these models is vastly improved by allowing a certain proportion of the amino acid sites to be invariant. An even more important, although disquieting, finding is that none of these models fits well, as judged by standard statistical criteria. The primary reason for this is that amino acid sites undergo substitution according to a process that is very heterogeneous. Because most phylogenetic inference is accomplished by choosing the optimal tree under the assumption that a homogeneous process is acting on the sites, the potential invalidity of some such conclusions is raised by this article's results. The seriousness of this problem depends upon the robustness of the phylogenetic inferential procedure to departures from the underlying model.


Man's place in Hominoidea revealed by mitochondrial DNA genealogy. Molecular biology has resurrected C. Darwin and T.H. Huxley's question about the origin of humans, but the precise branching pattern and dating remain controversial. To settle this issue, a large amount of sequence information is required. We determined mitochondrial (mt) DNA sequences for five hominoids; pygmy and common chimpanzees, gorilla, orangutan, and siamang. The common region compared with the known human sequence is 4759 bp long, encompassing genes for 11 transfer RNAs and 6 proteins. Because of the high substitution rates in mammalian mtDNA and an unprecedentedly large region compared, the sequence differences clearly indicate that the closest relatives to human are chimpanzees rather than gorilla. For dating the divergences of human, chimpanzee, and gorilla, we used only unsaturated parts of sequence differences in which the mtDNA genealogy is not obscured by multiple substitutions. The result suggests that gorilla branched off 7.7 +/- 0.7 million years (Myr) ago and human 4.7 +/- 0.5 Myr ago; the time difference between these divergences being as long as 3 Myr.


Unusual organizational features of the Drosophila Gart locus are not conserved within Diptera. The Drosophila Gart locus consists of two genes. One gene encodes three enzymes in the de novo purine nucleotide biosynthesis pathway [glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART)]. The second gene lies within an intron of the purine gene and encodes a cuticle protein. To investigate the evolution of the Gart locus, the Chironomus tentans homolog was cloned by screening a genomic DNA library with a polymerase chain reaction product. This study shows that the interesting structural features of this locus conserved in two distant Drosophila species are not found in the Chironomus homolog. These features include the cuticle protein gene nested within an intron and the existence of an alternative transcript to yield a monofunctional enzyme. In addition, the extremely rapid divergence of coding sequence seen for members of the tandemly duplicated AIRS domain in Drosophila is found to be much less rapid in Chironomus.


Finite-state models in the alignment of macromolecules. Minimum message length encoding is a technique of inductive inference with theoretical and practical advantages. It allows the posterior odds-ratio of two theories or hypotheses to be calculated. Here it is applied to problems of aligning or relating two strings, in particular two biological macromolecules. We compare the r-theory, that the strings are related, with the null-theory, that they are not related. If they are related, the probabilities of the various alignments can be calculated. This is done for one-, three-, and five-state models of relation or mutation. These correspond to linear and piecewise linear cost functions on runs of insertions and deletions. We describe how to estimate parameters of a model. The validity of a model is itself an hypothesis and can be objectively tested. This is done on real DNA strings and on artificial data. The tests on artificial data indicate limits on what can be inferred in various situations. The tests on real DNA support either the three- or five-state models over the one-state model. Finally, a fast, approximate minimum message length string comparison algorithm is described.


A statistical test of hypotheses on the organization and origin of the genetic code. Theories of the origin of the genetic code assign different weights to amino acid properties such as polarity and precursor-product relationship. Previous statistical work on the origin of the genetic code has produced controversial results. We analyze relationships between various amino acid and tRNA properties by one and the same statistical method. It is shown that polarities as well as precursor-product relationships are both likely to have been important in shaping the genetic code, together with codon swapping that left protein sequences intact.


Patterns of nucleotide substitutions inferred from the phylogenies of the class I major histocompatibility complex genes. Patterns of nucleotide substitutions in human major histocompatibility complex (MHC) class I genes were estimated by using phylogenetic trees of DNA sequences. The pattern is defined as a set of 12 parameters, each of which represents the relative frequency of substitutions from a particular nucleotide to another. The pattern at the antigen recognition sites (ARS) in functional MHC genes was remarkably different from that at the remaining coding region (non-ARS). In particular, the proportion of transitions among all the nucleotide substitutions (Ps) was extremely low at the third codon positions of ARS. In the HLA-A genes, Ps at the third codon positions was only 6% in ARS, whereas it was 69% in non-ARS. In HLA-B, the corresponding values were 30% in ARS and 80% in non-ARS, respectively. On the other hand, Ps in a class I pseudogene (HLA-H) was 57%, which was in good agreement with Ps in other pseudogenes. Because pseudogenes are selectively neutral, the pattern in pseudogenes is regarded as the pattern of spontaneous substitution mutations. In general, the pattern in functional genes that are subject to selective forces deviates from the pattern in pseudogenes. At the third codon positions in coding regions, transitions scarcely cause amino acid replacements, whereas about half of transversions do cause replacements. Accordingly, Ps at the third codon positions decreases if amino acid replacements are accelerated by natural selection but increases if amino acids are conserved by functional constraint. Our observations imply that the ARS region is subject to natural selection favoring amino acid replacements, whereas the non-ARS region is subject to functional constraint.


Conserved and variable repeat structures in the Balbiani ring gene family in Chironomus tentans. The four Balbiani ring (BR) genes, BR1, BR2.1, BR2.2, and BR6 in the midge Chironomus tentans constitute a gene family encoding secretory proteins with molecular weights of approximately 10(6) daltons. The major part of each gene is known to consist of tandemly organized composite repeat units resulting in a hierarchic repeat arrangement. Here, we present the sequence organization of the 5' part of the BR2.2 and BR6 genes and describe the entire transcribed part of the two genes. As the BR1 and BR2.1 genes were also fully characterized recently, this allows the comparison of all genes in the BR gene family. All four genes share the same exon-intron structure and have evolved by gene duplications starting from a common ancestor, having the same overall organization as the BR genes of today. The genes encode proteins that have an approximately 10,000-amino acid residue extended central domain, flanked by a highly charged, approximately 200-residue amino-terminal domain and a globular 110-residue carboxy-terminal domain. Exons 1-3 and the beginning of exon 4 encode the amino-terminal domain, which throughout contains many regions built from short repeats. These repeats are often degenerate as to repeat unit and sequence and are present in different numbers between the genes. In several instances these repeat structures, however, are conserved at the protein level where they form positively or negatively charged regions. Each BR gene has a 26-38-kb-long exon 4, which consists of an array of 125-150 repeat units and encodes the central domain. The number of repeat units appears to be largely preserved by selection and all repeat units in the array are very efficiently homogenized. Occasionally variant repeats have been introduced, presumably from another BR gene by gene conversion, and spread within the array. Introns 1-3 at the 5' end of the genes have diverged extensively in sequence and length between the genes. In contrast, intron 4 at the 3' end is virtually identical between three of the four genes, suggesting that gene conversion homogenizes the 3' ends of the genes, but not the 5' ends.


Evidence that mammalian glutamine-dependent carbamyl phosphate synthetase arose through gene fusion. On the basis of homology, the mammalian CAD (glutamine-dependent carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) gene appears to have arisen from the fusion of four separate ancestral genes. Evidence for two of these precursor genes is found in the carbamyl phosphate synthetase (CPSase) domain of CAD. In prokaryotes, such as Escherichia coli CPSase is encoded by two distinct cistrons of the carAB operon. Whereas carA and carB are separated by a short noncoding intercistronic region, the homologous sequences of the CAD gene encode an amino acid bridge. This bridge connects the subdomains of the CAD CPSase. We constructed a bacterial carAB fusion gene in which the intercistronic region codes for a hamster bridgelike sequence. The fused carAB gene directs the synthesis of a stable bifunctional polypeptide whose glutamine-dependent CPSase activity is comparable to the E. coli CPSase holoenzyme. The fusion in E. coli of the single gene counterparts of CAD demonstrates a potential model system to study the genetic events that lead to gene fusion and the creation of multienzymatic proteins.


Phylogeny and evolutionary rates of G protein alpha subunit genes. Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s alpha), inhibitory (i alpha), transducin (t alpha), Gx (x alpha), Gz (z alpha), G11 (alpha 11), G12 (alpha 12), G13 (alpha 13), G16 (alpha 16), Gq (q alpha), and other (o alpha) G protein alpha subunits have been constructed. The analysis shows that the G12 (alpha 12 and alpha 13), Gq (alpha 11, alpha 16, and q alpha), and Gs (s alpha genes) groups form one cluster, and the Gx (x alpha and z alpha genes), G(i) (i alpha genes), Gt (t alpha 1 and t alpha 2), and G(o) (o alpha genes) groups form another cluster. During mammalian evolution, the rates of synonymous substitutions for these genes were estimated to be between 1.77 x 10(-9)/site/year and 5.63 x 10(-9)/site/year, whereas those of non-synonymous substitutions were between 0.008 x 10(-9)/site/year and 0.067 x 10(-9)/site/year. These evolutionary rates are similar to those for histone genes, suggesting equally important biological functions of the G protein alpha subunits.


Statistical analysis and prediction of the exonic structure of human genes. Nonhomologous fully sequenced human protein-coding genes were studied. Three sets of exon-exon junctions were formed defined by the intron (shadow) position relative to the reading frame. For the analysis of intron shadow signals in exons, information content and discrimination energy approaches were used with the correction allowing one to ignore the influence of a protein-coding message. The corrected formulas allow one to define the consensuses for the three types of intron shadow signals as a AG/guwn, cAG/GUnn, and cAG/gunU, and provide better recognition than the original formulas. The analysis of the codon usage in the signal positions leads to the conclusion that the prevalence of some amino acids in corresponding protein sites is caused by the signal requirements and not vice versa. The distribution of potential intron shadow signals in exons contradicts the hypothesis of intron insertion into suitable preexisting sites. There exists a correlation between the intron types and/or the exon length modulo 3.


Estimating the fraction of invariable codons with a capture-recapture method. A codon-based approach to estimating the number of variable sites in a protein is presented. When first and second positions of codons are assumed to be replacement positions, a capture-recapture model can be used to estimate the number of variable codons from every pair of homologous and aligned sequences. The capture-recapture estimate is compared to a maximum likelihood estimate of the number of variable codons and to previous approaches that estimate the number of variable sites (not codons) in a sequence. Computer simulations are presented that show under which circumstances the capture-recapture estimate can be used to correct biases in distance matrices. Analysis of published sequences of two genes, calmodulin and serum albumin, shows that distance corrections that employ a capture-recapture estimate of the number of variable sites may be considerably different from corrections that assume that the number of variable sites is equal to the total number of positions in the sequence.


Chaos game representation of coding regions of human globin genes and alcohol dehydrogenase genes of phylogenetically divergent species. Chaos game representation (CGR) is a novel holistic approach that provides a visual image of a DNA sequence quite different from the traditional linear arrangement of nucleotides. Although it is known that CGR patterns depict base composition and sequentiality, the biological significance of the specific features of each pattern is not understood. To systematically examine these features, we have examined the coding sequences of 7 human globin genes and 29 relatively conserved alcohol dehydrogenase (Adh) genes from phylogenetically divergent species. The CGRs of human globin cDNAs were similar to one another and to the entire human globin gene complex. Interestingly, human globin CGRs were also strikingly similar to human Adh CGRs. Adh CGRs were similar for genes of the same or closely related species but were different for relatively conserved Adh genes from distantly related species. Dinucleotide frequencies may account for the self-similar pattern that is characteristic of vertebrate CGRs and the genome-specific features of CGR patterns. Mutational frequencies of dinucleotides may vary among genome types. The special features of CG dinucleotides of vertebrates represent such an example. The CGR patterns examined thus far suggest that the evolution of a gene and its coding sequence should not be examined in isolation. Consideration should be given to genome-specific differential mutation rates for different dinucleotides or specific oligonucleotides.


Delayed onset muscle soreness: mechanisms and management. This review describes the phenomenon of delayed onset muscle soreness (DOMS), concentrating upon the types of muscle contraction most likely to produce DOMS and the theories underlying the physiological mechanisms of DOMS. Ways of attempting to reduce the effects of DOMS are also summarized, including the application of physical and pharmacological therapies to reduce the effects of DOMS and training for reduction or prevention of DOMS.


The biomechanics of the triple jump: a review. The purpose of this paper is to review the published opinions of coaches and the findings of scientists concerning the techniques used in the triple jump, and to identify promising avenues for future research on the subject. A model is developed to identify those factors that have a causal role in determining the official distance of a triple jump. This model is then used as a basis for the subsequent review. The review itself considers each of the biomechanical factors identified in the model, some additional factors reported in the literature, and selected characteristics of triple jumping techniques. It is concluded that research on triple jump techniques has been sparse and has had little impact on practice. Identification of the individual attributes that determine the optimum ratio of the phase distances for a given athlete, the loads to which the supporting legs are subjected and the control of balance during a triple jump are seen as challenging and potentially useful topics of future research.


Group cohesion and starting status in successful and less successful elite volleyball teams. The main purpose of this study was to examine the relationship between members' perceptions of group cohesion and starting status in elite volleyball teams. The results of the study revealed that the form of the cohesion-starting status relationship was moderated by the variable of success. The results for less successful teams revealed that differences did emerge between specific measures of cohesion endorsed by starters and non-starters. No such differences in cohesion emerged when the starters and non-starters on successful teams were compared. These results provide initial support for the suggestion that the most successful teams are the ones where the perceptions of cohesiveness by starters and non-starters are similar. A secondary purpose of the study was to determine whether those teams that were the most successful and similar in their members' perceptions of cohesiveness, were also the teams whose members have the most positive outcome expectancy. The results supported this prediction.


Prognosis after first myocardial infarction. Comparison of Q-wave and non-Q-wave myocardial infarction in the Framingham Heart Study. To compare the short- and long-term prognosis following a first Q-wave or non-Q-wave myocardial infarction. Cohort study with a mean follow-up period of 5.1 +/- 4.9 years. Population-based. Framingham (Mass) Heart Study subjects with an initial recognized myocardial infarction during a 17-year period were studied, including 227 men and 136 women with a mean age of 67.2 years. Seventy-seven percent of first infarctions were Q-wave infarctions and 23% were non-Q-wave infarctions. Reinfarction and death from coronary heart disease. During the follow-up period, subjects with non-Q-wave infarctions had a significantly higher rate of reinfarction than subjects in the Q-wave group (P = .02 for the entire follow-up). The 10-year reinfarction rates were 44.8% vs 27.4%. When analyzed separately by age and sex, differences in reinfarction rates were only noted in men and in those under the age of 65 years. There were no differences in coronary heart disease death rates based on Q-wave status, even when examined separately by age and sex. Multivariate analysis revealed a 1.8-fold higher risk of reinfarction in the non-Q-wave group (95% confidence interval, 1.1 to 3.1), and also demonstrated that baseline hypertension was an independent risk factor for predicting reinfarction (relative risk, 1.8; 95% confidence interval, 1.1 to 3.2). There were no differences in the rates of sudden death or all-cause mortality following the two types of myocardial infarction. Additionally, subjects with a first Q-wave infarction had a higher rate of subsequent congestive heart failure, while those with non-Q-wave infarctions had a significantly higher rate of coronary insufficiency (unstable angina with transient ST-T wave abnormalities). These results confirm and extend findings from prior studies that have identified patients with first non-Q-wave myocardial infarctions as potentially unstable, with greater subsequent morbidity and similar mortality to their counterparts with Q-wave infarctions.


The comparative retention of National Health Service Corps and other rural physicians. Results of a 9-year follow-up study. To contrast the retention of physicians serving National Health Service Corps (NHSC) Scholarship Program obligations in rural settings to that of non-NHSC physicians working in the same or similar practices, and to identify promising retention-enhancing strategies. Cohort study. Four hundred twelve primary care physicians initially identified during an earlier study as working in a national stratified random sample of 178 externally subsidized rural clinics in 1981. Thirty-six percent were serving obligations to the NHSC, nearly all through the NHSC's Scholarship Program. The NHSC and non-NHSC inception cohorts (those first coming to their 1981 [or "index"] practices from May 1979 through December 1981) were created from within the entire group for use in most analyses. In 1990, physicians were resurveyed to learn of their backgrounds, experiences in their index practices, and their subsequent career moves. By 1984 and in each year thereafter, fewer NHSC than non-NHSC physicians of the entire respondent cohort remained (1) in their index practices, (2) in their index communities, and (3) in practice in any rural county (P less than .001). In the inception cohort, fewer NHSC than non-NHSC physicians were retained within all three settings by the third year after their initial dates of employment (P less than or equal to .01). After 8 years of employment, group retention rates for NHSC and non-NHSC inception cohort physicians were 12% vs 39% in the index practice and 29% vs 52% in nonmetropolitan practice. Physicians in both NHSC and non-NHSC groups who left their index practices generally left rural practice altogether. When compared to non-NHSC physicians working in comparable rural settings, the retention of rural NHSC physicians is seen to be poor and only partially explained by fixed physician, practice, or community variables. Long-term retention of NHSC providers is now receiving much needed attention at the federal level.


Improved cholesterol-related knowledge and behavior and plasma cholesterol levels in adults during the 1980s. To determine whether cholesterol-related knowledge and behavior and plasma cholesterol levels were stable until the inception of large-scale national interventions in the middle to late 1980s, whether they subsequently improved, and whether these levels varied by subgroups. Data were collected from 4173 adults aged 25 through 74 years in the two control cities (San Luis Obispo and Modesto, Calif) of the Stanford Five-City Project. Five separate, community-based surveys were conducted in 1979-1980, 1981-1982, 1983-1984, 1985-1986, and 1989-1990. Cholesterol-related knowledge and behavior and plasma cholesterol levels improved (P = .0001) in both cities after the early 1980s. Those who were more educated, female, older, or nonsmokers had significantly higher knowledge and behavior scores, and those who were younger, more educated, or normotensive had significantly lower plasma cholesterol levels. Improvements in this population's cholesterol-related knowledge and behavior and plasma cholesterol levels began in 1985-1986, suggesting that the extensive cholesterol interventions that began in the middle 1980s in the United States created positive cholesterol-related changes at the community level.


[Antimicrobial activity of cefetamet against fresh clinical isolates of Branhamella catarrhalis]. Against strains of Branhamella catarrhalis which were separated from various RTIs (respiratory tract infections) in 1991 antimicrobial activities (MICs) of cefetamet (CFMT) were determined, and the following conclusions were obtained. 1. The MIC80 of CFMT against B. catarrhalis was 0.39 microgram/ml, which was higher than that of cefixime (CFIX) by one dilution or twofold, but was lower than that of cefpodoxime (CPDX) by two dilutions or fourfold and that of cefotiam (CTM) by three dilutions or eightfold. 2. The fact that all of the 50 strains tested were beta-lactamase producers appeared to indicate that CFMT was stable against BRO-1 and BRO-2 beta-lactamases produced by B. catarrhalis. 3. Blood concentrations of the test drug, CFMT, and control drugs upon normal single doses were calculated using pharmacokinetic parameters. Lengths of time periods during which drug concentrations stayed above their MICs against B. catarrhalis obtained in this study were determined for CFMT, CFIX, CPDX and CTM. They were, respectively, 12 hours, 12 hours, 6 hours, and 2 hours, thus CFMT appeared to remain above MIC for sufficiently long time for the treatment of RTIs which are affected by B. catarrhalis directly or indirectly.


[Effect of cefclidin and E1077, new cephalosporins, on the alcohol-metabolizing system in rats]. Administration of latamoxef and cefoperazone, reported to act like disulfiram in humans, caused a depression of mitochondrial low Km aldehyde dehydrogenase (low Km ALDH) activity in rats. In addition, a marked increase of blood acetaldehyde concentration was observed when rats were given alcohol orally at 18 hours after administration of these cephalosporins. However, mitochondrial low Km ALDH activity and blood acetaldehyde level were not altered by repeated administration of 300 mg and 1,000 mg of cefclidin (CFCL, E1040) or E1077 per kg. From these results, it was concluded that neither CFCLn or E1077 affected the alcohol metabolizing-system.


Tuberculosis and HIV infection in sub-Saharan Africa. To review the epidemiologic, clinical, and pathological characteristics and the public health implications of human immunodeficiency virus (HIV)-associated tuberculosis in sub-Saharan Africa. Published medical literature (English and French) and proceedings of international and African conferences on the acquired immunodeficiency syndrome (AIDS). Selection by the authors of articles most pertinent to HIV infection and tuberculosis in Africa and internationally. Direct reporting of quantitative data (eg, HIV seroprevalence levels) and of qualitative descriptions and conclusions from selected literature. High rates (20% to 67%) of HIV infection in patients with tuberculosis have been reported from East, West, Central, and Southern Africa. An increase in tuberculosis cases has been reported at the same time as the emergence of AIDS in several countries. Autopsies in Abidjan, Ivory Coast (Côte d'Ivoire), have shown tuberculosis as the most frequent opportunistic infection in patients dying of AIDS. Clinical differences in patients with tuberculosis who were HIV-positive and HIV-negative are reviewed, the most important being a greatly increased mortality rate in HIV-associated disease. Access to HIV testing is required for firm diagnosis, for clinical care and counseling, and for public health surveillance. The epidemiology of tuberculosis has been profoundly influenced by the epidemic of HIV infection in sub-Saharan Africa. Greatly increased human and material resources are required for this neglected problem in international health.


[Laboratory and clinical studies on panipenem/betamipron in the field of pediatrics]. Laboratory and clinical studies on panipenem/betamipron (PAPM/BP), a new carbapenem antibiotic, were carried out in the field of pediatrics and the following results were obtained. 1. The antibacterial activities of panipenam (PAPM) against clinically isolated organisms in our department were high overall. 2. After 30 minutes intravenous infusion of PAPM/BP at a dose of 10 mg/10 mg/kg in 1 and of 20 mg/20 mg/kg in 2 children, peak plasma levels of PAPM ranged from 33.21 to 75.66 micrograms/ml at the end of the infusion. The half-lives were 0.81 to 0.93 hours. The cumulative urinary recovery rates in the first 6 hours after the start of drip infusion ranged from 10.7 to 40.4%. 3. PAPM/BP was administered to 16 pediatric patients with various infections. The clinical and bacteriological efficacy rates were both 100%. 4. No side effects were observed. Abnormal laboratory test results were also mild; slight elevation of GOT, GOT/GPT and eosinophylia in 1 each and thrombocytosis in 2.


[Studies on panipenem/betamipron in the field of pediatrics]. Pharmacokinetic and clinical studies in pediatrics were performed on panipenem/betamipron (PAPM/BP), a combined drug of a carbapenem antibiotic (panipenem) and organic ion inhibitor (betamipron) at a weight ratio of 1:1. 1. Plasma levels and urinary excretion were studied when PAPM/BP, at 10 mg/10 mg/kg (4 cases) or 20 mg/20 mg/kg (5 cases), was administered using intravenous drip infusion in 30 minutes to 9 children (4-14 years old). The plasma PAPM level at the end of drip infusion was 30.75 +/- 4.98 micrograms/ml in the cases administered with 10 mg/10 mg/kg and 68.72 +/- 5.73 micrograms/ml in the cases administered with 20 mg/20 mg/kg. Drug concentrations then gradually decreased with half-lives of 1.08 +/- 0.09 hours and 0.98 +/- 0.02 hour, and reached 0.39 +/- 0.14 micrograms/ml and 0.62 +/- 0.06 micrograms/ml, respectively, after 5.5 hours. Plasma BP levels at the end of drip infusion was 18.93 +/- 3.75 micrograms/ml in the cases administered 10 mg/10 mg/kg and 37.09 +/- 2.68 micrograms/ml in the cases administered 20 mg/kg, and half-lives were 0.55 +/- 0.07 hour and 0.61 +/- 0.03 hour, respectively; the plasma BP level could not be determined in any cases after 5.5 hours. Mean urinary recovery rates of PAPM in the first 6 hours after the start of intravenous drip infusion were 33.0 +/- 6.1% in the cases administered 10 mg/10 mg/kg and 21.8 +/- 2.3% in the cases administered 20 mg/20 mg/kg and those of BP were 77.0 +/- 2.4% and 76.6 +/- 7.3%, respectively. 2. When PAPM/BP, was administered at 31.3 mg/31.3 mg/kg thought by intravenous drip infusion in 30 minutes to 1 case of purulent meningitis, PAPM levels were 0.76 micrograms/ml at the end of drip infusion but varied between 0.80 to 1.97 micrograms/ml 30 minutes after the end of drip infusion during 8 days of treatment. 3. PAPM/BP was administered to 43 cases, 47 diseases of bacterial infections in the domain of pediatrics to study its clinical efficacy, bacteriological efficacy and adverse reactions. Single doses were 5.2mg/5.2mg to 31.3 mg/31.3 mg/kg; frequencies of administration were 3 to 4 times a day, and durations of administration were 3 1/3 to 11 days; and total dosages ranged between 1.125 g/1.125 g and 11.0 g/11.0 g.(ABSTRACT TRUNCATED AT 400 WORDS)


[Evaluation of panipenem/betamipron in pediatric field]. Panipenem/betamipron (PAPM/BP), a mixture of a newly synthesized carbapenem antibiotic panipenem (PAPM) and N-benzoyl-beta-alanine, betamipron (BP), was evaluated for pharmacokinetics, in vivo and in vitro antimicrobial effect, and clinical efficacy in pediatric patients. Intravenous drip infusion of either 10 mg/10 mg/kg or 20 mg/20 mg/kg of PAPM/BP for 30 minutes resulted in maximum plasma concentrations of 36.6 micrograms/ml and 92.5 micrograms/ml, half lives (T 1/2 beta) of 1.17 hours and 0.88 hours, and urinary excretion until 6 hours of 29% and 17.7%, respectively. Antibacterial activities of PAPM against Gram-positive cocci and Gram-negative rods isolated from pediatric patients were equal to or slightly stronger than those of imipenem, ceftazidime, cefoperazone, and piperacillin. Clinical effects of PAPM/BP evaluated in 17 patients were as follows; excellent in 8 cases, good in 8 cases, and fair in 1 case. The overall efficacy rate was 94.1%. Elevations of GOT and/or GPT were observed in 2 patients and transient eosinophilia was observed in 1 patient.


[Clinical studies on panipenem/betamipron in pediatric field]. We have carried out clinical studies on panipenem/betamipron (PAPM/BP, CS-976). The results are summarized as follows. Treatment with PAPM/BP was made in 21 cases of pediatric bacterial infections including 2 cases of tonsillitis, 15 cases of pneumonia and 1 case each of bronchitis, scarlet fever, urinary tract infection and otitis media. Results obtained were excellent in 15 cases, good in 6 cases. No significant side effects due to the drug were observed in any cases.


[Studies on efficacy and safety of panipenem/betamipron against infections in pediatrics and on its movement to cerebrospinal fluid including cases of penicillin-resistant Streptococcus pneumoniae meningitis]. The efficacy and the safety of panipenem/betamipron (PAPM/BP), a new carbapenem antibiotic against infections in pediatrics were studied. The obtain results are summarized as follows. 1. The transfer of PAPM/BP to cerebrospinal fluid (CSF) was studied in 2 cases of purulent meningitis. The PAPM/BP levels in CSF in a dose 26.1 mg/kg peaked at 3.21 micrograms/ml on sampling 30 minutes after administration, followed by decreasing gradually with the improvement in clinical symptoms and came to 0.86 micrograms/ml on the 12th day (30 minutes after administration). 2. PAPM/BP at dose levels of 50 mg/kg to 69 mg/kg a day (daily doses of 104 mg/kg, 175 mg/kg 4 times a day for 2 cases of purulent meningitis) was administered by intravenous drip infusion 3 times daily for 4 to 15 days to 2 cases of purulent meningitis (including 1 case of penicillin-resistant Streptococcus penumoniae meningitis), 3 cases of pneumonia, 2 cases of phlegmon, 2 cases of periproctal abscess and 2 cases of urinary tract infections for a total of 11 cases. As results, all the cases showed good responses including 5 excellent and 6 good responses. Bacteriological efficacies in all of the 9 eligible cases were assessed as "eradicated". 3. As for the safety, an increase in the platelet count and slight evaluation of GOT and GPT were seen in 1 case as abnormal changes in the laboratory findings, although no side-effect was observed. 4. The results above show that PAPM/BP is useful for the treatment of general infections in pediatrics and that a daily dose of about 60 mg/kg given in 3 divided doses in effective enough.(ABSTRACT TRUNCATED AT 250 WORDS)


[Clinical evaluation of panipenem/betamipron in pediatrics]. Studies were carried out on the clinical efficacy of panipenem/betamipron (PAPM/BP) against bacterial infections. The results are summarized as follows: 1. PAPM/BP were administered to total 21 patients (7 cases of pneumonia, 1 case of bronchitis, 3 cases of cellulitis, 2 cases of purulent lymphadenitis, 2 cases of otitis media, 1 case of purulent parotitis, 1 case of sinusitis, 1 case of mastoiditis, 2 cases of urinary tract infection and 1 case of purulent meningitis) by drip intravenous injection. 2. Clinical responses of PAPM/BP were excellent in 12 cases, good in 7, poor in 1 and unknown in 1 case. The overall efficacy rate was 95.0%. 3. Concentration of PAPM in cerebrospinal fluid after 1 hour drip intravenous administration in 1 case of purulent meningitis were 6.84 micrograms/ml at the acute stage and 3.28 micrograms/ml at the recovering stage. 4. Neither side effects nor abnormal laboratory findings were observed except 1 case of increase of thrombocytosis out of 19 cases. 5. From the results, PAPM/BP was determined to be an efficacious and safe drug for the therapy of pediatric infection.


[Clinical studies on panipenem/betamipron in pediatrics]. Panipenem/betamipron (PAPM/BP) was given by 30 minutes drip infusion to 15 children with acute bacterial infections including 11 with acute pneumonia, 2 each with staphylococcal scalded skin syndrome and urinary tract infections. Good to excellent clinical responses were obtained in all of the 15 patients and bacterial eradications were obtained for all 12 strains identified in these cases. Urticaria considered to be drug related was observed in 1 patient. Slight elevations of GOT and GPT and eosinophilia were observed in 1 case each. From the above clinical results, it appears that PAPM/BP is a useful antibiotic for treatment of pediatric patients with various bacterial infections.


[Clinical studies of panipenem/betamipron in pediatrics]. We conducted clinical studies on panipenem/betamipron (PAPM/BP), a newly developed parenteral carbapenem antibiotic, for its clinical application in the field of pediatrics. 1. A clinical study was performed on 13 children with infections, including 6 with acute bronchopneumonia, 1 each with acute pharyngitis, acute bronchitis, sepsis, staphylococcal scalded skin syndrome, urinary tract infection, subcutaneous abscess and furuncle. PAPM/BP was administered by intravenous drip infusion. Doses varied from 12 to 27 mg/kg body weight were given t.i.d. or q.i.d. Lengths of treatment ranged from 4 to 25 days. Clinical efficacies were excellent in 3 and good in 9 cases, with an efficacy rate of 92%. 2. No adverse reactions were observed. In laboratory tests, elevations of GOT, GPT and urobilinogen were observed in 3 cases. It was concluded that PAPM/BP was a promising drug for the treatment of bacterial infections in children.


[Pharmacokinetics and clinical studies of panipenem/betamipron in the pediatric field]. Panipenem/betamipron (PAPM/BP) is a mixture of panipenem (PAPM), carbapenem antibiotic, and betamipron (BP), N-benzoyl-beta-alanine. The adverse reaction to PAPM of the kidney is reduced by the addition of BP to PAPM which inhibits the anion transport in the kidney tubules. We studied the pharmacokinetics and the clinical efficacies of PAPM/BP in children and we evaluated the antibacterial activities of PAPM by determining MIC values of PAPM in vitro against organisms isolated in our children's hospital from January to December, 1990. 1. Pharmacokinetics 10 mg/kg of PAPM/BP (10 mg PAPM/10 mg BP) was administered intravenously by drip infusion to 7 children. The mean blood concentration of PAPM was 14.8 micrograms/ml at the peak, and the mean half life was 0.9 hours in blood. PAPM was not detected in blood 3 hours after the time when the peak values were attained. 2. Clinical studies 10 mg/kg of PAPM/BP was administered intravenously 3 times a day to 18 cases including 15 of respiratory infections, 2 of otitis media and 1 of sepsis. The clinical efficacies of PAPM/BP were excellent or good in 17 out of the 18 cases. All causative organisms isolated in 5 cases, Methicillin-sensitive Staphylococcus aureus (MSSA) (1 case), Streptococcus pneumoniae (1), Haemophilus influenzae (2) and Branhamella catarrhalis (1) were eradicated in a few days upon the administrations of PAPM/BP. No adverse reactions due to PAPM/BP were observed, but a slight elevation of platelet counts in blood was observed in 1 case, which was normalized soon after the end of the treatment. 3. Antibacterial activities in vitro(ABSTRACT TRUNCATED AT 250 WORDS)


[Diagnosis and classification of leukemia]. Modern diagnosis and classification of leukemia are reviewed. The FAB (French, American, British) classification, introduced in the late 1970s has been the basis for most studies to date. During the 15 years since then, new categories such as M7 and M0 were added to the classification. The MIC proposal (morphology, immunology, cytogenetics) has been an important development which emerged from the knowledge about chromosomal changes and immunophenotyping. Improvement in diagnosis and classification will emerge from studies employing all the above techniques, including DNA analysis, in the 1990s.


[A new system of diagnosis and classification--malignant lymphoma]. Malignant lymphoma is usually divided into Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) according to biological characteristics. Malignant lymphoma in Japan has such the characteristics as few incidence of HD, which is usually favorable in prognosis, and high incidence of NHLs, which have further distinctive features of less incidence of favorable follicular B cell lymphoma and of higher incidence of unfavorable diffuse T cell lymphoma including adult T cell leukemia/lymphoma (ATLL) in comparison with those in western countries. As a recent trend of progress in lymphoma diagnosis an introduction of immunological and molecular biological techniques has given an impact to the previous classification of malignant lymphoma based mainly on morphological criteria. Nowadays, the classification of pathologic type in HD seems to be settled in Rye classification. On the other hand in NHL the LSG classification has been well fixed in Japan and Working Formulation originated in USA has been well used internationally after a historical confusion. In this article a present state of working classification in malignant lymphoma is briefly described and discussed.


[Usefulness of monoclonal antibodies for immunophenotyping in leukemia]. Clinically useful monoclonal antibodies, applied for immunophenotyping of leukemias, are reviewed. With a combination of 15 antibodies, including CD2, CD3, CD4, CD5, CD7, and CD8 for T cell marker analysis, CD10, CD19, CD20, surface immunoglobulins, and cytoplasmic mu chain for B cell marker analysis, CD13 and CD33 for myeloid marker analysis, and HLA-DR and CD25 for other marker analysis, acute lymphoblastic leukemias of T cell type, cALL type, pre-B cell type and B cell type, acute myeloid leukemias, acute unclassified leukemias and adult T cell leukemias could be clearly diagnosed by immunophenotyping of cell membrane molecules. By using additional CD11b, CD14, and CD15 monoclonal antibodies, subclassification of acute myeloid leukemia was partially possible.


[Immunologic phenotype of malignant lymphoma]. Recent advances in clinical research on surface marker analysis of malignant lymphoma cells are reviewed. Malignant lymphoma can be classified into T-cell malignancy or B-cell malignancy, using flow cytometry or immunohistochemical analysis. Based on recent results of immunophenotypic analysis and clinical data, a new clinicopathologic classification of lymphoid malignancy is proposed. T-cell malignancy bearing T-cell receptor of gamma delta-type is discussed. Other recent topics on malignant lymphoma, such as B-cell lymphoma of the pleural cavity developing from long-standing pyothorax, mediastinal large-B-cell lymphoma with sclerosis, HIV-related B-cell lymphoma, and EB-virus genome carrying B-cell lymphoma in ATL are also discussed.


[Immunophenotypic analysis in acute leukemia]. Acute leukemias have been classified on French-American-British (FAB) criteria depending on the morphocytochemical features of blasts. Immunophenotyping and clonal rearrangement analysis of lineage-associated genes can decide a frozen stage of the hematopoietic differentiation process in blasts from acute leukemias. B-lineage acute lymphoblastic leukemia (ALL) and T-lineage ALL are systematically classified according to the sequential expression of differentiation-associated antigens. In acute myelogenous leukemia (AML), several new entities are proposed: AML-M0 is an AML without cytologic maturation, in which the myeloid commitment should be demonstrated by myeloperoxidase-positive microgranule on immunohistochemical staining or electron-microscopy, or by positive reaction for CD13 or CD33 antigens. CD7-positive AML is considered to be one of immature subtypes of AML, rather than hybrid leukemia. Thus, immunological studies on blasts enable us to discriminate a subgroup of leukemias, which will perhaps contribute to the improvement of treatment approach.


[Immunophenotype of chronic lymphocytic leukemia and prolymphocytic leukemia]. The immunophenotype of chronic lymphocytic leukemia (CLL) and prolymphocytic leukemia (PLL) is described, mainly according to the FAB classification. CLL and related diseases have been recognized as highly variable diseases and their classification is still confusing. Is there true T-CLL? How should we deal with non-T, non-B-CLL such as NK-GLL? What about the patient with borderline manifestations? Is the concept "MLUS" appropriate? Further examination and more detailed analysis is required in order to clarify the disease entity of CLL and related disorders.


[Large granular lymphocytic leukemia].

[Large granular lymphocytic leukemia]. We investigated the surface markers, cell-function, clonality, and the association of IL-2 receptors and a second messenger of src family of tyrosine kinase p56lck in IL-2 signal transduction of the leukemic cells of 12 patients with large granular lymphocytic leukemia (LGL leukemia). The leukemic cells of 5 patients were CD3+ and 5 of them were CD3-. In three patients with CD3- leukemia examined, one showed karyotype abnormality of 46, XY, -10, +mar and the delta gene of TCR was rearranged in one patient. The TCR of the leukemic cells of a patient MH with CD3+, CD4 and CD8 (double positive marker: DP) recognised rabbit IgG presented by macrophages. The recognition was class II restricted. We examined the expression pattern of CD8 subunits and found that DP leukemic cells commonly expressed CD8 alpha+ beta-. These results suggested that DP leukemic cells were CD4+ T cells and expressed CD8 alpha secondarily. The p75 IL-2 receptors were detected, however, the modulation of p56lck in the process of IL-2 signal transduction were not found out. There was no association between p75 and p56lck when leukemic LGL cells proliferated on stimulation with IL-2.


[Immunophenotyping analysis of acute undifferentiated leukemia]. The diagnosis of acute undifferentiated leukemia (AUL) is made when the leukemic cells do not have cytologic or cytochemical features of myeloid cells, and do not express myeloid antigens (CD13, CD14, CD33, CD41 etc.) or lymphoid antigens (CD2, CD3, CD19, CD20, Sm Ig etc.). Most of these cells are reported to be positive for CD7, CD34, TdT and HLA-DR, either alone or in combination. Cell lineage can be suspected in most AUL cells by genotypic analysis, phenotypic analysis after culturing with TPA, or peroxidase activity by ultrastructural or immunohistochemical analysis, which indicates the heterogeneity of AUL. The patients with AUL appear to have a poor prognosis with conventional chemotherapy.


[Bi-phenotypic leukemia and hybrid leukemia]. Phenotypes of leukemic cells can be determined through dual staining with pairs of FITC-labeled and PE-labeled monoclonal antibodies using a laser flow cytometer. Hybrid acute leukemia (HAL) was diagnosed when leukemic cells expressed 2 or more lymphoid markers and at least one myeloid maker simultaneously. Based on this criteria, forty out of 111 cases with untreated acute leukemia were diagnosed as HAL, 16 of 41 (39%) patients with acute lymphoblastic leukemia and 15 of 70 (21%) patients with acute non-lymphocytic leukemia were diagnosed as having HAL. We classified these HAL cells into 4 types by the following items. Type I; leukemic cells expressed only CD33 and 2 or more B-cell antigens, type II; only CD33 and 2 or more T-cell antigens, type III; CD13 and/or CD33 and 2 or more B-cell antigens, and type IV; CD13 and/or CD33 and 2 or more T-cell antigens. There were 7 type I cases and 16 type III cases. The leukemic cells of these two types were thought to be ALL cells expressing one or several myeloid lineage-associated antigens. There were 4 type II cases and 10 type IV cases. Type II and IV were regarded as lymphoid-lineage associated antigens positive AMLs. At least in adults, the expression of myeloid-associated antigens of ALL cells seems to identify a high risk group of ALL patients with a poorer response to standard ALL therapy.


[Immunogenotypes and surface marker analysis in Ph1 positive chronic myelogenous leukemia in the blastic crisis]. Immunophenotypic and immunogenotypic changes in 23 patients with Ph positive chronic myelogenous leukemia in blast crisis were determined using a panel of monoclonal antibodies and gene probes. According to the immunophenotypes, 9 patients were considered to be in lymphoid blast crisis, including 7 patients with lymphoblastic crisis and 2 patients with lymphoid/myeloid mixed blast crisis. Leukemia cells from the remaining 14 patients showed myeloid phenotypes and 10 of these had platelet-associated antigens. Rearrangement of the immunoglobulin (Ig) gene was observed in all the 9 patients with lymphoid blast crisis, and Ig gene rearrangement was associated with the expression of CD19 antigen. Two patients with myeloid blast crisis showed rearrangements of T-cell-receptor gene, but, dissociation between phenotypes and genotypes was not frequently observed in patients with blast crisis.


[Review: progress of cytogenetics in hematopoietic malignancies]. In 1960, Nowell and Hungerford found, for the first time, a minute chromosome at the metaphase in chronic myelocytic leukemia (CML) cells, which was called Philadelphia chromosome (9; 22 translocation) later. Ph1 chromosome was considered to be specific for the disease and was frequently used as an important marker for the definite diagnosis. In 1970s banding techniques revealed some other specific chromosome abnormalities, like 8; 14, 8; 21, and 15; 17 translocations, for acute leukemias. In 1980s, molecular-biology techniques were applied in the fields of leukemia research. As a result, many break point cluster regions were discovered in relation to the immunoglobulin chain genes and T cell receptor genes (Table 2). In this review, the specificity of chromosome abnormalities as well as genetic changes in types of leukemia is discussed.


[Ph1-positive leukemia: cytogenetic outline and prognosis]. Ph1-positive leukemias consist of acute leukemia (Ph1 AL) and CML. Cytogenetically, Ph1 AL is often associated with +6, -7, +8, +21, or +Ph1. CML is predominantly accompanied by +Ph1, +8, i (17q), +19 in myeloid crisis and +Ph1, +8, +21 in lymphoid crisis. Thus, i(17q) seems specific for myeloid crisis of CML. Ph1 constricts ABL/BCR within M-BCR in CML and in one half of the adult Ph1 AL. BCR breaks upstream to M-BCR in the other half of adult AL and in most of childhood AL. However, the breakpoint does not affect clinical and hematological features in AL. Consequently, there seems to be two types of Ph1 leukemia; one is AL representing m-BCR rearrangement and the other is CML and Ph1 AL showing M-BCR rearrangement.


[Correlation between karyotype and prognosis in adult lymphocytic leukemia]. Correlation between karyotype and prognosis in adults with lymphocytic leukemia are reviewed. Concerning acute lymphoblastic leukemia (ALL), patients with normal karyotype or with hyperdiploidy (greater than 50 chromosomes) show good prognosis. On the other hand, patients with structural abnormalities, such as t(8;14), t(4;11), t(11;14) or with hyperdiploidy (47 to 50 chromosomes), show poor prognosis. Above findings indicate that karyotype is a significant prognostic factor in adults as well as in children with ALL. Karyotype must be considered in designing clinical trials to improve the prognosis in patients with lymphocytic leukemia, especially in ALL.


[Chromosome abnormalities and prognosis in childhood acute leukemia]. The chromosomal classification system of childhood acute leukemia according to ploidy or structural chromosomal changes of leukemia cells is clinically useful for the prediction of the treatment outcome. While patients with hyperdiploid (greater than 50 chromosomes) ALL enjoy the best prognosis, those with hypodiploid or pseudodiploid ALL are expected to have unfavorable clinical outcome. Among various translocations or deletions in ALL, t(4;11), t(9;22) and t(8;14) predict the worst clinical outcome. AML patients with t(8;21) or inv (16) have better 50% survival than those with other chromosome changes or normal diploidy. Chromosome analysis of leukemia cells should be included in the diagnostic workup of childhood acute leukemia.


[Chromosome abnormalities and prognosis in non-Hodgkin's lymphoma]. Clonal chromosome abnormalities are found in most patients with non-Hodgkin's lymphoma. The role of the chromosome abnormalities in predicting the prognosis of lymphoma patients has not been fully clarified, because of different histological classifications being used in different areas and the complexity of the chromosome abnormalities often found in lymphoma. Recent studies have shown distinct correlation of rather rare abnormalities with specific histologic and immunologic phenotypes and prognoses. Many chromosome abnormalities seem to specifically correlate with these parameters of non-Hodgkin's lymphomas, as seen in leukemias. The chromosome data also seem to support the histological observations that some lymphomas may show uneven geographical distributions.


[Applications of FISH to chromosome analysis in hematological malignancies]. In situ hybridization provides a direct method to localize DNA sequences on metaphase chromosomes. Initially, nucleic acid probes were labeled isotopically and detected by autoradiography. Recently, fluorescent in situ hybridization (FISH) have been developed. FISH has resolved several problems, such as radiolysis of probes, long exposure times, high background noise, and unclear localization of silver-grains visualized by autoradiography. In addition, FISH allows us to study numerical and structural chromosome aberrations using chromosome-specific DNA probes, not only in metaphase chromosome, but also in interphase nuclei. Therefore, FISH can be applied to a broad spectrum of biological and clinical problems in hematological malignancies.


[Immune gene rearrangement as markers of hematological malignancies]. Molecular genetical analysis using Ig and TCR genes has been applied for hematological malignancies. In acute leukemias, this method is used to clarify the cellular lineage, and in malignant lymphomas to detect the monoclonal component. In this article, we describe the mechanism of immune gene rearrangement and the present status and future directions of immunogenetic analysis of leukemias and lymphomas.


[Phenotypic and genotypic analysis of acute leukemia--current status of lineage specific classification]. More detailed identification and understanding of the heterogeneity of leukemias using a broad panel of markers seems to be essential for the successful design of more sophisticated and effective treatments. Based on the FAB system, immunological phenotypes using a panel of monoclonal antibodies, and rearrangements of immunoglobulin and T-cell receptor genes, acute leukemia can be divided into six subtypes such as B-lineage, T-lineage, AML, NK-lineage, AUL and mixed lineage leukemia. The definition of B-lineage and T-lineage cells, a new classification for mixed lineage leukemia, incidence of dual rearrangements and their clinical significance are discussed.


[PCR analysis of T cell antigen receptor genes for human lymphoid malignancies]. T-cell receptor genes encode proteins related to their specific recognition structures. As is true for immunoglobulins, T-cell receptor genes consist of separate germ line segments that encode the variable and constant regions of the chain. The variable region genes are assembled from two or three separate germ line segments that somatically rearrange during differentiation. The diversity in the variable region of T-cell receptors results from germ line diversity of V, (D), and J sequences, combinatorial joining of the segments, junctional diversity leading to codon changes at the end of gene segments, N-region diversity arising from addition of template independent nucleotides at junction. Each T-cell clone has its own unique structure of the variable region, and in T-cell malignancies, this variable region is retained after malignant transformation. This enables the nucleotide sequences of the variable region genes to be used as a molecular marker for the malignant clone. By amplification of these sequences of variable genes, it is now possible to study the pathological features of the leukemic clones in vivo, as well as to follow the patients for minimal residual disease.


[Chromosomal translocations involving immunoglobulin or T-cell receptor genes in lymphoid malignancy]. Tumor-specific chromosomal translocations in lymphoid malignancy frequently involve the fusion of immunoglobulin (Ig) or T-cell receptor (TCR) gene loci with oncogenes, thereby activating the latter to contribute to the malignant phenotype. The preponderance of chromosomal translocations involving Ig or TCR genes suggests that the enzymatic machinery, the lymphocyte recombinase, is used in the formation of abnormal chromosomes. However, the role of ectopic recombination signals for recombinase in chromosomal translocations has been controversial. In this paper, molecular studies of genes adjacent to the breakpoints of these chromosomal translocation are reviewed, and the mechanism of chromosomal translocation and clinical characteristics of the tumor with specific chromosomal translocation are discussed.


[Activation of protooncogenes by point mutations in hematological malignancies]. The activation of protooncogenes (ras, fms and myc genes) by point mutations in hematological malignancies are described in this review. Ras mutations are found in a variety of human malignancies at codon 12, 13, and 61. Generally, N-ras mutations are frequent in hematological malignancies. Fms mutation at codon 301 and 969, which are seen in 10 to 20% cases of AML and MDS, increase tyrosine kinase activity of the fms products. Ras and fms mutations are postulated to influence leukemogenesis at rather early stages. Burkitt lymphomas are characterized by specific chromosomal translocations between c-myc gene and one of the immunoglobulin genes. Furthermore, mutations in the 3' border of the exon 1 of c-myc are frequent, and may play an additional role in pathogenesis of Burkitt lymphoma.


[Activation of oncogenes by chromosomal translocation in hematopoietic tumors]. Specific chromosomal translocations are one of the cytogenetic characteristics of hematopoietic malignancies. The application of molecular biological techniques has led to important insight into the molecular consequences, accompanied by the chromosomal translocations. Specific oncogenes and related genes activated by the translocations have been identified at the translocation breakpoints. Translocated oncogenes are deregulated by their juxtaposition to enhancers of Ig or TCR genes, resulting in their overexpressions in some lymphoid tumors. Alternatively, they are activated by gene fusion, resulting in a chimeric oncoprotein, as seen in Ph1-positive leukemia. The molecular consequences by chromosomal translocation in hematopoietic malignancies is focused here.


[Abnormalities of the retinoblastoma susceptibility gene (RB) in hematologic malignancies]. The retinoblastoma susceptibility gene (RB) is expressed in all lineages of normal hematopoietic cells and plays an important role in controlling cell cycle progression at G1/S. Abnormalities of the RB gene may, therefore, predispose to the development of hematologic malignancies. DNA rearrangement was reported to be present in 1.5-12.1% of cases with primary leukemias and the absence of the RB gene product was also observed in 6.3-23.2%. The abnormalities were frequently observed in blastic crisis of CML, especially of the megakaryoblastic phenotype, AML with monocytic differentiation and Ph1-positive leukemias. These results indicate that abnormalities of RB are relatively common in hematologic malignancies and loss of RB function may contribute to the altered growth of these cells.


[Mutations of the p53 gene in hematologic neoplasms]. The p53 gene encodes a nuclear phosphoprotein and is now considered as a tumor suppressor gene. Mutations of the p53 gene have frequently been observed in several types of solid tumors and are believed to be implicated in the development of these tumors. Recent studies have shown that the p53 gene is altered in chronic myelogenous leukemia (CML) in blast crisis. In CML, alterations of the p53 gene may play an important role in the development of blast crisis. More recently, p53 mutations have been reported in other types of hematologic neoplasms, such as acute leukemia, adult T-cell leukemia, and malignant lymphoma. These observations suggest that inactivation of the p53 gene is involved in the tumorigenesis of various types of hematologic neoplasms.


[DCC gene and leukemia].

[DCC gene and leukemia]. The deleted in colorectal carcinomas (DCC) gene, located in human chromosome band 18q21, was identified as a potential tumor suppressor gene by Fearon et al. in 1990. The DCC gene encodes a protein which is highly similar to neural cell adhesion molecules and other related cell surface glycoproteins. In colorectal carcinoma, the expression of the DCC gene is reduced or absent in 88% of cases. We examined the expression of the DCC gene using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Its expression was reduced or absent in some leukemias. Our findings suggest the possibility that this gene may play a role in leukemogenesis.


[Overexpression of PRAD1 gene in B-cell malignancy with t(11;14)(q13;q32) translocation]. PRAD1 (parathyroid adenoma 1) gene at chromosome 11q13 has been cloned from parathyroid adenomas as a putative oncogene, activated by translocation with the parathyroid hormone gene. 4.5 kb and 1.7 kb mRNA are transcribed and both have the same open reading frame of 885 bp encoding 34 kd protein of a cyclin gene family, cyclin D1. Recently, overexpression of PRAD1 gene has been reported to be correlated closely with the rearrangement of bcl-1 locus, particularly in centrocytic lymphoma. In our study, overexpression of PRAD1 gene was shown in five B cell lines with t(11;14)(q13;q32) including one centrocytic lymphoma line and 4 myeloma lines, when compared with other hematopoietic cell lines without translocation. One of the cell lines, SP-49, demonstrated a truncated mRNA of 3.4 kb, in addition to 1.7 kb of normal size. Southern blot analysis demonstrated a rearrangement with PRAD1 cDNA probe, suggesting that the gene is altered in this particular cell line. By cloning analysis, we confirmed that 1.8 kb deletion in 3' region of PRAD1 gene eliminating the destabilizing signal of PRAD1 mRNA, gave rise to the aberrant mRNA of 3.4 kb. These findings suggest that PRAD1 gene is most likely the candidate oncogene for bcl-1 activated by t(11; 14)(q13;q32) translocation. The gene alteration found in one cell line, SP-49, might also play an important role for deregulation of the gene.


[Structure and function of oligosaccharide chains of the envelope glycoprotein gp120 of human immunodeficiency virus type 1]. The surface of the human immunodeficiency virus (HIV-1), a causative agent for acquired immunodeficiency syndrome (AIDS), is covered with the major envelope glycoprotein gp120, of which the carbohydrate moiety accounted for 50% of the molecular mass. There is evidence that glycosylation of gp120 is prerequisite to the various stages of HIV infection. The oligosaccharide structures of gp120 have been determined using recombinant gp120 of HIV-1 (IIIB) produced in chinese hamster ovary cells and virus-derived gp120 isolated from H9 lymphocytes chronically infected with HIV-1 (IIIB). Three oligosaccharides have been suggested to be involved in the HIV-infection process. Occurrence of infection process which is independent of CD4 recognition and mediated by gp120 oligosaccharides, mannose-binding protein, and complement system has been suggested.


[Recent advances in EDRF research].

[Recent advances in EDRF research]. We have developed a new bioassay system for endothelium-derived relaxing factor (EDRF), using endothelial cells in culture. Bovine pulmonary arterial endothelial cells (BPAE) were grown on microcarrier beads and superfused in a cell column. The effluent was applied to bioassay tissues and biological and the chemical characters of EDRF were examined and compared with those of nitric oxide (NO). EDRF was vascular selective and negatively charged, whereas NO was a general relaxant and not charged. The hypothesis that EDRF is identical to NO is discussed. We also examined the interaction of some EDRF agonists with endothelial cells and biological characteristics of the agonists were discussed.


Expression of O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea resistance of human brain tumors. Northern blot analysis with O6-methylguanine-DNA methyltransferase (MGMT) cDNA as a probe was used to analyze the MGMT activity regulating drug resistance of human cells to chloroethylnitrosoureas (CENUs). By this method, the expression levels of MGMT mRNA in six human glioma cell lines and 12 human brain tumor tissues from surgical specimens were determined. These MGMT mRNA levels were compared with the SD10 values of the tumor cells, estimated by cell survival assay, which indicated their resistance to the anticancer drug, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). Human brain tumors that were highly resistant to ACNU, such as glioblastoma Gbl1 and metastatic brain tumor Col1 with SD10 values (microM) of above 100, expressed markedly increased amounts of 0.95 kb MGMT mRNA. In contrast, tumor cells such as U-87MG, U-251MG, U-343MG, U-373MG and SF-126 with SD10 values of under 14 indicating low resistance to ACNU scarcely synthesized any MGMT mRNA. These results indicated that the level of expression of MGMT mRNA in human brain tumors determined by Northern blot analysis truly reflects their cellular resistance to ACNU. Thus the Northern method with MGMT cDNA probe reported here is a practical and reliable method for estimation of cellular resistance to CENUs such as ACNU and for screening the chemotherapeutic response to CENUs of human brain tumors.


Three-dimensional computed tomography image of small pulmonary lesions. To evaluate diagnostic imaging of small lesions in the peripheral lung, three-dimensional (3D) reconstruction CT images were used to study 36 cases (including which had been 19 resected) of pulmonary nodules less than 3 cm in diameter. Rapid scanning was employed in 17 cases and helical scanning (in which the X-ray tube rotates continuously with simultaneous table sliding) in 19 cases. The rapid scanning could not be evaluated in two cases because respiratory motion resulted in discontinuous image data. We were able to evaluate 34 cases. The addition of 3D images to the conventional images made it possible for us to obtain useful information on visualizing relations between nodules and vascular structures in 32% (11/34) of the cases. The 3D images enabled us to visualize the lesions and pulmonary vessels three-dimensionally. It was, however, difficult to evaluate lesion shape since we were able to obtain useful information in only 3% (1/34) of the cases. In helical scanning, data are acquired from an extensive area within a short time, providing consecutive axial-slice images remain free of any respiratory artifacts. In this regard, helical scanning is suitable for generating 3D images. A diagnostic procedure based on 3D lesion analysis should be developed and standardized.


Phase I study of YK-176 (2'-deoxycoformycin) in patients with adult T-cell leukemia-lymphoma. The DCF Study Group. YK-176 is a newly isolated 2'-deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase, produced by Aspergillus nidulans. In a cooperative phase I study, YK-176 was administered to 22 patients, comprising 18 with adult T-cell leukemia-lymphoma (ATL), two with cutaneous T-cell lymphoma (CTCL), one with lymphoblastic lymphoma of T-cell type and one with carcinoma of the uterine cervix. Doses of YK-176 ranged from 3.0 to 9.0 mg/m2 and were given intravenously for three consecutive days. General malaise, anorexia, nausea, vomiting and low grade fever were frequently encountered, but were transient and not dose-related. At all dose levels hematological toxicities were mild. Two of seven patients receiving 7.0 mg/m2 for three consecutive days developed hepatocellular enzyme elevations (grade 2) and one patient, proteinuria (grade 2). One of two patients given 9.0 mg/m2 for three consecutive days manifested a life-threatening (grade 4) disturbance of consciousness and dyspnea, presumably ascribable to the drug-related toxicity of YK-176. The results suggest that 7.0 mg/m2 i.v. for three consecutive days is the maximum acceptable dose of YK-176. Central nervous system, pulmonary and possibly renal toxicities appeared to be dose-limiting. Out of the 20 patients evaluable for therapeutic response, partial remissions were observed in four, three with ATL and one with CTCL, who received less than 7.0 mg/m2 for three consecutive days. We conclude that YK-176 is an active agent against ATL at doses that may not be associated with prohibitive toxicity. A starting dose of 5.0 mg/m2 for three consecutive days is recommended for further phase II studies on ATL.


Phase II evaluation of cisplatin and 5-fluorouracil in advanced squamous cell carcinoma of the esophagus: a Japanese Esophageal Oncology Group Trial. Thirty-nine patients with advanced measurable squamous cell carcinomas were treated with two or more courses of 70 mg cisplatin/m2 on day 1 and 700 mg infused 5-fluorouracil/m2 on days 1-5 every 21 days. The overall response rate was 35.9 (95% confidence limits, 24.8-55.1%). Responses were seen in primary sites in the esophagus of five patients, in the lung of seven, the liver of one and the mediastinal lymph nodes of one. The average response duration was 3.5 (range 1-12) mo for patients who achieved partial response. The average survival time after the first administration was 9.5 mo for patients who responded to the treatment whereas, for those who did not, it was 5.6 mo. The major form of toxicity was myelosuppression and there were six patients with grade 3 toxicity and one with grade 4. The present study was designed to evaluate the effectiveness of combined cisplatin and 5-fluorouracil for advanced squamous cell carcinoma, and the results showed that it had a reasonable effect and might possibly be used as a postoperative chemotherapy because of its mild side effects.


Long-term clinical study on luteinising hormone-releasing hormone agonist depot formulation in the treatment of stage D prostatic cancer. The TAP-144-SR Study Group. The results of long-term administration of leuprorelin acetate depot (TAP-144-SR), a sustained-release preparation of luteinising hormone-releasing hormone (LH-RH) agonist, to 40 stage D prostate cancer patients who had had no previous hormonal therapy, have been analyzed. The drug was administered by subcutaneous injection once every four weeks at a dose of 3.75 or 7.5 mg. The mean duration of treatment was 480 (141-1,266) days, and treatment continued for more than one year for 60% of the 40 patients. Objective response was evaluated in accordance with Japanese Response Criteria. The best objective response in the overall evaluation was a partial response in 68.6% of the total patient group (3.75 mg, 64.3%; 7.5 mg, 71.4%). During the administration period, 21 patients were diagnosed as having progressive disease (PD), and the mean time to onset of PD was 308 days. The earliest diagnoses of PD were made on bone metastases (17 cases) and prostatic acid phosphatase levels (12 cases). The survival rate, as determined by Kaplan-Meier's method, was 81.2% (3.75 mg, 80.5%; 7.5 mg, 81.3%) two years after starting the regimen and 66.0% (3.75 mg, 65.1%; 7.5 mg, 66.0%) after three years. There was no dose dependency between either of the two drug doses, which we adopted as functions of the objective response rate, and patients' survival. Serum levels of testosterone, LH and follicle-stimulating hormone (FSH) were monitored from the first-dose until the 148th week of administration. In all patients receiving the drug as scheduled, the testosterone level was maintained at less than 1 ng/ml, and the LH and FSH levels were maintained at levels lower than their respective upper normal limits. There were no particular adverse reactions specific to the long-term administration subsequent to the foregoing 12-week administration study. In conclusion, LH-RH analogue treatment is thought to be a long-lasting, effective, palliative measure for patients with previously-untreated, advanced, prostatic cancer.


Feasibility study on quality-of-life questionnaires for patients with advanced lung cancer. In order to evaluate quality-of-life (QOL) questionnaires, we studied the reliability and validity of the self-rating forms: A, comprising 16 questions; B, comprising 39 questions (Table II and III), in a special study supported by the Ministry of Health and Welfare. Sixty-four patients with inoperable lung cancer and 50 patients with chronic, non-malignant disease were entered into the study. Form A was translated and modified to Japanese culture from Schipper's Functional Living Index-Cancer, and Form B was of our own design. The internal consistency of the forms was examined by Cronbach's alpha coefficient. Both forms except for aspects of physical and social concern on form B, had alpha values greater than 0.65. A concurrent validity with performance status was shown over all aspects of concern (Pearson's correlation, r greater than 0.27, P less than 0.05) except for social (forms A and B) and psychological (form B). Analysis of variance of the QOL scores revealed significantly lower scores for the cancer patients than for the non-malignant-disease patients, except for the social aspect of concern (P less than 0.001). Factor analysis, using the principal component method, identified four major factors to account for 60 and 42% of the total variance on forms A and B, respectively. The factors were: patient's mood, anxiety over disease and treatment, relationships with other people, physical capability. In conclusion, this has been the first feasible study to show that QOL forms from the West could be used in Japan with some modifications. We are at present studying successive changes in QOL for advanced lung cancer patients in clinical trials.


Primary lung cancer complicated with pneumothorax. Eighteen lung cancer patients with a pneumothorax complication were studied. Pneumothorax appears rarely in lung cancer patients, having been found in 18 out of 5567 (0.32%) at our hospital over a period of ten years. Of the 18 patients, eight had adenocarcinoma, seven epidermoid carcinoma and three alveolar cell carcinoma. Unlike those of previous reports, our results showed pneumothorax not to be found exclusively on the same side as the lung cancer. It was contralateral in five cases. Pneumothorax was the initial manifestation of lung cancer in three cases and occurred as a complication in another 15. Of these 15 patients, 11 were described as developing pneumothorax between one and twelve months after completion of radiation therapy for lung cancer. Another two patients developed pneumothorax following cytotoxic chemotherapy. Pneumothorax occurred prior to any treatment for lung cancer in the remaining two patients. The factors contributing to pneumothorax in the lung cancer patients were the rupture of the necrotic neoplastic tissue into the pleural cavity, the rupture of a subpleural bleb or the formation of interstitial air due to partial bronchial obstruction by the tumor, complications arising from radiation therapy and cytotoxic chemotherapy, or any combination of such factors. Pneumothorax was an ominous sign for the lung cancer patients. Most (12/14) died within six months of the onset of pneumothorax.


High endothelial venules in extranodal lymphomas with emphasis on their prognostic significance. In the present study, the frequency of occurrence of high endothelial venules (HEVs) in extranodal lymphomas at various sites was examined, and its prognostic implication subsequently estimated. The presence of HEVs was tested for by an immunohistochemical method using the monoclonal antibody, HECA-452, known to be specific for HEVs. The number of cases at each extranodal site were 13 in the thyroid, three in the salivary gland, 15 in the gastrointestinal(GI) tract, 20 in the brain, six in the lung, 12 in the pleura, nine in the testis, 10 in the bone, 19 in the skin and seven in the breast. Histologically, 26 of the 114 cases (23%) were of high grade malignancy according to the Working Formulation. Immunohistochemistry revealed 89 cases (78%) to be of B-cell type, eight (7%) T-cell type and 17 (15%) others. HEVs were observed frequently among the cases of lymphoma of the salivary gland (100%), thyroid (77%), GI tract (53%), lung (50%) and breast (43%). Evident were a preponderance of female patients (P less than 0.02) and those with histologically favorable lymphoma types (P less than 0.001), and a more favorable prognosis (P less than 0.001) for cases with HEVs over those without. There were no significant differences in age and immunologic type distributions between cases with and without HEVs. The findings suggest that the presence of HEVs could be used as an indicator for a favorable prognosis in cases of extranodal lymphoma.


Analysis of 13 postmortem examinations with regard to the effect of chemotherapy on neuroblastoma patients. Thirteen postmortem examinations on neuroblastoma patients were reviewed clinicopathologically. Commonly found metastatic sites were bone, liver and lung. The introduction of massive doses of cyclophosphamide and cisplatinum into chemotherapy protocol markedly reduced the tumor burden: no tumor focus in liver or lung, small tumor burden in bone. Lymph node metastasis remained, however, and was refractory to the chemotherapy. The intensive chemotherapy caused severe side effects: interstitial or bacterial pneumonia and intrathecal bleeding, which sometimes proved fatal. Such side effects must, therefore, be carefully controlled in order to maximize the survival rate.


Synchronous cancer of squamous cell carcinoma of the lung and lymphocytic leukemia (B-cell type): a case report. The patient was an 82-year-old male who consulted us over a lymph node enlargement in the right side of his neck. The WBC was 23,700/microliters (lymphocytes 80%), and a diagnosis of chronic lymphocytic leukemia (CLL) was given based on the findings of a bone marrow puncture and lymph node biopsy. During the observation period, abnormal shadows accompanied by cavitation appeared in the right lower lung field, and CT-guided percutaneous biopsy findings indicated a squamous cell carcinoma of the lung. No metastatic lesions were noted. No aggressive surgical or internal treatments were performed in consideration of the patient's age. The patient was admitted again and died of respiratory failure due to lung carcinoma. Reports of CLL and other malignant tumors arising in one patient are rare in Japan, and those of their occurring concurrently are even rarer. In the present report, a case of synchronous cancer of squamous cell carcinoma of the lung and CLL is described, together with a review of the literature.


Successful aggressive treatment against multiple intra-abdominal metastases from renal cell carcinoma 18 years after nephrectomy. The management of late metastases from renal cell carcinoma is often difficult because of multiple organ involvement. We report a case of multiple metastases from renal cell carcinoma in the duodenum, pancreas, intestine, falciform ligament and liver, 18 years after nephrectomy. The patient underwent a total pancreatectomy following a gastroduodenal arterial embolization to control duodenal bleeding, a resection of the ileum and falciform ligament at a second laparotomy and repeated hepatic arterial embolizations to control the growth of liver metastases. Aggressive treatment should be undertaken in cases of late recurrence of renal cell carcinoma after nephrectomy because of the possibly slow-growing biological character of the tumor.


[Psychosomatic aspects of stress].

[Psychosomatic aspects of stress]. We established the Bromocriptine test for the dopaminergic function of the hypothalamopituitary gland. The secretion patterns of plasma GH and PRL to 2.5 mg Bromocriptine, a dopamine receptor agonist, were classified into two types; a response type and a non-response type. The former showed an increase in plasma GH levels and suppression of PRL secretion; the latter showed no change in GH after Bromocriptine administration. The response type cases corresponded to psychosocial stress by neurotic and maladaptive behavior. The non-response type cases corresponded to psychosocial stress by alexithymic and over adaptive behaviors. Case Presentation 1. Essential Hypertension: a. 56-year old male, response type, blood pressure elevated by stress in daily life. Psychosomatic treatment: advice about blood pressure measurement at his home, brief psychotherapy and drug therapy. b. 53-year-old male, non-response type, type A behavior. Psychosomatic treatment: advice to increase awareness of body-mind relationships of his disorder, self-control training and drug therapy. 2. Gastric ulcers: a. 40-year-old male, response type, CMI IV region (Neurotic tendencies). Psychosomatic treatment: autogenic training and drug therapy. b. 28-year-old male, non-response type, high JAS scores(Over adaptative behavior). Psychosomatic treatment: advice to increase awareness of body-mind relationships of occurrence of his ulcers, to induce change in his perceptions of way of life, to encourage taking rest. 3. Technostress syndrome: a. 23-year-old female, response type, technoanxiety. Psychosomatic treatment: advice to make her take rest, and change in arrangements at her working place. b. 27-year-old male, non-response type, technodependent. Psychosomatic treatment: Fasting therapy. This therapy changed the non-response pattern to normal.


[Stress and gastroenterology: an immunoneuroendocrinological approach]. The numerous interactions between the immune and neuroendocrine systems are being studied in a rapidly expanding interdisciplinary field called "immunoneuroendocrinology" or "psychoneuroimmunology". We have recently reported that interleukin-1 (IL-1), a cytokine mainly produced by activated monocytes and macrophages, has various central nervous system (CNS) actions besides its immunological activities. Among these activities, for example, IL-1 activates the hypothalamic-pituitary-adrenal axis, a main hormonal response to stress, by stimulating the release of corticotropin-releasing factor from the hypothalamus. IL-1 also acts centrally in the brain to decrease food intake in a dose-related manner, suggesting that anorexia, a typical clinical manifestation frequently observed in acute infectious diseases, may be caused by IL-1, a cytokine that is released under such conditions as acute infection. Furthermore, we have very recently found that IL-1 has potent antisecretory and anti-ulcer actions mediated by the CNS. Based upon these results, we have proposed that there may be an "immune-brain-gut" axis, in which IL-1 acts as an afferent signal and gastric functions as efferent one. At this symposium, by presenting some of our data on the CNS effects of IL-1, we report the potential of these immunoneuroendocrinological studies for a better understanding of the pathophysiology of stress-induced gastrointestinal disorders.


[Considerations in clinical testing--implications of urinary 17-ketosteroid sulfate and 17-hydroxycorticosteroid measurements for stress research]. With the recent increase in number of patients with stress, the search is continuing for health-indicators that can be used to determine stress reliably and quickly. These indicators rely on subjective evaluations, and the establishment of an objective method of determining stress is necessary. We have developed a method involving the determination of the 17-Ketosteroid sulfates (17-KS-S) which has proved valuable for stress research. By simultaneous measurement of both 17-KS-S and 17-hydroxycorticosteroids (17-OHCS), which have different biological significance, and which originate from the adrenal gland, an organic Yin-Yang evaluation of the adrenocortical function can be made. The two values (mainly the 17-KS-S values) are closely correlated to the biological stress state as evaluated from psychosocial factors. This method should prove to be valuable in the clinic as an objective method of determining stress.


[Primary care considerations--17-ketosteroid sulfate-17-hydroxycorticosteroid method as a clinical stress indicator]. In primary care, the management of stress-related diseases occupies a wide spectrum of prevention and treatment, from holistic health care to final cure, centered on medical health and an organic integration of body, mind, and social parameters. Inquiries are being made at examinations and interviews to obtain information on the health condition of the patient, but, they are inadequate. There is a need for an objective indicator to help in the diagnosis. With the introduction of the Stress Barometer (measurements of 17-ketosteroid sulfates and 17-hydroxy-corticosteroids) it has become possible to obtain critical information on a patient in a very short time. This method allows early detection of stress, depression state and tiredness from work which is not presently possible from blood tests. It then becomes possible to call the patient's attention to these matters and to work at increasing awareness, energy levels, and responses to the conditions that are determined in this manner.


[Bleeding time].

[Bleeding time]. Bleeding time indicates the interaction of the platelets with the damaged vessel wall and the subsequent formation of the primary hemostatic plug. Bleeding time has been widely used in the diagnosis of bleeding disorders, especially thrombocytopenia, abnormalities in platelet function, vascular disorders, and von Willebrand's disease. There are a number of methods to perform the bleeding time test, but there are significant problems concerning sensitivity, specificity, and reproducibility. To study the inhibitory effects of monoclonal antibodies (anti-vWF, anti-GPIb, and anti-GPIIb/IIIa) on primary hemostasis, these antibodies were infused to normal pigs. Anti-vWF and anti-GPIb antibodies markedly prolonged the bleeding time and inhibited hemostatic plug formation. The anti-GPIIb/IIIa antibody completely inhibited ADP-and collagen-induced platelet aggregation, but no or only mild prolongation of bleeding time was observed. The quantitative bleeding time which measures both the time and the amount of blood loss is useful in the diagnosis of hemorrhagic disorders and in judging the efficacy of the treatment. It will provide important information to understand the mechanism of primary hemostasis.


[Platelet aggregations and glycoproteins]. Platelet membrane glycoproteins play important roles in platelet functions. In this symposium, we reported three types of deficiency of platelet membrane glycoproteins; Glanzmann's thrombasthenia, Bernard-Soulier syndrome, GPIV-deficiency and discussed the roles of GPIIb/IIIa, GPIb and GPIV, respectively, in the aggregation of these abnormal platelets. In a patient with Bernard-Soulier syndrome, the abnormality in the patient's GPIb was caused by the double heterozygote from her parents who were unrelated. Roles of GPIV were negligible in platelet aggregation since the GPIV-deficient platelets (Naka-) found in healthy donors showed normal platelet aggregations. We developed two monoclonal antibodies, TM83 and TM60 against GPIIIa and GPIb, respectively, and showed that these antibodies inhibited the function of GPIb and GPIIb/IIIa complex, respectively. We demonstrated that TM83 inhibited an epitope of GPIIIa and/or GPIIb/IIIa complex which changes its conformation due to Ca2+ deprivation and is essential for exposure of the fibrinogen-binding site.


[Evaluation of platelet intracellular calcium ion concentrations with flow cytometry]. Platelet intracellular Ca++ ([Ca++] i) was measured by flow cytometry, using a new Ca(++)-sensitive fluorescent dye, fluo 3. The acetoxymethyl derivative of fluo 3, fluo 3 AM, was incorporated into platelets most efficiently in the presence of 1.5 micrograms/ml pluronic F-127, a surfactant often used to facilitate intracellular incorporation of lipophilic agents. [Ca++] i measurement with flow cytometry proved to be more sensitive than that with ordinary fluorescence spectrophotometers, detecting cells with elevated [Ca++] i at much lower agonist concentrations. Two-dimensional analysis using forward scatter intensities and [Ca++] i more clearly defined a subset of platelets responsive to low concentrations of agonists. In normal subjects [Ca++] i hardly changed in response to low-dose thrombin (0.002 U/ml), while it invariably showed a marked increase in response to high-dose thrombin (0.02 U/ml). Thus, these dose of thrombin were used to evaluate clinically whether platelets was hyper-reactive or hypo-reactive. Of 44 patients with diabetes mellitus, 6 patients had hypo-responsive platelets, while platelets were sensitive to low concentrations of thrombin in 3 patients. Since such differences cannot be detected by conventional spectrophotometric methods, [Ca++] i measurement by flow cytometry may prove useful tool for clinical evaluation of platelet function.


[Analysis of platelet shape change, inositol metabolism, and Ca mobilization in patients with platelet dysfunction]. Agonists-induced platelet shape change, inositol metabolism, and Ca mobilization were investigated in patients with various platelet dysfunctions. The platelet shape change determined by our method revealed that arachidonate-induced platelet shape change was completely defective in patients with cyclo-oxygenase (CO) deficiency (A). STA2-induced platelet shape change was also defective in one of five patients with impaired aggregation to STA2 (B). Thrombin-induced platelet shape change was weak in patients with Bernard-Soulier syndrome. In patient with Hermansky-Pudlak syndrome, the platelets did not respond normally to STA2, arachidonate or PMA. These findings suggested that the determinations of platelet shape change by our method was useful in diagnosing platelet dysfunctions. Inositol metabolism and Ca mobilization in response to thrombin, STA2, or NaF were also investigated in patient A,B, and impaired aggregation to A23187 in patient C. The responses were normal in patient A, suggested that CO activity did not affect them. Inositol metabolism was also normal in patient C, although Ca mobilization in response to A23187 was delayed, and that in response to thrombin was defective in the absence of extracellular Ca2+. This suggests that the patient's platelets have a defective IP3-induced Ca mobilization pathway. STA2 selectively failed to induce IP3 formation and Ca mobilization in patient B, although 3H-labelled thromboxane ligand (3H-U46619) bound to the patient's platelets, normally. These findings suggested that the patient's platelets have a defect in postreceptor signal transduction, especially thromboxane receptor-mediated phospholipase C activation pathway.


[Determination of serum interleukin-6 concentration by an enzyme-linked immunosorbent assay in patients with paraproteinemia]. A sensitive enzyme-linked immunosorbent assay of human interleukin-6 (IL-6) was developed to measure the serum IL-6 by Fujirebio INC. Its sensitivity was 3 pg/ml and its analytical range was from 3 to 200 pg/ml. Its precision, reproducibility, and sensitivity were quite satisfactory. The serum IL-6 levels in 200 normal individuals were less than 3 pg/ml. Serum IL-6 concentration in patients with malignant and benign monoclonal gammopathy (BMG) was determined by an ELISA. Serum IL-6 concentration in patients with Bence Jones protein (BJP) type multiple myeloma (MM) (n = 12) was 12.3 +/- 12.7 (mean +/- SD) pg/ml, BJP and IgG type MM (n = 4) 11.5 +/- 5.8 pg/ml, IgM type MM (n = 11) 11.1 +/- 17.5 pg/ml and IgA type MM (n = 4) 4.0 +/- 1.4 pg/ml. They were significantly higher in BJP, BJP and IgG, and IgM types than in normal individuals. The cases with the serum IL-6 of more than 10 pg/ml were move frequent in MM (32.8%) than in BMG (16.3%). The correlation between the serum IL-6 and C-reactive protein (CRP) was r = 0.563 in patients with MM (n = 61) and r = 0.498 in BMG (n = 43). Besides, during the clinical course of a patient with IgG-lambda and BJP-lambda type MM, serum IL-6 concentration responded more sharply than CRP and WBC on candida infection. The measurement of serum IL-6 therefore, seemed not useful for differential diagnosis of monoclonal gammopathies, but it seemed useful as an acute phase protein as well as CRP.


[Inhibitory effect of ulinastatin on the alpha-thrombin activation of factors V and VIII]. Ulinastatin is a remedy of urine serinprotease inhibitors and also it inhibits enzymatic activities of several blood coagulation factors. The action of ulinastatin on alpha-thrombin shows noncompetitive inhibition with the formation of enzyme-inhibitor complex. In this study, the effects of ulinastatin on the alpha-thrombin activation of factors V and VIII are observed by 5-20% gradient gel SDS PAGE with or without Western blotting method. The addition of ulinastatin to the mixture of factor V and alpha-thrombin inhibits the production of active peptides proceeded in the alpha-thrombin activation process of factor V. Furthermore, using anti-factor VIII 43KDa peptide monoclonal antibody with Western blotting method, the addition of ulinastatin to the mixture of alpha-thrombin and factor VIII indicates to inhibit the release of 43KDa peptide from factor VIII in the process of factor VIII activation induced by alpha-thrombin.


[Analysis of factors affecting anaerobic threshold in healthy subjects]. The anaerobic threshold (AT) is used to determine the exercise capacity in patients with heart failure and healthy subjects. To determine the factors affecting AT, we determined the AT in healthy subjects, and examined the factors that determine AT in healthy subjects. One hundred and sixteen healthy subjects (79 men and 37 women) performed on a bicycle a stepwise increasing submaximal exercise. During the work test the parameters usually used in the detection of AT (Vo2, Vco2, VE), blood pressure, heart rate and oxygen saturation were recorded by a computerized system every minute. AT was determined from changes in ventilation and gas-exchange. The fat index was calculated from height and body weight measured at the beginning of the ventilatory function test. A significant correlation was obtained between AT and age, AT and fat index, AT and %VC, AT and maximum heart rate during exercise, AT and recovery rate of heart rate after exercise, and AT and Vo2 at rest. In addition, we examined the relationship among the parameters in 4 groups according to sex and age (30-49; younger, 50-69; older), because sex and age affected most parameters. We found a significant correlation between AT and fat index (older men and women), AT and %VC (younger and older women), AT and Vo2 at rest (younger and older men). We considered that the main factors that determined AT in healthy subjects were age, sex, fat index, %VC and Vo2 at rest.


[Serum HDL-cholesterol in patients with breast carcinoma]. To extend our previous findings on lipid metabolism in breast carcinoma, serum levels of cholesterol and HDL-cholesterol in 144 patients with breast carcinoma, 64 patients with cervix carcinoma, 52 patients with benign breast diseases and 83 healthy females were determined. Blood samples, obtained from patients with carcinoma of the breast and the cervix, were taken prior to radical surgery and at the time of sampling, none of them had received chemotherapy and/or total intravenous nutritional therapy. No significance was observed in the serum levels of cholesterol between the patients with carcinoma, benign diseases and healthy females. The serum levels of HDL-cholesterol, measured by the heparin-calcium-nickel precipitation method were lower among the patients with breast carcinoma than among the patients with cervix carcinoma and healthy females. The mean values and S.D.s of serum HDL-cholesterol of the patients in stage I (23), II (35) and (7) were 42.0 +/- 14.7, 36. 4 +/- 13.1 and 28.7 +/- 9.5 mg/100 ml, respectively, exhibiting a tendency to decrease with the increase in malignancy.


[Between- and within-subject variability of pulmonary function. Four year follow-up of adult males]. Maximal forced expiration of 326 adult males 30 to 55 yr of age was measured four times during a follow-up of 4 yr. Acceptable results were obtained in 270 subjects three times or more. Forced vital capacity (FVC), forced expiratory volume in one second, maximal expiratory flows at 50% and 25% FVC divided by squared height (CFVC, CFEV1, CVmax50, and CVmax25, respectively) were analyzed. Between- and within-subject variance were estimated by a linear model, including the effects of individual and age as explanatory variables. The goodness of fit of the model was satisfactory, since the coefficients of determination of the model were more than 0.96 for CFVC and CFEV1 and around 0.91 for CVmax50 and CVmax25. Estimated between-subject variance was far larger than within-subject variance, being more than ten times for CFVC and CFEV1, about eight times for CVmax50, and five times for CVmax25. At the same time, annual decline of pulmonary function determined cross-sectionally was significantly larger than that determined longitudinally. These results indicated the superiority of longitudinal analysis to cross-sectional analysis in evaluating relatively small effects on pulmonary function especially in the case of chronic exposure of toxic substances in low doses.


[Risk factors of cerebro-cardiovascular events in treated hypertensive male workers in the fifth decade]. The relationship between cerebro-cardiovascular events (CCE) and work-related factors was examined in a cohort of 899 treated hypertensive men who were aged 50-59 yr and working more than 7 portal to portal hours (PPH). During the follow-up of 2.8 yr (2,513 person-years), 27 cases of CCEs occurred, which were classified into 18 cases of stroke, 7 cases of acute myocardial infarction, and 2 cases of others. Using univariate analysis, it was found that managerial position and long PPH (more than 11 h) were significantly related to CCE (relative risk of 3.0 and 2.2, respectively) as well as risk factors such as emaciation, left ventricular hypertrophy, excessive sleeping hours, obesity, cigarette smoking, and inadequate control of systolic blood pressure. Using Cox proportional hazards general model, both managerial position and long PPH remained independently related to the risk of CCE (hazards ratio and 95% confidence interval, 4.1; 1.7-10.0 and 2.7; 1.1-6.2, respectively), after adjustment for other risk factors. These findings suggested that work-related factors, such as managerial position and long PPH, are independent risk factors of CCE among treated hypertensive male workers in the fifth decade.