Reduced serum levels of ALT and GGT and high carbohydrate intake among workers exposed to toluene below the threshold limit values.

We have found decreased serum alanine aminotransferase (ALT) and gamma-glutamyltransferase (GGT) levels in human males exposed to toluene at low levels, suggesting that toluene exposure or related factors in lifestyles were likely to have lowering effects on serum ALT and GGT activities between toluene-exposed and the control subjects. The present study was attempted to investigate whether there is any difference in nutrient intakes between the two groups. Biochemical results were also analysed together with nutrient intakes. In agreement with our previous reports, the present data also provided significant evidence of lowered serum ALT and GGT activities in toluene-exposed males after age matching (p < 0.05). Energy intakes from carbohydrate, fat and protein were 64.6%, 22.5% and 12.9% in the toluene-exposed group and 62.0% (p < 0.05), 24.8% (p < 0.05) and 13.2%, respectively, in the controls. Multiple regression analysis indicated that carbohydrate, protein and energy intakes are significantly associated with toluene exposure. No significant differences in alcohol consumption, tobacco use, work intensities and physical activities were found between the two groups. The present results suggest that the toluene exposure at levels generally considered to be safe, probably have some biological effects on the liver and related organs.

A case of possessive state with onset influenced by 'door-to-door' sales.

Recently in Japan, 'door-to-door sales' has become of concern because it has created numerous legal and social problems. In this paper, a 47 year old dissociative trance disorder case who presented with possession by God is discussed. Specific types of door-to-door sales is known to use superstition and folk beliefs as tools to lure customers. In this particular case, these religious factors seemed to have played an important role in the precipitation of the disorder and its presentation. In addition, the brain-washing environment observed in video lectures used in door-to-door sales seemed to play an important role in the development of the possessive state. We also performed social psychiatric analysis of the occurrence of the possessive state in a city area, which has been considered to develop within traditional culture. Phenomenological classification by one of the authors was useful for diagnosing underlying disorders in the possessive state.

Brief reactive psychosis induced by sensitivity training: similarities between sensitivity training and brainwashing situations.

Sensitivity training (ST), which originated in the USA during the late 1940s, has been used as part of training seminars in Japanese corporations since the late 1950s. The possibility of negative psychiatric effects of ST, and especially its role in inducing psychiatric symptoms, is yet to be clarified. A case of a 41-year-old male company worker whose brief psychosis was induced by a sensitivity training seminar held by the company he worked for is presented. In reviewing the psychopathology of the case with records from the ST seminar, we found similarities between the patient's ST seminar and brainwashing situations. Specifically, the patient experienced severe conflict (of thought process) between his Christian beliefs and being labeled a coward at the seminar. We conclude that monitoring of the ST programs is crucial in order to ensure the psychological safety of ST participants in Japan.

Relationship between solvent inhalation and antisocial behavior: special emphasis on two types of violence seen in solvent abusers.

Organic solvent inhalation is a serious problem among youths in Japan. It induces physical and mental disorders, and is related closely to crime and delinquency. The relationship between solvent inhalation and antisocial behavior was investigated in 75 youths. The subjects were divided into three groups according to the level of violent behavior and the time of appearance: a non-violent group, a late-onset group (violence occurred after the start of inhalation) and an early-onset group (violence had occurred before the start of inhalation). Various parameters were compared among the groups. The late-onset group showed the following characteristics; (i) the frequency of inhalation was high, and many subjects experienced hallucinations and mood changes caused by inhalation; (ii) the family environment was characterized by conflict; and (iii) the subjects had strong psychological conflicts and showed dissociative coping with frustration. The violence, psychological conflict and dissociation in this group were found to be related to the inhalation, with familial conflict as a background. The violence in the early-onset group was considered to be the manifestation of a violent personality as the frequency of inhalation and the incidence of mental symptoms were both low. The subjects in this group showed weak psychological conflict and tended to be demanding of others when attempting to cope with frustration. Two types of violent behaviors in the teenagers who inhaled solvents were identified. The violence of solvent inhalers should be managed according to the type.

[Social psychiatric study of the over-the-counter antitussive drug abuse; shifts resulting from modification of the formulae].

We surveyed 22 patients (of whom 4 were women) with a history of antitussive drug abuse at a psychiatric hospital in the suburbs of Tokyo and at the Clinical Institute for Addiction Problems from 1 July 1986 to 30 June 1991. for changes in their pattern of abuse after the composition was altered. Here are our findings: 1) Researchers have long differed on whether ephedrine or codeine was responsible for the abuse of over-the-counter antitussive agents. The present study revealed that although codeine was still present in the new Bron syrup, its abuse was clearly on the decline in Japan, leading us therefore to conclude that psychological dependence on ephedrine was central to the spate of over-the-counter antitussive abuse observed in the 1980s. Although we cannot ignore the mellow feeling codeine creates, it is difficult to give it more central role in the abuse of such drugs. 2) Users were divided into two groups, one that had used other agents before using antitussives, and another that had never used any other agent but antitussives. A comparison of these two groups revealed clear differences in learning behavior, social adaptation, familial environment and delinquent tendencies, prior to their drug abuse. 3) After the composition was altered, abuse spread to Tonin syrup manufactured by another laboratory, Bron tablets, new Bron syrup and ephedrine tablets. Relatively recently, there have been some new cases of abuse of Bron tablets and an increase in the number of cases who continued to use Bron syrup even after its composition was altered. The Medical literature also reveals that in many countries the abuse of both codeine and ephedrine is becoming a social problem, and clearly either agent may be abused separately.

Transcriptional attenuation control of the tylosin-resistance gene tlrA in Streptomyces fradiae.

The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase-coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome-mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD.

[A study of relationships among solvent inhalation, personality and expectancy; especially on affinity to hallucination, sensation seeking and neurotic tendency].

94 delinquents in two homes for resocialization were surveyed to elucidate the relationship among the status of volatile solvent inhalation, expectancy and personality. The subjects were classified into solvent-inhalation group and non-solvent-inhalation group, and the former was divided into solvent dependence group and abuse group according to DSM-III-R. Each group was given personality tests; general health questionnaire (GHQ), sensation seeking scale (SSS), the vividness of visual imagery (VVIQ), test of visual imagery control (TVIC) and Yatabe-Guilford test (YG). In addition we investigated expectancy and mental symptoms caused by inhalation in it. The results are summarized as follows; 1. Inhalation group scored higher on SSS than non-inhalation group. 2. Compared with abuse group, dependence group presented with 1) higher GHQ score meaning neurotic tendency; 2) higher TVIC score meaning imagery-control-ability; 3) a higher incidence of day-dream and hallucination, especially egosyntonic type; 4) higher expectancy of "enhancement of positive affection", "reduction of negative affection" and "hallucination seeking"; 5) lower expectancy of "association with friends". 3. Higher scored inhalers on TVIC showed visual hallucinations, especially egosyntonic type more frequently than lower ones. 4. Significant correlations were recognized between GHQ score and expectancy of "reduction of negative affection", and between lack of objectiveness score of YG subscale and "hallucination seeking" expectancy. These results suggested that 1) beginning of inhalation is associated with sensation seeking trait, 2) progression to solvent dependence is correlated with three expectancies, i.e., seeking egosyntonic hallucinations paralleling high imagery-control-ability and subjectivity, reducing negative affection paralleling neurotic tendency, and enhancing positive affection. According to the findings, indulgence to drug induced positive imagery led delinquents into splitting of personality. In their treatment we need to help them to integrate high imagery ability and sensation seeking for recovery.

Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins.

The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.

Depression in members of a new religious sect in Japan.

We investigated 226 people belonging to a regional church of a new religious sect (hereinafter referred to as religious group) and 899 ordinary people living in the rural area near this church (hereinafter referred to as nonreligious group) with a questionnaire using Zung's Self-Rating Depression Scale (SDS). In males ranging in age from 40 to 69, the mean scores of 7 SDS items out of 20 were significantly higher in the religious group than in the nonreligious group. Males at the age of 70 or older in both groups, however, did not show any significant difference in these items. It suggests that the religious affiliation exerted mentally good effects on the older male members.

Cloning genes for the biosynthesis of a macrolide antibiotic.

Macrocin-O-methyltransferase (MacOMeTase) catalyzes the final enzymatic step in the biosynthesis of tylosin in Streptomyces fradiae. A 44-base mixed oligonucleotide probe containing only guanosine and cytidine in the third position of degenerate codons was synthesized based on the amino acid sequence of the amino terminus of MacOMeTase. Plaque blot hybridization to a bacteriophage lambda library and colony blot hybridization to a cosmid library of S. fradiae DNA identified recombinants that contained overlapping fragments of chromosomal DNA. The nucleotide sequence of the cloned DNA verified that the DNA contained the coding sequence for MacOMeTase. Recombinant plasmids transformed mutants blocked in tylosin biosynthesis and complemented tylF (the structural gene for MacOMeTase) and tyl mutations of eight other classes.

The use of recombinant DNA techniques to study tylosin biosynthesis and resistance in Streptomyces fradiae.

A substantial amount of information on the biosynthesis of tylosin has been obtained over the past ten years. Physiological studies and experiments with tylosin-blocked (tyl) mutants have suggested the probable pathway by which tylactone is converted to tylosin. The development of recombinant DNA methodology for streptomycetes in general, and for Streptomyces fradiae in particular, has allowed us to apply gene cloning techniques in further studies of tylosin biosynthesis in S. fradiae. The macrocin O-methyltransferase (MOMT), which catalyzes the last step in tylosin biosynthesis, was purified, and the sequence of the 35 amino acids at its amino-terminus was determined. A synthetic 44 base oligonucleotide probe was constructed on the basis of the amino acid sequence. The probe was used to identify sequences containing the MOMT structural gene in bacteriophage and cosmid libraries of S. fradiae DNA. Complementation of tyl mutants with the cloned DNA sequences identified nine tyl biosynthetic genes (tylC, D, E, F, H, J, K, L, and M) in a 42 kb stretch of DNA. Genes complementing four mutant classes, tylA, B, G, and I were not found. A tylosin-resistance gene, tlrB, was located just left of the tyl gene cluster. Tylosin-sensitive mutants of S. fradiae, which were isolated from regenerated protoplasts and which have pleiotropic deficiencies in tylosin biosynthesis, contained deletions which included at least some of the identified tyl loci and one or both of two tylosin-resistance genes, tlrB and tlrC. Possible schemes for the functional organization of the tyl region of the S. fradiae genome are discussed.

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae.

A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyls) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage lambda Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes.

Genetic mapping, cloning and physiological aspects of the glucose kinase gene of Streptomyces coelicolor.

Glucose kinase in Streptomyces coelicolor has a molecular weight of about 110,000. In crude extracts, the enzyme exhibited apparent Km values of 0.20 mM for ATP, 0.27 mM for glucose, and 2.2 mM for the glucose analogue 2-deoxyglucose. Mutations (glk) to 2-deoxyglucose-resistance, which greatly reduce glucose kinase activity and result in relief of glucose repression of utilisation of various carbon sources, were mapped between proA and hisA in the S. coelicolor linkage map. Glucose kinase activity, 2-deoxyglucose-sensitivity, glucose utilisation and glucose repression, were all restored to glk mutants by a 3.5 kb DNA fragment cloned from S. coelicolor into a phage vector (phi C31 KC515), and by larger (10-30 kb) fragments cloned into a low copy number plasmid vector (pIJ916). The glk gene was further localised to a 2.9 kb BclI fragment of the cloned DNA by sub-cloning. Part or all of this fragment was present in each of five primary plasmid clones tested.

The glycerol utilization operon of Streptomyces coelicolor: genetic mapping of gyl mutations and the analysis of cloned gylDNA.

Glycerol-3-phosphate dehydrogenase (gylB) mutations (which cause glycerol sensitivity), and presumed glycerol kinase (gylA) and/or regulatory mutations eliminating both glycerol-3-phosphate dehydrogenase and glycerol kinase activities, map close to the argA locus of Streptomyces coelicolor A3(2). Using the plasmid vector pIJ702 and restriction enzymes BglII and SstI, extensively overlapping S. coelicolor DNA fragments of 2.74 kb and 2.84 kb were isolated, either of which could restore the wild-type phenotype to gylB and some gylA mutants. Genetic and biochemical analyses of mutants carrying the cloned gyl DNA suggested that a functional gyl promoter had not been cloned, and that restoration of the Gyl+ phenotype was achieved by recombination between the cloned and chromosomal gyl DNA sequences. After subcloning parts of this DNA into the phage vector phi C31 KC400, "gene disruption" analysis was carried out, which confirmed the absence of the gyl promoter, and indicated that a polycistronic mRNA traverses gylA and then gylB.

Glycerol catabolic enzymes and their regulation in wild-type and mutant strains of Streptomyces coelicolor A3(2).

Assay procedure were developed for a soluble glycerol kinase (apparent Km (glycerol) 9 microM) and a probably membrane-associated, NAD-independent L-glycerol-3-phosphate dehydrogenase [apparent Km (L-glycerol 3-phosphate) 7 mM] present in Streptomyces coelicolor A3(2). Both enzymes were cold sensitive. They were co-ordinately induced (about 35-fold) by addition of glycerol to cultures growing on arabinose as sole carbon source. Induction was rifampicin sensitive. The dehydrogenase was absent from glycerol-sensitive mutants, and both kinase and dehydrogenase were absent from glycerol non-utilizing (but glycerol-resistant) mutants, demonstrating that the two enzymes are part of the major pathway of glycerol catabolism in S. coelicolor. Circumstantial evidence suggested that their inducer is glycerol 3-phosphate rather than glycerol. The enzymes were subject to co-ordinate repression by various carbon sources, of which glucose exerted the strongest effect (a fivefold repression). Previously described mutants resistant to 2-deoxyglucose, shown here to have very low glucose kinase activity, were defective in glucose repression of the glycerol enzymes.

Biosynthesis of the macrolide antibiotic tylosin. A preferred pathway from tylactone to tylosin.

The efficiencies of bioconversion of twenty-three potential intermediates in the biosynthesis of tylosin were determined with a mutant strain blocked only in tylactone biosynthesis. The results indicated that tylactone, the first intermediate excreted by Streptomyces fradiae, is converted to tylosin by a preferred sequence of reactions which include: (1) addition of mycaminose to the C-5 hydroxyl position of the lactone; (2) hydroxylation of the C-20 methyl group to a hydroxymethyl group; (3) dehydrogenation of the C-20 hydroxymethyl group to a formyl group; (4) hydroxylation of the C-23 methyl group to a hydroxymethyl; (5) addition of 6-deoxy-D-allose to the C-23 hydroxymethyl group; (6) addition of mycarose to the 4'-hydroxyl group of mycaminose; (7) addition of a methyl group to the 2"'-hydroxyl position of demethylmacrocin, and (8) addition of a methyl group to the 3"'-hydroxyl position of macrocin to produce tylosin. The intermediates which lacked both neutral sugars (mycarose and 6-deoxy-D-allose) were biologically unstable, and substantial quantities of these compounds were degraded during standard bioconversion experiments. However, the amount of one such intermediate (O-mycaminosyltylonolide) degraded was substantially reduced when low concentrations of the compound were used for bioconversion, and under these conditions, much higher efficiencies of bioconversion to tylosin were obtained. We have shown that a mutant blocked in hydroxylation of the C-20 methyl group is also blocked in the further dehydrogenation of the C-20 hydroxymethyl group to a formyl group, and have confirmed in in vitro studies that the 2"'-O-methylation of demethylmacrocin must proceed the 3"'-O-methylation of macrocin to produce tylosin.

S-Adenosyl-L-methionine: macrocin O-methyltransferase activities in a series of Streptomyces fradiae mutants that produce different levels of the macrolide antibiotic tylosin.

A series of mutants of Streptomyces fradiae selected for increased production of the macrolide antibiotic tylosin was analyzed for levels of expression of macrocin O-methyltransferase, the enzyme which catalyzes the final step in the biosynthesis of tylosin. Increased tylosin production was accompanied by increased macrocin O-methyltransferase in some of the mutants. Increased expression of macrocin O-methyltransferase was due to more rapid early biosynthesis of the enzyme, to reduced decay of enzyme specific activity late in the fermentation, or to combinations of both. Mutant strains which showed rapid loss of enzyme specific activity late in the fermentation converted large amounts of tylosin to relomycin. The most productive mutants, which synthesized elevated levels of macrocin O-methyltransferase, also produced large amounts of macrocin, the substrate for the enzyme. Incomplete conversion of macrocin to tylosin by these mutants may be due to substrate and product inhibition (E. T. Seno and R. H. Baltz, Antimicrob. Agents Chemother. 20:370-377, 1981). The results suggest that both the levels of precursors and the levels of expression of tylosin biosynthetic enzymes are important for efficient production of tylosin.