Molecular cloning of the arylsulfate sulfotransferase gene and characterization of its product from Enterobacter amnigenus AR-37.

The gene encoding the Enterobacter amnigenus AR-37 arylsulfate sulfotransferase (ASST) was cloned, sequenced, and expressed in Escherichia coli NM522. Sequencing led to the identification of three contiguous open reading frames (ORFs) on the same strand. Based on amino acid sequence homology, ORF1, ORF2, and ORF3 are designated astA, dsbA, and dsbB, respectively. A multiple sequence alignment revealed conserved regions in ASST. An N-terminal amino acid sequence analysis of the purified ASST from E. coli NM522 (pEAST72) showed that it is subject to N-terminal processing. The specific activity of purified ASST is 436.5 U/mg of protein. The enzyme is a monomeric protein with a molecular mass of 64 kDa. Using phenol as an acceptor substrate, 4-methylumbelliferyl sulfate is the best donor substrate, followed by beta-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and alpha-naphthyl sulfate. For PNS, alpha-naphthol is the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine, and tyrosine. The enzyme has a different acceptor specificity than the enzyme purified from Eubacterium A-44. It is similar to Klebsiella K-36 and Haemophilus K-12. The apparent K(m) values for PNS using phenol as an acceptor and for phenol using PNS as a donor are 0.163 and 0.314 mM, respectively. The pI and optimum pH are 6.1 and 9.0, respectively.

The relationship between stress and absenteeism.

UNLABELLED: BACKGROUND OF STUDY: Three hundred and sixty three subjects from various civil service organisations were administered the SCOPE-i (Stress, Coping and Personality Inventory) as part of the Institute of Mental Health's efforts to promote mental health in the workplace. AIM OF STUDY: This study examines the relationship between stress-related factors and absenteeism. Absenteeism is measured by the number of days of medical leave taken (MC) and self-report of minor illnesses (MI) which are not severe enough to warrant the coverage of a medical certificate. RESULTS: In this study, we are interested in the differences between MI and MC, and their respective relationships with stress-related factors. We hypothesised that MI, rather than MC, is more related to poor workplace conditions. The findings of this study support this hypothesis. Thus if workplace environment is stressful, people are still likely to come to work despite their illness. An interesting observation in this study is the different ways in which environmental stressors and psychosocial aspects of the workplace environment affect the MC variable. Individuals are more likely to take MC when the environmental stressors are high (i.e., poor lighting, uncomfortable temperatures, etc) as compared to poor psychosocial environmental conditions (e.g., work overload, high organisational tension, career limitations and high personal constraints). On the other hand, when faced with poor psychosocial environmental conditions, MI increases instead of MC. CONCLUSION: These findings have implications on the types of changes in a workplace which employers should make in order to decrease MC and improve physical well-being. In addition, the study shows the usefulness of MI in future studies as a dependent variable.

Induction of ermAMR from a clinical strain of Enterococcus faecalis by 16-membered-ring macrolide antibiotics.

We cloned the MLSB resistance determinant by PCR from a clinical isolate of Enterococcus faecalis 373, which is induced more strongly by a 16-membered-ring macrolide, tylosin, than by erythromycin. To elucidate the molecular basis of resistance of E. faecalis 373, we analyzed the cloned gene, designated ermAMR, by site-directed mutagenesis and reporter gene assay. Our results showed that an arginine-to-cysteine change in the seventh codon of the putative leader peptide endowed tylosin with resistance inducibility and that TAAA duplication enabled the control region to express the downstream methylase gene at a drastically increased level.

Role of mRNA termination in regulation of ermK.

To study the role of mRNA termination in the regulation of ermK, we introduced mismatches into terminators by in vitro mutagenesis. In wild-type ermK, only truncated transcription products were detected in the absence of induction. In contrast, only the full-length transcript was synthesized in the terminator 1 and terminator 2 double mutants, even in the absence of erythromycin. These results indicate that the expression of ermK is primarily regulated by transcriptional attenuation rather than translational attenuation. We also tested the possible contribution of translational attenuation control to the regulation of ermK by constructing a triple mutant (terminator 1 plus terminator 2 plus the methylase Shine-Dalgarno region). A higher level of beta-galactosidase synthesis was seen in the triple mutant. Therefore, unlike with previously described attenuators, it can be concluded that both transcriptional and translational attenuation contribute to the regulation of ermK, although transcriptional attenuation plays a larger role.

Overexpression of arylsulfate sulfotransferase as fusion protein with glutathione S-transferase.

A procedure has been developed for the overexpression and purification of milligram quantities of the Klebsiella K-36 arylsulfate sulfotransferase (ASST). The structural gene was amplified by means of a polymerase chain reaction (PCR) technique and inserted into the plasmid vector pGEX-3X. The plasmid pGEX-100, carrying the Klebsiella K-36 astA structural gene under the control of the Escherichia coli tac promoter, was transformed into the E. coli strain BL21 (DE3). The ASST was produced in E. coli as a fusion with glutathione S-transferase. Conditions for protein production, isolation on glutathione Sepharose 4B, and Xa cleavage to generate active ASST were developed. The purification yielded approximately 0.7 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties almost the same as those of the enzyme purified from Klebsiella K-36 cells. The purification procedure was very rapid and is suitable for obtaining considerable amounts of enzyme at a relatively high yield compared with its purifying method from the culture of the Klebsiella K-36 strain.

Relationships between angiotensin I converting enzyme gene polymorphism and renal complications in Korean IDDM patients.

OBJECTIVES: The prognosis of IDDM is mainly dependent on complicated diabetic nephropathy which is probably determined by both metabolic abnormalities and genetic predisposition. Angiotensin I converting enzyme (ACE) regulates systemic and renal circulations through angiotensin II formation and kinins metabolism. The insertion(i)/deletion(D) polymorphism in intron 16 of ACE gene is strongly associated with ACE levels, and subjects homozygote for deletion (genotype DD) have the highest plasma values. Recently, it was reported that I/D polymorphism of ACE gene is associated with diabetic nephropathy in Caucasian IDDM patients. We studied the relationship between the ACE gene polymorphism and diabetic nephropathy in Korean IDDM patients. METHODS: The study population consisted of 59 IDDM patients (duration > 5 yrs) and 107 control subjects. IDDM subjects were divided into 2 groups according to the presence or absence of diabetic nephropathy (with nephropathy: n = 31, without nephropathy: n = 28). After extraction of genomic DNA from peripheral blood leukocytes, PCR was performed using the sense primer (5' -GCC CTG CAG GTG TCT GCA GC-3') and anti-sense primer (3'-TGC CCA TAA CAG TGC TTC ATA -5'), respectively. The PCR products were electrophoresed in 2% agarose gels, and DNA was visualized directly with ethidium bromide staining. RESULTS: Frequencies for II, ID and DD genotypes were similar in IDDM subjects and controls (23: 19:17 vs 49:41:17, p = 0.142) and derived allele frequencies for I and D alleles were similar in both groups (0.551:0.449 vs 0.649:0.351, p = 0.098). The ACE genotype distributions were not different in diabetic subjects with or without nephropathy (12:9:10 vs 11:10:7, p = 0.78) and derived allele frequencies were also similar (0.532:0.468 vs 0.571:0.429, p = 0.81). CONCLUSION: The I and D allele frequency in our controls was different compared to ACE allele frequencies of Caucasian populations, but very similar compared to those of Chinese or Japanese subjects. We found that I/D polymorphism of ACE gene is not implicated in the diabetic nephropathy of Korean IDDM patients and may be explained by ethnic differences.

Psychiatric referrals in the general hospital.

Psychiatric disorders and physical illnesses often coexist. Although there is evidence of high psychiatric morbidity in general hospital patients, only a small percentage are referred to the consultation-liaison psychiatrist. The paper describes and discusses the common psychiatric conditions encountered and referred in general hospitals. They include attempted suicides, psychiatric disorders presenting with physical symptoms, organic psychiatric disorders presenting with psychiatric symptoms and the psychiatric consequences of physical illness. The major diagnostic categories of these referrals are organic psychotic disorders, functional psychoses and neurotic disorders. Some broad guidelines are listed for the referring physician. Patients presenting with suicidal ideations or attempts, suspected emotional psychiatric disturbance, a history of psychiatric illness, an apparent incompetence in giving consent for medical procedures and those with diagnostic and management problems could be referred to the psychiatrist.

Psychological and psychiatric investigations of HIV infection in Singapore.

A psychosocial study was carried out on 10 out of the 18 HIV positive persons who were under surveillance and receiving counselling at either Middle Road Hospital or the Communicable Disease Centre in February 1988. The results of the psychological testing and psychiatric findings are presented. All 10 were male: 9 of them were homosexual and 1 heterosexual. Only one had significant depressive symptoms at the time of study. All had fears about their infection being found out by others and of the stigma that this might bring to their families. Psychometric tests on memory function did not show any signs of visuo-verbal memory impairment but did reveal a possible indication of audio-verbal memory dysfunction.