Purification and biochemical properties of an N-hydroxyarylamine O-acetyltransferase from Escherichia coli.

The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS-PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.

Purification and DNA-binding properties of the cro-type regulatory repressor protein cng encoded by the Lactobacillus plantarum phage phi g1e.

The putative repressor protein Cng (10kDa on an SDS gel) for the lytic pathway of Lactobacillus plantarum phage φg1e was purified using the Escherichia coli Pt7 system, and its DNA-binding ability for the seven operator-like sequences, the GATAC-boxes (Gb1 to Gb7), was investigated in vitro. In gel-shift assays, Cng selectively bound to the DNA fragments containing the GATAC-box(es). In addition, DNase I footprinting analysis with supercoiled DNA demonstrated that Cng can specifically cover about a 25bp region centered around each of the GATAC-boxes, although two boxes, Gb4 and Gb6, were only partially protected. Moreover, protein crosslinking experiments using glutaraldehyde suggested that Cng most likely functions as a dimer. On the other hand, the binding ability of Cpg for the GATAC-boxes in supercoiled DNA was also examined under the same conditions as in Cng; unlike Cng, Cpg covered Gb4 and Gb6 completely sufficiently as well as the other five boxes. Thus, the present and previous [Kakikawa et al., Gene 215 (1998) 371-379; 242 (2000) 155-166] results indicate a possibility that the two proteins Cng and Cpg selectively bind to the GATAC-boxes that act as operators, and can decide between the lytic or lysogenic pathways through repression of the promoter activity of P(R) as well as P(L).

The effect of managed care on the treatment outcome of substance use disorders.

This study examined the effect of managed care and other reimbursement mechanisms on the outcome of substance abuse treatment at a single treatment facility. A retrospective review of 1594 patient records yielded treatment utilization, diagnostic, and demographic data. Recidivism rates for intensive managed care, traditional managed care, private pay, and state-funded groups of patients were compared. Results showed that, contrary to expectations, recidivism rates were not different for managed vs nonmanaged care patients. In addition, recidivist patients had significantly more ICD-9 diagnoses than nonrecidivist patients. A discussion of future research suggests that other outcome measures need to be examined in addition to recidivism rate, such as psychosocial functioning following treatment and indicator(s) of severity of illness, to better determine the effect of managed care and other reimbursement mechanisms on treatment outcome.

Comparison of mutagenicity and theoretical reactivity of 2,4-dinitrobenzaldehyde and 2,6-dinitrobenzaldehyde in bacterial mutation assay and molecular orbital method.

The mutagenicities and theoretical reactivity indices of 2,4-dinitrobenzaldehyde (2,4-DNBAl) and 2,6-dinitrobenzaldehyde (2,6-DNBAl) were investigated using Salmonella typhimurium strains TA98, TA98NR, TA98/1,8-DNP6, and TA100, TA100NR and TA100/1,8-DNP6, by means of the modified intermediate neglect of differential overlap/3 (MINDO)/3) method. The mutagenic activities of 2,4-DNBAl in TA98NR and TA98/1,8-DNP6 were lower than in TA98, whereas the activity in TA100NR was higher than in TA100 and TA100/1,8-DNP6. The mutagenic activity of 2,6-DNBAl in TA100 and that in TA100 and TA100/1,8-DNP6 decreased. These results suggest that the mutagenicities of 2,4-DNBAl and 2,6-DNBAl are dependent either on the microbial nitroreduction and subsequent acetylation or the presence of an aldehyde group. Among the reactivity indices examined, the frontier electron density values were correlated to the mutagenicities of 2,4-DNBAl and 2,6-DNBAl in TA100, TA100NR and TA100/1,8-DNP6 and the values of energy of the lowest unoccupied molecular orbit were correlated to the mutagenicities of several substituted dinitrobenzenes.

Metabolism of 2,4-dinitrotoluene by Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6, and mutagenicity of the metabolites of 2,4-dinitrotoluene and related compounds to strains TA98 and TA100.

The products detected in the incubation of 2,4-dinitrotoluene (2,4-DNT) with Salmonella typhimurium strains TA98 and TA98/1,8-DNP6 were nitrosonitrotoluenes, hydroxylaminonitrotoluenes, aminonitrotoluenes and dimethyl dinitroazoxybenzene. The capacity of TA98NR to reduce 2,4-DNT was much lower than that of TA98 and TA98/1,8-DNP6. The bacterial products showed no mutagenic activity in the Ames assay using TA98 and TA100. These results indicate that the lack of mutagenic activity of 2,4-DNT is not due to low reductive metabolism of 2,4-DNT by the bacteria, but to the lack of mutagenic activity of the bacterial reductive products of 2,4-DNT, including dimethyl dinitroazoxybenzene.

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains.

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes<2,4-DNT<2,2'-dimethyl-5, 5'-dinitroazoxybenzene (2,2'-DM-5, 5'-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)<<4, 4'-dimethyl-3,3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB), and aminonitrotoluenes (2A4AT, 4A2NT)<2,4-DNT<4HA2NT4,4'-dimethyl-3, 3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2, 6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2'-dimethyl-3, 3'-dinitroazoxybenzene (2,2'-DM-3,3'-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.

Mutagenicity of 2,6-dinitrotoluene and its metabolites, and their related compounds in Salmonella typhimurium.

The mutagenic activities of 2,6-dinitrotoluene (2,6-DNT) and its 6 metabolites, and their 8 related compounds were examined using Salmonella typhimurium strains TA98 and TA100 in the absence or presence of S9 mix. 2,6-DNT itself showed no mutagenicity toward either strain, but 2,6-dinitrobenzaldehyde (2,6-DNBAl), one of the metabolites of 2,6-DNT, showed the highest mutagenic activity in strain TA100. 2,6-DNBAl was a direct-acting mutagen, not requiring metabolic activation. The other compounds containing nitro groups showed weak or no mutagenic activity. This result suggests that the direct-acting mutagenicity of 2,6-DNBAl is mainly due to the aldehyde group of the 2,6-DNBAl molecule.

Presence of nitrate-accumulating sulfur bacteria and their influence on nitrogen cycling in a shallow coastal marine sediment.

Nitrate flux between sediment and water, nitrate concentration profile at the sediment-water interface, and in situ sediment denitrification activity were measured seasonally at the innermost part of Tokyo Bay, Japan. For the determination of sediment nitrate concentration, undisturbed sediment cores were sectioned into 5-mm depth intervals and each segment was stored frozen at -30 degrees C. The nitrate concentration was determined for the supernatants after centrifuging the frozen and thawed sediments. Nitrate in the uppermost sediment showed a remarkable seasonal change, and its seasonal maximum of up to 400 microM was found in October. The directions of the diffusive nitrate fluxes predicted from the interfacial concentration gradients were out of the sediment throughout the year. In contrast, the directions of the total nitrate fluxes measured by the whole-core incubation were into the sediment at all seasons. This contradiction between directions indicates that a large part of the nitrate pool extracted from the frozen surface sediments is not a pore water constituent, and preliminary examinations demonstrated that the nitrate was contained in the intracellular vacuoles of filamentous sulfur bacteria dwelling on or in the surface sediment. Based on the comparison between in situ sediment denitrification activity and total nitrate flux, it is suggested that intracellular nitrate cannot be directly utilized by sediment denitrification, and the probable fate of the intracellular nitrate is hypothesized to be dissimilatory reduction to ammonium. The presence of nitrate-accumulating sulfur bacteria therefore may lower nature's self-purification capacity (denitrification) and exacerbate eutrophication in shallow coastal marine environments.

Enzyme-cytochemistry of human chorion laeve at term: enzyme localization on the chorionic trophoblast.

Is the chorion laeve merely a remnant of the chorion frondosum in placental development? Or is it metabolically active, having something to do with maternofetal interactions? In order to answer these questions at least in part, we determined the ultracytochemical localizations of some important enzymes such as nonspecific phosphatase (alkaline phosphatase), specific phosphatase (Ca(++)-ATPase and 5'-nucleotidase) and adenylate cyclase in the human chorion laeve at term. Strong activities of these enzymes were localized by ultracytochemistry on the plasma membrane of the trophoblast in the chorion laeve. These enzyme activities were confirmed by a series of cytochemical-control experiments, i.e., substrate-free control, heat-stability control, and inhibition control by inhibitors of alkaline phosphatase. These observations indicate that the chorionic trophoblast is probably metabolically active and that it might play an important role in the physiology of the fetal membrane.

Enzyme-cytochemistry of human chorion laeve at term: enzyme localization on the chorionic trophoblast.

Is the chorion laeve merely a remnant of the chorion frondosum in placental development? Or is it metabolically active, having something to do with maternofetal interactions? In order to answer these questions at least in part, we determined the ultracytochemical localizations of some important enzymes such as nonspecific phosphatase (alkaline phosphatase), specific phosphatase (Ca(++)-ATPase and 5'-nucleotidase) and adenylate cyclase in the human chorion laeve at term. Strong activities of these enzymes were localized by ultracytochemistry on the plasma membrane of the trophoblast in the chorion laeve. These enzyme activities were confirmed by a series of cytochemical-control experiments, i.e., substrate-free control, heat-stability control, and inhibition control by inhibitors of alkaline phosphatase. These observations indicate that the chorionic trophoblast is probably metabolically active and that it might play an important role in the physiology of the fetal membrane.

Behavior modification of abnormal gait and chronic pain secondary to somatization disorder.

An interdisciplinary team, which included a physiatrist, psychologist, physical therapist, occupational therapist, social worker, and nursing staff, undertook the treatment of a 33-year-old woman with a 16-year history of gait problems and multiple somatic complaints. Previously, she had been followed by a number of physicians and had undergone both invasive and noninvasive diagnostic procedures as well as several surgical procedures. After limited response to such treatment, she was referred to the outpatient PM&R clinic for evaluation. Physical and psychologic study led to a primary diagnosis of somatization disorder, leading to inpatient treatment which combined a systematic gait-training program, withdrawal of reinforcement for maladaptive disability-related behavior, and reinforcement of increases in normal activities. The patient attained all of the goals in her program in 11 weeks.

Cecal filling and defecation of chickens infected with Eimeria tenella.

White Leghorn cockerels, 11 to 22 days old, were inoculated each with a single oral dose of 4-5 X 10(4) sporulated oocysts of Eimeria tenella. Radiographic study of urinary backflow in infected chickens injected with sodium iothalamate subcutaneously indicated that retrograde movement of ceca was impaired particularly 7, 10, and 14 days after infection. No inflow was noted 7 days after infection when barium sulfate was inoculated into cloaca. Weight of cecal contents examined 7 days after infection was significantly smaller than uninfected control. Number of cecal feces was counted every 24 h beginning 4 through 14 days after infection. The counts in infected birds were significantly fewer than uninfected control 8, 9, and 10 days after infection. Outflow of cecal contents was studied in chickens surgically injected with barium sulfate into cecum 7 days after infection. Radiographic study indicated that most of uninfected control ceca excreted or evacuated the medium between 10 and 24 h after injection, while a few infected birds cleared ceca during the same period.

Studies on the permeability and enzyme-cytochemistry of the mouse hemotrichorial placenta.

The permeability of higher molecular weight substances was investigated in mouse chorioallantoic labyrinthine hemotrichorial placenta, using horseradish peroxidase as a tracer. At the same time, ultrastructural localizations of some important enzymes, such as alkaline phosphatase (ALP), acid phosphatase (ACP), Ca(++)-ATPase and guanylate cyclase were elucidated in this organ by means of the enzyme-cytochemical technique. Peroxidase easily entered the space between layers I and II, and no penetration of this tracer beyond layer II was observed. The reaction products for ALP activity were found mainly on the maternal side of the plasma membrane of the layer II trophoblast. ACP activity was confined to the lysosomes of this layer II cell. In short, peroxidase stopped at the cell surface of the layer II trophoblast, and both ALP and ACP coexisted in this layer II cell. These observations strongly suggest that the layer II trophoblast, especially the surface plasma membrane of this cell, may have an important role in regulating the materno-fetal transfer of substances in mouse chorioallantoic placenta.

The acceptability of prenatal diagnosis in Japan.

Prenatal diagnosis has been performed more frequently in Japan recently, but is still less popular than in the United States or Europe. Legal arrangements, insufficient economic support, and insufficient medical information provided to future parents may explain this difference. The acceptability of prenatal diagnosis, based on the concept of the cost of side effects and elective abortion, was found to be similar when examined through decision analysis and direct survey. Amniocentesis was considered useful for more than half of couples in Japan when the incidence of chromosomal abnormality is more than 0.5% and proper information about the decision to undergo this examination is provided to the pregnant woman and her family.

Mutagenicity of some hydroxylaminotoluene derivatives towards Salmonella typhimurium in esterification systems.

The mutagenicity of 2-hydroxylamino-4-nitrotoluene (2HA4NT), 4-hydroxylamino-2-nitrotoluene (4HA2NT), 2-hydroxylamino-6-nitrotoluene (2HA6NT) or 4-acetylamino-2-hydroxylaminotoluene (4AA2HAT) towards Salmonella typhimurium strains TA98 and TA100 was investigated in the absence and presence of uridine-5'-diphosphoglucuronic acid (UDPGA), acetyl CoA or 3'-phosphoadenosine-5'-phosphosulfate (PAPS) systems, or S9 mix. None of the hydroxylaminonitrotoluenes (2HA4NT, 4HA2NT or 2HA6NT) were mutagenic in both strains while 4AA2HAT was a base-pair substitution mutagen in the UDPGA and PAPS systems. The indirect mutagenic activity was markedly decreased by omission of microsomal fraction (MCF) or UDPGA from the UDPGA system and by addition of beta-glucuronidase to the system. Similarly, the mutagenic activity was markedly decreased either when 105000 X g supernatant fluid (S105), adenosine triphosphate (ATP) or Na2SO4 was omitted from the PAPS system or when pentachlorophenol (PCP) or aryl sulphatase was added to the system. Moreover, the mutagenic activity in either system was markedly decreased by the addition of glutathione (GSH). These results suggested that two esterifications with glucuronic acid and sulfuric acid may play an important role in the appearance of mutagenic activity of 4AA2HAT.

Intestinal transformation of 2,6-dinitrotoluene in male Wistar rats.

1. Metabolites formed by anaerobic incubation of 2,6-dinitrotoluene (2,6-DNT) with intestinal microflora of male Wistar rats were examined. Intestinal transformation of 2,4-dinitrotoluene (2,4-DNT) was also studied to determine whether azoxy compounds are produced in the anaerobic incubation. 2. 2,6-DNT was transformed by the intestinal microflora into 2-nitroso-, 2-hydroxylamino- and 2-amino-6-nitrotoluene, and 2,6-diaminotoluene. A time course study showed that 2-nitroso-, 2-hydroxylamino-, and 2-amino-6-nitrotoluene reached peaks at 2, 5 and 6 h of the anaerobic incubation; 2,6-diaminotoluene appeared at 12 h of the incubation. The formation of 2,6-diaminotoluene from 2-amino-6-nitrotoluene in the incubation was confirmed. 3. Two nitroazoxy compounds, namely, 2,2'-dimethyl-5,5'-dinitroazoxybenzene and 4,4'-dimethyl-3,3'-dinitroazoxybenzene, in addition to known metabolites (nitrosonitrotoluenes, hydroxylaminonitrotoluenes, aminonitrotoluenes and diaminotoluene), were detected in the incubation of 2,4-DNT with intestinal microflora. The formation of the two nitroazoxy compounds (2% dose in 24 h) was non-enzymic and merely involved mixing 2-hydroxylamino-4-nitrotoluene with 2-nitroso-4-nitrotoluene or 4-hydroxylamino-2-nitrotoluene with 4-nitroso-2-nitrotoluene in methanol, respectively.

The problem of relating fetal outcome with breech presentation to mode of delivery.

We retrospectively analyzed 546 consecutive singleton pregnancies with breech presentations that ended at > or = 36 weeks of gestation for the relationship between the intended mode of delivery and fetal outcome. Twelve patients were excluded from the analysis because these infants had major malformations. Of the 534 remaining patients, 124 (23%) were delivered by elective cesarean section. The other 410 women (77%) went into spontaneous labor. Intrapartum emergency cesarean section was required in 112 (27%) of these 410 women; the other 298 (73%) were delivered vaginally. There were 5 poor neonatal outcomes: 3 perinatal deaths and 2 cases of cerebral palsy probably due to intrapartum asphyxia. The risk of poor outcome was thus 1.2% (5/410), in the intended vaginal delivery group vs. no such outcome in the group of 124 patients that had an elective cesarean section. Three of 5 infants with poor outcome were actually born by emergency cesarean section and comparisons of results according to ultimate method of delivery rather than according to intended method of delivery may be misleading and in our case would have been biased against cesarean section.

Bacterial metabolism of 2,6-dinitrotoluene with Salmonella typhimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds.

1. Metabolites produced by the incubation of 2,6-dinitrotoluene (2,6-DNT) with Salmonella typhimurium strains TA 98, TA 98/1,8-DNP6 and TA 98NR were examined. Mutagenicities of bacterial products and related compounds were also examined in the Ames assay using TA 98 and TA 100. 2. 2,6-DNT was converted to 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotoluene and 2-amino-6-nitrotoluene, with concurrent spontaneous formation of 2,2'-dimethyl-3,3'-dinitroazoxybenzene, in the incubation with TA 98 and TA 98/1,8-DNP6. Capacity of TA 98NR to reduce 2,6-DNT was much lower than that of TA 98 and TA 98/1,8-DNP6. 3. Bacterial products, including 2,2'-dimethyl-3,3'-dinitroazoxybenzene, showed no mutagenic activity in the Ames assay. 4. Results indicate that the lack of mutagenic activity of 2,6-DNT is not due to low reductive metabolism of 2,6-DNT by bacteria, but due to the lack of mutagenic activity of the bacterial reductive products of 2,6-DNT.

Synthesis of pyridinium dinitrobenzyl sulfates and potassium (dinitrobenzyl beta-D-glucopyranosid)uronates.

Sulfates and glucuronides of 2,4-dinitrobenzyl alcohol 1a and 2,6-dinitrobenzyl alcohol 1b, which are major or putative metabolites of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT), were synthesized from 1a and 1b by reaction with pyridinium sulfonate and methyl (2,3,4-tri-O-acetyl-alpha-D-glucopyranosyl)uronate bromide 3, respectively, as their pyridinium salts (2a, 2b) and potassium salts (6a, 6b). These conjugates are important for the study of the carcinogenicity of 2,4-DNT and 2,6-DNT.

The clinical efficacy of gamete intrafallopian transfer by minilaparotomy versus in vitro fertilization and embryo transfer.

OBJECTIVE: To evaluate the clinical performance of gamete intrafallopian transfer (GIFT) and in vitro fertilization and embryo transfer (IVF-ET). METHODS: Infertile women were divided into 2 groups: for GIFT, 239 patients (326 cycles) with at least 1 patent tube; and, for IVF-ET, 125 patients (210 cycles) with bilateral tubal occlusion. A specially designed retractor was developed to replace the gametes into an appropriate section of the Fallopian tube accurately and safely. Several parameters, including the pregnancy and delivery rates of each group, were compared. RESULTS: The success rate per trial in the GIFT group was approximately 1.5 times higher than that in the IVF group (pregnancy rate: 44.2% vs. 31.0%, p < 0.01; delivery rate: 33.4% vs. 22.9%, p < 0.01). The pregnancy and delivery rates of GIFT decreased steadily with the number of trials. These apparent decreases were not observed up to the 3rd trial in IVF-ET cases. CONCLUSIONS: GIFT with a minilaparotomy procedure yielded significantly higher success rates than IVF-ET. Accordingly, GIFT is considered to be the first treatment choice in infertility cases with at least 1 patent Fallopian tube.