2,3-Dihydroxybenzoate meta-Cleavage Pathway is Involved in o-Phthalate Utilization in Pseudomonas sp. strain PTH10.

Pseudomonas sp. strain PTH10 can utilize o-phthalate which is a key intermediate in the bacterial degradation of some polycyclic aromatic hydrocarbons. In this strain, o-phthalate is degraded to 2,3-dihydroxybenzoate and further metabolized via the 2,3-dihydroxybenzoate meta-cleavage pathway. Here, the opa genes which are involved in the o-phthalate catabolism were identified. Based on the enzymatic activity of the opa gene products, opaAaAbAcAd, opaB, opaC, and opaD were found to code for o-phthalate 2,3-dioxygenase, dihydrodiol dehydrogenase, 2,3-dihydroxybenzoate 3,4-dioxygenase, and 3-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, respectively. Collectively, these enzymes are thought to catalyze the conversion of o-phthalate to 2-hydroxymuconate-6-semialdehyde. Deletion mutants of the above opa genes indicated that their products were required for the utilization of o-phthalate. Transcriptional analysis showed that the opa genes were organized in the same transcriptional unit. Quantitative analysis of opaAa, opaB, opaC, opaD, opaE, and opaN revealed that, except for opaB and opaC, all other genes were transcriptionally induced during growth on o-phthalate. The constitutive expression of opaB and opaC, and the transcriptional induction of opaD located downstream of opaB, suggest several possible internal promoters are existence in the opa cluster. Together, these results strongly suggest that the opa genes are involved in a novel o-phthalate catabolic pathway in strain PTH10.

Isolation and characterization of a bacterial strain that degrades cis-dichloroethenein the absence of aromatic inducers.

Bacteria capable of degrading cis-dichloroethene (cDCE) were screened from cDCE-contaminated soil, and YKD221, a bacterial strain that exhibited a higher growth on minimal salt agar plates in the presence of cDCE than in the absence of cDCE, were isolated. Phylogenetic studies of the 16S rRNA as well as gyrB, rpoD, and recA in YKD221 indicated that this strain is closely related to the type strains of Pseudomonas plecoglossicida, monteilii, and putida. An average nucleotide identity analysis indicated that YKD221 is most closely related to P. putida strains, including the type strain, which suggests that YKD221 belongs to P. putida. Although the genome of YKD221 was very similar to that of P. putida F1, a toluene-degrading strain, the YKD221 genome has 15 single-nucleotide polymorphisms and 4 insertions compared with the F1 genome. YKD221 caused the release of sufficient chloride ions from cDCE to suggest that the strain is able to completely dechlorinate and degrade cDCE. YKD221 also degraded trichloroethene but was unable to degrade trans-dichloroethene and tetrachloroethene. The degradation activity of YKD221 was elevated after growth on toluene. Inactivation of todC1, which encodes for a large subunit of the catalytic terminal component in toluene dioxygenase, resulted in a complete loss of growth on toluene and cDCE degradation activity. This is the first evidence of the involvement of todC1C2BA-coded toluene dioxygenase in cDCE degradation. YKD221 did not appear to grow on cDCE in a minimal salt liquid medium. However, YKD221 did exhibit an enhanced increase in cell concentration and volume of cells during growth on minimal salt agar plates with cDCE when first grown in LB medium. This behavior appears to have led us to misinterpret our initial results on YKD221 as an indication of improved growth in the presence of cDCE.

Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1.

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

γ-Resorcylate catabolic-pathway genes in the soil actinomycete Rhodococcus jostii RHA1.

The Rhodococcus jostii RHA1 gene cluster required for γ-resorcylate (GRA) catabolism was characterized. The cluster includes tsdA, tsdB, tsdC, tsdD, tsdR, tsdT, and tsdX, which encode GRA decarboxylase, resorcinol 4-hydroxylase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, an IclR-type regulator, a major facilitator superfamily transporter, and a putative hydrolase, respectively. The tsdA gene conferred GRA decarboxylase activity on Escherichia coli. Purified TsdB oxidized NADH in the presence of resorcinol, suggesting that tsdB encodes a unique NADH-specific single-component resorcinol 4-hydroxylase. Mutations in either tsdA or tsdB resulted in growth deficiency on GRA. The tsdC and tsdD genes conferred hydroxyquinol 1,2-dioxygenase and maleylacetate reductase activities, respectively, on E. coli. Inactivation of tsdT significantly retarded the growth of RHA1 on GRA. The growth retardation was partially suppressed under acidic conditions, suggesting the involvement of tsdT in GRA uptake. Reverse transcription-PCR analysis revealed that the tsd genes constitute three transcriptional units, the tsdBADC and tsdTX operons and tsdR. Transcription of the tsdBADC and tsdTX operons was induced during growth on GRA. Inactivation of tsdR derepressed transcription of the tsdBADC and tsdTX operons in the absence of GRA, suggesting that tsd gene transcription is negatively regulated by the tsdR-encoded regulator. Binding of TsdR to the tsdR-tsdB and tsdT-tsdR intergenic regions was inhibited by the addition of GRA, indicating that GRA interacts with TsdR as an effector molecule.

Essential thrombocythemia as a risk factor for stillbirth.

INTRODUCTION: The risk of abortion is known to be high in women with essential thrombocythemia (ET). However, a few studies have focused on the risk of stillbirth among fetuses reaching gestational age compatible with life. METHODS: Review of medical charts of pregnant women with ET who received cares at a single center between January 2003 and June 2013 and the English literature in which more than 20 pregnancies with ET were dealt with regarding outcomes. Outcomes were classified into three categories: spontaneous abortion or preterm delivery before GW 24, stillbirth at and after GW 24, and live birth (LB). Japan national statistics was used to estimate the risk of stillbirth among women with GW 22 or more. RESULTS: In all nine pregnancies in four women with ET at our hospital, two miscarriages, one stillbirth (intrauterine death at GW 35), and six LBs occurred. There were six reports in the English literature in which a total of 374 pregnancy outcomes were described: 110 miscarriages (29%), 14 stillbirths (3.7% of all 374 pregnancies and 5.3% of 264 pregnancies with GW≥24), and 250 LBs (67%) occurred. Japan national statistics between 1995 and 2011 indicated that the risk of stillbirth was less than 0.50% among women with GW≥22. CONCLUSIONS: The risk of stillbirth was extremely high among women with ET. More intensified monitoring of fetal wellbeing may be required to improve outcome of pregnancy complicated with ET.

Difference in the D-dimer rise between women with singleton and multifetal pregnancies.

INTRODUCTION: The differences in the d-dimer rise between women with singleton and multifetal pregnancies have not been studied extensively. MATERIALS AND METHODS: d-Dimer levels were determined in 1089 blood specimens from 1089 women in various stages of pregnancy, including 977 and 112 women with singleton and multifetal pregnancies, respectively. None of the 1089 women developed hypertension or clinical venous thromboembolism during pregnancy or in the postpartum period. RESULTS: The D-dimer levels were significantly and positively correlated with gestational week at examination in women with singleton or multifetal pregnancies. The d-dimer levels (µg/ml, mean ± SD [number of specimens]) determined at the 1st trimester did not differ significantly (0.81 ± 0.82 [102] for singleton vs. 1.20 ± 0.77 [7] for multifetal), but those at the 2nd (1.61 ± 1.45 [216] vs. 2.62 ± 2.26 [59]) and 3rd (2.37 ± 2.22 [659] vs. 4.02 ± 2.14 [46]) trimesters were significantly higher in women with multifetal than singleton pregnancies. The 90th percentile value was 4.31 µg/ml for 1089 specimens. A significantly greater number of women exceeded 4.31 µg/ml during the 2nd (16.9% vs. 5.6%, P=0.0043) and 3rd (34.8% vs. 10.6%, P < 0.0001) trimesters among those with multifetal than with singleton pregnancies. CONCLUSIONS: The degree of D-dimer rise in pregnancy was greater in women with multifetal than with singleton pregnancies.

Antithrombin activity, platelet count, hemoglobin concentration and hematocrit value determined immediately before vaginal delivery among healthy women.

AIMS: To determine the normal reference values for antithrombin (AT) activity, platelet count (Plt), hemoglobin concentration (Hb), and hematocrit value (Ht) immediately before vaginal delivery among healthy pregnant women with singleton pregnancies and to determine association of these blood parameters with fetal growth. METHODS: A complete blood count was performed and the AT activity was examined in 300 consecutive women admitted to hospital at > or = gestational week 36 for labor pains and/or the rupture of fetal membranes. All the women were normotensive and had singleton pregnancies, and none of the women had proteinuria, a weekly weight gain > or = 0.5 kg, or other specific complications upon admission. All the women attempted a vaginal delivery. RESULTS: The medians (5th-95th percentile) were 90% (71-110%) for AT activity, 234x10(9)/L (150-337x10(9)/L) for Plt, 11.0 g/dL (9.5-12.8 g/dL) for Hb, and 34.0% (30.4-38.6%) for Ht. Women with an Hb value of > or = the median (11.0 g/dL) gave birth to significantly smaller infants than their counterparts. CONCLUSIONS: A considerable number of healthy women exhibit a reduced AT activity and/or platelet count immediately before delivery. Hemoconcentration evidenced by a raised Hb value adversely effects on infant growth. Our data may be helpful when considering the normal ranges of these blood parameters for healthy parturient women.

Identification and characterization of uptake systems for glucose and fructose in Rhodococcus jostii RHA1.

Rhodococcusjostii RHA1 takes up and utilizes glucose and fructose as its sole carbon source. To address glucose and fructose transport systems in RHA1, an in silico analysis was performed. BLAST searches revealed that the ro02365 and ro06844 genes share 51 and 47% amino acid identity, respectively, with the major facilitator superfamily (MFS) glucose transporter, GlcP, of Streptomycescoelicolor A3(2) and the ro01549-01552 and ro06781-06785 loci share 43-51% amino acid identity with proteins of the phosphotransferase system (PTS) in S. coelicolor A3(2). Real-time reverse transcription-polymerase chain reaction analysis indicated that ro02365 and ro06781 were upregulated during growth on glucose and fructose, respectively. The ro06781 gene is an ortholog of the histidine-containing phosphocarrier protein in the PTS. Deletion mutants of ro02365 and ro06781 failed to grow on glucose and fructose, respectively, indicating that these genes are essential for growth on the corresponding sugar. They also failed to take up glucose and fructose, respectively. Hence ro02365 is responsible for the uptake of glucose as a MFS transporter and was designated as glcP, and ro06781 is responsible for the uptake of fructose as a component of the PTS and was designated as ptsH.

Glucose-mediated transcriptional repression of PCB/biphenyl catabolic genes in Rhodococcus jostii RHA1.

Rhodococcus jostii RHA1, a Gram-positive polychlorinated biphenyl (PCB) degrader, exhibited biphasic growth in a medium containing biphenyl (BPH) and glucose (Glc), with consumption of Glc and low 2,3-dihydroxybiphenyl 1,2-dioxygenase activity in the first growth stage. These results suggested the repression of BPH metabolism in the presence of Glc, in which RHA1 preferentially utilized Glc in the first growth stage and BPH in the second stage. A reporter assay using a transcriptional fusion of the bphAa promoter (P(bphAa)) and the luxAB luciferase genes was performed, and lower luciferase activity was observed during the first growth stage, indicating transcriptional repression of P(bphAa) activity in the presence of Glc. Transcription levels of the representative genes of five BPH catabolic enzyme gene clusters (bph/etb gene clusters) and their transcriptional regulatory genes (bphST) were examined by real-time reverse transcription PCR. The results obtained indicate that transcription of the targeted bph/etb genes, which were upregulated by BPH, was repressed in the presence of Glc. Among the substrates examined, fructose in addition to Glc induced the repression of P(bphAa) transcription. These results indicate that BPH utilization in RHA1 is under the control of carbon catabolite repression at the transcriptional level in addition to BphST-dependent transcriptional regulation.

Do uterotrophic drugs increase the risk of fatal hemorrhagic brain stroke?

OBJECTIVE: To evaluate whether uterotrophic agents increase the risk of fatal hemorrhagic brain stroke. METHODS: Between 1991 and 1992, there were 230 maternal deaths among 2,420,000 pregnant women in Japan and the causes of these deaths was investigated in 1994. Using information provided in this report, we identified 35 women who died from or were assumed to die from hemorrhagic brain stroke. We assumed that 93% of women would have tried vaginal delivery. The risk of fatal hemorrhagic brain stroke after uterotrophic agent use was calculated according to the assumption that 5.0-40% of women received uterotrophic agents. RESULTS: Use of uterotrophic agents for induction/augmentation of labor was confirmed in five (14.3%) of the 35 women who died from hemorrhagic brain stroke. The incidence of fatal brain stroke after the use of uterotrophic agents was only significantly higher than that for spontaneous hemorrhagic brain stroke if these agents were administered in ≤ 6.0% of women. CONCLUSIONS: Because more than 6.0% of women received uterotrophic agents, these agents are unlikely to increase the risk of fatal hemorrhagic brain stroke.

Evidence of the escape of antithrombin from the blood into the interstitial space in pregnant women.

OBJECTIVE: we investigated whether ascites samples obtained from pregnant women during cesarean sections contained antithrombin because it is unknown whether antithrombin escapes from the blood and passes into the interstitial space during pregnancy. METHODS: the concentration and activity levels of antithrombin were determined in six ascites samples obtained from six consecutive women who exhibited generalized edema, ascites, and a gradual decline in antithrombin activity. RESULTS: all six ascites samples contained antithrombin (mean ± SD, 4.9 ± 2.2 mg/dL; range, 2.7-8.8 mg/dL) and exhibited an antithrombin activity level of 15.5 ± 6.0% (range, 10-24%). CONCLUSIONS: antithrombin escapes from the blood into the interstitial space in pregnant women. This phenomenon partially explains the gradual decline in antithrombin activity observed in these six pregnant women with generalized edema and large volumes of ascites.

Marked gestational edema as a clinical sign of life-threatening condition.

A 35-year-old Japanese nulliparous woman exhibited rapid weight gain (6 kg/7 days), reduced antithrombin activity and platelet count at 37 weeks of gestation without hypertension or proteinuria, and underwent cesarean section. Postnatally, pulmonary edema developed for 7 days, with transient hypertension and proteinuria, and bodyweight loss (14.6 kg) by 14 days postpartum. Platelet count and antithrombin activity normalized promptly postpartum. Despite a life-threatening clinical condition due to enhanced vascular permeability, neither hypertension nor proteinuria appeared antenatally. Determining antithrombin activity and platelet count may be useful for distinguishing between women with pathological edema and physiological edema.

[Successful desensitization using cisplatin after carboplatin-associated hypersensitivity reactions in 3 cases with recurrent ovarian carcinoma].

The standard chemotherapy for ovarian carcinoma is paclitaxel and carboplatin concomitant therapy. This combination is so frequently administered that a carboplatin-associated hypersensitivity reaction may occur after long-term treatment. Recently, we experienced carboplatin-associated hypersensitivity reactions in three cases. They showed symptoms of tachycardia, chest tightness, and dyspnea. We report these cases that could be readministered platinum agent using the cisplatin desensitization method. This method was relatively safe and successful for patients with platinum-sensitive disease.

The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse.

Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1's 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O(2)-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.