The localized melting of mini-F origin by the combined action of the mini-F initiator protein (RepE) and HU and DnaA of Escherichia coli

Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50^° bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region. The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaC complex, much as has been documented for oriC- dependent replication.     

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.     

Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects

Isono, Shiroh, John E Remmers, Atsuko Tanaka, Yasuhide Sho,Jiro Sato, and Takashi Nishino. Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects.J. Appl. Physiol. 82(4):1319-1326, 1997.Anatomic abnormalities of the pharynx arethought to play a role in the pathogenesis of obstructive sleep apnea(OSA), but their contribution has never been conclusively proven. Thepresent study tested this anatomic hypothesis by comparing themechanics of the paralyzed pharynx in OSA patients and in normalsubjects. According to evaluation of sleep-disordered breathing (SDB)by nocturnal oximetry, subjects were divided into three groups: normalgroup (n = 17), SDB-1(n = 18), and SDB-2(n = 22). The static pressure-arearelationship of the passive pharynx was quantified under generalanesthesia with complete paralysis. Age and body mass index werematched among the three groups. The site of the primary closure was thevelopharynx in 49 subjects and the oropharynx in only 8 subjects.Distribution of the location of the primary closure did not differamong the groups. Closing pressure(PC) of the velopharynx forSDB-1 and SDB-2 groups (0.90 ± 1.34 and 2.78 ± 2.78 cmH₂O, respectively) wassignificantly higher than that for the normal group (3.77 ± 3.44 cmH₂O;P < 0.01). Maximal velopharyngealarea for the normal group (2.10 ± 0.85 cm²) was significantly greaterthan for SDB-1 and SDB-2 groups (1.15 ± 0.46 and 1.06 ± 0.75 cm², respectively). Theshape of the pressure-area curve for the velopharynx differed betweennormal subjects and patients with SDB, being steeper in slope nearPC in patients with SDB.Multivariate analysis of mechanical parameters and oxygen desaturationindex (ODI) revealed that velopharyngealPC was the only variable highly correlated with ODI. VelopharyngealPC was associated withoropharyngeal PC, suggestingmechanical interdependence of these segments. We conclude that thepassive pharynx is more narrow and collapsible in sleep-apneic patientsthan in matched controls and that velopharyngeal PC is the principal correlate ofthe frequency of nocturnal desaturations.

     

Application of bacterial magnetic particles as novel DNA carriers for ballistic transformation of a marine cyanobacterium

Summary Bacterial magnetite particles (BMPs) of 50 to 100nm diam were used as DNA carriers for the ballistic transformation of the marine cyanobacteriumSynechococcus. BMPs were bombarded into the cyanobacterial cells at several bombardment velocities using a particle gun. Successful transformation and gene expression were confirmed by Southern hybridization and CAT assay, respectively. The BMPs were also observed in the cyanobacterial cells by transmission electron microscopy. These results suggested that BMPs can be used as carriers for introducing DNA into bacterial cells.     

Photomicrobial sensors for selective determination of phosphate

A photomicrobial sensor consisting of immobilized Chlorella vulgaris and an oxygen electrode has been developed for selective determination of phosphate. When 40 mM phosphate was added to the sensor system, the photocurrent increased to a maximum under light irradiation with a response time of 1 min. The current increased with increasing phosphate concentration in the range 8–70 mM. Selectivity of the sensor was satisfactory. Good agreement was obtained between the phosphate concentrations in lake water determined by the photomicrobial sensor and by conventional colorimetry (correlation coefficient 0.96).     

Photomicrobial electrode for selective determination of sulphide

Summary A photomicrobial electrode, which uses the photosynthetic bacteria Chromatium sp. in conjunction with a hydrogen electrode, was developed for the determination of sulphide. The response time of the photomicrobial electrode was 5–10 min. A linear relationship was obtained between the current of the electrode and the sodium sulphide concentration below 3.5 mM. The minimum detectable concentration of sodium sulphide was 0.4 mM. Selectivity of the sensor is satisfactory. A good agreement was obtained between the photomicrobial electrode and the ethylene blue method (correlation coefficient: 0.90).     

A cold-labile acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver. Reactivation and reconstitution of the active species from the inactive monomer

An acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold-induced inactivation and dissociation of the enzyme: At a protein concentration of 14 micrograms/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37 degrees C appeared to be optimal for reactivation; warming at 25 degrees C or at 43 degrees C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme-protein concentration of 14 micrograms/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl-CoA (0.5 mM) restored the activity about 50%. High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity. Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4 degrees C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.     

Reexamination of paternal age effect in Down's syndrome

Summary Paternal age distribution for 1279 cases of Down's syndrome born in 1952–1968 was compared with the corresponding distribution for the general population, corrected for the maternal age as well as for the year of birth of the patients. Although there was no difference in the mean paternal age, the two distributions differed significantly, largely due to the excess of fathers aged 55 years and over and to the deficit of those aged 40–44 years in the patients born to mothers aged 30 years and over. The overall pattern of the relative incidence of Down's syndrome with advancing paternal age, with maternal age controlled, seems consistent with the hypothesis proposed by Stene et al. (1977). It increased from 0.8 for fathers aged 20–24 years slowly up to 1.2 for those aged 45–49 years, though with an intermediate drop to 0.8 at the age of 40–44 years, and then sharply to 2.4 for those aged 55 years and over. This rising pattern of the relative incidence with paternal age was essentially the same for the patients born in 1952–1960 and for those born in 1961–1968, although the slope was less steep in the latter than in the former group.This paper is dedicated to Professor Heinrich Schade in honor of his 70th birthday