Purification, characterization, and mechanism of a flavin mononucleotide-dependent 2-nitropropane dioxygenase from Neurospora crassa

A nitroalkane-oxidizing enzyme was purified to homogeneity from Neurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an 18O2 atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH3CH(NO2)CH3 + O2-->2CH3COCH3 + 2HNO2, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of the Neurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenula enzyme. Both of these enzymes are flavoenzymes; however, the Neurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.     

Molecular structure and stereoelectronic properties of sarmazenil--a weak inverse agonist at the omega modulatory sites (benzodiazepine receptors): comparison with bretazenil and flumazenil

X-ray diffraction and ab initio MO theoretical calculations were used in order to investigate the structural and electronic properties of sarmazenil, a weak inverse agonist at the omega modulatory sites (benzodiazepine receptors). This compound was compared to bretazenil, a partial agonist, and to the antagonist flumazenil on the basis of structural and electronic data. The conformational and theoretical properties (interatomic pi overlap populations, molecular electrostatic potential (MEP), the topology of frontier orbitals, and proton affinity) of these three imidazobenzodiazepinones were determined in order to analyse the stereoelectronic properties in relation with their distinct intrinsic efficacies at the omega modulatory sites.     

Candida fungemia in neonates treated with fluconazole: report of forty cases, including eight with meningitis

PURPOSE OF THE STUDY: To assess efficacy and safety of fluconazole in neonates with Candida fungemia. STUDY DESIGN: Multicenter prospective protocol of all fungemias appearing between January 1, 1993, and December 31, 1997, in four major university hospitals. RESULTS: Forty neonates, 28 of them with very low birth weight (<1500 g; 30.5 median gestation week), with documented Candida albicans fungemia were treated with intravenous fluconazole in a daily dosage of 6 mg/kg once daily for 6 to 48 days. Thirty-four received fluconazole as monotherapy and 6 received it in combination with amphotericin B. Thirty-two (80%) were cured; 4 of them relapsed despite at least 14 days of therapy, but they were ultimately cured without sequelae. Eight other neonates died, 4 because of fungal infection and 4 because of prematurity or hemorrhage or lung failure, with fungemia (20% overall and 10% attributable mortality). Two neonates had elevated liver enzymes during fluconazole therapy and 2 others had elevated serum creatinine during fluconazole monotherapy. In none of them did these abnormalities necessitate discontinuation of antifungal therapy. In 8 neonates fungal meningitis developed as a complication of fungemia. All but 3 fungemias were C. albicans; 3 were Candida parapsilosis. CONCLUSIONS: Fluconazole was safe and effective antifungal therapy even in complicated or Candida fungemia in neonates and in infants with very low birth weight.     

Molecular size-dependent leakage of intracellular molecules from frog skeletal muscle fibers permeabilized with beta-escin

The permeability of beta-escin-treated cell membrane was characterized in terms of the permeant molecular size, by monitoring the leak of cytoplasmic molecules in frog skeletal muscle fibers. With a low concentration of beta-escin (5 microM), most of the cellular ATP was lost within 30-40 min (as revealed by rigor force generation), whereas a fluorescence-labeled dextran injected into the cytoplasm (approximately 10 kDa) and cytoplasmic proteins (14-80 kDa) slowly leaked out of the cell. A high concentration of beta-escin (50-100 microM) accelerated the leak of large molecules. Therefore, low concentrations of beta-escin may be used as a means of permeabilizing the cell membrane to relatively small molecules, while retaining a major fraction of the cellular macromolecules.     

Study of 18-cm long single-sided AC-coupled silicon microstripdetectors

The SSC GEM silicon Central Tracker design incorporated 18-cm long single-sided AC-coupled silicon microstrip ladders. Compared to the 12-cm long ladders considered in the preliminary stages of the tracker design, the 18-cm long ladders have the advantage of reduced cost, channel count and overall power consumption, and led to a simplified tracker assembly. However, such long ships also present the challenge of maintaining satisfactory performance. The increased capacitance and series resistance contribute to lower signal-to-noise ratios, longer time walk, higher power consumption per channel and increased probability of crosstalk to neighboring channels. In this paper, an accurate method to calculate the geometric capacitance of the AC-coupled microstrips is presented and the calculated results are compared with measurements, SPICE simulations are performed to predict the noise, the extent of interstrip capacitive coupling and the dispersion of the detector signal due to the finite series resistance of the metal strips and the long length of the detector. The influence of the preamplifier current and the shaping time on the signal and noise levels is also presented. The study concludes that the 18-cm long ladders can successfully satisfy the performance goals of the GEM silicon Central Tracker     

Inhibition of G1 cyclin expression in normal rat kidney cells by inostamycin, a phosphatidylinositol synthesis inhibitor

We previously reported that inostamycin, an inhibitor of CDP-DG: inositol transferase, inhibited cell proliferation in normal rat kidney (NRK) cells by blocking cell cycle progression at the G1 phase. In the present paper, we report the effect of inostamycin on the serum-induced activation of Ser/Thr protein kinases that are involved in G1 progression. In quiescent NRK cells mitogen-activated protein kinase (MAP kinase) and casein kinase II were activated within 15 min after serum addition. Neither activation was affected by the treatment with inostamycin. However, in the inostamycin-treated cell, cyclin-dependent kinase 2 (CDK2) failed to be activated after serum stimulation. Since serum-induced expression of cyclin E was also suppressed by inostamycin, this inhibitor would appear to block CDK2 activation by inhibiting cyclin E expression. Furthermore, inostamycin also inhibited cyclin D1 expression induced by serum; and consequently, hyperphosphorylation of retinoblastoma protein (pRB) by RB-kinases such as CDK4 and CDK2 was abolished, which would result in elimination of functional inactivation of pRB. Thus, early G1 arrest in NRK cells by inostamycin is due to the inhibition of cyclin D1 and E expressions.     

De novo epileptic confusional status in a patient with cobalamin deficiency

A 80-year-old man with cobalamin deficiency and no history of epilepsy developed a partial complex epileptic confusional status (ECS) unresponsive to acute i.v. diazepam. Brain CT scan and MRI investigation ruled out a focal cerebral lesion. Therapy with high doses (10,000 micrograms i.m. daily) of cobalamin alone was started, and the patient fully recovered in the following 72-hour. Control EEGs repeatedly performed days and weeks later showed progressive disappearance of the frontal interictal spiking, while the patient was on monotherapy with cobalamin (5,000 micrograms i.m. weekly). A month later the patient unfortunately discontinued replacement therapy and 13 weeks later he developed a fatal convulsive epileptic status. To our knowledge the association of ECS and cobalamin deficiency has not been previously reported.