Reaction of neuronal nitric-oxide synthase with oxygen at low temperature. Evidence for reductive activation of the oxy-ferrous complex by tetrahydrobiopterin

The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at -30 degreesC. In the absence of substrate, the complex formed between ferrous NOS and O2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spectral characteristics as the oxyferrous complex of cytochrome P450 (lambda max = 416.5 nm). It then decomposed to the ferric state. The oxidation of the flavin components was much slower and could be observed only at temperatures higher than -20 degreesC. In the presence of substrate (L-arginine), another, 12-nm blue-shifted, intermediate spectrum was formed. The breakdown of the latter species resulted in the production of Nomega-hydroxy-L-arginine in a stoichiometry of maximally 52% per NOS heme. This product formation took place also in the absence of the reductase domain of NOS. Both formation of the blue-shifted intermediate and of Nomega-hydroxy-L-arginine required the presence of tetrahydrobiopterin (BH4). We propose that the blue-shifted intermediate is the result of reductive activation of the oxygenated complex, and the electron is provided by BH4. These observations suggest that the reduction of the oxyferroheme complex may be the main function of BH4 in NOS catalysis.     

Measuring the human body's microclimate using a thermal manikin

The human body is surrounded by a microclimate, which results from its convective release of heat. In this study, the air temperature and flow velocity of this microclimate were measured in a climate chamber at various room temperatures, using a thermal manikin simulating the heat release of the human being. Different techniques (Particle Streak Tracking, thermography, anemometry, and thermistors) were used for measurement and visualization. The manikin surface temperature was adjusted to the particular indoor climate based on simulations with a thermoregulation model (UCBerkeley Thermal Comfort Model). We found that generally, the microclimate is thinner at the lower part of the torso, but expands going up. At the head, there is a relatively thick thermal layer, which results in an ascending plume above the head. However, the microclimate shape strongly depends not only on the body segment, but also on boundary conditions: The higher the temperature difference between the surface temperature of the manikin and the air temperature, the faster the airflow in the microclimate. Finally, convective heat transfer coefficients strongly increase with falling room temperature, while radiative heat transfer coefficients decrease. The type of body segment strongly influences the convective heat transfer coefficient, while only minimally influencing the radiative heat transfer coefficient.     

Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. A dpp1Delta mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1Delta mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S. cerevisiae.     

Temperature reduction due to the application of phase change materials

Overheating is a major problem in many modern buildings due to the utilization of lightweight constructions with low heat storing capacity. A possible answer to this problem is the emplacement of phase change materials (PCM), thereby increasing the thermal mass of a building. These materials change their state of aggregation within a defined temperature range. Useful PCM for buildings show a phase transition from solid to liquid and vice versa. The thermal mass of the materials is increased by the latent heat. A modified gypsum plaster and a salt mixture were chosen as two materials for the study of their impact on room temperature reduction. For realistic investigations, test rooms were erected where measurements were carried out under different conditions such as temporary air change, alternate internal heat gains or clouding. The experimental data was finally reproduced by dint of a mathematical model.     

Hydroxyl radical production via the photo-Fenton reaction in the presence of fulvic acid

The photo-Fenton reaction, the reaction of photoproduced Fe(II) with H2O2 to form the highly reactive hydroxyl radical (OH*), could be an important source of OH* in sunlit natural waters. To determine if the photo-Fenton reaction is significant in mildly acidic surface waters, we conducted experiments simulating conditions representative of natural freshwaters using solutions of standard fulvic acid and amorphous iron oxide at pH 6.0. A probe method measuring 14CO2 produced by the reaction of 14C-labeled formate with OH* was used to detect small OH* production rates without otherwise influencing the chemical reactions occurring in the experiments. Net H2O2 accumulation was simultaneously measured using an acridinium ester chemiluminescence method. Measured losses of H2O2 by reaction with Fe(II) in dark experiments produced approximately the expected quantities of OH*. The difference between H2O2 accumulation in the presence and absence of Fe(III) in fulvic acid solutions exposed to light was interpreted as the loss of H2O2 by reaction with photoproduced Fe(II), consistent with measured OH* production rates. The Fe ligand desferrioxamine mesylate eliminated both OH* production and H2O2 photoloss induced by Fe. Our results imply that when Fe is a major sink of H2O2, the photo-Fenton reaction is likely to be the most important source of OH*, leading to a significant sink of organic compounds in a wide variety of sunlit freshwaters.     

Trafficking of newly synthesized surfactant protein A in isolated rat alveolar type II cells

We examined the synthesis, transport, and localization of surfactant protein A (SP-A) in primary cultures of alveolar type II cells. In type II cells maintained in culture for 6 h, 39% of the SP-A pool detected with an enzyme-linked immunosorbent assay (ELISA) was found in lamellar bodies (LBs). After 24 h in culture, 53% of the cellular SP-A pool was found in LBs. The absolute amount of SP-A in the LB compartment was almost identical at 6 and 24 h of culture. In contrast to the results obtained with ELISA, 35S labeling of newly synthesized SP-A revealed that less than 7% of the cellular SP-A pool was in LBs at either 6 or 24 h of culture. In the 6-h cultures, 17% of the total (i.e., cells and media) [35S]SP-A pool was extracellular. In the 24-h cultures, 70% of the [35S]SP-A pool was extracellular. The secretion of [35S]SP-A was blocked by brefeldin A at all times. When medium containing newly secreted [35S]SP-A was incubated with alveolar type II cells maintained in culture for 24 h, the protein was taken up and incorporated into the LB fraction. More than 80% of the internalized SP-A was associated with the LB compartment after a 6 h incubation. The uptake of [35S]SP-A was blocked at 4 degrees C and was promoted by addition of unlabeled SP-A at 37 degrees C. These findings support a pathway of extracellular routing of SP-A prior to its accumulation in LBs in cultured type II cells.     

Entirely intracanalicular meningioma: contrast-enhanced MR findings in a rare entity

We report an unusual case of an entirely intracanalicular meningioma in a 51-year-old woman. Contrast-enhanced MR images showed an enhancing lesion filling the lateral portion of the left internal auditory canal. Our findings suggest that meningiomas should be considered in the differential diagnosis of entirely intracanalicular masses.