Synthesis of 3,6α,16α-trihydroxy-1,3,5(10)-estratrien-17-one 6-hemisuccinate and [6,73H]-3,16α-dihydroxy-1,3,5(10)-estratrien-17-one

The preparation of high specific activity ³H labeled 16α-hydroxyestrone and of the 6-hemisuccinate of the ketol metabolite of estradiol is described. The synthetic procedures which were required to produce these essential ingredients of a radioimmunoassay for 16α-hydroxyestrone required multistep syntheses because of the multifunctional and labile nature of the metabolite.     

Poduction of Biotin by Microbial Transformation of dl-cis-Tetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI)

The conditions for biotin production were investigated. Urea was a more effective nitrogen source than ammonium chloride and ammonium sulfate. About 60% conversion from dl-cis-tetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI) to biotinol and biotin occurred using Corynebacterium sp. B–321. Strain M–6318 which derived from B–321 as a mutant incapable of assimilating n-alkane produced large amounts of dl-biotin from dl-TOPTI. The inability of the microbe to assimilate n-alkane resulted in repression of biotin degradation. Maximum conversion (80%) was obtained by growing cultures of strain M–6318 in the constant presence of n-paraffin.     

Identification of the siderophores from Vibrio hollisae and Vibrio mimicus as aerobactin

Abstract Hydroxamate siderophores were purified from low-iron cultures of Vibrio hollisae ATCC 33564 and Vibrio mimicus ATCC 33653. By a combination of ¹H and ¹³C NMR spectroscopy, fast atom bombardment mass spectrometry, and compositional analysis, both of the siderophores were identified as aerobactin, a citrate-based dihydroxamate siderophore, which is highly prevalent in species of the family Enterobacteriaceae . Four other clinical strains belonging to these species also produced aerobactin. In response to iron limitation, all strains expressed two high molecular mass outer membrane proteins. The protein with an apparent molecular mass of 77 kDa, which was common to all strains examined, may be the ferric aerobactin receptor.     

Nitrogen removal by co-occurring methane oxidation,denitrification, aerobic ammonium oxidation,and anammox

Quantification of microbial contaminant biodegradation based on stable isotope fractionation analysis (SIFA) relies on known, invariable isotope fractionation factors. The microbially induced isotope fractionation is caused by the preferential cleavage of bonds containing light rather than heavy isotopes. However, a number of non-isotopically sensitive steps preceding the isotopically sensitive bond cleavage may affect the reaction kinetics of a degradation process and reduce the observed (i.e., the macroscopically detectable) isotope fractionation. This introduces uncertainty to the use of isotope fractionation for the quantification of microbial degradation processes. Here, we report on the influence of bacterial cell density on observed stable isotope fractionation. Batch biodegradation experiments were performed under non-growth conditions to quantify the toluene hydrogen isotope fractionation by exposing Pseudomonas putida mt-2(pWWO) at varying cell densities to different concentrations of toluene. Observed isotope fractionation depended significantly on the cell density. When the cell density rose from 5 × 10⁵ to 5 × 10⁸cells/mL, the observed isotope fractionation declined by 70% and went along with a 55% decrease of the degradation rates of individual cells. Theoretical estimates showed that uptake-driven diffusion to individual cells depended on cell density via the overlap of the cells’ diffusion-controlled boundary layers. Our data suggest that biomass effects on SIFA have to be considered even in well-mixed systems such as the cell suspensions used in this study.     

Wound healing in the cornea of the chick embryo: III. The influence of pore size of millipore filters on the migration of isolated epithelial sheets in culture

The migratory activity of epithelia isolated from the cornea and the dorsal skin of chick embryos of different ages was examined in vitro. Five types of Millipore filters differing in pore size served as models to represent degrees of unevenness of the substrate instead of the natural wound beds of the corneal stroma and the dorsal dermis. Migration of the epithelium was rapid and extensive when the pore size was below 0.8 μm, but was inhibited or stopped when the pore size reached or exceeded 0.8 μm. The effect of surface properties of the substratum on the motility of the cell membrane and thus on the movement of the cells is discussed.     

Interaction of bilirubin with brain capillaries and its toxicity

The interaction of bilirubin with isolated brain capillaries, and the effect of bilirubin on the uptake of 2-deoxyglucose by the capillaries were investigated with 1-month-old Sprague-Dawley rats. The binding of bilirubin to the brain capillaries was observed only at a molar ratio of bilirubin to bovine serum albumin higher than 1.0. An absorption spectrum with a microspectrophotometer of the bilirubin-capillary complex showed a broad absorption maximum from 425 to 440 nm with a shoulder near 490 nm, but no shoulder was observed in the case of the bilirubin emulsion. The bilirubin binding activity was dependent on pH and temperature of the medium, but was not affected by sulfhydryl blocking agents such as p-chloromercuribenzoate and N-ethylmaleimide. Bilirubin saturation kinetics gave an apparent K_m for bilirubin of 61.7 μM. Release of the bilirubin from the brain capillaries to the medium was observed at 37°C but not at 4°C. The uptake of 2-deoxyglucose by the isolated brain capillaries was inhibited by bilirubin in a noncompetitive manner, giving an apparent K_i for the pigment of 137 μM.These results suggest that bilirubin may be responsible for the decreased 2-deoxyglucose uptake in the brain capillaries by disturbing the membrane structure of the capillary endothelial cells.     

Specific protein delivery to target cells by antibody-displaying bionanocapsules

Bionanocapsules (BNCs) are nanoparticles with a high biocompatibility composed of the L protein of the hepatitis B virus surface antigen. BNC can deliver bioactive molecules to hepatocytes efficiently and specifically. However, delivery is limited to hepatocytes and incorporation of proteins into BNC is quite troublesome. Here, in order to alter the specificity of BNC and to achieve efficient protein delivery, we developed engineered BNC displaying the ZZ domain of protein A and incorporating enhanced green fluorescent protein (EGFP) inside the particles using an insect cell expression system. The ZZ domain displayed on the surface of BNC binds to anti-epidermal growth factor receptor (EGFR) antibodies, allowing specific delivery of EGFP to HeLa cells. The engineered BNCs are a promising and powerful tool for efficient and cell-specific protein delivery.     

Radial dispersion of red blood cells in blood flowing through glass capillaries: the role of hematocrit and geometry

The flow properties of blood in the microcirculation depend strongly on the hematocrit (Hct), microvessel geometry, and cell properties. Previous in vitro studies have measured the radial displacement of red blood cells (RBCs) at concentrated suspensions using conventional microscopes. However, to measure the RBCs motion they used transparent suspensions of ghost red cells, which may have different physical properties than normal RBCs. The present study introduces a new approach (confocal micro-PTV) to measure the motion of labeled RBCs flowing in concentrated suspensions of normal RBCs. The ability of confocal systems to obtain thin in-focus planes allowed us to measure the radial position of individual RBCs accurately and to consequently measure the interaction between multiple labeled RBCs. All the measurements were performed in the center plane of both 50 and 100 microm glass capillaries at Reynolds numbers (Re) from 0.003 to 0.005 using Hcts from 2% to 35%. To quantify the motion and interaction of multiple RBCs, we used the RBC radial dispersion (D(yy)). Our results clearly demonstrate that D(yy) strongly depends on the Hct. The RBCs exhibited higher D(yy) at radial positions between 0.4 and 0.8R and lower D(yy) at locations adjacent to the wall (0.8-1R) and around the middle of the capillary (0-0.2R). The present work also demonstrates that D(yy) tends to decrease with a decrease in the diameter. The information provided by this study not only complements previous investigations on microhemorheology of both dilute and concentrated suspensions of RBCs, but also shows the influence of both Hct and geometry on the radial dispersion of RBCs. This information is important for a better understanding of blood mass transport mechanisms under both physiological and pathological conditions.     

Interleukin-1 beta up-regulates TACE to enhance alpha-cleavage of APP in neurons: resulting decrease in Abeta production

The proinflammatory cytokine interleukin (IL)-1β is up-regulated in microglial cells surrounding amyloid plaques, leading to the hypothesis that IL-1β is a risk factor for Alzheimer's disease. However, we unexpectedly found that IL-1β significantly enhanced α-cleavage, indicated by increases in sAPPα and C83, but reduced β-cleavage, indicated by decreases in sAPPβ and Aβ40/42, in human neuroblastoma SK-N-SH cells. IL-1β did not significantly alter the mRNA levels of BACE1, ADAM-9, and ADAM-10, but up-regulated that of TACE by threefold. The proform and mature form of TACE protein were also significantly up-regulated. A TACE inhibitor (TAPI-2) concomitantly reversed the IL-1β-dependent increase in sAPPα and decrease in sAPPβ, suggesting that APP consumption in the α-cleavage pathway reduced its consumption in the β-cleavage pathway. IL-1Ra, a physiological antagonist for the IL-1 receptor, reversed the effects of IL-1β, suggesting that the IL-1β-dependent up-regulation of α-cleavage is mediated by the IL-1 receptor. IL-1β also induced this concomitant increase in α-cleavage and decrease in β-cleavage in mouse primary cultured neurons. Taken together we conclude that IL-1β is an anti-amyloidogenic factor, and that enhancement of its signaling or inhibition of IL-1Ra activity could represent potential therapeutic strategies against Alzheimer's disease.     

Characterization of mycorrhizal fungi isolated from the threatened Cypripedium macranthos in a northern island of Japan: two phylogenetically distinct fungi associated with the orchid

We isolated Rhizoctonia-like fungi from populations of the threatened orchid Cypripedium macranthos. In ultrastructural observations of the septa, the isolates had a flattened imperforate parenthesome consisting of two electron-dense membranes bordered by an internal electron-lucent zone, identical to the septal ultrastructure of Rhizoctonia repens (teleomorph Tulasnella), a mycorrhizal fungus of many orchid species. However, hyphae of the isolates did not fuse with those of known tester strains of R. repens and grew less than half as fast as those of R. repens. In phylogenetic analyses, sequences for rDNA and internal transcribed spacer (ITS) regions of the isolates were distinct from those of the taxonomically identified species of Tulasnella. On the basis of the ITS sequences, the isolates clustered into two groups that corresponded exactly with the clades demonstrated for other Cypripedium spp. from Eurasia and North America despite the geographical separation, suggesting high specificity in the Cypripedium–fungus association. In addition, the two phylogenetic groups corresponded to two different plant clones at different developmental stages. The fungi from one clone constituted one group and did not belong to the other fungal group isolated from the other clone. The possibility of switching to a new mycorrhizal partner during the orchid’s lifetime is discussed.