Inhibition kinetics and affinity labeling of bacterial squalene:hopene cyclase by thia-substituted analogues of 2, 3-oxidosqualene

Five sulfur-containing analogues of 2,3-oxidosqualene (OS) were evaluated as inhibitors of squalene:hopene cyclase (SHC) from Alicyclobacillus acidocaldarius. In these analogues, sulfur replaces carbons at C-6, C-10, C-14, C-18, or C-19 of OS. Each analogue was a submicromolar inhibitor of SHC with IC50 values ranging from 60 to 570 nM. Enzyme inhibition kinetic analysis was performed using homogeneous recombinant A. acidocaldarius SHC. While analogues 9 (S-14, Ki = 109 nM, kinact = 0.058 min-1) and 11 (S-19, Ki = 83 nM, kinact = 0.054 min-1) were time-dependent inhibitors of SHC, analogues 7 (S-6, Ki = 127 nM) and 8 (S-10, Ki = 971 nM) showed no time dependency with SHC. Analogue 10 (S-18) was the most potent inhibitor and showed time-dependent irreversible inhibition (Ki = 31 nM, kinact = 0.071 min-1). Kinetic analysis for the five analogues with purified rat liver OSLC was conducted to compare the vertebrate and prokaryotic enzymes. Affinity labeling experiments, using either [17-3H]10 or [22-3H]10 with crude and with pure recombinant SHC, clearly showed specific labeling. A single major radioactive band at 72 kDa on SDS-PAGE indicated that irreversible covalent modification of SHC had occurred. These results suggest that the presence of sulfur at C-18 of OS can interrupt the cyclization and that an intermediate partially cyclized cation may be captured by a nucleophilic residue of the SHC active site.     

Reduction of ischemic brain injury by topical application of glial cell line-derived neurotrophic factor after permanent middle cerebral artery occlusion in rats

BACKGROUND AND PURPOSE: Glial cell line-derived neurotrophic factor (GDNF) plays important roles in the survival and recovery of some mature neurons under pathological conditions. However, the effect of GDNF in ameliorating ischemic brain injury has not been well documented. Therefore, we investigated a possible effect of GDNF on the changes of infarct size, brain edema, DNA fragmentation, and immunoreactivities for caspases after permanent middle cerebral artery occlusion (MCAO) in rats. METHODS: For the estimation of ischemic brain injury, we calculated the infarct size of MCA region and also measured the brain water content as edema formation at 24 hours after the MCAO. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick labeling (TUNEL) staining was performed for the detection of DNA fragmentation. Immunoreactivities for caspase-1 (ICE), caspase-2 (Nedd-2), and caspase-3 (CPP32) were stained. RESULTS: Both infarct size and brain edema after permanent MCAO were significantly reduced by topical application of GDNF (48% and 30% decreases, P=0.01). TUNEL staining and immunoreactivities for caspases were markedly induced at 12 hours after permanent MCAO in the vehicle-treated animals. However, the spatial distribution of those immunohistochemically positive cells was dissociative in each caspase. Induction of TUNEL staining and immunoreactivities for caspases-1 and -3 was greatly reduced with GDNF treatment, whereas the reduction of caspase-2 staining was only minimum. CONCLUSIONS: These data suggest that the reduction of infarct size and brain edema by GDNF was greatly associated with the reduction of DNA fragmentation and apoptotic signals predominantly through caspases-1 and -3 cascades.     

Model-based surgical planning and simulation of cranial base surgery

Plastic skull models of seven individual patients were fabricated by stereolithography from three-dimensional data based on computed tomography bone images. Skull models were utilized for neurosurgical planning and simulation in the seven patients with cranial base lesions that were difficult to remove. Surgical approaches and areas of craniotomy were evaluated using the fabricated skull models. In preoperative simulations, hand-made models of the tumors, major vessels and nerves were placed in the skull models. Step-by-step simulation of surgical procedures was performed using actual surgical tools. The advantages of using skull models to plan and simulate cranial base surgery include a better understanding of anatomic relationships, preoperative evaluation of the proposed procedure, increased understanding by the patient and family, and improved educational experiences for residents and other medical staff. The disadvantages of using skull models include the time and cost of making the models. The skull models provide a more realistic tool that is easier to handle than computer-graphic images. Surgical simulation using models facilitates difficult cranial base surgery and may help reduce surgical complications.     

Thin oxide films of silicon by high pressure oxidation

High pressure oxidation of silicon was performed at an oxidation temperature range of 650 to 800°C for thin oxide films with about 300 å thickness. The index of refraction was dependent on an oxidation temperature but independent of the oxidation pressure as 1.475 at 750°C. The dielectric breakdown strength of the oxide film was measured by the voltage ramping,method. The fixed oxide charge about 1.0 × 10¹¹ cm^?2 was also measured from the high frequency C-V measurement. The pulse scanning C-V measurement technique was used to measure the minority carrier generation rate in the depleted surface. The surface generation velocity was slightly dependent on the oxidation temperature and indicated that the fast surface states increased with decreasing oxidation temperature.     

Lung phosphatidylcholine and fasting in male and female rats

Lung from male and female rats fasted for 4 days were used. Phospholipid, phosphatidylcholine and its molecular species were analyzed in lungs from these rats and effects of fasting upon the biosynthesis of phosphatidylcholine in lungs from both sexes were determined using radioactive choline. The molecular species of phosphatidylcholine in both male and female rats did not differ with fasting except the monoenoic species. Incorporation of choline into phosphatidylcholine in both male and female rats significantly increased after fasting, but distribution of radioactivity in phosphatidylcholine yielded similar values in each group. These results suggest that the decrease of saturated phosphatidylcholine content after fasting may be not due to specific change in saturated phosphatidylcholine.     

Sero-epidemiological study on equine influenza in Japan

Ten male subjects performed five maximal treadmill running tests at 7 mph. Tests included two (test-retest) progressive, step increment (2 1/2% grade elevation), discontinuous tests (DCT); a progressive, step increment, continuous test (ct) and two constant load tests (CL and CL +2 1/2%). A DCT test was performed first for establishment of peak elevation levels as constant load tests were performed at the peak elevation level attained (CL) and at a level 2 1/2% higher (CL + 2 1/2%). The second DCT test and the remaining three tests were administered randomly. Peak preformance capability (operationally defined as duration at highest grade elevation) was markedly reduced during progressive tests as compared with constant load tests. There was a similar reduction in peak performance capability during the CT test as compared with DCT test. Maximal oxygen uptake (VO2max) was quite similar among the various tests. It was concluded that VO2max attained during progressive, step increment, tests is unaffected by cumulative submaximal work. Discontinuous and continuous progressive tests provide similar VO2max results.     

Mechanical properties of molybdenum neutron-irradiated at a high temperature

Molybdenum specimens prepared by two processes, powder-metallurgy (PM) and electron-beam melting (EB), were irradiated to a fast neutron fluence of 2.74 × 10²⁴n/m² (E_n? 1 MeV) at about 600°C (873 K), and their mechanical properties were studied in detail. It was shown that the degree of irradiation embrittlement in EB-Mo was smaller than that in PM-Mo, which might be caused by stronger grain-boundaries and probably smaller irradiation-hardening in the former. From the relation between the recovery of ductility and microstructural changes in post-irradiation annealed PM-Mo at 800 (1073 K), 1000 (1273 K) and 1200°C (1473 K), it was concluded that the recovery resulted from a decrease of irradiation hardening due to a rearrangement and a disappearance of depleted-zones, dislocation-loops and voids in order with increasing annealing temperature. An anomalous mode of fracture was observed in as-irradiated specimens, which consisted of inhomogeneous deformation, then brittle fracture not at the center but at the root of the deformation neck. This mode was observed in a narrow temperature range near the DBTT. A possible mechanism is discussed.     

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis

Three crystalliferous (Cry+) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry-) mutants, either induced or spontaneously derived from a single Cry+ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry+ and Cry- variants were analyzed by both a cleared lysate- and a modified Eckhardt lysate-electrophoresis technique. All of the Cry- mutants derived from the Cry+ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry- variants. All three Cry+ strains, including the parent of the Cry- strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry+ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry- derivatives differed in other respects from the pattern for their parent strain. The various Cry+ and Cry- strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.     

Incidence of tics in the offspring of childhood tiquers: a controlled follow-up study

A five-year follow-up study of children whose parents had manifested tic in their childhood was carried out to see if they are more prone to develop tic than control children. The prevalence of tics was 20 per cent in the index group and 10 per cent in the controls, a statistically significant difference. This result could not be explained by the children's imitation of their parents. The mothers of the index children and 1000 unselected mothers were also compared for the prevalence of psychological and psychosomatic symptoms when their children were three years of age. There was no indication that the former were more psychologically distressed. It is suggested that there is a genetic basis for the susceptibility to develop tic.     

Characteristics of chlorophyll formation of the aerial microalga Coelastrella striolata var. multistriata and its application for environmental biomonitoring

The growth and ammonium uptake of the aerial microalga Coelastrella striolata var. multistriata, which was isolated from the surface of rocks, were characterized in this study. The specific growth rate of the alga was mu=0.3 d(-1), as calculated in the growth logarithmic phase. The algal cells were able to remove almost 100% of the ammonium ions from medium in 5 d, with the removal rate of ammonium-N being 0.4 mg/l/h. It was shown that the alga has a unique ability to be a reddish orange to green color depending on the nitrogen source concentration in the medium. Astaxanthin, adonixanthin, canthaxanthin, and beta-carotene were found in the reddish orange cells of the alga. The assessment of water pollution was attempted using this aerial microalga. When the reddish orange alga was incubated in the experimental medium with added ammonium-, nitrate-, or urea-N as a nitrogen source, an approximately linear relationship existed between the nitrogen concentration and chlorophyll formation. Using the chlorophyll formation of the alga, for example, it was possible to estimate spectrophotometrically the total nitrogen content in water collected from aquatic systems. Biofunctional materials for environmental biomonitoring using photosynthetic microorganisms are called green devices in this study.