Evaluation of the possibility that free fatty acids cause false-positive result in diarrhetic shellfish poisoning mouse bioassay in actual use

Midgut glands of bivalves are used for the mouse bioassay of diarrhetic shellfish poison (DSP). A large quantity of free fatty acids (FFAs) causes a false positive outcome in the assay. To examine whether this is likely to occus under conditions of actual use, we analyzed the contents of the FFAs in the enlarged midgut glands during gametogenesis of Japanese scallops Patinopecten yessoensis that had been caught at two points in Hunka Bay on March 27, 2006, because the content of FFAs may increase with activation of lipogenesis for gametogenesis. Fatty acids (FAs) were measured with fluorometric high-performance liquid chromatography after derivatization with 9-anthryldiazomethane. The total FFAs (14 : 0, 16 : 0, 18 : 0, 16 : 1, 18 : 1, 18 : 4, 20 : 5 and 22 : 6) represented 3.3-4.2 wt% of the lipid. The toxic FFAs accounted for 40-43 wt% of the total FFAs. Content of each FFA (18 : 1, 2.7-5.0 mg/g lipid; 18 : 4, tr.-2.0 mg/g; 20 : 4, n.d.; 20 : 5, 8.0-9.1 mg/g and 22 : 6, 2.0-2.1 mg/g) was lower than the lethal dose tentatively calculated from the relative toxicity. It appears that the likelihood of FFAs causing false-positives in the mouse bioassay is low if the sample is fresh and is extracted immediately after homogenizing.     

Laboratory and field measurements of dry deposition of sulfur dioxide onto Chinese loess surfaces

Laboratory and field measurements were conducted to examine dry deposition of SO2 onto Chinese loess surfaces using native soil sampled in the loess plateau, China. The field tests were employed in Beijing and Lanzhou, China, by directly measuring the dry deposition of SO2 on soil, which uses soil put on a collector as an SO2 passive sampling medium. In the laboratory, a high rate of uptake to SO2 deposition for Chinese soil surfaces due to the highly alkalinity was found. The uptake of SO2 deposition was dependent on the pH soil and relative humidity. Furthermore, we evaluated some factors that affect the measurement precision: response of SO2 uptake, repeatability, recovery factor, and variability associated with the weight and the surface coverage on the collectors. As a result, it was shown that the measurement precision was primarily related to the ratio of the SO2 deposition amount relative to the sulfur content of the original soil. This result was consistent with the field observations. The laboratory and field results indicated an excellent agreement on the SO2 uptake inherent in the results from the soil surfaces in different regions.     

Cloning and characterization of multigenes encoding the immunodominant 30-kilodalton major outer membrane proteins of Ehrlichia canis and application of the recombinant protein for serodiagnosis

A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp. , p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.     

Effect of a new rifamycin derivative, rifalazil, on liver microsomal enzyme induction in rat and dog

1. The effect of a new rifamycin derivative, rifalazil (KRM-1648), on liver microsomal enzyme induction was studied in rat and dog with repeated oral administration of the compound. Relative liver weight, cytochrome b5 and P450 contents, enzyme activities of NADPH-cytochrome c reductase, aniline hydroxylase, p-nitroanisole O-demethylase, aminopyrine N-demethylase, and erythromycin N-demethylase were measured. 2. In rat, rifalazil treatment at 300 mg/kg/day for 10 days increased cytochrome b5 content but it did not affect liver weight, P450 content or enzyme activities. In contrast, rifampicin and rifabutin increased relative liver weights, cytochrome contents and enzyme activities under similar conditions. 3. In dog, rifalazil did not affect any parameters at 30 or 300 mg/kg/day for 13 weeks. 4. These findings indicate that rifalazil is not an enzyme inducer in rat and dog. This property differs from other rifamycin derivatives such as rifampicin and rifabutin.     

Effect of amezinium metilsulfate on the finger skin vasoconstrictor response to cold stimulation and venoconstrictor response to noradrenaline

Orthostatic hypotension can be caused by an inadequate vasoconstrictor response. The effects of amezinium on vasoconstrictor response to sympathetic stimulation and to exogenous noradrenaline were investigated and compared with those of midodrine. In 8 healthy men, the following experiments were performed after a single oral dose of 10mg of amezinium, 2mg of midodrine or a placebo. First, finger-tip blood flow (FTBF) was recorded using a laser Doppler flowmeter before and during the contralateral hand cooling and a reduction ratio of FTBF was calculated as an index of the vasoconstrictor response. Second, dose-response curves to increasing doses (1-512ng/min) of noradrenaline infused locally to the dorsal hand vein were determined using a linear variable differential transformer. The reduction ratio of FTBF was significantly increased (p<0.05) by amezimium [placebo, 75.9 +/- 9.8(mean +/- SD)%; amezinium, 85.1 +/- 7.9%; midodrine, 78.1 +/- 9.3%]. The infusion rate of noradrenaline producing a half-maximum venoconstriction was significantly decreased (p<0.05) by amezinium (placebo, 40.6 +/- 33.9 ng/min; amezinium, 21.0 +/- 21.3 ng/min; midodrine, 33.2 +/- 31.5 ng/min). These findings indicate that amezinium increases the vasoconstrictor response to sympathetic stimulation and to noradrenaline in normal subjects, and this mechanism might contribute to the improvement by amezinium of the symptoms of orthostatic hypotension.     

Transforming growth factor-beta1 and protein kinase C synergistically activate the c-fos serum response element in myocardial cells

Stimulation of NMDA receptor increases NO-dependent cGMP synthesis. A significantly higher cGMP level was observed in hippocampus (about 8-fold increase) than in cerebral cortex (2.5-fold increase), as compared to basal value. The activity of NO synthase (NOS) and the basal level of cGMP in unstimulated slices were only slightly higher in hippocampus than in the cortex. About 60% of NOS total activity was found in the brain membrane fraction. The enzyme activity was not affected by glucocorticoids, even after 20 days of hydrocortisone treatment in dose of 40 mg/kg b.w. Brain ischemia induced by ligation of the both common carotid arteries in gerbils (Meriones unquiculatus) significantly increased NOS activity as well as cGMP and putrescine concentrations but decreased mono-ADP-ribosolation of proteins. Changes of NOS activity and cGMP concentration evoked by ischemia were decreased by specific inhibitor of the neuronal form of NOS (nNOS), 7-nitrodazole and the inhibitor of guanylate cyclase, LY 83,583 administered respectively in a dose of 25 mg/kg b.w. and 6 mg/kg b.w. 5 min. before ischemia. The inhibitor of nNOS, 7NI, did not change the concentration of putrescine during ischemia and reperfusion. Our results indicated that these inhibitors could protect the brain against excessive production of nitric oxide and biochemical processes dependent on it. In this way they may offer a new strategy in the therapy of brain ischemia.     

Fidelity levels of DNA polymerases in tumorigenic state cells and serially transplantable tumor cells

The crystal structure of a calcium-bound form of bovine annexin VI has been determined with X-ray diffraction data to 2.9 A by molecular replacement. Six Ca2+ ions were found, five in AB loops, one in a DE loop. Two loops (II-AB, which binds calcium, and V-AB, which does not) have conformations that differ significantly from those in calcium-free, human recombinant annexin VI. There are only small differences between the calci- and the apo-annexin VI in the rest of the molecule. Calcium by itself does not promote a major conformational change.     

Localization of unique functional determinants in the calmodulin lobes to individual EF hands

We have investigated the functional interchangeability of EF hands I and III or II and IV, which occupy structurally analogous positions in the native I-II and III-IV EF hand pairs of calmodulin. Our approach was to functionally characterize four engineered proteins, made by replacing in turn each EF hand in one pair by a duplicate of its structural analog in the other. In this way functional determinants we define as unique were localized to the component EF hands in each pair. Replacement of EF hand I by III reduces calmodulin-dependent activation of cerebellar nitric oxide synthase activity by 50%. Replacement of EF hand IV by II reduces by 60% activation of skeletal muscle myosin light chain kinase activity. There appear to be no major unique determinants for activation of these enzyme activities in the other EF hands. Replacement of EF hand III by I or IV by II reduces by 50-80% activation of smooth muscle myosin light chain kinase activity, and replacement of EF hand I by III or II by IV reduces by 90% activation of this enzyme activity. Thus, calmodulin-dependent activation of each of the enzyme activities examined, even the closely related kinases, is dependent upon a distinct pattern of unique determinants in the four EF hands of calmodulin. All the engineered proteins examined bind four Ca2+ ions with high affinity. Comparison of the Ca2+-binding properties of native and engineered CaMs indicates that the Ca2+-binding affinity of an engineered I-IV EF hand pair and a native I-II pair are similar, but an engineered III-II EF hand pair is intermediate in affinity to the native III-IV and I-II pairs, minimally suggesting that EF hands I and III contain unique determinants for the formation and function of EF hand pairs. The residues directly coordinating Ca2+ ion appear to play little or no role in establishing the different Ca2+-binding properties of the EF hand pairs in calmodulin.     

Enhancement of BTI degradation in pMOSFETs under high-frequency bipolar gate bias

Negative bias temperature (NBT) instability of p-MOSFETs with ultrathin SiON gate dielectric has been investigated under various gate bias configurations. The NBT-induced interface trap density (/spl Delta/N/sub it/) under unipolar bias is essentially lower than that under static bias, and is almost independent of the stress frequency up to 10 MHz. On the contrary, /spl Delta/N/sub it/ under bipolar pulsed bias of frequency larger than about 10 kHz is significantly enhanced and exhibits a strong frequency dependence, which has faster generation rate and smaller activation energy as compared to other stress configurations. The degradation enhancement is attributed to the energy to be contributed by the recombination of trapped electrons and free holes upon the silicon surface potential reversal from accumulation to inversion.     

The generalized boundary element approach to Burgers' equation

The generalized boundary element method is presented for the numerical solution of Burgers' equation. The new method is based on the set of boundary integral equations derived for each subdomain by using the fundamental solution for the linearized differential operator of the equation. The resulting system of quasi-non-linear equations is solved implicitly with use of a simple iterative procedure. The adaptability and the accuracy of the proposed method are demonstrated by three examples and a comparison of the numerical results with the exact solution or other existing solutions is shown for the first example.