Seasonal changes in serum leptin of the feral raccoon (Procyon lotor) determined by canine-leptin-specific ELISA

Several reports have been published on blood leptin concentrations in feral animals, including members of the Carnivora, using a commercially available multi-species radioimmunoassay (RIA) kit with anti-human leptin antibody. However, we observed weak immunoreactivity between recombinant canine leptin and anti-human leptin antibody, suggesting a limitation in the applicability of the RIA kit for leptin assays in Carnivora species. We tested the applicability of RIA and sandwich enzyme-linked immunosorbent assay (ELISA) with anti-canine leptin antibody to assay blood leptin in the dog (Canis familiaris) and the raccoon (Procyon lotor). When RIA was used for recombinant canine leptin and dog sera, values were much lower than those determined by ELISA at higher concentrations (>10 ng/ml), while rather higher at lower concentrations (<2 ng/ml). A similar discrepancy between the two methods was found for serum leptin concentrations in raccoons. Clear seasonal variations were observed by ELISA, but not by RIA, with high values in autumn (3.46+/-0.45 ng/ml) and low values in spring and summer (0.71+/-0.07 ng/ml). Serum leptin concentrations in raccoons correlated positively with their body weight (r=0.753) and body mass index (r=0.755), corroborating our previous findings of a strong positive correlation between serum leptin concentrations and body fat content in dogs. Thus, the canine leptin ELISA is useful for assays of dog and raccoon leptin, and blood leptin is a good marker of nutritional condition in the species of Carnivora assayed in this study.     

Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase

The reactivity of immobilized glucose oxidase-containing liposomes (IGOL) prepared in our previous work (Wang et al. [2003] Biotechnol Bioeng 83:444-453) was considerably improved here by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane as well as by entrapping inside the liposome's aqueous interior not only glucose oxidase (GO), but also catalase (CA), both from Aspergillus niger. CA was used for decomposing the hydrogen peroxide produced in the glucose oxidation reaction inside the liposomes. The presence of OmpF enhanced the transport of glucose molecules from the exterior of the liposomes to the interior. In a first step of the work, liposomes containing GO and CA (GOCAL) were prepared and characterized. A remarkable protection effect of the liposome membrane on CA inside the liposomes at 40 degrees C was found; the remaining CA activity at 72 h incubation was more than 60% for GOCAL, while less than 20% for free CA. In a second step, OmpF was incorporated into GOCAL membranes, leading to the formation of OmpF-embedded GOCAL (abbreviated GOCAL-OmpF). The activity of GO inside GOCAL-OmpF increased up to 17 times in comparison with that inside GOCAL due to an increased glucose permeation across the liposome bilayer, without any leakage of GO or CA from the liposomes. The optimal system was estimated to contain on average five OmpF molecules per liposome. Finally, GOCAL-OmpF were covalently immobilized into chitosan gel beads. The performance of this novel biocatalyst (IGOCAL-OmpF) was examined by following the change in glucose conversion, as well as by following the remaining GO activity in successive 15-h air oxidations for repeated use at 40 degrees C in an airlift bioreactor. IGOCAL-OmpF showed higher reactivity and reusability than IGOL, as well as IGOL containing OmpF (IGOL-OmpF). The IGOCAL-OmpF gave about 80% of glucose conversion even when the catalyst was used repeatedly four times, while the corresponding conversions were about 60% and 20% for the IGOL and IGOL-OmpF, respectively. Due to the absence of CA, IGOL-OmpF was less stable and resulted in drastically inhibited GO.     

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.     

Exploring the subsite-structure of vimelysin and thermolysin using FRETS-libraries

Vimelysin is a metalloproteinase with high activity at low temperature and an unusual resistance to organic solvents. Substrate specificities of vimelysin and thermolysin were examined using FRETS-libraries, revealing a significant difference at the P3' position: vimelysin preferred acidic amino acid residues, whereas thermolysin preferred basic residues. Homology modeling of vimelysin suggests that oppositely charged residues in the S3' subsites (R215 in vimelysin and D213 in thermolysin) may be responsible for this specificity difference. This hypothesis was confirmed by examining the R215D mutant of vimelysin, which showed a substrate specificity profile intermediate between thermolysin and vimelysin.     

Genetic analysis of diet-induced hypercholesterolemia in exogenously hypercholesterolemic rats

The exogenously hypercholesterolemic (ExHC) rat is an established strain that exhibits a polygenic syndrome of hypercholesterolemia after feeding on a cholesterol-containing diet, and the extent of this differs between male and female rats in the strain. The present study was performed to determine the genetic background of diet-induced hypercholesterolemia in ExHC rats. We used quantitative trait locus (QTL) analyses of the F2 progeny derived from ExHC and Brown-Norway rats. Rats were fed a diet containing 1% cholesterol, and a genome-wide scan was then performed. Significant QTLs for serum total cholesterol levels were revealed on chromosomes 5 and 14 in the vicinity of markers D5Rat95 and D14Rat43, having maximum logarithm of the odds scores of 6.0 and 5.8, respectively. A suggestive QTL for the trait was also detected on chromosome 3 at D3Rat140. In particular, the QTL on chromosome 5 was specific for female rats. These loci were novel QTLs for post-dietary serum total cholesterol levels. In addition, cross-mating analysis in F1 generations suggested that the responsiveness to dietary cholesterol in ExHC rats is partly attributable to X-linked inheritance. Identifying such genetic factors may be useful in predicting the risks associated with diet-induced hypercholesterolemia in humans.     

Mitochondrial small heat-shock protein enhances thermotolerance in tobacco plants

To clarify the role of mitochondrial small heat-shock protein (MT-sHSP) in the heat-shock response, we introduced the tomato (Lycopersicon esculentum) MT-sHSP gene under the control of the 35S promoter into tobacco (Nicotiana tabacum), and examined the thermotolerance of the transformed plants. Irrespective of the orientation, sense or antisense, of the gene, the transgenic plants exhibited a normal morphology and growth rate in the vegetative growth stage. When 4-week-old seedlings were exposed to sudden heat stress, the sense plants which overexpress the MT-sHSP gene exhibited thermotolerance, whereas the antisense plants in which the expression of the gene is suppressed exhibited susceptibility.     

Systematic single base-pair substitution analysis of DNA binding by the cAMP receptor protein in cyanobacterium Synechocystis sp. PCC 6803

The cAMP receptor protein SYCRP1 in cyanobacterium Synechocystis sp. PCC 6803 is a regulatory protein that binds to the consensus DNA sequence (5'-AAATGTGATCTAGATCACATTT-3') for the cAMP receptor protein CRP in Escherichia coli. Here we examined the effects of systematic single base-pair substitutions at positions 4-8 (TGTGA) of the consensus sequence on the specific binding of SYCRP1. The consensus sequence exhibited the highest affinity, and the effects of base-pair substitutions at positions 5 and 7 were the most deleterious. The result is similar to that previously reported for CRP, whereas there were differences between SYCRP1 and CRP in the rank order of affinity for each substitution.     

N-octyl-beta-valienamine up-regulates activity of F213I mutant beta-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease

Gaucher disease (GD) is the most common form of sphingolipidosis and is caused by a defect of beta-glucosidase (beta-Glu). A carbohydrate mimic N-octyl-beta-valienamine (NOV) is an inhibitor of beta-Glu. When applied to cultured GD fibroblasts with F213I beta-Glu mutation, NOV increased the protein level of the mutant enzyme and up-regulated cellular enzyme activity. The maximum effect of NOV was observed in F213I homozygous cells in which NOV treatment at 30 microM for 4 days caused a approximately 6-fold increase in the enzyme activity, up to approximately 80% of the activity in control cells. NOV was not effective in cells with other beta-Glu mutations, N370S, L444P, 84CG and RecNciI. Immunofluorescence and cell fractionation showed localization of the F213I mutant enzyme in the lysosomes of NOV-treated cells. Consistent with this, NOV restored clearance of 14C-labeled glucosylceramide in F213I homozygous cells. F213I mutant beta-Glu rapidly lost its activity at neutral pH in vitro and this pH-dependent loss of activity was attenuated by NOV. These results suggest that NOV works as a chemical chaperone to accelerate transport and maturation of F213I mutant beta-Glu and may suggest a therapeutic value of this compound for GD.     

Environment- and mutation-dependent aggregation behavior of Alzheimer amyloid beta-protein

The deposition of amyloid beta-protein in the brain is a fundamental process in the development of Alzheimerís disease; however, the mechanism underlying aggregation of amyloid beta-protein remains to be determined. Here, we report that a membrane-mimicking environment, generated in the presence of detergents or a ganglioside, is sufficient per se for amyloid fibril formation from soluble amyloid beta-protein. Furthermore, hereditary variants of amyloid beta-protein, which are caused by amyloid precursor protein gene mutations, including the Dutch (E693Q), Flemish (A692G) and Arctic (E693G) types, show mutually different aggregation behavior in these environments. Notably, the Arctic-type amyloid beta-protein, in contrast to the wild-type and other variant forms, shows a markedly rapid and higher level of amyloid fibril formation in the presence of sodium dodecyl sulfate or GM1 ganglioside. These results suggest that there are favorable local environments for fibrillogenesis of amyloid beta-protein.     

Interleukin-18 induces serum amyloid A (SAA) protein production from rheumatoid synovial fibroblasts

Interleukin-18 (IL-18) is a novel proinflammatory cytokine that was recently found in synovial fluids and synovial tissues from patients with rheumatoid arthritis (RA). To investigate the role of IL-18 in rheumatoid synovitis, the levels of IL-18 and serum amyloid A (SAA) were measured in synovial fluids from 24 patients with rheumatoid arthritis (RA) and 13 patients with osteoarthritis (OA). The levels of IL-18 and SAA in the synovial fluids were elevated in RA patients. In contrast, the levels of IL-18 in synovial fluids from OA patients were significantly lower compared to those of RA patients. SAA was not detected in synovial fluids from OA patients. The expression of SAA mRNA in rheumatoid synovial cells was also examined. SAA4 mRNA, which was constitutively expressed by rheumatoid synovial cells, was not affected by IL-18 stimulation. Although acute phase SAA (A-SAA, SAA1 + 2) mRNA was not detected in unstimulated synovial cells, its expression was induced by IL-18 stimulation. By immunoblot, we demonstrated that IL-18 induced the SAA protein synthesis from rheumatoid synovial cells in a dose-dependent manner. These results indicate a novel role for IL-18 in rheumatoid inflammation through the synovial SAA production.