Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT2 receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.     

Techniques of SNP genotyping and remote sensing applied for elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype

We present a conceptual framework for applying techniques of SNP genotyping as a molecular biological approach and remote sensing as an ecological approach to elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype. Additionally, we discuss the obstacles that frustrate our efforts to identify how the human genome encodes the complex phenotype and suggest the use of computational methods designed for knowledge discovery within hereditary database.     

Surface-Based Analysis on Shape and Fractional Anisotropy of White Matter Tracts in Alzheimer's Disease

Background

White matter disruption has been suggested as one of anatomical features associated with Alzheimer''s disease (AD). Diffusion tensor imaging (DTI), which has been widely used in AD studies, obtains new insights into the white matter structure.

Methods

We introduced surface-based geometric models of the deep white matter tracts extracted from DTI, allowing the characterization of their shape variations relative to an atlas as well as fractional anisotropy (FA) variations on the atlas surface through large deformation diffeomorphic metric mapping (LDDMM). We applied it to assess local shapes and FA variations of twenty-three deep white matter tracts in 13 patients with AD and 19 healthy control subjects.

Results

Our results showed regionally-specific shape abnormalities and FA reduction in the cingulum tract and the sagittal stratum tract in AD, suggesting that disruption in the white matter tracts near the temporal lobe may represent the secondary consequence of the medial temporal lobe pathology in AD. Moreover, the regionally-specific patterns of FA and shape of the white matter tracts were shown to be of sufficient sensitivity to robustly differentiate patients with AD from healthy comparison controls when compared with the mean FA and volumes within the regions of the white matter tracts. Finally, greater FA or deformation abnormalities of the white matter tracts were associated with lower MMSE scores.

Conclusion

The regionally-specific shape and FA patterns could be potential imaging markers for differentiating AD from normal aging.     

Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog.

Capsiate is a nonpungent capsaicin analog, a recently identified principle of the nonpungent red pepper cultivar CH-19 Sweet. In the present study, we report that 2-wk treatment of capsiate increased metabolic rate and promoted fat oxidation at rest, suggesting that capsiate may prevent obesity. To explain these effects, at least in part, we examined uncoupling proteins (UCPs) and thyroid hormones. UCPs and thyroid hormones play important roles in energy expenditure, the maintenance of body weight, and thermoregulation. Two-week treatment of capsiate increased the levels of UCP1 protein and mRNA in brown adipose tissue and UCP2 mRNA in white adipose tissue. This dose of capsiate did not change serum triiodothyronine or thyroxine levels. A single dose of capsiate temporarily raised both UCP1 mRNA in brown adipose tissue and UCP3 mRNA in skeletal muscle. These results suggest that UCP1 and UCP2 may contribute to the promotion of energy metabolism by capsiate, but that thyroid hormones do not.     

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

Defense response of a pepper cultivar cv. Sy-2 is induced at temperatures below 24°C

Temperature is one of the most important environmental factors that influence plant growth and development. Recent studies imply that plants show various responses to non-extreme ambient temperatures. Previously, we have found that a pepper cultivar cv. Sy-2 (Capsicum chinense) shows developmental defects at temperatures below 24°C. In this study, to gain new insights into the temperature sensitivity of cv. Sy-2, temperature-sensitive genes were screened using microarray techniques. At restrictive temperature of 20°C, almost one-fourth of the 411 up-regulated genes were defense related or predicted to be defense related. Further expression analyses of several defense-related genes showed that defense-related genes in cv. Sy-2 were constitutively expressed at temperatures below 24°C. Moreover, accumulation of high level of salicylic acid (SA) in cv. Sy-2 grown at 20°C suggests that the defense response is activated in the absence of pathogens. To confirm that the defense response is induced in cv. Sy-2 below 24°C, we evaluated the resistance to biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria and necrotrophic fungal pathogen Cercospora capsici. Cv. Sy-2 showed enhanced resistance to X. campestris pv. vesicatoria, but not to C. capsici.     

Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS

Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.     

Identification of a neural cell specific variant of microtubule-associated protein 4

The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a region rich in proline and basic residues (proline-rich region). We searched the bovine adrenal gland for MAP4 isoforms, and identified a novel variant lacking 72 consecutive amino acid residues within the proline-rich region, as compared with the full-length MAP4. The amino acid sequence of the missing region was highly conserved (about 85% identity/similarity) among the corresponding regions of bovine, human, mouse, and rat MAP4, which suggested the functional significance of this region. A comparison of the genomic sequence with the cDNA sequence revealed that the missing region is encoded by a single exon. A MAP4 variant cDNA homologous to the bovine form was also detected in rat cells, suggesting that the new variant can be generated by alternative splicing, not only in bovine but also in other mammalian species. The mRNA expression of the novel isoform was restricted to the brain and the adrenal medulla, suggesting that this isoform is specific to a certain cell type. Using a bacterially expressed fragment corresponding to the microtubule-binding domain of the novel isoform, we analyzed its in vitro characteristics. The fragment induced microtubule assembly and bound to preformed microtubules, but the activities were slightly lower than those of the conventional MAP4 fragment, which carries the full-length proline-rich region. The microtubules assembled in the presence of the fragment failed to be bundled. Instead, a constant spacing between neighboring microtubules was observed.