Absorption of acylated anthocyanins in rats and humans after ingesting an extract of Ipomoea batatas purple sweet potato tuber

We evaluated the absorbability of anthocyanins in humans and rats administered with a beverage prepared from an extract of the tuber of purple sweet potato (Ipomoea batatas Cultivar Ayamurasaki), or with an anthocyanin concentrate. Two major anthocyanin components, cyanidin 3-O-(2-O-(6-O-(E)-caffeoyl-beta-D-glucopyranosyl)-beta-D-glucopyranoside)-5-O-beta-D-glucopyranoside) and peonidin 3-O-(2-O-(6-O-(E)-caffeoyl-beta-D-glucopyranosyl)-beta-D-glucopyranoside)-5-O-beta-D-glucopyranoside), were detected in the plasma and urine of both rats and humans by HPLC or liquid chromatography/mass spectrometry (LC/MS). The plasma concentration of anthocyanins in humans reached a maximum 90 minutes after ingestion, and the recovery of anthocyanins in the urine was estimated as 0.01-0.03%. These results indicate that acylated anthocyanins could be selectively absorbed after ingesting food.     

Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus beta-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.     

Nedd4 regulates egress of Ebola virus-like particles from host cells

Ebola virus budding is mediated by two proline-rich motifs, PPxY and PTAP, within the viral matrix protein VP40. We have previously shown that a Nedd4-like protein BUL1, but not Nedd4, positively regulates budding of type D retrovirus Mason-Pfizer monkey virus (J. Yasuda, E. Hunter, M. Nakao, and H. Shida, EMBO Rep. 3:636-640, 2002). Here, we report that the cellular E3 ubiquitin ligase Nedd4 regulates budding of VP40-induced virus-like particles (VLPs) through interaction with the PPxY motif. Mutation of the active site cysteine (C894A), resulting in abrogation of ubiquitin ligase activity, impaired the function of Nedd4 on budding. In addition, the WW domains of Nedd4 are essential for binding to the viral PPxY motif, and a small fragment of Nedd4 containing only WW domains significantly inhibited Ebola VLP budding in a dominant-negative manner. Our findings suggest that the viruses containing PPxY as an L-domain motif specifically use E3 in the process of virus budding. We also examined the effects of overexpression of Tsg101 and its mutant. As expected, Tsg101 enhanced VP40-induced VLP release, and TsgDeltaC, which lacks its C-terminal half, inhibited VLP release. These results indicate that Nedd4, together with Tsg101, plays an important role in Ebola virus budding.     

Cytologic features of hyalinizing trabecular adenoma of the thyroid

OBJECTIVE: To clarify the cytologic findings of hyalinizing trabecular adenoma (HTA) in order to reduce erroneous diagnoses of papillary carcinoma. STUDY DESIGN: Review of aspiration cytologic smears of 16 HTA cases and comparison with those of 20 papillary carcinoma cases. RESULTS: The smears from HTA were slightly cellular, and 5 of 16 cases were insufficient for evaluation. Vague, curved nuclear palisading, radiating arrangement surrounding hyaline materials and yellow bodies were observed in 9 (81.8%) of 11 cases that had sufficient material. The tumor cells were mainly spindled; elongated, polygonal and stellate cells were also seen. In 9 of 11 cases, tumor cells with cytoplasmic processes were occasionally observed. The cytoplasm was faintly stained and somewhat filamentous. The cell border was indistinct. Neither papillary nor follicular structures were seen. Intranuclear cytoplasmic inclusions were identified in 100% of HTA and 75% of papillary carcinomas. The incidences of nuclear grooves in HTA and papillary carcinoma were 81.8% and 100%, respectively. CONCLUSION: Cytologic findings indicating HTA are vague, curved nuclear palsiading; radiating arrangement surrounding hyaline material; elongated cells; cell processes; ill-defined cell border; faintly stained and filamentous cytoplasm; yellow bodies; and hyaline material in the background. All are useful cytologic characteristics in distinguishing HTA from papillary carcinoma. A lack of papillary architecture and sheetlike arrangement may also suggest HTA rather than papillary carcinoma.     

Identification of functional domains of the Escherichia coli SeqA protein

The Escherichia coli SeqA protein, a negative regulator of chromosomal DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated G-mA-T-C sequences in the replication origin, oriC. In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation. To study the functional domains responsible for the DNA-binding and self-association activities, we performed a deletion analysis of the SeqA protein and found that the N-terminal (amino acid residues 1-59) and the C-terminal (amino acid residues 71-181) regions form structurally distinct domains. The N-terminal domain, which is not involved in DNA binding, has the self-association activity. In contrast, the C-terminal domain, which lacks the self-association activity, specifically binds to the hemimethylated G-mA-T-C sequence. Therefore, two essential SeqA activities, self-association and DNA-binding, are independently performed by the structurally distinct N-terminal and C-terminal domains, respectively.     

Secondary structure analyses of protein films on gold surfaces by circular dichroism

In order to analyze the secondary structures of protein molecules adsorbed on gold surfaces, circular dichroism (CD) spectra were measured and the secondary structure contents of protein ultra-thin films were estimated quantitatively. A disulfide group was introduced to cytochrome b(562) (cyt.b562), which is a water-soluble b-type heme protein. The cyt.b562 molecules self-assembled to form an ultra-thin protein film both on a gold substrate modified with 2,2(')-dithiodiacetic acid and on a bare gold surface. CD measurements were carried out both in solution and in air, and these results were compared. The protein denaturation was partially prevented, not only in solution but also in air, by both the modification of the substrate and the introduction of the anchor group to the protein molecule. The secondary structure contents of ultra-thin protein films on flat gold surfaces were observed for the first time both in solution and in air by CD spectra.     

Protective effects of flavonoids on the cytotoxicity of linoleic acid hydroperoxide toward rat pheochromocytoma PC12 cells

The protective effects of nine flavonoids, including apigenin, eriodictyol, 3-hydroxyflavone, kaempherol, luteolin, quercetin, rutin, and taxifolin (Table 1), on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat pheochromocytoma PC12 cells were examined. The cytotoxicity was assessed by the trypan blue exclusion test and so-called MTT assay. When cells were preincubated with each flavonoid prior to LOOH exposure, quercetin, 3-hydroxyflavone, or luteolin decreased LOOH cytotoxicity toward undifferentiated cells, while only luteolin decreased efficiently LOOH cytotoxicity toward differentiated cells. On the other hand, when cells were coincubated with each flavonoid and LOOH, kaempherol, eriodictyol, quercetin, 3-hydroxyflavone, luteolin, or taxifolin decreased LOOH cytotoxicity toward undifferentiated and differentiated cells. On both preincubation prior to LOOH exposure and coincubation with LOOH, luteolin acted as the most efficiently protective agent against LOOH cytotoxicity. Further, these flavonoids showed protective effects on coincubation rather than preincubation. Flow cytometry using the fluorescence probe 2',7'-dichlorofluorescin diacetate revealed that LOOH increases the intracellular level of reactive oxygen species in undifferentiated cells in a dose-dependent manner, and that desferrioxamine mesylate suppresses the LOOH-induced increase in the level. These flavonoids suppress the LOOH-induced increase. Further, the protective effect of flavonoids on LOOH cytotoxicity correlates with the suppression of the LOOH-induced increase. These results suggest that such flavonoids are beneficial for neuronal cells under oxidative stress.     

Imprint cytology of epithelioid angiomyolipoma in a patient with tuberous sclerosis. A case report

BACKGROUND: Angiomyolipoma composed predominantly of epithelioid cells has been referred to as epithelioid angiomyolipoma. As this subtype shows considerable cellular atypia, it may be erroneously diagnosed as malignant epithelioid tumor, such as renal cell carcinoma and hepatocellular carcinoma. So far, only one report describing the cytologic findings of epithelioid angiomyolipoma has been documented, and epithelioid angiomyolipoma occurring in the peritoneal cavity has not been reported. CASE: Eleven years after resection of a renal epithelioid angiomyolipoma in a 34-year-old male with tuberous sclerosis, a tumor appeared in the peritoneal cavity and three masses in the liver. The intraoperative smears imprinted from part of the peritoneal mass revealed many large, atypical cells. The well-preserved atypical cells showed abundant, round to polyhedral, granular cytoplasm. Bizarre, giant nuclei with hyperchromasia and huge nucleoli were occasionally seen. Intranuclear cytoplasmic inclusions and mitotic figures were occasionally observed. As the epithelioid cells were markedly pleomorphic, we could not rule out hepatocellular carcinoma, cytologically and histologically, in the intraoperative consultation. In permanent sections the tumor was composed predominantly of epithelioid cells showing an alveolar pattern or sheetlike arrangement. Mitotic counts were zero to one per 10 high-power fields. Immunohistochemically, the epithelioid tumor cells were positive for vimentin, alpha-smooth muscle actin and HMB-45, consistent with epithelioid angiomyolipoma. MIB-1-labeling index was 1.6%. CONCLUSION: When one sees atypical epithelioid tumor cells in a tuberous sclerosis patient during an intraoperative consultation, one must consider epithelioid angiomyolipoma.     

Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex

The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity. The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.     

A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.