Enhanced immobilization of hexa-arginine-tagged esterase on gold nanoparticles using mixed self-assembled monolayers

Mixed self-assembled monolayers (MSAMs) composed of diverse ligands offer a mechanism for the specific binding of biomolecules onto solid surfaces. In this study, we examined the formation of MSAMs on gold nanoparticles (AuNPs) and the immobilization of hexa-arginine-tagged esterase (Arg₆-esterase) on the surfaces of the resulting particles. The functionalization of AuNPs with MSAMs was achieved by introducing a mixture of tethering and shielding ligands into an AuNP solution. The formation of self-assembled monolayers (SAMs) on the AuNP surface was characterized by UV/visible spectroscopy, transmission electron microscopy, and Fourier-transform infrared spectroscopy. Arg₆-esterase was immobilized in a highly specific manner onto AuNPs treated with mixed SAMs (MSAM–AuNPs) by providing a shielding ligand which reduce the non-specific adsorption of enzymes caused by hydrophobic interaction compared to AuNPs treated with single-component SAMs (SSAM–AuNPs). Moreover, Arg₆-esterase immobilized on MSAM–AuNPs showed substantially enhanced catalytic activity up to an original activity compared to that on SSAM–AuNPs (58%).     

Fine needle aspiration cytology of epithelioid angiosarcoma. Report of a case with nuclear grooves and indentations

BACKGROUND: Angiosarcoma, because of its rarity and histologic diversity, has been a persistent challenge to cytopathologists. Epithelioid angiosarcomas are often confused with carcinomas, melanomas and other epithelioid sarcomas, both cytologically and histologically. Here we report the cytopathologic features of a case of epithelioid angiosarcoma with prominent nuclear grooves. CASE: A 61-year-old male visited the hospital with a neck mass. On fine needle aspiration the hypercellular smear, with a lymphocytic bloody background, showed variable-sized, round to oval, pleomorphic cells with frequent nuclear grooves, indentations and plump cytoplasm. The cells were diffusely scattered or loosely aggregated, with occasional acinar and cell-in-cell configuration. The case was diagnosed as epithelioid angiosarcoma on excisional biopsy. CONCLUSION: Whenever fine needle aspiration cytology, especially in the head and neck area, suggests malignancy composed predominantly of epithelioid cells, epithelioid angiosarcoma should be considered. The differential diagnosis should include metastatic tumors, such as carcinoma, melanoma and primary sarcoma. Nuclear grooves and indentations are 1 of the important diagnostic features of epithelioid angiosarcoma.     

Two highly divergent mitochondrial DNA lineages within Pseudogobio esocinus populations in central Honshu, Japan

To elucidate the genetic population structure of Pseudogobio esocinus (Cyprinidae) in central Honshu, Japan, we performed phylogeographic analysis based on partial mitochondrial cytochrome b gene sequences. We found not only differentiation of mtDNA lineages between basins (uncorrected p ≈ 2%), presumably associated with uplifting of the Suzuka Mountains in the early Pleistocene, but also more diverged two mtDNA lineages within basins (p ≈ 8%). No evidence of mtDNA introgression from related sympatric species was found. The phylogeographic history of these two lineages should be elucidated by further analysis based on the specimens of P. esocinus from the entire distribution range.     

A Single Recurrent Mutation in the 5'-UTR of IFITM5 Causes Osteogenesis Imperfecta Type V

Osteogenesis imperfecta (OI) is a heterogenous group of genetic disorders of bone fragility. OI type V is an autosomal-dominant disease characterized by calcification of the forearm interosseous membrane, radial head dislocation, a subphyseal metaphyseal radiodense line, and hyperplastic callus formation; the causative mutation involved in this disease has not been discovered yet. Using linkage analysis in a four-generation family and whole-exome sequencing, we identified a heterozygous mutation of c.-14C>T in the 5'-untranslated region of a gene encoding interferon-induced transmembrane protein 5 (IFITM5). It completely cosegregated with the disease in three families and occurred de novo in five simplex individuals. Transfection of wild-type and mutant IFITM5 constructs revealed that the mutation added five amino acids (Met-Ala-Leu-Glu-Pro) to the N terminus of IFITM5. Given that IFITM5 expression and protein localization is restricted to the skeletal tissue and IFITM5 involvement in bone formation, we conclude that this recurrent mutation would have a specific effect on IFITM5 function and thus cause OI type V.     

Cloning and functional annotation of rare mRNA species from drought-stressed hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

To better understand gene expression at very low levels, we have designed a method to eliminate cDNA clones representing abundant mRNAs. A cDNA library for drought-stressed hot pepper (Capsicum annuum) (Choi et al., 2002) underwent double-negative screening, once with probes made from a drought-stressed plant, the second time, with probes from a non-stressed plant. The cDNA clones that showed very weak or negative signals were isolated for further analysis, which resulted in 1399 cDNA clones from about 20,000 screened clones. When nucleotide sequences were determined, we obtained 1142 tentative unique genes, with a redundancy rate of 20.41%. An homology database search for the deduced amino acid sequences revealed that about 79% of the cDNA clones could not be matched for functioning with previously characterized sequences. However, when these uncategorized clones were subjected to classification based on functional domains, most could be cited. Notably, clones with possible functions in RNA transport, protein synthesis, and regulation of protein activity showed a dramatic increase in appearance while those coding for transposable elements, viral proteins, and plasmid proteins occupied a much smaller portion compared with those in theArabidopsis thaliana genome. In addition, those coding for proteins targeted to the endoplasmic reticulum were dramatically more abundant in our clones compared with theArabidopsis database.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

C-type natriuretic peptide system in rabbit colon

C-type natriuretic peptide (CNP) is mainly distributed in the brain and vascular endothelium and is considered to act as a local regulator in many tissues. The present study was aimed to determine the presence of CNP system and its biological function in rabbit colon. The serial dilution curves of tissue extracts were parallel to the standard curve of CNP-22. With gel permeation chromatography and reverse-phase HPLC, the major immunoreactive peak of CNP was observed at the same elution time corresponding to the synthetic CNP-53. The concentration of CNP in the mucosal layer of colon was 212.49 ± 30.44 pg/g tissue wet weight (n = 7), which was significantly higher than that in the muscular layer. The presence of CNP mRNA was also detected by RT-PCR and Southern blot analysis. Production of cGMP by the activation of particulate guanylyl cyclase stimulated by BNP and CNP was higher in membranes obtained from the muscular layer than from mucosal layer. More cGMP was produced by CNP than by ANP. Both natriuretic peptide receptor-A and -B mRNAs were detected by RT-PCR and specific binding sites to ¹²⁵I-[Tyr⁰]-CNP-22 were mainly localized to the muscular layer. Synthetic CNP inhibited basal tension, frequency and amplitude of basal motility of taenia coli of the right colon. This study showing the presence of CNP system and its biological function in colon suggests that endogenous CNP synthesized in the mucosal layer may have a paracrine function as a local regulator of colonic motility.     

Stimulation of Epstein-Barr virus-infected human B cell growth by physiological concentrations of 4-hydroxynonenal

We had previously shown that cyclosporin A (CsA) directly promoted the immortalization of Epstein-Barr virus (EBV)-infected human B cells (EBV-B cells) via an oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end-product of lipid peroxidation. We hypothesized that HNE may mediate a direct oxidative stress-promoting effect of CsA on EBV-B cells. HNE-protein adducts in CsA-treated EBV-B cell extracts were assayed immunochemically using a Slot-Blot method. Cell proliferation was assayed by [(3)H]-thymidine incorporation. EBV oncogene latent membrane protein-1 (LMP1) expression was assayed by using PE-conjugated anti-LMP1 antibody in flow cytometry. We found that CsA at 500 ng ml(-1) and 1000 ng ml(-1) significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units +/- SE) by 251.3 +/- 7.5 to 361.3 +/- 9.7 and 342.7 +/- 10.7, respectively (p < 0.05, n = 3). EBV-B cells treated with a physiological concentration of HNE (1 microM) for 0.5 and 1 h and cultured for 2 and 4 weeks showed significantly increased [(3)H]-thymidine incorporation. EBV-B cells treated with HNE (1 microM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher ( > 2.0 times) LMP1-positive cell population over the control. In conclusion, in accordance with our previous findings, we show that CsA treatment of EBV-B cells results in increased production of the lipid peroxidation reactive end-product HNE that directly promotes EBV-B cell proliferation and LMP1 expression. This observation provides evidence for further understanding the mechanism of CsA-induced oxidative stress on EBV-related post-transplant lymphoproliferative disorder (PTLD).