Structural characterisation of printable noble metal/poly(vinyl-alcohol) nanocomposites for optical applications.

In order to enable exploitation of noble metal/poly(vinyl-alcohol) nanocomposites for device fabrication, solutions of poly(vinyl-alcohol) suitable for piezo-driven inkjet printing techniques are identified and discussed in terms of their material properties. The printable poly(vinyl-alcohol) medium is then exploited as a host material through the formation of silver or gold nanoparticles in order to create nanocomposites that exhibit a surface plasmon resonance behaviour associated with the small metallic inclusions. To mitigate some of the material redistribution effects associated with the drying of printed droplets containing finely divided materials, the metallic nanoparticles are formed after the printing and drying process is completed, by way of an in situ reduction of an appropriate metal salt by the poly(vinyl-alcohol)-host matrix itself, which takes place at modest temperatures compatible with most substrate materials. An obvious application for such nanocomposites is in optical elements whereby the surface plasmon resonance associated with the metal is the functional aspect of devices such as sensors or active optical elements. High Resolution Transmission Electron Microscopy was used to examine the dimensions, distribution, morphology and crystal structure of the silver and gold nanoparticles in detail allowing discussion of their suitability for these applications and what further optimisation may be necessary to adequately control their formation.

Redox properties of nanostructured lanthanide-doped ceria spheres prepared by microwave assisted hydrothermal homogeneous co-precipitation.

In this work, nanostructured LnxCe(1-x)O2-δ (Ln: Gd and Pr; x = 0.1 and 0.2) spheres were synthesized by microwave assisted hydrothermal homogeneous co-precipitation and their properties were characterized by synchrotron radiation XRD, X-ray absorption near-edge spectroscopy (XANES) and scanning and high-resolution electron microscopy (SEM and HRTEM). In situ XRD and XANES experiments were carried out under reducing and oxidizing conditions in order to investigate the redox behaviour of these materials. The nanostructured mixed oxide spheres were found to have a cubic crystal structure (Fm3m space group). The spheres were composed of nanoparticles with an average crystallite size of about 10 nm. The Ln(0.1)Ce(0.9)O2-δ compositions exhibited the highest specific surface area (∼ 60 m(2) g(-1)). In situ XRD experiments showed an increase in lattice parameters upon reduction, which was attributed to the reduction of Ce(4+) and Pr(4+) cations to Ce(3+) and Pr(3+), which have larger radii, and to the associated increase in VO concentration. This increase in lattice parameters was considerably more pronounced for PrDC than GDC, and was explained by the considerably larger change in ionic radius for Pr upon reduction. XANES absorption experiments at the Ce and Pr L3-edge showed that the changes observed upon reduction of the Pr-containing samples resulted mostly from the formation of Pr(3+) rather than Ce(3+), and supported the previously reported proposal that Pr(3+) has a stabilizing effect on Ce(4+).

Soybean meal alters autochthonous microbial populations, microvilli morphology and compromises intestinal enterocyte integrity of rainbow trout, Oncorhynchus mykiss (Walbaum).

Abstract Rainbow trout were fed either a diet containing fishmeal (FM) as the crude protein source or a diet containing 50% replacement with soybean meal (SBM) for 16 weeks. An enteritis-like effect was observed in the SBM group; villi, enterocytes and microvilli were noticeably damaged compared with the FM group. The posterior intestine microvilli of SBM-fed fish were significantly shorter and the anterior intestine microvilli significantly less dense than the FM-fed fish. Electron microscopy confirmed the presence of autochthonous bacterial populations associated with microvilli of both fish groups. Reduced density of microvilli consequently led to increased exposure of enterocyte tight junctions, which combined with necrotic enterocytes is likely to diminish the protective barrier of the intestinal epithelium. No significant differences in total viable counts of culturable microbial populations were found between the groups in any of the intestinal regions. A total of 1500 isolates were tentatively placed into groups or genera, according to standard methods. Subsequent partial 16S rRNA sequencing revealed species that have not been identified from the rainbow trout intestine previously. Compared with the FM group levels of Psychrobacter spp. and yeast were considerably higher in the SBM group; a reduction of Aeromonas spp. was also observed.

Association of UNP, a ubiquitin-specific protease, with the pocket proteins pRb, p107 and p130.

The murine Unp gene encodes a widely expressed ubiquitin-specific protease. The predicted sequence of the UNP protein features motifs common to viral oncoproteins through which these proteins interact with the retinoblastoma gene product pRb, as well as the related 'pocket proteins' p107 and p130. We have explored the possibility that UNP interacts with pocket proteins, and report here that such associations can be detected in vitro and in cells. Associations of UNP and pocket proteins are sensitive to site-directed mutations in a manner directly analogous to those documented in viral oncoproteins. We conclude that within cells UNP does physically associate with pRb, and can also associate with p107 and p130.

The de-ubiquitinating enzyme Unp interacts with the retinoblastoma protein.

The ubiquitin pathway is involved in the proteolytic turnover of many short-lived cellular regulatory proteins. Since selective degradation of substrates of this system requires the covalent attachment of a polyubiquitin chain to the substrates, degradation could be counteracted by de-ubiquitinating enzymes (or isopeptidases) which selectively remove the polyubiquitin chain. Unp is a human isopeptidase with still poorly understood biological functions. Here, we show that cellular Unp specifically interacts with the retinoblastoma gene product (pRb).

Imidacloprid treatment of marsupials for fleas (Pygiopsylla hoplia).

Two eastern barred bandicoots (Perameles gunnii), two eastern quells (Dasyunrus viverrinus), four fat-tailed dunnarts (Sminthopsis crassicaudata), a Leadbeater's possum (Gymnobelideus leadbeateri), a yellow-bellied glider (Petaurus australis), and a ring-tailed possum (Pseudocheirus peregrinus) were treated with imidacloprid (10.7 +/- 0.6 mg/kg topically, minimum dose 0.5 mg) after observation of fleas (Pygiopsylla hoplia) on animals and in their nest boxes. No live fleas or adverse reactions to imidacloprid were observed for 27 days following treatment. The ease of application, absence of toxicity, and long residual effect makes imidacloprid a useful drug for treating flea infestations in these marsupials.

Characterization of the ubiquitin-specific protease activity of the mouse/human Unp/Unph oncoprotein.

The ubiquitin-specific proteases (Ubps) are a family of largely dissimilar enzymes with two major conserved sequence regions, containing either a conserved cysteine residue or two conserved histidine residues, respectively. The murine Unp oncoprotein and its human homologue, Unph, both contain regions similar to the conserved Cys and His boxes common to all the Ubps. In this study we show that Unp and Unph are active deubiquitinating enzymes, being able to cleave ubiquitin from both natural and engineered linear ubiquitin-protein fusions, including the polyubiquitin precursor. Mutation of the conserved Unp Cys and His residues abolishes this activity, and identifies the likely His residue in the catalytic triad. Unp is tumorigenic when overexpressed in mice, leading to the suggestion that Unp may play a role in the regulation of ubiquitin-dependent protein degradation. We have demonstrated here that the high-level expression of Unp in yeast does not disrupt the degradation of the N-end rule substrate Tyr-beta-galactosidase (betagal), the non-N-end rule substrate ubiquitin-Pro-betagal, or the degradation of abnormal, canavanine-containing proteins. These data suggest that Unp is not a general modulator of ubiquitin-dependent proteolysis. However, Unp may have a role in the regulation of the degradation of a specific, as yet undescribed, substrate(s).

Characterization and chromosomal localization of USP3, a novel human ubiquitin-specific protease.

Conjugation to the small eukaryotic protein ubiquitin can functionally modify or target proteins for degradation by the proteasome. Removal of the ubiquitin modification, or deubiquitination, is performed by ubiquitin-specific proteases and is an important mechanism regulating this pathway. Here we describe a novel human ubiquitin-specific protease, USP3, initially identified as a partial cDNA clone similar to one of two highly conserved sequence regions common to all ubiquitin-specific proteases. We have isolated a complete USP3 cDNA clone containing both of these conserved sequence regions. The USP3 gene appears to be single copy and maps to human chromosome 15q22.3. A USP3 probe detects two mRNA transcripts, one of which corresponds in length to the cDNA. Both are expressed at low levels in all tissues examined, with highest expression in pancreas. The USP3 protein is a functional ubiquitin-specific protease in vitro, and is able to inhibit ubiquitin-dependent degradation of both an N-end Rule substrate and abnormal endogenous proteins in yeast. USP3 is also only the second known ubiquitin-specific protease capable of efficiently cleaving a ubiquitin-proline bond.

Identification, functional characterization, and chromosomal localization of USP15, a novel human ubiquitin-specific protease related to the UNP oncoprotein, and a systematic nomenclature for human ubiquitin-specific proteases.

We have identified a novel gene, USP15, encoding a human ubiquitin-specific protease (USP). The USP15 protein consists of 952 amino acids with a predicted molecular mass of 109.2 kDa and contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes. USP15 shares 60.5% sequence identity and 76% sequence similarity with the human homolog (UNP/Unph/USP4) of the mouse Unp proto-oncogene. Recombinant USP15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to beta-galactosidase and glutathione S-transferase. USP15 can also cleave the ubiquitin-proline bond, a property previously unique to Unp/UNP. Chromosomal mapping by fluorescence in situ hybridization and radiation hybrid analyses localized the USP15 gene to chromosome band 12q14, a different location than that of UNP (3p21.3). Analysis of expressed sequence tag databases reveals evidence of alternate polyadenylation sites in the USP15 gene and also indicates that the gene may possess an exon/intron structure similar to that of the Unp gene, suggesting they have descended from a common ancestor. A systematic nomenclature for the human USPs is proposed.

The Ubp6 family of deubiquitinating enzymes contains a ubiquitin-like domain: SUb.

A sequence motif that is Similar to Ubiquitin (SUb) has been identified in the Saccharomyces cerevisiae ubiquitin-specific protease Ubp6. SUb is conserved in all known Ubp6 homologues from a spectrum of eukaryotic species and is also present in a group of hypothetical proteins of unknown function (Unk1-3) present in sequence databases. An N-terminal deletion mutant of Ubp6 that lacks SUb is still capable of cleaving alpha-linked ubiquitin fusions, suggesting that SUb forms a separate domain to the catalytic core of Ubp6 and demonstrating that it is not required for in vitro cleavage activity. A homology model of the 78 N-terminal amino acids of human Ubp6, based on the known fold of ubiquitin, is presented. In human Ubp6, SUb shares only 20% sequence identity with ubiquitin. Even weaker similarity occurs between S. cerevisiae SUb and ubiquitin. The homology model supports a ubiquitin-like fold for SUb and suggests that two conserved Lys residues, corresponding to Lys48 and Lys63 of ubiquitin, are functionally important.

Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis.

The endoglycosidase heparanase is an important in the degradation of the extracellular matrix by invading cells, notably metastatic tumor cells and migrating leukocytes. Here we report the cDNA sequence of the human platelet enzyme, which encodes a unique protein of 543 amino acids, and the identification of highly homologous sequences in activated mouse T cells and in a highly metastatic rat adenocarcinoma. Furthermore, the expression of heparanase mRNA in rat tumor cells correlates with their metastatic potential. Exhaustive studies have shown only one heparanase sequence, consistent with the idea that this enzyme is the dominant endoglucuronidase in mammalian tissues.

A novel family of ubiquitin-specific proteases in chick skeletal muscle with distinct N- and C-terminal extensions.

We have recently identified a cDNA for a ubiquitin-specific protease (UBP), UBP41, that encodes the smallest functional UBP identified to date, using an Escherichia coli-based in vivo screening method. In the present study we isolated highly related cDNAs encoding a new family of UBP enzymes, named UBP46, UBP52 and UBP66. These UBPs have virtually identical catalytic domains spanning the sequence of UBP41 between the active-site Cys and the His box (95% identity). However, they possess distinct N- and/or C-terminal extensions. Moreover, they are more closely related to each other than to any other members of the UBP family. Thus these chick UBPs must define a novel family of de-ubiquitinating enzymes and should represent the first example among the UBP family enzymes, whose multiplicity is achieved by variation in their N- and C-terminal extensions. The chick UBPs were expressed in E. coli, and purified from the cells to apparent homogeneity using 125I-labelled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. Each of the purified UBP46, UBP52 and UBP66 enzymes behaved as proteins of similar sizes under both denaturing and non-denaturing conditions, suggesting that all of them consist of a single polypeptide chain. The UBP enzymes cleaved the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes and thus are active against ubiquitin-beta-galactosidase as well as a ubiquitin C-terminal extension protein of 80 amino acids. All UBPs except UBP66 released free ubiquitin from poly-His-tagged di-ubiquitin. However, the isopeptidase activity for hydrolysing polyubiquitinated lysozyme conjugates was not detected from these UBPs, which makes these UBPs distinct from UBP41. These results suggest that the chick UBPs may play an important role in production of free ubiquitin from linear polyubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins.

A novel Asn344 deletion in the core domain of coagulation factor XIII A subunit: its effects on protein structure and function.

In this study a previously undescribed 3 bp deletion, AAT1030-1032, in the factor XIII A subunit gene, has been detected in a Thai patient. The inframe deletion results in the translation of a factor XIII A subunit that lacks Asn344. This is the first inframe deletion to be identified in the factor XIII A subunit gene because six previously reported deletions have all caused frameshifts. The deletion has been introduced into a factor XIII A subunit cDNA and the deleted polypeptide expressed in yeast. The mRNA encoding the mutant enzyme appears to have normal stability but the translated protein is subject to premature degradation. In addition, the mutated enzyme exhibited very little transglutaminase activity compared with the wild-type enzyme. Structural modeling of the deleted enzyme suggests that the absence of Asn344 would have a potent impact on the catalytic activity by reorienting the residues associated with the catalytic center. Thus, the Asn344 deletion strongly confirms the significance of the residues surrounding the catalytic center of the factor XIII A subunit.

Genomic structure of Unp, a murine gene encoding a ubiquitin-specific protease.

The murine Unp gene encodes a ubiquitin-specific protease, a member of a family of enzymes that includes the product of the human tre-2 oncogene. The Unp gene has previously been mapped to chromosome 9. We have cloned in bacteriophage a 50 kilobase region of chromosome 9 containing the Unp gene, and have determined the nucleotide sequence of the gene. The gene has 22 exons, distributed over 47.4 kb. A processed ribosomal S2 pseudogene was identified in the third intron of the Unp gene. Expression of Unp is driven by a GC-rich, 'housekeeping' type promoter.

Molecular cloning of a novel ubiquitin-specific protease, UBP41, with isopeptidase activity in chick skeletal muscle.

A cDNA encoding a new ubiquitin-specific protease, UBP41, in chick skeletal muscle was cloned using an Escherichia coli-based in vivo screening method. Nucleotide sequence analysis of the cDNA containing an open reading frame of 1,071 base pairs revealed that the protease consists of 357 residues with a calculated molecular mass of 40,847 Da, and is related to members of the UBP family containing highly conserved Cys and His domains. Chick UBP41 was expressed in E. coli and purified from the cells to apparent homogeneity, using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. The purified enzyme behaved as an approximately 43-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. Like other deubiquitinating enzymes, it was sensitive to inhibition by ubiquitin-aldehyde and sulfhydryl blocking agents, such as N-ethylmaleimide. The UBP41 protease cleaved at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes; thus, it is active against ubiquitin-beta-galactosidase as well as ubiquitin C-terminal extension protein of 80 amino acids. UBP41 also released free ubiquitin from poly-His-tagged di-ubiquitin. Moreover, it converted poly-ubiquitinated lysozyme conjugates to mono-ubiquitinated forms of about 24 kDa, although the latter molecules were not further degraded to free ubiquitin and lysozyme. These results suggest that UBP41 may play an important role in the recycling of ubiquitin by hydrolysis of branched poly-ubiquitin chains generated by the action of 26 S proteasome on poly-ubiquitinated protein substrates, as well as in the production of free ubiquitin from linear poly-ubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins.

Purification and characterization of UBP6, a new ubiquitin-specific protease in Saccharomyces cerevisiae.

Ubiquitin-specific protease-6 (UBP6) in Saccharomyces cerevisiae was expressed in Escherichia coli and purified from the cells using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a model substrate. The purified UBP6 behaved as a 58-kDa under both nondenaturing and denaturing conditions, indicating that the enzyme comprises a single polypeptide. It was maximally active at pH levels between 8.5 and 9, but showed little or no activity at pH below 7 and above 9.5. As with other UBPs, its activity was strongly inhibited by sulfhydryl-blocking reagents, such as N-ethylmaleimide, and by ubiquitin-aldehyde. In addition to the model substrate, UBP6 hydrolyzed ubiquitin-alphaNH-protein extensions, such as the ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids and ubiquitin-alphaNH-dihydrofolate reductase, but not poly-His-tagged diubiquitin. It was also capable of releasing free ubiquitin from branched polyubiquitin chains that are ligated to proteins through epsilonNH-isopeptide bonds, although to a limited extent. These results suggest that UBP6 may play an important role in the generation of free ubiquitins and certain ribosomal proteins from ubiquitin-ribosomal fusion proteins as well as in deubiquitination of certain polyubiquitinated proteins targeted for degradation by the 26S proteasomes.

Interdisciplinary documentation of patient education: how collaboration can effect change.

Changing healthcare trends are affecting all healthcare providers today, including those at Hillside Rehabilitation Hospital. Computerizing the interdisciplinary documentation of patient information was one change Hillside's leaders implemented to remain competitive and cost-effective. The benefits of moving from a handwritten documentation system to a computerized system were many. However, unforeseen difficulties with retrieving data became evident because the system was not developed to accommodate information about patient education. Through interdisciplinary collaboration, staff identified areas for improvement and made changes to the documentation process. They developed a "traveling card" for documenting patient education and then met a new set of challenges. The new documentation system met departmental and Joint Commission on Accreditation of Healthcare Organizations requirements for comprehensive patient education and documentation.

Zeta, a novel class of glutathione transferases in a range of species from plants to humans.

Sequence alignment and phylogenetic analysis has identified a new subgroup of glutathione S-transferase (GST)-like proteins from a range of species extending from plants to humans. This group has been termed the Zeta class. An atomic model of the N-terminal domain suggests that the members of the Zeta class have a similar structure to that of other GSTs, binding glutathione in a similar orientation in the G site. Recombinant human GSTZ1-1 has been expressed in Escherichia coli and characterized. The protein is a dimer composed of 24.2 kDa subunits and has minimal glutathione-conjugating activity with ethacrynic acid and 7-chloro-4-nitrobenz-2-oxa-1, 3-diazole. Although low in comparison with other GSTs, GSTZ1-1 has glutathione peroxidase activity with t-butyl and cumene hydroperoxides. The members of the Zeta class have been conserved over a long evolutionary period, suggesting that they might have a role in the metabolism of a compound that is common in many living cells.

A ubiquitin-specific protease that efficiently cleaves the ubiquitin-proline bond.

Ubiquitin is a small eukaryotic protein that is synthesized naturally as one of several fusion proteins, which are processed by ubiquitin-specific proteases to release free ubiquitin. The expression of heterologous proteins as fusions to ubiquitin in either prokaryotic or eukaryotic hosts often dramatically enhances their yield, and allows the exposure of any amino acid following cleavage of ubiquitin. The single exception is when proline is the amino acid immediately following ubiquitin; the ubiquitin-proline bond is poorly cleaved by presently studied ubiquitin-specific proteases. We show that the mouse ubiquitin-specific protease Unp, and its human homolog Unph, can efficiently cleave the ubiquitin-proline bond in ubiquitin fusion proteins of different sizes. N-terminal sequencing of the cleavage products reveals that cleavage occurs precisely at the ubiquitin-proline junction. The biological significance of this cleavage activity is unclear, as ubiquitin-proline fusions do not occur naturally. However, it may indicate a different catalytic mechanism for these ubiquitin-specific proteases and/or that they can cleave ubiquitin-like proteins. Unp and Unph thus represent versatile ubiquitin-specific proteases for cleaving ubiquitin-fusion proteins in biotechnology and basic research, regardless of both the amino acid immediately following ubiquitin, and the size of the fusion partner.

Protein expression using ubiquitin fusion and cleavage.

Expressing proteins and polypeptides as fusions to ubiquitin offers the advantage of an often dramatic increase in yield, and the ability to produce any desired amino-terminal residue upon ubiquitin cleavage. The recent availability of cloned ubiquitin-cleaving enzymes has enhanced this technique for both bacterial and eukaryotic host systems.