Atomic and Molecular Layer Deposition of Chiral Thin Films Showing up to 99% Spin Selective Transport.

Spin electronics is delivering a much desired combination of properties such as high speed, low power, and high device densities for the next generation of memory devices. Utilizing chiral-induced spin selectivity (CISS) effect is a promising path toward efficient and simple spintronic devices. To be compatible with state-of-the-art integrated circuits manufacturing methodologies, vapor phase methodologies for deposition of spin filtering layers are needed. Here, we present vapor phase deposition of hybrid organic-inorganic thin films with embedded chirality. The deposition scheme relies on a combination of atomic and molecular layer deposition (A/MLD) utilizing enantiomeric pure alaninol molecular precursors combined with trimethyl aluminum (TMA) and water. The A/MLD deposition method deliver highly conformal thin films allowing the fabrication of several types of nanometric scale spintronic devices. The devices showed high spin polarization (close to 100%) for 5 nm thick spin filter layer deposited by A/MLD. The procedure is compatible with common device processing methodologies.

Biophysical and biological characterisation of collagen/resilin-like protein composite fibres.

Collagen type I, in various physical forms, is widely used in tissue engineering and regenerative medicine. To control the mechanical properties and biodegradability of collagen-based devices, exogenous cross-links are introduced into the 3D supramolecular structure. However, potent cross-linking methods are associated with cytotoxicity, whilst mild cross-linking methods are associated with suboptimal mechanical resilience. Herein, we assessed the influence of resilin, a super-elastic and highly stretchable protein found within structures in arthropods where energy storage and long-range elasticity are needed, on the biophysical and biological properties of mildly cross-linked extruded collagen fibres. The addition of resilin-like protein in the 4-arm poly(ethylene glycol) ether tetrasuccinimidyl glutarate cross-linked collagen fibres resulted in a significant increase of stress and strain at break values and a significant decrease of modulus values. The addition of resilin-like protein did not compromise cell metabolic activity and DNA concentration. All groups are supported parallel to the longitudinal fibre axis cell orientation. Herein we provide evidence that the addition of resilin-like protein in mildly cross-linked collagen fibres improves their biomechanical properties, without jeopardising their biological properties.

The Role of Autumn Infections in the Progression of Fire Blight Symptoms in Perennial Pear Branches.

The role of autumn infections in the progression of fire blight (caused by Erwinia amylovora) symptoms in perennial pear branches was studied in orchard-grown trees in Israel. The extent of symptom progression and the final length of fire blight cankers in perennial branches were variably affected by the vigor of the trees and the season of infection. Following spring infections, when all trees supported active shoot growth, fire blight symptoms progressed more rapidly and to longer distances in trees that exhibited high vigor (i.e., with numerous annual shoots on most terminal branches) than in low-vigor trees (i.e., few or no annual shoots on terminal branches). Irrespective of the vigor of the trees, the progression of fire blight symptoms in perennial branches ceased between mid-May and mid-July, and only a small proportion (0 to 14.2%) of the infections had invaded main limbs or trunks of trees. Progression of fire blight symptoms following autumn infections was related to the preceding summer (August to No-vember) shoot regrowth: in trees in which the shoots did not restore their growth in the summer, the rate of symptom progression in perennial branches was higher in trees with a low vigor than in those with a high vigor, whereas for those with summer regrowth the relationship between rates of symptom expression was reversed. Irrespective of the vigor group and of whether there was summer regrowth, symptoms in perennial branches continued to progress through the winter until the following spring. Most of the autumn infections (50 to 78.5%) that developed in susceptible trees had invaded main limbs or trunks of trees. The results of this study indicate that factors related to host phenology and physiology, rather than factors related to environmental influences (such as temperature), govern the extent, rate, and duration of fire blight progression in perennial pear branches. Furthermore, it turned out that autumn infections play a substantial role in fire blight epidemiology in Israel.

Expression, refolding and indirect immobilization of horseradish peroxidase (HRP) to cellulose via a phage-selected peptide and cellulose-binding domain (CBD).

We examined the potential immobilization of horseradish peroxidase (HRP) to cellulose with cellulose-binding domain (CBD) as a mediator, using a ligand selected from a phage-displayed random peptide library. A 15-mer random peptide library was panned on cellulose-coated plates covered with CBD in order to find a peptide that binds to CBD in its bound form. The sequence I/LHS, which was found to be an efficient binder of CBD, was fused to a synthetic gene of HRP as an affinity tag. The tagged enzyme (tHRP) was then immobilized on microcrystalline cellulose coated with CBD, thereby demonstrating the indirect immobilization of a protein to cellulose via three amino acids selected by phage display library and CBD.

Expression, purification and applications of staphylococcal protein A fused to cellulose-binding domain.

Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purification and diagnostics. Immobilization is required for most of these applications. Here we describe a genetic-engineering approach to immobilizing ProtA on cellulose, by fusing it to cellulose-binding domain (CBD) derived from the cellulose-binding Protein A of Clostridium cellulovorans. The bifunctional fusion protein was expressed in Escherichia coli, recovered on a cellulose column and purified by elution at alkaline pH. ProtA-CBD was used to purify IgG from rabbit serum and its ability to bind IgG from different sources was determined. The bifunctional chimaeric protein can bind up to 23.4 mg/ml human IgG at a ratio of 1 mol of ProtA-CBD/2 mol of human IgG, and can purify up to 11.6 mg/ml rabbit IgG from a serum. The ability to bind functionally active CBD-affinity reagents to cellulosic microtitre plates was demonstrated. Our results indicate that a combination of CBD-affinity reagents and cellulosic microtitre plates is an attractive diagnostics matrix for the following reasons: (i) cellulose exhibits very low non-specific binding; and (ii) CBD-fusion proteins bind directly to cellulose at high density. A unique signal-amplification method was developed based on the ability of ProtA-CBD to link stained cellulose particles to primary antibody in a Western blot.

Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase.

The beta-glucosidase from Aspergillus niger (CMI CC 324262) was purified, and an N-terminal sequence and two internal sequences were determined. BglI genomic gene and the cDNA were cloned from a genomic library and by reverse transcriptase-polymerase chain reaction, respectively. The cDNA was successfully expressed in Saccharomyces cerevisiae and Pichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member of glycosidase family 3. (1)H-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enzyme hydrolyzes with net retention of anomeric configuration. Accordingly, the enzyme was inactivated by 2-deoxy-2-fluoro beta-glucosyl fluoride, with kinetic parameters of k(i) = 4.5 min(-1), K(I) = 35.4 mM, through the trapping of a covalent glycosyl enzyme intermediate. The catalytic competence of this intermediate was demonstrated by the fact that incubation with linamarin resulted in reactivation, presumably via a transglycosylation mechanism. Peptic digestion of the 2-deoxy-2-fluoroglucosyl enzyme and subsequent analysis of high pressure liquid chromatography eluates by electrospray ionization triple quadrupole mass spectrometry in the neutral loss mode allowed the localization of a 2-deoxy-2-fluoroglucosyl-peptide. Sequence determination of this labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Asp-261 as the catalytic nucleophile within the sequence VMSDW. Asp-261 is fully conserved within this family, consistent with its key role, and aligns with the aspartic acid residue previously identified in the Aspergillus wentii enzyme by labeling with conduritol B epoxide (Bause, E., and Legler, G. (1974) Hoppe-Seyler's Z. Physiol. Chem. 355, 438-442).

Immobilization of recombinant heparinase I fused to cellulose-binding domain.

Immobilization of biologically active proteins is of great importance to research and industry. Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins. We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD. Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD). The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies. The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column. The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin. C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose. CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity. By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled. The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared. The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization. Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step.

A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization.

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques. All forms of A-protein were found to activate the secretion of tumor necrosis factor alpha from murine macrophage. To date, this represents the first large-scale production of biologically active recombinant A-protein.

A cellulose-binding domain-fused recombinant human T cell connective tissue-activating peptide-III manifests heparanase activity.

The chemokine connective tissue-activating peptide (CTAP)-III, which belongs to the leukocyte-derived growth factor family of mediators, was previously shown to be mitogenic for fibroblasts. However, it has recently been shown that CTAP-III, released from platelets, can act like a heparanase enzyme and degrade heparan sulfate. This suggests that CTAP-III may also function as a proinflammatory mediator. We have successfully cloned CTAP-III from a lambdagt11 cDNA library of PHA-activated human CD4(+) T cells and produced recombinant CTAP-III as a fusion protein with a cellulose-binding domain moiety. This recombinant CTAP-III exhibited heparanase activity and released degradation products from metabolically labeled, naturally produced extracellular matrix. We have also developed polyclonal and monoclonal antibodies, and these antibodies against the recombinant CTAP-III detected the CTAP-III molecule in human T cells, polymorphonuclear leukocytes, and placental extracts. Thus, our study provides tools to examine further immune cell behavior in inflamed sites rich with extracellular moieties and proinflammatory mediators.

Production and purification of a recombinant human hsp60 epitope using the cellulose-binding domain in Escherichia coli.

The heat shock protein hsp60 plays a functional role in insulin-dependent diabetes mellitus. The hsp60 epitope p277 (aa 437-aa 460) is effective in vaccinating mice against diabetes. A synthetic peptide gene (p277) that encodes the human hsp60 epitope was cloned to the 3' end of the cellulose-binding domain gene (cbd). CBD-p277 was overexpressed in Escherichia coli and purified on a cellulose column. A methionine at the C-terminal end of CBD enabled CNBr cleavage between CBD and p277. After CNBr cleavage, free CBD and residual uncleaved CBD-p277 were recovered by cellulose chromatography. The p277 peptide was further purified on a RPC-FPLC column. The molecular weight of the recombinant peptide was confirmed by electrospray mass spectrometry. The recombinant peptide was found to be biologically active in assays involving clone C9 T-cell proliferation, lymph-node cell proliferation, and antibody production. Thus the use of CBD as an affinity tag and the utilization of affordable cellulose matrices offers an attractive method for the production and purification of recombinant peptides.

Bacterial cellulose-binding domain modulates in vitro elongation of different plant cells

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 &mgr;g mL-1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 &mgr;g mL-1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.

Cloning and characterization of elongation specific endo-1,4-beta-glucanase (cel1) from Arabidopsis thaliana.

The isolation of an elongation-specific endo-1,4-beta-glucanase-cel1 from Arabidopsis thaliana was made possible by the fact that considerable homology exists between different endo-1,4-beta-glucanase (EGase) genes from different plants. Degenerate primers were synthesized based on two conserved regions from the avocado and tomato cellulase amino acid sequences. The A.thaliana cel1 cDNA gene was found to encode a 54kDa protein; sequence comparison with the avocado EGase revealed 56% identity. Northern blot analysis of cel1 suggested its developmental regulation. RNA transcripts were undetectable in fully expanded leaves as well as at the basal internode of flowering stems. However, a strong transcript signal was detected in the elongating zone of flowering stems of normal plants. The RNA transcript level of cel1 in the elongating zone of dwarf flowering stems was significantly lower than in the corresponding zone in normal plants. This suggests cel1's involvement in cell elongation in A. thaliana. Transgenic tobacco plants transformed with the putative cel1 promoter region fused to the gus reporter gene, showed a significant GUS staining both in shoot and root elongating zones. These results further substantiate the link between cel1 expression and plant cell elongation.

Synthesis of isopropyl-1-thio-beta-D-glucopyranoside (IPTGlc), an inducer of Aspergillus niger B1 beta-glucosidase production.

Production of beta-glucosidase in Aspergillus niger B1 is subjected to catabolic repression by glucose. Aspergillus niger B1 grown on bran as a carbon source secreted beta-glucosidase. The maximum level of the enzyme was reached after 7 d of fermentation. Addition of 1% glucose to the medium suppressed beta-glucosidase production to undetectable levels. In this study, the organic synthesis of a potential inducer of beta-glucosidase production by A. niger B1's reported. Isopropyl-1-thio-beta-D-glucopyranoside (IPTGlc) was synthesized using a two-step organic synthesis protocol. The H-NMR data agreed with those reported previously for the galactoside analog. When IPTGlc was added 24 h after inoculation at a final concentration of 0.4 mM, similar levels of beta-glucosidase were reached 3 to 4 d earlier as compared to fermentation without IPTGlc induction. In practice, this may translate to a more efficient method of producing beta-glucosidase from this fungus.

Characterization of BspA, a major boiling-stable, water-stress-responsive protein in aspen (Populus tremula).

We identified a novel 66 kDa boiling-stable protein (BspA) in cultured shoots of aspen (Populus tremula L.) which was highly expressed in response to gradual water stress. The BspA protein, which was highly expressed as early as 1 h after initiation of a drought treatment, accumulated during progressive water stress, decreased on rehydration, and was expressed in response to abscisic acid (ABA) application, as detected by SDS-PAGE protein analysis and Western blotting. Anti-BspA antibodies also cross-reacted with a 119 kDa protein. The 119 kDa protein was also induced by water stress, but it was detected only in the total protein fraction and not in the heat-stable fraction. The BspA protein cross-reacted with antibodies raised against a water-stress-responsive protein isolated from the African resurrection plant Craterostigma plantagineum Hochst. The N-terminal amino acid sequence of BspA was determined and exhibited high homology with the wheat germins GF-2.8 and GF-3.8. The BspA protein was the only major, water-stress-responsive boiling-stable protein detected in aspen.

A disaccharide that inhibits tumor necrosis factor alpha is formed from the extracellular matrix by the enzyme heparanase.

The activation of T cells by antigens or mitogens leads to the secretion of cytokines and enzymes that shape the inflammatory response. Among these molecular mediators of inflammation is a heparanase enzyme that degrades the heparan sulfate scaffold of the extracellular matrix (ECM). Activated T cells use heparanase to penetrate the ECM and gain access to the tissues. We now report that among the breakdown products of the ECM generated by heparanase is a trisulfated disaccharide that can inhibit delayed-type hypersensitivity (DTH) in mice. This inhibition of T-cell mediated inflammation in vivo was associated with an inhibitory effect of the disaccharide on the production of biologically active tumor necrosis factor alpha (TNF-alpha) by activated T cells in vitro; the trisulfated disaccharide did not affect T-cell viability or responsiveness generally. Both the in vivo and in vitro effects of the disaccharide manifested a bell-shaped dose-response curve. The inhibitory effects of the trisulfated disaccharide were lost if the sulfate groups were removed. Thus, the disaccharide, which may be a natural product of inflammation, can regulate the functional nature of the response by the T cell to activation. Such a feedback control mechanism could enable the T cell to assess the extent of tissue degradation and adjust its behavior accordingly.

Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.

Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A.

The cbpA gene for the Clostridium cellulovorans cellulose binding protein (CbpA), which is part of the multisubunit cellulase complex, has been cloned and sequenced. When cbpA was expressed in Escherichia coli, proteins capable of binding to crystalline cellulose and of interacting with anti-CbpA were observed. The cbpA gene consists of 5544 base pairs and encodes a protein containing 1848 amino acids with a molecular mass of 189,036 Da. The open reading frame is preceded by a Gram-positive-type ribosome binding site. A signal peptide sequence of 28 amino acids is present at its N terminus. The encoded protein is highly hydrophobic with extremely high levels of threonine and valine residues. There are two types of putative cellulose binding domains of approximately 100 amino acids that are slightly hydrophilic and eight conserved, highly hydrophobic beta-sheet regions of approximately 140 amino acids. These latter hydrophobic regions may be the CbpA domains that interact with the different enzymatic subunits of the cellulase complex.

Analysis of functional domains of endoglucanases from Clostridium cellulovorans by gene cloning, nucleotide sequencing and chimeric protein construction.

The nucleotide sequence of engD, an endo-beta-1,4-glucanase gene from Clostridium cellulovorans was determined (Genbank Accession No. M37434). The COOH-terminal part of the gene product, EngD, contained a Thr-Thr-Pro repeated sequence followed by a region that has homology to the exoglucanase of Cellulomonas fimi. EngD and EngB, another C. cellulovorans endoglucanase, show 75% amino acid sequence homology at their NH2-termini, in contrast to their carboxyterminal domains which show no homology. EngD had endoglucanase activity on carboxymethylcellulose (CMC), cellobiosidase activity on p-nitrophenyl-cellobioside (p-NPC), and partial hydrolytic activity on crystalline cellulose (Avicel), while EngB showed hydrolytic activity against only CMC. Chimeric proteins between EngB and EngD were constructed by exchanging the non-homologous COOH-terminal regions. Chimeric proteins that contained the NH2-terminus of EngD retained cellobiosidase activity but chimeras with the EngB NH2-terminus showed no cellobiosidase activity. Hydrolysis of crystalline cellulose (Avicelase activity) was observed only with the enzyme containing the EngD NH2-terminus and EngD COOH-terminus.

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans.

An endoglucanase gene, engB, from Clostridium cellulovorans, previously cloned into pUC19, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichia coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosome-binding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.