Disorganized intercellular junctions are critical for maintaining the integrity of solid epithelial tumors and prevent the infiltration of oncological therapies into the bulk of the malignancy. We ...have developed small, recombinant proteins which bind a critical junction protein, desmoglein 2, triggering the transient and specific opening of tumor tight junctions allowing for infiltration of the tumor with immune cells, oncolytic viruses, drugs, and other therapeutics. Our new molecule, JOC-x, is a promising candidate for a new class of tumor-targeting agents that accumulate both around and within tumors and remodel the tumor microenvironment. Native cysteines were removed from the parental protein, JO-4, followed by addition of a single cysteine to allow for convenient attachment of various payloads that can be targeted directly to the tumor. Our tumor-targeting protein exhibits high avidity, minimal aggregation, and is easily purified at good yields from E. coli. For proof of concept, we demonstrate effective conjugation to biotin as a model for flexible co-targeting, addition of metal ion chelators as models for imaging and radiotherapy, and linkage of the TLR3 agonist poly(I:C) as a model immune-oncologic agent. This second-generation cancer co-therapeutic protein is optimized for activity and primed for cGMP manufacture in preparation for upcoming clinical studies.
Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, can significantly ...increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon and C. J. Oberg, J. Dairy Sci. 80:799-805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 and Lactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, the epsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticus LH100 plus the non-EPS-producing mutant S. thermophilus DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of D-galactose, L-rhamnose, and L-fucose in a ratio of 5:2:1
Alternanase is an enzyme which endo-hydrolytically cleaves the α-(1→3), α-(1→6)-linked
d-glucan, alternan. The main products are isomaltose, α-
d-Glc
p-(1→3)-α-
d-Glc
p-(1→6)-
d-Glc and the cyclic ...tetrasaccharide
cyclo{→6)-α-
d-Glc
p-(1→3)-α-
d-Glc
p-(1→6)-α-
d-Glc
p-(1→3)-α-
d-Glc
p-(1→}. It is also capable of acting on oligosaccharide substrates. The cyclic tetrasaccharide is slowly hydrolyzed to isomaltose. Panose and the trisaccharide α-
d-Glc
p-(1→6)-α-
d-Glc
p-(1→3)-
d-Glc both undergo transglycosylation reactions to give rise to the cyclic tetrasaccharide plus
d-glucose, with panose being converted at a much faster rate. The tetrasaccharide α-
d-Glc
p-(1→3)-α-
d-Glc
p-(1→6)-α-
d-Glc
p-(1→4)-
d-Glc is hydrolyzed to
d-glucose plus the trisaccharide α-
d-Glc
p-(1→3)-α-
d-Glc
p-(1→6)-
d-Glc. Alternanase does not act on isomaltotriose, theanderose (6
Glc-
O-α-
d-Glc
p sucrose), or α-
d-Glc
p-(1→6)-α-
d-Glc
p-(1→6)-α-
d-Glc
p-(1→4)-α-
d-Glc. The enzyme releases 4-nitrophenol from 4-nitrophenyl α-isomaltoside, but not from 4-nitrophenyl α-
d-glucopyranoside, 4-nitrophenyl α-isomaltotrioside, or 4-nitrophenyl α-isomaltotetraoside.
Graphic
Characterization of a novel modified alternan Leathers, Timothy D.; Nunnally, Melinda S.; Ahlgren, Jeffrey A. ...
Carbohydrate polymers,
10/2003, Letnik:
54, Številka:
1
Journal Article
Recenzirano
A novel modified alternan, produced by using newly isolated strains of
Penicillium sp., was physically characterized. High molecular weight native alternan was progressively modified to lower ...molecular weight heterodisperse forms, associated with a reduction in absorbance at 225 nm, light scattering, and opalescence. Methylation analysis indicated that modified alternan has a linkage pattern similar to that of native alternan. The solution viscosity properties of modified alternan resemble those of ultrasonicated alternan and commercial gum arabic. However, alternan lacks the emulsification capacity of gum arabic. Alternan solutions are stable for at least 7 days under all conditions tested, from 4 to 70 °C and from pH 3–9. Dry preparations of alternan are bright white powders that are not highly hygroscopic. Thus, modified alternan is promising for further development as a gum arabic substitute, particularly in food applications requiring a low-viscosity bulking agent.
The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by ...alternating alpha(1-->6), alpha(1-->3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure. When followed by preparative isoelectric focusing, a single band of 117 kDa was measured when the purified protein was analyzed by HPLC size-exclusion chromatography/multiangle light scattering. The purification procedure can be scaled to permit large quantities of enzyme to be purified in high (36%) yield.
ABSTRACT
Soluble protein extracts of germinating maize seedlings exhibited a limited ability to hydrolyze purified xylans, and specific assays were unable to confirm the presence of ...endo‐β‐1,4‐xylanase activity. However, extracts contained a variety of aryl‐glycosidase activities, including β‐glucosidase, β‐xylosidase, and α‐l‐arabinofuranosidase. These activities peaked in three‐ to four‐day seedlings and were particularly concentrated in shoot and root tissues. Maximal levels of β‐glucosidase were two orders of magnitude greater than those of β‐xylosidase or α‐l‐arabinofuranosidase. Isoelectric focusing gels revealed multiple forms of these enzymes. The principal β‐glucosidase and α‐l‐arabinofuranosidase protein species were clustered at pI 4.8–4.9 and pI 5.8–6.0, respectively. β‐Xylosidase activity appeared to be associated with both of these enzymes, and no evidence was obtained for a distinct β‐xylosidase.
Alternanase, an endoglucanase that hydrolyzes the bacterial exopolysaccharide alternan, will also hydrolyze the trisaccharide, panose, to produce glucose and a disaccharide that can be formed into a ...novel, cyclic tetrasaccharide. The glucose can then be selectively and quantitatively measured by enzyme-based reaction which forms the basis of a coupled enzyme assay to quantitate alternanase activity. By this method a preparation of alternanase purified by affinity chromatography on immobilized isomaltose had a maximum reaction rate (V^sub max^) of 0.75 μmol glucose min^sup -1^ and a K^sub m^ of 34 mM for panose. Two competitive inhibitors of alternanase activity were also evaluated using this coupled enzyme assay: isomaltose had a K^sub i^ of 94 mM while the cyclic tetrasaccharide had a K^sub i^ of 66 mM.PUBLICATION ABSTRACT
Functional interleukin 7 (IL-7) receptors are expressed on the surface of multiphenotypic, biphenotypic, and immature B-lineage human lymphoid precursor cells with germ-line immunoglobulin ...heavy-chain genes but not on more mature B-lineage lymphoid cells with rearranged and/or expressed immunoglobulin heavy-chain genes. Thus, IL-7 may have an important regulatory role during the earliest stages of human B-cell ontogeny. The engagement of the surface IL-7 receptors on immature B-cell precursor cells with recombinant human IL-7 (rhIL-7) results in enhanced tyrosine phosphorylation of multiple phosphoproteins, stimulates inositol phospholipid turnover and DNA synthesis, and promotes their clonal proliferation. These effects are (i) specific for rhIL-7, since rhIL-3, rhIL-4, rhIL-5, rhIL-6, and recombinant human granulocyte colony-stimulating factor do not elicit similar activities on IL-7 receptor-positive human pro-B cells; and (ii) mediated by IL-7 receptors, since they are not observed in IL-7 receptor-negative B-lineage lymphoid cell populations. rhIL-7-induced tyrosine phosphorylation on the 35-, 53-, 55-, 62-, 69-, 76-, 94-, 150-, 170-, and 190-kDa substrates as well as rhIL-7-induced stimulation of inositol phospholipid turnover are abrogated by the tyrosine kinase inhibitor genistein. These results demonstrate that the IL-7 receptor on immature human B-cell precursor populations is intimately linked to a functional tyrosine kinase pathway and tyrosine phosphorylation is an important and perhaps mandatory step in the generation of the IL-7 receptor-linked transmembrane signal.
A xanthanase complex secreted by a consortium of heat-stable, salt-tolerant bacteria includes a lyase that specifically removes terminal pyruvated beta-D-mannose residues from the side chains of ...xanthan gum. The enzyme was purified to homogeneity from the culture broth following ion-exchange chromatography and gel permeation chromatography. It consists of a single subunit of molecular weight 33,000. The enzyme is stable to 55 degrees C for more than 6 h in 20 millimoles sodium phosphate buffer (pH 5.0) containing 0.25 M NaCl. Optimal enzyme activity was observed at 0.05 M NaCl and a pH of 5. The enzyme has a pI of 3.7. It does not remove unsubstituted terminal beta-D-mannose residues from xanthan side chains nor does it hydrolyze p-nitrophenyl-p-D-mannose. Treatment of xanthan with purified lyase results in a polysaccharide containing side chains terminating in an unsaturated 4,5-ene-glucuronic acid
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