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The Journal of biological chemistry  2007;282(13):9834-9845.
Anthrax toxin, a three-component protein toxin secreted by Bacillus anthracis, assembles into toxic complexes at the surface of receptor-bearing eukaryotic cells. The protective antigen (PA) protein binds to receptors, either tumor endothelial cell marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), and orchestrates the delivery of the lethal and edema factors into the cytosol. TEM8 is reported to be over-expressed during tumor angiogenesis, whereas CMG2 is more widely expressed in normal tissues. To extend prior work on targeting of tumor with modified anthrax toxins, we used phage display to select PA variants that preferentially bind to TEM8 as compared to CMG2. Substitutions were randomly introduced into residues 605-729 of PA, within the C-terminal domain 4 of PA, which is the principal region that contacts receptor. Candidates were characterized in cellular cytotoxicity assays with CHO cells expressing either TEM8 or CMG2. A PA mutant having the substitutions R659S and M662R had enhanced specificity toward TEM8 over-expressing CHO cells. This PA variant also displayed broad and potent tumoricidal activity to various human tumor cells, especially to HeLa and A549/ATCC cells. By contrast, the substitution N657Q significantly reduced toxicity to TEM8 but not CMG2 over-expressing CHO cells. Our results indicate that certain amino acid substitutions within PA domain 4 create anthrax toxins that selectively kill human tumor cells. The PA R659S/M662R protein may be useful as a therapeutic agent for cancer treatment.
PMCID: PMC2530824  PMID: 17251181
2.  Expression of cmg1, an Exo-β-1,3-Glucanase Gene from Coniothyrium minitans, Increases during Sclerotial Parasitism 
During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains β-1,3-glucan as its major component. A PCR-based strategy was used to clone a β-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-β-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiae INVSc1 expressing the cmg1 gene secreted a ∼100-kDa β-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth of S. sclerotiorum by 35 and 85% at concentrations of 300 and 600 μg ml−1, respectively. A single copy of the cmg1 gene is present in the genome of C. minitans. Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum.
PMCID: PMC92660  PMID: 11157256
3.  1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) Inhibits Angiogenesis via Inhibition of CMG2 
Journal of medicinal chemistry  2013;56(5):1940-1945.
CMG2 is a transmembrane extracellular matrix binding protein that is also an anthrax toxin receptor. We have shown that high affinity CMG2 binders can inhibit angiogenesis and tumor growth. We recently described a high throughput FRET assay to identify CMG2 inhibitors. We now report the serendipitous discovery that PGG (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose) is a CMG2 inhibitor with anti-angiogenic activity. PGG is a gallotannin produced by a variety of medicinal plants that exhibits a wide variety of anti-tumor and other activities. We find that PGG inhibits CMG2 with a submicromolar IC50 and it also inhibits the migration of human dermal microvascular endothelial cells at similar concentrations in vitro. Finally, oral or intraperitoneal administration of PGG inhibits angiogenesis in the mouse corneal micropocket assay in vivo. Together, these results suggest that a portion of the in vivo anti-tumor activity of PGG may be the result of antiangiogenic activity mediated by inhibition of CMG2.
PMCID: PMC3600088  PMID: 23394144
pentagalloyl glucose; 5GG; angiogenesis; corneal neovascularization; cancer; polyphenol
4.  Phenolic Compounds as Antiangiogenic CMG2 Inhibitors from Costa Rican Endophytic Fungi 
Targeting and inhibiting CMG2 (Capillary Morphogenesis Gene protein 2) represents a new strategy for therapeutic agents for cancer and retinal diseases due to CMG2’s role in blood vessel growth (angiogenesis). A high throughput FRET (Förster Resonance Energy Transfer) assay was developed for the identification of CMG2 inhibitors as anti-angiogenetic agents. Bioassay-guided separation led to the isolation and identification of two new compounds (1 and 2) from CR252M, an endophytic fungus Coccomyces proteae collected from a Costa Rican rainforest, and one known compound (3) from CR1207B (Aurapex penicillata). Secondary in vitro assays indicated anti-angiogenic activity. Compound 3 inhibited the endothelial cell migration at 52 µM, but did not show any endothelial cell antiproliferative effect at 156 µM. The structure of the two new compounds, A (1) and B (2), were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR experiments.
PMCID: PMC3563091  PMID: 22910038
Fungus; Coccomyces proteae; Aurapex penicillata; CMG2; Phenolic
5.  104 Immunogenicity and Safety Aspects of Adeno-Associated Virus–Like Particles (AAVLPS) as Carriers for B-Cell Vaccines 
The World Allergy Organization Journal  2012;5(Suppl 2):S51-S52.
Adeno-associated viruses (AAV) are non-human pathogenic and replication defective ssDNA viruses. The surface of AAV consists of 60 capsomers, which can be exploited for high density display of recombinant peptides. AAV-like particles (AAVLP) can be generated via assembly of the recombinant capsid protein VP3. The aim of this study was to evaluate the uptake mechanism, immunogenicity and safety aspects of an AAVLP-displayed B-cell epitope, taking ovalbumin (OVA) as a model antigen/allergen.
An OVA derived linear B-cell epitope and for control purposes OVA-non related peptide TP18 (cholesterol-ester transfer protein 18) were inserted into capsid protein VP3 of AAVLPs.
Life cell microscopy indicated that AAVLP internalized into HeLa epithelial cells and remained in intracellular vesicles up to 18 hours. When we immunized BALB/c subcutaneously, sera of AAVLP-OVA immunized mice showed similar titres of OVA-specific IgG1 compared to mice immunized with OVA protein. However, in OVA immunized mice high OVA-specific IgE levels could be recorded, whereas immunizations with OVA-AAVLP rendered background IgE levels only. In accordance, sera of OVA mice which permitted mast cell degranulation upon OVA trigger in a specific β-hexosaminidase release assay, whereas sera of OVA-AAVLP mice did not contain anaphylactogenic antibodies. In an in vivo anaphylaxis experiment, upon intravenous OVA challenge OVA-immunized mice presented significant drop of body temperature, whereas AAVLP-OVA mice remained unaffected.
Our study demonstrates the immunogenicity, safety and efficacy of AAVLP as display system of B-cell epitopes for vaccination.
PMCID: PMC3512624
6.  Fabrication and characterization of nuclear localization signal-conjugated glycol chitosan micelles for improving the nuclear delivery of doxorubicin 
Supramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS)-modified polymeric micelles were studied with the aim of improving nuclear drug delivery.
In this research, cholesterol-modified glycol chitosan (CHGC) was synthesized. NLS-conjugated CHGC (NCHGC) was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa and HepG2 cells.
The degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC) micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater cellular uptake and higher amounts of DOX in the nuclei of HeLa cells than free DOX and DOX-loaded CHGC (DCHGC) micelles. The half maximal inhibitory concentration (IC50) values of free DOX, DCHGC, and DNCHGC micelles against HepG2 cells were 4.063, 0.591, and 0.171 μg/mL, respectively. Moreover, the IC50 values of free DOX (3.210 μg/mL) and the DCHGC micelles (1.413 μg/mL) against HeLa cells were nearly 6.96- and 3.07-fold (P < 0.01), respectively, higher than the IC50 value of the DNCHGC micelles (0.461 μg/mL).
The results of this study suggest that novel NCHGC micelles could be a potential carrier for nucleus-targeting delivery.
PMCID: PMC3459689  PMID: 23049255
polymeric micelles; drug delivery; nucleus-targeting delivery
7.  The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo 
Journal of Bacteriology  1998;180(7):1947-1950.
A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels of Bradyrhizobium japonicum holocytochrome c550 expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.
PMCID: PMC107112  PMID: 9537397
1. It has been shown in this paper that while non-ionized gelatin may exist in gelatin solutions on both sides of the isoelectric point (which lies for gelatin at a hydrogen ion concentration of CH = 2.10–5 or pH = 4.7), gelatin, when it ionizes, can only exist as an anion on the less acid side of its isoelectric point (pH > 4.7), as a cation only on the more acid side of its isoelectric point (pH < 4.7). At the isoelectric point gelatin can dissociate practically neither as anion nor as cation. 2. When gelatin has been transformed into sodium gelatinate by treating it for some time with M/32 NaOH, and when it is subsequently treated with HCl, the gelatin shows on the more acid side of the isoelectric point effects of the acid treatment only; while the effects of the alkali treatment disappear completely, showing that the negative gelatin ions formed by the previous treatment with alkali can no longer exist in a solution with a pH < 4.7. When gelatin is first treated with acid and afterwards with alkali on the alkaline side of the isoelectric point only the effects of the alkali treatment are noticeable. 3. On the acid side of the isoelectric point amphoteric electrolytes can only combine with the anions of neutral salts, on the less acid side of their isoelectric point only with cations; and at the isoelectric point neither with the anion nor cation of a neutral salt. This harmonizes with the statement made in the first paragraph, and the experimental results on the effect of neutral salts on gelatin published in the writer's previous papers. 4. The reason for this influence of the hydrogen ion concentration on the stability of the two forms of ionization possible for an amphoteric electrolyte is at present unknown. We might think of the possibility of changes in the configuration or constitution of the gelatin molecule whereby ionized gelatin can exist only as an anion on the alkaline side and as a cation on the acid side of its isoelectric point. 5. The literature of colloid chemistry contains numerous statements which if true would mean that the anions of neutral salts act on gelatin on the alkaline side of the isoelectric point, e.g. the alleged effect of the Hofmeister series of anions on the swelling and osmotic pressure of common gelatin in neutral solutions, and the statement that both ions of a neutral salt influence a protein simultaneously. The writer has shown in previous publications that these statements are contrary to fact and based on erroneous methods of work. Our present paper shows that these claims of colloid chemists are also theoretically impossible. 6. In addition to other physical properties the conductivity of gelatin previously treated with acids has been investigated and plotted, and it was found that this conductivity is a minimum in the region of the isoelectric point, thus confirming the conclusion that gelatin can apparently not exist in ionized condition at that point. The conductivity rises on either side of the isoelectric point, but not symmetrically for reasons given in the paper. It is shown that the curves for osmotic pressure, viscosity, swelling, and alcohol number run parallel to the curve of the conductivity of gelatin when the gelatin has been treated with acid, supporting the view that these physical properties are in this case mainly or exclusively a function of the degree of ionization of the gelatin or gelatin salt formed. It is pointed out, however, that certain constitutional factors, e.g. the valency of the ion in combination with the gelatin, may alter the physical properties of the gelatin (osmotic pressure, etc.) without apparently altering its conductivity. This point is still under investigation and will be further discussed in a following publication. 7. It is shown that the isoelectric point of an amphoteric electrolyte is not only a point where the physical properties of an ampholyte experience a sharp drop and become a minimum, but that it is also a turning point for the mode of chemical reactions of the ampholyte. It may turn out that this chemical influence of the isoelectric point upon life phenomena overshadows its physical influence. 8. These experiments suggest that the theory of amphoteric colloids is in its general features identical with the theory of inorganic hydroxides (e.g. aluminum hydroxide), whose behavior is adequately understood on the basis of the laws of general chemistry.
PMCID: PMC2140289  PMID: 19871727
9.  pH-Responsive Nanoparticle Vaccines for Dual-Delivery of Antigens and Immunostimulatory Oligonucleotides 
ACS nano  2013;7(5):3912-3925.
Protein subunit vaccines offer important potential advantages over live vaccine vectors, but generally elicit weaker and shorter-lived cellular immune responses. Here we investigate the use of pH-responsive, endosomolytic polymer nanoparticles that were originally developed for RNA delivery as vaccine delivery vehicles for enhancing cellular and humoral immune responses. Micellar nanoparticles were assembled from amphiphilic diblock copolymers composed of an ampholytic core-forming block and a re-designed polycationic corona block doped with thiol-reactive pyridyl disulfide groups to enable dual-delivery of antigens and immunostimulatory CpG oligodeoxynucleotide (CpG ODN) adjuvants. Polymers assembled into 23 nm particles with simultaneous packaging of CpG ODN and a thiolated protein antigen, ovalbumin (ova). Conjugation of ova to nanoparticles significantly enhanced antigen cross-presentation in vitro relative to free ova or an unconjugated, physical mixture of the parent compounds. Subcutaneous vaccination of mice with ova-nanoparticle conjugates elicited a significantly higher CD8+ T cell response (0.5% IFN-ɣ+ of CD8+) compared to mice vaccinated with free ova or a physical mixture of the two components. Significantly, immunization with ova-nanoparticle conjugates electrostatically complexed with CpG ODN (dual-delivery) enhanced CD8+ T cell responses (3.4% IFN-ɣ+ of CD8+) 7-, 18-, and 8-fold relative to immunization with conjugates, ova administered with free CpG, or a formulation containing free ova and CpG complexed to micelles, respectively. Similarly, dual-delivery carriers significantly increased CD4+IFN-ɣ+ (Th1) responses, and elicited a balanced IgG1/IgG2c antibody response. Intradermal administration further augmented cellular immune responses, with dual-delivery carriers inducing ~7% antigen-specific CD8+ T cells. This work demonstrates the ability of pH-responsive, endosomolytic nanoparticles to actively promote antigen cross-presentation and augment cellular and humoral immune responses via dual-delivery of protein antigens and CpG ODN. Hence, pH-responsive polymeric nanoparticles offer promise as a delivery platform for protein subunit vaccines.
PMCID: PMC4042837  PMID: 23590591
vaccine; pH-responsive polymer; micelle; CpG adjuvant; endosomal escape; antigen cross-presentation
10.  RGD peptide-mediated chitosan-based polymeric micelles targeting delivery for integrin-overexpressing tumor cells 
Solid tumors need new blood vessels to feed and nourish them as well as to allow tumor cells to escape into the circulation and lodge in other organs, which is termed “angiogenesis.” Some tumor cells within solid tumors can overexpress integrins αvβ3 and αvβ5, which can specifically recognize the peptide motif Arg-Gly-Asp (RGD). Thus, the targeting of RGD-modified micelles to tumor vasculature is a promising strategy for tumor-targeting treatment.
RGD peptide (GSSSGRGDSPA) was coupled to poly(ethylene glycol)-modified stearic acid-grafted chitosan (PEG-CS-SA) micelles via chemical reaction in the presence of N,N′-Disuccinimidyl carbonate. The critical micelle concentration of the polymeric micelles was determined by measuring the fluorescence intensity of pyrene as a fluorescent probe. The micelle size, size distribution, and zeta potential were measured by light scattering and electrophoretic mobility. Doxorubicin (DOX) was chosen as a model anticancer drug to investigate the drug entrapment efficiency, in vitro drug-release profile, and in vitro antitumor activities of drug-loaded RGD-PEG-CS-SA micelles in cells that overexpress integrins (ανβ3 and ανβ5) and integrin-deficient cells.
Using DOX as a model drug, the drug encapsulation efficiency could reach 90%, and the in vitro drug-release profiles suggested that the micelles could be used as a controlled-release carrier for the hydrophobic drug. Qualitative and quantitative analysis of cellular uptake indicated that RGD-modified micelles could significantly increase the DOX concentration in integrin-overexpressing human hepatocellular carcinoma cell line (BEL-7402), but not in human epithelial carcinoma cell line (Hela). The competitive cellular-uptake test showed that the cellular uptake of RGD-modified micelles in BEL-7402 cells was significantly inhibited in the presence of excess free RGD peptides. In vitro cytotoxicity tests demonstrated DOX-loaded RGD-modified micelles could specifically enhance the cytotoxicity against BEL-7402 compared with DOX-loaded PEG-CS-SA and doxorubicin hydrochlorate.
This study suggests that RGD-modified PEG-CS-SA micelles are promising drug carriers for integrin-overexpressing tumor active targeting therapy.
PMCID: PMC3265222  PMID: 22282676
cellular uptake; chitosan polymeric micelles; cytotoxicity; doxorubicin; integrin; RGD peptide
11.  The pH-Sensitive Fusogenic 3-Methyl-Glutarylated Hyperbranched Poly(Glycidol)-Conjugated Liposome Induces Antigen-Specific Cellular and Humoral Immunity 
We examined the ability of a novel liposome, surface modified by 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG), to enhance antigen-specific immunity in vitro and in vivo and to function as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) encapsulated in MGlu-HPG-modified liposomes more effectively than free OVA or OVA encapsulated in unmodified liposomes. Immunization of mice with OVA-containing MGlu-HPG-modified liposomes induced antigen-specific splenocyte proliferation and production of gamma interferon (IFN-γ) more strongly than did immunization with free OVA or OVA encapsulated in unmodified liposomes. The immune responses induced by OVA encapsulated in MGlu-HPG-modified liposomes were significantly suppressed by addition of anti-major histocompatibility complex (MHC) class I and class II monoclonal antibodies, indicating the involvement of antigen presentation via MHC class I and II. Furthermore, delayed-type hypersensitivity responses and OVA-specific antibodies were induced more effectively in mice immunized with OVA encapsulated by MGlu-HPG-modified liposomes than with unencapsulated OVA or OVA encapsulated in unmodified liposomes. These results suggested that MGlu-HPG-modified liposomes effectively induced both cell-mediated and humoral immune responses. Collectively, this study is the first to demonstrate the induction of both cell-mediated and humoral immune responses in vivo by MGlu-HPG-modified liposomes.
PMCID: PMC3428382  PMID: 22815149
12.  The CMG (CDC45/RecJ, MCM, GINS) complex is a conserved component of the DNA replication system in all archaea and eukaryotes 
Biology Direct  2012;7:7.
In eukaryotes, the CMG (CDC45, MCM, GINS) complex containing the replicative helicase MCM is a key player in DNA replication. Archaeal homologs of the eukaryotic MCM and GINS proteins have been identified but until recently no homolog of the CDC45 protein was known. Two recent developments, namely the discovery of archaeal GINS-associated nuclease (GAN) that belongs to the RecJ family of the DHH hydrolase superfamily and the demonstration of homology between the DHH domains of CDC45 and RecJ, show that at least some Archaea possess a full complement of homologs of the CMG complex subunits. Here we present the results of in-depth phylogenomic analysis of RecJ homologs in archaea.
We confirm and extend the recent hypothesis that CDC45 is the eukaryotic ortholog of the bacterial and archaeal RecJ family nucleases. At least one RecJ homolog was identified in all sequenced archaeal genomes, with the single exception of Caldivirga maquilingensis. These proteins include previously unnoticed remote RecJ homologs with inactivated DHH domain in Thermoproteales. Combined with phylogenetic tree reconstruction of diverse eukaryotic, archaeal and bacterial DHH subfamilies, this analysis yields a complex scenario of RecJ family evolution in Archaea which includes independent inactivation of the nuclease domain in Crenarchaeota and Halobacteria, and loss of this domain in Methanococcales.
The archaeal complex of a CDC45/RecJ homolog, MCM and GINS is homologous and most likely functionally analogous to the eukaryotic CMG complex, and appears to be a key component of the DNA replication machinery in all Archaea. It is inferred that the last common archaeo-eukaryotic ancestor encoded a CMG complex that contained an active nuclease of the RecJ family. The inactivated RecJ homologs in several archaeal lineages most likely are dedicated structural components of replication complexes.
This article was reviewed by Prof. Patrick Forterre, Dr. Stephen John Aves (nominated by Dr. Purificacion Lopez-Garcia) and Prof. Martijn Huynen.
For the full reviews, see the Reviewers' Comments section.
PMCID: PMC3307487  PMID: 22329974
1. It is shown by volumetric analysis that on the alkaline side from its isoelectric point gelatin combines with cations only, but not with anions; that on the more acid side from its isoelectric point it combines only with anions but not with cations; and that at the isoelectric point, pH = 4.7, it combines with neither anion nor cation. This confirms our statement made in a previous paper that gelatin can exist only as an anion on the alkaline side from its isoelectric point and only as a cation on the more acid side of its isoelectric point, and practically as neither anion nor cation at the isoelectric point. 2. Since at the isoelectric point gelatin (and probably amphoteric colloids generally) must give off any ion with which it was combined, the simplest method of obtaining amphoteric colloids approximately free from ionogenic impurities would seem to consist in bringing them to the hydrogen ion concentration characteristic of their isoelectric point (i.e., at which they migrate neither to the cathode nor anode of an electric field). 3. It is shown by volumetric analysis that when gelatin is in combination with a monovalent ion (Ag, Br, CNS), the curve representing the amount of ion-gelatin formed is approximately parallel to the curve for swelling, osmotic pressure, and viscosity. This fact proves that the influence of ions upon these properties is determined by the chemical or stoichiometrical and not by the "colloidal" condition of gelatin. 4. The sharp drop of these curves at the isoelectric point finds its explanation in an equal drop of the water solubility of pure gelatin, which is proved by the formation of a precipitate. It is not yet possible to state whether this drop of the solubility is merely due to lack of ionization of the gelatin or also to the formation of an insoluble tautomeric or polymeric compound of gelatin at the isoelectric point. 5. On account of this sudden drop slight changes in the hydrogen ion concentration have a considerably greater chemical and physical effect in the region of the isoelectric point than at some distance from this point. This fact may be of biological significance since a number of amphoteric colloids in the body seem to have their isoelectric point inside the range of the normal variation of the hydrogen ion concentration of blood, lymph, or cell sap. 6. Our experiments show that while a slight change in the hydrogen ion concentration increases the water solubility of gelatin near the isoelectric point, no increase in the solubility can be produced by treating gelatin at the isoelectric point with any other kind of monovalent or polyvalent ion; a fact apparently not in harmony with the adsorption theory of colloids, but in harmony with a chemical conception of proteins.
PMCID: PMC2140300  PMID: 19871741
14.  The Effects of Salt on the Physicochemical Properties and Immunogenicity of Protein Based Vaccine Formulated in Cationic Liposome 
Recently, we have developed a simple and potent therapeutic cancer vaccine consisting of a cationic lipid and a peptide antigen. In this report, we expanded the utility of this formulation to a protein based vaccine. First, we formulated the human papillomavirus (HPV) 16 E7 protein (E7) in different doses of DOTAP liposome. The results showed that these formulations failed to regress an established tumor. However, when sodium chloride (30 mM) was added to the DOTAP (100 nmol) / E7 (20 μg) formulation, anti-tumor activity was generated in the immunized mice. Correlatively, 30 mM NaCl in the DOTAP/E7 protein formulation increased the particle size from ∼350 to 550 nm, decreased the protein loading capacity (from 95 to 90%), and finally increased the zeta potential (from 29 mV to 38 mV). Next, a model protein antigen ovalbumin (OVA) was formulated in different doses of DOTAP liposomes. Similarly, the results showed that 20 μg OVA formulated in 200 nmol DOTAP with 30 mM NaCl had the best OVA- specific antibody response, including both IgG1 and IgG2a, suggesting both Th1 and Th2 immune responses were generated by this formulation. In conclusion, we have expanded the application of cationic DOTAP liposome formulation to protein based vaccines and also identified that small amounts of salt could change the physicochemical properties of the vaccine formulation and enhance the activity of the DOTAP/protein based vaccine. The enhancement of immune responses by salt is possibly due to its interference of the electrostatic interaction between the cationic lipid and the protein antigen to facilitate the antigen release from the carrier and at the same time activate the antigen presenting cells.
PMCID: PMC2841293  PMID: 18992312
Cationic liposome; Protein antigen; Immunogenicity; Salt; Vaccine
15.  The Role of Bloom Index of Gelatin on the Interaction with Retinal Pigment Epithelial Cells 
Biocompatible materials are of considerable interest in the development of cell/drug delivery carriers for therapeutic applications. This paper investigates the effects of the Bloom index of gelatin on its interaction with retinal pigment epithelial (RPE) cells. Following two days of culture of ARPE-19 cells with gelatin samples G75-100, G175, and G300, the in vitro biocompatibility was determined by cell proliferation and viability assays, and glutamate uptake measurements, as well as cytokine expression analyses. The mitochondrial dehydrogenase activity in the G300 groups was significantly lower than that of G75-100 and G175 groups. The Live/Dead assays also showed that the gelatin samples G300 induced mild cytotoxicity. In comparison with the treatment of gelatins with low Bloom index, the exposure to high Bloom strength gelatins markedly reduced the glutamate uptake capacity of ARPE-19 cells. One possible explanation for these observations is that the presence of gelatin samples G300 with high viscosity in the medium may affect the nutrient availability to cultured cells. The analyses of pro-inflammatory cytokine IL-6 expression at both mRNA and protein levels showed that the gelatins with low Bloom index caused less cellular inflammatory reaction and had more acceptable biocompatibility than their high Bloom strength counterparts. These findings suggest that the Bloom index gives influence on cellular responses to gelatin materials.
PMCID: PMC2812822  PMID: 20111679
gelatin; Bloom index; in vitro biocompatibility; retinal pigment epithelial cells
16.  Recombinant collagen for animal product-free dextran microcarriers 
Manufacturers of vaccines and other biologicals are under increasing pressure from regulatory agencies to develop production methods that are completely animal-component-free. In order to comply with this demand, alternative cell culture substrates to those now on the market, primarily collagen or gelatin, must be found. In this paper, we have tested a number of possible substitutes including recombinant collagen, a 100-kDa recombinant gelatin fragment and a peptide derived from a cell-binding region of type I collagen. The small 15-amino acid peptide did not support attachment of human fibroblasts in monolayer culture. The 100-kDa gelatin fragment supported cell attachment in monolayer culture, but was significantly less active than intact porcine gelatin. Recombinant type I collagen was as successful in promoting cell attachment as native collagen, and both were more effective than porcine gelatin. Based on these data, dextran microspheres were treated with the same attachment proteins—porcine gelatin, native collagen, or recombinant collagen. The same trends were observed as in monolayer culture. Concentrations of the recombinant collagen (as well as native collagen) supported cell attachment on dextran microspheres at concentrations as low as 0.01 μg/cm2. Treatment of the dextran with a low level of polyethylenimine, a cationic moiety, further enhanced attachment when used in conjunction with the low concentration of recombinant collagen. Where there was increased cell attachment, increased proliferation followed. We are confident, based on these findings, that a fully recombinant substitute could replace gelatin in current microcarrier preparations without losing the cell growth benefits provided by the native protein.
PMCID: PMC2833965  PMID: 18815845
Microcarrier; Animal component-free; Recombinant collagen; Dextran; PEI
17.  Negatively charged liposomes show potent adjuvant activity when simply admixed with protein antigens 
Molecular pharmaceutics  2011;8(4):1174-1185.
Liposomes have been investigated extensively as a vaccine delivery system. Herein the adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when admixed with protein antigens, ovalbumin (OVA, pI = 4.7), Bacillus anthracis protective antigen protein (PA, pI = 5.6), or cationized OVA (cOVA). Mice immunized subcutaneously with OVA admixed with different liposomes generated different antibody responses. Interestingly, OVA admixed with net negatively charged liposomes prepared with DOPA was as immunogenic as OVA admixed with positively charged liposomes prepared with DOTAP. Immunization of mice with the anthrax PA protein admixed with the net negatively charged DOPA liposomes also induced a strong and functional anti-PA antibody response. When the cationized OVA was used as a model antigen, liposomes with net neutral, negative, or positive charges showed comparable adjuvant activities. Immunization of mice with the OVA admixed with DOPA liposomes also induced OVA-specific CD8+ cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA tumors in mice. However, not all net negatively charged liposomes showed a strong adjuvant activity. The adjuvant activity of the negatively charged liposomes may be related to the liposome’s ability (i) to up-regulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development.
PMCID: PMC3148289  PMID: 21615153
Protein antigen; antibody response; CTL; anti-tumor activity; antigen uptake; APC activation
18.  The effect of the controlled release of basic fibroblast growth factor from ionic gelatin-based hydrogels on angiogenesis in a murine critical limb ischemic model 
Biomaterials  2007;28(16):2646-2654.
The localized delivery of exogenous, angiogenic growth factors has become a promising alternative treatment of peripheral artery disease (PAD) and critical limb ischemia. In the present study, we describe the development of a novel controlled release vehicle to promote angiogenesis in a murine critical limb ischemic model. Ionic, gelatin-based hydrogels were prepared by the carbodiimide-mediated amidation reaction between the carboxyl groups of gelatin or poly-L-glutamic acid molecules and the amine groups of poly-L-lysine or gelatin molecules, respectively. The degree of swelling of the synthesized hydrogels was assessed as a function of EDC/NHS ratios and the pH of the equilibrating medium, while the release kinetic profile of basic fibroblast growth factor (FGF-2) was evaluated in human fibroblast cultures. The degree of swelling (DS) decreased from 26.5 ±1.7 to 18.5 ±2.4 as the EDC concentration varied from 0.75 to 2.5 mg/ml. 80% of the FGF-2 was released at controlled rates from gelatin-polylysine (gelatin-PLL) and gelatin-polyglutamic acid (gelatin-PLG) hydrogel scaffolds over a period of 28 days. Cell adhesion studies revealed that the negatively charged surface of the gelatin-PLG hydrogels exhibited superior adhesion capabilities in comparison to gelatin-PLL and control gelatin surfaces. Laser Doppler Perfusion Imaging as well as CD31+ capillary immunostaining demonstrated that the controlled release of FGF-2 from ionic gelatin-based hydrogels is superior in promoting angiogenesis in comparison to the bolus administration of the growth factor. Over four weeks, FGF-2 releasing gelatin-PLG hydrogels exhibited marked reperfusion with a Doppler ratio of 0.889 (± 0.04) which was 69.3% higher than in the control groups.
PMCID: PMC1945227  PMID: 17320947
angiogenesis; hydrogels; FGF-2; controlled delivery; critical limb ischemia
1. Our results show clearly that the Hofmeister series is not the correct expression of the relative effect of ions on the swelling of gelatin, and that it is not true that chlorides, bromides, and nitrates have "hydrating," and acetates, tartrates, citrates, and phosphates "dehydrating," effects. If the pH of the gelatin is taken into considertion, it is found that for the same pH the effect on swelling is the same for gelatin chloride, nitrate, trichloracetate, tartrate, succinate, oxalate, citrate, and phosphate, while the swelling is considerably less for gelatin sulfate. This is exactly what we should expect on the basis of the combining ratios of the corresponding acids with gelatin since the weak dibasic and tribasic acids combine with gelatin in molecular proportions while the strong dibasic acid H2SO4 combines with gelatin in equivalent proportions. In the case of the weak dibasic acids he anion in combination with gelatin is therefore monovalent and in the case of the strong H2SO4 it is bivalent. Hence it is only the valency and not the nature of the ion in combination with gelatin which affects the degree of swelling. 2. This is corroborated in the experiments with alkalies which show that LiOH, NaOH, KOH, and NH4OH cause the same degree of swelling at the same pH of the gelatin solution and that this swelling is considerably higher than that caused by Ca(OH)2 and Ba(OH)2 for the same pH. This agrees with the results of the titration experiments which prove that Ca(OH)2 and Ba(OH)2 combine with gelatin in equivalent proportions and that hence the cation in combination with the gelatin salt with these two latter bases is bivalent. 3. The fact that proteins combine with acids and alkalies on the basis of the forces of primary valency is therefore not only in full agreement with the influence of ions on the physical properties of proteins but allows us to predict this influence qualitatively and quantitatively. 4. What has been stated in regard to the influence of ions on the swelling of the different gelatin salts is also true in regard to the influence of ions on the relative solubility of gelatin in alcohol-water mixtures. 5. Conductivity measurements of solutions of gelatin salts do not support the theory that the drop in the curves for swelling, osmotic pressure, or viscosity, which occurs at a pH 3.3 or a little less, is due to a drop in the concentration of ionized protein in the solution; nor do they suggest that the difference between the physical properties of gelatin sulfate and gelatin chloride is due to differences in the degree of ionization of these two salts.
PMCID: PMC2140423  PMID: 19871862
20.  Compensatory Thio-redox Interactions Between DsbA, CcdA and CcmG Unveil the Apocytochrome c Holdase Role of CcmG During Cytochrome c Maturation 
Molecular microbiology  2008;70(3):652-666.
During cytochrome c maturation (Ccm), the DsbA-dependent thio-oxidative protein-folding pathway is thought to introduce a disulfide bond into the heme-binding motif of apocytochromes c. This disulfide bond is believed to be reduced through a thio-reductive pathway involving the Ccm components CcdA (DsbD), CcmG and CcmH. Here, we show in Rhodobacter capsulatus that in the absence of DsbA cytochrome c levels were decreased and CcdA or CcmG or the putative glutathione transporter CydDC were not needed for Ccm. This decrease was not due to overproduction of the periplasmic protease DegP as a secondary effect of DsbA absence. In contrast, CcmH was absolutely necessary regardless of DsbA, indicating that compensatory thio-redox interactions excluded it. Remarkably, the double (DsbA-CcmG) and triple (DsbA-CcmG-CcdA) mutants produced cytochromes c at lower levels than the DsbA-null mutants, unless they contained a CcmG derivative (CcmG*) lacking its thio-reductive activity. Purified CcmG* can bind apocytochrome c in vitro, revealing for the first time a thiol-independent, direct interaction between apocytochrome c and CcmG. Furthermore, elimination of the thio-redox components does not abolish cytochrome c production, restricting the number of Ccm components essential for heme-apocyt c ligation per se during Ccm.
PMCID: PMC2581645  PMID: 18786143
cytochrome c maturation; thio-oxidation; thio-reduction; Rhodobacter capsulatus; photosynthetic and respiratory electron transfer
21.  Induction of ovalbumin-specific cytotoxic T cells by in vivo peptide immunization 
CTL recognize peptide forms of processed, foreign antigens in association with class I molecules encoded by the MHC and are usually directed against endogenously synthesized "cellular antigens," such as those expressed by virus-infected cells. In vitro studies have shown that small exogenous peptides can directly associate with class I molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. We have recently generated OVA-specific, H-2Kb-restricted CTL by immunizing C57BL/6 mice with a syngeneic tumor line transfected with the OVA cDNA. The CTL recognize the OVA transfectant E.G7-OVA and the synthetic peptide OVA258-276, but fail to recognize the native protein. We reasoned that given the potential for direct peptide/class I association observed in vitro, OVA258-276 may induce CTL after in vivo priming. However, we found that this is not the case. OVA258-276 and peptides of increasing lengths up to OVA242-276 and OVA242-285, which are all able to form the target complex in vitro, are inefficient at priming E.G7-OVA-specific CTL responses after intravenous injection. This is also true for both native and denatured OVA. In contrast to these results the synthetic peptide OVA229-276 corresponding to a peptide in a partial tryptic digestion of OVA can efficiently prime C57BL/6 mice in vivo after intravenous injection. This peptide elicits CTL that appear identical to those derived from animals immunized with syngeneic cells producing OVA endogenously. These results are discussed in terms of separate class I and class II antigen presentation pathways and the ability of only certain, exogenous antigens to enter the cytoplasmic, class I pathway.
PMCID: PMC2189277  PMID: 2784478
22.  3D cell entrapment in crosslinked thiolated gelatin-poly(ethylene glycol) diacrylate hydrogels 
Biomaterials  2011;33(1):48-58.
The combined use of natural ECM components and synthetic materials offers an attractive alternative to fabricate hydrogel-based tissue engineering scaffolds to study cell-matrix interactions in three-dimensions (3D). A facile method was developed to modify gelatin with cysteine via a bifunctional PEG linker, thus introducing free thiol groups to gelatin chains. A covalently crosslinked gelatin hydrogel was fabricated using thiolated gelatin and poly(ethylene glycol) diacrylate (PEGdA) via thiol-ene reaction. Unmodified gelatin was physically incorporated in a PEGdA-only matrix for comparison. We sought to understand the effect of crosslinking modality on hydrogel physicochemical properties and the impact on 3D cell entrapment. Compared to physically incorporated gelatin hydrogels, covalently crosslinked gelatin hydrogels displayed higher maximum weight swelling ratio (Qmax), higher water content, significantly lower cumulative gelatin dissolution up to 7 days, and lower gel stiffness. Furthermore, fibroblasts encapsulated within covalently crosslinked gelatin hydrogels showed extensive cytoplasmic spreading and the formation of cellular networks over 28 days. In contrast, fibroblasts encapsulated in the physically incorporated gelatin hydrogels remained spheroidal. Hence, crosslinking ECM protein with synthetic matrix creates a stable scaffold with tunable mechanical properties and with long-term cell anchorage points, thus supporting cell attachment and growth in the 3D environment.
PMCID: PMC3282186  PMID: 21955690
ECM protein; crosslinking modality; gelatin; PEG; 3D cell entrapment
23.  Robust Stimulation of Humoral and Cellular Immune Responses following Vaccination with Antigen-Loaded β-Glucan Particles 
mBio  2010;1(3):e00164-10.
β-Glucan particles (GPs) are purified Saccharomyces cerevisiae cell walls treated so that they are primarily β1,3-d-glucans and free of mannans and proteins. GPs are phagocytosed by dendritic cells (DCs) via the Dectin-1 receptor, and this interaction stimulates proinflammatory cytokine secretion by DCs. As the hollow, porous GP structure allows for high antigen loading, we hypothesized that antigen-loaded GPs could be exploited as a receptor-targeted vaccine delivery system. Ovalbumin (OVA) was electrostatically complexed inside the hollow GP shells (GP-OVA). Incubation of C57BL/6J mouse bone marrow-derived DCs with GP-OVA resulted in phagocytosis, upregulation of maturation markers, and rapid proteolysis of OVA. Compared with free OVA, GP-OVA was >100-fold more potent at stimulating the proliferation of OVA-reactive transgenic CD8+ OT-I and CD4+ OT-II T cells, as measured by in vitro [3H]thymidine incorporation using DCs as antigen-presenting cells. Next, immune responses in C57BL/6J mice following subcutaneous immunizations with GP-OVA were compared with those in C57BL/6J mice following subcutaneous immunizations with OVA absorbed onto the adjuvant alum (Alum/OVA). Vaccination with GP-OVA stimulated substantially higher antigen-specific CD4+ T-cell lymphoproliferative and enzyme-linked immunospot (ELISPOT) responses than that with Alum/OVA. Moreover, the T-cell responses induced by GP-OVA were Th1 biased (determined by gamma interferon [IFN-γ] ELISPOT assay) and Th17 biased (determined by interleukin-17a [IL-17a] ELISPOT assay). Finally, both the GP-OVA and Alum/OVA formulations induced strong secretions of IgG1 subclass anti-OVA antibodies, although only GP-OVA induced secretion of Th1-associated IgG2c antibodies. Thus, the GP-based vaccine platform combines adjuvanticity and antigen delivery to induce strong humoral and Th1- and Th17-biased CD4+ T-cell responses.
Most licensed vaccines work by promoting protective antibody responses. However, for many infectious diseases, antibody-mediated protection appears to play a relatively minor role, and vaccination has met with limited success. While live-attenuated organisms generally elicit T-cell responses, their use in vaccines is limited by the potential for causing disease. Thus, there is an urgent need for new vaccine platforms that deliver antigens in such a manner as to promote strong T-cell-mediated responses. Here we designed a novel vaccine platform consisting of yeast-derived β-glucan particles (GPs) that combines antigen delivery and adjuvant activity. GPs loaded with the model antigen ovalbumin (OVA) stimulated robust humoral and T-cell responses in mice. In addition, the cellular response was Th1 and Th17 biased. This work has implications for the design of vaccines that stimulate biased T-cell responses as well as for understanding how immunity to fungal pathogens develops.
PMCID: PMC2925077  PMID: 20802824
24.  Cholesterol-Peptide Hybrids to Form Liposome-Like Vesicles for Gene Delivery 
PLoS ONE  2013;8(1):e54460.
In this paper, four amphiphilic cholesterol-peptide conjugates (Ch-R5H5, Ch-R3H3, Ch-R5 and Ch-R5) were designed and synthesized, and their properties in gene delivery were evaluated in vitro with an aim of developing more efficient gene delivery carriers. These amphiphilic cholesterol-peptide conjugates are composed of hydrophobic cholesterol and positively charged peptides. They were able to self-assemble into micelles at low concentrations and their critical micelle concentrations in phosphate buffered saline (pH 7.4) are ≤85 µg/mL. Amphiphilic cholesterol-peptide conjugates condensed DNA more efficiently than a hydrophilic cationic oligoarginine (R10) peptide with no hydrophobic segment. Their transfection efficiencies were at least two orders of magnitude greater than that of R10 peptide in HEK-293 cells. Moreover, the introduction of histidine residues in cholesterol-peptide conjugates led to higher gene expression efficiency compared with cholesterol-peptides without histidine (Ch-R5 and Ch-R3), and the luciferase expression level was comparable or even higher than that induced by PEI at its optimal N/P ratio. In particular, Ch-R5H5 condensed DNA into smaller nanoparticles than Ch-R3H3 at higher N/P ratios, and the minimum size of Ch-R5H5/DNA complexes was 180 nm with zeta potential of 23 mV, achieved at the N/P ratio of 30. This liposome-like vesicle may be a promising gene delivery carrier for intravenous therapy.
PMCID: PMC3559823  PMID: 23382899
25.  A Polymeric Protein Induces Specific Cytotoxicity in a TLR4 Dependent Manner in the Absence of Adjuvants 
PLoS ONE  2012;7(9):e45705.
Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA257–264 and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA257–264 in vivo. BLS-OVA257–264 immunization induced the proliferation of OVA257–264-specific CD8+ lymphocytes and also increased the percentage of OVA257–264-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA257–264-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA257–264 induced the generation of CD8+ effector cells. BLS-OVA257–264 was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA257–264 without adjuvants induced a strong OVA257–264-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor. These data show that TLR4 signaling is necesary for the induction of a cytotoxic response but not for antigen cross presentation.
PMCID: PMC3454435  PMID: 23029192

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