Crystalline Biohybrid Materials Based on Protein Cages.

Highly ordered superstructures of nanomaterials can be synthesized using protein cages as templates for the assembly of inorganic nanoparticles. Here, we describe in detail the creation of these biohybrid materials. The approach involves computational redesign of ferritin cages, followed by recombinant protein production and purification of the new variants. Metal oxide nanoparticles are synthesized inside the surface-charged variants. The composites are assembled using protein crystallization to yield highly ordered superlattices, which are characterized, for example, with small angle X-ray scattering. This protocol provides a detailed and comprehensive account on our newly established strategy for the synthesis of crystalline biohybrid materials.

Multipotent Adult Progenitor Cells, but Not Tissue Inhibitor of Matrix Metalloproteinase-3, Increase Tissue Sparing and Reduce Urological Complications following Spinal Cord Injury.

Following spinal cord injury (SCI), inflammation amplifies damage beyond the initial insult, providing an opportunity for targeted treatments. An ideal protective therapy would reduce both edema within the lesion area and the activation/infiltration of detrimental immune cells. Previous investigations demonstrated the efficacy of intravenous injection of multipotent adult progenitor cells (MAPC®) to modulate immune response following SCI, leading to significant improvements in tissue sparing, locomotor and urological functions. Separate studies have demonstrated that tissue inhibitor of matrix metalloproteinase-3 (TIMP3) reduces blood-brain barrier permeability following traumatic brain injury in a mouse model, leading to improved functional recovery. This study examined whether TIMP3, delivered alone or in concert with MAPC cells, improves functional recovery from a contusion SCI in a rat model. The results suggest that intravenous delivery of MAPC cell therapy 1 day following acute SCI significantly improves tissue sparing and impacts functional recovery. TIMP3 treatment provided no significant benefit, and further, when co-administered with MAPC cells, it abrogated the therapeutic effects of MAPC cell therapy. Importantly, this study demonstrated for the first time that acute treatment of SCI with MAPC cells can significantly reduce the incidence of urinary tract infection (UTI) and the use of antibiotics for UTI treatment.

Carbazole-, Aspidofractinine-, and Aspidocarpamine-Type Alkaloids from Pleiocarpa pycnantha.

Three new alkaloids, janetinine (1a), pleiokomenine A (2), and huncaniterine B (3a), and 13 known compounds, pleiomutinine (3b), huncaniterine A (3c), 1-carbomethoxy-ß-carboline (4), evoxanthine (5), deformyltalbotine acid lactone (6), pleiocarpamine (7), N4-methyl-10-hydroxygeissoschizol (8), spegatrine (9), neosarpagine (10), aspidofractinine (11), N1-methylkopsinin (12), pleiocarpine (13), and N1-methylkopsinin- N4-oxide (14), were isolated from the stem bark of Pleiocarpa pycnantha. Janetinine (1a) is a carbazole alkaloid; in pleiokomenine A (2), two aspidofractinine-type alkaloids are bridged by a methylene unit in an unprecedented way, and huncaniterine B (3a) is a pleiocarpamine-aspidofractinine-type dimer. The structures and relative configurations of these compounds were elucidated on the basis of NMR and MS analyses. Their absolute configurations were defined by means of experimental and calculated ECD data, and additionally, the structures of 5 and 13 were determined by single crystal X-ray diffraction. Compounds 1a, 2, 3b, 4, 6, 9, and 12 displayed cancer chemopreventive properties through either quinone reductase induction ( CD = 30.7, 30.2, 29.9, 43.5, and 36.7 µM for 1a, 4, 6, 9, and 12, respectively) and/or NF-κB inhibition with IC50 values of 13.1, 8.4, 9.4, and 8.8 µM for 2, 3b, 6, and 12, respectively.

Treatment of Burn and Surgical Wounds With Recombinant Human Tropoelastin Produces New Elastin Fibers in Scars.

Tropoelastin (TE), the soluble precursor of insoluble elastin fibers, is produced in minimal amounts in adults. Burn injuries result in inflexible collagen-rich scars because of lack of elastin fiber formation. We studied the feasibility of using recombinant human tropoelastin to enable elastin fiber production in burn and surgical scars to improve skin flexibility. In a swine hypertrophic burn scar model, normal skin and 3 × 3-cm partial thickness thermal burns underwent dermatome resection at 1 week post burn and randomized to four subcutaneous injections of saline or TE (either 0.5, 5, or 10 mg/ml) spaced 3 days apart. Two burn sites received TE injections after wound closure (0.5 or 10 mg/ml). At 90 days, skin hardness, flexibility, and histology were evaluated. All injury sites developed hypertrophic scars. New elastin fibers were found in burn scars in all injuries injected after skin closure with low (5/5) and high (6/6) TE doses (P < .05). No elastin fibers were observed without TE treatment. No significant differences in skin hardness, flexibility, or inflammation were observed. This is the first report demonstrating that subcutaneous injections of TE into surgical and burn injuries can safely produce new elastin fibers in scars. Despite the development of new elastin fibers, skin flexibility was not improved, possibly because of insufficient elastin fiber maturation or the hypertrophic model used. The ability to restore elastin fiber formation in adult skin after burns, trauma, and surgery may improve skin regeneration and reduce disabling complications of scar formation.

Rapid assay of stem cell functionality and potency using electric cell-substrate impedance sensing.

Regenerative medicine studies using autologous bone marrow mononuclear cells (BM-MNCs) have shown improved clinical outcomes that correlate to in vitro BM-MNC invasive capacity. The current Boyden-chamber assay for testing invasive capacity is labor-intensive, provides only a single time point, and takes 36 hours to collect data and results, which is not practical from a clinical cell delivery perspective. To develop a rapid, sensitive and reproducible invasion assay, we employed Electric Cell-substrate Impedance Sensing (ECIS) technology. Chemokine-directed BM-MNC cell invasion across a Matrigel-coated Transwell filter was measurable within minutes using the ECIS system we developed. This ECIS-Transwell chamber system provides a rapid and sensitive test of stem and progenitor cell invasive capacity for evaluation of stem cell functionality to provide timely clinical data for selection of patients likely to realize clinical benefit in regenerative medicine treatments. This device could also supply robust unambiguous, reproducible and cost effective data as a potency assay for cell product release and regulatory strategies.

Development of a functional schwann cell phenotype from autologous porcine bone marrow mononuclear cells for nerve repair.

Adult bone marrow mononuclear cells (BM-MNCs) are a potential resource for making Schwann cells to repair damaged peripheral nerves. However, many methods of producing Schwann-like cells can be laborious with the cells lacking a functional phenotype. The objective of this study was to develop a simple and rapid method using autologous BM-MNCs to produce a phenotypic and functional Schwann-like cell. Adult porcine bone marrow was collected and enriched for BM-MNCs using a SEPAX device, then cells cultured in Neurobasal media, 4 mM L-glutamine and 20% serum. After 6-8 days, the cultures expressed Schwann cell markers, S-100, O4, GFAP, were FluoroMyelin positive, but had low p75(NGF) expression. Addition of neuregulin (1-25 nM) increased p75(NGF) levels at 24-48 hrs. We found ATP dose-dependently increased intracellular calcium [Ca(2+)](i), with nucleotide potency being UTP = ATP > ADP > AMP > adenosine. Suramin blocked the ATP-induced [Ca(2+)](i) but α, ß,-methylene-ATP had little effect suggesting an ATP purinergic P2Y2 G-protein-coupled receptor is present. Both the Schwann cell markers and ATP-induced [Ca(2+)](i) sensitivity decreased in cells passaged >20 times. Our studies indicate that autologous BM-MNCs can be induced to form a phenotypic and functional Schwann-like cell which could be used for peripheral nerve repair.

Bismuth subsalicylate increases intracellular Ca2+, MAP-kinase activity, and cell proliferation in normal human gastric mucous epithelial cells.

Clinical and laboratory studies have shown that bismuth subsalicylate (BSS) is helpful in the healing of gastric ulcers because of the bactericidal effects of bismuth (Bi3+) on H. pylori. Bismuth or BSS has also been reported to possess other nonbactericidal or "gastroprotective" effects in the stomach. It is known in other cell types that the effects of extracellular divalent or trivalent cations (e.g., Ca2+) can activate a plasma membrane-bound calcium-sensing receptor (CaSR). In a previous study, we found the existence of a CaSR which was activated by extracellular Ca2+ and found to increase intracellular Ca2+ [Ca2+]i, MAP-kinase activity, and gastric epithelial cell proliferation. In the present study, we were interested in determining whether the effects of the trivalent cation Bi3+ (in the form of BSS) on [Ca2+]i, MAP-kinase activity, and proliferation of gastric cells. We found that BSS dose dependently increased [Ca2+]i, p44/p42 and p38 MAP-kinase activites, and gastric mucous epithelial cell growth. The addition of BAPTA to chelate intracellular Ca2+ blocked BSS-induced p44/p42 MAP-kinase activities but not p38 MAP-kinase activity. The p44/p42 MAP-kinase inhibitor PD98059 and the p38 MAP-kinase inhibitor SB203580 dose dependently decreased gastric mucous cell growth over a 24 hr. All of the BSS-induced changes in [Ca2+]i, MAP-kinase activity, and gastric cell proliferation could be reproduced with the CaSR-agonist gadolinium (Gd3+). Our data suggest that BSS may possess additional novel effects by increasing gastric mucous epithelial cell growth through a Ca2+/MAP-kinase-dependent pathway.

Effects of Helicobacter pylori on intracellular Ca2+ signaling in normal human gastric mucous epithelial cells.

In stomach, Helicobacter pylori (Hp) adheres to gastric mucous epithelial cells (GMEC) and initiates several different signal transduction events. Alteration of intracellular Ca2+ concentration ([Ca2+]i) is an important signaling mechanism in numerous bacteria-host model systems. Changes in [Ca2+]i induced by Hp in normal human GMEC have not yet been described; therefore, we examined effects of Hp on [Ca2+]i in normal human GMEC and a nontransformed GMEC line (HFE-145). Cultured cells were grown on glass slides, porous filters, or 96-well plates and loaded with fura 2 or fluo 4. Hp wild-type strain 60190 and vacA-, cagA-, and picB-/cagE- isogenic mutants were incubated with cells. Changes in [Ca2+]i were recorded with a fluorimeter or fluorescence plate reader. Wild-type Hp produced dose-dependent biphasic transient [Ca2+]i peak and plateau changes in both cell lines. Hp vacA- isogenic mutant produced changes in [Ca2+]i similar to those produced by wild type. Compared with wild type, cagA- and picB-/cagE- isogenic mutants produced lower peak changes and did not generate a plateau change. Preloading cultures with intracellular Ca2+ chelator BAPTA blocked all Hp-induced [Ca2+]i changes. Thapsigargin pretreatment of cultures to release Ca2+ from internal stores reduced peak change. Extracellular Ca2+ removal reduced plateau response. Hp-induced peak response was sensitive to G proteins and PLC inhibitors. Hp-induced plateau change was sensitive to G protein inhibitors, src kinases, and PLA2. These findings are the first to show that H. pylori alters [Ca2+]i in normal GMEC through a Ca2+ release/influx mechanism that depends on expression of cagA and picB/cagE genes.

Helicobacter pylori induces apoptosis in Barrett's-derived esophageal adenocarcinoma cells.

Helicobacter pylori may protect against the development of dysplasia in Barrett's epithelium of patients with gastroesophageal reflux disease. The aim of this study was to determine whether H. pylori preferentially induces apoptosis in Barrett's-derived cancer cells compared to normal cells. A Barrett's-derived adenocarcinoma cell line (OE33) was grown. H. pylori wild-type, isogenic vacA-, cagA(-), and picB-/cagE- mutant strains were grown on agar plates. Intact or sonicated bacteria were used to treat normal and OE33 cells for 24 hours, and Hoechst dye binding was performed to measure apoptosis. FAS protein expression was determined by Western immunoblotting. OE33 cells treated with intact H. pylori wild-type strains produced significant (P < 0.05) dose-dependent increases in apoptosis compared to normal esophageal cells. H. pylori wild-type and vacA- isogenic strains were more effective than cagA- and picB-/cage- isogenic strains in inducing apoptosis in OE33 cells. In OE33 cells, H. pylori sonicates produced lower levels of apoptosis than intact bacteria. Wild-type H. pylori strains increased Fas protein expression in OE33 cells at 18 hours. H. pylori induced apoptosis at a higher rate in the Barrett's-derived human esophageal adenocarcinoma cells than in normal esophageal cells. The H. pylori-induced apoptosis was primarily dependent on intact bacteria and the presence of the cagA and picB/cagE gene products. H. pylori-induced apoptosis may involve the Fas-caspase cascade.