Colchicine modulates calcium homeostasis and electrical property of HL‐1 cells

Colchicine is a microtubule disruptor that reduces the occurrence of atrial fibrillation (AF) after an operation or ablation. However, knowledge of the effects of colchicine on atrial myocytes is limited. The aim of this study was to determine if colchicine can regulate calcium (Ca²⁺) homeostasis and attenuate the electrical effects of the extracellular matrix on atrial myocytes. Whole‐cell clamp, confocal microscopy with fluorescence, and western blotting were used to evaluate the action potential and ionic currents of HL‐1 cells treated with and without (control) colchicine (3 nM) for 24 hrs. Compared with control cells, colchicine‐treated HL‐1 cells had a longer action potential duration with smaller intracellular Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ content by 10% and 47%, respectively. Colchicine‐treated HL‐1 cells showed a smaller L‐type Ca²⁺ current, reverse mode sodium–calcium exchanger (NCX) current and transient outward potassium current than control cells, but had a similar ultra‐rapid activating outward potassium current and apamin‐sensitive small‐conductance Ca²⁺‐activated potassium current compared with control cells. Colchicine‐treated HL‐1 cells expressed less SERCA2a, total, Thr17‐phosphorylated phospholamban, Cav1.2, CaMKII, NCX, Kv1.4 and Kv1.5, but they expressed similar levels of the ryanodine receptor, Ser16‐phosphorylated phospholamban and Kv4.2. Colchicine attenuated the shortening of the collagen‐induced action potential duration in HL‐1 cells. These findings suggest that colchicine modulates the atrial electrical activity and Ca²⁺ regulation and attenuates the electrical effects of collagen, which may contribute to its anti‐AF activity.     

In vitro study of using calcium phosphate cement as immunoisolative device to enclose insulinoma/agarose microspheres as bioartificial pancreas

In this study, the feasibility of using calcium phosphate cement (CPC) as immunoisolative device to enclose insulinoma/agarose microspheres as bioartificial pancreas was evaluated. We fabricated a chamber by CPC and utilized X-ray diffraction, Scanning electron microscope and Mercury intrusion porosimetry to identify the characters of the CPC chamber. The nominal molecular weight cut-off and cytotoxicity of CPC chamber were also evaluated. An insulinoma cell line (RIN-m5F) was chosen as insulin source and encapsulated in agarose microspheres and then enclosed in preformed CPC chamber. Insulin secretion was analyzed by Enzyme-linked immunosorbant assay to evaluate the function of insulinoma enclosed in CPC chamber. Results showed that the CPC chamber was non-cytotoxicity to insulinoma and can block the penetration of molecules which molecular weight larger than 12.4 kDa. Insulinoma inside the CPC chamber can secrete insulin in stable level for 30 days. This study indicated that we may use CPC as immunoisolative material to enclose insulinoma/agarose microspheres as bioartificial pancreas.     

Optimal thermotolerance of Bifidobacterium bifidum in gellan-alginate microparticles

The purpose of this research was to encapsulate Bifidobacterium bifidum using gellan, sodium alginate and prebiotics as coating materials, and to maximize the thermotolerance of the probiotics with an optimal combination of the coating materials. The optimal ratio of the coating materials for the microparticles under heat treatments (75 degrees C, 1 min) was obtained by using the response surface method and the sequential quadratic programming technique. Optimization results indicated that 2% sodium alginate mixed with 1% gellan gum as coating materials would produce the highest thermotolerance in terms of B. bifidum count. The verification experiment yielded a result close to the predicted values, with no significant difference (P > 0.05). The results of heat treatments also demonstrated that the addition of gellan gum in the walls of probiotic microcapsules provided improved protection for B. bifidum. These probiotic counts remained at 10(5)-10(6) CFU/g for the microcapsules stored for 2 months, then treated in heat and in simulated gastric fluid.     

Effects of fibrillin-1 degradation on microfibril ultrastructure

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.     

Heptad repeats regulate protein phosphatase 2a recruitment to I-kappaB kinase gamma/NF-kappaB essential modulator and are targeted by human T-lymphotropic virus type 1 tax

The switching on-and-off of I-kappaB kinase (IKK) and NF-kappaB occurs rapidly after signaling. How activated IKK becomes down-regulated is not well understood. Here we show that following tumor necrosis factor-alpha stimulation, protein phosphatase 2A (PP2A) association with IKK is increased. A heptad repeat in IKKgamma, helix 2 (HLX2), mediates PP2A recruitment. Two other heptad repeats downstream of HLX2, termed coiled-coil region 2 (CCR2) and leucine zipper (LZ), bind HLX2 and negatively regulate HLX2 interaction with PP2A. HTLV-1 transactivator Tax also binds HLX2, and this interaction is enhanced by CCR2 but reduced by LZ. In the presence of Tax, PP2A-IKKgamma binding is greatly strengthened. Interestingly, peptides spanning CCR2 and/or LZ disrupt IKKgamma-Tax and IKKgamma-PP2A interactions and potently inhibit NF-kappaB activation by Tax and tumor necrosis factor-alpha. We propose that when IKK is resting, HLX2, CCR2, and LZ form a helical bundle in which HLX2 is sequestered. The HLX2-CCR2-LZ bundle becomes unfolded by signal-induced modifications of IKKgamma or after Tax binding. In this conformation, IKK becomes activated. IKKgamma then recruits PP2A via the exposed HLX2 domain for rapid down-regulation of IKK. Tax-PP2A interaction, however, renders PP2A inactive, thus maintaining Tax-PP2A-IKK in an active state. Finally, CCR2 and LZ possibly inhibit IKK activation by stabilizing the HLX2-CCR2-LZ bundle.     

Irreversible block of cardiac mutant Na+ channels by batrachotoxin

Batrachotoxin (BTX) not only keeps the voltage-gated Na(+) channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na(+) channels, how Na(+) ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na(+) ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNa(v)1.5 Na(+) channels. We found that BTX at 5 microM indeed elicited a strong block of hNa(v)1.5-N927K currents (approximately 70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na(+) channel gating. Once occurred, this unique use-dependent block of hNa(v)1.5-N927K Na(+) channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels albeit with a faster on-rate; approximately 90% of peak Na(+) currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/H/D/S/Q/G/E substitutions in fast inactivation-deficient hNa(v)1.5 Na(+) channels, only N927K/R Na(+) currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNa(v)1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na(+) ions.     

Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.