[Study on preparation of 3D printing degradable tissue engineering ossicles].

Objective: In combination with 3D printing technology and degradable composite materials, to discuss the preparation method of tissue engineering ossicles for middle ear hearing reconstruction. Methods: Domestic polymer (polylactic acid-glycolic acid copolymer, PLGA) and degradable ceramic material (ß-tricalcium phosphate, ß-TCP) were selected and prepared by low temperature deposition method according to the design ratio to Program according to the outline design code of the required scaffold to generate appropriate print files, and then the self-developed low-temperature deposition printing device was used to prepare tissue-engineered osseous scaffolds in accordance with the print files in a low-temperature environment. The scaffolds was freeze-dried and sterilized for later use after printing. Light microscopy and scanning electron microscopy were used to observe the apparent characteristics and internal structure of the scaffolds and to check its pore size, porosity and mechanical properties. Results: After printing, a degradable scaffold was obtained. Under the optical microscope, it was a small cylindrical shape with a diameter of 1.5 mm and a length of 6.0 mm, and its surface had micropores. The degradable scaffold had a horizontal and vertical interlaced warp and weft structure, the wire spacing was 1.2 mm, and the pores were connected to each other. The surface could see circular or quadrangular pores with a pore size of about 100-400 µm. The diameter of the inter-pore cross-linked channels was about 50 µm and the diameter of the surrounding circular micropores was about 10-40 µm. ß-TCP particles with a size of about 700 nm were attached to the surface of the PLGA material. The average porosity of the whole scaffolds was (83.43±0.01)%, and the content of BMP-2 loaded was about 0.7 µg/mm(3). After freeze-drying, the mechanical strength of the scaffold was moderate, and there was no obvious deformation during stretching and compression, which met the mechanical requirements of tissue engineering ossicles. Conclusions: Using the low-temperature deposition printing method and strictly controlled processes and conditions, a polymer-degradable ceramic ossicle tissue engineering scaffold can be prepared for implantation experiments. The scaffold has suitable porosity and mechanical properties, and can be loaded with osteoinductive factors.

[Effect of Rab11 gene expression on the invasion and migration of cervical cancer cell line SiHa in hypoxia].

Objective: To explore the expression of Ras-related protein 11 (Rab11) in hypoxia, the effect of Rab11 on the invasion and migration of cervical cancer cell line SiHa and its possible mechanism. Methods: SiHa cells were divided into 4 groups, the normoxic blank group (normal culture in normoxia), the hypoxic blank group (normal culture in hypoxia), the negative control group [transfection of negative control small interfering RNA (siRNA) in hypoxia], the Rab11-siRNA group (transfection of Rab11 siRNA in hypoxia). Western blot was used to examine the expression of Rab11, integrin α5, integrin ß3, phosphorylated focal adhesion kinase (p-FAK), phosphorylated phosphatidylinositol 3 kinase (p-PI3K) protein, together with the expression of Ras correlative C3 creotoxin substrate 1 (Rac1), which was critical in regulating cell invasion. The mRNA expression of Rab11 in the 4 groups was detected by realtime-qPCR. The cell invasion was detected by matrigel assay, while the cell migration was detected by transwell assay. Immunofluorescence was used to identify intracellular location of Rac1 in SiHa cell. Results: (1) The expression of Rab11, intergrin α5, intergrin ß3, p-FAK, p-PI3K and Rac1 in the normoxic blank group were 0.56±0.04, 0.33±0.03, 0.32±0.03, 0.36±0.03, 0.35±0.03 and 0.47±0.03, respectively. In the hypoxic blank group, they were 0.73±0.03, 0.74±0.03, 0.61±0.03, 0.62±0.03, 0.60±0.03 and 0.73±0.03, respectively. In the negative control group, their expressions were 0.72±0.03, 0.73±0.03, 0.59±0.03, 0.61±0.03, 0.59±0.03 and 0.72±0.03, respectively. While in the Rab11-siRNA group, they were 0.44±0.03, 0.30±0.03, 0.29±0.03, 0.30±0.03, 0.30±0.03 and 0.34±0.04, respectively. The expressions of Rab11, α5, ß3, p-FAK, p-PI3K and Rac1 were significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and were significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (2) The expressions of Rab11-mRNA were 1.000±0.000, 1.454±0.114, 1.442±0.101, 0.570± 0.046 in the normoxic blank group, the hypoxic blank group, the negative control group and the Rab11-siRNA group, respectively. It was significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (3) By Matrigel, the invasion cell number in the normoxic blank group, the hypoxic blank group,the negative control group and the Rab11-siRNA group were 65±12, 106±16, 104± 17 and 50±11, respectively. The invasion capacity was significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (4) By transwell assay, the migration cells in the normoxic blank group, the hypoxic blank group, the negative control group and the Rab11-siRNA group were 127±12, 169±15, 161±13 and 77±13, respectively. The capacity of invasion was significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (5) The immunofluorescence showed that the red fluorescence intensity around nucleus was significantly increased in the normoxic blank group, the hypoxic blank group and the negative control group than in the Rab11-siRNA group. Conclusions: Hypoxia could promote the invasion and migration of SiHa cells. In hypoxia, the down regulation of Rab11 expression could inhibit the invasion and migration of SiHa cells. This might be due to the decreased expression of the intergrin α5, intergrin ß3, p-FAK, p-PI3K and Rac1 protein.

Cyclin A and B functions in the early Drosophila embryo.

In eukaryotes, mitotic cyclins localize differently in the cell and regulate different aspects of the cell cycle. We investigated the relationship between subcellular localization of cyclins A and B and their functions in syncytial preblastoderm Drosophila embryos. During early embryonic cycles, cyclin A was always concentrated in the nucleus and present at a low level in the cytoplasm. Cyclin B was predominantly cytoplasmic, and localized within nuclei only during late prophase. Also, cyclin B colocalized with metaphase but not anaphase spindle microtubules. We changed maternal gene doses of cyclins A and B to test their functions in preblastoderm embryos. We observed that increasing doses of cyclin B increased cyclin B-Cdk1 activity, which correlated with shorter microtubules and slower microtubule-dependent nuclear movements. This provides in vivo evidence that cyclin B-Cdk1 regulates microtubule dynamics. In addition, the overall duration of the early nuclear cycles was affected by cyclin A but not cyclin B levels. Taken together, our observations support the hypothesis that cyclin B regulates cytoskeletal changes while cyclin A regulates the nuclear cycles. Varying the relative levels of cyclins A and B uncoupled the cytoskeletal and nuclear events, so we speculate that a balance of cyclins is necessary for proper coordination during these embryonic cycles.

Ca2+ binding to occluded sites in the CrATP-ATPase complex of sarcoplasmic reticulum: evidence for two independent high-affinity sites.

Occluded Ca2+ sites in the CrATP-ATPase complex are studied by first forming the complex in the presence of EGTA so that the sites can be occluded while vacant. 45Ca2+ binding to the occluded sites is then studied under equilibrium conditions. Binding curves are produced for two independent Ca2+ sites with Kd(1) = 0.2 microM and Kd(2) = 1.6 microM. When both sites are saturated, only the Ca2+ bound to the lower affinity site can exchange with free Ca2+. On addition of EGTA (15 vs 0.5 mM Ca2+) all bound Ca2+ dissociates, the net dissociation rate of one-half of the Ca2+ being approximately 10-fold greater than that of the other one-half (at 37 degrees C). When Ca2+ is bound only to the higher affinity site, this Ca2+ will exchange slowly if the concentration of free Ca2+ is below the saturation level of the lower affinity site. An ionophore dependency of the rates of binding and dissociation indicates that the access to the sites is through the interior of the vesicle. Solubilization in C12E9 releases the Ca2+ in the higher affinity site. Our observations are consistent with a model of the ATPase where the lower affinity of two transport sites is associated with the interior position (closest to the lumen) in a transmembrane channel. It is further evident that when in the occluded state, the higher affinity site is available without Ca2+ first being bound to the lower affinity site, eliminating cooperativity from the binding mechanism. In turn, this implies a connection between the integrity of the high-affinity binding site and the linking of sections of the catalytic site by CrATP.

Sarcoplasmic reticulum calcium ATPase. Labeling of a putative Mg2+ site by reaction with a carbodiimide and a spin-label.

The sarcoplasmic reticulum Ca(2+)-ATPase loses hydrolytic activity and the ability to be phosphorylated by Pi following incubation with EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]. 4 nmol of tempamine per mg SR protein can be coupled to either a glu or an asp side chain through the EDC reaction. Mg2+ protects against loss of activity and tempamine labeling with a mid-point of about 3 mM in the absence of Ca2+. This is similar to the Kd for a Mg2+ that serves as a cofactor in enzyme phosphorylation. The Mg2+ protection constant is lowered by an order of magnitude when Ca2+ is bound to the transport sites. It is suggested that control of the Mg2+ binding site affinity may be part of the mechanism of enzyme activation by Ca2+.

Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides.

Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mM tert-butyl hydroperoxide in the presence of 200 microM equine myoglobin or to 100 mM hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.