Key Engineering Materials Vols. 277-279

Abstract: The fission yeast, Schizosaccharomyces pombe is a single-celled free-living fungus that shares many features with cells of more complicated eukaryotes. Many of the genes required for the cell-cycle control, proteolysis, protein modification, and RNA splicing are highly conserved with those of higher eukaryotes. Moreover, fission yeast has the merit of genetics and its genetic system is already well characterized. As such, the current study evaluated the use of a fission yeast system as a tool for the functional study of mammalian genes and attempted to set up an assay system for novel genes. Since the phenotypes of a deletion mutant and the overexpression of a gene are generally analyzed for a functional study of specific genes in yeast, the present study used overexpression phenotypes to study the functions of mammalian genes. Therefore, based on using a thiamine-repressive promoter, two mammalian genes were expressed in fission yeast, and their overexpressed phenotypes compared with those in mammalian cells. The phenotypes resulting from overexpression were analyzed using a FACS, which analyzes the DNA contents, and a microscope. One of the selected genes was the mammalian Polo-like kinase 1 (Plk1), which is activated and plays a role in the mitotic phase of the cell division cycle. The overexpression of various constructs of Plk1 in the HeLa cells caused cell cycle defects, suggesting that the ectopic Plk1s blocked the endogenous Plk1 in the cells. As expected, when the constructs were overexpressed in the fission yeast system, the cells were arrested in mitosis and defected at the end of mitosis. As such, this data suggests that the Plk1-overexpressed phenotypes were similar in the mammalian cells and the fission yeast, thereby enabling the mammalian Plk1 functions to be approximated in the fission yeast. The other selected gene was the N-Myc downstream-regulated gene 2 (ndrg2), which is upregulated during cell differentiation, yet still not well characterized. When the ndrg2 gene was overexpressed in the fission yeast, the cells contained multi-septa. The septa were positioned well, yet their number increased per cell. Therefore, this gene was speculated to block cell division in the last stage of the cell cycle, making the phenotype potentially useful for explaining cell growth and differentiation in mammalian cells. Accordingly, fission yeast is demonstrated to be an appropriate species for the functional study of mammalian genes.

Abstract: The translational enhancement of the 5′-leader sequence of the tobacco mosaic virus (TMV) and tobacco etch viral 5′-leader sequence (TEV) was compared in relation to the expression of the human calcitonin (hCT) gene in transgenic potato plants. The synthetic human calcitonin tetrameric genes were cloned under the control of the 35S Cauliflower mosaic virus (CaMV)promoter linked to the TMV or TEV leader sequences. The resulting constructs were cloned into the binary vector Bin19 and potato minituber discs transformed using an agrobacterium strain. The expression of the hCT gene was studied by mRNA-DNA hybridization, a RT-PCR, ELISA and RIA. The results showed that the TMV omega element increased the yield of the expressed recombinant protein two to three-fold in comparison with the TEV leader sequence.

Abstract: Human skin substitutes are needed for implantation and wound repair based on the new concept of tissue engineering in combination with biomaterials and cell biological technology. However, failure sometimes occurs when the wound healing is delayed in vivo due to acute inflammation resulting from the early degradation of the transplanted biomaterials. Accordingly, the current study modified conventional biomaterials to overcome early degradation and strong inflammation. In a conventional skin substitute, the animal origin collagenous materials have a slight antigenicity as xenogenic materials, however, the modified method was able to obtain a low antigenicity and anti-inflammation effect using atelo-collagen and an amniotic component. The tyrosine content in the developed atelo-collagen, representing the antigenicity, was reduced from 0.590% to 0.046% based on an HPLC analysis. In addition, to reduce the inflammation and foreign material reaction, an amniotic component was applied to the atelo-collagen materials. While, to enhance the wound healing, the modified skin substitute was developed as a composite matrix of an atelo-collagen scaffold with an amniotic membrane component. A quantitative analysis of hEGF in the amniotic membrane was also performed using different processing methods. Finally, a tissueengineered skin substitute was constructed by cultivating skin cells in the collagen scaffold attached to an amniotic membrane.

Abstract: The luminometry assay using the wild-type Vibrio harveyi BB120 was evaluated as a possible detection method for quorum sensing inhibitors. The effects of the concentration of the quorum sensing signal molecule (AHL) as well as the cell density of the reporter strain and the different AHL analogues on luminescence expressed as relative light units (RLU) were examined. Inhibition of V. harveyi luminescence was observed in a dose dependent manner for all five AHL analogues. The RLU values exhibited linearity within the range of 2.9 x 102 ~ 3.2 x 105. Detection up to 102nM was possible for dodecanoyl-homoserine lactone and AHLs with alkyl chain lengths of C-8~C-14 were more active than the shorter chain-lengthed hexanoyl-homoserine lactones. Lipophilicity of the AHL seems to affect the sensitivity of the assay.

Abstract: A series of 5-substituted 2-(p-methoxyphenyl)-1H-benzimidazoles was synthesized and evaluated for cytotoxicity against 4 human cancer cell lines, HCT 15, PC-3, A549, and ACHN. Except for the 5-chloro analogue, most of the 5-substituted compounds showed significant cytotoxicities in these cell lines. However, the structure activity relationship study revealed that neither the electronic nor the lipophilic parameters of the 5-substituents were related to cytotoxicity. Moreover, none of the analogues showed significant NF к-β inhibition activity implying that cytotoxicity was not related to this mechanism. The 5-methyl analogue was the most potent compound in this series with a GI50 of 0.9 µM in the A549 cell line.

Abstract: Autonomic derangement can be assessed by depressed baroreflex sensitivity in a restrictive left ventricular diastolic filling pattern. However, it is unclear how reflex or other compensations affect the inspiratory fall of systolic arterial pressure (IFSAP) during acute airway obstruction. In an in-vivo situation, it is particularly difficult to isolate individual effects of heart rate, vascular tone, pleural pressure variation, and ventricular interdependence. For this reason, a computer model study was undertaken. A previously validated computer model of the cardiopulmonary system was adapted for this study, relating mechanisms such as baroreflex regulation of heart rate to IFSAP. The model provided time-course simulations of hemodynamics by numerically integrating 28 nonlinear, time-varying differential equations. Two models for baroreflex gain were tested, including a simple 1st-order relationship between systolic arterial pressure (SAP) and R-R interval (RRI) and the autoregressive moving average (ARMA) model reported in literature. The ARMA model predicted the next RRI based on SAP from several previous cardiac cycles and previous RRI predictions. Experimental data were obtained retrospectively from 22 patients with chronic airway obstruction before and during breathing through an external resistance. The computer model provided good fits to arterial pressure waveforms: correlation coefficients (r) ranging from 0.71 to 0.96 (mean±SD: 0.87±0.06) with a simple 1st-order SAP-RRI model. The ARMA model did not improve the goodness of fit.

Abstract: Various concentrations of either lidocaine or tetracaine, plus combinations of lidocaine and tetracaine were formulated into liposomes to improve topical anesthesia. The topical anesthetic effects of these liposomal mixtures of local anesthetics (Lipo-MLA) were then compared with those of EMLA (Eutectic Mixture of Local Anesthetics) and single local anesthetic liposomes using a pinprick test on healthy adult volunteers. The Lipo-MLA exhibited significantly improved anesthetic effects compared to the EMLA and single local anesthetic liposomes with a faster onset time of approximately thirty minutes and duration of at least four hours. A dermal toxicity study using rats revealed that Lipo-MLA was safe at greater than 2,000 mg/kg bodyweight.