A novel expression system for recombinant marine mussel adhesive protein Mefp1 using a truncated OmpA signal peptide

To express an increased level of recombinant Mefp1 (marine mussel adhesive protein) in soluble form, we constructed expression vectors encoding truncated OmpA signal peptide-Mefp1 fusion proteins. OmpA signal peptide (OmpASP) is the 21 residue peptide fragment of the 23 residue OmpA signal sequence cleavable by signal peptidase I. We successfully produced increased levels of soluble recombinant Mefp1 (rMefp1) with various deletions of OmpASP, and found that the increased expression was caused by the increased pI of the N-terminus of the fusion proteins (> or = 10.55). All the OmpA signal peptide segments of 3-21 amino acids in length had the same pI value (10.55). Our results suggest that the pI value of the truncated OmpASP (OmpASP(tr)) play an important role in directional signaling for the fusion protein, but we found no evidence for the presence of a secretion enhancer in OmpASP. For practical applications, we increased the expression of soluble rMefp1 with OmpASP(tr) peptides as directional signals, and obtained rMefp1 with the native amino terminus (nN-rMefp1) using an OmpASP(tr)| Xa leader sequence that contains the recognition site for Xa protease.     

Functional equivalence of translation factor eIF5B from Candida albicans and Saccharomyces cerevisiae

Eukaryotic translation initiation factor 5B (eIF5B) plays a role in recognition of the AUG codon in conjunction with translation factor eIF2, and promotes joining of the 60S ribosomal subunit. To see whether the eIF5B proteins of other organisms function in Saccharomyces cerevisiae, we cloned the corresponding genes from Oryza sativa, Arabidopsis thaliana, Aspergillus nidulans and Candida albican and expressed them under the control of the galactose-inducible GAL promoter in the fun12Delta strain of Saccharomyces cerevisiae. Expression of Candida albicans eIF5B complemented the slow-growth phenotype of the fun12Delta strain, but that of Aspergillus nidulance did not, despite the fact that its protein was expressed better than that of Candida albicans. The Arabidopsis thaliana protein was also not functional in Saccharomyces. These results reveal that the eIF5B in Candida albicans has a close functional relationship with that of Sacharomyces cerevisiae, as also shown by a phylogenetic analysis based on the amino acid sequences of the eIF5Bs.     

A MAPK pathway is involved in the control of cortical granule reaction and mitosis during bovine fertilization

In order to understand the mechanism by which mitogen-activated protein kinase (MAPK) regulates fertilization, we examined the effect of the MAPK pathway inhibitor U0126 on polyspermy, cortical granule reaction and mitosis in bovine oocytes during and after fertilization. Oocytes were treated with 30 microM U0126 for 30 min prior to insemination, or from 15 to 27 hr following insemination. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK activity was decreased in U0126 treated oocytes. Oocytes that were treated with U0126 before insemination displayed a significantly higher incidence of polyspermic penetration and incomplete cortical granule reaction than that observed in untreated oocytes (P < 0.05). Exposure of oocytes to 30microM U0126 15-27 hr after insemination induced aberrant microtubule assembly and cell division, often resulting in the formation of two or three daughter cells with altered shapes and sizes. These results suggest that an ERK-like cascade is part of a mechanism that controls cortical granule reaction and the formation of the mitotic spindle following sperm penetration in the bovine.     

Cytoprotective activity of annphenone against oxidative stress-induced apoptosis in V79-4 lung fibroblast cells

We have elucidated the cytoprotective effect of annphenone (2,4-dihyroxy-6-methoxy-acetophenone 4-O-beta-d-glucopyranoside) against oxidative stress-induced apoptosis. Annphenone scavenged intracellular reactive oxygen species (ROS) and increased antioxidant enzyme activities. It thereby prevented lipid peroxidation and DNA damage, which was demonstrated by the inhibition of the formation of thiobarbituric acid reactive substance (TBARS), inhibition of the comet tail and decreased phospho-H2A.X expression. Annphenone protected Chinese hamster lung fibroblast (V79-4) cells from cell death via the inhibition of apoptosis induced by hydrogen peroxide (H(2)O(2)), as shown by decreased apoptotic nuclear fragmentation, decreased sub-G(1) cell population and inhibited mitochondrial membrane potential (Deltapsi) loss. Taken together, these findings suggest that annphenone exhibits antioxidant properties by inhibiting ROS generation and thus protecting cells from H(2)O(2)-induced cell damage.     

Epinephrine increases DNA synthesis via ERK1/2s through cAMP, Ca(2+)/PKC, and PI3K/Akt signaling pathways in mouse embryonic stem cells

Epinephrine is a catecholamine that plays important roles in regulating a wide variety of physiological systems by acting through the adrenergic receptors (ARs). The cellular responses to AR stimulation are mediated through various signaling pathways. Therefore, this study examined the effects of epinephrine on DNA synthesis and related signaling molecules in mouse embryonic stem cells (ESCs). Epinephrine increased DNA synthesis in a dose- and time-dependent manner, as determined by the level of [(3)H]-thymidine incorporation. AR subtypes (alpha1(A), alpha2(A), beta1, beta2, and beta3) were expressed in mouse ESCs and their expression levels were increased by epinephrine. In this experiment, epinephrine increased cAMP levels, intracellular Ca(2+) concentration ([Ca(2+)](i)), and translocation of protein kinase C (PKC) from the cytosol to the membrane compartment. In addition, we observed Akt phosphorylation in response to epinephrine; this was stimulated by phosphorylation of the epidermal growth factor receptor (EGFR). Epinephrine also induced phosphorylation of ERK1/2 (p44/42 MAPKs), while inhibition of PKC or Akt blocked this phosphorylation. Epinephrine increased the mRNA levels of proto-oncogenes (c-fos, c-jun, c-myc), while inhibition of ERK1/2 decreased these mRNA levels. In experiments aimed at examining the involvement of cell cycle regulatory proteins, epinephrine increased the levels of cyclin E/cyclin-dependent kinase 2 (CDK2) and cyclin D1/cyclin-dependent kinase 4 (CDK4). In conclusion, epinephrine stimulates DNA synthesis via ERK1/2 through cAMP, Ca(2+)/PKC, and PI3K/Akt signaling pathways in mouse ESCs.     

Mesenchymal stem cells for enhancing biological healing after meniscal injuries

The meniscus is a semilunar fibrocartilage structure that plays important roles in maintaining normal knee biomechanics and function. The roles of the meniscus, including load distribution, force transmission, shock absorption, joint stability, lubrication, and proprioception, have been well established. Injury to the meniscus can disrupt overall joint stability and cause various symptoms including pain, swelling, giving-way, and locking. Unless treated properly, it can lead to early degeneration of the knee joint. Because meniscal injuries remain a significant challenge due to its low intrinsic healing potential, most notably in avascular and aneural inner two-thirds of the area, more efficient repair methods are needed. Mesenchymal stem cells (MSCs) have been investigated for their therapeutic potential in vitro and in vivo. Thus far, the application of MSCs, including bone marrow-derived, synovium-derived, and adipose-derived MSCs, has shown promising results in preclinical studies in different animal models. These preclinical studies could be categorized into intra-articular injection and tissue-engineered construct application according to delivery method. Despite promising results in preclinical studies, there is still a lack of clinical evidence. This review describes the basic knowledge, current treatment, and recent studies regarding the application of MSCs in treating meniscal injuries. Future directions for MSC-based approaches to enhance meniscal healing are suggested.     

Water and nonelectrolyte permeability of plant cell membranes after short term application of amino acids and phosphorylated amino acids

The effects of amino acids (aa) and N-(diisopropyloxyphosphoryl)-amino acids (DIPP-aa) on cell membranes were investigated by evaluating water and methyl urea permeability. Permeability coefficients P_f and P_s were determined by standard osmotic methods for cells ofPisum sativum stem base epidermis after 20 min exposure to a 5 mM solution of each aa and DIPP-aa. The P_f value ofP. sativum epidermal cells (untreated controls) was 1.3 ± 0.4 × 10^-3 μm s^-1. Treat ments with the diisopropyl-oxyphosphoryl derivatives of three one charged and three polar amino acids (serine, threonine, asparagine, and aspartic acid) and unsubstituted (free) serine and threonine increased water permeability up to about two fold of the control value. Serine and threonine and their DIPP-derivatives increased methyl urea permeability (controls 1.03 ± 0.09 × 10^-3 μm s^-1) 30 to 80 percent Other amino acids and their DIPP-derivatives caused small or insignificant changes of water permeability. Only certain polar amino acids and their DIPP-derivatives increased the osmotic water and methyl urea permeation through the plasma membrane. The specificity of these molecules on plasma membranes suggests that the active amino acids (serine and threonine) and their DIPP-derivatives interact with charged membrane molecules. The relatively small changes in water and methyl urea permeability may indicate that the effective aa’s and their DIPP-derivatives interact with phospholipids rather than aquaporin. A concurring alteration of water channel proteins, however, cannot excluded.     

A novel composite scaffold for cardiac tissue engineering

Summary One approach to the engineering of functional cardiac tissue for basic studies and potential clinical use involves bioreactor cultivation of dissociated cells on a biomaterial scaffold. Our objective was to develop a scaffold that is (1) highly porous with large intereconnected pores (to facilitate mass transport), (2) hydrophilic (to enhance cell attachment), (3) structurally stable (to withstand the shearing forces during bioreactor cultivation), (4) degradable (to provide ultimate biocompatibility of the tissue graft), and (5) elastic (to enable transmission of contractile forces). The scaffold of choice was made as a composite of poly(Dl-lactide-co-caprolactone), poly(Dl-lactide-co-glycolide) (PLGA), and type I collagen, with open interconnected pores and the average void volume of 80±5%. Neonatal rat heart cells suspended in Matrigel were seeded into the scaffold at a physiologically high density (1.35×10⁸ cells/cm³) and cultivated for 8 d in cartridges perfused with culture medium or in orbitally mixed dishes (25 rpm); collagen sponge (Ultrafoam^⋆m) and PLGA sponge served as controls. Construct cellularity, presence of cardiac markers, and contractile properties were markedly improved in composite scaffolds as compared with both controls.     

Enhanced yield and homogeneity of buffalo growth hormone by an improved chromatographic protocol

Loss of buffalo Growth Hormone (buGH) in the various side fractions of standard buGH purification protocol has been determined quantitatively by direct binding ELISA and qualitatively by SDS-PAGE and Western blot analysis. Accounting result indicated that there was a considerable loss of buGH in the side fractions. An alternative protocol to prevent loss and to obtain a high yield of buGH has been developed by introducing anion exchange chromatography, QAE-Sephadex. This has resulted in a simple, reproducible three-step protocol. In this protocol, an extract obtained at 250 mM (NH4)2 SO4, pH 5.5, was loaded onto the QAE-Sephadex column in 0.1 M NH4 HCO3. At this salt concentration, the bulk of the buGH came as QAE unbound fraction. Some amount of buGH, together with contaminating proteins, was bound to QAE-Sephadex and these could be eluted with 1 M KCl. The immunopotency of the enriched buGH preparation "QUB" (QAE unbound fraction) in a direct binding ELISA was similar to that of the semi-pure buGH (ECS/APECS) preparation obtained using the standard protocol, but the yield was 4 times higher. The SDS-PAGE data showed that the banding pattern of standard semi-pure buGH and QUB were quite similar and QUB can be loaded onto the Sephacryl S-200 gel filtration chromatography to yield a highly purified buGH. SDS-PAGE and Western blot analyses showed the major band of buGH in QUB at the same position as in the case of standard buGH. It has also been demonstrated here that it is possible to separate buffalo prolactin (buPRL) and buGH on QAE-Sephadex.     

Oxoiron(IV) porphyrin π-cation radical complexes with a chameleon behavior in cytochrome P450 model reactions

There is an intriguing, current controversy on the involvement of multiple oxidizing species in oxygen transfer reactions by cytochromes P450 and iron porphyrin complexes. The primary evidence for the multiple oxidants theory was that products and/or product distributions obtained in the catalytic oxygenations were different depending on reaction conditions such as catalysts, oxidants, and solvents. In the present work, we carried out detailed mechanistic studies on competitive olefin epoxidation, alkane hydroxylation, and C=C epoxidation versus allylic C–H hydroxylation in olefin oxygenation with in situ generated oxoiron(IV) porphyrin -cation radicals (1) under various reaction conditions. We found that the products and product distributions were markedly different depending on the reaction conditions. For example, 1 bearing different axial ligands showed different product selectivities in competitive epoxidations of cis-olefins and trans-olefins and of styrene and para-substituted styrenes. The hydroxylation of ethylbenzene by 1 afforded different products, such as 1-phenylethanol and ethylbenzoquinone, depending on the axial ligands of 1 and substrates. Moreover, the regioselectivity of C=C epoxidation versus C–H hydroxylation in the oxygenation of cyclohexene by 1 changed dramatically depending on the reaction temperatures, the electronic nature of the iron porphyrins, and substrates. These results demonstrate that 1 can exhibit diverse reactivity patterns under different reaction conditions, leading us to propose that the different products and/or product distributions observed in the catalytic oxygenation reactions by iron porphyrin models might not arise from the involvement of multiple oxidizing species but from 1 under different circumstances. This study provides strong evidence that 1 can behave like a chameleon oxidant that changes its reactivity and selectivity under the influence of environmental changes.Electronic Supplementary Material Supplementary material is available for this article at .