Construction and Characterization of Cell-Penetrating Peptide-Fused Fibroblast Growth Factor and Vascular Endothelial Growth Factor for an Enhanced Percutaneous Delivery System.

The exogenous administration of growth factors has been examined for treating wounds and such factors serve as cosmetic agents for skin regeneration. However, the topical application of growth factors is hampered by their limited percutaneous absorption. In this study, we genetically modified basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) using a cell-penetrating peptide to facilitate their permeation into skin and to avoid multiple steps in the chemical or physical modification of biomaterials. Low-molecular-weight protamine (LMWP)-fused bFGF and VEGF-A (LMWP-bFGF and LMWP-VEGF-A) were designed by serial polymerase chain reaction (PCR)-mediated addition of the LMWP codons onto the bFGF and VEGF-A genes and then produced in Escherichia coli. The purified LMWP-bFGF and LMWP-VEGF-A represented >90% of the total proteins, as determined by SDS-PAGE and densitometric quantification, and their actual molecular weights, determined by mass spectroscopy, were 19,026 and 15,715 Da, respectively, corresponding to the theoretical values. N-terminal amino acid sequencing analyses confirmed the N-terminal conjugation of LMWP to bFGF and VEGF-A. Immunoblotting analyses using antibodies against bFGF and VEGF-A, together with the proliferative effects of LMWP-bFGF and LMWP- VEGF-A on human keratinocytes and fibroblasts, demonstrated that the original biological activity of each growth factor was not altered by LMWP conjugation. In addition, the LMWP conjugation did not induce further cytotoxic effects on the skin cells, while the cell membrane-penetrating activities of LMWP-bFGF and LMWP-VEGF-A were significantly enhanced compared with the respective unconjugated growth factors. These results suggest that LMWP-bFGF and LMWP-VEGF-A can be used as effective topical therapeutic or cosmetic agents for skin regeneration and anti-aging treatments.

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana. Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana. The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana. The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana-derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli-derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana-derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana-derived recombinant human acidic fibroblast growth factor effectively protects skin cell from UVB, suggesting its potential use as a cosmetic or therapeutic agent against skin photoaging.

Calumenin, a multiple EF-hands Ca2+-binding protein, interacts with ryanodine receptor-1 in rabbit skeletal sarcoplasmic reticulum.

Calumenin is a multiple EF-hand Ca2+-binding protein located in endo/sarcoplasmic reticulum of mammalian tissues. In the present study, we cloned two rabbit calumenin isoforms (rabbit calumenin-1 and -2, GenBank Accession Nos. SY225335 and AY225336, respectively) by RT-PCR. Both isoforms contain a 19 aa N-terminal signal sequence, 6 EF-hand domains, and a C-terminal ER/SR retrieval signal, HDEF. Both calumenin isoforms exist in rabbit cardiac and skeletal muscles, but calumenin-2 is the main isoform in skeletal muscle. Presence of calumenin in rabbit sarcoplasmic reticulum (SR) was identified by Western blot analysis. GST-pull down and co-immunoprecipitation experiments showed that ryanodine receptor 1 (RyR1) interacted with calumenin-2 in millimolar Ca2+ concentration range. Experiments of gradual EF-hand deletions suggest that the second EF-hand domain is essential for calumenin binding to RyR1. Adenovirus-mediated overexpression of calumenin-2 in C2C12 myotubes led to increased caffeine-induced Ca2+ release, but decreased depolarization-induced Ca2+ release. Taken together, we propose that calumenin-2 in the SR lumen can directly regulate the RyR1 activity in Ca2+-dependent manner.

Mesenchymal stem cells feeder layer from human umbilical cord blood for ex vivo expanded growth and proliferation of hematopoietic progenitor cells.

Ex vivo expansion of hematopoietic stem cells was suggested as the best way of overcoming problems caused by limited hematopoietic cell number for cord blood transplantation. In this study, we quantified and characterized an ex vivo expansion capacity of umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) as a cell feeder layer for support of UCB-derived committed hematopoietic progenitor cells (HPCs) in the absence or presence of recombinant cytokines. The UCB-derived MSCs used in the study differentiated into osteoblast, chondrocytes, and adipocytes under proper conditions. Frequencies in colony forming unit-granulocyte, macrophage, colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte, burst forming unit-erythrocyte, and colony forming unit-erythrocyte increased to 3.46-, 9.85-, 3.64-, and 2.03-folds, respectively, only in culture supplemented by UCB-derived MSCs as a cell feeder layer without recombinant cytokines (culture condition C). Identified expansion kinetics in all kinds of committed HPCs showed plateaus at 7 culture days, suggesting some consumable components were required for the expansion. Physiological importance and different roles for different committed HPCs of UCB-derived MSCs as a cell feeder layer were revealed by a distinguished expansion capacity for colony forming unit-megakaryocyte. The preferred maintenance of CD33(-)CD34(+) in culture condition C was also identified. The presence of cobblestone-like areas as hematopoietic microenvironment and various cell feeder layer-originated hematopoietic cytokines including interleukin-1beta and granulocyte, macrophage-colony stimulating factor were suggested as underlying mechanisms for the identified expansion capacity. The present numeric and biological information about intrinsic expansion capacity for UCB-derived committed HPCs will increase further biological and clinical applications of UCB-derived MSCs.

Molecular properties of excitation-contraction coupling proteins in infant and adult human heart tissues.

Excitation-contraction coupling (ECC) proteins in the human heart were characterized using human atrial tissues from different age groups. The samples were classified into one infant group (Group A: 0.2-7 years old) and three adult groups (Group B: 21-30; Group C: 41-49; Group D: 60-66). Whole homogenates (WH) of atrial tissues were assayed for ligand binding, 45Ca2+ uptake and content of ECC proteins by Western blotting. Equilibrium [3H]ryanodine binding to characterize the ryanodine receptor (RyR) of the sarcoplasmic reticulum (SR) showed that the maximal [3H]ryanodine binding (Bmax) to RyR was similar in all the age groups, but the dissociation constant (kd) of ryanodine was higher in the infant group than the adult groups. Oxalate-supported 45Ca2+ uptake into the SR, a function of the SR SERCA2a activity, was lower in the infant group than in the adult groups. Similarly, [3H]PN200-110 binding, an index of dihydropyridine receptor (DHPR) density, was lower in the infant group. Expression of calsequestrin and triadin assessed by Western blotting was similar in the infant and adult groups, but junctin expression was considerably higher in the adult groups. These differences in key ECC proteins could underlie the different Ca2+ handling properties and contractility of infant hearts.

Characterization of isoforms and genomic organization of mouse calumenin.

Calumenin is a multiple EF-hand protein located in endo/sarcoplasmic reticulum of mammalian heart and other tissues [J. Biol. Chem. 272 (1997) 18232; Genomics 49 (1998) 331; Biochim. Biophys. Acta 1386 (1998) 121]. In the present study, a new isoform of mouse calumenin (mouse calumenin 2) was cloned by RT-PCR and genomic DNA PCR. The deduced amino acid sequence of mouse calumenin 2 is 315 aa long with the calculated MW of 37,064 and pI of 4.26. It has 92% aa sequence identity to previously identified mouse calumenin [J. Biol. Chem. 272 (1997) 18232] (mouse calumenin 1). The difference in the aa sequence was restricted to the first two EF-hand regions (residues 74-138). Northern blot analysis shows that mouse calumenin 2 is highly expressed in heart, lung, testis and unpregnant uterus. The expression of mouse calumenin 2 appears to decrease when fetal development is progressed. Genomic DNA PCR, sequencing and data mining of mouse genome database were utilized to examine the exon-intron boundaries of mouse calumenin genes. Both mouse calumenin 1 and 2 genes encompass six exons, and five of them (Exon1, 3, 4, 5 and 6) are identical. However, mouse calumenin 1 contains Exon2-1, whereas mouse calumenin 2 contains a neighboring Exon2-2. The calumenin genes are localized on mouse chromosome 6 having conserved synteny with human chromosome 7q32. For comparison, the genomic organization of human calumenin was also examined using the published human genome database (UCSC Genome Bioinformatics at ). Like mouse calumenin genes, two human calumenin genes also consist of five identical exons (Exon1, 3, 4, 5 and 6) and a different Exon2. The present study suggests that the genomic organization of calumenin genes is well conserved between human and mouse.