To control the sustained release of melatonin and modulate the osteogenic differentiation of mesenchymal stem cells (MSCs), melatonin was firstly loaded onto TiO nanotubes by direct dropping method, and then a multilayered film was coated by a spin-assisted layer-by-layer technique, which was composed of chitosan (Chi) and gelatin (Gel). Successful fabrication was characterized by field emission scanning electron microscopy, atomic force microscope, X-ray photoelectron spectroscopy and contact angle measurement, respectively. The efficient sustained release of melatonin was measured by UV-visible-spectrophotometer. After 2 days of culture, well-spread morphology was observed in MSCs grown on the Chi/Gel multilayer-coated melatonin-loaded TiO nanotube substrates as compared to different groups. After 4, 7, 14 and 21 days of culture, the multilayered-coated melatonin-loaded TiO nanotube substrates increased cell proliferation, increased alkaline phosphatase (ALP) and mineralization, increased expression of mRNA levels for runt-related transcription factor 2 (Runx2), ALP, osteopontin (OPN) and osteocalcin (OC), indicative of osteoblastic differentiation. These results demonstrated that Chi/Gel multilayer-coated melatonin-loaded TiO nanotube substrates promoted cell adhesion, spreading, proliferation and differentiation and could provide an alternative fabrication method for titanium-based implants to enhance the osteointegration between bone tissues and implant surfaces.
Phosphorylated forms of Tetrahymena macronuclear histone H1 were separated from each other and from dephosphorylated H1 by cation-exchange HPLC. A homogeneous fraction of hyperphosphorylated macronuclear H1 was then used to generate novel polyclonal antibodies highly selective for phosphorylated H1 in Tetrahymena and in human cells. These antibodies fail to recognize dephosphorylated forms of H1 in both organisms and are not reactive with most other nuclear or cytoplasmic phosphoproteins including those induced during mitosis. The selectivity of these antibodies for phosphorylated forms of H1 in Tetrahymena and in HeLa argues strongly that these antibodies recognize highly conserved phosphorylated epitopes found in most H1s and from this standpoint Tetrahymena H1 is not atypical. Using these antibodies in indirect immunofluorescence analyses, we find that a significant fraction of interphase mammalian cells display a strikingly punctate pattern of nuclear fluorescence. As cells enter S-phase, nuclear staining becomes more diffuse, increases significantly, and continues to increase as cells enter mitosis. As cells exit from mitosis, staining with the anti-phosphorylated H1 antibodies is rapidly lost presumably owing to the dephosphorylation of H1. These immunofluorescent data document precisely the cell cycle changes in the level of H1 phosphorylation determined by earlier biochemical studies and suggest that these antibodies represent a powerful new tool to probe the function(s) of H1 phosphorylation in a wide variety of eukaryotic systems.
Standard environmental features of the neonatal intensive care unit (NICU) may be stressful and not optimal for the maturation of very low birth weight premature infants (VLBWPIs). The present study investigated whether structured no-touch periods and reducing periods of light and sound stimulation may influence the developmental indices of VLBWPIs. Between June 2012 and June 2013, 60 consecutive VLBWPIs were equally apportioned to either an experimental or control group. The groups were statistically comparable with regard to sex ratio, gestational age and birth weight. Each group received routine nursing care, but infants in the experimental group were additionally cared for in a separate room with 3 h of rest every 8 h, and reduced light and sound from staff and instruments. At 7 and 14 days following birth, plasma insulin-like growth factor 1 (IGF-1) levels of the experimental group were significantly higher than that of the control group. Furthermore, at day 7 and 14, the body weight and crown-to-heel lengths were significantly increased in the experimental group compared with the control group. In summary, during the first 2 weeks following birth, the reduction of touch, sound and light stimulation in the NICU were associated with higher plasma IGF-1 levels and physical growth of VLBWPIs. These results may have implications for the better management of VLBWPIs in the NICU.
To evaluate the therapeutic potential of insulin-like growth factor-I (IGF-I) as an anabolic agent during aging, we determined its effects on IGF binding proteins (BPs) in male rats of 2, 8, 16, and 24 months of age. In control animals, a striking increase (143%) in the predominant 39-45 kDa serum IGFBP (BP-3), with little change in serum IGF-I, accompanied the marked deceleration of growth which occurred between 2 and 8 months; the levels of IGF-I and its BPs declined by 15% and 34%, respectively, later in life. Infusion of IGF-I (1.2 mg/kg/day) for 2 weeks produced progressively larger increases in circulating IGF-I with age, from 24% to 95% between 2 and 24 months, consistent with an age-related decrease in exogenous IGF-I clearance. We attributed these results to the large increase in IGFBPs that occurred with maturation, as well as an induction of IGFBP-3 (34-68%) and a larger increase in the 30-34 kDa IGFBP (BP-2; 136-235%) following IGF-I treatment in the older (16-24 months) animals. Anabolic actions of IGF-I, which were seen only in the older rats, included modes increases in weight velocity (5.2 +/- 1.2 g/week), serum phosphorous (20%), and alkaline phosphatase (26%) compared to age-matched controls. In conclusion, differential changes in the relative levels of the different IGFBPs with IGF-I treatment in older animals appeared to profoundly influence both the half-life and tissue accessibility of exogenous IGF-I, thus modulating the potential benefits of IGF-I as an anabolic agent during aging.
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