When polypeptide chains fold into a protein, hydrophobic groups are compacted in the center with exclusion of water. We report the crystal structure of an alanine-rich antifreeze protein that retains ~400 waters in its core. The putative ice-binding residues of this dimeric, four-helix bundle protein point inwards and coordinate the interior waters into two intersecting polypentagonal networks. The bundle makes minimal protein contacts between helices, but is stabilized by anchoring to the semi-clathrate water monolayers through backbone carbonyl groups in the protein interior. The ordered waters extend outwards to the protein surface and likely are involved in ice binding. This protein fold supports both the anchored-clathrate water mechanism of antifreeze protein adsorption to ice and the water-expulsion mechanism of protein folding.

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ATP-citrate lyase (ACLY, EC 2.3.3.8) 3 catalyzes the reaction, citrate ϩ CoA ϩ ATP 3 acetyl-CoA ϩ oxaloacetate ϩ ADP ϩ P i , in the presence of magnesium ions (1). ACLY is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids. Acetyl-CoA produced in the mitochondria cannot be exported to the cytoplasm. Instead, acetylCoA in mitochondria is transformed to citrate by citrate synthase, and citrate is exported to the cytoplasm where ACLY regenerates acetyl-CoA. This acetyl-CoA is an important precursor for fatty acid synthesis. In addition, ACLY has been shown to signal the metabolic state of cells, likely by providing acetyl-CoA for histone acetyltransferases in the cells' nuclei (2). The physiological importance of the enzyme is supported by knock-out experiments in which mice embryos lacking ATPcitrate lyase could not be obtained (3).Our understanding of the reaction mechanism of ACLY has been based on studies of this enzyme and of two enzymes with sequence similarity to ACLY. The first is succinyl-CoA synthetase (SCS), which catalyzes the formation of succinyl-CoA from succinate and CoA using ATP (4). The second is citrate synthase, the enzyme that generates citrate from acetyl-CoA and oxaloacetate in mitochondria. The reaction catalyzed by ACLY is thought to occur in four steps (Reactions 1-4):where E represents ACLY. Like SCS, ACLY is phosphorylated by ATP on an active site histidine residue to give E-P in step 1 (Reaction 1) (5, 6). The phosphoryl group is transferred to citrate in step 2 (Reaction 2). Citryl-phosphate is thought to remain bound to the enzyme, symbolized by E⅐citryl-P. Phosphate is released in the attack by CoA to form the citryl-CoA thioester bond in step 3 (Reaction 3) (7). In the last step (Reaction 4), citryl-CoA is cleaved to give acetyl-CoA and oxaloacetate, the reverse reaction to that catalyzed by citrate synthase.

Human umbilical cord mesenchymal stem cells (hUCMSCs) are considered to be an ideal replacement for bone marrow MSCs. However, up to date, there is no convenient and efficient method for hUCMSC isolation and culture. The present study was carried out to explore the modified enzyme digestion for hUCMSC in vitro. Conventional enzyme digestion, modified enzyme digestion, and tissue explant were used on hUCMSCs to compare their efficiencies of isolation and culture, to observe primary cell growth and cell subculture. The results show that the cells cultured using the tissue explant method had a longer culture cycle (P \ 0.01) and lower yield of primary cells per centimetre of umbilical cord (P \ 0.01) compared with the two enzyme digestion methods. Subculture adherence and cell doubling took significantly less time with the tissue explant method (P \ 0.05) than with the conventional enzyme digestion method; however, there was no significant difference between the tissue explant method and the modified enzyme digestion method (P [ 0.05). Comparing two enzyme digestion methods, the modified method yielded more cells than did the conventional method (P \ 0.01), and primary cell adherence took significantly less time with the modified method than with the conventional method (P \ 0.05). Cell cycle analysis of the third-generation hUCMSCs cultured by modified enzyme digestion method indicated that most cells were quiescent. Immunofluorescence staining showed that these cells expressed MSC markers CD44 and CD90. And Von Kossa and oil red O staining detection showed that they could be differentiated into 123 Cytotechnology (2013) 65:819-827 DOI 10.1007 osteoblasts and adipocytes with induction medium in vitro. This study suggests that hUCMSC isolation and culture using 0.2 % collagenase II at 37°C for digestion of 16-20 h is an effective and simple modified enzyme digestion method.

ObjectiveDifficulty in wound healing is one common complication of diabetes mellitus. The study explored whether the therapeutic effect of human umbilical cord mesenchymal stem cells (hUCMSCs) on diabetic ulcer wound was enhanced by the activation of the Wnt signaling pathway.MethodsRat diabetic model was established by intraperitoneal injection of Streptozotocin (STZ). hUCMSCs were purified and seeded on the collagen–chitosan laser drilling acellular dermal matrix (CCLDADM) scaffold, which was subsequently implanted into the cutaneous wound of normal and diabetic rats, followed by daily injection of Wnt signaling pathway agonist (Wnt3a) or antagonist (sFRP3) at the edge of the scaffold. Wound healing was checked on days 7, 14, and 21, and the fibrous tissue deposition, capillaries, and epidermal regeneration at the wound were examined by hematoxylin–eosin staining. The hUCMSCs-CCLDADM scaffold was cultured in vitro and treated with Wnt3a or sFRP3, followed by evaluation of cell proliferation, cell proliferation rate, survival status, and altered protein levels in the Wnt signaling pathway using BrdU staining, CCK-8 assay, live/dead staining, and Western blotting, respectively.ResultsOn days 7 and 14 postoperatively, the speed of wound healing was significantly lower in diabetic rats than that in normal control rats. This phenomenon was significantly improved by the activation of the Wnt signaling pathway that also elevated the fibrous protein deposition and the abundance of capillary in the granulation tissue. Conversely, blockade of Wnt signaling slowed the healing of skin wound in diabetic rats. The activation of Wnt signaling pathway promoted the proliferation and differentiation and decreased the apoptosis of hUCMSCs, thereby elevating the number of living hUCMSCs on the CCLDADM scaffold, while the suppression exerted a contrary effect.ConclusionThe activation of the Wnt signaling pathway promotes the healing of diabetic skin wound by the regulation of proliferation and differentiation of hUCMSCs on the CCLDADM scaffold.