A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.     

In vitro differentiation of human mesenchymal stem cells to epithelial lineage

Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.     

Phosphorylation driven motions in the COOH-terminal Src kinase,CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS)

Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate function in the COOH-terminal Src kinase. We have employed enhanced methods of hydrogen-deuterium exchange-mass spectrometry (DXMS) to probe conformational changes on CSK in the absence and presence of nucleotides and thereby provide a structural framework for understanding phosphorylation-driven conformational changes. High quality peptic fragments covering approximately 63% of the entire CSK polypeptide were isolated using DXMS. Time-dependent deuterium incorporation into these probes was monitored to identify short peptide segments that exchange differentially with solvent. Regions expected to lie in loops exchange rapidly, whereas other regions expected to lie in stable secondary structure exchange slowly with solvent implying that CSK adopts a modular structure. The ATP analog, AMPPNP, protects probes in the active site and distal regions in the large and small lobes of the kinase domain, the SH2 domain, and the linker connecting the SH2 and kinase domains. The product ADP protects similar regions of the protein but the extent of protection varies markedly in several crucial areas. These areas correspond to the activation loop and helix G in the kinase domain and several inter-domain regions. These results imply that delivery of the gamma phosphate group of ATP induces unique local and long-range conformational changes in CSK that may influence regulatory motions in the catalytic pathway.     

A new strategy for the preparation of supramolecular neutral hydrogels

This paper demonstrates that miscible blends from water-insoluble polymers, such as poly(2,4,4-trimethylhexamethylene terephthalamide) (1), methylamine imidized poly(methyl methacrylate) (2), and aromatic poly(ether sulfone) (3) and water-soluble polymers, such as poly(2-ethyl-2-oxazoline) (4) and poly(N-vinyl pyrrolidone) (5), respectively, represent a new class of supramolecular hydrogels. When the degree of polymerization (DP) of the water-soluble polymer is larger than that of water-insoluble polymer, the resulting hydrogels adsorb extremely high amounts of water (i.e., 229 wt % in the case of the hydrogel 1/4) and remain mechanically tough. The high water uptake capability of these blends is explained by a supramolecular network structure generated by H-bonding and/or other noncovalent interactions between the water-insoluble hydrophobic polymer and water-soluble hydrophilic segments as reversible cross-linking points interconnected by hydrophilic water soluble segments. The glass transition temperatures of these hydrogels are tailored via the ratio between the weight percent of the two polymers and by the glass transition temperature of the parent polymers. These supramolecular hydrogels can be processed from melt or solution and maintain excellent mechanical properties both in dry and in the water swollen state. This class of hydrogels is of interest for areas such as membranes, contact lenses, tissue engineering, and other biomedical applications.     

Expression of somatostatin,cortistatin, and somatostatin receptors in human monocytes,macrophages, and dendritic cells

Increasing evidence suggests that neuropeptides play a role in the regulatory mechanisms between the neuroendocrine and immune systems. A differential expression of the five known somatostatin (SS) receptors (sst1-5) has been demonstrated in human immune cells and tissues. However, little is known concerning regulation and expression of sst1-5 and the peptide SS. Therefore, we investigated the expression and the time-dependent regulation of sst1-5, SS, and cortistatin (CST), a novel SS-like peptide, in human monocytes (MO), monocyte-derived macrophages (MP), and dendritic cells (DC) in the basal and lipopolysaccharide (LPS)-activated state. MO, MP, and DC selectively expressed sst2 mRNA. SS mRNA was not detectable, whereas all samples expressed CST mRNA. Expression levels of sst2 and CST mRNA showed marked differences and were in the rank order of MP>DC>MO. LPS stimulation did not induce expression of SS or sst1,3,4,5. However, sst2 mRNA expression was upregulated significantly by stimulation with LPS. CST mRNA was upregulated as well. During differentiation of MO in MP or DC, time-dependent, significantly increasing sst2 and CST mRNA levels were found. By confocal microscopy, the presence of sst2 receptors was demonstrated on MP, but not on DC. This study demonstrates for the first time a selective and inducible expression of the recently discovered CST, as well as sst2, in human monocyte-derived cells, suggesting a role for a CST-sst2 system rather than a SS-sst2 system in these immune cell types.