The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells

HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a G(i/o) protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.     

Synthesis of hexose-related imidazolidinones: Novel glycation products in the Maillard reaction

Carbohydrate-peptide esters which mimic the reactivity of sugar 6-phosphates in nonenzymatic glycations were used as model compounds for the study of the Maillard reaction in vitro. We found that intramolecular cyclization of the monosaccharide ester in which the sugar moiety (D-glucose or D-galactose) is linked, through the C-6 hydroxy group, to the C-terminal carboxy group of the endogenous opioid pentapeptide leucine-enkephalin, in methanol as the solvent, resulted in the formation of imidazolidinone diastereoisomers having cis or trans relative geometry of the substituents at the imidazolidinone ring moiety. The diastereoisomeric imidazolidinones were separated and each transformed by hydrolysis into the corresponding D-gluco- and D-galacto-related imidazolidinone products of leucine-enkephalin. Along with the previous evidence that, from the same sugar-peptide esters by changing the reaction conditions Amadori rearrangement products could be obtained [Horvat et al. (1998) J Chem Soc Perkin Trans 1:99–13], the presented results point to the possibility that similar carbohydrate-related imidazolidinones may also be generated in the early stage of the Maillard reaction in vivo.     

In vitro nonenzymatic glycation enhances the role of myoglobin as a source of oxidative stress

Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO₂ and GMbO₂, respectively. H₂O₂-induced iron release was significantly higher from GMbO₂ than that from MbO₂. This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H₂O₂ was studied. GMbO₂ degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO₂. Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H₂O₂ with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO₂. Compared to MbO₂, GMbO₂ was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO₂. GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H₂O₂. These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.     

AGE-breakers cleave model compounds,but do not break Maillard crosslinks in skin and tail collagen from diabetic rats

Advanced glycation end products (AGE), formed by nonenzymatic Maillard reactions between carbohydrate and protein, contribute to the increase in chemical modification and crosslinking of tissue proteins with age. Acceleration of AGE formation in collagen during hyperglycemia, with resultant effects on vascular elasticity and basement membrane permeability, is implicated in the pathogenesis of diabetic complications. AGE-breakers, such as N-phenacylthiazolium (PTB) and N-phenacyl-4,5-dimethylthiazolium (PMT) halides, have been proposed as therapeutic agents for reversing the increase in protein crosslinking in aging and diabetes. We have confirmed that these compounds, as well as the AGE-inhibitor pyridoxamine (PM), cleave the model AGE crosslink, phenylpropanedione, and have studied the effects of these compounds in reversing the increased crosslinking of skin and tail collagen isolated from diabetic rats. Crosslinking of skin collagen, measured as the half-time for solubilization of collagen by pepsin in 0.5M acetic acid, was increased approximately 5-fold in diabetic, compared to nondiabetic rats. Crosslinking of tail tendon collagen, measured as insolubility in 0.05 N acetic acid, was increased approximately 10-fold. Collagen preparations were incubated in the presence or absence of AGE-breakers or PM in phosphate buffer, pH 7.4, for 24h at 37 degrees C. These treatments did not decrease the half-time for solubilization of diabetic skin collagen by pepsin or increase the acid solubility of diabetic tail tendon collagen. We conclude that, although AGE-breakers and PM cleave model crosslinks, they do not significantly cleave AGE crosslinks formed in vivo in skin collagen of diabetic rats.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

Regional variations in intestinal brush border membrane fluidity and function during diabetes and the role of oxidative stress and non-enzymatic glycation

The physical state (fluidity) of lipids modulates the activities of several membrane bound enzymes and transport proteins. Alteration of brush border membrane (BBM) fluidity is one of the several changes exhibited by the small intestine during diabetes. In the present study, an investigation of the diabetes induced regional changes in fluidity, oxidative damage, non-enzymatic glycation as well as the activities and the kinetic parameters of the enzymes alkaline phosphatase and -glutamyl transpeptidase was carried out on the intestinal BBM. At the end of 6 weeks of diabetes, significant increases in the extent of both oxidative damage and non-enzymatic glycation were observed along the length of the intestine along with a simultaneous decrease in membrane fluidity. A significant correlation between the decrease in BBM fluidity and increase in non-enzymatic glycation was observed in the duodenum and jejunum. Additionally regional variations in the activities and kinetic parameters of both the enzymes were observed.     

Advanced glycation end-products (AGEs) induce concerted changes in the osteoblastic expression of their receptor RAGE and in the activation of extracellular signal-regulated kinases (ERK)

An increase in the interaction between advanced glycation end-products (AGEs) and their receptor RAGE is believed to contribute to the pathogenesis of chronic complications of Diabetes mellitus, which can include bone alterations such as osteopenia. We have recently found that extracellular AGEs can directly regulate the growth and development of rat osteosarcoma UMR106 cells, and of mouse calvaria-derived MC3T3E1 osteoblasts throughout their successive developmental stages (proliferation, differentiation and mineralisation), possibly by the recognition of AGEs moieties by specific osteoblastic receptors which are present in both cell lines. In the present study we examined the possible expression of RAGE by UMR106 and MC3T3E1 osteoblastic cells, by immunoblot analysis. We also investigated whether short-, medium- or long-term exposure of osteoblasts to extracellular AGEs, could modify their affinity constant and maximal binding for AGEs (by 125I-AGE-BSA binding experiments), their expression of RAGE (by immunoblot analysis) and the activation status of the osteoblastic ERK 1/2 signal transduction mechanism (by immunoblot analysis for ERK and P-ERK). Our results show that both osteoblastic cell lines express readily detectable levels of RAGE. Short-term exposure of phenotypically mature osteoblastic UMR106 cells to AGEs decrease the cellular density of AGE-binding sites while increasing the affinity of these sites for AGEs. This culture condition also dose-dependently increased the expression of RAGE and the activation of ERK. In proliferating MC3T3E1 pre-osteoblasts, 24–72 h exposure to AGEs did not modify expression of RAGE, ERK activation or the cellular density of AGE-binding sites. However, it did change the affinity of these binding sites for AGEs, with both higher- and lower-affinity sites now being apparent. Medium-term (1 week) incubation of differentiated MC3T3E1 osteoblasts with AGEs, induced a simultaneous increase in RAGE expression and in the relative amount of P-ERK. Mineralising MC3T3E1 cultures grown for 3 weeks in the presence of extracellular AGEs showed a decrease both in RAGE and P-ERK expression. These results indicate that, in phenotypically mature osteoblastic cells, changes in ERK activation closely follow the AGEs-induced regulation of RAGE expression. Thus, the AGEs-induced biological effects that we have observed previously in osteoblasts, could be mediated by RAGE in the later stages of development, and mediated by other AGE receptors in the earlier pre-osteoblastic stage.     

Does enhanced expression of the Na+-CA2+ exchanger increase myocardial vulnerability to ischemia/reperfusion injury in rabbit hearts?

The present study focused on examining the efficacy of feeding a rutin-glucose derivative (G-rutin) to inhibit glycation reactions that can occur in muscle, kidney and plasma proteins of diabetic rats. Both thiobarbituric acid-reactive substance levels and protein carbonyl contents in muscle and kidney were significantly (p < 0.05) reduced in streptozotocin-induced diabetic rats fed G-rutin supplemented diet, compared to diabetic rats fed control diet. The N -fructoselysine content in muscle and kidney, a biomarker of early glycation reaction, was markedly (p < 0.05) increased by diabetes, but significantly (p < 0.05) reduced in diabetic rats fed G-rutin. Advanced glycation end-products (AGEs) in serum and kidney protein were measured by immunoblot using anti-AGE antibody, and were also reduced in diabetic rats fed dietary G-rutin. Feeding G-rutin also slightly inhibited aldose reductase activity in these animals. These results demonstrate for the first time that dietary G-rutin consumption can provide potential health benefits that are related to the inhibition of tissue glycation reactions common to diabetes.     

CRE-decoy oligonucleotide-inhibition of gene expression and tumor growth

The authors prepared water-soluble (WSF), urea-soluble (USF), alkali-soluble (ASF), sonicated (SF), sonicated insoluble (SIF) and membrane (MF) fractions of lens proteins from human senile and diabetic cataractous lenses and age-matched clear lenses. Levels of advanced glycation end products (AGEs) including carboxymethyl lysine (CML), a glycoxidation product, were determined by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA). Distribution of AGEs in the various protein fractions was ascertained by SDS-PAGE and Western blotting. An overall increase in the levels of AGEs in diabetic cataractous lenses as compared to senile cataractous lenses and clear lenses has been observed. ASF and SF , both of which originated from the urea-insoluble fraction, showed the highest levels of AGEs. However, no clear-cut differences in CML levels were seen among clear lenses and senile and diabetic cataractous lenses. AGEs were found to be distributed mostly in the high molecular aggregates in all the fractions. These data suggest that AGEs contribute to protein aggregation and subsequent insolubilization.     

Molecular markers for resistance to Heterodera glycines in advanced soybean germplasm

Germplasm line J87-233 is resistant to soybean cyst nematode (SCN) races 1, 2, 3, 5 and moderately resistant to race 14 with resistance derived from 3 primitive sources, Peking, PI 88788 and PI 90763. F_2:3 progeny of J87-233 and SCN-susceptible Hutcheson cross were evaluated for response to SCN races 1, 2, 3, 5 and 14. Linkage groups (LG) A, B, F, G, J, M, N, S were tested with 215 genomic clones and 45 decamers for parental genotypes. QTL for race 1 and QTL for race 3 were detected on LG A2, the region of BLT65V and SCAR 548/563_{1100/1025,975.} The cluster analysis of 12 soybean cultivars and 38 plant introductions confirmed association of SCAR_{1100/1025,975} with resistance to races 1 and 3, and suggested possible DNA rearrangements that might give rise to new resistance specificities in the region. The highly significant association of K69T marker with SCN race 1 resistance in conjunction with its location, 18.5 cM from the reported QTL, exemplifies the importance of the QTL locus on LG G and suggests expansion of the linkage map in the LG G-terminal region. Detected interaction between loci on LG A2 and LG G, and also with loci on LG F and LG M, may play a significant role in the genotype-specific response to SCN. Identification of two major regions on LG A2 and LG G for SCN resistance shows their applicability to advanced germplasm, however, transmission of molecular marker alleles indicates that applied markers are not yet reliable in revealing all possible recombination events in breeding for SCN resistance.