Glutamine decreases lipopolysaccharide-induced intestinal inflammation in infant rats

Using a gastrostomy-fed (GF) rat infant "pup-in-a-cup" model, the effects of protein deprivation and supplemental glutamine (Gln) and glutamate (Glu) were examined to test the hypothesis that Gln decreases the proinflammatory response induced by LPS in the developing infant rat small intestine. Four groups of 6- to 7-day-old pups were fed a rat milk substitute (RMS), one providing 100% and three providing 25% of normal protein intake for another 6 days. Two of the 25% protein-fed groups received supplemental Gln or Glu. GF and LPS treatment blunted body growth and intestinal villus height and increased intestinal cytokine-induced neutrophil chemoattractant (CINC) mRNA in the protein-deprived, non-Gln-treated group compared with mother-fed pups (P < 0.05). Gln blunted intestinal CINC mRNA (P < 0.05), but Glu did not. Intestinal CINC peptide in the LPS-treated pups provided 100 and 25% protein was elevated approximately 13-fold compared with the mother-reared pups (P < 0.001). Gln and Glu decreased intestinal CINC peptide by 73 and 80%, respectively. GF, LPS-treated pups also had a higher level of plasma CINC peptide (P < 0.05). Gln but not Glu decreased plasma CINC peptide (P < 0.05). An approximate sixfold elevation of intestinal MPO activity in the GF, LPS-treated rats was decreased by Gln and Glu by 92% (P < 0.001) and 54% (P < 0.05), respectively. Intestinal and plasma TNF-alpha were increased in GF, LPS-treated pups (P < 0.01), and Gln and Glu both blunted this increase (P < 0.05) in the intestine but not in the plasma. The results indicate that Gln decreases the LPS-induced inflammatory response in infant rat intestine under different conditions of protein intake.     

Phosphodiesterase type 5 inhibition improves early memory consolidation of object information

The nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway is assumed to play an important role in processes underlying learning and memory. We used phosphodiesterase type 5 (PDE5) inhibitors to study the role of cGMP in object- and spatial memory. Our results and those reported in other studies indicate that elevated hippocampal cGMP levels are required to improve the memory performance of rodents in object recognition and passive avoidance learning, but not in spatial learning. The timing of treatment modulates the effects on memory and strongly supports a role for cGMP in early stages of memory formation. Alternative explanations for the improved memory performance of PDE5 inhibitors are also discussed. Immunocytochemical studies showed that in vitro slice incubations with PDE5 inhibitors increase NO-stimulated cGMP levels mainly in hippocampal varicose fibers. Reviewing the available data on the localization of the different components of the NO-cGMP signaling pathway, indicates a complex interaction between NO and cGMP, which may be independent of each other. It is discussed that further studies are needed, immunocytochemical and behavioral, to better understand the cGMP-mediated molecular mechanisms underlying memory formation.     

Dual role of CCR2 during initiation and progression of collagen-induced arthritis: evidence for regulatory activity of CCR2+ T cells

Chemokines play an important role in the recruitment of leukocytes and have recently been shown to also attract regulatory T cells. Using blocking mAbs, we analyzed the role of the chemokine receptor CCR2 during initiation and progression of collagen-induced arthritis in mice. Blockade of CCR2 from days 0 to 15 markedly improved clinical signs of arthritis and histological scores measuring leukocyte infiltration, synovial hyperplasia, and bone and cartilage erosion. CCR2 blockade during disease initiation significantly reduced plasma titers of collagen Abs in vivo. In vitro CCR2 blockade also interfered with collagen-specific activation and proliferation of T cells. Surprisingly, CCR2 blockade from days 21 to 36 markedly aggravated clinical and histological signs of arthritis and increased the humoral immune response against collagen. We show that CCR2 is expressed on regulatory T cells. Purified CCR2+ T cells are fully anergic toward polyclonal and collagen-specific activation and potently suppress activation of other T and B cells. The subpopulation of CCR2+ CD25+ regulatory T cells increases approximately 5-fold in the progression phase, while CCR2 expression on other leukocyte populations remains unchanged. These findings identify CCR2+ T cells as regulatory T cells and indicate that CCR2 also plays an important role in down-modulating an inflammatory response.     

Differential activation of M26-containing meiotic recombination hot spots in Schizosaccharomyces pombe

Certain genomic loci, termed hot spots, are predisposed to undergo genetic recombination during meiosis at higher levels relative to the rest of the genome. The factors that specify hot-spot potential are not well understood. The M26 hot spot of Schizosaccharomyces pombe is dependent on certain trans activators and a specific nucleotide sequence, which can function as a hot spot in a position- and orientation-independent fashion within ade6. In this report we demonstrate that a linear element (LE) component, Rec10, has a function that is required for activation of some, but not all, M26-containing hot spots and from this we propose that, with respect to hot-spot activity, there are three classes of M26-containing sequences. We demonstrate that the localized sequence context in which the M26 heptamer is embedded is a major factor governing whether or not this Rec10 function is required for full hot-spot activation. Furthermore, we show that the rec10-144 mutant, which is defective in full activation of ade6-M26, but proficient for activation of other M26-containing hot spots, is also defective in the formation of LEs, suggesting an intimate link between higher-order chromatin structure and local influences on hot-spot activation.     

The effects of the insulin-like growth factor-I aptamer, NBI-31772, on glucose homeostasis in the mouse

The majority of insulin-like growth factor-I (IGF-I) in the adult rodent circulation is bound to high affinity IGF binding proteins. We investigated the changes in IGF-I clearance, blood glucose and plasma insulin levels, and tissue 2-deoxyglucose uptake after intravenous administration of the IGF aptamer, NBI-31772, which selectively competes with IGF-I for binding to the IGFBPs, but has no effect at the IGF-I receptor. Clearance of 125I-IGF-I was significantly increased in NBI-31772-treated mice compared with vehicle-treated mice (t1/2 = 45.0 +/- 1.9 vs. 56.3 +/- 3.9 min, respectively; p = 0.021). However, NBI-31772 had no significant effect on glucose levels, and no insulin sparing effect was apparent neither under basal conditions nor during an intravenous glucose challenge. The decline in the specific activity after 3H-2-deoxyglucose administration was significantly less rapid in NBI-31772-treated mice compared with controls, suggesting that the IGF-I aptamer had an inhibitory effect on hepatic gluconeogenesis. In contrast, no insulin-like effect was apparent in other tissues examined. 3H-2-deoxyglucose accumulation was similar in all tissues analyzed, including skeletal muscle, which is thought to be particularly sensitive to IGF-I. These data suggest that the IGF-I aptamer affects clearance of radiolabeled IGF-I from the circulation, but has no marked effects on glucose nor insulin homeostasis. The search for hydrophilic IGF aptamers with longer duration of action that could be used in the treatment of diabetes may be rewarding.     

Stability of proteins: temperature, pressure and the role of the solvent

We focus on the various aspects of the physics related to the stability of proteins. We review the pure thermodynamic aspects of the response of a protein to pressure and temperature variations and discuss the respective stability phase diagram. We relate the experimentally observed shape of this diagram to the low degree of correlation between the fluctuations of enthalpy and volume changes associated with the folding-denaturing transition and draw attention to the fact that one order parameter is not enough to characterize the transition. We discuss in detail microscopic aspects of the various contributions to the free energy gap of proteins and put emphasis on how a cosolvent may either enlarge or diminish this gap. We review briefly the various experimental approaches to measure changes in protein stability induced by cosolvents, denaturants, but also by pressure and temperature. Finally, we discuss in detail our own molecular dynamics simulations on cytochrome c and show what happens under high pressure, how glycerol influences structure and volume fluctuations, and how all this compares with experiments.     

Immunohistochemical analysis of steroid receptors and glycodelin A (PP14) in isolated glandular epithelial cells of normal human endometrium

Highly purified fractions of isolated endometrial cells can be useful for investigating endometrial function. After a first collagenase digestion, normal human endometrial stromal and epithelial cells were separated by filtration. Glands were purified further by two collagenase digestion steps, filtration, differential sedimentations, and Ficoll gradient centrifugation. Epithelial cells were polyhedral and grew as islands in a whorl-like wavy pattern around glandular fragments. High cell culture purity was confirmed with the positive immunohistochemical reaction against cytokeratin 7,8,18,19. Isolated human glands had a similar distribution pattern of estrogen receptor (ER) and progesterone receptor (PR) as observed in vivo, suggesting that glands have a functional hormone receptor system at the time of plating. Using a specific monoclonal antibody against glycodelin A (GdA), a characteristic cyclical expression was demonstrated during the menstrual cycle. The GdA reaction was weak in the proliferative phase, increasing significantly till the late secretory phase, suggesting a similar GdA concentration in vitro as observed in vivo glands. In conclusion, this method could be a model for studying endometrial glandular cells from different menstrual phases, endometrial cell interactions, implantation mechanisms, GdA regulation mechanisms, and pharmacological or other influences on ER and PR alteration.