Modulation of cerebral acetylcholine metabolism by the dorsal diencephalic conduction system in the rat

We investigated the effects of interruption of the impulse flow in the habenulopeduncular pathways by local infusion of tetrodotoxin on the acetylcholine and choline content in selected dopamine rich regions in the forebrain and midbrain in rats. The tetrodotoxin infusion caused a marked increase in acetylcholine content in the medial frontal cortex, striatum and ventral tegmental area+interpeduncular nucleus, but not in the limbic area or the substantia nigra, whereas choline content was reduced only in both the striatum and ventral tegmental area+interpeduncular nucleus. There was an increase in 3,4-dihydroxyphenylacetic acid content in the striatum after the manipulation. These findings suggest that the dorsal diencephalic conduction system may be involved in the integration of the activity of cholinergic neurons in the forebrain and midbrain regions and striatal dopanine neurons may play a role in the modulation of cholinergic neurons.     

Oxidation of Arg-410 promotes the elimination of human serum albumin

The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs. The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice after labeling with 111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase in carbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in alpha-helical content as the result of oxidation was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in the pharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A and R410A mutants were affected (extent of effect: R410A > K199A > H146A). The liver clearance of these proteins is closely correlated to hydrophobicity (r = 0.929, P < 0.01) and the net charge of the proteins (r=0.930, P < 0.01). The rate of elimination of HSA is closely related to the hydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.     

Serum concentrations of androstenediol and androstenediol sulfate, and their relation to cytokine production during and after normal pregnancy

Since it is known that androstenediol (ADIOL) has potent immunoregulatory effects, changes in ADIOL levels during and after pregnancy might affect the maternal immune system. We examined serum concentrations of ADIOL and androstenediol 3-sulfate (ADIOLS) together with IFN-gamma and IL-4 production levels during pregnancy and after delivery up to 10-11 months postpartum. The subjects were 73 normal pregnant, 76 normal postpartum, and 28 normal non-pregnant women. ADIOL and ADIOLS were measured using EIA and GC/MS, respectively. The cytokine levels in the supernatant of whole-blood cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin were measured using ELISA. ADIOL levels significantly decreased compared to non-pregnant levels in the first trimester (P < 0.05) and were reversed in the third trimester (P < 0.05). After pregnancy, ADIOL levels gradually declined, and a significant decrease was observed at 10-11 months postpartum (P < 0.05). ADIOLS levels were significantly lower in the third trimester (P < 0.05) and significantly higher at the first month postpartum (P < 0.001) compared to non-pregnant women. IFN-gamma and IL-4 levels decreased during pregnancy and subsequently increased postpartum. On the other hand, we found significant negative correlations between ADIOL concentrations and production levels of IFN-gamma (P < 0.05) or IL-4 (P < 0.05). These findings suggest that ADIOL may be involved in modifying the maternal immune response during and after pregnancy.     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults

Lipid peroxidation has been implicated in a variety of pathophysiological processes, including inflammation, atherogenesis, neurodegeneration, and the ageing process. Phospholipid hydroperoxide glutathione peroxidase (GPX4) is the only major antioxidant enzyme known to directly reduce phospholipid hydroperoxides within membranes and lipoproteins, acting in conjunction with alpha tocopherol (vitamin E) to inhibit lipid peroxidation. Here we describe the generation and characterization of GPX4-deficient mice by targeted disruption of the murine Gpx4 locus through homologous recombination in embryonic stem cells. Gpx4(-/-) embryos die in utero by midgestation (E7.5) and are associated with a lack of normal structural compartmentalization. Gpx4(+/-) mice display reduced levels of Gpx4 mRNA and protein in various tissues. Interestingly, cell lines derived from Gpx4(+/-) mice are markedly sensitive to inducers of oxidative stress, including gamma-irradiation, paraquat, tert-butylhydroperoxide, and hydrogen peroxide, as compared to cell lines derived from wild-type control littermates. Gpx4(+/-) mice also display reduced survival in response to gamma-irradiation. Our observations establish GPX4 as an essential antioxidant enzyme in mice and suggest that it performs broad functions as a component of the mammalian antioxidant network.     

Keratinocyte growth factor

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex sterorid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.     

Electro-transfer of small interfering RNA ameliorated arthritis in rats

RNA interference provides the powerful means of sequence-specific gene silencing. Particularly, small interfering RNA (siRNA) duplexes may be potentially useful for therapeutic molecular targeting of human diseases, although novel delivery systems should be devised to achieve efficient and organ-specific transduction of siRNA. In the present study, we demonstrated that electro-transfer of a siRNA-polyamine complex made efficient and specific gene knockdown possible in the articular synovium. Targeted suppression of the tumor necrosis factor-alpha gene through this procedure significantly ameliorated collagen-induced arthritis in rats. Our results suggest the potential feasibility of therapeutic intervention with RNA medicines for treatment of rheumatoid and other locomotor diseases.     

Inhibition of D-serine accumulation in the Xenopus oocyte by expression of the rat ortholog of human 3'-phosphoadenosine 5'-phosphosulfate transporter gene isolated from the neocortex as D-serine modulator-1

D-serine in mammalian brains has been suggested to be an endogenous co-agonist of the NMDA-type glutamate receptor. We have explored the molecules regulating D-serine uptake and release from the rat neocortex cDNA library using a Xenopus oocyte expression system, and isolated a cDNA clone designated as dsm-1 (D-serine modulator-1) encoding a protein that reduces the accumulation of D-serine to the oocyte. dsm-1 is the rat orthologue of the human 3'-phosphoadenosine 5'-phosphosulfate transporter 1 (PAPST1) gene. The hydropathy analysis of the deduced amino acid sequence of the Dsm-1 protein predicts the 10 transmembrane domains with a long hydrophobic stretch in the C-terminal like some amino acid transporters. The dsm-1 mRNA is predominantly expressed in the forebrain areas that are enriched with D-serine and NMDA receptors, and in the liver. The transient expression of dsm-1 in COS-7 cells demonstrates a partially Golgi apparatus-related punctuate distribution throughout the cytoplasm with a concentration near the nucleus. dsm-1-expressing oocytes diminishes the sodium-dependent and -independent accumulation of D-serine and the basal levels of the intrinsic D-serine and increases the rate of release of the pre-loaded D-serine. These findings indicate that dsm-1 may, at least in part, be involved in the D-serine translocation across the vesicular or plasma membranes in the brain, and thereby control the extra- and intracellular contents of D-serine.     

Osteopontin as a positive regulator in the osteoclastogenesis of arthritis

We examined the role of osteopontin (OPN) in the osteoclastogenesis of arthritis using collagen-induced arthritis (CIA). Cells from arthritic joints of wild-type (OPN +/+) mice spontaneously developed bone-resorbing osteoclast-like cells (OCLs). The cultured cells showed an enhanced expression of receptor activator of nuclear factor kappaB ligand (RANKL) and a decreased expression of osteoprotegerin (OPG). The addition of OPG reduced the number of OCLs, indicating that the osteoclastogenesis depends on the RANK/RANKL/OPG system. The cells also produced OPN abundantly and anti-OPN neutralizing antibodies suppressed the development of OCLs. Moreover, the addition of OPN increased the expression of RANKL and augmented differentiation of OCLs from OPN-deficient (OPN -/-) cells. OPN, like the combination of 1alpha,25-dihydroxyvitamin D(3) and dexamethasone, also enhanced the RANKL expression and decreased OPG expression in a stromal cell line, ST2. These results suggest that OPN acts as a positive regulator in the osteoclastogenesis of arthritis through the RANK/RANKL/OPG system.