Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein

The chemistry of Maillard or browning reactions of glycated proteins was studied using the model compound, N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in protein. Incubation of fFL (15 mM) at physiological pH and temperature in 0.2 M phosphate buffer resulted in formation of N epsilon-carboxymethyllysine (CML) in about 40% yield after 15 days. CML was formed by oxidative cleavage of fFL between C-2 and C-3 of the carbohydrate chain and erythronic acid (EA) was identified as the split product formed in the reaction. Neither CML nor EA was formed from fFL under a nitrogen atmosphere. The rate of formation of CML was dependent on phosphate concentration in the incubation mixture and the reaction was shown to occur by a free radical mechanism. CML was also identified by amino acid analysis in hydrolysates of both poly-L-lysine and bovine pancreatic ribonuclease glycated in phosphate buffer under air. CML was also detected in human lens proteins and tissue collagens by HPLC and the identification was confirmed by gas chromatography/mass spectroscopy. The presence of both CML and EA in human urine suggests that they are formed by degradation of glycated proteins in vivo. The browning of fFL incubation mixtures proceeded to a greater extent under a nitrogen versus an air atmosphere, suggesting that oxidative degradation of Amadori adducts to form CML may limit the browning reactions of glycated proteins. Since the reaction products, CML and EA, are relatively inert, both chemically and metabolically, oxidative cleavage of Amadori adducts may have a role in limiting the consequences of protein glycation in the body.     

Expression of the cytochrome b-URF6-URF5 region of the mouse mitochondrial genome

The nature of RNA coded by the only light-strand (L-strand) open-reading frame unidentified reading frame 6 (URF6) was studied by using a variety of single- and double-strand DNA subclones derived from the 3.6-kilobase (kb) cytochrome b (cyt b)-URF5 coding region of the mouse mitochondrial genome. Northern blot experiments using single-strand-specific M13 clones indicate that both the heavy (H) and L strands of this genomic region are symmetrically transcribed and processed into poly(adenylic acid) [poly(A)] RNAs of comparable size. The 1.2- and 2.4-kb RNAs coded by the H strand, putative mRNAs for cyt b and URF5 reading frames, respectively, are derived from a common precursor of 3.6-kb RNA. The L-strand-coded 1.15-kb RNA, on the other hand, is derived from a short-lived precursor of 3.6-kb RNA by a multiple-step processing involving a 2.4-kb intermediate RNA. The S1 nuclease protection experiments using both the 3'- or 5'-end-labeled DNA probes and also affinity-purified 32P-labeled RNA probes indicate that the 1.15-kb RNA maps between the start of the URF6 reading frame (3' end) and a region 590-600 nucleotides to the 5' end of this reading frame. The 1.15-kb RNA thus contains the entire URF6 coding sequence and an about 590-nucleotide-long 3' untranslated region. The molar abundance of the three mRNAs in the steady-state mitochondrial RNA varies markedly. The 1.15-kb URF6 mRNA is only one-tenth the level of 1.2-kb cyt b mRNA, although it is nearly as abundant as the 2.4-kb URF5 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and release of sulfated glycoproteins by cultured glial cells

Both primary cultured glial cells and cloned (C-6) glioma cells have been shown to synthesize and release sulfated glycoproteins. It was found that N-linked tri- and tetra-antennary glycopeptides recovered from the glycoproteins contained most of the (35S) sulfate label. C-6 glial cells showed a higher rate of oligosaccharide sulfation than the primary glial cultures. Both cell types exhibited a high rate of release of sulfated glycoproteins into the medium. The ratio of 35S/3H incorporated from (35S) sulfate and (3H) glucosamine in the released material was higher than that of the glycoproteins associated with the cell, indicating an enrichment of sulfated glycoproteins in the secreted materials. Monensin inhibited both the synthesis and the release of sulfated glycoproteins.     

Effects of short-term administration of sex hormones on normal and autoimmune mice

The effects of short-term administration (2 to 4 wk) of sex hormones on the immune system of normal (C57BL/6) and autoimmune (C57BL/6-lpr, C3H/lpr, B/W) strains of mice were investigated. Both estrogen (E2) and testosterone (Te) had significant effects on the numbers of T and B cells as well as on the density of cell surface antigens as demonstrated by flow cytometry. For example, Te depleted Thy-1.2+ thymocytes in normal mice and brought about a shift to lower density cells. Lyt-2+ cells appeared to be the main target cells of hormonal modulation in normal and autoimmune mice. Both sex hormones significantly depleted these cells in the thymus but had differential effects in the peripheral lymphoid organs, particularly in the spleen. In general, E2 depleted Lyt-2+ cells, whereas Te increased or maintained this subpopulation of cells in spleen and lymph nodes. Similarly, the suppressor cell activity and IL 2 production on a per cell basis in E2-treated animals was diminished, whereas Te-treated animals had normal or enhanced activity. The relevance of these findings to differential sex susceptibility in autoimmune diseases is discussed.     

Inhibition of plasma prolactin in the rat by amantadine

A single iv injection of 15 or 30 but not 7.5 mg/kg BW of an antiviral drug, amantadine, significantly (P less than 0.05) decreased plasma prolactin (PRL) concentrations in male rats. This effect was dose-dependent, with the highest dose producing a longer-lasting decrease in plasma PRL. The amantadine-induced decrease was unaffected by a simultaneous injection of 5-hydroxytryptophan (30 mg/kg BW) but was completely blocked by a simultaneous injection of haloperidol (0.05 mg/kg BW). It is concluded that this novel effect of amantadine on PRL is produced by an interaction with the dopaminergic system.     

Asplenetin,a flavone and its glycoside from Launaea asplenifolia

A new flavone, asplenetin, has been isolated from Launea asplenifolia and characterized as 5,7,3′,4′,5′-pentahydroxy-3-(3-methylbutyl)flavone. Its glycoside, asplenetin 5-O-neohesperidoside, is also reported.     

Traditional preparation and uses of cassava in Nigeria

Various methods of cassava preparation are practised by different ethnic groups in Nigeria. These methods involve peeling cassava roots, soaking roots in streams, grating cassava, and pressing grated cassava. Other methods include heating sieved, grated cassava, boiling peeled cassava roots, and pounding boiled or dried cassava roots. The traditional, cassava-based products aregari, fufu, akpu, cassava flour, edible starch, and tapioca. Detoxification of fresh cassava roots is partly achieved through cell rupture during cutting and grating, soaking in running or standing water in earthen pots for 3–5 days, heating, drying, and boiling.     

Solvent influence on rate and mechanism of oxidation of ethylenediaminetetraacetatocobaltate(II) by periodate

The kinetics of the oxidation of Co^II(EDTA)²⁻ by IO₄⁻ were studied in various ethanol + water mixtures covering the range 7.9 to 58.0 wt% ethanol, at five different temperatures in the range 15–35 °C. The effect of solvent on the rate and mechanism of the reaction was investigated. An inner-sphere mechanism for the reaction was proposed and supported by the calculated activation parameters.     

Subcellular Distribution and Developmental Expression of Cholesterol Ester Hydrolases in Fetal Rat Brain Cultures

Abstract: Cholesterol ester hydrolase activities previously have been identified in brain and linked to the production of myelin, which has very low levels of esterified cholesterol. We have studied two cholesterol ester hydrolase activities (termed the pH 6.0 and pH 7.2 activities) in cultures derived from 19- to 21-day-old dissociated fetal rat brains and in developing rat brain. In vivo the levels of both the pH 6.0 and pH 7.2 activities began to increase by about 10 postnatal days, reached maximal levels at 20 days (20 and 1.5 nmol/h/mg protein, respectively), and thereafter remained nearly constant (pH 6.0) or decreased somewhat before becoming constant (pH 7.2). In contrast, in the cultures the pH 6.0 cholesterol ester hydrolase activity was low until 21 days in culture (DIC; 20 nmol/h/mg protein), increased to a peak activity at 31 DIC (60 nmol/h/mg protein), remained high for 24 days, and finally decreased (18 nmol/h/mg protein at 63 DIC); the pH 7.2 cholesterol ester hydrolase activity was very low until 20 DIC, increased to a peak activity at 31 days (3 nmol/h/mg protein), and thereafter decreased to a lower level (2 nmol/h/mg protein) that was maintained for about 24 days before decreasing (0.7 nmol/h/mg protein at 63 DIC). Therefore, (a) the time courses of appearance of both cholesterol ester hydrolase activities were delayed by 10–14 days relative to that seen in vivo, and (b) the specific activities observed in the cultures were transiently two- to three-fold higher than in rat brain, but then declined to levels characteristic of whole brain homogenates. Subcellular fractionation of the cultures demonstrated that the pH 7.2 cholesterol ester hydrolase activity, along with myelin basic protein and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity, was enriched in a membrane fraction collected at an interface between 0.32 M and 0.9 M sucrose; the pH 6.0 cholesterol ester hydrolase activity, in contrast, was enriched in the microsomal fraction.