Hydrogen peroxide involvement in the rhodanese inactivation by dithiothreitol.

The conditions required to obtain rhodanese inactivation in the presence of dithiothreitol indicate the involvement of hydrogen peroxide produced by metal-ion catalyzed oxidation of dithiothreitol. Inhibition of dithiothreitol oxidation by a chelating agent, or by removal of hydrogen peroxide by catalase prevents the enzyme inactivation. The inactivated enzyme contains a disulfide bond resulting from the oxidation of the catalytic sulfhydryl group and another sulfhydryl group close to it. This disulfide might be formed via a sulfenic intermediate.     

Apurinic and/or apyrimidinic endonuclease activity in ataxia telangiectasia cell extracts.

The possibility that the increased sensitivity of ataxia telangiectasia towards ionizing radiation is related to a DNA-repair deficiency has been examined further. When compared to unaffected controls, 6 lines of fibroblast cells derived from ataxia patients demonstrated a slightly reduced endonucleolytic activity (165 +/- 12 units vs. 214 +/- 28 units) towards apurinic and/or apyrimidinic sites as determined in a "nicking" assay.     

Biochemical changes in mouse liver after palmitoyl-ethanolamide (PEA) administration

PEA (palmitoylethanolamide; N-(2-hydroxyethyl)palmitamide) daily and orally administered to male mice caused: (1) increased incorporation of labelled orotic acid into DNA and RNA, (2) an increase in the activity of uridine kinase and decrease of tryptophan pyrrolase, (3) decreased ribonuclease activity of isolated liver ribosomes, (4) raising of specific radioactivity after injection of labelled amino acids in both mitochondrial and microsomal fractions of liver homogenate, (5) increased incorporation of [¹⁴C]-palmitic acid and ³²P into liver phospholipids.     

Aflatoxin B1-2,3-oxide: evidence for its formation in rat liver in vivo and by human liver microsomes in vitro

Injection of [³H]aflatoxin B₁ into rats yielded covalently bound derivatives in hepatic DNA, rRNA, and protein. Mild acid hydrolysis of the DNA and rRNA adducts formed a derivative indistinguishable from 2,3-dihydro-2,3-dihydroxy-aflatoxin B₁. The data indicate that approximately 60% of the nucleic acid adducts were derived from reactions $\text{in vivo}$ with aflatoxin B₁-2,3-oxide. Acid hydrolysis of rRNA-[³Haflatoxin B₁ adduct formed by human liver microsomes $\text{in vitro}$ also liberated the dihydrodiol in significant amount. The 2,3-oxide of aflatoxin B₁ is a probable ultimate carcinogenic metabolite.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.     

Lead uptake and the effects of EDTA on lead-tissue concentrations in the desert species mesquite (Prosopis spp.)

Experimental results have shown that the desert plant species mesquite (Prosopis spp.) is capable of accumulating high levels of lead in the roots, translocating it to the aerial portion of the plant. One-week-old mesquite seedlings were treated for 7 d in a hydroponic culture using a modified Hoagland solution. Six treatments were used; three treatments contained only Pb [as Pb(NO3)2] at 25-, 50-, and 75-mg L(-1) levels and three treatments contained the same levels of Pb, but with equimolar concentrations of disodium ethylenediamine tetraacetic acid (EDTA). Our results showed that the plants exposed to 25-, 50-, and 75-mg Pb L(-1) treatments without EDTA concentrated in stems 524, 3726, and 1417 mg kg(-1), respectively. However, the plants treated with Pb-EDTA concentrated in stems 480-, 607-, and 1247-mg Pb kg(-1) for the 25-, 50-, and 75-mg Pb L(-1) treatments, respectively. Results for the roots followed a similar trend; without EDTA the Pb levels ranged from 16,055, 89,935, and 63,396 for the 25-, 50-, and 75-mg Pb L(-1) treatments, respectively, and with EDTA these levels were 9,562, 49,902, and 39,181 mg kg(-1) for the three treatments. However, the addition of EDTA increased lead movement to the leaves. The levels of Pb without EDTA were 20, 35, and 51 mg kg(-1) for the 25-, 50-, and 75-mg Pb L(-1) levels, respectively. Treatments with EDTA showed uptake levels of 105, 124, and 313 for the 25-, 50-, and 75-mg Pb L(-1) treatments. Further, the percent Pb in dry leaf tissues for all EDTA treatments were greater than 0.1%. However, only the 25-mg Pb L(-1) treatment was greater than 0.1%, compared to 0.04 and 0.08% for the 50- and 75-mg Pb L(-1) treatments, respectively. Preliminary transmission and scanning electron microscopy corroborate the presence of lead.     

Immunological analysis of a glycoprotein (contact sites A) involved in intercellular adhesion of dictyostelium discoideum

We have prepared antisera in rabbits to the “contact sites A” glycoprotein (gp80) purified from Dictyostelium discoideum. IgG isolated from these anti-sera reacts with a number of different proteins in D discoideum lysates, as analyzed by immune precipitation and by antibody staining of gel electropherograms transferred to nitrocellulose. Blocking experiments indicate that this cross-reactivity reflects the presence of common antigeneic determinants on gp80 and other cellular proteins, rather than the presence of extraneous antibodies in the antisera. The spectrum of reactive proteins is different a: different stages of development. In particular, gp80 itself is synthesized only for a restricted period during the cell aggregation phase. The protein persists throughout development and can be detected in spores. Anti-gp80 Fab fragments bind to the surface of developing D discoideum cells and specifically block their developmentally regulated adhesion. After absorption with vegetative cells, the IgG stains only gp80 and (to a lesser extent) one other band in lysates of aggregation-competent cells. The absorbed antibodies also can block adhesion. Several proteins that appear late in development also arc stained by the absorbed IgG.     

Glucocorticoid-induced reduction of poly (ADP-ribose) synthetase in nuclei from chick embryo liver.

The effect of glucocorticoid hormone administration on the nuclear poly(ADP-ribose) synthetase activity of chick embryoliver was investigated. Compared with the values obtained with control nuclei, the enzyme activity was markedly reduced in the nuclei of liver prepared from chick embryo treated with 0.1 mg hydrocortisone for 12 hours or longer. The possible relationship between the reduction of poly(ADP-ribose) synthetase activity and decrease in DNA synthesis is discussed.     

Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion.

Monoclonal and polyclonal L1 antibodies react by indirect immunofluorescence with the cell surface of cultured tetanus toxin-positive neurons from post-natal cerebella of mice, but not with glial fibrillary acidic protein-positive astrocytes, O4 antigen-positive oligodendrocytes or fibronectin-positive fibroblasts or fibroblast-like cells. During cerebellar development L1 antigen is detectable on tetanus toxin-positive cells as early as embryonic day 13 after 3 days in culture. In sections of the early post-natal cerebellum, L1 antigen is found on pre-migratory neurons in the internal, but not in the external part of the external granular layer. In the adult cerebellum, L1 antigen is predominantly localized in the molecular layer and around Purkinje cells. Fibers in white matter and the granular layer are also L1 antigen-positive. Granule cell bodies and synaptic glomeruli are weakly antigen-positive. Several cell lines derived from neuroblastoma C1300 also express L1 antigen. The antigen is not detectable by enzyme-linked immunosorbent assay in tissue homogenates of liver, kidney, lung, heart, sperm or thymus. With polyclonal L1 antibodies, cross-reactive determinants are found in brains of rat, guinea pig, hamster, chicken, rabbit and man, but not in frog, while monoclonal antibody reacts detectably only with mouse brain. The molecular species recognized by both monoclonal and polyclonal antibodies display two prominent bands by SDS-PAGE under reducing and non-reducing conditions with apparent mol. wts. of 140 and 200 kd. L1 antigen isolated from cultured cerebellar cells consists mainly of a band in the 200-kd range and a faint one at 140 kd. L1 antigen from neuroblastoma N2A shows two bands with slightly higher apparent mol. wts. All molecular forms of L1 antigen can be labeled by [3H]fucose and [3H]glucosamine. Ca2+-independent re-aggregation of cerebellar cells from early post-natal C57BL/6J mice and of the continuous cell line N2A derived from the murine neuroblastoma C1300 is inhibited by Fab fragments of the polyclonal, but not of monoclonal antibody, both of which are known to react with the surface membrane of these cells.     

An arabinogalactan protein(s) is a key component of a fraction that mediates local intercellular communication involved in tracheary element differentiation of zinnia mesophyll cells

Local intercellular communication is involved in tracheary element (TE) differentiation of zinnia (Zinnia elegans L.) mesophyll cells and mediated by a proteinous macromolecule, which was designated xylogen. To characterize and isolate xylogen, a bioassay system to monitor the activity of xylogen was developed, in which mesophyll cells were embedded in microbeads of agarose gel at a low (2.0-4.3x10(4) cells ml(-1)) or high density (8.0-9.0x10(4) cells ml(-1)) and microbeads of different cell densities were cultured together in a liquid medium to give a total density of 2.1-2.5x10(4) cells ml(-1). Without any additives, the frequency of TE differentiation was much smaller in the low-density microbeads than in the high-density microbeads. This low level of TE differentiation in the low-density microbeads was attributable to the shortage of xylogen. When cultures were supplemented with conditioned medium (CM) prepared from zinnia cell suspensions undergoing TE differentiation, the frequency of TE differentiation in the low-density microbeads increased remarkably, indicating the activity of xylogen in the CM. The xylogen activity in CM was sensitive to proteinase treatments. Xylogen was bound to galactose-specific lectins such as Ricinus communis agglutinin and peanut agglutinin, and precipitated by beta-glucosyl Yariv reagent. These results indicate that xylogen is a kind of arabinogalactan protein.     

The correlation between benzo[a]pyrene-induced mutagenicity and DNA adduct formation in Salmonella typhimurium TA100

In an attempt to stabilize the dose response in the Salmonella typhimurium test (STT), the use of DNA-bound products from BP was evaluated as a measure of the biologically effective dose. In addition to the previously documented interlaboratory variation, we observed a 3-fold difference in the dose response of TA100 to BP even when the assay was repeated with the same experimental conditions. When overall BP-DNA adduct formation was related to the level of His+ revertants, a series of responses emerged with two predominating. In the first type of response around 70 revertants per plate were generated for every BP molecule bound per 10(6) nucleotides of cellular DNA. The second response gave about 1400 revertants per plate for one BP bound in every 10(6) nucleotides. Several intermediates curves were also detected. The variation in the mutational response to binding levels occurred regardless of the source of S9 or the growth stage of the cells. These experiments indicate that there was no constant level of DNA damage that would lead to a specified number of revertants of TA100 by BP and that DNA modification was not solely responsible for mutagenic potency. It is possible that an induction of an error-prone repair function of the muc gene carried by the plasmid pKM101 in TA100 may be affecting the relationship between the measured adduct level and reversion frequency.     

Potential mechanism(s) involved in the regulation of glycogen synthesis by insulin

Stimulation of glycogen synthesis is one of the major physiological responses modulated by insulin. Although, details of the precise mechanism by which insulin action on glycogen synthesis is mediated remains uncertain, significant advances have been made to understand several steps in this process. Most importantly, recent studies have focussed on the possible role of glycogen synthase kinase-3 (GSK-3) and glycogen bound protein phosphatase-1 (PP-1G) in the activation of glycogen synthase (GS) - a key enzyme of glycogen metabolism. Evidence is also accumulating to establish a link between insulin receptor induced signaling pathway(s) and glycogen synthesis. This article summarizes the potential contribution of various elements of insulin signaling pathway such as mitogen activated protein kinase (MAPK), protein kinase B (PKB), and phosphatidyl inositol 3-kinase (PI3-K) in the activation of GS and glycogen synthesis.