Enzymatic Regulation of Glycoprotein Synthesis in Peripheral Nervous System Myelin

The enzyme UDP-N-acetylglucosamine: dolichyl phosphate, N-acetylglucosamine-1-phosphate transferase initiates the synthesis of the oligosaccharide chain of complex-type glycoproteins. In view of the high content of glycoprotein in peripheral nerve myelin, the properties of this enzyme, its changes with age, and the effect of the specific inhibitor tunicamycin were investigated. The enzyme activity in rat peripheral nerve homogenate was completely dependent on the presence of exogenous dolichyl phosphate as well as Mg2+ and a detergent (Triton X-100) and was also greatly stimulated by a high salt concentration (0.4 M KCl) and AMP. The highest specific activity was present in the postmitochondrial membranes. The specific activity in postmitochondrial membranes in the presence of exogenous dolichyl phosphate reached a maximum at 17 days and remained relatively high throughout development, up to 2 years of age, but the activity was much lower when dolichyl phosphate was not added. This indicates that the enzyme level does not decrease with age, but that the content of the lipid cofactor may limit glycoprotein synthesis in vivo. Tunicamycin (5 micrograms) was injected intraneurally into 24-day-old rat sciatic nerve, and the enzyme was assayed from 1 to 24 days after injection. The specific activity of the transferase remained at low levels (5-40% of the level in control nerve) in most injected nerves assayed throughout this postinjection period. A protein previously identified as the unglycosylated P0 protein was synthesized in vitro by the tunicamycin-injected nerve and could be demonstrated to be incorporated into myelin in large amounts at 2 days and in small amounts at 6 days after injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional properties of palmaris longus muscles of rhesus monkeys transplanted as index finger flexors

This experiment with skeletal muscle autografts in monkeys was designed to retest previous findings that transplanted skeletal muscle can regenerate to a functional degree in primates without predenervation and to test a new hypothesis that increased functional demands on regenerated muscle grafts in monkeys may result in improved functional capacity of the grafts. Rhesus monkey index flexors were replaced with free palmaris longus muscle autografts with microneural anastomoses between the graft motor nerve and the severed profundus motor nerve. One monkey was taught selective index flexion before grafting and continued with this program after grafting to test the effect of training on the graft. Mature grafts were evaluated for in vivo contractile properties and by histology and histochemistry and were compared with a group of normal Rhesus palmaris longus muscles. The results reconfirm the capacity of nonpredenervated monkey skeletal muscle grafts to regenerate and to achieve some contractile ability and suggest that training of free muscle grafts may enhance recovery of their functional and structural properties.     

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.     

Absence of genotoxic effects of nonasbestos mineral fibers

The biological activity of natural and synthetic mineral fibers has been examined. Natural attapulgite [(Mg, Al)₂Si₄O₁₀(OH).4H₂0], synthetic xonotlite [Ca₃Si₃O₈(OH)₂] and natural sepiolite [Mg₂Si₃O₈.2H₂O] were selected. Genotoxic effects were investigated by means of a well established cellular model based upon the measurement of unscheduled DNA synthesis (UDS) in rat hepatocytes in primary culture. The intrinsic capacity of the fibers (1 and 10 µ/ml) to induce UDS was first tested. None of the fiber types showed detectable UDS-eliciting activity. Also, the possible modulation of the cellular response to genotoxic agents by the materials was examined by exposing the cells to mixtures of 2-acetylaminofluorene (AAF) (0.05 and 0.25 µg/ml) and fibers (1 and 10 µg/ml). In these experiments, the UDS response was significantly diminished in the presence of xonotlite. This phenomenon may reflect changes in the uptake and/or metabolism of AAF or may result from an inhibition of DNA repair processes, the latter suggesting a possible cocarcinogenic potential for this synthetic silicate. These results point to the immediate necessity of studying more extensively the biological effects of fibrous materials that can be used as substitutes for asbestos.Abbreviations AAF 2-acetylaminofluorene - DMSO dimethyl-sulfoxide - FBS fetal bovine serum - IRDA Institut de Recherche et de Développement sur l'Amiante - LDH lactate dehydrogenase - UDS unscheduled - DNA synthesis - WME Williams' Medium E This work was supported by the Institut de Recherche et de Développement sur l'Amiante (IRDA), Sherbrooke, Canada.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.     

The presence of LHRH-like receptors in Dunning R3327H prostate tumors

Quantitative analyses of LH-RH-like membrane receptors were performed in five tumors from the transplantable Dunning R3372H rat prostatic adenocarcinoma. The binding of D-Trp⁶-LH-RH, an agonist of LH-RH, was observed in all 5 tumors. The antagonist [Ac-Dp-Cl-Phe^1,2,D-Trp³,D-Lys⁶,D-Ala¹⁰]-LH-RH was bound to 4 tumors. The apparent equilibrium dissociation constant (K_d) for D-Trp⁶-LH-RH receptor was from 2.6–3.9 × 10^?10 M. The apparent equilibrium B_max values (maximum number of binding sites) were from 17.2–86.0 fmol/mg membrane protein for D-Trp⁶-LH-RH receptor. The K_d for the antagonist was from 2.4–2.7 × 10^?10 M and the B_max values were from 35.5–66.0 fmol/mg membrane protein. Similar binding studies performed in 6 normal rat prostates showed no binding capacities.     

Major polypeptide of duck hepatitis B surface antigen particles

The 40- to 50-nm pleomorphic particles found in the sera of domestic Pekin ducks infected with duck hepatitis B virus were purified by rate zonal and isopycnic centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide analysis of these particles, called duck hepatitis B surface antigen particles, revealed the major component to be a single 17,500-dalton polypeptide. This result is in contrast to polypeptide analyses of the surface antigens of related mammalian viruses, including hepatitis B, in which a major doublet of polypeptides is seen with molecular weights ranging from 23,000 to 29,000. Tryptic maps of 17,500-dalton polypeptide resembled that of the major non-glycosylated polypeptide of the adw subtype of hepatitis B surface antigen. A serological assay for antibody to the purified duck virus particles is also described.     

Effect of experimentally induced calcium deficiency on the developmental expression of collagen types in chick embryonic skeleton

In order to investigate the effect of embryonic calcium deficiency on the cellular differentiation processes in embryonic skeletogenesis, chick embryos were maintained in long-term shell-less cultures in vitro. The absence of the eggshell, which normally provides over 120 mg of calcium to the embryo during the course of development, resulted in severely retarded and anomalous skeletal formation. The pattern of cytodifferentiation in the skeletal elements during development was assessed by examining collagen type synthesis in both endochondral and intramembranous bones of normal and shell-less embryos as a function of developmental age. Skeletal tissues obtained from these embryos at various developmental stages were maintained in short-term organ culture in medium containing [³H]Pro. The metabolically labeled collagen was isolated from these tissues and typed biochemically based on electrophoresis, ion-exchange chromatography, differential salt fractionation, zone precipitation chromatography, and CNBr peptide mapping. The results indicate that, compared to chronologically equivalent normal controls, calcium-deficient skeletal elements from shell-less embryos appeared to fail to mature into complete bony tissues and instead exhibited partial cartilage phenotype with the expression of cartilage-specific type II collagen.