A factor derived from chick embryo retina which inhibits DNA synthesis of retina itself

Chick embryo retinas contain a peptide factor that inhibits DNA synthesis in explants of chick embryo retina. The inhibitory factor, obtained by acid/ethanol extraction from 15-day-old chick embryo retinas, was partially purified by affinity chromatography on heparin-sepharose CL-6B and gel filtration on Sephadex G-100. The inhibitor reduced DNA synthesis with maximal effects observed in retinal explants from 7 to 8-day-old chick embryos. The inhibitory effect became apparent after 10 h of incubation and reached the maximum levels after 16 h. DNA-inhibiting activity was heat and acid-stable and was destroyed by trypsin and alkaline treatments. The inhibitory effect was observed in retinal explants incubated in a medium free froml-glutamine, and the addition of this compound to the medium reduced the inhibitory effect in a concentration-dependent manner.     

Cultivation and growth of dissociated neurons from chick embryo cerebral cortex in the presence of different substrates

Summary Dissociated cells from 7-day old chick embryo cerebral hemispheres were cultivated for one month in Rose chambers. Four different culture conditions were employed in the composition of the matrix on which the cells were cultivated: collagen alone, collagen plus embryonic extract, collagen plus plasma and collagen plus plasma and embryonic extract.Within the first 48 hours of cultivation the cells formed processes under all four culture conditions. In the presence of plasma the dissociated cells remained well isolated; in the other culture conditions many cells reassociated into clumps.After 2–3 weeks in cultures on collagen or collagen plus embryonic extract many polygonal cells developed and formed a layer upon which typical neurons and oligodendrocyte-like cells were observed. After 3 weeks the polygonal cells began to transform into astrocyte-like cells. In the presence of plasma the cell bodies of the neuroblasts remained small and round. The processes developed generally consisted of one long and many short thick fibres; all processes had a bulbous appearance. In 3–4-weeks old cultures the cells which remained viable, were morphologically unchanged.The differences in the morphological aspects of the cells cultivated on plasma and those cultivated on collagen alone or with embryonic extract are discussed.This work was supported in part by the Délégation Générale à la Recherche Scientifique et Technique and the Mind Science Foundation. Thanks to Prof. Dr. Z. Lodin, Czechoslovak Academy of Sciences, for his continuing help. We are grateful to Mrs. M. F. Knoetgen for technical assistance.     

Changes induces by haloperidol (antidepressant drug) on the developing retina of the chick embryo

Morphological and histological studies were done on the retina of chick embryos of 6th, 10th and 16th days of incubation by a single dose of haloperidol (0.25 mg/egg), injected on day zero and 5 of incubation. To get an idea about the extent of the teratogenic effect of this drug on the development retina. Sign of malformation in the chick embryo after administration of haloperidol were seen as absent of the ear vesicles, eyes or decreased size of them. Retardation of growth of the retina at 6th, 10th and 16th days treated chick embryo were observed as evidenced of reduction of the size of the retina, associated with sign of degeneration of the retina cells.

Conclusion

The injection of haloperidol drug give rise to several side effects as retardation of growth and degeneration of the cells. The decrease of the thickness of the layers and less density of the cells was related to direct effect of the drug on the cells of this organ. Also on the DNA formation and on the retardation of cellular mitotic activates, therefore the retina appeared decrease in thickness and less cell density with degeneration its cells.     

De novo synthesis of poly(dAT) by the cell extract of a thermophile Bacillus stearothermophilus carrying a plasmid pTB913

Abstract In the absence of added template and primer, DNA synthesis activity which required dATP, dTTP, magnesium ion, and ATP was detected in the cell extracts prepared from a thermophile Bacillus stearothermophilus carrying a plasmid pTB913, but not from the strain without plasmid. Polymer synthesis was detectable only after a lag period and then proceeded at an exponential rate. The DNA synthesized in vitro was the alternating copolymer of dAMP and dTMP, poly(dAT). This reaction was very similar to the de novo DNA synthesis by DNA polymerase I of Escherichia coli, Bacillus subtilis , and Micrococcus luteus , except for the requirement of ATP and thermostability.     

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.     

Purification of Saccharomyces cerevisiae RNase H(70) and identification of the corresponding gene

We purified Saccharomyces cerevisiae RNase H(70) to homogeneity, using an optimized chromatographic purification procedure. Renaturation gel assay assigned RNase H activity to a 70 kDa polypeptide. Sequencing of tryptic peptides identified the open reading frame YGR276c on chromosome VII of the S. cerevisiae genome as the corresponding gene, which encodes a putative polypeptide of molecular mass of 62849. We therefore renamed this gene RNH70. Immunofluorescence microscopy using a RNH70-EGFP fusion construct indicates nuclear localization of RNase H(70). Deletion of RNH70 from the yeast genome did not result in any serious phenotype under the conditions tested. Homology searches revealed striking similarity with a number of eukaryotic proteins and open reading frames, among them the chimpanzee GOR protein, a homolog of a human autoimmune antigen, found to elicit autoimmune response in patients infected with hepatitis C virus.     

A preliminary report on the establishment of pregnancies in an in vitro fertilization (IVF) programme at the University of British Columbia (UBC)

Between July 1982 and November 1983, two pregnancies were established using in vitro fertilization and embryo transfer (IVF and ET) procedures with three different schedules to induce follicular maturation. All women were cycling normally and had inoperable or absent fallopian tubes. Of 83 oocytes aspirated from 24 patients (31 cycles), 75% were considered mature and 25% immature by the morphological characteristics of the oocytes and cumulus cells. Oocytes were preincubated for 6–24 hours, and after insemination, 60% cleaved to the two-to-four-cell stage. The superovulation induction schedule employing hMG administered according to the individually adjusted treatment scheme established two pregnancies. This schedule was considered the superior regimen, as it gave the highest proportion of mature oocytes (89%) which cleaved (78%). The pregnancy-attaining follicle showed a high progesterone:estradiol-17β ratio (P₄/E₂) in its microenvironment of aspirated follicular fluid, culture media of granulosa cells, and oocyte-cumulus complex. Our observations indicate a high P₄/E₂ ratio in the pregnancy-attaining follicle, and thereby reflect a further parameter in influencing maturation of the oocytes most likely to implant.     

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Picornavirus infectivity is dependent on the RNA poly(A) tail, which binds the poly(A) binding protein (PABP). PABP was reported to stimulate viral translation and RNA synthesis. Here, we studied encephalomyocarditis virus (EMCV) and poliovirus (PV) genome expression in Krebs-2 and HeLa cell-free extracts that were drastically depleted of PABP (96%-99%). Although PABP depletion markedly diminished EMCV and PV internal ribosome entry site (IRES)-mediated translation of a polyadenylated luciferase mRNA, it displayed either no (EMCV) or slight (PV) deleterious effect on the translation of the full-length viral RNAs. Moreover, PABP-depleted extracts were fully competent in supporting EMCV and PV RNA replication and virus assembly. In contrast, removing the poly(A) tail from EMCV RNA dramatically reduced RNA synthesis and virus yields in cell-free reactions. The advantage conferred by the poly(A) tail to EMCV synthesis was more pronounced in untreated than in nuclease-treated extract, indicating that endogenous cellular mRNAs compete with the viral RNA for a component(s) of the RNA replication machinery. These results suggest that the poly(A) tail functions in picornavirus replication largely independent of PABP.     

The effects of succinylacetone (4,6-dioxoheptanoic acid) on δ-aminolevulinate synthase activity and the content of heme in monolayers of chick embryo liver cells

Succinylacetone was shown to inhibit aminolevulinate dehydratase (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24) to reduce cellular heme and porphyrins and to induce δ-aminolevulinate synthase (succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37) in monolayers of chick embryo liver cells. Marked synergistic effects on δ-aminolevulinate synthase activity were obtained by combining succinylacetone with levulinate and porphyrogenic drugs. The time course of δ-aminolevulinate synthase activity showed a delayed synergistic response.     

Poly(A)+ RNA from Tetrahymena: Stimulation of Protein Synthesis in Vitro*†

SYNOPSIS Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at -17S and 25S. After heat denaturation all the 25S RNA was converted to 17S. indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A)–RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15–fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28.000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.     

Diverse patterns of poly(A) tail elongation and shortening of murine maternal mRNAs from fully grown oocyte to 2-cell embryo stages

We previously succeeded in constructing a cDNA library, CPF7, enriched with cDNA derived from maternal RNAs with the extended poly(A) tail in mouse fertilized eggs. In this study, we performed RNA blot analysis to examine the elongation in maternal RNAs using 20 representative clones isolated from CPF7 as probes. Various patterns of elongation, shortening, and/or degradation of maternal RNAs were observed from fully grown oocytes to early 2-cell embryos and could be roughly classified into three types and seven subtypes. These findings indicate that poly(A) elongation and shortening of maternal RNAs are not restricted to certain types of maternal RNAs but occur in many of them, and suggest a complex mechanism governing modification of the 3' end of maternal RNAs during the oocyte-to-embryo transition.     

Distinct roles of the R3H and RRM domains in poly(A)-specific ribonuclease structural integrity and catalysis

Deadenylases specifically catalyze the degradation of eukaryotic mRNA poly(A) tail in the 3′- to 5′-end direction with the release of 5′-AMP as the product. Among the deadenylase family, poly(A)-specific ribonuclease (PARN) is unique in its domain composition, which contains three potential RNA-binding domains: the catalytic nuclease domain, the R3H domain and the RRM domain. In this research, we investigated the roles of these RNA-binding domains by comparing the structural features and enzymatic properties of mutants lacking either one or two of the three RNA-binding domains. The results showed that the R3H domain had the ability to bind various oligonucleotides at the micromolar level with no oligo(A) specificity. The removal of the R3H domain dissociated PARN into monomers, which still possessed the RNA-binding ability and catalytic functions. Unlike the critical role of the RRM domain in PARN processivity, the removal of the R3H domain did not affect the catalytic pattern of PARN. Our results suggested that both R3H and RRM domains were essential for the high affinity of long poly(A) substrate, but the R3H domain did not contribute to the substrate recognition of PARN. Compared to the RRM domain, the R3H domain played a more important role in the structural integrity of the dimeric PARN. The multiple RNA-binding domain architecture endows PARN the property of highly efficient catalysis in a highly processive mode.     

Effect of magnesium ions on the thermal stability of human poly(A)-specific ribonuclease

Poly(A)-specific ribonuclease (PARN), a member of the DEDD family, is a key enzyme involved in the deadenylation of mRNA in higher eukaryotic cells. In this research, it was found that Mg(2+) could protect PARN against thermal inactivation by increasing the midpoint of inactivation and decreasing the inactivation rate. This protective effect was unique to Mg(2+) in a concentration-dependent manner. However, the thermal unfolding and aggregation was promoted by the addition of Mg(2+) at high temperatures. These results revealed that Mg(2+) might have dual effects on PARN stability: protecting the active site but endangering the overall structural stability.     

小麦返白系返白期间Poly(A)RNA与蛋白质水平的变化

比较了小麦返白系与其原始品种矮变1号在返白期间Poly(A)RNA与蛋白质水平的变化。在心叶变白过程中。返白系的蛋白质含量下降,随着复绿又回升;而RNA含量变化与此大致呈平行关系。在全白阶段,返白系的Poly(A)RNA含量大幅度下降。体外合成的蛋白质也少得多,但其翻译活性/Poly(A)RNA及各期的蛋白质/Poly(A)RNA比例并不比对照低。可能返白系在返白期间蛋白质水平的变化与转录水平的基因调控有关。     

Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K⁺ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K⁺, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.     

Effect of phospholipids on the activity of sialosyl lactosylceramide (GM3): N-acetylgalactosaminyl transferase from chick embryo brain

Summary A preparation of UDP-Ga1NAc:sialosyl lactosylceramide N-acetylgalactosaminyl transferase (E.C. 2.4.1.92) obtained from 14-day chick embryo brain was delipidated partially by treatment with cold acetone in the presence of varying amounts of sodium dodecyl sulphate (SDS). The lipid content and the enzyme activity of the preparation decreased as the concentration of SDS increased. At 0.3% SDS the lipid content was about 30% and the enzyme activity about 15% of the original. The activity could be restored up to 60% of the original by added phospholipids, provided the removal of endogenous lipids did not exceed 70%. Phospholipids with diferent composition showed different abilities to restore the enzyme activity. Among phosphatidylcholines the decreasing order of effectivity was dilauroyl dimiristoyl dipalmitoyl distearoyl-choline. Dimiristoyl phosphatidylcholine, dimiristoyl phosphatidylglycerol and dipalmitoyl phosphatidylglycerol activated the enzyme more effectively than dimiristoyl phosphatidyl ethanolamine, dimiristoyl phosphatidic acid or brain phosphatidylserine. No correlation was found between the activating ability and the charge in the polar head group of the lipid added. Addition of dilauroyl phosphatidylcholine to the delipidated preparation increased about 5-fold the Vmax without affecting the apparent Km for both the donor nucleotide and acceptor glycolipid. The data suggest that the lipid composition of the enzyme environment constitutes a potential level of regulation of the activity of this key enzyme of ganglioside biosynthesis.     

Inhibitory effect of naked neural BC1 RNA or BC200 RNA on eukaryotic in vitro translation systems is reversed by poly(A)-binding protein (PABP)

Regulated protein biosynthesis in dendrites of neurons might be a key mechanism underlying learning and memory. Neuronal dendritic BC1 RNA and BC200 RNA and similar small untranslated RNAs inhibit protein translation in vitro systems, such as rabbit reticulocyte lysate. Likewise, co-transfection of these RNAs with reporter mRNA suppressed translation levels in HeLa cells. The oligo(A)-rich region of all active small RNAs were identified as the RNA domains chiefly responsible for the inhibitory effects. Addition of recombinant human poly(A)-binding protein (PABP) significantly compensated the inhibitory effect of the small oligo(A)-rich RNA. In vivo, all BC1 RNA appears to be complexed with PABP. Nevertheless, in the micro-environment of dendritic spines of neuronal cells, BC1 RNPs or BC200 RNPs might mediate regulatory functions by differential interactions with locally limited PABP and/or directly or indirectly, with other translation initiation factors.