Control of mRNA translation in oocytes and developing embryos of giant moths. I. Function of the 5' terminal "Cap"in the tobacco hornworm, Manduca sexta

Injection of labeled leucine into oocytes and developing embryos of the tobacco hornworm, Manduca sexta, revealed that the rate of protein synthesis increases dramatically after fertilization and continues to rise until gastrulation. Cell-free preparations of oocytes and developing embryos show a similar pattern of in vitro incorporation. When messenger RNA extracted from unfertilized oocytes was examined by gradient density centrifugation under denaturing conditions, a broad peak was observed which centered around 15 S. In contrast to mRNA extracted from oocytes, that from embryos was found to be capped by 7-methylguanosine at the 5′ terminus. When translation of oocyte mRNA was compared with that of embryo mRNA in a cell-free translation system derived from wheat germ, oocyte RNA translated less efficiently. In the presence of an inhibitor of methylation, S-adenosylhomocysteine, the differences were further widened. In competition with a cap analog, 7-methylguanosine 5′-monophosphate, embryo mRNA translation was inhibited more than oocyte at low concentrations of analog. These results are taken to indicate that the lack of a cap at the 5′ terminus could be one mechanism to inhibit translation prior to fertilization.     

Specific inhibitor of complement (C5)-derived chemotactic activity in systemic lupus erythematosus related antigenically to the Bb fragment of human factor B

Serum and plasma from patients with active systemic lupus erythematosus contain a specific inhibitor of complement (C5)-derived chemotactic activity. We found that the inhibitor is antigenically related to the Bb fragment of complement factor B. Lupus plasma and purified inhibitor significantly reduced the chemotactic activity of zymosan-treated normal serum, an effect that was abolished by antibodies to factor B. Similar results were obtained when purified Bb was used. Neither purified inhibitor nor Bb inhibited the chemotactic activity of purified human C5a or C5a des Arg. As reported previously, the chemotactic activity of C5a des Arg was enhanced significantly by the addition of an anionic polypeptide (cochemotaxin) present in normal serum and plasma. Interestingly, both purified lupus inhibitor and Bb inhibited the chemotactic activity exhibited by mixtures of C5a des Arg and its cochemotaxin. This effect was due, most likely, to their ability to neutralize the enhancing effect of the cochemotaxin on the chemotactic activity of C5a des Arg. Immunoelectrophoresis and western blots revealed that the purified inhibitor reacted with anti-factor B and exhibited a similar charge and molecular weight as purified Bb.     

Effect of pupation environment on the quality of pupae and adults of the Mediterranean fruit fly

When mature larvae of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) pupated in bran rather than naked there was an increase in adult eclosion, but no difference in 9 day pupal weight, 9 day lipid content, or flight ability. There was no difference when pupation occurred at 20 or 25°C. Pupation in sand at 20°C caused severe mortality, while pupation in sand at 25°C did not. Data are presented which favour naked pupation over pupation in any medium, and evidence is presented which indicates that collection of mature larvae in a rotary tumbler rather than by the conventional popping method is economically preferable and is without significant biologically adverse effects.

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Résumé Si des larves mûres de Ceratitis capitata se nymphosent dans du son ou à l'air libre, dans le premier cas le taux d'éclosions est plus élevé, mais le poids et la teneur en lipides au neuvième jour de nymphose ainsi que l'aptitude au vol sont identiques dans les deux. Il n'y a pas de différences pour les pupaisons à 20°C et 25°C, mais la pupaison dans le sable à 20°C provoque une forte mortalité, tandis qu'à 25°C le sable n'a aucun effet. Les résultats obtenus sont favorable à une pupaison à nu plutôt que dans un substrat, et des arguments sont apportés montrant que la collecte des larves mûres dans un culbuteur rotatif ne provoque significativement pas plus d'effets nocifs que la méthode classique du saut.

     

Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells

The deletion mutation in the HPRT-deficient mouse embryonic stem (ES) cell line E14TG2a has been corrected by gene targeting. The presence of plasmid sequences in the correcting vector DNA did not affect the frequency of correction. We have characterized three different HPRT gene structures in correctants. Cells from one corrected clone have been introduced into mouse blastocysts, and germ line transmission of the ES cell-derived corrected gene has been achieved. The corrected gene has the same pattern of expression as the wild-type gene, with the characteristic elevated level of expression in brain tissue. Hence, we have demonstrated the feasibility of introducing targeted modifications into the mouse germ line by homologous recombination in ES cells.     

Ectoenzymes of the kidney microvillar membrane. Isolation and characterization of the amphipathic form of renal dipeptidase and hydrolysis of its glycosyl-phosphatidylinositol anchor by an activity in plasma.

Renal dipeptidase (EC 3.4.13.11) has been solubilized from pig kidney microvillar membranes with n-octyl-beta-D-glucopyranoside and then purified by affinity chromatography on cilastatin-Sepharose. The enzyme exists as a disulphide-linked dimer of two identical subunits of Mr 45,000 each. The purified dipeptidase partitioned into the detergent-rich phase upon phase separation in Triton X-114 and reconstituted into liposomes consistent with the presence of the glycosyl-phosphatidylinositol membrane anchor. The N-terminal amino acid sequence of the amphipathic, detergent-solubilized, form of renal dipeptidase was identical with that of the hydrophilic, phospholipase-solubilized, form, locating the membrane anchor at the C-terminus of the protein. The glycosyl-phosphatidylinositol anchor of both purified and microvillar membrane renal dipeptidase was a substrate for an activity in pig plasma which displayed properties similar to those of a previously described phospholipase D. The cross-reacting determinant of the glycosyl-phosphatidylinositol anchor was generated by incubation of purified renal dipeptidase with bacterial phosphatidylinositol-specific phospholipase c, whereas the anchor-degrading activity in plasma failed to generate this determinant.     

Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme.

Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.     

Case finding in the elderly: does the primary care team already know enough?

The needs of an elderly population in one general practice were assessed by the primary health care team. A simple questionnaire was filled in as far as possible by members of the team on known social and functional criteria for each patient aged over 75. The questionnaire was complete for 100 of 118 elderly patients. The information was accurate for over 95% of the criteria as verified by information held by the health authority. This information will now be used for yearly reviews of each patient based on opportunistic contacts. This is a simple method of collating information that is already known. If such information was available to health authorities it would give them an idea of the type and extent of the support services needed for elderly people.     

Profiles of hypoxanthine guanine phosphoribosyl transferase and adenine phosphoribosyl transferase activities measured in single preimplantation human embryos by high-performance liquid chromatography

The profiles of hypoxanthine guanine phosphoribosyl transferase (HGPRT) and adenine phosphoribosyl transferase (APRT) activities were examined in normally fertilized human embryos developing at the normal rate in vitro between the 2-4-cell stage on Day 2 and the blastocyst stage on Day 6 after insemination. The activities of both enzymes were assayed simultaneously in extracts of single embryos by measuring the rate of production of the reaction products, inosine monophosphate (IMP) and adenine monophosphate (AMP), separated by high-performance liquid chromatography (HPLC). The activity profiles of the two enzymes over this period showed marked differences. The activity of HGPRT, coded by the X chromosome, increased between Days 2 and 4 (P less than 0.01) but declined sharply by Day 6 (P less than 0.001), whereas autosome-coded APRT activity remained low between Days 2 and 5, but increased on Day 6 (P less than 0.05). The profile of HGPRT activity may reflect a combination of decreasing levels of maternal enzyme inherited from the oocyte and the initiation of embryonic gene expression followed by X inactivation at the blastocyst stage on Day 6.