Molecular differentiation of the rabbit ovum. I. During oocyte maturation in vivo and in vitro

The normality of nuclear and cytoplasmic maturation of rabbit oocytes, matured in vivo and in vitro, has been assessed by cytogenetic and electrophoretic criteria. The findings indicate not only that nuclear maturation in vivo and in vitro are directly comparable, but also, as observed by high-resolution, two-dimensional polyacrylamide gel electrophoresis, (1) that both qualitative and quantitative changes in the pattern of polypeptide synthesis occur during maturation, (2) that these patterns are directly comparable in oocytes that had been matured either in vivo or in vitro, and (3) that each stage of maturation is associated with the appearance of specific polypeptides in the autoradiographic patterns. The major differences observed between oocytes matured under these two conditions are (1) that several polypeptides fail to appear in in vitro matured oocytes at the time they are detected in vivo and (2) that the synthesis of some polypeptides is prolonged in vitro compared to in vivo matured oocytes.     

Variations in the amounts of RNA polymerase forms I, II and III during preimplantation development in the mouse.

DNA-dependent RNA polymerase has been measured at various stages of preimplantation development in mouse embryos. The total RNA polymerase activity per embryo increases rapidly from the 8-cell stage to the blastocyst stage. Studies with low α-amanitin concentrations, which inhibit form II RNA polymerase, and high α-amanitin concentrations, which inhibit both form II and III RNA polymerases indicate that the relative proportions of the three forms change significantly during preimplantation development. The changes which occur in the types and levels of RNA polymerase appear to parallel corresponding changes in the synthesis of the major classes of RNA.     

Ouabain binding to preimplantation rabbit blastocysts

Ciliary ganglia (CG) from 8-day-old chick embryos were cultured as explants on a highly adhesive collagen substratum in the presence of the ciliary neuronotrophic factor (CNTF). A remarkable correlation was found between the formation of an outgrowth of ganglionic nonneuronal cells and the timing and extent of neuritic development outside the ganglion. Neurites were not seen to emerge from the ganglion before the onset (24 hr after explantation) of a nonneuronal cell outgrowth. After nonneurons began to migrate over the collagen substratum, neurites could be seen to extend up to, but not beyond the distal limit of the nonneuronal outgrowth. Time-lapse analysis showed that neuritic growth cones could move in synchrony with a nonneuron with which they were in contact as well as over the nonneuronal cell surface, but not on the collagen located distally to the external edge of the nonneuronal outgrowth.Freshly dissected CGs were also grown as secondary explants on preformed host monolayers of ganglionic nonneurons. These secondary explants showed considerable neuritic development within 24 hr, while control ganglia explanted on collagen had not produced neurites. Autoradiographic experiments indicated that this neuritic outgrowth occurred on nonneuronal cells emerging precociously from the secondary explant, rather than on the preexisting host nonneurons. Electron microscopy of 24-hr explants demonstrated that, inside the ganglion, neurites were also very closely associated with the surface of nonneuronal cells.Neuritic behavior in this nonneuron/collagen terrain is compared with previously described observations of CG explants on polyornithine (PORN) or dissociated CG neurons on PORN or collagen. These observations led to the identification of a PORN-bindable neurite promoting factor (PNPF) which does not bind to, and is not active on, collagen. The hypothesis is discussed that PNPF molecules are present on the surface of nonneuronal cells and that the cells owe to those molecules their competence as a suitable terrain for the elongation of neuritic processes.     

Studies on bacterial cell wall inhibitors: II. Inhibition of peptidoglycan synthesis in vivo and in vitro by amphomycin

Amphomycin has been reported by the present authors to be a selective inhibitor of cell wall peptidoglycan synthesis in Bacillus cereus T (ōmura, S., Tanaka, H., Shinohara, M., ōiwa, R. and Hata, T. (1975) Chemotherapy 5, 365–369). Investigations were carried out to clarify the target of amphomycin.Amphomycin (10 μg/ml) lysed growing cells of B. cereus T, and inhibited peptidoglycan synthesis, accompanied by accumulation of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc) peptides. The nucleotide precursors that accumulated in cells of Staphylococcus aureus FDA 209P in the presence of amphomycin were identified as UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala, UDP-MurNAc-L-Ala and UDP-MurNAc. In the experiments using a particulate enzyme system of Bacillus megaterium KM, amphomycin inhibited the polymerization of UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) and UDP-N-acetylglucosamine, and also inhibited the formation of lipid intermediates, but did not inhibit the cross-linking, the last step of peptidoglycan synthesis. Unlike bacitracin, amphomycin did not lyse protoplasts of B. megaterium KM.We conclude that the site of action of amphomycin is the formation of MurNAc-(pentapeptide)-P-P-lipid from MurNAc-pentapeptide and undecaprenol (lipid) phosphate.     

Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes

An extensive array of cortical microtubules in oocytes of the starfish Pisaster ochraceus undergoes multiple cycles of disappearance and reappearance during maturation and early development. These events were studied in isolated fragments of the oocyte cortex stained with antitubulin antibodies for indirect immunofluorescence. The meshwork of long microtubules is present in the cortex (a) of immature oocytes, i.e., before treatment with the maturation-inducing hormone 1-methyladenine, (b) for 10-20 min after treatment with 1-methyladenine, (c) after formation of the second polar body (in reduced numbers in unfertilized oocytes), and (d) in the intermitotic period between first and second cleavage divisions. The array of cortical microtubules is absent in oocytes (a) undergoing germinal vesicle breakdown, (b) during the two meiotic divisions (polar body divisions), and (c) during mitosis of the first and, perhaps, subsequent cleavage divisions. The cycle of assembly-disassembly of cortical microtubules is synchronized to the cycle of nuclear envelope breakdown and reformation and to the mitotic cycle; specifically, cortical microtubules are present when a nucleus is intact (germinal vesicle, female pronucleus, zygote nucleus, blastomere nucleus) and are absent whenever a meiotic or mitotic spindle is present. These findings are discussed in terms of microtubule organizing centers in eggs, possible triggers for microtubule assembly and disassembly, the eccentric location of the germinal vesicle, and the regulation of oocyte maturation and cell division.     

Precardiac mesoderm differentiation in vitro

The differentiation of precardiac mesoderm into beating heart tissue was examined during explant culture. Explanted tissue forms tubular heart-like vesicles and initiates rhythmic contractility within 18-24 h in vitro, a developmental time-course approximating that observed during in vivo development. Electron-microscopic observations reveal that beating heart cells are rich in cytoplasmic myofibrils in varying degrees of order, with some regions containing highly organized myofibrillar arrays. The analysis of actin-isotype biosynthesis, using metabolic labeling with [35S]-methionine and isoelectric-focusing resolution of the synthesized radioactive polypeptides, demonstrates that the initiation of cardiac alpha-actin synthesis and the pattern of transition in the synthesis of alpha-, beta-, and gamma-actin isotypes is equivalent to the initiation time and pattern observed in vivo. A possible collagen involvement in the differentiation process was investigated by assessing the effects of collagen-synthesis inhibitors on the development of the explant cultures. Two different agents, alpha, alpha'-dipyridyl and L-azetidine-2-carboxylic acid, exhibited a dose-dependent ability to inhibit the formation of beating heart tissue. When examined by electron microscopy, the nonbeating tissue exhibited a drastic depression of myofibrillogenesis, but otherwise appeared healthy. Further examination of the effect of L-azetidine-2-carboxylic acid demonstrated that the inhibition of myofibril formation and heartbeat was correlated with a 60% inhibition of native collagen synthesis; however, the time-course and pattern of actin-isotype biosynthesis was completely unaffected. The data suggest a possible involvement in heart differentiation that is necessary for either the synthesis of non-actin cardiac contractile proteins or the assembly of cardiac contractile proteins into myofibrils.     

Eukaryotic ribosomal proteins. Molecular weights of proteins from rabbit liver subunits.

The molecular weights of the proteins from rabbit liver ribosomal 40 S and 60 S subunits were determined after preliminary separation of these proteins by two-dimensional electrophoresis: each spot present in the polyacrylamide slab was cut off, eluted and rerun in a SDS one-dimensional polyacrylamide gel. The molecular weights range from 9,000 to 35,000 with a number-average molecular weight of 19,600 for the 40 S proteins, and from 9,400 to 52,000 with a number-average molecular weight of 23,600 for 60 S proteins.     

Resistance and susceptibility to infection in inbred murine strains. II. Variations in the effect of treatment with thymosin fraction 5 on the release of lymphokines in vivo

Of nine inbred murine strains sensitized intravenously with killed lyophilized Candida albicans and challenged 3 weeks later with a C. albicans filtrate, four strains were low responders and five were high responders in the in vivo release of migration inhibitory factor (MIF) and gamma interferon (IFN-gamma). An identical distribution of high- and low-responder strains occurred in response to sensitization with Mycobacterium bovis BCG and subsequent challenge with old tuberculin. Treatment of the murine strains with thymosin fraction 5 prior to sensitization and challenge had different effects: (a) the high-responder strains had a decrease in their release in vivo of the two lymphokines; (b) three of five of the low-responder strains had a striking increase in the in vivo release of MIF and IFN-gamma; and (c) one low-responder strain did not have its response altered. A parallelism existed between the capacity of a murine strain to release the two lymphokines in vivo on stimulation with C. albicans antigens and the capacity of that strain to resist intravenous infection with living C. albicans.     

Molecular basis of negative co-operativity in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

The interactions of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with NAD⁺ and with its fluorescent derivative 1, N⁶-etheno-adenine dinucleotide were investigated using a variety of spectroscopic methods. These techniques included: difference spectroscopy, circular dichroism, fluorescence and circular polarized luminescence. It was found that the greatest structural change in the protein tetramer occurs upon binding of the first mole of coenzyme. We have also demonstrated that progressive structural changes occur at the adenine subsite in the NAD⁺ binding site as a function of coenzyme saturation. These conformational changes are probably responsible for the progressive decrease in the affinity towards the coenzyme. It was also found that every NAD⁺ molecule induces the same conformational change of the nicotinamide subsite. These results offer a molecular explanation for the negative co-operativity in the binding of the coenzyme, without a change in the catalytic power of the NAD⁺ site as a function of coenzyme saturation. These results also offer a new explanation for the fact that enzyme exhibits half-of-the-sites reactivity towards certain ligands and full-site reactivity towards others. It is suggested that those ligands interacting at the adenine subsite of the NAD⁺ binding site induce the half-of-the-sites reactivity.Our results support the view that both the negative co-operativity in coenzyme binding and half-of-the-sites reactivity are due to ligand-induced conformational changes on an a priori symmetric glyceraldehyde-3-phosphate dehydrogenase molecule.     

Identification of beta-lymphotoxin as the predominant molecular class of in vitro and in vivo Syrian hamster lymphotoxin

The molecular class of Golden Syrian hamster lymphotoxin produced in vitro and in vivo was determined by size-exclusion high-performance liquid chromatography using silica-based protein separation columns eluted with a 0.1 M sodium phosphate, pH 7.4 buffer containing 0.1% Mr 4000 polyethylene glycol. Lymphotoxin cytolytic activity was quantitated in the column effluent by measuring the ability of the fractions to lyse alpha-L929 cells as indicated by [3H]TdR release. Lymphotoxin activity induced by an 8- or 24-hr or 5-day phytohemagglutinin stimulation of peritoneal leukocytes, by 24-hr phytohemagglutinin-coated alpha-L929-cell stimulation of peritoneal leukocytes, or by 24-hr phytohemagglutinin stimulation of spleen cells occurred in the Mr range of 20,000-56,000, with major components in the 35,000-50,000 beta-lymphotoxin region. No activity was present in the complex (greater than 200,000) region and only minimal activity was detectable in the alpha (70,000-160,000) and gamma (12,000-20,000) regions. In vivo-induced lymphotoxin, obtained by peritoneal lavage 48 hr after intraperitoneal administration of phytohemagglutinin, was entirely beta-lymphotoxin and was not detectable in the plasma. Lymphotoxin produced in vitro and injected simultaneously with the gamma-emitting radionuclide 99mtechnetium, inhibited in vivo development of radiation-induced transplacental carcinogenesis. Thus, Syrian hamster lymphotoxin with antitumor activity consists of glycoproteins with isoelectric points of 4.8-5.2, Mr of 20,000-56,000, and major in vitro and in vivo forms in the beta-lymphotoxin range.     

Three-dimensional hair follicular differentiation of a trichilemmoma cell line in vitro

A cell line was established in vitro from a benign hair follicular tumor of human trichilemmoma. Individual and organized cellular differentiation of this cell line was studied. When these cells were cultured for a long time (more than 3 weeks) without subculture, they started to pile up spontaneously. A part of the pile became indented and simultaneously the opposite side of the indentation budded out. The bud slowly elongated 2 to 3 mm in length in 8 to 12 weeks in culture. Light and electron microscopy revealed the internal structure of piles and elongated buds to be a three-dimensional hair follicular structure. The cells in the outermost layer were least mature. These were cuboid in shape and contained glycogen. The cells in the middle layer were more differentiated with a decreased amount of glycogen and an increased number of tonofilaments and desmosomes. The cells in the innermost layer were most differentiated. Cells were flat in shape and highly convoluted. The cell membrane was thickened as observed in cornified cells in vivo. These organized differentiations were also confirmed by histochemical and immunocytochemical studies; using a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide method, free sulfhydryl groups were detected but disulfide bonds were absent in the early cell culture. Disulfide bonds increased slowly and accumulated in the innermost layer of piles. Accumulation of keratin substances, detected by indirect immunofluorescence method using anti-human keratin antibody, was also observed specifically in the piles. These results suggest that an established cell line of human trichilemmoma spontaneously produced, without stromal influence, hair follicular structures as well as individual cell differentiations in vitro as do trichilemmal (hair follicular) cells in vivo.     

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1⁺ Ly-1⁺2⁺ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia⁺ B lymphocytes were not required, and Ia⁺ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1⁺2⁺ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.     

The induction of forward gene mutation and gene conversion in yeasts by treatment with cyclophosphamide in vitro and in vivo

The present paper assesses the most suitable conditions for metabolic activation with yeasts in vitro, at least as far as cyclophosphamide (Cy) is concerned. These include treatment time, incubation temperature, the amounts of S9 and cofactors. Particular attention is devoted to the use of various solvents, showing that their use can considerably affect the mutagenic response of the chemical being tested. It also examines the effects of enzyme inducers (by using S9 from rats and mice) such as phenobarbital (PB) and 5,6-benzoflavone (BF) administered separately or together. The metabolizing capability of other organs such as the lungs and kidneys is also determined. All these data are compared with Cy genotoxicity (in vivo) evaluated by the intrasanguineous host-mediated assay and by recovering the yeast target cells from the liver, lungs and kidneys. The most striking effects are that, in vitro, PB greatly enhances Cy genotoxicity, whilst in vivo it substantially reduces it.     

Cyclic nucleotide phosphodiesterase and protein activator in fetal rabbit tissues.

Cyclic nucleotide phosphodiesterase activity (EC 3.1.4.17) was studied in fetal and newborn rabbit brain, heart, liver, kidney, and lung. Kinetic analysis of phosphodiesterase activity from homogenates of organs from the 25-day embryo suggested the presence of a high K_m and a low K_m activity for both cyclic AMP and cyclic GMP hydrolysis. The addition of 1 μm cyclic GMP to the assay stimulated the hydrolysis of cyclic AMP by whole homogenates of liver, brain, lung, and kidney, but not heart, at all of the ages studied. The addition of micromolar levels of calcium ion stimulated cyclic GMP hydrolysis by homogenates of fetal brain, heart, and kidney, with or without added protein activator. Cyclic GMP phosphodiesterase activity was not stimulated by the addition of calcium ion in homogenates of early fetal rabbit liver and lung, but stimulation was detected in the late embryo and newborn. The presence of the heat-stable protein activator was demonstrated in brain, heart, kidney, liver, and lung tissue at all of the fetal ages studied, and in the newborn rabbit. DEAE-cellulose chromatography demonstrated the presence of three separable enzymes in brain and liver at 15 days, heart at 19 days, and lung and kidney at 25 days of gestation, with no changes in the kinetic properties of the isolated enzymes during development. These experiments suggest that all of the organs studied have the mature array of phosphodiesterases early in development, but an enzyme from liver and lung becomes sensitive to regulatory control by calcium only late in gestation.     

Collagen fibrillogenesis in vitro: comparison of types I, II, and III

The self-assembly of pepsin-extracted types I, II, and III collagen was studied to determine how differences in the triple-helical structure between collagen types influence in vitro collagen fibrillogenesis. Collagen types I, II, and III were extracted and purified from bovine sources, and were studied in solution by laser light scattering, pH titration, and determination of turbidity-time curves. The molecular weights were between 280,000 and 289,000, while the translational diffusion coefficients and particle scattering factors at 175.5 degrees were consistent with those expected for single collagen molecules. Titration of collagen types I, II, and III between pH 7.0 and 2.0 using HCl indicated that type I collagen had the most titratable carboxylic groups with type II and III having significantly fewer titratable groups. The self-assembly of these collagens was studied in vitro in phosphate-buffered saline. The time course and extent of fibril formation were studied turbidimetrically, and were found to be dependent on collagen type. Apparent rate constants were determined for both the lag and growth phases of fibril formation. The rates of both phases were greater for type III than for type I collagen, with the rates for type II collagen being intermediate. The extent of fibril formation was based on the turbidity per unit concentration (specific turbidity) extrapolated to zero concentration (intrinsic turbidity), which was found to be greater for type I than for type III collagen. Type II collagen had the smallest intrinsic turbidity. The specific and intrinsic turbidity values were consistent with the relative fibril diameters seen in dermis and cartilage by transmission electron microscopy. These observations indicate that helix-helix interactions are important in the regulation of the rate and extent of collagen fibrillogenesis and may be involved in the determination of fibril structure.     

The in vitro cellular synthesis of hemoglobin components in various media and its relevance to the concept of stenogenic and eurygenic states of differentiation.

The increase above normal in the rate of synthesis of human fetal hemoglobin in incubated cells from adults, as observed previously, is only relative. The addition of serum to the incubation medium tends to reestablish the normal ratio of rates. In the absence of serum the decrease in rates of synthesis affects most strongly the hemoglobin component, whichever it be, that is synthesized more intensively at the cell stage under consideration. This is brought out by the comparison of the effects of different media on fetal and adult cells. The data lead to the definition of the concept of stenogenic and eurygenic states of differentiation.     

RNA transcription and translation in sea urchin oocytes and eggs

The steady-state concentrations and absolute rates of synthesis of ribosomal RNA (rRNA) molecules were measured in oocytes, eggs, embryos, and larvae of the Hawaiian sea urchin Tripneustes gratilla. The steady-state concentration per genome of the RNA precursor sequences measured by hybridization to a cloned rDNA fragment was approximately 100- to 300-fold greater in the RNA obtained from oocytes and eggs than in the RNA extracted from embryos and larvae. Since the rate of processing of the rRNA precursor at different stages is not greatly different, the rates of rRNA synthesis must be considerably greater in oocytes than in embryo cells. The absolute rate of RNA synthesis in oocytes and embryos was determined from the incorporation of [³H]guanosine into cellular GTP pools and into both precursor and mature rRNA species. The data indicate an approximately 40-fold higher rate of rRNA synthesis in oocytes than that measured in embryos or previously in larvae (J. Griffith and T. Humphreys, 1979, Biochemistry18, 2178–2185). Together these results indicate that the ribosomal genes are transcribed much more rapidly during sea urchin oogenesis than during embryogenesis or larval stages.     

In vitro synthesis of simian virus 40 DNA. II. Evidence for a repair mechanism.

The technique of density labeling of DNA by BrdU was used to characterize the material synthesized in vitro by cytoplasmic extracts of SV40 infected cells incubated in the presence of simian virus 40 (SV40) DNA component I molecules (Girard et al, Biochimie, this volume). In a first experiment, the template was labeled beforehand in vivo using [14C]-BrdU, and the in vitro incubation was carried out in the presence of [3H]-dGTP and [3H]-dTTP. In a second experiment, the template was labeled in vivo with 32P, and the in vitro incubation was in the presence of [3H]-dGTP and BrdUTP. After digestion with the restriction endonuclease Hind II + III, the fragments from the end products of the reaction were analyzed by density gradient centrifugation, at pH 7 and pH 13. In both experiments the DNA product molecules had the same density as the resepctive DNA templates. Cellular enzymes seem to be responsible for this in vitro synthesis of DNA, since cytoplasmic extracts from uninfected cells were almost as active as those from SV40 infected cells. The system was proved efficient in the conversion of "open circular" molecules (component II DNA molecules) to covalently closed circular DNA molecules (relaxed component I molecules). The use of DNA complexed with histones did not impart viral specificity to the system. It is concluded that the cytoplasmic extract is only capable of supporting the repair synthesis of added viral DNA.