Nuclear Magnetic Resonance of Sodium-23 Linoleate-Water: Basis for an Alternative Interpretation of Sodium-23 Spectra within Cells

The ²³Na spectrum from liquid crystals of sodium linoleate in water has been studied by nuclear magnetic resonance (NMR) techniques. The integrated intensity of the visible central spectral line was 34-39% of the intensity of a reference sample containing an equal quantity and concentration of ²³Na nuclei. Since satellite signals were clearly demonstrable, the effect reflected a nuclear quadrupolar interaction rather than a splitting of the ²³Na into two populations of bound and free nuclei. It is proposed that a similar quadrupolar effect may be the basis for the apparent binding of the ²³Na observed in biological systems.     

Effects of inhibition of basement membrane collagen deposition on rat mammary gland development

DNA biosynthesis by a system containing giant nuclei isolated from rat trophoblast cells at Day 13 of pregnancy has been studied. A method for the isolation of giant nuclei in good yield has been described. These nuclei were capable of incorporating [³H]dTTP into DNA for 2 hr and the incorporation was proportional to the amount of DNA template (nuclei). The system was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg²⁺ and was stimulated by monovalent ions such as K⁺. The optimum pH was 8.6. The product of the reaction was insensitive to RNase, sensitive to DNase, and banded at 1.710 g/ml in neutral CsCl together with bulk rat trophoblast DNA. Pulse-chase and density labeling experiments utilizing bromodeoxyuridine have indicated that replicative, discontinuous synthesis was taking place at sites previously active in vivo. DNA polymerases α, β, and γ were shown to be present in the nuclei. Experiments utilizing selective inhibitors of polymerases have demonstrated that DNA replication by trophoblast nuclei in vitro was insensitive to the specific α-polymerase inhibitor, aphidicolin, but almost completely inhibited by 2′, 3′-dideoxythymidine 5′-triphosphate as well as by N-ethylmaleimide suggesting that DNA replication observed in these trophoblast nuclei in vitro may be carried out by DNA polymerase γ.     

FTIR spectra signatures reveal different cellular effects of EGFR inhibitors on nonsmall cell lung cancer cells

ATP‐analogue inhibitors, Gefitinib (Iressa) and Erlotinib (Tarceva) had been approved for advanced and metastatic nonsmall cell lung cancer (NSCLC) cells against tyrosine kinase domain of epidermal growth factor receptor (EGFR). Many techniques have been developed to better understand the drug mechanism which is multistep, time‐consuming and expensive. Herein, we performed Fourier‐transform infrared (FTIR) microscopy for evaluating the biochemical change on NSCLC (A549) cells after treatment. At levels that produced equivalent effects, Gefitinib dramatically induced cell apoptosis via impaired mitochondrial transmembrane potential. Whereas, Erlotinib had a slight effect on A549. Principal component analysis was performed to distinguish the effect of EGFR inhibitors on A549. FTIR spectra regions were divided into three regions: lipids (3000‐2800 cm^?1), proteins (1700‐1500 cm^?1) and carbohydrates and nuclei acids (1200‐1000 cm^?1). Biochemical changes can be evaluated by these spectral regions. This work may be a novel concept for utilizing FTIR spectroscopy for high‐throughput discriminative effects of a drug or compound and its derivatives on cells.     

Deoxyribonucleic acid synthesis in isolated polytene nuclei of Drosophila hydei

DNA synthesis has been studied in polytene nuclei isolated from larval salivary glands of Drosophila hydei. The incubation conditions employed promote maximum incorporation of TTP-H³ and retention of normal polytene chromosome morphology. The chromosome structure is sensitive to the Mg²⁺ concentration; a normal banding pattern is observed between 4 and 10 mM Mg²⁺. At the optimum pH of 7.8, incorporation continues for over an hour. All four deoxyribonucleoside triphosphates are required for maximum incorporation. The reaction is stimulated by 0.6 mmATP and strongly inhibited at higher ATP concentrations. Competition experiments demonstrate that either TDP or TTP is the effective labeled precursor. The labeled product is sensitive to DNase and has a density identical to that of nuclear DNA. Autoradiographs prepared from spread chromosomes demonstrate that discontinuous and continuous labeling patterns observed in vivo are also produced with isolated nuclei in the absence of cytoplasmic factors. Incubation of the isolated nuclei results in a low level of uniform incorporation that is superimposed on the normal autoradiographic pattern obtained after in vivo labeling. This background incorporation can be greatly increased by prior irradiation of the glands. The presence of exogenous DNA during nuclear incubation stimulates total incorporation. These observations demonstrate that the isolated nuclei possess a reserve synthetic capacity. About 20% of the isolated nuclei are inactive in DNA synthesis.This investigation was supported by PHS Research Grant No. 5 R01 GM 16298 from the National Institute of General Medical Sciences.     

Nucleo-cytoplasmic exchange of non-histone proteins in amphibian embryos

As a basis for understanding the role of non-histone proteins in nuclear differentiation, we have identified one period during embryogenesis when intense accumulation of non-histones occurs in nuclei of Rana pipiens. We then demonstrated, experimentally, the loss of non-histones from nuclei after transplantation into enucleated eggs. ³H-tryptophan or ³H-lysine was injected into blastocoeles of mid-blastulae and into archenterons of late gastrulae; embryos were subsequently studied autoradiographically. Nuclei of animal hemisphere cells from blastulae accumulated only small amounts of ³H-tryptophan within 3 h, whereas a large accumulation occurred in endodermal nuclei of gastrulae as early as 1 h, and after 3 h 95.9% ( ) of the nuclei were densely labelled. Significant accumulation of ³H-lysine occurred in the majority of nuclei of both types within 3 h (blastulae ; gastrulae ). Controls, involving RNase or boiling TCA, demonstrated that the ³H-amino acids have been incorporated mainly into proteins. Endodermal nuclei labelled either with ³H-tryptophan or ³H-lysine after a 3 h incubation were transplanted singly into enucleated eggs. Autoradiograms demonstrated that most non-histones leave the nucleus during its reprogramming in the egg cytoplasm prior to first cleavage; whereas other types of proteins labelled with ³H-lysine remain for the most part in the nucleus. Cytochemical studies indicated that some of the non-histones which leave transplanted nuclei are acidic proteins; whereas some of those proteins which remain in the nucleus are histones.In addition to the above findings, the results of these studies demonstrate the feasibility in the future of studying the nucleocytoplasmic migration of different kinds of macromolecules in a developmental metazoan system and determining their roles in the establishment of nuclear differentiation.     

The presence of F3-F2a1 dimers and F1 oligomers in chromatin.

The oligomeric structure of histones in nuclei and chromatin has been studied by crosslinking nuclei and chromatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Crosslinked histones were detected as new high molecular weight components on SDS gels, and the protomers of the crosslinked histones were identified by their characteristic ¹²⁵I-fingerprints. The results show that a considerable portion of histones F3 and F2a1 exist in nuclei and chromatin as an F3-F2a1 dimer. Evidence is presented that histone F1 probably exists in chromatin as large oligomers.     

INCORPORATION OF TRITIATED CYTIDINE INTO RIBONUCLEIC ACID BY ISOLATED PEA NUCLEI

A new method for the isolation of the nuclei from plant tissue has been devised. This method consists in passing the plant tissue through a set of spring-loaded, counter-rotating rollers and collecting the liberated nuclei in sucrose solution containing calcium ions. A semi-automatic machine which couples the rollers with a special tissue chopping device has been constructed. It has been shown that nuclei obtained in this way actively incorporate cytidine-H³ into RNA. This incorporation is increased in the presence of nucleoside triphosphates and an energy-regenerating system.     

INCORPORATION OF TRITIATED THYMIDINE IN THE CELLS OF CAENORHABDITIS BRIGGSAE (NEMATODA) REARED IN AXENIC CULTURE

In the rhabditid nematode Caenorhabditis briggsae the incorporation of thymidine-H³ has been studied by autoradiography after Feulgen staining, with animals maintained under axenic conditions in a medium of only partly defined composition. Labeling has been followed in adults left in the presence of thymidine-H³ for periods of from ½ to 24 hours, as well as in adults reared from larvae in the presence of the tritiated nucleoside. A massive incorporation is found in the nuclei of the gonads and intestine; also a less intense particulate cytoplasmic incorporation is clear in certain cells, especially those of the intestine. In general, all labeling has proved to be sensitive to DNase, but resistant to RNase. The label's stability has been tested by the transfer of adults into a medium containing "cold" thymidine. They remain there for up to 48 hours. A transfer for 24 hours results in a considerable decrease in the intensity of nuclear and cytoplasmic labeling; a stay of 48 hours leads to its complete disappearance from non-dividing (intestinal) as well as dividing (gonadal) nuclei. A phenomenon of DNA turnover is envisaged and discussed as a possible physiological attribute of C. briggsae.     

The effect of sodium butyrate on acetylation in vitro of chromosomal proteins in three classes of liver nuclei from different ages of rats

The optimum conditions of in vitro incorporation of sodium [³H]acetate into sliced rat liver were studied. The incubations with sliced liver from three different ages of rats were performed in the presence of sodium n-butyrate. It was found that butyrate decreases the incorporation of sodium [³H]acetate into the homogenate, isolated nuclei, non-histone chromosomal proteins and histones for all age groups. The acetylations of non-histone chromosomal proteins and histones increase with age upto 2-months and decrease in 4-month-old rats both in the absence and presence of butyrate. Liver nuclei were fractionated by the simple method of zonal centrifugation into three classes, namely diploid stromal, diploid parenchymal and tetraploid parenchymal nuclei. The acetylations of non-histone chromosomal proteins and histones in three classes of nuclei of three ages of rats were studied in the presence and absence of butyrate. Butyrate can decrease the overall acetylations of non-histone chromosomal proteins and histones but increase the amount of polyacetylated histone H4 in all classes of nuclei of the three ages.     

Inhibition of DNA synthesis by isolated liver nuclei from frog virus 3 infected mice

Incorporation of [³H] dTTP into acid insoluble material by liver nuclei from mice infected for 3 hours with 5 × 10⁸ PFU of Frog Virus 3 was markedly decreased. The incorporation was noticeably stimulated by the addition of calf thymus DNA. It has been verified that the inhibition of incorporation by the nuclei from infected animals was not related to an increase of deoxyribonuclease activity and that the product of the reaction showed the properties of DNA.     

Autoradiographs of Pollen Tube Nuclei with Calcium-45

Autoradiography with Ca⁴⁵ has been used to obtain information about the relation between calcium and chromosomes. Labelled pollen from the Easter lily, Lilium longiflorum, was allowed to develop into pollen tubes between 5 and 6 cm. long in the styles of non-radioactive flowers. All of the nuclei, namely the tube nucleus and the two sperm nuclei, retain Ca⁴⁵ after this period of growth and development. Since the two sperm nuclei have formed during this interval by the mitotic division of the generative nucleus and growth of the tube has occurred under the influence of the tube nucleus, it is inferred that the calcium was bound in a stable nuclear component, the chromosomes.     

Long-lasting effects of electroconvulsive shock on the pattern of poly(A)-RNA synthesis in rabbit cerebral cortex

The pattern of poly(A)-associated [poly(A)⁺] RNA synthesis was studied in rabbit cerebral cortex in the period following a single electroconvulsive shock (ECS). Labeled uridine was injected into the brain 2 and 4 hr after ECS and the animals sacrificed 1 hr later. Total and poly(A)⁺ RNA were then prepared from cortical nuclei and microsomes and analyzed. The amounts of newly synthesized total and poly(A)⁺ RNA in nuclei and microsomes appeared to be close to the control. However, the pattern of newly synthesized poly(A)⁺ nuclear RNA appeared to be still displaced toward the high molecular weights as it was in the early post-ECS period. The result indicates a long-lasting disturbance of brain poly(A)⁺-RNA metabolism by ECS.     

Stable nuclear RNA returns to post-division nuclei following release to the cytoplasm during mitosis

When nuclei from ³H-RNA-containing amebae (A. proteus), chased for as many as 8 cell generations, are implanted into unlabeled enucleate cells, the nuclei retain 30% or more of the cellular ³H-RNA (or at least 15 times the cytoplasmic concentration of ³H-RNA). After such cells divide, the daughter nuclei retain approximately the same proportion of total cellular ³H-RNA—although all (or almost all) of the nuclear RNA is liberated to the cytoplasm during mitosis. Thus, we conclude that RNA stably associated with the interphase nucleus has a particular affinity for the nucleus despite the fact it is in the cytoplasm when the chromosomes are condensed and the nuclear envelope is not intact.     

THE EFFECT OF ELECTROCONVULSIVE SHOCK ON PROTEIN SYNTHESIS IN MOUSE BRAIN

The effect of a single electroconvulsive shock on protein synthesis in mouse brain cortex was studied by observing the incorporation into protein of intraperitoneally injected [³H]- or [¹⁴C]leucine. When the precursor was injected immediately after the electroshock there was a 50 per cent inhibition of the incorporation which was not seen with injections at times later than 10 min. To investigate a possible specificity, the cerebral cortices of experimental and sham control animals which had been injected with different isotopes were homogenized together and fractionated by differential centrifugation. Cell fractions were then separately extracted with phosphate buffer and with Triton X-100. The ratio of ³H to ¹⁴C in each fraction was compared with that of the total homogenate to reveal any specific effects due to the electroconvulsive shock. The treatment produced a slight inhibition of the incorporation of the isotope into the heavier particulate fractions (i.e. nuclei, mitochondria, synaptosomes) relative to that in the microsome and cell sap fractions. A possible explanation of these results is given with a discussion of the limitations of the technique.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Synchrony of Nuclear Replication in Individual Hyphae of Aspergillus nidulans

The synchrony of nuclear replication in individual, multinucleate hyphae of Aspergillus nidulans has been investigated. Samples were taken from cultures of germinating conidiospores, and the relative frequency of hyphae containing two to eight nuclei was determined. Because the conidiospores are mononucleate, complete synchrony will yield populations of hyphae containing only 2ⁿ nuclei, n being the number of doublings after germination. The appearance of hyphae with total numbers of nuclei other than 2ⁿ will indicate lack of synchrony. The relative frequency of hyphae not having 2ⁿ nuclei will depend on the degree of synchrony in the individual hyphae; numerical aspects of this relation are discussed. In two different strains, replication of the nuclei in any one hypha was highly synchronized when the dry weight doubling time was 1.4 to 1.8 hr. As the doubling time was made longer by changing the nitrogen or carbon source, synchrony was progressively lost. At the slowest growth rate tested, the interval between the division of the fastest and the slowest nucleus equaled 48% of the dry weight doubling time. The active replication of some nuclei in a hypha where other nuclei were resting suggested that nuclear duplication in this eukaryotic organism may be controlled by specific initiators.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

Role of nuclear proteins on [3H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Nuclear DNA in normal and refed Amoeba proteus

Amoeba proteus synthesizes DNA in G2 phase of the cell cycle upon feeding after starvation. The characteristics of the DNA synthesized in G2 have been studied by microscope photometry of individual Feulgen-stained nuclei and by buoyant density centrifugation of nuclear DNA in CsCl. Amoeba nuclei were found to contain 42.8 pg of DNA. This DNA bands in CsCl at a density of 1.693 g/cm³ with a satellite at 1.714 g/cm³ which makes up 24% of nuclear DNA. DNA from whole cells has an additional non-nuclear satellite at 1.726 g/cm³. When cells are starved and re-fed with food labeled with [³H]thymidine, the DNA synthesized is predominantly the 1.714 satellite. The amount of DNA synthesized in G2 is small since there is no measurable difference in Feulgen dye binding to nuclei of starved vs starved and re-fed cells. The data suggest that refeeding induces a resumption of late S phase DNA synthesis, or the preferential synthesis of specific DNA sequences such as rRNA genes.