The effects of chlorphentermine and phentermine on certain metabolic parameters associated with development of rat kidney and liver.

Daily oral administration of the anorexigenic agents chlorphentermine or phentermine (60 mg/kg) to rats for either 1, 3, 5 or 7 days resulted in a significant fall in the incorporation of [¹⁴C]thymidine into renal and hepatic DNA throughout the course of the experiment. Although 24 h after treatment with either drug there was no dramatic change in the incorporation of [¹⁴C]orotic acid into liver RNA, a statistically significant reduction was noted after 3, 5 and 7 days. In rat kidney, the incorporation of [¹⁴C]orotic acid into RNA was only significantly depressed by chlorphentermine at 5 days and by phentermine at 3 days. In general, treatment with either anorexigenic agent tended to significantly lower or not affect the endogenous concentrations of renal and hepatic putrescine, spermidine and spermine. The chlorphentermine-induced decrease in liver and kidney nucleic acid synthesis was accompanied by depression in the levels of cyclic AMP in both tissues as well as a reduction in the activity of adenylate cyclase in renal tissue. In contrast, chlorphentermine produced a rise in hepatic adenylate cyclase at 5 days followed by a return to control values after 7 days. The phentermine-induced alterations in nucleic acid metabolism appeared generally to occur independent of any changes in the adenylate cyclase-cyclic AMP system of renal and hepatic tissues. In view of the fact that nucleic acids, polyamines and cyclic AMP constitute integral components of the growth process, our data suggest that chlorphentermine and phentermine interfere with certain biochemical parameters associated with the development of kidney and liver.     

The time and duration of the premeiotic inteprhase in microsporocytes ofTrillium kamtschaticum

The time and duration of each phase of the premeiotic interphase were determined in microsporocytes of two clones (S and K clones) ofTrillium kamtschaticum. After collectionTrillium plants were stored at 3 C or 7 C prior to completion of premeiotic mitosis in archesporial cells. For autoradiography, cells were explanted in the presence of³H-thymidine to identify the interval of the premeiotic DNA synthesis. Approximate durations of the G₁, S and G₂ phases for the K clone stored at 3 C were estimated to be 12, 12 and 14 days, respectively. The interval of premeiotic development was markedly different between clones. A high degree of synchrony in meiotic development, which is usually observed within anthers up to late meiotic prophase, was confirmed at the S phase, suggesting that synchrony is established during the G₁ interval.     

Distribution of cellulosic wall in the anthers of Arabidopsis during microsporogenesis

Key message

Cellulose-specific staining revealed that tapetal cells and microsporocytes lose cellulosic walls before the onset of meiosis. Cellulosic wall degradation in microsporocytes might be independent of tapetal cells (or TPD1).

Abstract

Some cell types in a variety of angiosperms have been reported to lack cell walls. Here, we report that the tapetal cells of the anther of Arabidopsis thaliana did not appear to have a cellulosic wall based on staining with Calcofluor and Renaissance 2200. During sporogenous cell formation, cellulosic wall was present in all anther tissues. However, before meiosis it was almost absent on the tapetal cells and on the microsporocytes. In a sporocyteless/nozzle (spl/nzz) mutant, which lacks several components (microsporocytes, tapetum, middle layer and endothecium), cellulosic wall was detected in all anther cells. In another mutant, tapetum determinant1 (tpd1), which lacks tapetum and has more microsporocytes, cellulosic wall was almost absent on the microsporocytes before meiosis, similar to the wild type. These results suggest that the tapetum cells and microsporocytes lose cellulosic walls during microsporocyte formation, and that cell wall degradation occurs downstream of SPL/NZZ and is independent of TPD1.     

Nutrient media for plant-parasitic nematodes. VI. Nucleic acids content and nucleotide synthesis in Aphelenchoides rutgersi

The nucleic acids content of Aphelenchoides rutgersi, Hooper and Myers, was 0.9% DNA and 2.6% RNA dry weight. The DNA contained 29.5% adenine, 29.3% thymine, 22.5% guanine, and 18.8% cytosine, while the RNA was composed of 22.8% adenine, 23.0% uracil, 31.4% guanine, and 22.9% cytosine on a molar basis.The nematodes needed folic acid for reproduction regardless of the presence or absence of nucleic acid supplements in the culture medium. This was shown by including aminopterin, a folic acid antagonist in the culture medium. A 2-hr incubation of nematodes with glycine-¹⁴C (U) and orotic-5-³H acid resulted in the incorporation of ³H-label into both DNA and RNA. Only the RNA fraction contained a significant amount of ¹⁴C-label. When this RNA was fractionated, the adenine and guanine accounted for the ¹⁴C-label, while cytidylic and uridylic acids contained the ³H-label, thereby demonstrating purine and pyrimidine synthesis by A. rutgersi. The incorporation of orotic acid into the pyrimidines was 8 times higher than that of glycine into purines.     

A transient accumulation of poly(A)-containing RNA in the tapetum of Hyoscyamus niger during microsporogenesis

The distribution of poly(A)-containing RNA in the tapetal cells of Hyoscyamus niger during microsporogenesis was followed by in situ hybridization with [³H]poly(U) as a probe. Although no poly(A)-containing RNA accumulated in the premeiotic tapetum, [³H]poly(U) binding sites were detected in the tapetum as meiosis was completed in the microsporocytes. Accumulation of poly(A)-containing RNA in the tapetal cells reached a peak before the first haploid mitosis in the pollen grains. With the onset of tapetal senescence at the late uninucleate stage of the pollen grain, [³H]poly(U) binding sites gradually decreased and they completely disappeared in the tapetum at the binucleate pollen stage. The significance of the results is discussed, particularly with regard to the possible role of tapetum in the synthesis of informational macromolecules during microsporogenesis.     

DNA and RNA Syntheses by Intraerythrocytic Stages of Plasmodium knowlesi*

Incorporation of the nucleic acid precursors, orotic acid, adenosine, thymidine, and uridine, was studied in various stages of intraerythrocytic Plasmodium knowlesi from infected rhesus monkeys. Incubation of the parasitized erythrocytes with the precursors was for 3 hr periods using a plasma-free culture medium. The samples containing primarily rings, early trophozoites, or late trophozoites incorporated orotic acid, adenosine, and uridine into RNA; however, these stages exhibited negligible or very low levels of incorporation of any of the precursors into DNA. The sample containing late trophozoite and schizont stages incorporated orotic acid, adenosine, and uridine into RNA, and orotic acid, adenosine, and very low levels of thymidine into DNA. These results indicate that DNA synthesis (the S phase of the cell cycle) occurs very close to the time of nuclear division, and that either the G1 or G2 phase is very short in P. knowlesi. It was also observed that adenosine and orotic acid, 2 precursors which are incorporated into both DNA and RNA, are utilized differently by the intraerythrocytic parasites. Incorporation of orotic acid into RNA and DNA and adenosine incorporation into DNA were continuous for the entire incubation period, whereas incorporation of adenosine into RNA was very low during the last 2 hr of each period. It was further demonstrated that the parasites utilized exogenous uridine for synthesis of RNA, and that the older parasite stages incorporated thymidine into DNA.     

Onset of nucleic acid synthesis during germination of Pisum sativum L.

Summary Measurments of total nucleic acid content of the embryonic axis indicated that massive net synthesis of both DNA and RNA was initiated at approximately 30 h after the onset of germination. The onset of net nucleic acid synthesis was marked by an increase in the rate of incorporation of [³H]thymidine into DNA, and of [³H]orotic acid and [³H]uridine into both DNA and RNA. rRNA was usually more heavily labelled than tRNA, but was not preferentially accumulated, suggesting a grater rate of turnover of rRNA than tRNA. Some incorporation of precursors occurred prior to the onset of net nucleic acid synthesis, particularly into RNA. This was taken to represent nucleic acid turnover. There was no evidence that the scavenging pathways for nucleotide biosynthesis were more important than the normal pathways in contributing precursors for net nucleic acid synthesis.     

Nucleolar RNA Synthesis during Meiosis of Lily Microsporocytes

SEVERAL authors have reported a decrease in nucleolar incorporation of ³H-uridine into RNA in male gametocytes of maize, locusts and mammals during meiotic prophase^1–4, but the inactive nucleolus often persists. In the microsporocytes of Liliutn henryi the cytoplasmic ribosomes reportedly decrease in number during the extended meiotic prophase as the cellular RNA concentration also decreases⁵. Stern (personal communication) has also observed a decrease in RNA content in meiotic cells of Lilium longiflorum. We have examined the RNA synthetic activities of lily microsporocytes to see if the large nucleolus present is engaged in the synthesis of ribosomal RNA.     

Polyamines and nucleic acid metabolism in chick embryo. Incorporation of labelled precursors into nucleic acids of subcellular fractions and polyribosomal patterns

1. An increase in polyamine concentration, caused by inhibiting the amine oxidase activities with iproniazid, increased the incorporation of [³H]orotic acid into chick-embryo RNA and DNA. On the other hand, a decrease in polyamine concentration, obtained by causing an increase in amine oxidase activities, decreased [³H]orotic acid incorporation into nucleic acids. This was particularly evident for nuclear DNA and ribosomal RNA. 2. Polyribosomal patterns obtained by sucrose-density-gradient centrifugation showed highest radioactivity in the regions of 259s and 280s aggregates in those embryos in which the polyamine contents were enhanced, whereas a decrease in the radioactivity was observed when the polyamine concentrations were decreased. 3. The activity of DNA-dependent RNA polymerase, assayed in the same experimental conditions, also varied in the same fashion with changes in polyamine concentration.     

The effect of 2-thiouracil on RNA synthesis in pollen tubesof Nicotiana alata

The level of RNA in pollen is approximately 20 mg g^-1 and remains constant during 6 h pollen germinationin vitro also in the presence of 2-thiouracil which stimulates pollen tube elongation. The synthesis of RNA in pollen tubes was investigated according to the incorporation of the label from uracil-2-¹⁴C, 2-thiouracil-2-¹⁴C, orotic acid-5-³H, fructose-U-¹⁴C and from³²PO₄ ^3- into RNA fractions separated by methylated albumine kieselguhr chromatography. The distribution of radioactivity on elution profiles was different according to the radioactivity source, however it was not changed by the presence of 2-thiouracil in cultivation medium. 2-Thiouracil incorporates into pollen tube RNA at about 50% the rate of uracil. It inhibited the incorporation of orotic acid, of fructose and of phosphate into all RNA fractions. It is suggested that the analogue inhibits the enzymes involved in RNA synthesis essentially as 2-thiouridine-5’-phosphate.     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

An Improved Screening Method for Inhibitors of Nucleic Acid Synthesis

A screening method for the antibiotics capable of inhibiting synthesis of nucleic acid in bacterial cells or mammalian tumor cells was investigated. The DNA and RNA syntheses in Bacillus subtilis 168 thymine^?, indole^? were studied by the assay of incorporations of ³H-thymine and ¹⁴C-uracil into the cells, respectively. With known antibiotics against nucleic acid synthesis, the adequacy of the method was examined, and the result proved that this method is more sensitive and specific than the conventional assay methods.

It was found as a new fact that cellocidin is a potent and specific inhibitor to the thymine incorporation into DNA.

By an almost similar procedure, an inhibitory effect of several antibiotics on the incorporations of ³H-thymidine and ¹⁴C-uridme into Ehrlich ascites carcinoma cells was also studied.     

Effects of Cycloheximide upon Formation of Ribonucleic Acid Cytidylic and Uridylic Acids

Concentrations of cycloheximide as low as 3 μg/ml inhibited incorporation of labeled orotic acid or uridine into RNA cytidylic acid of soybean (Glycine max) hypocotyl sections. Even lower concentrations of this well known protein synthesis inhibitor interfered with conversion of labeled cytidine into RNA uridylic acid. Both cycloheximide and puromycin inhibited absorption of ³H-phenylalanine and its incorporation into protein, but puromycin did not significantly affect the labeling patterns of RNA cytidylic and uridylic acids when orotic acid-6-¹⁴C was fed. Results give further support to the hypothesis that cycloheximide inhibits the interconversion of uridine and cytidine nucleotides, presumably by acting as a glutamine antagonist in the glutamine-dependent reaction catalyzed by cytidine triphosphate synthetase.     

Tapetum determinant1 is required for cell specialization in the Arabidopsis anther

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. The microsporocytes generate microspores, whereas the tapetal cells support the development of microspores into mature pollen grains. Despite their importance to plant reproduction, little is known about the underlying genetic mechanisms that regulate the differentiation and interaction of these highly specialized cells in the anther. Here, we report the identification and characterization of a novel tapetum determinant1 (TPD1) gene that is required for the specialization of tapetal cells in the Arabidopsis anther. Analysis of the male-sterile mutant, tpd1, showed that functional interruption of TPD1 caused the precursors of tapetal cells to differentiate and develop into microsporocytes instead of tapetum. As a results, extra microsporocytes were formed and tapetum was absent in developing tpd1 anthers. Molecular cloning of TPD1 revealed that it encodes a small protein of 176 amino acids. In addition, tpd1 was phenotypically similar to excess microsporocytes1/extra sporogenous cells (ems1/exs) single and tpd1 ems1/exs double mutants. These data suggest that the TPD1 product plays an important role in the differentiation of tapetal cells, possibly in coordination with the EMS1/EXS gene product, a Leu-rich repeat receptor protein kinase.     

Commitment of Mitotic Cells to Meiosis during the G2 Phase of Premeiosis

In the developing anther, archesporial cells that proliferateby mitotic division are converted into meiotic cells duringthe premeiotic interphase. Experiments with explanted microsporocytesof Lilium and Trillium were made to obtain evidence for theconversion of mitotic to meiotic cells during the premeioticperiod. Explanted premeiotic cells were cultured through thedivision cycle at relatively high division frequencies and showeda variety of division types with respect to chromosomal events.The type of division depended on the premeiotic stage at whichthe cells were explanted. Cells in the G₁, S and early G² phasesunderwent mitotic division and formed a diad or binucleate monad.Cells explanted at the late G₂ phase were cultured throughoutthe normal meiotic cycle, which resulted in typical tetrad configuration. In microsporocytes explanted during the main part of the G₂interval, centromere behavior was meiotic, but chromosome pairingand chiasma formation were disturbed. Thus, she G₂ intervalwas shown to be critical for the commitment of mitotic cellsto meiotic division. Detailed analysis showed that the intracellularchanges that commit the cells to meiosis begin shortly aftercompletion of premeiotic DNA synthesis and that these changesare progressive and cumulative. (Received February 2, 1982; Accepted May 24, 1982)     

BIOCHEMICAL CHANGES IN YEAST DURING SPORULATION. I. FATE OF NUCLEIC ACIDS AND RELATED COMPOUNDS

Changes in nuclear figures and in activities of nucleic acid and protein syntheses were observed mainly on Saccharomyces cerevisiae G2-2 during sporogenesis. Patterns of DNA synthesis and of meiosis show that the sporogenic process in yeast was divided into an induction phase (I-phase), a DNA-synthesizing phase (S-phase) and a maturation phase (M-phase). Meiotic figures appeared most frequently at the end of the S-phase at approximately 12 hr in sporulation culture. In M-phase visible spores formed. The amount of protein increased in the initial 7 hr culture of 1-phase, then decreased in the S- and M-phases. But in sporulation culture of the asporogenic diploid strain 3c × a, protein did not decrease. RNA increased within 3 hr of the I-phase then stopped increasing. DNA synthesis occurred critically during S-phase, i.e. between 7 and 12 hr. and was somewhat resumed during the later part of M-phase. Oligodeoxyri-bonucleotide content decreased in the I- and M-phases and increased temporarily. Deoxyribosides decreased linearly during the sporogenic processes. Based on these results and results of experiments estimating the incorporation of ¹⁴C-uracil into nucleic acid and ¹⁴C-amino acid mixture into protein fractions, the roles of nucleic acid synthesis activities in meiosis and in sporulation are discussed.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

RELATIONSHIP BETWEEN RNA SYNTHESIS, CELL DIVISION, AND MORPHOLOGY OF MAMMALIAN CELLS : I. Puromycin Aminonucleoside As An Inhibitor of RNA Synthesis and Division in HeLa Cells

Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P³² and H³-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division within the period studied (3 generation times). The cell content of RNA returned to normal when the inhibitor was removed.     

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C¹⁴ and uridine-H³ were used as ribonucleic acid (RNA) precursors, thymidine-H³ as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C¹⁴ as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.     

The effect of kinetin on the incorporation of labelled orotate into various fractions of ribonucleic acid of excised radish leaf discs

Summary Discs from senescing radish leaves were floated either on water or on a solution of kinetin and incubated in the dark. The kinetin-treated discs were fed with orotic acid-5-T and the control discs with orotic acid-6-C¹⁴. The two lots of discs were combined and the RNA extracted by the phenol method. The H³:C¹⁴ ratio of the RNA after fractionation on sucrose density gradient was compared with the H³:C¹⁴ ratio of RNA extracted from two lots of control discs, one fed with orotic acid-5-T and the other with orotic acid-6-C¹⁴ and then extracted together. In this way it was found that 21 hours treatment with kinetin causes a small but consistent stimulation of labelled precursor incorporation into all classes of RNA investigated.