Synthesis of nucleic acids and localization of atrial natriuretic peptide in crayfish haemocytes

Three types of cells circulate in the haemolymph of the crayfish Astacus astacus, i.e., agranular haemocytes (HCs I), small-granule haemocytes (HCs II), and large-granule haemocytes (HCs III). Their proliferation, differentiation, and function remain poorly understood. Using light and electron microscopic autoradiography with [³H]-thymidine, we found that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To verify whether HCs I are proliferating progenitor cells for granular HCs, we have analyzed autographs of the HC population 1, 2, 7, and 21 days after a single [3H]-thymidine administration. Contrary to our expectations, we have failed to find labeled HCs II and HCs III. These findings have raised doubts as to the capacity of HCs I to differentiate into two other types of HCs. With the use of ³H-uridine autoradiography, it was found that RNA synthesis was the most active in HCs I and 2 and 4 times lower in HCs II and HCs III, respectively. ANP-like immunoreactivity was revealed in large granules of the HCs III by electron microscopic immunocytochemistry. We assume that the presence of ANP in secretory granules extends the possible functions of crayfish HCs and suggests their participation in the regulation of the watersalt balance and immune response.     

Immunocytochemical localization of atrial natriuretic peptide in endothelial and granular cells of the heart of lophotrochozoa

In parallel with contraction, vertebrate cardiomyocytes perform endocrine function and produce natriuretic peptides (NP)--ANP and BNP--involved in cardiovascular homeostasis maintenance. ANP-like peptides have been reported also in hearts of some invertebrates, however, their cellular localization was not determined. By electron microscopical immunocytochemistry with polyclonal monospecific antibodies raised against ANP and protein A-gold technique, we have localized ANP-like immunoreactivity in granules within endothelial cells in the heart of the brachiopod Rhynchonella psittacea, the polychaete Arenicola marina, and the gastropod mollusc Achatina fulica--all being representatives of the major phylogenetic group Lophotrochozoa. ANP-like immunoreactivity was also revealed in one of 3 morphologically distinguishable types of granules in the snail heart granular cells. By electron microscopical autoradiography with the use of [3H]-thymidine, the ability for DNA synthesis was demonstrated in heart endothelial cells of the investigated animals. Forms of NP-system organization in hearts of Lophotrochozoa and Vertebrates, and close histogenetic relationships of endothelial and granular cells in the snail heart are discussed.     

In vitro synthesis of a lipid-linked trisaccharide involved in synthesis of enterobacterial common antigen.

The heteropolysaccharide chains of enterobacterial common antigen (ECA) are made up of linear trisaccharide repeat units with the structure----3)-alpha-D-Fuc4NAc-(1----4)- beta-D-ManNAcA-(1----4)-alpha-D-GlcNAc-(1----, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The assembly of these chains involves lipid-linked intermediates, and both GlcNAc-pyrophosphorylundecaprenol (lipid I) and ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) are intermediates in ECA biosynthesis. In this study we demonstrated that lipid II serves as the acceptor of Fuc4NAc residues in the assembly of the trisaccharide repeat unit of ECA chains. Incubation of Escherichia coli membranes with UDP-GlcNAc, UDP-[14C]ManNAcA, and TDP-[3H]Fuc4NAc resulted in the synthesis of a radioactive glycolipid (lipid III) that contained both [14C]ManNAcA and [3H]Fuc4NAc. The oligosaccharide moiety of lipid III was identified as a trisaccharide by gel-permeation chromatography, and the in vitro synthesis of lipid III was dependent on prior synthesis of lipids I and II. Accordingly, the incorporation of [3H]Fuc4NAc into lipid III from the donor TDP-[3H]Fuc4NAc was dependent on the presence of both UDP-GlcNAc and UDP-ManNAcA in the reaction mixtures. In addition, the in vitro synthesis of lipid III was abolished by tunicamycin. Direct conversion of lipid II to lipid III was demonstrated in two-stage reactions in which membranes were initially incubated with UDP-GlcNAc and UDP-[14C]ManNAcA to allow the synthesis of radioactive lipid II. Subsequent addition of TDP-Fuc4Nac to the washed membranes resulted in almost complete conversion of radioactive lipid II to lipid III. The in vitro synthesis of lipid III was also accompanied by the apparent utilization of this lipid intermediate for the assembly of ECA heteropolysaccharide chains. Incubation of membranes with UDP-[3H]GlcNAc, UDP-ManNAcA, and TDP-Fuc4NAc resulted in the apparent incorporation of isotope into ECA polymers, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In addition, the in vitro incorporation of [3H]Fuc4NAc into ECA heteropolysaccharide chains was demonstrated with ether-treated cells that were prepared from delta rfbA mutants of Salmonella typhimurium. These mutants are defective in the synthesis of TDP-Fuc4NAc; as a consequence, they are also defective in the synthesis of lipid III and they accumulate lipid II. Accordingly, incubation of ether-permeabilized cells of delta rfbA mutants with TDP-[3h]Fuc4NAc resulted in the incorporation of isotope into both lipid III and ECA heteropolysaccharide chains.     

Immunohistochemical identification of proliferating cells in organ culture using bromodeoxyuridine and a monoclonal antibody

The ability to measure cell proliferation is important in the study of cancer biology. The usual technique for quantitating proliferating cells in tissue explant and organ culture by detection of [3H]-thymidine incorporation into DNA by autoradiography is tedious and time-consuming. We have developed a technique for identification and quantitation of bromodeoxyuridine (an analogue of thymidine) in cultured tissue explants. Fetal mouse colon explants were exposed in vitro to bromodeoxyuridine (BUdR) or [3H]-thymidine for 3 to 72 hr and then for various periods to unlabeled thymidine. The tissues were stained with a monoclonal anti-bromodeoxyuridine antibody and in parallel [3H]-thymidine incorporation was detected by autoradiography. Incorporation of BUdR was measured by quantitating the amount of pigment deposited over nuclei after immunohistochemical staining, using an optical data digitizer. It was found that both techniques identified proliferating cells. Dividing cells were present both in crypts and in the surrounding stroma in Day 14 fetal mouse colon cultures. The immunohistochemical technique was more rapid and less cumbersome than autoradiography.     

Effects of soluble factors and extracellular matrix on DNA synthesis and surfactant gene expression in primary cultures of rat alveolar type II cells

Proliferation and differentiation of epithelial cells are thought to be regulated by soluble factors in extracellular fluid and insoluble components of the extracellular matrix. We have examined the combined effects of soluble factors and an extracellular matrix (EHS matrix) on DNA synthesis, cell proliferation, and surfactant protein gene expression in primary cultures of alveolar type II epithelial cells. Cells on EHS matrix cultured in DMEM containing insulin, cholera toxin, EGF, aFGF, 5% rat serum, and 15-fold concentrated bronchoalveolar lavage fluid (D-GM) formed larger aggregates than cells cultured on the same substratum in DMEM containing 5% rat serum (D-5). Cells cultured in D-GM on EHS matrix incorporated more [³H]-thymidine than cells on the same substratum in D-5, with an eight-fold increase seen on day 4 of culture. This increase in [³H]-thymidine incorporation was accompanied by a labeling index of greater than 65% of the cells. Cell counts showed that exposure of type II cells on EHS matrix to D-GM resulted in increased cell number on day 4 of culture. [³H]-thymidine autoradiography combined with immunostaining with anti-cytokeratin, anti-SP-A, and anti-vimentin antibodies demonstrated that the proliferating cells were epithelial cells that contained SP-A. Type II cells cultured on plastic in D-GM also showed increased [³H]-thymidine incorporation compared to cells cultured in D-5. The level of [³H]-thymidine incorporation by cells on plastic, however, was significantly less than that seen in cells cultured in the same medium on EHS matrix. Type II cells cultured on EHS matrix in D-GM had a decreased abundance of mRNAs for SP-A and SP-C than cells cultured on EHS matrix in D-5 as determined by Northern analysis. This inhibition was reversed by switching from D-GM to D-5 on day 4 and culturing the cells for an additional 4 days. In contrast, SP-B mRNA was increased in response to D-GM. This increase was not reversed by switching from D-GM to D-5 on day 4. These results suggest that the interaction of soluble factors and extracellular matrix components has a strong influence on type II cell proliferation, which were partially associated with the reversible inhibition of lung tissue-specific protein mRNAs. Their dynamic interplay among the type II cell, the extracellular matrix, and growth factors may determine multicellular functions and play an important role in normal lung development and in the repair of the lung epithelium following injury.     

Use of a double-label method to detect rapid changes in the rate of cell proliferation.

We developed a double-label method to directly measure the rate at which cells enter S-phase of the cell cycle. All cells in S-phase were first labeled with a short pulse of [3H]-thymidine. This was followed by a longer incubation in bromodeoxyuridine (BrdU), a thymidine analogue. Nuclei labeled with [3H]-thymidine were detected by autoradiography and those labeled with BrdU by immunocytochemistry. Cells labeled only with BrdU must have entered S-phase at some time after the end of the [3H]-thymidine pulse. Thus, the rate of entry of cells into S-phase could be determined. This method was shown to be more accurate and more sensitive than determining changes in the rate at which cells entered S-phase with a continuous labeling protocol. It was possible to detect changes in proliferative activity that occurred in less than 1 hr. We used this double-label technique to study changes in the cell cycle during the terminal differentiation of chicken embryo lens fiber cells. These studies revealed differences in the effects of several treatments known to stimulate fiber cell differentiation. They also demonstrated the presence in the embryonic eye of factors that stimulate and prevent lens cell proliferation and differentiation.     

Palladium(II) salt and complexes of spermidine with a six-member chelate ring. Synthesis, characterization, and initial DNA-binding and antitumor studies.

By reaction of spermidine trihydrochloride with K2PdCl4 and PdCl2 at different pH's, we have synthesized the [sperH3]2[PdCl4]3 (I), [PdCl2(sperH)]2[PdCl4] (II), and [(PdCl2)3(sper)2] (III) compounds. The structure of these compounds was studied by IR and 1H NMR; complex II was analyzed by x-ray diffraction. In this complex the spermidine is attached to the PdCl2 group forming a six-member chelate ring with a protonated terminal amine group. The crystal of [PdCl2(sperH)]2[PdCl4] x 2H2O (II) is monoclinic, P2(1)/n, with a = 7.023(1) A, b = 12.662(1) A, c = 18.435(3) A, and beta = 99.95(1) degrees, Z = 4, R = 0.051, and Rw = 0.058 on the basis of 2690 independent reflections. We have compared the antitumor activity in vitro against the isolated human breast carcinoma MDA-MB 468 cell line of compounds I, II, and III with that of cis-diamminedichloroplatinum(II), cis-DDP. The results show that compounds III and III have values of ID50 similar (0.74 microgram/ml) or even lower (0.56 microgram/ml) than cis-DDP (0.80 microgram/ml). We also observed that compounds I, II, and III have the ability to induce conformational changes in covalently closed circular (ccc) form of the pUC8 plasmid DNA. Compounds II and III also induce conformational changes in the open circular (oc) form of this plasmid.     

Simultaneous preparation of both enantiomers of juvenile hormones labeled at C-10 with tritium at high specific activity

We report an improved method for the synthesis of high specific activity insect [10-(3)H]juvenile hormones (JH) I, II, and III which affords both enantiomers of each in high optical purity. A synthetic route for JH I was modified to give higher yields and purity. We increased the specific activity of the synthetic [10-(3)H]JHs using normal phase liquid chromatography optimized to give near baseline resolution of [10-(3)H]JHs and unlabeled JHs. Racemic [10-(3)H]JHs and their corresponding diol metabolites were enantiomerically separated using a chiral column eluted with 2-propanol:hexane. Acidic hydration of the unnatural antipode of the [10-(3)H]JHs gives the diol antipode with the same stereochemistry as that from epoxide hydrolase action on the natural JH antipode. The [10-(3)H]JH diol enantiomers can also be resolved with the same chiral column using a more polar solvent. The synthesis of high specific activity chiral ethyl ester analogs of JH I and II can also be accomplished using this synthetic route.     

Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

A specific photoaffinity label for hemolymph and ovarian juvenile hormone-binding proteins in Leucophaea maderae

A tritium-labeled diazocarbonyl juvenile hormone (JH) analog, (10-[10,11-3H]epoxyfarnesyl diazoacetate, [3H]EFDA), covalently bound to proteins in both hemolymph and ovarian extracts when reaction mixtures were irradiated with UV light. The addition of various concentrations of unlabeled JH III selectively inhibited [3H]EFDA photoattachment to proteins. Using the Scatchard method of analysis, [3H]EFDA bound specifically and with relatively high affinity (KD = 1.5 X 10(-6) M) to a macromolecule in each extract, although nonspecific binding to other molecules was also present (20-50%). To determine if [3H]EFDA bound at the JH III-binding site on the binding proteins, radioactive [3H]JH III or [3H]EFDA was complexed with proteins in the presence of various concentrations of either unlabeled JH III or JH I under equilibrium conditions. The results demonstrated that the natural hormone, JH III, displaced both bound labeled ligands 4.1 +/- 0.5 times better than the homolog JH I. Thus, the photoaffinity label [3H]EFDA bound at the same site on the protein as [3H] JH III. Fluorescent autoradiography of [3H]EFDA-labeled proteins separated by sodium dodecyl sulfate electrophoresis revealed that several proteins in both hemolymph and ovarian extracts bound [3H]EFDA. To determine the specificity of binding, extracts were irradiated with UV light in the presence of unlabeled JH III and [3H]EFDA. The results demonstrated that JH III prevented photoattachment of [3H]EFDA to a major protein in each extract. The molecular weight of these proteins was estimated at approximately 200,000 for both the hemolymph protein and the ovarian protein.     

Regional distribution and pharmacological characteristics of [3H]N-acetyl-aspartyl-glutamate (NAAG) binding sites in rat brain

Autoradiographical studies revealed that 10 nM [3H]N-acetyl-aspartyl-glutamate (NAAG) labelled grey matter structures, particularly in the hippocamus, cerebral neocortex, striatum, septal nuclei and the cerebellar cortex. The binding was inhibited by (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)-glycine (DCG IV), an agonist at group II metabotropic glutamate receptors (mGluR II). (RS)-alpha-Methyl-4-tetrazolylphenylglycine (MTPG), (RS)-alpha-cyclopropyl-4-phosphonoglycine (CPPG) and (RS)-alpha-methylserine-O-phosphate monophenyl ester (MSOPPE), all antagonists at mGluR II and mGluR III, also inhibited [3H]NAAG binding. Other inhibitors were (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD), a broad-spectrum mGluR agonist with preference for groups I and II and the mGluR I agonists/mGluR II antagonists (S)-3-carboxy-4-hydroxyphenylglycine (3,4-CHPG) and (S)-4-carboxy-3-hydroxyphenylglycine (4,3-CHPG). Neither the mGluR I specific agonist (S)-dihydroxyphenylglycine nor any of the ionotropic glutamate receptor ligands such as kainate, AMPA and MK-801 had strong effects (except for the competitive NMDA antagonist CGS 19755, which produced 20-40% inhibition at 100 microM) suggesting that, at low nM concentrations, [3H]NAAG binds predominantly to metabotropic glutamate receptors, particularly those of the mGluR II type. Several studies have indicated that NAAG can interact with mGluR II and the present study supports this notion by demonstrating that sites capable of binding NAAG at low concentrations and displaying pharmacological characteristics of mGluR II exist in the central nervous tissue. Furthermore, the results show that autoradiography of [3H]NAAG binding can be used to quantify the distribution of such sites in distinct brain regions and study their pharmacology at the same time.     

Passage in monolayer influences the response of chondrocytes to dynamic compression

This study tests the hypothesis that expansion by passage in monolayer influences the response of isolated articular chondrocytes to dynamic compression. Chondrocytes, isolated from bovine articular cartilage, were seeded in monolayer and passaged 4 times (P1-4). For assessment of chondrocytic and fibroblastic phenotype, freshly isolated and passaged cells were seeded on glass coverslips or in 2% alginate beads and cultured for 7 days in DMEM + 10% FCS. Samples were assayed for DNA and GAG content and stained for collagen types I and II. In separate experiments, freshly isolated or passaged chondrocytes were seeded at 10 x 10(6) cells.ml(-1) in 4% cylindrical agarose constructs and subjected to 15% dynamic compressive strain at 1 Hz for 24 hours. [(3)H]-thymidine incorporation, SO(4) incorporation and nitrite release were analysed. Immediately following isolation (P0), chondrocytes seeded in alginate expressed high levels of type II collagen, but did not stain for type I collagen. Following repeat passage the cells expressed enhanced levels of type I collagen, with an associated reduction in type II collagen staining. These data indicate a modulation to a fibroblastic phenotype during monolayer expansion which was not rapidly reversed by culture in a 3D hydrogel. Dynamic compression down-regulated SO(4) incorporation at P0, but did not affect [(3)H]-thymidine incorporation. By contrast the incorporation of both SO(4) and [(3)H]-thymidine was enhanced by dynamic compression at both P1 and to a lesser extent P2. SO(4) and [(3)H]-thymidine incorporation were inhibited at P3 and P4. Nitrite release was down-regulated by dynamic compression at all passages. These data demonstrate a clear modulation in the response of bovine articular chondrocytes to dynamic compression following passage in monolayer.     

Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues

Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidin-biotin-peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin-Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol-fixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanol-fixed tissues. The distribution of the positive nuclei in the two latter cases coincided with the proliferative compartment. On the other hand, double labelling with [3H]-thymidine and with the cyclin/PCNA antibody revealed that in methanol-fixed tissues the cyclin/PCNA labelling index did not differ by more than 6% from the [3H]-thymidine index. Besides the two labels overlapped in a proportion of labelled cells that was in reasonable agreement with expectation considering cells flow in and out of S phase since the time of [3H]-thymidine injection. This indicates that both labels recognize the same cells in this material. In contrast, in formaldehyde-fixed tissues, the cyclin/PCNA labelling index markedly exceeded the [3H]-thymidine labelling index. From this it is concluded that cyclin/PCNA immunostaining can be used: (1) In formaldehyde-fixed tissues (including existing material stored as paraffin blocks): for defining and mapping the proliferative (or germinative) compartment. (2) In methanol-fixed tissues as a substitute to the [3H]-thymidine autoradiographic labelling index. From this, a method is proposed (derived from classical 'double-labelling' technique) for measuring S phase duration in tissues fixed at a known interval time after a single labelling with [3H]-thymidine (or BrdU) and submitted to cyclin/PCNA immunocytochemical detection and to autoradiography (or to BrdU immunostaining).     

Evidence for differential DNA endoreplication during the development of a molluscan brain

The DNA content of neurons in the cerebral ganglion of Achatina fulica was determined by the two-wavelength method of microspectrophotometry using Feulgen-stained sections. DNA measurements of mouse hepatocytes were used as a control of the methods. All sampled neurons with a nuclear diameter greater than 7 microns were polyploid. The shape of the frequency histogram of DNA contents is not compatible with an interpretation that assumes one or more simple duplications of the genome. It is suggested, instead, that the results are due to either the underreplication of some DNA sequences or the selective amplification of genes. Additional experiments using [3H]-thymidine autoradiography showed that the incidence of neuronal DNA synthesis is highest during the period of the animal's greatest growth and then declines rapidly near the onset of sexual maturity. The mesocerebrum incorporated greater amounts of [3H]-thymidine than the rest of the brain, whereas the procerebrum remained diploid throughout the course of the study.     

Double labelling with bromodeoxyuridine and [3H]-thymidine of proliferative cells in small intestinal epithelium in steady state and after irradiation

The simultaneous immunohistochemical detection of bromodeoxyuridine (BrdU) and [3H]-thymidine ([3H]TdR), by conventional autoradiography, was performed on the mouse small intestine (ileum). Proliferation was studied under normal conditions as well as after 3 Gy of gamma-rays. The BrdU method in conjunction with [3H]TdR autoradiography appears to be reliable and useful for the study of cell kinetics especially in disturbed states, on condition that [3H]TdR is delivered to the animals before BrdU. It has been found that cells in the crypt are delayed by irradiation in their progression through the cell cycle predominantly in late S phase. The cells at the bottom of the crypt are more affected than the more differentiated but proliferating cells in the upper part of the crypt.     

Maternal phenytoin administration affects DNA and protein synthesis in embryonic primary palates

Recent studies have shown that phenytoin (Dilantin) administration to pregnant A/J mice on day 10 causes reduced growth in embryonic primary palates. The current investigation concentrates on biochemical and autoradiographic changes toward the end of primary palate formation (gestational day 11), which coincides with the developmental period used for the previously conducted morphological studies. On gestational day 10, one group of pregnant A/J mice was injected intraperitoneally (IP) with 60 mg/kg phenytoin and the other group with vehicle. Twenty-three hours after phenytoin administration, all animals were injected (IP) with either [3H]-thymidine or [3H]-leucine. After one hour of incorporation, animals were sacrificed, embryos removed and placed in ice-cold Eagle's minimum essential medium containing 0.02% NaN3 for biochemical assay or fixed immediately in Bouin's solution for autoradiography. For biochemical analyses, palates and limb buds were removed, homogenized, TCA precipitated, lyophilized, and acid hydrolyzed. Examination of the data revealed that DNA synthesis in control palates was 3.8-fold greater than in primary palates from embryos of phenytoin-treated mothers. Results were similar for limb buds from control embryos and from embryos of phenytoin-treated mothers. Experiments utilizing [3H]-leucine indicated that protein synthesis was 2.6-fold greater in primary palates from phenytoin-treated mothers than in control primary palates. Similar results were obtained for protein synthesis in limb-bud tissue from controls and embryos of phenytoin-treated mothers. Autoradiographic data supported the biochemical findings. DNA synthesis in primary palates from embryos of phenytoin-treated mothers decreased 3-fold; protein synthesis increased 2.2-fold compared with control primary palates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutants of the mini-F plasmid pML31 thermosensitive in replication.

Hydroxylamine mutagenesis was used for the induction of thermosensitive replication mutants of the mini-F plasmid pML31. Replication mutants were characterized by studying the segregation kinetics and the incorporation of [3H]-thymidine into plasmid deoxyribonucleic acid at the nonpermissive temperature. Based on these experiments two types of mutants could be distinguished. Mutants of type I are fast segregating with the kinetics expected if plasmid replication was blocked immediately. Double-label experiments showed a rapid shut-off of replication in these mutants at 42 degrees C. Mutants of type II segregate slower, showing only a partial inhibition of plasmid deoxyribonucleic acid synthesis at the nonpermissive temperature. The label incorporated at 42 degrees C was predominantly found in open circular plasmid molecules.     

Metabolic-morphologic characteristics of the integument of teleost fish with mature lymphocystis nodules

Summary Normal and virus-infected (lymphocystis disease) integument from five species of teleosts was examined by light and TEM autoradiography and SEM to establish metabolic-morphologic characteristics of integument with mature lymphocystis cells (LC's). LC's with numerous morphologic attributes of a late developmental stage showed highest incorporation of [³H]-thymidine in vivo (1–91 h) above the intracytoplasmic inclusion body (ci) with little radiolabel in nuclei, cytoplasmic icosahedral deoxyriboviruses (ICDVs) or capsule. Analysis by quantitative autoradiography revealed that the % total cell label in ci and cytoplasm did not vary appreciably from 1–91 h and was corroborative with morphologic criteria of maturity. A possible phylogenetic difference was noted between teleosts, wherein normal integument showed uptake of [³H]-thymidine in vivo (1 h) by cells at all levels of the epidermis, and cyclostomes (Spitzer et al. 1979) wherein labeling was confined to the basal third of the epidermis. Among four infected teleost species, the mean diameters of the ICDVs measured under the same conditions, ranged from 259.5 nm to 290.0 nm with the mean for each species differing significantly (p < 0.01) from each of the other means. Ruptured LC's were shown by TEM and SEM to have released ICDVs onto the lesions and integument. Various stages of LC degeneration, host response, and integumental repair processes were documented. An evaluation of labeling in vivo of the capsular matrix was compatible ([³H]-D-galactose> [³H]-L-lysine [³H]-L-fucose) with a glycosaminoglycan-protein structure.     

Increased uptake of thymidine in the activation of sea urchin eggs. III. Effects of aphidicolin

Uptake and phosphorylation of externally supplied [3H]-thymidine are fully stimulated in fertilized sea urchin eggs exposed to 5.0 micrograms/ml aphidicolin. As in untreated controls, the rate of uptake in aphidicolin-treated eggs increases greater than 50-fold shortly after fertilization, and greater than 85% of the transported thymidine is immediately phosphorylated to the triphosphate. The intracellular levels of [3H]-thymidine triphosphate (3H-dTTP) resulting from an external supply of [3H]-thymidine is therefore equal in aphidicolin-treated and untreated fertilized eggs. Under the same experimental conditions, the incorporation of externally supplied [3H]-thymidine into newly synthesized DNA of fertilized eggs is 90% inhibited by exposure to aphidicolin. The full availability of 3H-dTTP in these eggs further suggests that aphidicolin inhibits specifically at the level of DNA synthesis. This inhibitory effect is proportional to the concentration of aphidicolin between 0 and 5.0 micrograms/ml. In the continuous presence of 5.0 micrograms/ml aphidicolin, fertilized eggs fail to undergo mitotic chromosome condensation, nuclear envelope breakdown, and cytokinesis, suggesting a dependent link between these processes and the completion of nuclear DNA synthesis.