Methacrylate polymerization by AzoRNA: potential usefulness for chromosomal localization of genes

An azo pyrimidine nucleotide has been prepared and enzymatically attached to oligo(A) primers. The nucleotide's azo pyrimidine group has previously been shown to initiate polymerization of methacrylate esters designed to bind marker groups for visualization by microscopy. When attached to RNA molecules complementary to a chromosomal DNA segment, these nucleotides may allow localization of the DNA segment following in situ hybridization of the probe, methacrylate polymerization, and marker attachment. Since mRNA molecules of potential interest as probes bear a 3′-poly(A) tail, the modified nucleotides were added to oligo(A) primers as models. First, N⁴-ureidocytosine nucleotides were enzymatically added to ApApA, (Ap)₉A, or [5′-³²P]-(pA)₁₀, using the modified cytidine 5′-diphosphate and “primer-dependent” polynucleotide phosphorylase (M. luteus). In the case of the ApApA-primed reaction, the N⁴-ureidocytosine nucleotides in the product polynucleotide were converted into azo nucleotides by oxidation with N-bromosuccinimide. The other two primers were employed to study the time course of polynucleotide formation and to verify that primer was indeed being utilized by the enzyme. The suitability of the modified nucleotide for in situ hybridization studies was examined. Poly(N⁴-ureidocytidylic acid) was prepared from poly(C) and semicarbazide by the bisulfite-catalyzed transamination reaction. It was found that 95% of the N⁴-ureidocytosine nucleotides in this polynucleotide survive the elevated temperatures typically required for DNA:DNA denaturation and RNA:DNA annealing. When poly(N⁴-ureidocytidylic acid) was mixed with poly(I) in buffered aqueous salt solutions, no evidence for hybridization was found, so binding of the probe RNA to the denatured chromosomal DNA molecule via the modified nucleotides is not expected. Upon oxidation of poly(N⁴-ureidocytidylic acid) with N-bromosuccinimide, the azo nucleotides were formed, as judged by the appearance of a characteristic peak at approximately 350 nm in the uv-absorption spectrum of the yellow-orange product, azoRNA. The azo nucleotides in azoRNA exhibited the expected acid lability, which is known to be accompanied by 1-glyceryl methacrylate polymerization in the case of the simple azo pyrimidine. Because 1-glyceryl metharcylate bears substituent glycol groups for attaching heavy atoms or fluorescent markers, it is possible that probe RNA molecules bearing azo nucleotides may be useful for localizing low-multiplicity genes along eukaryotic chromosomes.     

Activation and inactivation of thyroxine by cultured rat hepatoma cells

The metabolism of thyroxine, 3,3′,5-triiodothyronine and 3,3′,5′-triiodothyronine was investigated in rat hepatoma cell cultures (R117-21B). These iodothyronines were labeled with ¹²⁵I in the phenolic ring and the metabolites were analyzed by ion-exchange column chromatography.When thyroxine was incubated with the cells at 37°C, its glucuronide was the major product and a little increase in ¹²⁵I^? was detected. Although 3,3′,5-triiodothyronine was not observed in the incubation medium, this metabolite was clearly identified in the ethanol extract obtained from the cell homogenates after 24 h incubation.This cell line also metabolized labeled 3,3′,5-triiodothyronine added to culture medium. After 24 h incubation, 3,3′,5-triiodothyronine glucuronide was the major metabolite and iodothyronine sulfates were also formed. The sulfates contained, 3,3′,5-triiodothyronine and 3,3′-diiodothyronine sulfates and an unknown component.In the metabolism of 3,3′,5′-triiodothyronine, the cells were very active in carrying out glucuronidation and phenolic ring deiodination, and this metabolism yielded 3,3′,5′-triiodothyronine and 3,3′-diiodothyronine glucuronides. The iodide fraction contained a small amount of 3,3′-diiodothyronine sulfate.These results show that the R117-21B rat hepatoma cells metabolize the thyroid hormones and their analogs by phenolic and nonphenolic ring deiodinations, by glucuronidation and by sulfation.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

DNA synthesis in cultured human fibroblasts: Regulation by 3′ : 5′-cyclic amp

The addition of serum to density-inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [³H] thymidine incorporation into acid-precipitable material, beginning after 8–12 hr and reaching maximum levels at 16–24 hr. Addition of dibutyryl-3′ : 5′-cyclic AMP (DBcAMP) together with serum inhibited [³H] thymidine incorporation by 75–95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3′ : 5′-cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [³H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G₁ was inhibited by high concentrations of 3′ : 5′-cyclic AMP.     

Use of diacylclycerol kinase to quantitate picomole levels of 1,2-diacylglycerol.

An extremely sensitive and specific analytic procedure is described for quantitating 1,2-diacylglycerol (DAG). Escherichia coli DAG kinase (EC 2.7.1.-) catalyzed the formation of [³²P]phosphatidic acid by the transfer of ³²PO₄ from [γ-³²P]adenosine 5′-triphosphate to DAG in linear proportion to the quantity of added DAG from 10 to 1000 pmol. This technique allowed reliable detection of as little as 2 pmol of added DAG. To assess levels of DAG in tissue lipid extracts a miniaturized method for silicic acid column chromatography was developed to separate DAG from triglycerides and phospholipids. When these procedures were applied to erythrocytes, lysis in the presence of Ca²⁺ caused a 10.6-fold rise in cellular DAG confirming not only the results obtained in an earlier investigation (1), but also the utility of this technique in the analysis of exceedingly small quantities of cellular DAG.     

Conversion of Purine Bases and Their Ribosides to 5′-Inosinic Acid by Bacillus subtilis

Among various nutritional mutants with weak 5′-nucleotidase derived from Bacillus subtilis IAM 1145, the adenine-requiring mutants could convert exogenously added hypo- xanthine, guanine, xanthine and their ribosides to 5′-inosinic acid (IMP) and accumulate it in the medium. Synthesis of IMP from purine derivatives was observed predominantly in an early stage of the cultivation. The conversion was stimulated by Fe²⁺ or Mn²⁺, and markedly depressed by an excess amount of adenine in the production-medium.     

EFFECTS OF ATP ON THE ACTIVITY OF NUCLEOSIDE 3'',5''-CYCLIC MONOPHOSPHATE PHOSPHODIESTERASE FROM BRAIN

ATP is known to inhibit the phosphodiesterase activity in the supernatant fraction of the brain homogenate. Results showed that, when enzyme activity was assayed by determining the change in the concentration of substrate, the magnitude of the inhibition by 2 ～ 3 mm -ATP was not more than 20% and this effect of ATP can be explained mainly, if not entirely, on the basis of chelation of ATP with Mg²⁺ and Ca²⁺in vitro, both of which are necessary for enzyme activity. When brain phosphodiesterase was assayed by measuring 5′-AMP (product), the effect of ATP was erroneously exaggerated. This is due to ATP-dependent conversion of 5′-AMP to inosinic acid catalysed by adenylate deaminase in the crude preparation.     

Fluorescence of nucleic acid-terbium (3) complexes

Terbium(III) binds to nucleic acids and acts as an acceptor of electronic excitation energy. The transfer appears to be primarily from guanosine residues. Fluorescence from Tb³⁺ bound to 5′-GMP is very much larger than fluorescence from either free 5′-GMP or free Tb³⁺ When bound to 5′-AMP, Tb³⁺ shows an intense excitation band at 414 nm, where free Tb³⁺ has no excitation band. Fluorescence from bound Tb³⁺ appears to be potentially useful in nucleic acid studies.     

High-performance liquid chromatographic assay for acid and alkaline phosphatase in serum

High-performance liquid chromatography was used to assay serum acid and alkaline phosphatase. Samples were incubated with adenosine-5′-monophosphoric acid (AMP) in a buffer of required pH, 5′-nucleotidase was inhibited with Ni²⁺ ions, and the phosphatase activity was determined by measuring the concentration of the reaction product, adenosine. The analysis time, after the incubation is terminated, is short (7 min), and the assay is quantitative and reproducible. Complete separation of the reaction product from the substrate and the naturally occurring serum constituents and the high sensitivity of the ultra-violet detection system eliminate some of the problems commonly encountered in spectrophotometric assays.     

Inhibition by cholera toxin of parathyroid hormone-induced calcium release from bone in culture.

Cholera toxin was added to the culture of fetal rat limb bone and its effect on calcium release as well as on adenylate cyclase activity was examined. Cholera toxin increased the content of adenosine 3′:5′-monophosphate (cAMP) in bone. The effect on cAMP was of slower onset and of longer duration as compared with parathyroid hormone (PTH) effect. PTH added to the tissue which had been stimulated by cholera toxin increased cAMP further but the effect was partially additive. In contrary to PTH which caused a clear calcium mobilization, cholera toxin by itself had no effect or rather inhibited the release of ⁴⁵Ca from the prelabeled bone. When the toxin (0.1–1 μg/ml) was combined with PTH (10 U/ml), calcium release stimulated by PTH was completely abolished.     

On the role of cellular calcium in the response of the parotid to dibutyryl and monobutyryl cyclic AMP.

The role of intracellular Ca in the exocytosis of α-amylase stimulated by derivatives of cyclic AMP was investigated. Partial depletion of cellular Ca stores was accomplished by prolonged (100–120 min) incubation in media containing no added Ca and 5.0 mM ethyleneglycolbis (aminoethylether)-N,N′-tetraacetic acid (EGTA). Release of α-amylase in response to the N⁶, O²′-dibutyryl or N⁶-monobutyryl derivatives of cyclic adenosine-3′,5′-monophosphate (cyclic AMP) was significantly inhibited by this procedure. When [K⁺]° was increased from 5.0 mM to 25.0 mM, Ca-depletion was accelerated, as was the inhibition of the response to the monobutyryl derivative. The Ca-depletion regimen did not affect the cellular content of other cations, suggesting that the effects were specific for Ca. The effects of the cyclic AMP derivatives on release of Ca was also investigated. Both monobutyryl and dibutyryl cyclic AMP significantly enhanced the rate of release of ⁴⁵Ca from pre-loaded parotid slices. These observations lend support to the hypothesis previously set forth suggesting that in the parotid, cyclic AMP acts to release Ca from intracellular stores. It is this rise in cytosolic Ca which may catalyze the events ultimately leading to exocytosis.     

The Origin of Carbons of Dihydrofuran Ring and C-6 in the Biosynthesis of Rotenone

For the investigation of rotenone biosynthesis, acetate-2-¹⁴C, mevalonic acid-2-¹⁴C lactone and methionine-methyl-¹⁴C were administered to Derris elliptica plants, respectively, and the distribution of carbon-14 in the labeled rotenone was determined by degradation. When mevalonic acid-2-¹⁴C lactone was incorporated into rotenone, the radioactivity was found equally in the carbons at both C-7′ and C-8′, indicating that these carbons are derived from the carbon-2 of mevalonic lactone. In the case of methionine-methyl-¹⁴C about 80% of the total radioactivity was found to enter two methoxyl groups. This result demonstrates that methionine is an efficient precursor of the methoxyl group. Furthermore, it is also suggested that methionine may be a precursor of the carbon at C-6.     

Metabolism of nicotine in Nicotiana glauca

A mixture of (?)-nicotine-[2′-³H] and (±)-nicotine-[2′-¹⁴C] was administered to Nicotiana glauca plants for 3 days, resulting in the formation of radioactive nornicotine (49·5% incorporation) and myosmine (2·05% incorporation). Negligible activity was detected in anabasine, cotinine, or 3-acetylpyridine, the last two compounds being added as carriers to the harvested plants. The radioactive nornicotine consisted of 48% (?)-nornicotine-[2′-¹⁴C,³H] and 52% (+)-nornicotine-[2′-¹⁴C]. Thus if (+)-nornicotine is formed from (?)-nicotine the transformation must involve loss of the hydrogen from C-2′. Myosmine is presumably formed from nicotine via nornicotine. However by feeding myosmine-[2′-¹⁴C] to N. glauca it was shown that the dehydrogenation is not reversible, no activity being detected in nornicotine. Nicotinic acid (0·14% incorporation) was a metabolite of myosmine-[2′-¹⁴C]. Essentially all the activity of the nicotinic acid was located on its carboxyl group, indicating that myosmine was a direct precursor.     

Conversion of 5' xanthylic acid to guanine and guanine nucleotides by a mutant of Brevibacterium ammoniagenes

A decoyinine resistant, KY 13501, isolated after nitrosoguanidine treatment from Brevibacterium ammoniagenes ATCC 6872 converted 5′XMP added in fermentation media to guanine derivatives and accumulated them in the media. The converted substances were identified as guanine, 5′GMP, 5′GDP, and 5′GTP. The conditions for the conversion were examined and the following points were clarified. (1) Very low concentration of manganese ion (Mn²⁺) showed profound effects on the conversion and the excessive amounts of the ion severely repressed the conversion. (2) Under limitation of Mn²⁺, 5′XMP was converted most efficiently when added at inoculation time. (3) The inhibition of the conversion by excessive amount of Mn²⁺ was completely released by addition of a surface activating agent, polyoxyethylene stearylamine. (4) For the conversion, it was essential to maintain pH of the media at 7.5 to 8.0 and supply ammonium ion.     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

Cytidylate cyclase: the product isolated by the method of Cech and Ignarro is not cytidine 3',5'-monophosphate.

When mouse liver homogenates were incubated with [α-³²P]-CTP according to the method of Cech and Ignarro (4,5), a [³²P] product (100–120 pmoles/mg protein/minute) was isolated by chromatography on alumina column, in the same fraction as [³H]-cyclic CMP.¹ However this product did not behave as cyclic CMP in various chromatographic systems. Moreover the amount of this [³²P] product isolated was markedly reduced by removal of protein prior to chromatography on alumina. Chromatography of the enzymatic reaction product either before or after alumina purification on Dowex 1 — formate column indicated that 5′-CMP and CDP are the major products, with no formation of cyclic CMP. These results indicate that the [³²P] labeled material isolated by Cech and Ignarro is mainly if not entirely 5′-CMP and CDP.     

Study on the Optical Rotatory Dispersion of Di-Peptides and its Application to the Recognition of Di-Peptides from Higher Peptides and Amino Acids

During the cource of the investigation of ribotidation of purine and pyrimidine bases by Brevibacterium ammoniagenes ATCC 6872, it was found that a large amount of uridine 5′-monophosphate (UMP) was accumulated in the culture broth when the organism was incubated in a medium containing uracil or orotic acid. The yields of UMP were 83% (4.8 mg/ml) from uracil and 100% (4.3 mg/ml) from orotic acid when each substrate was added at the concentration of 2 mg/ml.

Addition of 6-azauracil or 5-hydroxyuracil to the culture of the organism during cultivation led to the accumulation of both orotidine 5′-monophosphate (OMP) and UMP. The accumulation of OMP seemed to be due to the inhibition of OMP decarboxylase (E. C. 4.1.1.23) by the ribotide formed from each base. The OMP accumulation was enhanced by the addition of orotic acid in addition to 6-azauracil. When 6-azauracil was added to the medium before inoculation, UMP was predominantly accumulated, and when it was added after one day incubation, OMP was predominantly accumulated. A largest accumulation (3.6 mg/ml) of OMP was obtained when 6-azauracil was added on the 1st day and orotic acid was added on the 3rd day.

UMP and OMP accumulated in the medium were isolated from the cultured broth and identified by usual methods.     

The influence of cellular amino acids and the Na+: K+ pump on the membrane potential of the Ehrlich ascites tumor cell

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na⁺: K⁺ pump. The membrane potential (monitored by the fluorescent dye, 3,3′-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na⁺ but not cellular ATP. Na⁺ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na⁺ and amino acids.Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na⁺ (approx. 30 mM), the Na⁺: K⁺ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na⁺ was raised to 60 mM, the activity of the pump changed the membrane potential from the range ?25 to ?30 mV to ?44 to ?63 mV. This hyperpolarization required external K⁺ and was inhibited by ouabain.     

Nuclear magnetic resonance studies on pyridine dinucleotides. Carbon-13 assignments and structure determination of NADHX and the primary acid product of NADH.

The ¹³C spectra of β-NADH, NADHX, and the primary acid product of NADH were obtained and assigned. The conversion of the NADHX isomers to the two isomers of NADH acid product is demonstrated through the use of ¹³C-enriched compounds. The structure of NADHX is assigned as β-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide and the structures of the primary acid products of NADH are assigned as α-O^2′-6B-cyclotetrahydronicotinamide adenine dinucleotide and α-O^2′-6A-cyclotetrahydronicotinamide adenine dinucleotide.The structures of NADHX and the major isomer of the primary acid product, derived from studies of model compounds, are consistent with those proposed by Oppenheimer and Kaplan [Biochemistry (1974) 13, 4675, 4685]. However, the spectra of ¹³C-enriched primary acid product also demonstrated the existence of the A isomer which was not observed in the latter ¹H study. The A and B isomers were found to exist in the same ratio even when the primary acid product was formed directly from NADHX. This observation is discussed in terms of the previously proposed mechanism for the acid decomposition of NADH.     

N-(5'-phospho-4'-pyridoxyl)amines as substrates for pyridoxine (pyridoxamine) 5'-phosphate oxidase

A preparation of pyridoxine (pyridoxamine) 5′-phosphate oxidase, with a specific activity of 9,400 nmoles/hr/mg protein, 10-fold higher than that previously reported, was used to study the oxidation of various N-(5′-phospho-4′-pyridoxyl)amines. Values for K_m, from 3.1 × 10^?5 M to 1.6 × 10^?3 M, and for V_max, relative to pyridoxamine-P, of 20 to 140% were obtained. Compounds lacking a 5′-phosphate were not substrates, and the enzymic reaction was dependent on the presence of both FMN and O₂. N-(phosphopyridoxyl)-L-amino acids had lower K_m's than the corresponding -D-amino acid compounds. When 1-¹⁴C-N-(phosphopyridoxyl)glycine was used as a substrate, no ¹⁴CO₂ was evolved, and 1-¹⁴C-glycine was detected in the incubation mixture.