5' Cap methylation of homologous poly A(+) RNA by a RNA (guanine-7) methyltransferase from Neurospora crassa.

RNA (guanine-7) methyltransferase, partially purified from $\text{N.}\text{crassa}$ mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both $\text{N.}\text{crassa}$ poly A(+) RNA and reovirus unmethylated mRNA. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated poly A(+) RNA from $\text{N.}\text{crassa}$ yielded the “cap” structures m ⁷G(5′)pppAp and m ⁷G(5′)pppGp in a ratio of 2:1 respectively. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated reovirus mRNA yielded m ⁷G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in $\text{N.}\text{crassa}$ mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. $\text{187}$, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.     

The synthesis of freezing-point-depressing protein of the winter flounder Pseudopleuronectusamericanus in Xenpuslaevis oocytes

The serum of the winter flounder $\text{Pseudopleuronectus}$$\text{americanus}$ contains a freezing-point-depressing protein of a molecular weight approximately 10,000 with 60% alanine in its composition. When injected into Xenopus o?cyte, a 6–10 S, poly A-rich RNA preparation isolated from the fish liver polysomes stimulated 3–4 fold the incorporation of [³H] alanine into 10% trichloroacetic acid-soluble, non-dialysable proteins. Analysis of the protein fractions showed a translation product similar in molecular weight and electrophoretic mobility to flounder freezing-point-depressing protein. These observations indicated that the 6–10 S RNA from the flounder contained mRNA for the synthesis of flounder's freezing-point-depressing protein.     

Synthesis and antitumor activity of platinum complexes of protonated diamines

Complexes of the formula cis-[Pt(H$\text{N}\text{+}$^～N)(L)Cl₂], where (H$\text{N}\text{+}$^～N) are the protonated diamines including 3-aminoquinuclidine, N-aminopiperidine, piperazine, N-methylpiperazine, 1,1,4-trimethylpiperazine, and N-methyl-1,4-diazabicyclo [2,2,2] octane (N-methyl-dabco) and L = SCN^?, NO₂^?, Br^?, and F^?, were synthesized from the protonated diamine complexes, [Pt(H$\text{N}\text{+}$^～N)Cl₃]. The antitumor activities of the complexes were evaluated in vitro against L1210 murine leukemia cells, and ID₅₀ values for the L-substituted complexes were compared to values of the parent complexes. In each case it was found that replacement of a chloride ion by SCN^?, NO₂^?, Br^?, or F^?, either reduced or completely eliminated antitumor activity. This effect is explained in terms of the trans-directing ability of the ligand, L, compared to chloride. The NO₂-substituted complex of 3- aminoquinuclidine was tested in vivo and found to exhibit little or no antitumor activity.     

An mRNA decapping enzyme from ribosomes of Saccharomyces cerevisiae

By use of [³H]methyl-5′-capped [¹⁴C]mRNA from yeast as a substrate, a decapping enzyme activity has been detected in enzyme fractions derived from a high salt wash of ribosomes of $\text{Saccharomyces cerevisiae}$. The product of the decapping reaction is [³H]m⁷GDP. That the enzyme is not a non-specific pyrophosphatase is suggested by the finding that the diphosphate product, m⁷GpppA(G), and UDP-glucose are not hydrolyzed.     

Half-lives and relative amounts of stored and polysomal ribosomes and poly(A)+ RNA in mouse oocytes

Growing mouse oocytes were labeled in vitro with [³H]uridine and chased for 2 or for 7 days to estimate the relative amounts of RNA appearing in different fractions and to follow their turnover. Oocytes were lysed and thoroughly dispersed in the presence of 1% DOC, and centrifuged on sucrose gradients to separate polysomes from smaller components not engaged in translation. After the short chase, one-third of the labeled ribosomes appeared in EDTA-sensitive polysomes. The proportion of ribosomes in both fractions remained stable during the long chase, demonstrating no net flow from one fraction to the other. When gradient fractions were analyzed by poly(U) Sepharose chromatography, it was found that about 20% of the labeled poly(A)+ RNA appeared in polysomes after the short chase. The half-lives of stored and translated mRNA were followed relative to stable rRNA during the long chase. Stored mRNA was completely stable, but translated mRNA turned over with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 6 days. Other methods for separating stored from translated components were not successful, including sedimentation of putative large complexes (fibrillar lattices) containing stored components, or chromatography of lysates on oligo(dT)-cellulose. Results presented here combined with our previous results demonstrate that, during meiotic maturation, the percent of labeled stable RNA which is polyadenylated declines from 19 to 10%, suggesting deadenylation or degradation of half of the accumulated maternal mRNA.     

Catecholamine receptors and cyclic AMP formation in the central nervous system: effects of tetrahydroisoquinoline derivatives.

The ability of a series of tetrahydroisoquinoline (THIQ) alkaloids to inhibit the binding of radioligands to catecholamine receptors in the CNS has been examined. $\text{(+)}$ THP was the most potent inhibitor of [³H] dihydroalprenolol binding to β-adrenergic receptors and of [³H] haloperidol to dopaminergic receptors and was the least potent inhibitor of [³H] WB-4101 binding to α-adrenergic receptors. Other THIQ alkaloids examined such as salsoline, salsolinol, and reticuline were less potent than $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic and dopaminergic receptors, and more potent than $\text{(+)}$ THP in inhibiting radioligand to α-adrenergic receptors. The marked potency of $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic receptors (IC₅₀ ～ 10^?7 M) was confirmed by the potency of this compound in inhibiting (?) isoproternol elicited accumulations of cyclic AMP in brain slice preparations. These data indicate that, if formed $\text{in}$$\text{vivo}$ during alcohol consumption, THIQ derivatives such as THP may affect catecholamine neurons in the CNS.     

On the active site of hydrogenase from Chromatium vinosum

Isotope substitution of ⁵⁷Fe ($\text{I =}\text{1}\text{2}$) for ⁵⁶Fe has a pronounced effect on the two EPR signals of hydrogenase of Chromatium vinosum. It is proposed that signal 1, the intensity of which is increased several-fold by a deoxygenation-oxygenation cycle with a simultaneous increase of a signal from Fe³⁺, is due to a [3Fe-xS] cluster. It is further proposed that signal 2 is caused by a magnetic interaction of a [4Fe-4S]³⁺ cluster with an unidentified paramagnet. The addition of 10 μM Ni to the culture medium (already containing 1 μM Ni) increased the enzyme activity 3–6-fold, without effect on the growth of the bacterium. Addition of ⁶¹Ni ($\text{I =}\text{3}\text{2}$) to the medium did not change the EPR spectrum of hydrogenase. From a comparison of the EPR signal intensities and the enzyme activities it is concluded that, in the hydrogenase preparation as isolated, molecules containing a [3Fe-xS) cluster are not active, and that active molecules have a [4Fe-4S]^3+(3+,2+) cluster plus an as yet unidentified paramagnetic redox component. The latter is thought to be the primary site of interaction of the enzyme with H₂. Ni is considered as a possible candidate for this component.     

Studies on the metabolism of guanidine compounds in mammals

[¹⁴C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-¹⁴C]arginine or l-[guanidino-¹⁴C]canavanine. [${}^{14}\text{C}$]Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-¹⁴C]canavanine. These ${}^{14}\text{C}$ metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [¹⁴C]hydroxyguanidine to [¹⁴C]guanidine, and by repeated recrystallization of isolated urinary [${}^{14}\text{C}$]guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat.     

Competition between bacteriophage f2 RNA and bacteriophage T4 messenger RNA

In an $\text{Escherichia coli}$ cell-free protein synthesis assay, mRNA isolated from cells late after infection by phage T4 out-competes bacteriophage f2 RNA. Addition of a saturating or subsaturating amount of T4 mRNA inhibits translation of f2 RNA, while even an excess of f2 RNA has no effect on translation of T4 mRNA. Peptide mapping of reaction products labeled with formyl-[³⁵S]-methionyl-tRNA was used to quantitate f2 and T4 protein products synthesized in the same reaction. We suggest that messenger RNA competition might be one mechanism by which T4 superinfection of cells infected with phage f2 blocks translation of f2 RNA and possibly host mRNA.     

Determination of the multiplicity of the silk fibroin gene and detection of fibroin gene-related DNA in the genome of Bombyx mori.

The number of silk fibroin genes per genome in the silkworm Bombyx mori has been determined by hybridization using fibroin [¹²⁵I]mRNA. The purified [¹²⁵I]mRNA had an oligonucleotide pattern after RNAase T₁ digestion which was characteristic of fibroin mRNA (Suzuki &; Brown, 1972) and it hybridized specifically to DNA with a G + C content expected for a fibroin gene. Thermal denaturations indicated that these hybrids were mismatched by about 3%, which probably indicates some variation among the sequences encoding the internal repetitions of the fibroin protein.The concentration of fibroin gene sequences in B. mori DNA was measured by saturation hybridization of [¹²⁵I]mRNA to filter bound DNA. The same saturation level of 1.8 × 10^?5 μg mRNA per μg DNA was calculated from data obtained with unfractionated DNA and with fibroin gene sequences which had been separated from bulk B. mori DNA by actinomycin $\text{D}\text{CsCl}$ centrifugation. Scatchard plots of the subsaturation data extrapolated to an identical saturation value. Internal reiteration of the fibroin mRNA molecule was apparent from the high association constant of hybridization. An exhaustive hybridization experiment showed that such repetitions comprise at least 90% of each mRNA molecule. The saturation value, in conjunction with the genome DNA content and the mRNA size, indicated the presence of only one fibroin gene per haploid B. mori genome.Hybridization of actinomycin $\text{D}\text{CsCl}$ fractionated DNA indicated that fibroin mRNA can form hybrids with DNA that bands with bulk B. mori DNA. These hybrids appear to involve DNA which is related to, but distinguishable from, true fibroin gene sequences. The fibroin gene-related sequences form mismatched hybrids with the mRNA, are much shorter than the fibroin gene and are dispersed in B. mori DNA of much lower G + C content, and there are many copies of these sequences per B. mori genome.     

Concerning the specificity of heme oxygenase: the enzymatic oxidation of synthetic hemins.

Hemin XIII $\text{4}\text{～}$, hemin III $\text{5}\text{～}$, and iron $\text{1,4-di(β-hydroxyethyl)porphyrin}\text{6}\text{～}$ were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. These are all better substrates of the oxygenase than the natural substrate, hemin IX $\text{1}\text{～}$. The enzymatic oxidation was selective for the α-methine bridge and in every case only the α-biliverdins were obtained. The latter were readily reduced by biliverdin reductase to the corresponding α-bilirubins. The absence of isomers in addition to the α-bilirubins was established by preparing the derived azopigments and by using [α-¹⁴C]$\text{6}\text{～}$ and [α-¹⁴C]$\text{4}\text{～}$ as substrates. The chemical oxidation of $\text{4}\text{～}$, $\text{5}\text{～}$, and $\text{6}\text{～}$ gave the expected mixture of biliverdins. It is concluded that heme oxygenase is not specific for hemin IX. On the other hand, the enzyme is highly selective for the α-methine bridge, defined as the methine opposed to that flanked by the 6,7-propionic acid residues.     

Procedure for measurement of amino acid transport from blood to brain in small animals

The exponential plasma specific activity curve 2.5 to 12.5 min after injection (sc) of [¹⁴C]tyrosine was integrated and divided by time to obtain the mathematical relationship between the average equivalent specific activity $\text{S}$ and the measured specific activity S in any individual animal. $\text{S}$ is the constant, average value of S that is equivalent to the curvllinearly varying quantity that the body tissues are actually exposed to. Dividing the total brain radioactivity by $\text{S}$ gave the tissue Tyr uptake U. The function $\text{dU}\text{dt}$ is linear from 2.5 to 12.5 min and represents the rate of uptake of the amino acid. Incorporation into protein was similarly measured. Brain uptake of Tyr averaged 7.06, and the apparent protein incorporation was 1.99 nmol/g of brain per min. The γ-glutamyl cycle inhibitor l-methionine-RS-sulfoximine reduced total brain uptake of tyrosine by 42.8% and the apparent rate of protein incorporation by 39.0%.     

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

Molecular characterization of sinistirin

The molecular weight distribution of sinistrin (Inutest ®, Laevosan Ges., Linz, Austria), determined by analytical gel-permeation chromatography, using narrow fractions ($\text{M}$_w$\text{M}$_n< 1.07) obtained by preparative gel-permeation chromatography, covered the range 800–16,000 with $\text{M}$_n  2,500 and $\text{M}$_w  3,500. From viscosity measurements on dilute, aqueous solutions, the relation [η]  0.28 X M^0.3 was obtained, indicating a branched molecular structure; the largest molecules can be described by a sphere with r  23 Å. Comparison of the content of glucose and reducing sugars in the fractions with the molecular weight determined by vapour-pressure osmometry indicated that a glucose end-group is present in the majority of the molecules. The percentage of glucose end-groups is higher in the fractions of lower molecular weight. From this finding, speculations on the biosynthesis of sinistrin are made. The specific optical rotation of sinistrin fractions decreases linearly with 1/$\text{M}$_n.     

Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [¹⁴C]- or [³H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·10⁴ and 2.1·10⁴ were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with $\text{N-[}{}^3\text{H]ethylmaleimide}$ or $\text{N-[}{}^{14}\text{C]ethylmaleimide}$, and the ³H to ¹⁴C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.     

A study on the stability of an estrogen-protein conjugate in vivo and under in vitro conditions

[³ H] Estradiol-17β-succinyl bovine serum albumin conjugate ([³H]-E₂-BSA) was synthesized with a specific activity of 1.92 × 10⁷ cts/min/mg. The conjugate was administered iv to ovariectomized rats and the quantity of free [³H] steroid in the uterus was determined. Radioactive material was detected in all of the subcellular fractions of the uterus and identified as estradlol-17β. Similar subcellular distribution of the radioactivity was observed when [³H]E₂-BSA was added $\text{in vitro}$ to uterine homogenate. Free estradlo1-17β was released when the conjugate was Incubated with rat uterine homogenate or with serum. The results of the present study suggest that E₂-BSA is hydrolyzed $\text{in vivo}$ and under $\text{in vitro}$ conditions. It is recommended that the stability of a hormone-protein conjugate be established before use.     

Compartmentation of cytosolic dehydrogenases studied by transfer of tritium from labelled substrates into lactate in rat hepatocytes

This paper describes the transfer of tritium from [2-³H]xylitol or (1R)-[1-³H]ethanol into lactate in cells from fed rats either untreated or triiodothyronine-treated. The labelling pattern of lactate during the metabolism of [2-³H]xylitol or (1R)-[1-³H]ethanol follows the equation $\text{L = K(1?e}{}^{\mathrm{<mtext>?</mtext><mtext>t</mtext><mtext>\tau </mtext>}}\text{)}$ (μmol tritium/μmol lactate). The yield in lactate together with the minimum value of the total flux of reducing equivalents are used to estimate the specific radioactivity of NADH. We have calculated the lactate dehydrogenase-catalysed oxidation rate of NADH from the experimental values of lactate labelling and the specific radioactivity of NADH. We found the calculated flux of reducing equivalents from NADH to pyruvate to be of the same order of magnitude whether labelled ethanol or labelled xylitol was metabolized. We found the flux to be only a few percent of the maximal activity of lactate dehydrogenase. The results obtained suggest that the cytoplasm can be regarded as one compartment, containing a single pool of NAD(H).     

L-Proline site for triggering Bacillus megaterium spore germination.

Germination of $\text{Bacillus megaterium}$ QM B1551 spores can be triggered by L-proline chloromethyl ketone at ～ 10 fold lower concentrations than L-proline. [³H] L-proline chloromethyl ketone bound to several protein fractions, one of which was decreased in a mutant (JV137) that cannot be triggered by L-proline. Treatment of spores with [³H] acetic anhydride specifically inhibited L-proline triggered germination, and also covalently modified the same protein fraction which appears to be bound to the spore membrane. These results indicate that it is possible to identify a protein fraction in spores that may play a key role in triggering spore germination.     

The use of bacitracin as an inhibitor of the degradation of thyrotropin releasing factor and luteinizing hormone releasing factor

Bacitracin was found to be an effective inhibitor of the $\text{in}\text{vitro}$ degradation of both thyrotropin releasing factor¹ (TRF) and luteinizing hormone releasing factor (LRF) by guinea pig hypothalamic and whole brain homegenates and rat hypothalamic homogenates and subcellular fractions. Bacitracin was effective in inhibiting the degradation of TRF and LRF, as determined by radioimmunoassay, where it exhibited no interference with the assays. Kinetic studies of the degradation of exogenous synthetic [³H]-TRF demonstrated non-competitive inhibition by bacitracin with K_i = 1.9 × 10^?5 M, while studies on the degradation of [³H] LRF indicated competitive inhibition with K_i = 1.7 × 10^?5 M. Electrophoretic and amino acid analysis revealed that bacitracin itself was not degraded during the course of the $\text{in}\text{vitro}$ incubation.