Reductive activation of the methyl-tetrahydromethanopterin: coenzyme M methyltransferase from Methanobacterium thermoautotrophicum strain ΔH

The enzymatic conversion of formaldehyde to CH₃S-CoM in crude extracts of Methanobacterium thermoautotrophicum was used as a means to investigate the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction. All components necessary for formaldehyde conversion were shown to be present in a soluble protein fraction. This soluble cell fraction still contained a major amount of corrinoids. Apart from tetrahydromethanopterin no other soluble cofactors were required for formaldehyde conversion. The dependence of the system on catalytic amounts of ATP was shown to be specific. Several nucleoside triphosphates or ADP were unable to substitute for ATP. Remarkably, various strong reducing systems, especially titanium(III)citrate could replace ATP to a large extent. The ATP-dependent formaldehyde conversion to CH₃S-CoM was inhibited in the presence of nitrous oxide, detergents or 2,3-dialdehyde-ATP. The results support a role for a corrinoid protein in the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction at which ATP is involved in the activation of this protein, probably in the conversion of inactive B_12a or B_12r to active B_12s.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - BES 2-bromoethanesulfonate - BCE boiled cell-free extract - DTT dithiothreitol - TCS 3,3,4,5-tetrachlorosalicylanilide - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - AMP-PNP 5-adenylyl imidophosphate     

β-Ether cleavage of the lignin model compound 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether and derivatives by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 4-ethoxy-3-methoxyphenyl-glycerol--guaiacyl ether (V) in low nitrogen, stationary cultures under which conditions the ligninolytic enzyme system is expressed. 4-Ethoxy-3-methoxyphenylglycerol XIII, guaicol and 4-ethoxy-3-methoxybenzyl alcohol (II) were isolated as metabolic products. Exogenously added XIII was rapidly converted to 4-ethoxy-3-methoxybenzyl alcohol indicating that it is an intermediate in the metabolism of V. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-hydroxypropane VI. The degradation pathway for this dimer also included initial -ether cleavage and -hydroxylation of the diol product 1-(4-ethoxy-3-methoxyphenyl) 2,3 dihydroxypropane (XI) to yield the triol XIII which was cleaved at the , bond to yield 4-ethoxy-3-methoxybenzyl alcohol. Finally P. chrysosporium also cleaved the dimer 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1-hydroxypropane (VIII) at the -ether linkage yielding 1-(4-ethoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IX) which was subsequently cleaved at the , bond to yield II. All of the results indicate that oxidative -ether cleavage is an important initial reaction in the metabolism of -aryl ether lignin substructure dimeric compounds. Metabolities were identified after comparison with chemically synthesized standards by gas liquid chromatography-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography     

DNA variants within the 5′-flanking region of milk-protein-encoding genes II. The β-lactoglobulin-encoding gene

For the detection of polymorphisms within the 5-flanking region of the -lactoglobulin (-LG) -encoding gene a nucleotide sequence containing 795 bp of the promoter and 59 bp of exon I was cloned and sequenced. After comparing the sequence from the DNA of 11 diverse cows (different breeds and milk-protein yields), 14 singlebp substitutions were identified within the 5-flanking region and two in the 5-untranslated region (5-UTR) of exon I. Some of the variants are located in potential binding sites for trans-acting factors or in the 5-UTR. A PCR-based RFLP analysis was performed, and the genotypes of an additional 60 cows were identified at five variable 5-flanking sites. The results reveal three frequent combinations between the A and B alleles of the protein-coding region and the novel 5-flanking DNA variants. This finding may explain the differences of the protein-variant-dependent -LG synthesis (A>B) observed in vivo. A sequence comparison of the bovine and ovine promoters reveals an homology of 92.8% and shows a higher degree of conservation between positions -600 and -300.     

The reduction of tetrazolium salts by plant mitochondria

Summary Data has been obtained concerning the reduction of tetrazolium salts by mitochondria isolated from Jerusalem artichoke tubers with succinate as the substrate using a direct recording spectrophotometric method of assay. ATP was found to increase the rate of reduction of the tetrazolium salts, this being independent of the effect ATP had on the rate of oxygen uptake. The magnitude of the stimulation by ATP depended on the concentration of tetrazolium salts present and under certain circumstances was suppressed by the addition of azide and cyanide. The sites at which the tetrazolium salts were reduced along the electron transport chain were investigated. The role of ATP has been discussed in relation to the mechanism of tetrazolium reduction.Abbreviations TTC 2,3,5-triphenyl-2,1,3,4-tetrazolium chloride - BT 5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-diphenylene)-ditetrazolium chloride - NT 2,2,5,5-tetraphenyl-3,3-(p-diphenylene)-ditetrazolium chloride - MTT 3-(4,5-dimethyl thiozolyl-2)-2,5-diphenyl tetrazolium bromide - INT 2-(p-iodophenyl)-3-p-dinitrophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride - NBT 2,2-dinitrophenyl-5,5-diphenyl-3,3-dimethoxy-4,4-diphenylene)-ditetrazolium chloride - TNBT 2,2-5,5-tetra-p-nitrophenyl-3,3-dimethoxy-4,4-diphenylene) ditetrazolium chloride     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

Comparison of-nitro versus-amino 4, 4′-substituents of disulfonic stilbenes as chloride channel blockers

We showed previously that the disulfonic stilbene DNDS (4, 4-dinitrostilben-2, 2-disulfonic acid) was a potent blocker of outwardly rectifying chloride channels (ORCC). The studies reported here were designed to quantify the relationship between electron withdrawal by the 4, 4-substituents and blocker potency. Specifically we compared the blocking effects and molecular properties of the symmetrically substituted 4, 4-diaminostilben-2, 2-disulfonic acid (DADS) and the hemi-substituted 4-amino, 4-nitrostilben-2, 2-disulfonic acid (ANDS) with those of DNDS. Blockade was studied using outwardly rectifying colonic chloride channels incorporated into planar lipid bilayers. DADS was 430-fold and ANDS 44-fold less potent than DNDS as blockers of ORCC. Amplitude distribution analysis revealed that all three disulfonic stilbenes act as open channel blockers. Furthermore, this kinetic analysis indicated that the lower potency of DADS and ANDS was due to an increase in off rate. These results support the conclusion that the 4, 4-substituents make an important contribution to blockade by stabilizing the channel-blocker complex. Isopotential electron contour maps illustrated the dramatic shift in charge at the 4, 4-poles of the disulfonic stilbene molecule from electronegative in DNDS to electropositive in DADS as well as the bipolar contour of ANDS. Thus, the greater potency of DNDS results from the symmetric electronegative regions at the 4, 4-poles of the molecule. We hypothesize that the channel protein has two corresponding electropositive areas at the blocker binding site.     

Synthesis of oligoguanylates on oligocytidylate templates

Summary Short oligocytidylates can act as templates for the self-condensation of guanosine 5-phosphorimidazolide. In the absence of a catalytic metal ion or in the presence of Pb²⁺ a noticeable template effect is already observed with the dimer and the yield of long oligomers reaches a plateau with a hexamer template. Short templates give oligomers longers than the template length. The products are predominantly 2-5 linked for the Pb²⁺-catalyzed reaction while mixed linkages are observed in the uncatalyzed reaction.In the presence of Zn²⁺, a template effect is first observed with the pentamer and is maximal by the heptamer. The products are predominantly 3-5 linked. Oligomers shorter than or as long as the template are obtained in substantial yield, and longer products in much lower yields.Abbreviations G Guanosine - Gp guanosine 2(3)-phosphate - pG guanosine 5-phosphate - Gp! guanosine cyclic 2,3-phosphate - ImpG guanosine 5-phosphorimidazolide - ImpG^* [8-¹⁴C]-guanosine 5-phosphorimidazolide - pGp 5-phosphoguanosine 2(3)-phosphate - G²pG guanylyl-[2-5]-guanosine - G³pG guanylyl-[3-5]-guanosine - ImpGpG 5-phosphorimidazolide of GpG - (pG)_n (n = 2,3) oligomers of pG - GppG P₁, P₂-diguanosine 5-diphosphate - GppGpG 5-[guanosine 5-pyrophosphate] of GpG - NH₂pG guanosine 5-phosphoramidate - (pG)₄₊ tetramer and higher oligoguanylates with 5 terminal phosphate - oligo(G) oligoguanylate - Cp cytidine 2(3)-phosphate - Cp! cytidine cyclic 2,3-phosphate - (Cp)_n–1 Cp! (n= 2,3,4) oligocytidylates terminated by 5-OH groups and 2,3-cyclic phosphates - oligo(C) oligocytidylate - poly(C) polycytidylic acid - poly(U) polyuridylic acid - poly(C,G) random copolymer of C and G - BAP bacterial alkaline phosphatase (E. coli) - EDTA ethylenediaminetetraacetic acid - R_f chromatographic mobility     

Nucleic acid metabolism in yeast VI. Utilisation of exogenous dAMP

Summary It is shown that mutants of Saccharomyces cerevisiae able to efficiently utilise exogenous dTMP can also utilise exogenous dAMP. Under extracellular conditions permissive for dTMP uptake label stemming from offered [8-³H]dAMP is incorporated preferentially into alkali-resistant, high molecular weight material (putative DNA): only about 30% of high molecular weight cell-bound dAMP label was found to be sensitive towards mild alkali hydrolysis. This putative RNA label can be minimised to practically zero when mM Ade is employed in a dAMP labelling assay. Exogenous dAMP at 10 M was found to be cytostatic similarly to M dTMP and similarly to inhibit effectively import of exogenous P_i. We conclude from our results that there exists a yeast cytoplasmic membrane permease able to import dAMP. A model of this hypothetical permease system is presented.Abbreviations S. cerevisiae Saccharomyces cerevisiae - TIP yeast cytoplasmic membrane permease importing dTMP under permisive conditions - AIP hypothetical yeast cytoplasmic membrane permease importing dAMP under permissive conditions - tlr symbol for the recessive genetic trait to utilise effeciently exogenous dTMP - tmp symbol for the recessive genetic trait leading to dTMP auxotrophy - dTMP 2-deoxythymidine 5-monophosphate - dAMP 2-deoxyadenosine 5-monophosphate - (d)NMP (deoxy)ribonucleoside 5-monophosphate - dThd 2-deoxythymidine - Thy thymine - dAdo 2-deoxyadenosine - Ado adenosine - Ade adenine - Ura uracil - P_i inorganic phosphate Apart from discrete abbreviations we followed the rules of nomenclature as recommended by the IUPAC-IUB commission of biochemical nomenclature (CBN)     

Geometry of the dry-state oligomerization of 2′,3′-cyclic phosphates

Summary Evaporation of a solution of thymidine plus either theexo or theendo diastereomer of uridine cyclic 2,3-O, O-phosphorothioate (U > p(S) in 1,2-diaminoethane hydrochloride buffer gave the 2,5 and 3,5 isomers of (P-thio) uridylylthymidine (Up(S)dT) in a ratio of 1:2 with a combined yield of about 20%. These isomers were re-converted to U > p(S) and dT by a reaction that is known to proceed by an in-line mechanism. Both the 2,5 and 3,5 isomers gave as product the same diastereomer of U > p(S) that had been used originally in their formation. These dry-state prebiotic reactions (Verlander, Lohrmann, and Orgel 1973) are thus shown to be stereospecific, and both the 2,5 and 3,5 internucleotide bonds are formed by an in-line mechanism.Abbreviations DAE 1,2-diaminoethane - HPLC high pressure liquid chromatography - RNase bovine pancreatic ribonuclease A, EC 3.1.4.22 - TEAB triethylammonium bicarbonate - tris tris(hydroxymethyl)aminomethane - UMP(S) uridine monophosphorothioate - U > p uridine cyclic 2,3-phosphate - U > p(S) uridine cyclic 2,3-O, O-phosphorothioate - Up(S)dT (P-thio)uridylylthymidine - U2p(R_p-S)5dT (P-thio)uridylylthymidine with theR configuration at phosphorous, and a 2,5 internucleotide linkage     

A cytokinin-sensitive mutant of the moss,Physcomitrella patens,defective in chloroplast division

Summary An X-ray induced mutant (PC22) of the moss,Physcomitrella patens was analysed with respect to its morphology, physiology and suitability for microculture techniques. The mutant protonemata are defective in bud formation and in chloroplast division. As a consequence of the latter, giant chloroplasts are formed which disturb the development of the phragmoplast, the formation of regular cross walls, and cell division. Abnormal cross walls are rich in callose. The actin cytoskeleton was found to be less regularly developed in the mutant than in the wild type. Three-dimensional analysis of the microtubular arrangement with confocal laser scan microscopy demonstrates a close association between spindle- or phragmoplast- and interphase-microtubules. The deficiencies in chloroplast division and in bud formation can partly be compensated for by exogeneously applied cytokinin. The suitability of this particular developmental mutant for further studies was shown by regeneration of protoplasts in microculture and microinjection of the fluorochrome Lucifer yellow into the chloroplast.Abbreviations CLSM confocal laser scan microscope - DAPI diamidinophenyl indole - DiOC 3,3-dihexyloxacarbocyanine iodide - EGTA ethylene glycol-bis-(-amino-ethylether-N,N,N,N-tetraacetic acid - i⁶Ade N⁶-(²-isopentenyladenine) - PIPES piperazine-N, N-bis-2-ethanesulfonic acid - ptDNA chloroplast DNA Devoted to the memory of Prof. Dr. O. Kiermayer, our colleague and friend.     

Pollen tube extract supports the movement of actin filaments in vitro

Summary Muscle actin filaments labeled with rhodamine-phalloidin were observed to move on the surface coated with a crude extract of pollen tubes ofLilium longiflorum with an average velocity of 1.99±0.55 m/sec. The movement required both Mg²⁺ and ATP. These results indicate that the extract of pollen tubes contains a myosin-like translocatorAbbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride     

Physical properties and metabolite regulation of ribulose bisphosphate carboxylase from Thiobacillus A2

Purified ribulose-bisphosphate carboxylase (EC 4.1.1.39) was strongly and equally inhibited either by ADP or GDP and to a lesser extent by IDP. AMP or ATP exerted little effect on activity. Inhibition by the nucleotide diphosphates was competitive with respect to RuBP and non-competitive with respect to CO₂ and Mg²⁺, respectively. Treatment of the enzyme with urea or guanidine-HCl resulted in rapid loss of activity that was not restored by dialysis even in the presence of Mg²⁺ and cysteine. Sodium dodecyl sulfate electrophoresis of 8.0 M urea treated enzyme revealed the presence of a fast-moving (small) sub-unit with molecular weight 14150 and a slower moving (large) sub-unit with molecular weight 68000. Examination of native enzyme by sodium dodecyl sulfate electrophoresis gave sub-units of 13700 and 55500 respectively. The amino acid content standardized to phenylalanine was essentially similar to that from other sources. Arrhenius plots showed a break at 29°C with an E _a of 12.34 kcal per mole for the steeper part of the curve and a H of 11.43 kcal per mole while for the less steep region, the E _a was 1.04 kcal per mole and the H 1.92 kcal per mole.Abbreviations ADP adenosine-5-diphosphate - AMP adenosine-5-monophosphate - ATP adenosine-5-triphosphate - CDP cytidine-5-diphosphate - CMP cytidine-5-monophosphate - CTP cytidine-5-triphosphate - FDP fructose-1,6-diphosphate - F6P fructose-6-phosphate - GDP guanosine-5-diphosphate - GMP guanosine-5-monophosphate - G6P glucose-6-phosphate - GTP guanosine-5-triphosphate - IDP inosine-5-diphosphate - IMP inosine-5-monophosphate - PEP phosphoenolpyruvate - 6PG 6-phosphogluconate - R1P ribose-1-phosphate - R5P ribose-5-phosphate - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecyl sulfate - TDP thymidine-5-diphosphate - TMP thymidine-5-monophosphate - TTP thymidine-5-triphosphate - UDP uridine-5-diphosphate - UMP uridine-5-monophosphate - UTP uridine-5-triphosphate     

Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5′-azido-salicylamido)-undecanoic acid derivatives

Summary Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5-azido-salicylamido)-undecanoic acid (5 ASU) and its acetyl ester (Ac5 ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5 ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34×10^–7 M, evidenced a slightly higher affinity than that reported for C16–C20 fatty acids. Consistent with the binding curve, 5 ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 gmM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of¹²⁵I-5 ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t_1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5 ASU and Ac5 ASU respectively. In turn, irradiation of L-FABP incubated with 5ASU or Ac5 ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid. Both results, taken together, suggested that the derivatives are linked to the protein through the sites for fatty acids. When cross-linking of¹²⁵I-5 ASU was performed after incubation with delipidated cytosol and products were analyzed by SDS-PAGE, a band was visualized in a position similar to that of purified L-FABP.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Hepatic FABP - I-FABP Intestinal FABP - C-FABP Cardiac FABP - 5 ASU-11 (5-azido-salicylamido)-undecanoic acid - Ac5 ASU-11 (O-acetyl-5-azido-salicylamido)-undecanoic acid     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

Biochemical and immunological responses of hairy cell leukemia patients to interferonβ

Summary Ten hairy-cell leukemia patients were treated with interferon (IFN-) at a dose rate of 2 × 10⁶ IU/m² × 5 days for 4 weeks (induction therapy) and, thereafter, at the same dose three times a week for 11 months (maintenance therapy). The effect of this treatment on serum neopterin, 2-microglobulin, (2–5)oligoadenylate [(2–5)A _n ] levels, intracellular (2–5)A _n values and human Mx protein synthesis was analysed. There were significant rises in serum neopterin and (2–5)A _n levels during both induction and maintenance, whereas 2-microglobulin levels rose only during induction. Rises in intracellular (2–5)A _n were documented mainly during induction, but they were not significantly higher than pretherapy values. IFN provoked an increase in human Mx protein synthesis over the entire induction — maintenance period, but was only significantly higher than baseline during induction. All markers proved useful for monitoring the effects of IFN dose schedules, but were not predictive of clinical outcome. Natural killer activity and IFN production, which were initially defective, followed a different trend from that of the other factors studied, in that increases were documented only late in the course of therapy when the disease was already in remission.     

Heteropolynucleotides as templates for non-enzymatic polymerizations

Summary We have studied a number of condensation reactions involving ImpU, ImpT, ImpC, ImpA, ImpG, ImpUpG and ImpCpA as activated nucleotide donors and a variety of homo- and hetero-polynucleotides as templates. We did not obtain any evidence of a template effect with ImpU and ImpT, but observed some condensation of ImpC with GpG on appropriate templates. ImpA and ImpG take part in a number of more or less efficient template-directed reactions, as do ImpUpG and ImpCpA.Our results suggest that, on the primitive Earth, pyrimidine nucleotides could most easily have been incorporated into polymers as constituents of short oligomers, which contained one or more purine nucleotide. The linkage of the product depends strongly on the nature of the substrates; the percentage of the natural 3-5-linkage was, in some cases, less than 10% and, in others, as high as 70%. Wobble-pairing was often very effective in promoting condensations, suggesting that transition mutations would have been very frequent in prebiotic polynucleotide replication.Abbreviations and Conventions U uridine - T thymidine - C cytidine - A adenosine - G guanosine - pN nucleoside-5-phosphate - Np a mixture of 2- and 3-phosphates of a nucleoside - pNp a mixture of the 2-5-diphosphate and 3-5-diphosphate of a nucleoside - N₁ ² pN₂ a 2-5-linked dinucleoside monophosphate - N₁ ³ pN₂ a 3-5-linked dinucleoside monophosphate - N⁵ ppN a pyrophosphate derived from a nucleoside-5-phosphate. ImpN and ImpN₁pN₂ are 5-phosphorimidazolides of nucleosides and 3-5-linked dinucleoside monophosphates, respectively - poly(N) a homopolynucleotide - poly (U₁ C₂ A₄ G₃) a random copolymer derived from a substrate mixture containing U, C, A, G in ratio 1:2:4:3 - ODU optical density units measured at 260 nm     

Isolation and characterization of a multienzyme complex containing DNA replicative enzymes from mitochondria ofS. cerevisiae

A 40 S multienzyme complex containing mtDNA polymerase was isolated from mitochondria ofS. cerevisiae by density gradient centrifugation and by gel filtration chromatography. Besides DNA polymerase, RNA polymerase, primase, 35 exonuclease and an ATPase activities were found to be associated with it. The presence of some of these enzymes were confirmed by Western blot. This high molecular weight multienzyme complex containing DNA has most of the attributes of a putative replisome.Abbreviations BCIP 5 bromo 4 chloro 3 indolyl phosphate - mtDNA mitochondrial DNA - NBT Nitroblue tetrazolium - PBS phosphate buffered saline     

The composition of Erythrosins, Fluorescein, Phloxine and Rose Bengal: a study using thin-layer chromatography and solvent extraction

Synopsis Commercial samples of Erythrosin B (CI 45430), Erythrosin Y (CI 45425), Fluorescein (CI 45350), Phloxine (CI 45410) and Rose Bengal (CI 45440) have been analysed by thin-layer chromatography. The Erythrosins were found to be mixtures consisting in the main of 4-iodofluorescein, 4,5-di-iodofluorescein, 2,4,5-triiodofluorescein and 2,4,5,7-tetraiodofluorescein, in some instances together with 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein. Samples of Fluorescein were mixtures of the nominal dye usually with traces of several unidentified, fluorescent components. Those of Phloxine consisted mainly of mixtures of 4-bromo-4,5,6,7-tetrachlorofluorescein, 4,5-dibromo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tribromo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetrabromo-4,5,6,7-tetrachlorofluorescein, often with 4,5,6,7-tetrachlorofluorescein Samples of Rose Bengal were mixtures of 4-iodo-4,5,6,7-tetrachlorofluorescein, 4,5-di-iodo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein together with some unidentified components.Most of the commercial dye samples gave an insoluble residue when extracted with methanol. This residue was usually inorganic carbonate or halide. Some possible practical consequences of the various impurities are discussed.     

Sequence- and Regio-Selectivity in the Montmorillonite-Catalyzed Synthesis of RNA

The six binary montmorillonite clay-catalyzed reactions of the5-phosphorimidazolides of adenosine, cytidine, guanosine anduridine were performed and the eight dimers from each reactionwere separated and analyzed by HPLC. A 16–51-fold higher yieldof the 5-purine-pyrimidine dimers over that of the5-pyrimidine-purines was observed. The total yield of the5-purine-pyrimidine dimers was in the 50–70% range while thatof the 5-pyrimidine-purine dimers was 1.3–7.0%. Less sequenceselectivity was observed in the homodimers formed.Regioselectivity for the formation of 3, 5-phosphodiesterbonds over that found in the absence of clay was observed. The5-purine-pyrimidine, 5-pyrimidine-pyrimidine and5-purine-purine dimers had 3, 5-links in about half of theirphosphodiester bonds. The percent phosphodiester links in the5-pyrimidine-pyrimidine dimers was 18%, a value close to thatobserved in the absence of the montmorillonite catalyst. Themontmorillonite-catalyzed reaction of all four activatednucleotides was performed and the 24 products were separated andanalyzed. The trends observed in the binary reactions wereconfirmed and the results also showed that the relativereactivity of the activated monomers was A>G>C>U in theratio 8.2: 4.8: 1.3: 1 respectively. No 5-pyrimidine-purineswith a 5-U and pG³pU, pC³pAand pC³pG weredetected. These studies suggest that a limited population ofRNAs would have formed in catalyzed prebiotic reactions.