C′3 polymorphism in Italy

Summary C'3 phenotype and gene frequencies observed in two Italian samples are reported. The allele frequencies resemble those reported for other Caucasian populations. Five different rare variants are described.     

Deuteration and Tritiation of 2′-Deoxyribonucleosides in the 5′ and 4′ Positions

Abstract

The deuterations of 2′-deoxyguanosine in the 4′ and 5′ positions have been described elsewhere (1). The starting material is the 5′-aldehyde formed by mild oxidation with N,N-dicyclohexyl carbodiimide in dimethyl sulphoxide of the fully protected nucleoside with free 5′-alcoholic function. The 5′4euteration was achieved by reduction with deuterated sodium borohydride. Incorporation of deuterium in the 4′-position was achieved v i a an enhanced keto-enol tautomerim by heating the aldehyde in 50/50 D20/pyridine, with subsequent reduction of the aldehyde with NaBH4. The 6-furanoid form was isolated from the I-lyxo by-product by reverse phase HPLC. Applied to pyrimidine 2′-deoxyribonucleosides, this method was shown to give deuterated 2′-deoxycytidine and thymidine in good yield.     

Occurrence of Guanosine-5′-diphosphate-3′-diphosphate and Adenosine-5′-triphosphate-3′-diphosphate in Streptomyces galilaeus

Synthetic activity and existence of ppGpp and pppApp in an anthracycline-producing strain Streptomyces galilaeus were determined by radioimmunoassay and ³²P-labeling method during cultivation under both the antibiotic productive and non-productive conditions. The cellular ppGpp(pppGpp)-synthesizing activity was highest at the end of exponential growth, and 3-fold higher in the antibiotic-productive cultivation than in non-productive cultivation. The intracellular level of ppGpp determined by radioimmunoassay was high at the end of exponential growth, and afterwards its level decreased by one fifth. The low level of cellular ppGpp during the period of intense antibiotic production suggests that ppGpp consumption may play an important role in antibiotic production of S. galilaeus. The concentration of pppApp was not determined intracellularly by radioimmunoassay.     

Cyclisches Adenosin-3′:5′-monophosphat in pflanzlichen Leitbahnen?

Summary Sieve tube sap of the secondary phloem of Robinia pesudoacacia L. and exudate from the trumpet hyphae of the cauloids of Laminaria saccharina (L.) Lamour. were analyzed for the occurrence of cyclic adenosine-3:5-monophosphate with the help of the radio isotope dilution test (Boehringer, Mannheim). The concentration was about 0,1 M in the Laminaria exudate and 9,0 nM in the Robinia sap. Pretreatment of the saps with phosphodiesterase removes the activity of the A-3:5-MP. It therefore seems probable that A-3:5-MP occurs in the translocation tissues of plants.     

Adrenaline Increases Cyclic 3′5′-AMP Formation in Hamster Epidermis

CATECHOLAMINES probably influence cell proliferation by delaying cells in the premitotic phase^1,2. Bullough and Laurence found that crude skin extracts contained a tissue-specific protein (chalone) which inhibited epidermal cell proliferation and that the action of this extract was augmented by adrenaline³. They later found that adrenaline alone (0.00025 µg/ml.) reduced epidermal mitotic activity in mouse ears by about 50% in vitro⁴.     

Conserved Putative Signals in 3′ Intron Junctions in Rodents

Abstract

Several 3′ splice signals are known todate. At the 3′ splice site an AG doublet is frequently found. Just upstream of the splice site there is a string of 6–11 pyrimidines. More recently it has been found that one of the stages in the splicing process involves formation of a lariat, in which the 5′ end of the intron forms a 2′-5′ branch with an A residue located 18–37 nucleotides upstream of the 3′ splice site. The branching-point consensus is weakly defined and consists of the sequence YNYTRAY, where Y is a pyrimidine, R a purine and N any base. The A in the sixth position is the one with which branching occurs. Here we present the results of extensive searches for additional putative signals around the branching-point consensus and the 3′ splice site in rodent nuclear precursor mRNAs. The signals obtained for the over 370 rodent introns are compared with those found in a larger eukaryotic sample containing over 900 nuclear pre-mRNA introns. Of particular interest are GGGA and CCCA In both analyses GGGA occurs about 60 nucleotides upstream and CCCA is found 3–40 nucleotides downstream from the 3′ splice site. A model explaining some of the putative signals discussed here is also proposed. This model involves formation of alternate stem-loop structures around the branching point and 3′ splice site. Such signals and structures can possibly aid in protein or nucleoprotein branching point and splice site recognition.     

Studies on 5′-Phosphodiesterases in Microorganisms

5′-Phosphodiesterase, which degrades RNA into nucleoside-5′-monophosphates but does not attack DNA, is present not only in mycelium but also in culture filtrate of Penicillium citrinum Thorn 1131. For the formation of this enzyme pH of the culture medium must be kept below 7.0 during culture, as this enzyme is inactivated rapidly in alkaline solution. The pH optimum of this enzyme is in the region of pH 5. Cysteine, Mg⁺⁺, sodium fluoride, and inorganic ortho- or pyrophosphate are without appreciable effect on this enzyme. Nucleoside-5′-monophosphates, which have been regarded as new chemical seasonings, can be produced economically in a large scale by using the microbial 5′-phosphodiesterase.     

Diurnal variation in 5′-nucleotidase activity in rat liver

Summary The diurnal variation of 5-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.     

5′-Nucleotidase in spinal meningeal compartments in the rat

Summary The distribution of 5-nucleotidase (5-Nu) is reported in spinal meninges of the rat on the basis of an immunohistochemical and enzyme histochemical investigation. Strong immunoreactivity was found in the arachnoid membrane and in the sheaths of the spinal roots as well as in septa subdividing the roots. Also the superficial layer of the ligamentum denticulatum showed enzyme staining. No immunoreactivity could be detected in the pia mater or along the spinal nerve roots outside the subarachnoid space. Within the arachnoid mater the immunoreactivity was concentrated in the basal zone of the arachnoid membrane, thus appearing as a narrow fluorescent band near the border of the dura. An accentuation of immunoreactivity could be observed in areas where small dural blood vessels approach the subarachnoid space. It is well known that adenine nucleotides released from neural and glial cells of the central nervous system finally reach the cerebrospinal fluid. We presume that 5-Nu in the arachnoid membrane and spinal root sheaths is responsible for the conversion of adenine nucleotides into adenosine and that this conversion is associated with the reabsorption process of cerebrospinal fluid which most probably also takes place in spinal meninges. Adenosine, the product of 5-nucleotidase, could play a role in the reabsorption process by its vasodilatatory effect on dural and epidural vessels.     

Gene-dependent flavonoid 3′-hydroxylation in maize

The influence of the gene Pr on flavonoid 3-hydroxylase activity in maize is described. Specific activities are presented for the hydroxylase in seedlings and aleurone tissue homozygous dominant and recessive and heterozygous for Pr. Specific activity levels in both tissues increased in a nearly direct proportion with the increase in Pr dosage, which is consistent with Pr being the structural gene for the hydroxylase. Regression analysis of the gene dosage:enzyme activity comparison yielded correlation coefficients of 0.979 and 0.959 for the seedlings and aleurone, respectively. Quantitative identification of the cyanidin and pelargonidin in the aleurone indicated that cyanidin increased with an increase in dominant Pr, while pelargonidin decreased, although the increases and decreases observed were not directly proportional to the gene dosage. Comparison of the cyanidin/pelargonidin ratio to the gene dosage ratio in the different tissues showed a strong correlation (0.998), which demonstrates that the dosage of Pr controls the ratio of cyanidin to pelargonidin. Cyanidin was found at a low concentration in aleurone homozygous for pr. Hydroxylase activity in maturing field plants reaches its peak concentration near anthesis and is present at an appreciable concentration in mature plant tissue homozygous for pr, as well as in seedlings homozygous for pr. Suggestion is made that pr could be a hypomorphic allele or that a duplicate gene for Pr could exist to account for the hydroxylase activity in homozygous pr tissue. Evidence for the hydroxylase in the aleurone and the seedlings and the pigment ratio data from the aleurone suggest that Pr is indeed a structural gene for NADPH:flavonoid 3-hydroxylase.Cooperative Investigations, Agricultural Research Service, United States Department of Agriculture, and Missouri Agricultural Experiment Station, Columbia, Missouri 65211. Journal Series No. 9958.     

N,N′-carbonyldiimidazole-induced diketopiperazine formation in aqueous solution in the presence of adenosine-5′-monophosphate

Summary 3(2)-O-glycyl-adenosine-5-monophosphate is an intermediate in the conversion of N-[imidazolyl-(1)-carbonyl]-glycine to diketopiperazine in the presence of adenosine-5-monophosphate. The significance of these observations to prebiotic chemistry is discussed.Abbreviations AMP adenosine-5-monophosphate - A adenosine     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.     

Defect in 3′-phosphoadenosine 5′-phosphosulfate synthesis in brachymorphic mice: I. Characterization of the defect

Biosynthesis of the undersulfated proteoglycan found in brachymorphic mouse (bm/ bm) cartilage has been investigated. Similar amounts of cartilage proteoglycan core protein, as measured by radioimmune inhibition assay, and comparable activity levels of four of the glycosyltransferases requisite for synthesis of chondroitin sulfate chains were found in cartilage homogenates from neonatal bm/bm and normal mice, suggesting normal production of glycosylated core protein acceptor for sulfation. When incubated with ³⁵S-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS), bm/bm cartilage extracts showed a higher than control level of sulfotransferase activity. In contrast, when synthesis was initiated from ATP and ³⁵SO₄^2?, mutant cartilage extracts showed lower incorporation of ³⁵SO₄^2? into endogenous chondroitin sulfate proteoglycan (19% of control level) and greatly reduced formation of PAPS (10% of control level). Results from coincubations of normal and mutant cartilage extracts exhibited intermediate levels of sulfate incorporation into PAPS and endogenous acceptors, suggesting the absence of an inhibitor for sulfate-activating enzymes or sulfotransferases. Degradation rates of ³⁵S]PAPS and of ³⁵S-labeled adenosine 5′-phosphosulfate (APS) were comparable in bm/bm and normal cartilage extracts. Specific assays for both ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) showed decreases in the former (50% of control) and the latter (10–15% of control) enzyme activities in bm/bm cartilage extracts. Both enzyme activities were reduced to intermediate levels in extracts of cartilage from heterozygous brachymorphic mice (ATP-sulfurylase, 80% of control; APS kinase, 40–70% of control). Furthermore, the moderate reduction in ATP sulfurylase activity in bm/bm cartilage extracts was accompanied by increased lability to freezing and thawing of the residual activity of this enzyme. These results indicate that under-sulfation of chondroitin sulfate proteoglycan in bm/bm cartilage is due to a defect in synthesis of the sulfate donor (PAPS), resulting from diminished activities of both ATP sulfurylase and APS kinase, although the reduced activity of the latter enzyme seems to be primarily responsible for the defect in PAPS synthesis.     

Comparison of 3′ and 5′ biotin labelled oligonucleotides for in situ hybridisation

Oligonucleotide probes enzymatically labelled at the 3-end with biotin have been used successfully to detect target RNA and DNA in combination with in situ hybridisation. Addition of multiple biotin residues to the 3-end increases the hybridisation signals, but it is not known whether the same principle is applicable to the 5-end. We have labelled a 35-base oligonucleotide during synthesis with 1, 5 and 12 biotin molecules at the 5-end and compared it to conventional 3-labelling. In additional experiments the probes were labelled at both ends. Probes were applied to histological sections obtained from paraffin-embedded cell-clot-complexes that contain uninfected and Rhinoviral-infected cells, using a standard in situ hybridisation protocol with appropriate controls. Hybridisation signals were compared for intensity of cytoplasmic signal and sensitivity as number of positive cells. Both parameters increased in parallel with higher numbers of biotin residues attached to the 5-end and 12 biotin residues were almost as effective as 3-enzymatic tailing. The sensitivity could be increased above that of either 3- or 5-labelling by the addition of residues at both ends of the probe. The 5-attachment of biotin residues can extend the value of oligonucleotide probes employed for in situ hybridisation and yield increased sensitivity when combined with 3-enzymatic labelling.     

2′,3′-Cyclic nucleotide 3′-phosphohydrolase in rat liver mitochondrial membranes

2′,3′-Cyclic nucleotide 3′-phosphohydrolase (nucleoside-2′:3′-cyclic-phosphate 2′-nucleotidohydrolase, EC 3.1.4.37) activity has been demonstrated in rat liver mitochondria. The enzyme was localized in both the outer and inner mitochondrial membranes but was absent from the intermembrane space and matrix. The mitochondrial (cyclic nucleotide) phosphohydrolase was activated by freezing and thawing and by treatment with digitonin or detergents. It is suggested that (cyclic nucleotide) phosphohydrolase is an integral membrane protein which is buried to a significant degree within the membrane. Atractyloside was found to be a noncompetitive inhibitor of the enzyme both in intact mitochondria and in preparations of the mitochondrial membranes. The enzyme substrate, 2′,3′-cyclic adenosine monophosphate, had no effect on the oxidation of exogenous β-hydroxybutyrate or succinate by intact mitochondria. These findings suggest that 2′,3′-cyclic nucleotide 3′phosphohydrolase is more widely distributed than was previously thought and that the enzyme may play a fundamental role in membranes, independent of their specialized structure or functions.     

6′-Mercaptoguanosine-resistance is related with purF gene encoding 5′-phosphoribosyl-1′-pyrophosphate amidotransferase in inosine-5′-monophosphate overproducing Brevibacterium ammoniagenes

From the gene library constructed with the chromosomal DNA of 6-mercaptoguanosine (MGS)-resistant strain Brevibacterium ammoniagenes IPR-1, a DNA fragment which conferred MGS-resistance to the wild-type strain B. ammoniagenes ATCC6872 was cloned. The purF gene encoding 5-phosphoribosyl-1-pyrophosphate amidotransferase was identified from this fragment and its nucleotide sequence was determined. Wild type purF gene was also cloned by polymerase chain reaction using chromosomal DNA of ATCC6872 as the template and its sequence was determined. Two nucleotides, 583 A and 1065 A, of MGS-resistant purF gene had been changed from 583 G and 1065 G by mutagenesis, respectively. Both changes at position 583 and 1065 were proved to be responsible for MGS-resistance by site-directed mutagenesis.     

Biological activities of human interferon and 2′–5′ oligoadenylates in plants

Exogenous human interferon 2 (IFN) and 2–5 oligoadenylates (2–5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1–1 u/ml and 2–5A at 1–10 nM.     

Adenosine 3′:5′-cyclic monophosphate and catabolite repression in Escherichia coli

1. Both permanent and transient catabolite repression of beta-galactosidase synthesis in Escherichia coli are abolished by 5mm-3':5'-cyclic-AMP when elicited by glucose, but not when caused by a mixture of glucose, glucose 6-phosphate, gluconate and casein hydrolysate (casamino acids). 2. Glucose uptake is slightly increased by 3':5'-cyclic-AMP. 3. No significant effects of the nucleotide were found on the synthesis of protein and RNA, either in exponential growth on one substrate, or during a growth shift from glycerol to glycerol plus glucose. 4. Marked changes in the soluble-protein profiles of cells growing in glycerol and glucose were caused by the presence of 3':5'-cyclic-AMP. 5. Measurements of (14)CO(2) release from specifically-labelled glucose showed that 3':5'-cyclic-AMP greatly stimulated glycolytic activity while having a minor depressing effect on the metabolic flow through the pentose phosphate cycle. 6. The concentrations of several metabolic intermediates, particularly fructose 1,6-diphosphate, were greatly affected by the presence of 3':5'-cyclic-AMP. 7. Several metabolites partially relieved glucose repression of beta-galactosidase synthesis in EDTA-treated cells; three out of five of these metabolites reversed the effect more effectively than did 3':5'-cyclic-AMP. 8. The evidence for and against a direct role for 3':5'-cyclic-AMP is discussed. It is concluded that the evidence for indirect action is at least as strong as that for direct action.     

The Presence of 3′–5′ Exonuclease Activity in Rat Brain Neurons and Its Role in Template-Driven Extension of 3′-Mismatched Primers by DNA-Polymerase β in Aging Neurons

A three-step reaction strategy has been developed to examine the mechanism of extension of a mismatched primer in an oligoduplex substrate by rat neuronal extracts and DNA polymerase beta. The results revealed that in the case of duplexes with a mismatch at 3'-end of primer, significant extension by DNA polymerase beta has taken place only after the removal of the mismatched base, thus indicating the presence of a proof reading 3'-5' exonuclease activity in neuronal extracts of all ages. A closer examination of the neuronal exonuclease activity revealed that bases are excised from the 3' end in a sequential and nonspecific manner, although initial excision of a mismatched base was slightly faster. Further, the excision efficiency is seen to decrease with the age of the animal but apparently does not go below a critical level so as to become a rate-limiting factor for the DNA-repair activity.