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To achieve the goal of poliovirus eradication, surveillance of endemic areas is a crucial step in the poliovirus eradication program. Currently, six countries still have endemic poliovirus. We have tested a novel method which uses SDS/EDTA-treated chromatography paper strips to collect and transport poliovirus-containing stool samples. The SDS/EDTA-treated paper strips were soaked with different dilutions of poliovirus-containing feces and stored at different temperatures. After storing the SDS/EDTA paper strips for 5 months at 37 degrees C, poliovirus RNA could be successfully amplified using RT-PCR. Infectivity of wild-type poliovirus type 1, 2, and 3 was lost upon contact with the SDS/EDTA-treated strips. This easy, inexpensive, and biosafe chromatography paper strip method for the collection and transportation of poliovirus samples can be of use in poliovirus surveillance and polio vaccination programs.

A novel oligosaccharide has been isolated from human milk and identified as a trisialylated derivative of lacto-N-hexaose. This compound was purified by combining ion-exchange chromatography, preparative paper chromatography and high performance liquid chromatography. Structural analyses by methylation, fast atom bombardment-mass spectrometry and proton nuclear magnetic resonance spectroscopy are consistent with the following structure: formula: see text.

The structure of in vivo [3H]myo-inositol-labeled phosphatidylinositols in barley seeds were investigated by chemical degradation. In this report we present data that suggests the presence of scyllo-inositol-containing phosphatidylinositol in addition to the commonly occurring myo-inositol-containing phosphatidylinositol.

A rubredoxin-oxygen oxidoreductase, a homodimer with a molecular weight of 43 kDa per monomer, was found to be a component of an electron transfer chain that couples the reduction of oxygen to water with NADH oxidation. This FAD-containing protein appears to contain a new type of heme group. The electron transfer chain is not inhibited by cyanide and azide. In contrast, CO decreases NADH oxidation rate and also induces release of the prosthetic groups from the native terminal reductase.

The chain length of [3H]hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of [3H]glucosamine was investigated. [3H]Hyaluronic acid obtained from the matrix fraction was excluded from a Sepharose CL-2B column irrespective of the incubation period, whereas that from the medium was depolymerized into a constant chain length (Mr = 40,000). The reducing and non-reducing terminals of the depolymerized hyaluronic acid were N-acetylglucosamine and glucuronic acid, respectively. Prolonged incubation produced no oligosaccharides as shown by examination of hyaluronidase digests, suggesting the presence of a novel endo-beta-N-acetylglucosaminidase in cultured human skin fibroblasts.

3-Hydroxy-5-methylproline has been identified in hydrolysates of actinomycin Z 1 by ion-exchange and paper chromatography, paper electrophoresis, gas chromatography and mass spectrometry in comparison with the synthetic compound. The stereochemistry of this amino acid is under investigation. The amino acid composition of actinomycin Z 1 thus consists of threonine, hydroxythreonine, D-valine(2), 4-oxo-5-methylproline, 3-hydroxy-5-methylproline, sarcosine(2), N-methylalanine and N-methylvaline.

Phosphatidyl glycerolphosphate was found to serve as the glycerolphosphate donor for polymer synthesis. When CDP-diglyceride and radiolabeled glycerolphosphate were incubated with the membrane enzyme prepared from Streptococcus sanguis, active syntheses of radiolabeled lipids and polymers were observed. The synthesis of polymer was not inhibited by low concentration of unlabeled phosphatidylglycerol. When [3H, 32P]glycerolphosphate was used, the polymer synthesized contained both 3H and 32P. The lipids formed were characterized as phosphatidylglycerol and phosphatidyl glycerolphosphate. The polymers formed from the latter were characterized as lipoteichoic acid like compounds by sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Chitin, the naturally occurring polymer of N-acetyl-glucosamine, has been used as a ligand matrix for affinity chromatography of wheat germ agglutinin in a new purification method which is well suited for large scale preparations. The agglutinin obtained is homogeneous with respect to polypeptide chain molecular weight, has a blocked amino terminus and is free of proteolytic and β-N-acetyl-glucosaminidase activities.

Evidence is presented for the presence in filtrates of Diplococcus pneumoniae of an endo-glycosidase capable of acting on the O-glycosidic linkage between N-acetyl galactosamine and serine or threonine residues. The glycosidase was partially purified by chromatography on Affi-Gel 202. The resulting preparation acted on glycopeptides from mouse melanoma, fetuin and pig submaxillary mucin to release a disaccharide characterized as galactosyl-N-acetyl galactosamine. The enzyme had no action on phenyl α-N-acetyl-D-galactosaminide, asialo ovine submaxillary mucin or monosialoganglioside. A similar activity was detected in a commercial preparation of Clostridium perfringens neuraminidase.

The net synthesis of cAMP by an adenine auxotroph of Escherichia coli was measured by assaying the incorporation of tritium from [3H]-adenine into cyclic [3H] AMP during exponential growth. Synthesis of cAMP ceased abruptly when glucose was added to cells growing in glycerol and then recovered to an intermediate rate of synthesis after 0.5–1.0 generation. Cyclic AMP appeared to be synthesized from a precursor pool that turned over more rapidly than total cellular ATP. The rates of cAMP synthesis measured by this technique are compatible with the cellular levels of cAMP previously measured in this strain(3).

Cyclic inositol-1,2-phosphate was found to be present in tissue culture cells -SV40 transformed- at an approximate concentration of 10−6 molar. Of the water soluble radioactive split products of phosphatidylinositol metabolism more than 90% represents glycerylphosphorylinositol. In pulse chase experiments with [3H]glycerol and 32P-phosphate changes in the 3 H 32 P ratio of phosphatidylinositol were investigated during the biphasic decomposition of this lipid. On the basis of the results obtained, phosphatidylinositol turnover and a model of information exchange between membranes are discussed.

Summary An enzyme activity has been detected in bacterial cell extracts and liver homogenates which removes the isopentenyl side chain from isopentenylated species of transfer RNA. The site of cleavage does not involve the allylic double bond in the side chain but seems to involve C-N bond cleavage. The enzyme shows no activity when the free nucleoside is used as a substrate. The presence of this enzyme suggests that cells may have the ability in vivo to demodify species of tRNA which contain isopentenyl adenosine nucleotides.

When [14C]-asparagine was supplied to growing pea leaves aspartate and other compounds were formed, but after 4 hours more than half of the metabolised carbon skeleton was present as one compound, identified as 2-hydroxysuccinamate. This compound was also formed, with a high rate of conversion, when 2-ketosuccinamate (the product of transamination of asparagine) was supplied to the leaves. There was some synthesis of amino acids from 2-hydroxysuccinamate, and in the dark it was metabolised to release some carbon dioxide. Accumulation and metabolism of 2-hydroxysuccinamate has also been observed in soybean leaves. It is suggested that 2-hydroxysuccinamate is a major intermediate in the metabolism of the carbon skeleton of asparagine following transamination, an important route for asparagine ultilisation.

Chemically synthesized selenobiotin is, like sulfur biotin, able to bind to avidin. This observation was used to help identify biologically synthesized selenobiotin as an excretion product of Phycomyces blakesleeanus . The identification of [75Se]selenobiotin was based on the highly specific binding of biotin to avidin used as an affinity ligand to Sepharose, on its release from the complex by proteolytic treatment, and its chromatographic behavior relative to [14C]biotin standards. These results represent the first evidence of a biological synthesis of a heterocyclic ring that contains selenium in place of sulfur.

Summary 8-(6-aminohexyl)-amino-adenosine 3′,5′-monophosphate coupled to agarose has been utilized as affinity chromatography media for separation of the subunits of cAMP-dependent protein kinase and for purification of the regulatory cAMP receptor subunit. Quantitative recovery of highly purified cAMP receptor protein has been achieved using this method. The purified cAMP receptor protein is fully functional in binding cAMP and in reversibly combining with protein kinase catalytic subunits to generate the cAMP-dependent enzyme.

All-trans retinoic acid increased the incorporation of D-[ 3 H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[ 3 H]galactose was not blocked by tunicamycin. This effect was specific for D-[ 3 H]galactose since the incorporation of D-[ 3 H]glucosamine and L-[ 14 C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[ 3 H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[ 3 H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.

Hen oviduct membranes catalyze the transfer of xylose-[ 14 C] from UDP-xylose-[ 14 C] into a xylosyl lipid; a product tentatively identified as an oligosaccharide lipid; and glycoprotein(s). The synthesis of the xylosyl lipid is stimulated by exogenous dolichol phosphate. This finding, along with studies on the chemical properties of the xylosyl lipid, suggests that it has the structure xylosyl phosphoryl polyisoprenol. When exogenous, partially purified [ 14 C]-xylosyl lipid is incubated with the membrane preparation, label is found in the oligosaccharide lipid and in glycoprotein(s).

Summary The activities of four glycolipid glycosyltransferases involved in the biosynthesis in vitro of globoside and Forssman hapten were measured in normal mouse adrenal tissues, Y-1 mouse adrenal tumor cells, and tumors derived therefrom. These enzyme activities, found in Golgi-rich membranes isolated on a sucrose density gradient, were higher in the tumor cell cultures than in normal mouse adrenal tissue. The ratio between long chain oligoglycosylceramides and short chain glycosphingolipids was higher in the case of Y-l-K cells treated with dibutyryl cAMP than in concanavalin A, colchicine treated, or untreated control cells in culture.

Summary A method for the enzymatic preparation of radioactive carbamylaspartic acid (ureidosuccinic acid) from radioactive aspartic acid and carbamyl phosphate is described. It involves the use of extracts from the trypanosomid flagellate, Crithidia fasciculata , which contain active aspartate transcarbamylase (aspartate carbamoyltransferase). Two methods for the isolation of the carbamylaspartate from the incubation mixtures are described, one involving paper chromatography or paper electrophoresis and the other making use of ion-exchange chromatography on Dowex 50W (H+). The incubation allows the conversion of aspartate to carbamylaspartate of 90–98% within 2 hours and recovery of as much as 73% of the original aspartate carbons.

2-Phenethyl alcohol (2-PEA) and 2-phenyllactic acid (2-PLA) were isolated from the culture filtrates of Candida species grown in media containing peptone or phenylalanine as nitrogen source. These compounds were characterized by comparing their UV, IR, and NMR spectral properties with authentic samples. Candida species differed markedly in their production of 2-PEA and 2-PLA. Experiments using [14C]-phenylalanine indicated that both 2-PEA and 2-PLA are synthesised from L-phenylalanine. A pathway for the biosynthesis of 2-PEA from L-phenylalanine has been proposed.

Summary The isolation and partial characterization of two heparitinases and a heparinase from induced extracts from F. heparinum are reported. The heparinase acts upon heparin, heparitin sulfates C and D producing as the main degradation product a trisulfated disaccharide. The heparitinase I degrades only heparitin sulfate A and B producing as main products two N-acetylated disaccharides, one of them O-sulfated. The heparitinase II degrades heparitin sulfate B producing an N-O-sulfated disaccharide as the main product. Based on these results, the mode of action of the three enzymes as well as the structure of heparin and heparitin sulfates are proposed.

Commercial spleen phosphodiesterase preparations contain an activity (presumably enzymatic) that will deaminate adenosine, deoxyadenosine and demethylaminate N6-methyldeoxyadenosine but not their 3′ -or 5′-phosphates. The product obtained by the enzymic demethylamination of N6-methyldeoxyadenosine was identified as deoxyinosine on the basis of its electrophoretic mobility and its ultraviolet spectrum. The presence of a deaminase activity in spleen phosphodiesterase preparations must be considered when using the enzyme in nucleotide sequence studies.

Summary Porcine pepsin and penicillopepsin catalyze transpeptidation reactions which involve the transfer in high yield of N-terminal leucine from Leu-Tyr-Leu, and Leu-Tyr amide to intact substrate with the subsequent release of Leu-Leu. With the latter substrate porcine pepsin also forms Leu-Leu-Leu. With penicillopepsin small amounts of Leu-Leu-Tyr-Leu and Leu-Leu-Tyr amide resp. were obtained as intermediates from Leu-Tyr-Leu and Leu-Tyr amide. No radioactivity was incorporated into Leu-Leu when Leu-Tyr-Leu and [14C]-leucine (1:6) were incubated with the enzymes. It was concluded that the transpeptidation proceeds via covalent acyl intermediate.

Analysis of 35S labled nucleosides prepared from tRNA of Pseudomonas aeruginosa by phosphocellulose column chromatography, thin layer chromatography and Sephadex LH-20 column chromatography revealed the presence of 2-methylthioribosylzeatin in it. 2iPA, 6-(3-methyl-2-butenylamino)-9-β-D-ribofuranosyl purine; ms-2iPA, 6-(3-methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine; ribosyl-cis-zeatin, 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-β-D-ribofuranosylpurine; ribosyl-trans-zeatin, 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9-β-D-ribofuranosylpurine; ms-ribosylzeatin, 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine; s4U2, 4-thiouridine; s2U*, 5-methylaminomethyl-2-thiouridine; s2C, 2-thiocytidine; TLC — thin layer chromatography.

A nitrogen-free neutral mannooligosaccharide, similar in structure to the polysaccharide component of yeast mannoproteins, has been isolated from Mycobacterium smegmatis ATCC-356. It has a molecular weight of 3200 and is terminated at the reducing end by mannose. The evidence from nuclear magnetic resonance spectroscopy, methylation analysis, selective enzymic degradation and acetolysis indicates that the molecule consists of an α1→6-linked backbone to which single mannose units are attached in α1→2 linkage as sidechains.

We have recently shown that the monomeric subunit of mesodiaminopimelic acid containing bacterial peptidoglycans, i. e. the disaccharide tetrapeptide N-acetyl glucosaminyl-N-acetyl muramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine can replace whole killed mycobacteria in Freund's adjuvant. We now report further structural simplifications of the active molecule; natural N-acetyl-muramyl-tripeptides have been found active; the smallest adjuvant molecule found is a synthetic N-acetyl muramyl-dipeptide prepared by Sinay et al. (1).

Protein A24 is of interest because of the rapid reduction in its content in hypertrophic liver nucleoli. Although its amino acid composition is similar to that of histone 2A, protein A24 has a higher molecular weight and a higher content of glutamic acid, threonine and proline. Inasmuch as the tryptic and chymotryptic peptide fingerprints of protein A24 contain almost all the peptides of the corresponding fingerprints of histone 2A, protein A24 may be either a unique protein composed in large part of amino acid sequences of histone 2A or a “prohistone” for histone 2A.

Three 2-substituted isoxazolin-5-ones have been isolated from Lathyrus odoratus seedlings. Structural analysis by spectrometric and chemical methods determined their formulas as 2-(2-cyanoethyl)-3-isoxazolin-5-one, 2-(β,D-glucopyranosyl)-3-isoxazolin-5-one and 2-carboxymethyl-3-isoxazolin-5-one. The structures of the first two compounds were confirmed by their chemical synthesis.

This paper reports the separation and preliminary characterization of the products formed by the reaction of the antitumor compound cis -Pt(NH 3 ) 2 Cl 2 with DNA. Electrophoresis of the acid hydrolysed platinum-DNA complex gave a profile of platinum concentration which contained 5 peaks whose relative intensities varied with the amount of cis -Pt(NH 3 ) 2 Cl 2 fixed on the DNA. Similar analysis of the products formed between DNA and trans -Pt(NH 3 ) 2 Cl 2 or [Pt(dien)Cl]Cl, which are not active antitumor agents, indicated that these compounds bound to DNA in a different manner than cis -Pt(NH 3 ) 2 Cl 2 . DNA isolated from Escherichia coli which had been treated with cis -Pt(NH 3 ) 2 Cl 2 or [Pt(dien)Cl]Cl did not give the same electrophoresis profiles as the corresponding platinum-DNA complexes formed in vitro .

BCNU has been reacted with polycytidylic acid and two derivatives of CMP, 3-hydroxyethyl-CMP and 3,N4-ethano-CMP, have been identified in the acid hydrolysate of the polymer. Their formation accounts for some of the reaction of BCNU with nucleic acids, and may be related to the mechanism of action of this compound.

Summary When S . sanguis was pulsed with [2- 3 H]glycerol for 10 min and chased for 60 min, 52% of the [ 3 H]lipids were chased into exocellular compounds (34%) in the culture medium and cold phenol-extractable cell bound complex saccharides (18%). The apparent rapid turnover of the lipid fraction was found to be due to phosphatidylglycerol. The phenol extractable compounds have been purified and characterized as lipoteichoic acids. The exocellular compounds were fractionated with a Bio-Gel P-2 column into three peaks (Pk's 1, 2 and 3, based on descending order of molecular size). Pk 1 was characterized as a lipoteichoic acid-like compound containing glycerolphosphate and possibly glucose; Pk 2 as a mixture of glycerolphosphate and a small molecular weight compound, and Pk 3 as glycerol.

Summary An enzyme activity has been detected in barley leaves that converts cytokinin isopentenyladenosine to isopentenyladenine. This reaction seems to be cytokinin specific and represents an activation step in cytokinin metabolism.

D-biotin-d-sulfoxide can fulfill the growth requirement for E. coli mutants deficient in biotin biosynthesis. In vitro reduction of D-biotin-d-sulfoxide to biotin requires a protein cell-free extract and NADPH. Preliminary experiments suggest that more than one protein participates in the reaction.

Biosynthesis of glucagon was studied using isolated pigeon islets of Langerhans. H3-leucine and H3-tryptophan were found to be actively incorporated into glucagon and a large molecular weight glucagon-related component. Incorporation into both components was inhibited by cycloheximide and significantly reduced by glucose. The possibility of a precursor molecule for the biosynthesis of glucagon was suggested.

A dialyzable tRNA methylase inhibitor has been isolated from rat liver. By chromatographic and electrophoretic data as well as ultraviolet and mass spectral data, the inhibitor is identified as nicotinamide. Kinetic evidence indicates that nicotinamide is an inhibitor of tRNA methylase.

Several naturally-occurring lipids but not n-propanol, guanidine-HCl or a variety of synthetic detergents stimulate the 3′,5′-cyclic AMP-phosphodiesterase activities of a supernatant fraction of brain at 1.25 × 10 −7 M cAMP. The time courses of the reaction are linear in the presence and absence of lipid. On the other hand, lipid has different effects on various phosphodiesterase activities in fractions obtained after gel filtration of the crude extract. It stimulates the phosphodiesterase activities measured at 1.25 × 10 −7 M and 10 −4 M 3′,5′-cyclic-AMP and 1.25 × 10 −7 M 3′,5′-cyclic GMP in two of the fractions partially retained in the gel. However, lipid has little effect on the enzymatic hydrolysis of low concentrations of cAMP or cGMP and markedly inhibits the hydrolysis of high concentrations of cAMP by the fraction excluded from the gel.

An enzyme has been partially purified from E. coli which catalyzes in vitro the transfer of the Δ2-isopentenyl group from Δ2-isopentenyl- pyrophosphate to an adenosine residue in Mycoplasma sP. (Kid) tRNA. The product of the reaction has been identified as N6-(Δ2-isopentenyl)adenosine (iA), which is known to be absent in Kid tRNA. The enzyme has an approximate molecular weight of 55,000 daltons and is specific for tRNA.

Four pteridines have been isolated from two species of methane-oxidizing bacteria. They are: 2-amino-4-hydroxy-6-carboxypteridine, D- erythro -neopterin, 2-amino-4-hydroxypteridine, and 2-amino-4-hydroxy-6-methylpteridine. This latter compound has not previously been shown to occur naturally.

Summary Cell-free extracts of Claviceps spec. , strain SD 58, were obtained which catalyze the formation of L-dimethylallyltryptophan from dimethylallylpyrophosphate and L-tryptophan. The enzyme catalyzing this reaction has been partially characterized. It is absent from young mycelia, but begins to appear at the end of the growth phase, preceding alkaloid formation by about one day.

The inducibility of retinal glutamine synthetase (GS) by dibutyryl cyclic-3′,5′-AMP (DB-cAMP) was re-examined in view of conflicting reports. Various lots of DB-cAMP were compared for a) ability to induce GS in cultures of embryonic chick neural retina, and b) their composition as visualized by paper chromatography. Chromatographically purified DB-cAMP did not induce retinal GS, nor did cAMP, DB-cGMP, epinephrine, or norepinephrine; none of these enhanced the induction of GS by hydrocortisone. Some of the agents occasionally caused small increases in GS activity; however, these were invariably below the GS levels induced consistently by hydrocortisone. A single lot of DB-cAMP was found which significantly raised GS activity in the retina; it contained a contaminant which when isolated was found to be responsible for this effect.

An inhibitor of colicin B excreted into the culture media by E. coli strains, and hyperproduced by exb mutants, is shown to be the iron chelator enterochelin (= enterobactin). Production of the inhibitor by E. coli exb + is regulated by the concentration of iron in the medium. The exb mutants fall into two classes: exbA , also having the properties of tonB mutants, and exbB which have a partial requirement for methionine.

A mixed photoproduct of thymine and cysteine has been produced by irradiating a solution of thymine-2- 14 C (2.5 mM) and cysteine·HCl (10 mM) with ultraviolet light (2537A). On the basis of chemical and NMR studies its structure has been assigned as 5-S-cysteine, 6-hydrothymine. The mechanism favored for the formation of this photoproduct involves the addition of a thiyl radical to the 5-position of a thymyl radical.

Summary An enzymatic activity to synthesize N-acetyl-L-glutamate, known as an essential allosteric activator of the ammonia-dependent carbamyl phosphate synthetase, from L-glutamate and acetyl-CoA was demonstrated in the extracts of rat and mouse liver mitochondria. A partially purified enzyme had a strict substrate specificity: various amino acids and acyl-CoA analogues could not substitute for the both substrates. A notable finding is that the enzyme activity was markedly stimulated by L-arginine. The effect of arginine was specific, and all other amino acids tested, including usual components of proteins and intermediates of urea cycle as well as several guanidino compounds were totally ineffective. Arginine also stimulated the acetylglutamate synthesis in intact mitochondria. The occurrence of an enzyme specific for acetylglutamate synthesis and control of its activity by arginine may provide an important regulatory mechanism for the production of carbamyl phosphate as the first step of urea biosynthesis.

The mannose moiety of GDP- D -mannose- 14 C is transferred by a particulate enzyme from mung bean seedlings into a glucomannan. The product was characterized by isolation of a number of oligosaccharides after treatment of the product with a partially purified β-mannanase. Several of these oligosaccharides contained glucose and mannose in the approximate ratio of 1:1, 1:2 and 1:3 or 4, respectively. Since glucose- 14 C was found in the hydrolysis products of the oligosaccharides, it can be assumed that the particulate enzyme contains an epimerase that converts GDP- D -mannose- 14 C to GDP- D -glucose- 14 C.