A study of the auxins in cabbage using countercurrent distribution

The counter-current distribution technique has been applied to the study of the identity of the auxins present in fresh cabbage. The results indicate that the major fraction of the auxin activity is probably due to 3-indoleacetic acid, but this is not established conclusively. In addition to the material presumed to be 3-indoleacetic acid, two other biologically active fractions were obtained from cabbage. Whether these are other auxins or “precursors” of 3-indoleacetic acid has not been established. It has been demonstrated that the colorimetric test which has been used by other workers is not specific for 3-indoleacetic acid, and conclusions which have been based on the use of this test must be reconsidered.     

Sequence analysis of cohesive ends of the actinophage RP3 genome and construction of a transducible shuttle vector

Abstract The sequence of the cohesive ends of actinophage RP3 DNA has been determined. As with all other phages of Gram-positive bacteria that have been studied sofar, RP3 DNA has 3'-protruding ends. A shuttle cosmid has been constructed containing this cos area which can be efficiently transduced by phage RP3 to host cells of Streptomyces rimosus .     

Preparation of azacrown-functionalized 2'-O-methyl oligoribonucleotides, potential artificial RNases

An improved synthesis for 3-(3-aminopropyl)- and 3-(3-mercaptopropyl)-1,5,9-triazacyclododecane has been developed and alternative methods for their conjugation to oligonucleotides have been described. Accordingly, the 3-aminopropyl azacrown and its N-(3-aminopropanoyl)-3-aminopropyl analogue have been tethered to the 3'-terminus of a 2'-O-methyloligoribonucleotide by aminolytic cleavage of the thioester linker utilized for the chain assembly. Studies on a monomeric model compound verify that the reaction proceeds solely by the attack of the primary amino group. 5'-Conjugation has been achieved by introducing a 2-benzylthio-2-oxoethyl group to the 5'-terminus as a phosphoramidite reagent and cleaving the thioester bond with the 3-aminopropyl azacrown. For intrachain conjugation, a phosphoramidite reagent derived from 1-deoxy-1-(2-benzylthio-2-oxoethyl)-beta-d-erythro-pentofuranose has been inserted in a desired position within the chain and subjected to on-support aminolysis with the 3-aminopropyl azacrown or its N-(3-aminopropanoyl)-3-aminopropyl and N-(6-aminohexanoyl)-3-aminopropyl analogues. The 3-mercaptopropyl-derivatized azacrown has been tetherd by a disulfide bond to a 3'-(3-mercaptoalkyl)phosphate-tailed oligonucleotide. The 3'- and intrachain-tethered conjugates have been shown to cleave as their Zn(II) chelate complementary oligoribonucleotide sequences.     

Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.     

The synthesis of phenyl(2-3H)glyoxal.

A simple inexpensive method has been developed for the synthesis of [2-3H]acetophenone, which has been converted into phenyl[2-3H]glyoxal. The latter compound has been used to modify arginine residues in alkaline phosphatase from two sources, and also a sialidase.     

Genetic and cytogenetic studies of four glycolytic enzymes in Drosophila melanogaster: Aldolase,triosephosphate isomerase, 3-phosphoglycerate kinase,and phosphoglucomutase

Four glycolytic enzymes in Drosophila melanogaster have been genetically and/or cytogenetically mapped. The structural gene for aldolase (Ald) has been genetically mapped to 3-91.5 and cytogenetically localized to 97A-B. Tpi, the structural gene for triosephosphate isomerase, has been genetically mapped to 3-101.3 and cytogenetically localized to 99B-E. Utilizing closer-flanking markers than the previous mapping, Pgk, the structural gene for 3-phosphoglycerate kinase, has been mapped to 2-5.9; cytogenetically it was found to lie in the interval between 22D and 23E3. The cytogenetic location of Pgm, the structural gene for phosphoglucomutase which has been located genetically at 3-43.4, was determined to be in 72D1-5.     

Synthesis and biological activity of vitamin D3-sulfate

Vitamin D3-3 beta-sulfate has been synthesized using pyridine sulfur trioxide as the sulfate donor. It has been shown to be pure by high performance liquid chromatography and spectral methods. Unlike previous reports, the product has been identified unambiguously as the 3 beta-sulfate ester of vitamin D3 by its ultraviolet, nuclear magnetic resonance, infrared, and mass spectra. The biological activity of vitamin D3-sulfate was then determined in vitamin D-deficient rats. Vitamin D3-sulfate has less than 5% of the activity of vitamin D3 to mobilize calcium from bone and approximately 1% of the ability of vitamin D3 to stimulate calcium transport, elevate serum phosphorus, or support bone calcification. These results disprove previous claims that vitamin D3-sulfate has potent biological activity, and they further do not support the contention that vitamin D-sulfate represents a potent water-soluble form of vitamin D in milk.     

Effect of ion-binding and chemical phospholipid structure on the nanomechanics of lipid bilayers studied by force spectroscopy

The nanomechanical response of supported lipid bilayers has been studied by force spectroscopy with atomic force microscopy. We have experimentally proved that the amount of ions present in the measuring system has a strong effect on the force needed to puncture a 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer with an atomic force microscope tip, thus highlighting the role that monovalent cations (so far underestimated, e.g., Na(+)) play upon membrane stability. The increase in the yield threshold force has been related to the increase in lateral interactions (higher phospholipid-phospholipid interaction, decrease in area per lipid) promoted by ions bound into the membrane. The same tendency has also been observed for other phosphatidylcholine bilayers, namely, 2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dioleoyl-sn-3-phosphocholine, and also for phosphatidylethanolamine bilayers such as 1-palmitoyl-2-oleoyl-sn-3-phosphoethanolamine. Finally, this effect has been also tested on a natural lipid bilayer (Escherichia coli lipid extract), showing the same overall tendency. The kinetics of the process has also been studied, together with the role of water upon membrane stability and its effect on membrane nanomechanics. Finally, the effect of the chemical structure of the phospholipid molecule on the nanomechanical response of the membrane has also been discussed.     

Graft copolymerization of ethyl acrylate onto cellulose using ceric ammonium nitrate as initiator in aqueous medium

Ceric ammonium nitrate (CAN) in the presence of nitric acid has been used as efficient initiator for graft copolymerization of the ethyl acrylate onto cellulose at 35.0 +/- 0.1 degrees C. Graft copolymerization of ethyl acrylate onto cellulose has taken place through the radical initiation process. The graft yield and other grafting parameters have been evaluated by varying concentration of ethyl acrylate from 2.5 x 10(-1) to 15.0 x 10(-1) mol dm(-3) and ceric ammonium nitrate from 5.0 x 10(-3) to 25.0 x 10(-3) mol dm(-3) at constant concentration of the nitric acid (8.0 x 10(-2) mol dm(-3)). The rate of graft copolymerization has shown 1.5 order with respect to the concentration of the ceric ammonium nitrate. The graft copolymerization data obtained at different temperatures were used to calculate the energy of activation, which has been found to be 28.9 kJ mol(-1) within the temperature range from 20 to 50 degrees C. The effect of addition of cationic and anionic surfactants on graft copolymerization has also been studied. On the basis of the experimental observations, reaction steps have been proposed and a suitable rate expression for graft copolymerization has been derived.     

Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells

3-Phosphoglycerate kinase (3-PGK) has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The enzyme has been partially characterized and compared with the characteristics of this enzyme of other normal and malignant cells. The EAC cell 3-PGK is composed of a single subunit of 47 kDa. It has a broad pH optimum (pH 6.0-7.5) for its enzymatic activity. The apparent Km values of 3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 mM and 0.1 mM respectively. Similar to 3-PGK of other cells, the EAC enzyme requires either Mg2+ or Mn2+ for full activity; the optimum concentrations of Mg2+ and Mn2+ are 0.8 mM and 0.5 mM respectively. When ATP and 3-PGA act as substrates, ADP, the reaction product of 3-PGK-catalyzed reaction has been found to inhibit this enzyme. Kinetic studies were made on the inhibition of ADP in presence of the substrates ATP and 3-PGA. Attempts to hybridize 3-PGK and glyceraldehyde-3-phosphate dehydrogenase of EAC cells by NAD or glutaraldehyde were unsuccessful.     

Energy balance: a new role for PPARalpha

The nuclear receptor PPARα has been implicated in lipid and glucose homeostasis, although high affinity endogenous ligands have been elusive. The lipid oleylethanolamide has now been shown to be a true natural ligand for PPARα, and to participate in the regulation of food intake and body weight.     

Single-strand and double-strand cleavage at half-modified and fully modified recognition sites for the restriction nucleases Sau3a and Taqi

The influence of cytosine methylation on the cleavage of DNA by the restriction nucleases Sau3A and TaqI has been investigated. Bovine satellite DNA fragments containing a GATCGA sequence, i.e. a Sau3A site overlapping with a TaqI site have been used in this study. The methylation of these fragments has been determined by sequence analysis. It has been found that a TaqI site (TCGA) methylated at cytosine in both DNA strands is still sensitive to double-strand cleavage. A Sau3A site (GATC), however, is rendered resistant to double-strand cleavage by methylation of a single cytosine. Fragments containing the "half-modified" Sau3A site are nicked in the unmethylated DNA strand. It has been shown by sequence analysis of nicked DNA that the single-strand break occurs at the same position which is cleaved in unmodified DNA.     

In vivo repair of the 3''terminus of transfer RNA injected into amphibian oocytes.

Yeast transfer RNA specific for phenylalanine has been treated chemically to remove either one or two nucleotides of its 3' terminus and has been injected into Xenopus laevis oocytes to test whether this RNA can be repaired in vivo. The results obtained showed that oocytes could aminoacylate and thus repair tRNAPhe that has lost both its terminal adenosine and 3' phosphate. A similar result was obtained with tRNAPhe that had undergone two full cycles of 3' terminal nucleotide removal. The oocytes cannot aminoacylate tRNAPhe whose 3' terminal ribose has been oxidized with periodate or the derivative that retains a 3' phosphate after adenosine removal. In vitro assays show that the Xenopus ovary contains a tRNA nucleotidyl transferase with the properties similar to enzymes obtained from other sources which may be responsible for the 3' terminal repair observed in vitro.     

Postoperative hypoparathyroidism: risk of complications]

For assessing the risk of adverse complications of surgery the group of 130 patients with post-operational hypoparathyroidism was analysed. Surgical hypoparathyroidism has been diagnosed in 51% of operated on thyroid gland patients. Laryngeal nerves have been damaged in 46.6% of patients. The injury to laryngeal nerves has been irreversible in 2/3 of patients, and reversible in the remaining 1/3. Cataract, nephrolithiasis and vitamin D3 intoxication have been observed in some cases before surgery. Their incidence increased in severe surgical hypoparathyroidism. Osteoporosis of the spine has been diagnosed in 49% of patients including some with vertebral fractures. No correlation between the degree of spine osteoporosis and diagnosis before surgery, number of operations on thyroid gland, and type of therapy has been noted. The symptoms of hypercalcemia have been diagnosed in 5 patients out of which hypercalcemia has been transient in 2 patients, and lasted for 1-5 months in the remaining 3 patients. The results of 7,873 analyses of mineral metabolism have been assessed. Hypocalcemia has been found in 38.4%, hypercalcemia in 1.6%, hypomagnesemia in 25.7%, hyperphosphatemia in 41.5%, decreased alkaline phosphatase serum activity in 28.7%, and hypercalciuria in 22.4% of cases. Surgical hypoparathyroidism is frequently accompanied by surgical hypothyroidism and injury to the recurrent laryngeal nerves.     

A family of repetitive nucleotide sequences (Sau 3A family) in the genome of 2 forms of the sockeye salmon Oncorhynchus nerka

A family of DNA sequences (Sau3A repeat sequences) has been revealed in genomes of two forms of O. nerka. A DNA fragment belonging to this "family" has been cloned in bacterial plasmid. Its copy number (in genomes of normal (570 +/- 33) and dwarf (66 +/- 12) forms) has been counted. The structural organization of sequences of Sau3A family in genomes of males and females of normal form is not the same. The organization of Sau3A sequences is the same in genomes of dwarf males and dwarf females of normal form, but the quantity of Sau3A sequences copies is smaller. It is supposed that the DNA sequences of Sau3A family bear the sex determination of dwarf form of O. nerka. The fragment of rRNA gene of nerka has been cloned and the number of rRNA gene copies in genomes of normal (200 +/- 62) and dwarf (1690 +/- 24) forms has been defined.     

Factors required for the Uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro

The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.     

The yeast multicopper oxidase Fet3p and the iron permease Ftr1p physically interact

High affinity iron uptake in yeast is carried out by a multicomponent system formed by the ferroxidase Fet3p and the iron permease Ftr1p. The currently accepted model predicts that Fet3p and Ftr1p are functionally associated, however, a structural interaction between these two proteins has not been proven yet. The methylotrophic yeast Pichia pastoris has been used to perform cross-linking studies aimed to demonstrate the existence of a Fet3p-Ftr1p complex. Cross-linking of membrane suspensions with the membrane-impermeable reagents DTSSP and BS(3) has evidenced the presence of a high molecular weight band with Fet3p oxidase activity. This band has been purified and subjected to N-terminal sequence analysis. Two sequences were found in the cross-linked species, one of which could be assigned to Fet3p and the other to Ftr1p. This is the first experimental demonstration that Fet3p and Ftr1p are physically associated.