Rapid determination of chain length pattern in poly(ADP-ribose) samples

(³H)poly(ADP-ribose) synthesized from nuclei by incubation with (³H)NAD was released from protein by alkaline treatment and electrophoresed in dodecyl sulfate gels. Individual polymers up to at least 33 units were completely separated according to their chain length. Size distribution was visualized by fluorography of the gels, and quantified by radioactivity determination of sliced gels The method could be applied to crude nuclear extracts. It showed that nuclei of Ehrlich ascites tumor cells produced a poly(ADP-ribose) pattern distinctly different from that of rat liver nuclei.     

Aldolases of the lactic acid bacteria

Reciprocal qualitative and quantitative immunological experiments employing an anti-Pediococcus cerevisiae aldolase serum confirmed many of the interspecific relationships demonstrated previously among lactic acid bacteria with antisera prepared against the Streptococcus faecalis fructose diphosphate aldolase. The extent of immunological relatedness observed between the Lactobacillus and Pediococcus aldolases was markedly greater than that noted between Pediococcus and Streptococcus aldolases indicating that the pediococci share closer phylogenetic ties with the rod-shaped lactobacilli than with their spherical counterparts in the streptococci. In addition to confirming the existence of definitive, but distant, relationships between the lactic acid bacteria and certain gram positive nonsporeforming anaerobes, immunological cross-reactivity was also demonstrated between the pediococcal aldolases and those of Aerococcus viridans.This paper is dedicated with deepest appreciation to Prof. Roger Y. Stanier on the occasion of his 60th birthday in token of what his friendship and guidance have meant to me. — J. L.     

fMet-tRNA F Met binding and peptidyl transferase function in free and bound ribosomes from normal and puromycin aminonucleoside-treated rats.

Treatment of rats with the aminonucleoside of puromycin, which increases the incorporation of labelled phenylalanyl-tRNA into polypeptide chains in liver ribosome preparations studied in vitro, did not change the factor-dependent binding of fMet-tRNA f Met to ribosomes nor the peptidyl transferase function of the ribosomes. Peptidyl transferase function, as measured by fMet-tRNA f Met-puromycin formation, was comparable in the free and bound ribosome preparations. Similarly, the factor-dependent binding of fMet-tRNA f Met to ribosomes was the same in free ribosome preparations obtained from rat liver as it was in bound ribosome preparations that had been freed of membranes by puromycin incubation and high salt wash.     

Physical properties of arabinofuranosylcytosine diphosphate diacylglycerol,an antitumor liponucleotide

Dispersed from a dry film into buffer (5 mM phosphate, 0.15 M NaCl, pH 7.4), the liponucleotide 1-β-d-arabinofuranosylcytosine 5′-diphosphate l-1,2-diacylglycerol (ara-CDPdiacylglycerol) spontaneously forms vesicles which are several microns in diameter and probably unilamellar. Their average size immediately begins to decrease, and after 2 h none can be seen in the light microscope. During 1–2 days in unstirred solutions at 25°C, the vesicles are transformed to spherical or nearly spherical micelles having an apparent partial specific volume of 0.835 ml·g^?1, a maximum possible aggregation number of about 150, and an anhydrous radius of about 37 Å. The critical micelle concentration (CMC) is about 10 μM in buffer and 20 μM in distilled water, but micelle-monomer equilibration requires at least 1 week at a total concentration of 66 μM. This exceedingly slow equilibration is unique among reported detergents. The standard enthalpy and entropy of micellization are ?13 kJ·mol^?1 and 87 J·mol^?1·K^?1, respectively. These values are within the range reported for other detergents. Sonication accelerates the vesicle-micelle transformation to 30 min.     

Detecting clinical activity in systemic lupus erythematosus with an archaeal poly(ADP-ribose) polymerase-like thermozyme: a pivotal study

The clinical usefulness of an immunotest was evaluated by using purified poly(adenosine diphosphate (ADP)-ribose) polymerase from Sulfolobus solfataricus (PARPSso) as an antigen to detect the presence of abnormal anti-PARP antibodies in the sera of patients with systemic lupus erythematosus (SLE) at different clinical stages. Sera from 44 patients with SLE, subgrouped on the basis of disease activity (16 with inactive disease, 28 with active disease) were analysed with a new immunotest to detect anti-PARP antibodies, and with an immunofluorescent (IIF) assay for antinuclear antibodies (ANA) detection. ANA detection by IIF revealed that sera of healthy subjects were negative, whereas sera from patients with SLE were positive in all cases (13 positive at 1:80, 15 at 1:160, 15 at 1:320, 1 at 1:640, v/v). Anti-PARP activity was higher in ANA-positive patients than in controls (p?=?0.005). Within the group of SLE sera, disease and anti-PARP activity was increased more significantly in patients with active than in those with inactive disease (p?p?=?0.001, respectively). Correlation between anti-PARP and disease activity in SLE patients was statistically significant (p?Sso seems to be suitable for detecting anti-PARP antibodies and could play a role as a serological marker of disease activity in patients with SLE.     

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.     

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.     

Evolution of the salivary apyrases of blood-feeding arthropods

Phylogenetic analyses of three families of arthropod apyrases were used to reconstruct the evolutionary relationships of salivary-expressed apyrases, which have an anti-coagulant function in blood-feeding arthropods. Members of the 5′nucleotidase family were recruited for salivary expression in blood-feeding species at least five separate times in the history of arthropods, while members of the Cimex-type apyrase family have been recruited at least twice. In spite of these independent events of recruitment for salivary function, neither of these families showed evidence of convergent amino acid sequence evolution in salivary-expressed members. On the contrary, in the 5′-nucleotide family, salivary-expressed proteins conserved ancestral amino acid residues to a significantly greater extent than related proteins without salivary function, implying parallel evolution by conservation of ancestral characters. This unusual pattern of sequence evolution suggests the hypothesis that purifying selection favoring conservation of ancestral residues is particularly strong in salivary-expressed members of the 5′-nucleotidase family of arthropods because of constraints arising from expression within the vertebrate host.