A rapid method for determination of ribonucleotide reductase activity

Ribonucleoside diphosphate reductase activity is determined in centrifuged homogenates by following the conversion of cytosine ribonucleotide to cytosine deoxyribonucleotide. The enzymatic reaction is measured by monitoring the radioactivity of the reaction products separated by thin layer chromatography on PEI-cellulose plates. The method is rapid and permits the simultaneous processing of multiple samples.     

DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.     

New crystalline derivatives of bovine liver rhodanese

Mitochondrial bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase) has been crystallized in the form deprived of the transferable sulfur. The essential condition for crystallization was the removal of oxygen. Crystals of the sulfur-free enzyme are isomorphous with the previously characterized crystals of the sulfur-substituted enzyme. The new crystal species can react with either thiosulfate or selenosulfate to form the catalytic intermediate and, subsequently, with cyanide to form the corresponding product. Furthermore, the enzyme active site can be alkylated by iodoacetic acid.     

The α-<Emphasis Type="SmallCaps">l</Emphasis>-fucosidase from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups α-l-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first α-l-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed −1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for β and α glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.     

Evaluation of the mutagenic, antimutagenic and antiproliferative potential of Croton lechleri (Muell. Arg.) latex.

Sangre de Drago is a red viscous latex extracted from Croton lechleri (Euphorbiaceae) cortex, renowned in South American popular medicine for its wound-healing properties. The in vitro antiproliferative effects were determined on the human myelogenous leukemia K562 cells line (IC50 = 2.5 +/- 0.3 microg ml(-1)). The mutagenic and antimutagenic activity of C. lechleri sap was examined by means of the Ames/Salmonella test. No mutagenic activity was found on the Salmonella typhimurium strains T98 and T100, either with or without S9 activation. On the other hand, the sap showed an inhibitory effect against the mutagenic activity of the indirectly acting mutagen 2-Aminoanthracene in presence of S9 and a moderate protective activity against directly acting mutagens Sodium Azide and 2-Nitrofluorene. Therefore we suggest that C. lechleri sap interacts with the enzymes of the S9 mix, thereby inhibiting the transformation of 2-Aminoantracene into its active forms.     

Mediatietherapie bij ouderen met een persoonlijkheidsstoornis: Een verpleegkundige benadering

Behavioural counselling by nurses can enhance quality of life in elderly with a personality disorder. Although nurses have a crucial role in day-to-day treatment, there is a lack of evidence-based approaches. Based on the cognitive therapy, the treatment protocol Cognitive Model for Behavioural Interventions (CoMBI) provides an alternative nursing approach for personality disorders.     

A novel alpha-D-galactosynthase from Thermotoga maritima converts beta-D-galactopyranosyl azide to alpha-galacto-oligosaccharides

The large-scale production of oligosaccharides is a daunting task, hampering the study of the role of glycans in vivo and the testing of the efficacy of novel glycan-based drugs. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, are becoming important chemo-enzymatic tools for the production of oligosaccharides. However, while β-glycosynthase can be produced with a rather well-established technology, examples of α-glycosynthases are thus far limited only to enzymes from glycoside hydrolase 29 (GH29), GH31 and GH95 families. α-L-Fucosynthases from GH29 use convenient glycosyl azide derivatives as a strategic alternative to glycosyl fluoride donors. However, the general applicability of this method to other α-glycosynthases is not trivial and remains to be confirmed. Here, β-D-galactopyranosyl azide was converted to α-galacto-oligosaccharides with good yields and high regioselectivity, catalyzed by a novel α-galactosynthase based on the GH36 α-galactosidase from the hyperthermophilic bacterium Thermotoga maritima. These results open a new avenue to the practical synthesis of biologically interesting α-galacto-oligosaccharides and demonstrate more widespread use of β-glycosyl-azide as donors, confirming their utility to expand the repertoire of glycosynthases.     

Gamma interferon is able to enhance the oxidative metabolism of human neutrophils

The oxidative metabolism of human neutrophils has been studied after incubation of the cells with recombinant interferon-y. Neutrophils incubated for 2-4 hours with 2-50 U/ml recombinant interferon-y undergo a higher respiratory burst measured both as Oz consumption and Oz- production when stimulated with formyl-methionyl-leucyl-phenylalanine, Concanavalin A or phorbol myristate acetate. The potentiating effect of interferon-y requires more than one hour of incubation, is optimal at 20-50 U/ml and depends on the presence of serum in the incubation medium. The interferon effect depends on new protein synthesis. Cycloheximide at doses which do not alter the respiratory response of normal cells completely prevents the potentiating effect of interferon.     

Synaptic destabilization by neuronal Nogo-A

Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and β-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.