The Italian Journal of Biochemistry

V O L. 5 6, N. 2 JUNE 2007

DNA Protein Interactions at the rRNA of Saccharomyces cerevisiae
Francesco Cioci*, Francesca Di Felice, Francesco Chiani and Giorgio Camilloni
*Dipartimento di Genetica e Biologia Molecolare, Università di Roma “La Sapienza”

The Italian Journal of Biochemistry: 2007; 56(2): 81-90

Abstract. The rDNA cluster is the genetic locus encoding the ribosomal RNAs and physically defines where ribosomes begin to be assembled. In the yeast Saccharomyces cerevisiae, the highly repetitive structure of this locus makes it a very interesting target for studies about genome stability, chromatin-mediated transcriptional silencing and progression of aging. In fact, recombination among the repeated units is suppressed in a WT cell. Moreover, when genes transcribed by RNA polymerase II are inserted in the rDNA cluster, their transcription is silenced. Finally, the formation of rDNA minicircles (ERCs) has been shown to be one of the causes of aging in yeast. DNA topoisomerase I have been shown to suppress recombination specifically at the rDNA of S.cerevisiae. Moreover, also the chromatin structure of this locus is affected in a top1 strain, because rDNA specific transcriptional silencing is abolished. Nonetheless, the molecular basis of how this enzyme interferes with these functions is yet unknown. Here are reported results obtained by in vivo studies of DNA protein interactions occurring on the rDNA locus. The analyses include a fine mapping of nucleosome positioning; RNA polymerase I transcription factors and DNA topoisomerase I cleavage sites. Important conclusions can be drawn: i) nucleosome positioning in the Non Transcribed Spacer is not affected by RNA polymerase I transcription; ii) the RNA polymerase I transcription factors bind DNA in vivo with a defined hierarchy; iii) the DNA topoisomerase I cleaves the NTS in very specific sites, but cleavage is not induced by RNA polymerase I transcription. These in vivo studies help to characterize the molecular basis of important phenomena as the transcriptional silencing and genome stability in yeast.

Human DNA Topoisomerase IB: structure and functions
Erica Cretaio*, Luca Pattarello, Yari Fontebasso, Piero Benedetti1 and Carmen Losasso
*Department of Biology, University of Padova, Padova

The Italian Journal of Biochemistry: 2007; 56(2): 91-102

Abstract. Human DNA Topoisomerase I is a 765aa monomeric enzyme composed of four domains: the N-terminal domain, highly charged and responsible for several protein-protein interactions, the core domain that embraces the DNA during catalysis, the highly charged linker domain and the C-teminal domain containing the active site. The enzyme promotes the relaxation of supercoiled DNA by nicking and rejoining one of the strands of the DNA. Its activity is critical for many biological processes including DNA replication, transcription, and recombination. The aim of this review is to analyze the enzyme activity in terms of structure-function relationship.

Reverse gyrase: an unusual DNA manipulator of hyperthermophilic organisms
Anna D’Amaro, Mosè Rossi and Maria Ciaramella
Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Naples, Italy

The Italian Journal of Biochemistry: 2007; 56(2): 103-109

Abstract. Reverse gyrase is the only DNA topoisomerase capable of introducing positive supercoiling into DNA molecules. This unique activity reflects a distinctive arrangement of the protein, which is composed of a topoisomerase IA module fused to a domain containing sequence motives typical of helicases; however, reverse gyrase works neither like a canonical topoisomerase IA nor like a helicase. Extensive genomic analysis has shown that reverse gyrase is present in all organisms living above 70°C and in some of those living at 60- 70°C, but is invariably absent in organisms living at mesophilic temperatures. For its peculiar distribution and biochemical activity, the enzyme has been suggested to play a role in maintenance of genome stability at high temperature. We review here recent phylogenetic, biochemical and structural data on reverse gyrase and discuss the possible role of this enzyme in the biology of hyperthermophilic organisms.

Role of flexibility and long range communication on the function of human topoisomerase I
Giovanni Chillemi, Paola Fiorani, Alessandro Bruselles, Silvia Castelli, Alessia Campagna, Ofelia Sarra, Cinzia Tesauro, Monica Fiorentini, Oscar Vassallo, Ilda D’Annessa, Sabrina Santoleri, Alessandro Desideri* *CNR National Research Council, INFM National Institute for the Physics of Matter and Department of Biology, University of Rome, Tor Vergata, Rome, Italy

The Italian Journal of Biochemistry: 2007; 56(2): 110-114

Abstract. Eukaryotic topoisomerase I is an essential enzyme that regulates the changes in DNA topology, relaxing the superhelical tension associated with DNA replication, transcription and recombination. Human topoisomerase I is of significant medical interest being the only target of the antitumor drug camptothecin. The enzyme undergoes large conformational changes during its catalytic cycle and the knowedge of the degree of flexibility of the different regions provides an useful guide to the understanding of such movements. Molecular dynamics simulation is a well consolidated method for the investigation of structural and dynamic properties of proteins and nucleic acids and has been successfully applied to study the dynamical properties of the DNA-human topoisomerase complex. This review highlights some structural and dynamic properties of topoisomerase, obtained by MD simulations, that permits to explain the importance of flexibility in the modulation of the functional properties of the enzyme and in the transmission of communication between domains located far away one from each other.

RecQ helicases and topoisomerases: implications for genome stability in humans
Annapaola Franchitto
Section of Experimental and Computational Carcinogenesis, Istituto Superiore di Sanità, Rome

The Italian Journal of Biochemistry: 2007; 56(2): 115-121

Abstract. In recent years growing evidence has suggested that the RecQ helicases play a crucial role in preserving genome stability. The importance of this observation in humans is substantiated by the fact that the absence of a functional RecQ helicase is associated with genetic syndromes characterised by elevated predisposition to a wide variety of cancers. It is well recognized that maintenance of genome integrity relies on the accurate execution of the DNA replication process as well as on efficient DNA repair. A number of studies have described interactions of RecQ helicases with topoisomerases and it has been proposed that this cooperation may be essential for cell viability and the avoidance of cancer development in human cells.

DNA topoisomerase i as a transcription protein and a lethal cellular toxin
Luca Lotito, Francesca Ferri, Alessandra Russo, and Giovanni Capranico
Department of Biochemistry, University of Bologna, Bologna, Italy

The Italian Journal of Biochemistry: 2007; 56(2): 122-129

Abstract. DNA topoisomerase I constitutes a significant relaxing activity in nuclei of eukaryotic cells. The enzyme acts during several DNA transactions involving the generation of torsional stress in the DNA template. Moreover, antitumor agents targeting DNA topoisomerase I are used in the treatment of human cancers with significant clinical outcome. Major progress has been attained in recent years in the understanding of the basic cellular functions of DNA topoisomerase I. In particular, the consequences of topoisomerase I activity during transcription have been extensively investigated and constitute still a very active research area. Understanding of topoisomerase I inhibitors emphasizes drug activity against the enzyme, however the high drug potency cannot be explained by the DNA damage outcome only. Even though the understanding of enzyme structure has progressed in last years, however more insights into the activity of topoisomerase I poisons have not been achieved yet. Here, we will review landmark investigations on topoisomerase I involvement in different stages of the transcription process, addressing both enzyme functions as well as drug effects on molecular processes. Moreover, we will discuss recent findings on the targeting of topoisomerase I to pre-selected sites in transcribed chromatin by fusion to a sequence-specific DNA-binding protein domain.

Interplay between wrn and the checkpoint in s-phase
Pietro Pichierri
Genome Stability group at Section of Experimental and Computational Carcinogenesis, Istituto Superiore di Sanità, Rome, Italy

The Italian Journal of Biochemistry: 2007; 56(2): 130-140

Abstract. Stability of the genome is crucial for survival and faithful transmission of the genetic blueprint to progenitors. During DNA replication chromosome integrity can be challenged by a variety of exogenous and endogenous damaging agents and by the process of duplication itself. Thus, eukaryotic cells have evolved a sophisticated response called replication checkpoint supervising the accurate and complete genome replication. The replication checkpoint response bridges together replication, repair and cell cycle proteins in a coordinated network having the ATR kinase as culprit. ATR-mediated phosphorylation events control that stalled replication forks are properly sensed and stabilised, cell cycle progression halted and replication eventually recovered. In the recent years, the Werner syndrome protein (WRN) emerged as a central actor of the replication checkpoint being instrumental for correct recovery from arrested replication and a substrate of ATR. In this review, how WRN and the replication checkpoint could cross-talk and contribute to faithful recovery of stalled replication forks will be discussed.

The interplay among chromatin dynamics, cell cycle checkpoints and repair mechanisms modulates the cellular response to dna damage
Federico Lazzaro, Michele Giannattasio, Marco Muzi-Falconi and Paolo Pievani
Genome Stability group at Section of Experimental and Computational Carcinogenesis, Istituto Superiore di Sanità, Rome, Italy

The Italian Journal of Biochemistry: 2007; 56(2): 141-148

Abstract. Cells are continuously under the assault of endogenous and exogenous genotoxic stress that challenges the integrity of DNA. To cope with such a formidable task cells have evolved surveillance mechanisms, known as checkpoints, and a variety of DNA repair systems responding to different types of DNA lesions. These lesions occur in the context of the chromatin structure and, as expected for all DNA transactions, the cellular response to DNA damage is going to be influenced by the chromatin enviroment. In this review, we will discuss recent studies implicating chromatin remodelling factors and histone modifications in the response to DNA double-strand breaks (DSBs) and in checkpoint activation in response to UV lesions.

Purification, kinetic properties and physicochemical characterization of a novel acid phosphatase (ap) from germinating peanut (arachis hypogaea) seed
Jean Tia Gonnety*, Sébastien Niamké, Betty Meuwiah Faulet, Eugène Jean-Parfait N’guessan Kouadio, Lucien Patrice Kouamé
*Laboratoire de Biochimie et Technologie des Aliments de l’Unité de Formation et de Recherche en Sciences et Technologie des Aliments de l’Université d+’Abobo-Adjamé, Côte d’Ivoire

The Italian Journal of Biochemistry: 2007; 56(2): 149-157

Abstract. Acid phosphatase activity was detected in peanut (Arachis hypogaea) cotyledons during germination. Four (4) to six (6) days of germination was the meantime corresponding to maximum hydrolytic activity of this enzyme. The understanding of the role of acid phosphatase activity during germination led to purify this enzyme by successive chromatography separations on DEAE-Sepharose CL-6B, Sephacryl S-100 HR and Phenyl-Sepharose HP to apparent homogeneity from germinated peanut cotyledon five days old. This enzyme designated peanut cotyledon acid phosphatase (AP) had native molecular weight of 24 kDa by gel permeation. SDS-PAGE of the purified acid phosphatase resolved a single protein band that migrated to approximately 21.5 kDa. Thus, this acid phosphatase likely functions as a monomer. The enzyme had optimum pH (5.0) and temperature (55 °C), and appeared to be stable in the presence of anionic, cationic and non-ionic detergents. Substrate specificity indicated that the purified acid phosphatase hydrolyzed a broad range of phosphorylated substrates. However, natural substrates such as ADP and ATP were the compounds with highest rate of hydrolysis for the enzyme. Moreover, the purified acid phosphatase exhibited phytase activity. These results showed that this enzyme played a peculiar role during germination, notably in reducing the rate of phytic acid, an antinutritional substance contained in peanut seed.

Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4
Shah Ali Ul Qader*, Afsheen Aman, Noman Syed, Saeeda Bano, Abid Azhar
*Pharmaceutical Research Centre, PCSIR Laboratories complex, Karachi, Pakistan

The Italian Journal of Biochemistry: 2007; 56(2): 158-162

Abstract. Immobilization of dextransucrase from Leuconostoc mesenteroides PCSIR-4 on alginate is optimized for application in the production of dextran from sucrose. Dextransucrase was partially purified by ethanol upto 2.5 fold. Properties of dextransucrase were less affected by immobilization on alginate beads from soluble enzyme. Highest activities of both soluble and immobilized dextransucrase found to be at 35°C and optimum pH for activity remain 5.00. Substrate maxima for immobilized enzyme changed from 125 mg/ml to 200 mg/ml. Incubation time for enzyme-substrate reaction for maximum enzyme activity was increased from 15 minutes to 60 minutes in case of immobilized enzyme. Maximum stability of immobilized dextransucrase was achieved at 25°C with respect to time.

Simultaneous Purification and Polymerization Method for Bovine Serum Albumin Preparation
Fatemeh Yari, Kamran Mousavi Hosseini
Iranian Blood Transfusion Organization, Research Center, Tehran

The Italian Journal of Biochemistry: 2007; 56(2): 163-165

Abstract. Bovine serum albumin (BSA) has various applications in blood group serology and different research purposes. In this study purification of BSA has been compared with human serum albumin (HSA) using modified ethanol precipitation method based on the method of Cohn. The purification process was carried out under controlled conditions, particularly of ethanol concentration, pH, ionic strength and temperature. It was revealed that the produced BSA and HSA have purity more than 95%. It is obvious that HSA can be used, as a drug when the amount of its polymers is less than 5% whereas polymer generation is required in order to enhance the potentiating properties of BSA in agglutination of red cells. We propose here a simple and rapid two-step method for simultaneously purification and polymerization of BSA. By this method simply BSA with desired amount of polymers was obtained by 40% ethanol concentration.

Nitrogen effects
On proteins, chlorophylls and fatty acids during the growth
Of arthrospira platensis
Samah Ayachi*, Amor El Abed, Wissal Dhifi and Brahim Marzouk
*Centre de Biotechnologie, Technopole de Borj-Cedria, Unité des Plantes Aromatiques et Médicinales, Tunisie

The Italian Journal of Biochemistry: 2007; 56(2): 166-170

Abstract. Spirulina platensis (=Arthrospira platensis) is a tunisian strain which has been isolated for the first time in Oued Essed (Sousse, Sidi Bou Ali). Biomass evolution, proteins, chlorophylls and fatty acids composition of this alga were monitored by varying nitrogen concentrations in the culture medium. Nitrogen stress was provoked by adding sodium nitrate (NaNO3) in the culture medium with concentrations varying from 0 to 5 g/l. Results obtained showed that nitrogen depletion increased total proteins and total chlorophylls. The addition of NaNO3 (5g/l) led to an increase of total fatty acids amounts and modify fatty acids composition. Optimal quantities of palmitic, g -linolenic and oleic acids were obtained with NaNO3 free-cultures. Thus, the tunisian strain has valuable biological substances, worthy to determine the optimal conditions for its propagation.

Estimation of total and direct serum bilirubin using modified micro assay method
Afsheen Aman, Shah Ali Ul Qader & Saeeda Bano
Pharmaceutical Research Center, PCSIR Laboratories Complex, Karachi

The Italian Journal of Biochemistry: 2007; 56(2): 171-175

Abstract. Estimation of total and direct bilirubin in serum plays an important role in differential diagnosis of hyperbilirubinemia. Several direct spectrophotometric methods are commercially available for total and direct bilirubin estimation in which the amount of the sample (serum) varies from 200 ml to 800 ml. It is difficult to collect such amount of serum from infants, as neonatal jaundice is the most common problem in this age group. To overcome this problem modified micro assay method was developed using dimethylsulfoxide (DMSO). The amount of the serum sample is reduced from 100 ml to 20 ml per test for both total and direct bilirubin. A method comparison study was performed using 100 consecutive serum samples, by modified micro assay method and a reference Jendrassik-Grof method. Total bilirubin in these human serum samples ranged from 0.4-15.0 mg/dl and direct bilirubin ranged from 0.05-12.0 mg/dl. The results conclude that modified micro assay method had significant correlation with r-value of 0.99989 for total serum bilirubin and with r-value of 0.99971 for direct serum bilirubin. Linearity of the method is 20 mg/dl and 15 mg/dl for total and direct bilirubin, respectively. Monoreagent used during the assay is stable for 24 hours at 2-8°C while the kit is stable for one year at 2-8°C. In conclusion this micro assay method is rapid, reliable, simple and accurate for the estimation of total and direct bilirubin with small serum quantities. It is equally reliable for manual; semi automated and automated chemistry analyzers.

Biochemical evaluation of borage (Borago officinalis) rosette leaves through their essential oil and fatty acid composition
Baya Mhamdi, Wissem Aidi Wannes and Brahim Marzouk
Aromatic and Medicinal Plants Unit, Biotechnologic Center in Technopark Borj-Cedria, Hammam-Lif, Tunisia

The Italian Journal of Biochemistry: 2007; 56(2): 176-179

Abstract. Borago officinalis rosette leaves were sampled in the region of Amdoun (Tunisia) during different stages of their development. Essential oil contents varied from 0.01% to 0.13% respectively in young and adult leaves. Twenty three volatile compounds were identified. Hydrocarbons, mainly represented by nonadecane (29.8%), tetracosane (11.3%) and heptacosane (4.7%), constituted the major class in the young leaves (45.8%), followed by aldehydes (22.4%). The percentages of these two classes decreased to reach respectively 15% and 8.1% in adult leaves in favour of alcohols (57.9%) where cis-3-hexenol (29.6%) and hexanol (14.5%) were the main compounds. Total fatty acids amounts increased from 5.03 mg/g DW in young leaves to 32.23 mg/g DW in adult ones. The predominant fatty acids were a-linolenic (C18:3 n-3), stearidonic (C18:4 n-3), g-linolenic (C18:3 n-6), palmitic (C16: 0) and linoleic (C18:2 n-6) acids.

Essential oil composition of two Myrtus communis L. varieties grown in North Tunisia
Wissem Aidi Wannes*, Baya Mhamdi, Brahim Marzouk
Unité des Plantes Aromatiques et Médicinales, Centre de Biotechnologie du Technopole de Borj Cédria, Hammam-Lif, Tunisie

The Italian Journal of Biochemistry: 2007; 56(2): 180-186

Abstract. Two Myrtus communis varieties (var. italica and baetica) were studied in order to investigate their essential oil yield and composition. Essential oil yield varied in leaves, fruits and stems. So, in leaves, it was 0.5% for italica and 0.3% for baetica and was higher than in fruits and stems with respectively 0.1% and 0.04% for italica and 0.07% and 0.03% for baetica. The essential oil analysis performed by GC and GC/MS showed a composition characterized by a high percentage of monoterpene hydrocarbons in leaves, largely due to a-pinene with 51.3% for italica and 27.7% for baetica; 1,8-cineole, the alone compound of ether class, was predominant in fruits and stems with respectively 31.6% and 34.7% for italica and 19.8% and 25.8% for baetica.

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