Functional and structural analysis of cis-proline mutants of Escherichia coli aspartate aminotransferase.
Publication Date: 19/01/1999, on Biochemistry
by Birolo L, Malashkevich VN, Capitani G, De Luca F, Moretta A, Jansonius JN, Marino G
To elucidate the role of the two conserved cis-proline residues of aspartate aminotransferase (AspAT), one double and two single mutants of the enzyme from Escherichia coli (EcAspAT) were prepared: P138A, P195A and P138A/P195A in which the two prolines were replaced by alanine. The crystal structures of P195A and P138A/P195A have been determined at 2.3-2.1 A resolution. The wild-type geometry, including the cis conformation of the 194-195 peptide bond is retained upon substitution of proline 195 by alanine, whereas the trans conformation is adopted at the 137-138 peptide bond. Quite surprisingly, the replacement of each of the two prolines by alanine does not significantly affect either the activity or the stability of the protein. All the three mutants follow the same pathway as the wild type for unfolding equilibrium induced by guanidine hydrochloride [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913]. The kinetics of renaturation of P195A, where the alanine retains the wild-type cis conformation, is faster than wild type, whereas renaturation of P138A, which adopts the trans conformation, is slower. We conclude that cis-prolines seem to have been retained throughout the evolution of aspartate aminotransferase to possibly play a subtle role in directing the traffic of intermediates toward the unique structure of the native state, rather than to respond to the needs for a specific catalytic or functional role.
Glycolipids from sponges. VII. Simplexides, novel immunosuppressive glycolipids from the Caribbean sponge Plakortis simplex.
Publication Date: 18/01/1999, on Bioorganic & medicinal chemistry letters
by Costantino V, Fattorusso E, Mangoni A, Di Rosa M, Ianaro A
The new glycolipids simplexides (1) have been isolated from the marine sponge Plakortis simplex, and their structure determined by spectroscopic data and microgram-scale chemical degradation. Simplexides are composed of long-chain secondary alcohols glycosylated by a disaccharide chain, and represent a new structural kind of glycolipids. Simplexides strongly inhibit proliferation of activated T-cells by a non-cytotoxic mechanism and can be regarded as simple model molecules for designing immunosuppressive drugs.
Topology of the thyroid transcription factor 1 homeodomain-DNA complex.
Publication Date: 05/01/1999, on Biochemistry
by Scaloni A, Monti M, Acquaviva R, Tell G, Damante G, Formisano S, Pucci P
The topology of the thyroid transcription factor 1 homeodomain (TTF-1HD)-DNA complex was investigated by a strategy which combines limited proteolysis and selective chemical modification experiments with mass spectrometry methodologies. When limited proteolysis digestions were carried out with the protein in the absence or presence of its target oligonucleotide, differential peptide maps were obtained from which the amino acid residues involved in the interaction could be inferred. Similarly, selective acetylation of lysine residues in both the isolated and the complexed homeodomain allowed us to identify the amino acids protected by the interaction with DNA. Surface topology analysis of isolated TTF-1HD performed at neutral pH was in good agreement with the three-dimensional structure of the molecule as determined by NMR studies under acidic conditions. Minor differences were detected in the C-terminal region of the protein which, contrary to NMR data, showed no accessibility to proteases. Analysis of the complex provided an experimental validation of the model proposed on the basis of the homology with the homeodomain structures described so far. An increased accessibility of the C-terminal region was observed following the interaction, suggesting its displacement from the protein core by the oligonucleotide molecule. Comparative experiments with DNA fragments differing in sequence and binding capabilities highlighted structural differences among the complexes, mainly located in the N-terminal region of the homeodomain, thus accounting for their different dissociation constants.
The tbf-1 gene from the white truffle Tuber borchii codes for a structural cell wall protein specifically expressed in fruitbody.
Publication Date: 01/11/1998, on Fungal genetics and biology : FG & B
by De Bellis R, Agostini D, Piccoli G, Vallorani L, Potenza L, Polidori E, Sisti D, Amoresano A, Pucci P, Arpaia G, Macino G, Balestrini R, Bonfante P, Stocchi V
This paper reports the purification and localization of a Tuber borchii Vittad, fruitbody protein (TBF-1) and the cloning of the encoding gene. TBF-1 is detectable by SDS-PAGE analyses only in this white truffle species and presents a molecular mass of 11,994 Da. TBF-1 was purified by one-step Reversed-Phase HPLC and its complete amino acid sequence was determined after digestion with trypsin and N-Asp endoproteinase. Polyclonal antibodies were produced and tested in immunofluorescence and immunogold experiments, providing information about the protein localization. It was detected mostly on the hyphal walls, where it was colocalized with beta-1,3-glucans and chitin. The sporal wall was not labeled. The encoding gene (tbf-1) was cloned using several techniques involving PCR. The coding region consists of a 360-bp open reading frame interrupted by an intron, with another intron following the stop codon. A putative signal peptide of 12 amino acids was found at the N-terminal. Northern blot analysis revealed that tbf-1 is highly expressed in unripe and ripe fruitbodies and was not detectable in culture mycelium or ectomycorrhizal roots.
New 9,11-secosterols from gorgonia Subergorgia suberosa of the Indian Ocean.
Publication Date: 01/11/1998, on Steroids
by Aknin M, Costantino V, Mangoni A, Fattorusso E, Gaydou EM
Three new 9,11-secosterols, 3 beta,6 alpha,11-trihydroxy-9,11-seco-5 alpha-cholest-7-ene-9-one (1), 24S- (2a), and 24R-methyl-3 beta,6 alpha, 11-trihydroxy-9,11-seco-5 alpha-cholest-7,22E-diene-9-one (2b), were isolated from the Indian Ocean gorgonia, Subergorgia suberosa. Their structures were established by spectroscopic data.
Lysosomal segregation of a mannose-rich glycoprotein imparted by the prosequence of myeloperoxidase.
Publication Date: 01/11/1998, on Journal of cellular biochemistry
by Bening U, Castino R, Harth N, Isidoro C, Hasilik A
The role of the N-terminal sequence of myeloperoxidase in the intracellular targeting was examined by using glycosylated lysozyme as a reporter. A fusion protein was constructed in which the presequence residues-18 through -6 of the lysozyme moiety had been replaced by residues 1-158 of prepromyeloperoxidase. Expression of the fusion protein in Chinese hamster ovary cells demonstrated its partial secretion and partial intracellular retention. The latter was accompanied by trimming the myeloperoxidase prosequence off the lysozyme moiety. The rate of the retention of the lysozyme fusion protein was higher than that of glycosylated lysozyme that had been expressed in cells transfected with cDNA of glycosylated lysozyme. The retention was insensitive to NH4Cl. In the secreted protein, lysozyme contained predominantly complex oligosaccharides as demonstrated by a proteolytic fragmentation in vitro and resistance to endo-beta-N-acetylglucosaminidase H. In contrast, when targeted to lysosomes, the lysozyme moiety of the fusion protein contained predominantly mannose-rich oligosaccharides. In baby hamster kidney cells, the trimming of the oligosaccharides in the lysozyme fragment was less vigorous, and a selective targeting of molecules bearing mannose-rich oligosaccharides to lysosomes was more apparent than in Chinese hamster ovary cells. In the presence of monensin, the formation of complex oligosaccharides in the fusion protein and its secretion were strongly inhibited, whereas the intracellular fragmentation was not. We suggest that the prosequence of myeloperoxidase participates in the intracellular routing of the precursor and that this routing operates on precursors bearing mannose-rich rather than terminally glycosylated oligosaccharides and diverts them from the secretory pathway at a site proximal to the monensin-sensitive compartment of the Golgi apparatus.
Surgical management of the neck in squamous cell carcinoma of the floor of the mouth.
Publication Date: 01/11/1998, on Oral oncology
by Zupi A, Califano L, Mangone GM, Longo F, Piombino P
Nodal involvement in squamous cell carcinoma considerably lowers survival rate. Despite its importance, neck management has still not been adequately explored. The Authors have retrospectively reviewed the records of 112 cases. Unilateral N+ were treated with a homolateral therapeutic and a controlateral prophylactic neck dissection; bilateral N+ were treated with a bilateral therapeutic neck dissection. On first observation the majority of cases (66.1%) were T1-2, N+ patients accounted for 45.5%. Among N- patients, 21.3% of occult nodal metastases were observed. The 5-year survival rate was 52.7%. With N+ lesions, a radical neck dissection should be performed; the dissection should be performed bilaterally. With N- lesions a prophylactic modified radical neck dissection is recommended in T2-4 lesions.
Protein synthesizing units in presynaptic and postsynaptic domains of squid neurons.
Publication Date: 01/11/1998, on Journal of cell science
by Martin R, Vaida B, Bleher R, Crispino M, Giuditta A
Putative protein synthesizing domains, called plaques, are characterized in the squid giant synapse and axon and in terminals of squid photoreceptor neurons. Plaques are oval-shaped formations of about 1 microm in size, which (1) generate signals that have spectroscopic electron energy loss characteristics of ribosomes, (2) exhibit ribonuclease-sensitive binding of YOYO-1, a fluorescent RNA/DNA dye, and (3) in part hybridize with a poly(dT) oligonucleotide. In the giant synapse plaques are abundant in the postsynaptic area, but are absent in the presynaptic terminal. In the cortical layer of the optic lobes, plaques are localized in the large carrot-shaped presynaptic terminals of photoreceptor neurons, where they are surrounded by synaptic vesicles and mitochondria. Biochemical and autoradiographic data have documented that the protein synthetic activity of squid optic lobe synaptosomes is largely due to the presynaptic terminals of the photoreceptor neurons. The identification of ribosomes and poly(A+)-mRNA in the plaques indicates that these structures are sites of local protein synthesis in synaptic domains.
Perineural invasion of the lower alveolar nerve by oral cancer: a follow-up study of 12 cases.
Publication Date: 01/10/1998, on Journal of cranio-maxillo-facial surgery : official publication of the European Association for Cranio-Maxillo-Facial Surgery
by Zupi A, Mangone GM, Piombino P, Califano L
Twelve previously untreated cases of oral cancer with perineural infiltration were studied retrospectively. Age, sex, site, clinical stage and outcome were evaluated. Management of the neoplasm in each case involved surgical removal and six patients required adjuvant radiotherapy. The most frequent site was the lip. At the time of diagnosis, five patients had sensory complaints and palpable lymphadenopathy was observed in three patients. The 5-year crude survival rate was 16.7%. In the cases with postoperative assessment of perineural infiltration, a median survival time of 30.8 months was observed; while in the case of preoperative assessment of nerve infiltration, extensive surgery was performed with a consequent median survival time of 44.5 months. The perineural infiltration of the lower alveolar nerve is more common (6.3%) than is generally thought. This frequency is due to the relationship with the lower lip and the mandibular region. In carcinoma of the lip, spread is generally limited to 10-15 mm along the lower alveolar nerve. In carcinoma of the mandibular region, spread is entirely dependent on the location of the tumour; the absence of clinical fixation to the bone and the small size of the carcinoma does not preclude the possibility of bone involvement. Neurological symptoms should be evaluated carefully, and a radiographic investigation of the nerve canal is mandatory.
Structural characterization and independent folding of a chimeric glycoprotein comprising granulocyte-macrophage colony stimulating factor and erythropoietin sequences.
Publication Date: 01/08/1998, on Glycobiology
by Amoresano A, Andolfo A, Siciliano RA, Mele A, Coscarella A, De Santis R, Mauro S, Pucci P, Marino G
MEN 11300 is a hybrid glycoprotein of 297 amino acids obtained by fusion of the cDNA encoding GM-CSF with the cDNA encoding EPO followed by transfection of the hybrid gene into CHO cells. The oligonucleotide construct incorporated a spacing sequence between the two individual cDNAs which encodes eight amino acids constituting a linker peptide intended to separate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein was submitted to a detailed structural characterization including the verification of the entire amino acid sequence, the assignment of the disulfide bridges pattern, the identification of the glycosylation sites and the definition of the glycosidic moiety, including site-specificity. Partial processing of the C-terminal Arg residue and the occurrence of N-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established. Moreover, O-glycosylation at Ser257 and at the N-terminal region was also detected. A large heterogeneity was observed in the N-glycans due to the presence of differently sialylated and fucosylated branched complex type oligosaccharides whereas O-linked glycans were constituted by GalGalNAc chains with a different number of sialic acids. The disulfide bridges pattern was established by direct FABMS analysis of the proteolytic digests or by ESMS analysis of HPLC purified fractions. Pairing of the eight cysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, and Cys160-Cys164. This S-S bridges pattern is identical to that occurring in the individual natural GM-CSF and EPO, thus showing that the two protein moieties in MEN 11300 can independently acquire their native three-dimensional structure.
Types of purinoceptors and phospholipase A2 involved in the activation of the platelet-activating factor-dependent transacetylase activity and arachidonate release by ATP in endothelial cells.
Publication Date: 01/08/1998, on Prostaglandins & other lipid mediators
by Balestrieri ML, Malik KU, Balestrieri C, Lee TC
Acyl analogs of PAF are the major products synthesized during agonist stimulation of endothelial cells. We have previously shown that PAF: 1-acyl-2-lyso-sn-glycero-3-phosphocholine transacetylase in calf pulmonary artery endothelial cells is activated by ATP through protein phosphorylation, and the increase in transacetylase activity by ATP contributes to the biosynthesis of acyl analogs of PAF (J. Biol. Chem. 272, 17431-17437, 1997). To understand the mechanisms(s) by which ATP stimulates acyl analogs of PAF production, we have identified the subtypes of the purinergic receptor that are linked to the activation of two enzymes involved in the generation of acyl analogs of PAF, namely, transacetylase and phospholipase A2. Experiments with transient transfection of the cells with antisense and sense thio-oligonucleotide to cytosolic phospholipase A2 (cPLA2) were also performed to evaluate whether downstream activation of cPLA2 is involved in ATP-receptor mediated induction of arachidonate release and synthesis of radylacetyl-GPC. We found that the P2u/P2Y2 receptor, which recognizes a pyrimidine nucleotide, UTP, as well as purine nucleotides, shows a potency profile of UTP > ATP = ATP gamma S > 2-methylthio-ATP in mediating the activation of PAF: lysophospholipid transacetylase. On the other hand, ADP beta S and 2-methylthio-ATP have similar potencies as ATP but have lower potencies than UTP and ATP gamma S in stimulating the release of arachidonate. These results suggest that both P2u/P2Y2 and P2y/P2Y1 receptor subtypes promote arachidonate release. In addition, transient transfection of endothelial cells with cPLA2 antisense but not the sense thio-oligonucleotide inhibited the stimulation of arachidonate release and [3H]acetate incorporation into radyl[3H]acetyl-GPC. Thus, our data suggest that a receptor-mediated process is involved in the activation of transacetylase for the induced synthesis of acyl analogs of PAF in endothelial cells. Furthermore, it is likely that cPLA2 supplies the lysophospholipids as substrates for the transacetylation reaction.
A quantitative cytochrome oxidase mapping study, cross-regional and neurobehavioural correlations in the anterior forebrain of an animal model of Attention Deficit Hyperactivity Disorder.
Publication Date: 01/07/1998, on Behavioural brain research
by Papa M, Berger DF, Sagvolden T, Sergeant JA, Sadile AG
The aim of this study was to trace by molecular imaging techniques the neural substrates of attention deficit hyperactivity disorder (ADHD) using the spontaneously hypertensive rat (SHR) as animal model. Adult SHR and Wistar-Kyoto (WKY) controls were used throughout this study. In experiment 1, naive male SHR and WKY were used, whereas in experiment 2 SHR and WKY rats of both genders were trained on a multiple fixed interval (FI (120 s for water, 5-min extinction)) paradigm and sacrificed 6 months later. In both experiments coronal sections of the anterior forebrain were processed for quantitative cytochrome oxidase (COase) histochemistry by the method of Gonzalez-Lima. Optical density values were transformed into actual enzyme activity units by using tissue-calibrated standards. In experiment 1, non-trained male rats of the SHR line showed lower COase activity in the medial and lateral prefrontal cortices, compared with WKY controls. In experiment 2, there was a line x treatment interaction effect in the pole of the nucleus accumbens (ACB). Regional correlative analyses revealed that: (i) under basal conditions, SHR are more synchronized than WKY rats in the COase level of different brain regions; and (ii) the training desynchronizes COase activity in the WKY, further synchronizes it and increases the cross-talk between hemispheres in male SHR only. Neurobehavioral covariations between behavioural scores and metabolic capacity in the medial and lateral prefrontal/frontal cortices, the caudate-putamen complex (CPU), the pole, core, and shell of the accumbal complex (ACB), and the ventral pallidum (VP), indicated that, in the WKY rats, the frequency of lever pressing covaried positively with the COase activity in the CPU, whereas in the SHR covaried with both medial and lateral prefrontal/frontal cortices. The bursts of activity during the 1-1.33-s segment was positively correlated, in the WKY rats only, with the core and shell of the ACB, and with the VP. Finally, the correlative profiles showed significant gender differences with effects in male SHR only. Thus, the results lend support to the involvement of the cortico-striato-pallidal system in ADHD.
Reduced transduction mechanisms in the anterior accumbal interface of an animal model of Attention-Deficit Hyperactivity Disorder.
Publication Date: 01/07/1998, on Behavioural brain research
by Papa M, Sergeant JA, Sadile AG
The aim of this study was to map the neural substrates of attention-deficit hyperactivity disorder (ADHD) in the spontaneously hypertensive rat (SHR), which is thought to be a model for ADHD. To this aim, the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and transcription factors (TF) were used as markers. The focus of interest was the nucleus accumbens complex (ACB) which is thought to be an interface between limbic and motor systems. Juvenile, male rats of the SHR line and Wistar-Kyoto (WKY) controls were perfused and the brains processed for immunocytochemistry for CaMKII and the TF peptides of the FOS, JUN-B and ZIF-268 families. The results revealed that: (i) in both groups there were more CaMKII-positive neurones in the shell than in the core of the ACB; (ii) SHR had a reduced number of CaMKII-positive elements in anterior portions of the shell; and (iii) SHR had a lower expression of peptide products of the FOS family (c-FOS, in particular) and ZIF-268. In addition, there was a lower expression of c-FOS and zif-268 in the core of the ACB in the SHR. In contrast, there was an increased basal level of JUN-B in the core of the ACB of SHR. The reduced number of CaMKII and TF-positive elements in the most rostral portions of the accumbal complex of SHR, associated to the higher number of binding sites for the DA D-1/D-5 subtype, appears as a discrete alteration in the prosomeric development of the anterior basal forebrain and could be the key to the understanding of ADHD.
Ancient DNA in human bone remains from Pompeii archaeological site.
Publication Date: 29/06/1998, on Biochemical and biophysical research communications
by Cipollaro M, Di Bernardo G, Galano G, Galderisi U, Guarino F, Angelini F, Cascino A
aDNA extraction and amplification procedures have been optimized for Pompeian human bone remains whose diagenesis has been determined by histological analysis. Single copy genes amplification (X and Y amelogenin loci and Y specific alphoid repeat sequences) have been performed and compared with anthropometric data on sexing.
The selective inability to draw horizontal lines: a peculiar constructional disorder.
Publication Date: 01/06/1998, on Journal of neurology, neurosurgery, and psychiatry
by Grossi D, Fragassi NA, Giani E, Trojano L
A patient is described who was affected by degenerative dementia and who developed severe constructional apraxia. She showed a dissociation between the construction of horizontal lines (impaired) and oblique or vertical lines (spared) which has never been reported previously. A battery of tests disclosed that this phenomenon was consistent across a range of experimental conditions and that a similar dissociation was evident in perceptual and representational domains. This peculiar clinical finding suggests that mental representations of horizontal and vertical spatial relations in an egocentric coordinate system are functionally dissociated.