• An "ecological" constructional test.

    Publication Date: 01/08/1997, on Perceptual and motor skills
    by Trojano L, Angelini R, Gallo P, Grossi D
    DOI: 10.2466/pms.1997.85.1.51

    We describe a simple, three-dimensional constructional test (the Box test), which reflects common daily-living activities, to be used for the assessment of constructional disability in elderly brain-lesioned patients. Subjects are required to put as may of 12 objects of varied shape and volume as they can into a box. To carry out the task successfully subjects have to arrange the items according to an efficient constructional strategy. We administered this test to 68 normal subjects and to 50 brain-damaged patients. Analysis indicated the Box test is easy and simple to administer and can be used without difficulty by elderly patients having focal brain damage. Performance correlated well with general intelligence and other bidimensional, conventional constructional tasks. Right or left brain lesions have a similar, significant detrimental effect on performance but probably through different mechanisms.

  • Oestrogen control of the sexual dimorphism in the Harderian gland of Xenopus laevis.

    Publication Date: 01/08/1997, on The Journal of steroid biochemistry and molecular biology
    by Varriale B, Chieffi P

    Xenopus laevis shows a sexual dimorphism of the electrophoretic pattern of Harderian gland (HG) proteins. The male pattern displays three protein fractions whose molecular sizes are approx. 205, 180 and 78 kDa, respectively, and which are absent in the female pattern. Conversely, the female pattern displays two protein fractions of approx. 190 and 76 kDa, respectively. This sexual dimorphism led us to hypothesize a sex steroid control of the HG. Administration of 17beta-oestradiol to male Xenopus converts the male protein pattern into the female one, while the administration of testosterone to the female has no effect. In this respect neither Northern analysis nor the RNase-protection assay performed using a 213 bp encoding for the androgen-binding domain reveals the presence of an androgen receptor mRNA in Xenopus HG. Conversely, Northern analysis has shown an oestrogen receptor mRNA whose size is approx. 6.5 kb and the RNase-protection assay performed by using a 197 bp encoding for the oestrogen-binding domain has also displayed the presence of an oestrogen receptor mRNA in the female HG but not in the male one. In addition, the oestrogen administration to male Xenopus induces the appearance of an oestrogen receptor mRNA. Androgen administration to female toad is ineffective. Taken together, all these findings suggest that in Xenopus laevis oestrogens are involved into the HG physiology. The appearance of an oestrogen receptor mRNA in the oestradiol treated males supports the hypothesis of the occurrence of autoinduction of oestrogen receptor mRNA expression in the HG.

  • Structural characterization of four genetic variants of human serum albumin associated with alloalbuminemia in Italy.

    Publication Date: 15/07/1997, on European journal of biochemistry
    by Minchiotti L, Watkins S, Madison J, Putnam FW, Kragh-Hansen U, Amoresano A, Pucci P, Cesati R, Galliano M

    A long-term electrophoretic survey on plasma proteins, which was carried out in several clinical laboratories in Italy, identified 28 different genetic variants of human serum albumin and four cases of analbuminemia. We have previously characterized 16 point mutations, 3 C-terminal mutants, and the genetic defects in two analbuminemic subjects. Here, we report the molecular defects of four alloalbumins that have been characterized by protein structural analysis. Of these, three represent new single-point mutations: albumins Tregasio, Val122-->Glu, Bergamo, Asp314-->Gly, and Maddaloni, Val533-->Met. The fourth, albumin Besana Brianza, has the same Asp494-->Asn mutation that introduces a glycosylation site which has been previously reported in a variant from New Zealand, albumin Casebrook. However, in contrast to albumin Casebrook, albumin Besana Brianza is only partially glycosylated and the oligosaccharide is heterogeneous, consisting of a biantennary complex type N-glycan with either two or one sialic acid residue(s) on the antennae. Both albumin Maddaloni and Besana Brianza represent mutations at hypermutable CpG dinucleotide sites; albumin Maddaloni is a mutant that does not involve a charged amino acid.

  • Molecular cloning and characterization of a novel mRNA present in the squid giant axon.

    Publication Date: 15/07/1997, on Journal of neuroscience research
    by Chun JT, Gioio AE, Crispino M, Eyman M, Giuditta A, Kaplan BB

    Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library. The pA6 mRNA is relatively small (550 nucleotides in length) and is expressed in both nervous tissue and skeletal muscle. The axonal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative RT-PCR analysis indicate that there are 1.8 x 10(6) molecules of pA6 mRNA (approximately 0.45 pg) in the analyzed segment of the giant axon and suggest that the level of pA6 mRNA in the axonal domain of the giant fiber system might be equal to or greater than the level present in the parental cell soma. Sequence analysis of pA6 suggests that the mRNA encodes an integral membrane protein comprising 84 amino acids. The putative protein contains a single transmembrane domain located in the middle of the molecule and a phosphate-binding loop situated near the N terminus. The C-terminal region of the protein contains two potential phosphorylation sites. These four structural motifs manifest striking similarity to domains present in the ryanodine receptor, raising the possibility that pA6 represents a cephalopod intracellular calcium release channel protein.

  • The role of platelet-activating factor-dependent transacetylase in the biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine by stimulated endothelial cells.

    Publication Date: 11/07/1997, on The Journal of biological chemistry
    by Balestrieri ML, Servillo L, Lee T

    Acyl analogs of platelet-activating factor (PAF) (1-acyl-2-acetyl-sn-glycero-3-phosphocholine, acylacetyl -GPC) are the predominant products synthesized during thrombin or ionophore A23187-mediated activation of endothelial cells. However, the biosynthetic pathway responsible for the production of acylacetyl-GPC is not well understood. In the present investigation, we have demonstrated that the acyl analogs of PAF are also the major products from calf pulmonary artery endothelial cells in response to a time-dependent stimulation of ATP (10(-3) M), bradykinin (10(-8) M), or ionophore A23187 (2 microM). In addition, we have found that the CoA-independent PAF:acyllyso-GPC transacetylase recently identified by us is concurrently and transiently induced with maximal 4-fold enhancement at 5 min and returned to near basal level by 10 min treatment of endothelial cells with ATP. Acid phosphatase reduces the increased PAF:acyllyso-GPC transacetylase activity from the homogenates of ATP-activated endothelial cells. Reduced PAF:acyllyso-GPC transacetylase activity can be restored by incubating the acid phosphatase-treated homogenates with ATP (5 mM) and Mg2+ (10 mM). Furthermore, okadaic acid, a protein phosphatase 1 and 2A inhibitor, incubated with endothelial cells in a dose-dependent manner (1-100 nM) for 10-min potentiates and sustained the stimulation of PAF:acyllyso-GPC transacetylase activity by ATP. On the other hand, genistein, tyrphostin-25 (inhibitors of tyrosine-specific protein kinase), and calphostin C (an inhibitor of protein kinase C) block the activation of PAF:acyllyso-GPC transacetylase by ATP. These results are consistent with the notion that ATP regulates the transacetylase activity by reversible activation and inactivation via the phosphorylation and dephosphorylation cycle. ATP also augments the activities of alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferase. However, the activation of the acetyltransferases precedes that of the transacetylase with peak activation occurring at 1-2 min of the ATP treatment. In addition, sodium vanadate, also an inhibitor of protein phosphatase, stimulates the increase in the incorporation of [3H]acetate into acyl[3H]acetyl-GPC of the ATP-treated endothelial cells. Collectively, our data show that both acetyltransferases and transacetylase participate in and contribute to the biosynthesis of acyl analogs of PAF in a coordinate fashion in endothelial cells.

  • Design and solution structure of a partially rigid opioid antagonist lacking the basic center--models of antagonism.

    Publication Date: 01/07/1997, on European journal of biochemistry
    by Crescenzi O, Fraternali F, Picone D, Tancredi T, Balboni G, Guerrini R, Lazarus LH, Salvadori S, Temussi PA

    To discriminate between two general models of antagonism (participation and allosteric), an opioid antagonist lacking the basic nitrogen of tyramine was designed and characterized. Cyclo-[Tyr(Me)2-Tic-], the diketopiperazine of 2,6-dimethyltyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, is a partially rigid opioid antagonist; its pA2 (5.8) is one smaller than that of N,N-bisallyl-enkephalin but it has a very high binding affinity (10 nM) and has a delta selectivity (66 with respect to the binding to mu receptors) higher than that of naltrindole. The conformational state of this diketopiperazine, studied under a variety of solvent and temperature conditions by NMR and molecular dynamics, can be described in terms of only three conformers whose relative populations vary widely with solvent. Only one of the three conformers, characterized by a 90 degree arrangement of the aromatic rings of Tyr(Me)2 and Tic similar to those of rigid agonists and of the bioactive conformation of the corresponding linear antagonist, is consistent with the antagonist activity. This finding favors the participation model among the general mechanisms proposed to explain antagonism. Due to the simple composition of the conformational mixture and to the rigidity of the molecule, it is possible to propose a quantitative explanation for the discrepancy between the very high binding affinity (10 nM) and the fairly small in mouse vas deferens value (1.5 microM).

  • DSC studies on bovine serum albumin denaturation. Effects of ionic strength and SDS concentration.

    Publication Date: 01/06/1997, on International journal of biological macromolecules
    by Giancola C, De Sena C, Fessas D, Graziano G, Barone G

    This work analyzed the thermal denaturation process of defatted bovine serum albumin (BSA). DSC measurements were performed on changing the pH, the ionic strength and the sodium dodecyl sulfate (SDS) concentration. These data have been compared with those previously obtained by us and other authors. The purpose of these measurements was to study the correlation between the three-dimensional organization of BSA native protein structure and its thermodynamic stability and to clarify the non-covalent interactions between the globular proteins and amphipathic molecules. These measurements have shown that the thermal denaturation is always irreversible regardless of pH, ionic strength and SDS concentration. The nature of the irreversible process superimposed on the protein unfolding is discussed. The strong stabilizing effect of NaCl on the BSA native structure has been found for the range 0-1.0 M. It is worth noting that the calorimetric curves, confined to the pH region studied, could not be represented by a two-state transition model; they were deconvoluted as the sum of two independent two-state transitions. These transitions were correlated to the domain structure of BSA. Sodium dodecyl sulfate has a net stabilizing effect up to a molar ratio of 10:1 (ligand to protein). In this range of concentrations the presence of SDS cause a biphasic profile of excess heat capacity. A simple thermodynamic model was developed in attempt to reproduce the experimental DSC profiles and collect information regarding the binding equilibrium of SDS.

  • Differential expression of transcription factors in the accumbens of an animal model of ADHD.

    Publication Date: 06/05/1997, on Neuroreport
    by Papa M, Sergeant JA, Sadile AG

    Transcription factors have been used as neuronal markers in the nucleus accumbens (ACB) of male juvenile spontaneously hypertensive rats (SHR), an animal model of attention-deficit hyperactivity disorder (ADHD), to trace putative neural substrates. In SHR, immunocytochemistry and PC-assisted image analysis showed lower expression of pan-fos, c-fos, zif/268 in the shell, and the c-fos and zif/268 in the core, with an increased level of Jun-B in the core. The differential lower basal expression of transcription factors in the ACB of an animal model of ADHD implies a reduced number of modules and might represent a neural substrate of the attention deficits seen in SHR and children with ADHD at low motivational levels.

  • Solution conformation of nociceptin.

    Publication Date: 28/04/1997, on Biochemical and biophysical research communications
    by Salvadori S, Picone D, Tancredi T, Guerrini R, Spadaccini R, Lazarus LH, Regoli D, Temussi PA
    DOI: 10.1006/bbrc.1997.6285

    Nociceptin, a novel heptadecapeptide, interacts with ORL1 a G protein-coupled receptor whose sequence is closely related to that of the kappa opioid receptor but has no opioid activity. We have investigated the conformational preferences of Nociceptin also in comparison to Dynorphin A. The N-terminal part of Nociceptin has the same conformational preferences of the message of endogenous opioids but the C-terminal part of the sequence is more flexible than the corresponding address of Dynorphin A. [Tyr1]-Nociceptin, while retaining nociceptive activity, has also an opioid activity comparable to that of enkephalins.

  • The differential effects of poly(2-hydroxyethyl methacrylate) and poly(2-hydroxyethyl methacrylate)/poly(caprolactone) polymers on cell proliferation and collagen synthesis by human lung fibroblasts.

    Publication Date: 05/03/1997, on Journal of biomedical materials research
    by Peluso G, Petillo O, Anderson JM, Ambrosio L, Nicolais L, Melone MA, Eschbach FO, Huang SJ

    Because of its chemical versatility and demonstrated biocompatibility, poly(2-hydroxyethyl methacrylate) (pHEMA) has been widely used as a polymer for biomedical applications. Since this hydrophilic material shows a poor interface with cells, blendings with other polymers were done to improve cytocompatibility. In our polymer, the presence of hydrophobic dominions on the material surface, due to the interpenetrating polymerization of pHEMA with poly(caprolactone) (PCL), seems to ameliorate the cytocompatibility in terms of cell adhesion and metabolism. For our experiments, we used IMR-90 human fibroblasts, as these cells strongly regulate DNA, RNA, and protein synthesis as anchorage-dependent variables. Cell attachment on a pHEMA/PCL interpenetrating polymer network was optimal, suggesting a strong adhesion between the cells and the polymer surface. Cell adhesion was weaker on pHEMA, as a significant fraction of the fibroblasts revealed a lack of spreading, with most cells remaining spherical. Moreover, only fibroblasts seeded on pHEMA significantly decreased mRNA synthesis; collagen production and cell shapes ranged from fully flat and proliferating, to minimally spread and nonproliferating. Finally, DNA synthesis, as a measure of cell proliferation, was markedly inhibited in cells cultured on pHEMA but not on pHEMA/PCL. In conclusion, our results suggest that control of cell growth and metabolism by biomedical polymers is based on physicochemical mechanism(s) in which the hydrophilicity/hydrophobicity ratio of the material surfaces may play an important role.

  • Structural characterisation of human recombinant glycohormones follitropin, lutropin and choriogonadotropin expressed in Chinese hamster ovary cells.

    Publication Date: 15/12/1996, on European journal of biochemistry
    by Amoresano A, Siciliano R, Orrù S, Napoleoni R, Altarocca V, De Luca E, Sirna A, Pucci P

    The alpha and beta chains from human recombinant gonadotropins follitropin, lutropin and choriogonadotropin expressed in CHO cells have been structurally characterised both at the protein and at the carbohydrate level by using advanced mass spectrometric procedures. The three alpha chains share the same amino acid sequence while they display different glycosylation patterns. The oligosaccharide structures detected belong to the complex-type glycans with different degree of sialylation. Partial proteolytic processing occurred at the N-terminus of the follitropin beta chain and at the C-terminus of the lutropin beta chain. The N-linked glycans from the three beta chains were found to contain fucosylated and sialylated diantennary and triantennary complex-type structures. The follitropin beta chain showed the presence of N-acetyllactosamine repeats on the antennae. The overall structure of the recombinant glycohormones corresponds to their natural counterparts with the exception that sulphated terminal glycosylation is missing.

  • Baseline transition sleep and associated sleep episodes are related to the learning ability of rats.

    Publication Date: 01/12/1996, on Physiology & behavior
    by Vescia S, Mandile P, Montagnese P, Romano F, Cataldo G, Cotugno M, Giuditta A

    The EEGs of 18 adult male Wistar rats were recorded during a baseline session lasting 7 h (day 1). The following day, rats were trained for a 2-way active avoidance task in an automated shuttle-box. A retention test was scheduled on the third day. On the basis of the number of avoidances scored during the training and retention sessions, rats were assigned to a fast-learning group (FL; achieving criterion during the training session), a slow-learning group (SL; achieving criterion in the retention test session), and a nonlearning group (NL; failing to achieve criterion). Vigilance states were determined by analyzing EEG data in 5-s epochs and calculating EEG power spectra of consecutive time intervals as short as 1 s. This high-resolution method led to the identification of transition sleep episodes that followed slow-wave sleep (SS) and were followed by waking (TS-->W) or by paradoxical sleep (TS-->PS). Comparison of the baseline sleep variables of the 3 behavioral groups revealed the presence of several significant differences. These observations were confirmed by the results of correlative analyses between baseline sleep variables and number of avoidances scored during the training and retention sessions. The most reliable indices of the capacity to learn the avoidance task were the amounts of SS preceding the TS-->W or the TS-->PS sequence, and the amounts of either component of the latter sequence. These variables displayed markedly higher values in FL rats. In addition, the amount of SS preceding TS-->W and the amount of TS-->(W) were significantly correlated with the number of avoidances scored during the training session. On the other hand, 1' SS-->(PS) and (SS)-->PS episodes were longer in NL rats than in SL or FL rats, respectively; and 2, the duration of SS-->(PS) episodes was inversely correlated with the number of avoidances of the first training period. The data are interpreted to suggest that TS and associated sleep episodes may predict the acquisition of the avoidance task, and the episodes of SS-->PS not associated with TS may predict the retention of innate responses, such as freezings or escapes.

  • Hormonal regulation of FHG22 mRNA in Syrian hamster harderian glands: role of estradiol.

    Publication Date: 29/11/1996, on Molecular and cellular endocrinology
    by Varriale B, Alvarez J, Prieto F, Domínguez P

    The regulation of the FHG22 gene by sex steroids has been studied in Syrian hamster Harderian gland, an organ with sexual dimorphism in which the FHG22 mRNA is female-specific. Testosterone treatment of females caused irregular inhibitory effects on the FHG22 mRNA levels, whereas male castration originated transitory increases during less than 2 weeks. Treatment of 15 day-castrated males for 1 or 2 days with beta-estradiol-3-benzoate caused a marked stimulation in the FHG22 mRNA levels. The results found in vivo may be explained considering those found in female Harderian gland serum-free primary cell cultures. In the absence of hormones, the FHG22 mRNA levels decreased along the time and neither progesterone, testosterone, or 5 alpha-dihydrotestosterone affected the expression. However, estradiol stimulated the FHG22 mRNA expression in a time and dose-dependent manner: increasing effects were detected between 8-96 h of treatment and the EC50 was about 10(-9) M. The estradiol effect was reverted by the antiestrogen ICI 164,384 or by cycloheximide. We conclude that estradiol stimulates FHG22 mRNA expression in Harderian gland, although other agents may also control the expression in vivo.

  • Reduced CaMKII-positive neurones in the accumbens shell of an animal model of attention-deficit hyperactivity disorder.

    Publication Date: 25/11/1996, on Neuroreport
    by Papa M, Sagvolden T, Sergeant JA, Sadile AG

    THIS study aimed at investigating putative neural substrates of attention-deficit hyperactivity disorder in children using the spontaneously hypertensive rat (SHR) as animal model and the Ca2+/calmodulin-dependent protein kinase II (CaMKII) as a marker in the nucleus accumbens, an interface between limbic and motor systems. In prehypertensive male SHR and Wistar-Kyoto rats image analysis of CaMKII immunocytochemistry showed more positive elements in the shell than in the core, and in the former a lower level in SHR. The data indicate a reduced number of nucleus accumbens modules available for limbic-motor integration revealing putative substrates of the altered attentional and reinforcement mechanisms demonstrated in the SHR and in children with attention-deficit hyperactivity disorder.

  • A comment on: 'Molecular diagnosis of transthyretin Met30 mutation in an Italian family with familial amyloidotic polyneuropathy' by Paola Strocchi et al., FEBS Letters 359 (1995) 203-205.

    Publication Date: 18/11/1996, on FEBS letters
    by Di Iorio G, Sanges G, Sampaolo S, Bonavita V