Autoregulation of estrogen and androgen receptor mRNAs and downregulation of androgen receptor mRNA by estrogen in primary cultures of lizard testis cells.
Publication Date: 01/06/1998, on General and comparative endocrinology
by Cardone A, Angelini F, Varriale B
Steroid hormones regulate many developmental and physiological processes via specific receptors whose number can be up- or downregulated. The regulation of estrogen (ER) and androgen (AR) receptor mRNAs in primary cultures of lizard testis is described. The high degree of homology between the probes used and the receptor mRNAs in lizard testis was consistent with the high-stringency hybridisation conditions and the molecular size of ER mRNAs (7.4 and 4.5 kb) and AR mRNA (9.5 kb). Primary cultures of testis cells revealed a time- and drug-dependent relationship between ER and AR mRNAs. 17beta-oestradiol (E) autoregulated ER mRNA and downregulated AR mRNA. The antiestrogen ICI 164,384 reversed the latter effect. Cycloheximide (Cy), to inhibit protein synthesis, in combination with E, impaired the AR mRNA expression. Testosterone (T) autoregulated the expression of its own receptor mRNA whereas this effect was reversed by both flutamide (F) and Cy. Dose-response experiments showed that low concentrations of steroids (E or T 10(-12) M) increased ER or AR mRNA levels, respectively. These results suggest that both estrogen and androgen may autoregulate the expression of their own receptor mRNAs. Since in lizard testis androgens are significantly involved in meiosis and spermiogenesis and E dramatically impairs the AR mRNA expression, the latter effect may be key in regulating certain phases of reproduction.
Conformational sampling of bioactive conformers: a low-temperature NMR study of 15N-Leu-enkephalin.
Publication Date: 01/06/1998, on Journal of peptide science : an official publication of the European Peptide Society
by Amodeo P, Naider F, Picone D, Tancredi T, Temussi PA
Conformational studies of enkephalins are hampered by their high flexibility which leads to mixtures of quasi-isoenergetic conformers in solution and makes NOEs very difficult to detect in NMR spectra. In order to improve the quality of the NMR data, Leu-enkephalin was synthesized with 15N-labelled uniformly on all amide nitrogens and examined in a viscous solvent medium at low temperature. HMQC NOESY spectra of the labelled Leu-enkephalin in a DMSOd6/H2O) mixture at 275 K do show numerous NOEs, but these are not consistent with a single conformer and are only sufficient to describe the conformational state as a mixture of several conformers. Here a different approach to the structure-activity relationships of enkephalins is presented: it is possible to analyse the NMR data in terms of limiting canonical structures (i.e. beta- and gamma-turns) and finally to select only those consistent with the requirements of delta selective agonists and antagonists. This strategy results in the prediction of a family of conformers that may be useful in the design of new delta selective opioid peptides.
Determinants of cognitive disorders in Autosomal Dominant Cerebellar Ataxia type 1.
Publication Date: 07/05/1998, on Journal of the neurological sciences
by Trojano L, Chiacchio L, Grossi D, Pisacreta AI, Calabrese O, Castaldo I, De Michele G, Filla A
We assessed neuropsychological performances of 22 patients affected by Autosomal Dominant Cerebellar Ataxia type 1. All subjects completed a comprehensive battery of standardized tests requiring a verbal response, without time constraints. In order to verify the hypothesis that disease severity is the major factor in determining the cognitive status in this syndrome, patients were divided into three groups according to the severity of the clinical picture, as evaluated by the Inherited Ataxias Progression Scale (IAPS). Statistical analysis of the three groups' raw scores showed a significant decrement in patients with more severe clinical pictures on verbal short-term memory tasks. A similar trend, but not significant, was seen for general intelligence tests and verbal learning tasks. The decrement of verbal short-term memory could be related to motor speech problems. On the other hand, the decline of cognitive abilities over the course of the Autosomal Dominant Cerebellar Ataxia type 1 was not homogeneous enough to ensure statistically reliable trends. Therefore, this cross-sectional study suggests that the progression of the disease is a necessary factor in determining cognitive decline, but it is not sufficient. Other disease-related factors (age at onset, genotypic variety) could play a critical role: among these, the size of the expanded CAG repeats is significantly related to a decline of verbal intelligence and short-term memory in SCA2 patients.
Analysis of human serum albumin variants by mass spectrometric procedures.
Publication Date: 23/04/1998, on Biochimica et biophysica acta
by Amoresano A, Andolfo A, Siciliano RA, Cozzolino R, Minchiotti L, Galliano M, Pucci P
A new strategy for the structural characterisation of human albumin variants has been developed which makes extensive use of mass spectrometric methodologies. The rationale behind the method is to provide a rapid and effective screening of the entire albumin structure. The first step in this strategy consists in the attempt to determine the accurate molecular mass of the intact variant by electrospray mass spectrometry often providing a first indication on the presence of the variant. An HPLC procedure has been developed io isolate all the seven fragments generated by CNBr hydrolysis of HSA in a single chromatographic step. A rapid screening of the entire albumin structure is achieved by the ESMS analysis of the peptide fragments and the protein region(s) carrying the structural abnormality is identified by its anomalous mass value(s). Mass mapping of the corresponding CNBr peptide, either by Fast Atom Bombardment Mass Spectrometry (FABMS) or by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDIMS), leads to the definition of the site and the nature of the variation. This combined strategy was applied to the structural characterisation of three HSA genetic variants and provided to be an effective procedure for the rapid assessment of their structural modifications showing considerable advantages over the classical approach.
Tissue transglutaminase-catalyzed formation of high-molecular-weight aggregates in vitro is favored with long polyglutamine domains: a possible mechanism contributing to CAG-triplet diseases.
Publication Date: 15/04/1998, on Archives of biochemistry and biophysics
by Gentile V, Sepe C, Calvani M, Melone MA, Cotrufo R, Cooper AJ, Blass JP, Peluso G
To investigate possible biochemical mechanisms underlying the "toxic gain of function" associated with polyglutamine expansions, the ability of guinea pig liver tissue transglutaminase to catalyze covalent attachments of various polyamines to polyglutamine peptides was examined. Of the polyamines tested, spermine is the most active substrate, followed by spermidine and putrescine. Formation of covalent cross links between polyglutamine peptides and polyamines yields high-M(r) aggregates--a process that is favored with longer polyglutamines. In the presence of tissue transglutaminase, purified glyceraldehyde-3-phosphate dehydrogenase (a key glycolytic enzyme that binds tightly to the polyglutamine domains of both huntingtin and dentatorubral-pallidoluysian atrophy proteins) is covalently attached to polyglutamine peptides in vitro, resulting in the formation of high-M(r) aggregates. In addition, endogenous glyceraldehyde-3-phosphate dehydrogenase of a Balb-c 3T3 fibroblast cell line overexpressing human tissue transglutaminase forms cross-links with a Q60 polypeptide added to the cell homogenate. Possibly, expansion of polyglutamine domains (thus far known to occur in the gene products associated with at least seven neurodegenerative diseases) leads to increased/aberrant tissue transglutaminase-catalyzed cross-linking reactions with both polyamines and susceptible proteins, such as glyceraldehyde-3-phosphate dehydrogenase. Formation of cross-linked heteropolymers may lead to deposition of high-M(r) protein aggregates, thereby contributing to cell death.
A kindred affected by cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). A 2-year neuropsychological follow-up.
Publication Date: 01/04/1998, on Journal of neurology
by Trojano L, Ragno M, Manca A, Caruso G
We report a 2-year prospective neuropsychological study of five asymptomatic subjects with magnetic resonance imaging (MRI) abnormalities from an Italian kindred affected by cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These subjects completed tests for attention capacities, processing speed, abstract thinking, short-term memory, learning and constructional praxis. Seven normal subjects matched for age and education, belonging to the same pedigree and not having MRI hyperintensities were examined as controls. The results did not show significant differences between asymptomatic subjects and normal controls. Cognitive performance of asymptomatic subjects did not deteriorate during a 2-year follow-up. Our findings suggest that, at this stage of the disease process, the presence of diffuse leukoencephalopathy does not imply subtle cognitive defects.
Identification and characterization of a novel member of the dystrobrevin gene family.
Publication Date: 20/03/1998, on FEBS letters
by Puca AA, Nigro V, Piluso G, Belsito A, Sampaolo S, Quaderi N, Rossi E, Di Iorio G, Ballabio A, Franco B
A new member of the dystrobrevin gene family was identified using a bioinformatics approach. Sequence analysis indicates that this gene, named DTN-B, is highly homologous to the rabbit A0, the previously described dystrobrevin (DTN), Torpedo 87 kDa and to the C-terminus of dystrophin. The coiled-coil domain, shown to be the site of interaction between dystrobrevins and dystrophin, is highly conserved. Immunostaining studies indicate that DTN-B and DTN expression is absent in affected muscle fibers from DMD patients and carriers.
Caveolin transfection results in caveolae formation but not apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins in epithelial cells.
Publication Date: 09/02/1998, on The Journal of cell biology
by Lipardi C, Mora R, Colomer V, Paladino S, Nitsch L, Rodriguez-Boulan E, Zurzolo C
Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The "raft" hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI-anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI-anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42-53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI-anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.
Antisense oligonucleotides and myotonin gene expression in C2 mouse cells.
Publication Date: 01/02/1998, on Antisense & nucleic acid drug development
by Melone MA, Galderisi U, Iacomino G, Cipollaro M, Di Bernardo G, Cotrufo R, Peluso G, Cascino A
By describing the behavior of myotonin mRNA levels, from the quiescent to the differentiated state in C2 mouse myoblasts, we produced evidence bearing on the role of myotonin gene product in the control of cell growth and differentiation. To study the role of myotonin in myotonic dystrophy (DM) pathogenesis, we developed a suitable cellular model where myotonin gene expression was modulated by phosphorothioate antisense oligonucleotides in C2 cultured cells. Furthermore, an isoform of the gene product, similar to that described in humans and not yet described in the mouse, was found.
'Pure' constructional apraxia - a cognitive analysis of a single case.
Publication Date: 01/01/1998, on Behavioural neurology
by Trojano L, Grossi D
We report on a patient affected by selective drawing disabilities. The patient could correctly reproduce and draw simple geometric figures on request, but when he tried to reproduce more complex drawings or to draw common objects he performed very poorly. To identify the cognitive impairment in this patient, we adopted two test batteries based on recent information-processing models of drawing. Results showed that the patient's drawing disabilities were independent of visuo-perceptual and executive impairments. These findings support recent cognitive models of drawing abilities: some intermediate stages of drawing exist at which information is processed to prepare and guide motor output, and which may be selectively disrupted after discrete cerebral lesions.
Dystrophin localization and gene expression in the developing nervous system of the chick.
Publication Date: 01/01/1998, on Journal of neuroscience research
by Perrone-Capano C, Crispino M, Oteri G, Tata AM, Vignoli AL, Poiana G
The presence and distribution of dystrophin was studied in selected areas of the chick embryo nervous system and in primary cultures. Dystrophin was examined at the protein level by immunocytochemistry and at the transcriptional level by a semiquantitative reverse transcriptase-polymerase chain reaction analysis. Immunofluorescence staining shows that dystrophin is present early during embryogenesis in dorsal root ganglia, spinal cord, and ciliary ganglia and colocalizes with neurofilament subunits. Cultured dorsal root ganglion, spinal cord, and ciliary ganglion neurons show immunoreactivity for dystrophin, both in cell bodies and along fibers. Dystrophin mRNA level in ciliary and dorsal root ganglia is higher than in spinal cord throughout development and shows a tissue-specific pattern of expression. In primary cultures of dorsal root ganglia and ciliary ganglia, dystrophin mRNA level increases with time in vitro. However, in spinal cord cultures, dystrophin mRNA drastically decreases with time in vitro, but it is significantly increased when embryonic muscle extract is added to the cultures. Our results show that dystrophin is present in neurons from different areas of embryonic chick nervous system and that its mRNA level is developmentally regulated both in vivo and in vitro.
CTG repeat number in the nonaffected allele of myotonic dystrophy patients is not critical for disease expression.
Publication Date: 01/12/1997, on Human biology
by Cipollaro M, Galderisi U, Iacomino G, Galano G, Di Bernardo G, Lus G, Cotrufo R, Orsini A, Santoro L, Pastore L, Sarrantonio C, Salvatore F, Cascino A
To investigate whether unusual allele segregation might explain the dominant negative effect of the expanded allele for myotonic dystrophy on myotonin protein kinase mRNA metabolism, which is suggested to cause the disease, we determined the number of CTG repeats at the DM locus in the nonaffected alleles of 64 DM (dystrophia myotonia) patients. The relative distribution was then compared with the distributions obtained from alleles of the normal parents and normal siblings of DM patients. Comparison was also made with the allele distribution of normal subjects from the same geographic area. It appears that the CTG repeat number of the nonaffected allele in DM patients is not critical for the expression of the disease.
Long-term and low-dose treatment with cabergoline induces macroprolactinoma shrinkage.
Publication Date: 01/11/1997, on The Journal of clinical endocrinology and metabolism
by Colao A, Di Sarno A, Landi ML, Cirillo S, Sarnacchiaro F, Facciolli G, Pivonello R, Cataldi M, Merola B, Annunziato L, Lombardi G
Cabergoline (CAB), a long-lasting dopamine-agonist, specific for the D2 receptor, is effective in normalizing serum PRL levels in most patients with microprolactinoma or idiopathic hyperprolactinemia. Because few data are presently available on the effects of CAB treatment in macroprolactinomas, the aim of this open-label study was to investigate whether this drug was effective in producing tumor shrinkage, as well as in normalizing PRL levels. Twenty-three patients with macroprolactinoma entered this study 15 patients had had no treatment, whereas the remaining 8 patients had been previously treated with bromocriptine, which was with-drawn because of intolerance. Three of 23 patients had undergone unsuccessful surgery. Pretreatment serum PRL levels ranged from 100-3860 micrograms/L. CAB was administered at a dose of 0.5-3 mg once or twice a week for 12-24 months. Magnetic resonance imaging (MRI) scans were performed before and 3, 6, 12, and 24 months after the beginning of treatment, to evaluate tumor shrinkage, defined as a decrease of at least 80% of baseline tumor volume. After 3-6 months of treatment with a low dose (0.5-1 mg/week), serum PRL levels normalized in 18 patients. In the remaining 5 patients, whose serum PRL levels were not normalized, the dose was increased to 2-3 mg/week. This schedule caused the normalization of PRL levels in 1 patient, whereas in the remaining 4 patients, PRL levels were reduced to 30-82 micrograms/L. A tumor volume reduction greater than 80% at MRI occurred in 14 of 23 patients (61%) after CAB treatment (from 2609.4 +/- 534.7 to 530.1 +/- 141.3 mm3 at the 12-24th month follow-up, P < 0.001). A volume reduction of 41.8 +/- 3.4% was already evident after 3 months (1436 +/- 285.9 mm3; P < 0.001). The complete disappearance of the tumor mass at MRI occurred after 6 months of treatment with CAB in 1 patient, and in 5 patients after 1 yr of treatment. An improvement of visual field defects was obtained in 9 of the 10 patients presenting visual impairment before CAB treatment. The drug was tolerated well by all patients. Only 1 patient experienced mild nausea, which disappeared spontaneously after the 2nd day of treatment. Long-term, a low dose of the D2 receptor agonist CAB significantly reduced tumor volume and normalized serum PRL levels in a great majority of patients bearing macroprolactinoma. This treatment met with excellent patient compliance. This study suggests that CAB can be used as a first choice drug treatment in macroprolactinomas, as already shown for microprolactinomas and idiopathic hyperprolactinemia.
Active polysomes are present in the large presynaptic endings of the synaptosomal fraction from squid brain.
Publication Date: 15/10/1997, on The Journal of neuroscience : the official journal of the Society for Neuroscience
by Crispino M, Kaplan BB, Martin R, Alvarez J, Chun JT, Benech JC, Giuditta A
Previous data have suggested that the large nerve terminals present in the synaptosomal fraction from squid optic lobe are capable of protein synthesis (Crispino et al., 1993a,b). We have further examined this issue by comparing the translation products of synaptosomal and microsomal polysomes. Both preparations programmed an active process of translation, which was completely abolished by their previous treatment with EDTA. After immunoabsorption of the newly synthesized neurofilament (NF) proteins, the labeling ratio of the 60 and 70 kDa NF proteins was found to differ, in agreement with comparable differences obtained with intact synaptosomes. These observations indicate that the set of mRNAs translated by synaptosomes differs from that translated by nerve cell bodies. Hence, because NF proteins are neuron-specific, they support the view that the active synaptosomal polysomes are mostly localized in the large nerve terminals that represent the most abundant neuronal component of the fraction. This hypothesis was confirmed (1) by electron spectroscopic data demonstrating the presence of ribosomes and polysomes within the large nerve endings of the synaptosomal fraction, as well as in the carrot-like nerve endings of the retinal photoreceptors that constitute the only large terminals in the optic lobe, and (2) by light and high resolution autoradiography of synaptosomal samples incubated with [3H]leucine, showing that most labeled proteins are associated with the large nerve endings. This response was abolished by cycloheximide. Taken together, the data provide the first unequivocal demonstration that presynaptic nerve terminals are capable of protein synthesis.
Post-trial sleep in old rats trained for a two-way active avoidance task.
Publication Date: 01/10/1997, on Physiology & behavior
by Ambrosini MV, Bruschelli G, Mariucci G, Mandile P, Giuditta A
Nine male Wistar rats aged 27 months were trained for a two-way active avoidance task and tested for retention the following day. At variance with young adult rats, most of which succeed in mastering the task, all old rats displayed a large majority of freezing responses throughout the training and the retention sessions, thereby confirming the condition of learning impairment of aged rats. Comparison of baseline and post-trial sleep indicated the presence of a transient, but marked, increment in the average duration and total amount of post-trial slow-wave sleep followed by waking, and of a decrease in total amount of quiet waking. On the other hand, variables of paradoxical sleep and of slow-wave sleep followed by paradoxical sleep or by transition sleep did not show significant variations. Because these sleep variables are known to undergo significant variations in learning in young adult rats, the present data confirm that the latter effects are related to memory-processing events rather than to nonspecific effects of training. An additional outcome of training consisted in a marked post-trial decrement in the number of spike-wave discharges, which are known to occur in old rats during periods of quiet waking.