Transglutaminase from rat coagulating gland secretion. Post-translational modifications and activation by phosphatidic acids.
Publication Date: 01/11/1996, on The Journal of biological chemistry
by Esposito C, Pucci P, Amoresano A, Marino G, Cozzolino A, Porta R
Structural and biochemical characteristics of transglutaminase purified by a rapid chromatographic procedure from the rat coagulating gland (anterior prostate) secretion are reported. Fast atom bombardment mapping and automated Edman degradation experiments allowed us to verify that at least 85% of the entire transglutaminase amino acid sequence is identical to that derived from the cDNA of the major androgen-dependent rat prostate protein called DP1. The enzyme was found NH2 terminally blocked and largely post-translationally modified, since the presence of N-linked oligosaccharides, as well as of complex lipidic structures, was observed. Mass spectral analysis showed that Asn-408 and -488 are the glycosylated sites, the N-linked structures identified belonging to both high-mannose and complex type glycans. The presence of myo-inositol, of glycerol bound fatty acids, and the high content of mannose residues, are in agreement with previous observations suggesting that a lipid anchor is bound to coagulating gland secretion transglutaminase. Furthermore, two tightly bound calcium ions per molecule of enzyme were detected. Finally, a strong stimulation of the enzyme activity in vitro by both SDS and a variety of phosphatidic acids was observed. The reported structural and functional peculiarities should definitively lead to consider the prostate enzyme as a new member (type IV) of the transglutaminase family.
Differential compartmentalization of mRNAs in squid giant axon.
Publication Date: 01/11/1996, on Journal of neurochemistry
by Chun JT, Gioio AE, Crispino M, Giuditta A, Kaplan BB
Previously, we reported that the squid giant axon contains a heterogeneous population of mRNAs that includes beta-actin, beta-tubulin, kinesin, neurofilament proteins, and enolase. To define the absolute levels and relative distribution of these mRNAs, we have used competitive reverse transcription-PCR to quantify the levels of five mRNAs present in the giant axon and giant fiber lobe (GFL), the location of the parental cell soma. In the GFL, the number of transcripts for these mRNAs varied over a fourfold range, with beta-tubulin being the most abundant mRNA species (1.25 x 10(9) molecules per GFL). Based on transcript number, the rank order of mRNA levels in the GFL was beta-tubulin > beta-actin > kinesin > enolase > microtubule-associated protein (MAP) H1. In contrast, kinesin mRNA was most abundant in the axon (4.1 x 10(7) molecules per axon) with individual mRNA levels varying 15-fold. The rank order of mRNA levels in the axon was kinesin > beta-tubulin > MAP H1 > beta-actin > enolase. The relative abundance of the mRNA species in the axon did not correlate with the size of the transcript, nor was it directly related to their corresponding levels in the GFL. Taken together, these findings confirm that significant amounts of mRNA are present in the giant axon and suggest that specific mRNAs are differentially transported into the axonal domain.
Protein synthesis in the presynaptic endings of the squid photoreceptor neuron: in vitro and in vivo modulation.
Publication Date: 01/10/1996, on The Biological bulletin
by Benech JC, Crispino M, Martin R, Alvarez J, Kaplan BB, Giuditta A
Epigenetic factors and midbrain dopaminergic neurone development.
Publication Date: 01/10/1996, on BioEssays : news and reviews in molecular, cellular and developmental biology
by Perrone-Capano C, di Porzio U
In the mammalian brain dopamine systems play a central role in the control of movement, hormone release, emotional balance and reward. Alteration of dopaminergic neurotransmission is involved in Parkinson's disease and other movement disorders, as well as in some psychotic syndromes. This review summarises recent findings, which shed some light on signals and cellular interactions involved in the specification and maturation of the dopaminergic function during neurogenesis. In particular we will focus on three major issues: (1) the differentiation of dopaminergic neurones triggered by direct contact with the midbrain floor plate cells through the action of sonic hedgehog; (2) the neurotrophic factors acting on dopaminergic neurones; and (3) the role of target striatal cells on the survival and the axonal growth of developing or grafted dopaminergic neurones.
Modulation of cytokine production in activated human monocytes by somatostatin.
Publication Date: 01/10/1996, on Neuropeptides
by Peluso G, Petillo O, Melone MA, Mazzarella G, Ranieri M, Tajana GF
The immunosuppressor effects of the widely distributed neuropeptide somatostatin were examined on purified peripheral blood human monocytes. Somatostatin, at concentrations thought to be physiologic (10(-10)-10(-7) M), regulated monocyte/macrophage responses to (LPS) stimulation, as reflected by interleukin production. In particular, somatostatin had direct inhibitory effects on TNF-alpha, IL-1 beta, and IL-6 secretion by LPS-activated monocytes, while the decrease on IL-8 synthesis was modulated mainly by the action of somatostatin on TNF-alpha and IL-1 beta. In fact, the addition of these two inflammatory cytokines to the monocyte culture medium was able to induce IL-8 expression, as demonstrated by mRNA analysis, also in presence of the neuropeptide. Although somatostatin affected IL-8 production in an indirect way, it suppressed directly the chemotactic response of neutrophils to IL-8. Finally, somatostatin downregulation of monocyte activation was confirmed by the decrease of HLA-DR expression on cell plasma membranes (52% versus 33%). Our results confirm that somatostatin exerts preferential effects on the suppression of immunoreactions by modulating cytokine production and activity.
Comparison of SPT and NPT in the ascertainment of nasal mucosa as shock organ.
Publication Date: 01/09/1996, on Rhinology
by Testa B, Mesolella C, Testa F, Gallo LV, Testa D
The Skin Prick Test (SPT) is the principal tool in allergic diagnosis, but in allergic rhinitisan immunological disease which affects 12% of the total population-the Nasal Provocation Test (NPT) allows more reliable results to be obtained. In our study a positive response to NPT has been found in four subjects with a history of symptoms suggesting allergic rhinitis, who had a negative SPT. Subjects with a positive SPT for two or more inhalant antigens have a significantly reduced number of antigen responses to NPT. Moreover, in two cases, the antigen than induced a positive response to NPT was different from the antigen that induced positive SPT. So. NPT is a reliable way of diagnosing allergic rhinitis. A more specific and reliable ascertainment of the antigen responsible for allergic reaction avoids unnecessary and ineffective immunotherapeutical attempts based on false assumptions.
Delta-selective opioid peptides containing a single aromatic residue in the message domain: an NMR conformational analysis.
Publication Date: 01/09/1996, on Journal of peptide science : an official publication of the European Peptide Society
by Crescenzi O, Amodeo P, Cavicchioni G, Guerrini R, Picone D, Salvadori S, Tancredi T, Temussi PA
The sequence of deltorphin I, a delta-selective opioid agonist, has been systematically modified by inserting conformationally constrained C alpha, alpha disubstituted apolar residues in the third position. As expected, substitution of Phe with Ac6c, Ac5c and Ac3c yields analogues with decreasing but sizeable affinity. Surprisingly, substitution with Aib yields an analogue with almost the same binding affinity of the parent compound but with a greatly increased selectivity. This is the first case of a potent and very selective opioid peptide containing a single aromatic residue in the message domain, that is, only Tyr1. Here we report a detailed conformational analysis of [Aib3]deltorphin I and [Ac6c3]deltorphin I in DMSO at room temperature and in a DMSO/water cryomixture at low temperature, based on NMR spectroscopy and energy calculations. The peptides are highly structured in both solvents, as indicated by the exceptional finding of a nearly zero temperature coefficient of Val5 NH resonance. NMR data cannot be explained on the basis of a single structure but it was possible to interpret all NMR data on the basis of a few structural families. The conformational averaging was analysed by means of an original computer program that yields qualitative and quantitative composition of the mixture. Comparison of the preferred solution conformation with two rigid delta-selective agonists shows that the shapes of [Aib3]deltorphin I and [Ac6c3]deltorphin I are consistent with those of rigid agonists and that the message domain of opioid peptides can be defined only in conformational terms.
Dopamine transporter gene expression in rat mesencephalic dopaminergic neurons is increased by direct interaction with target striatal cells in vitro.
Publication Date: 01/07/1996, on Brain research. Molecular brain research
by Perrone-Capano C, Tino A, Amadoro G, Pernas-Alonso R, di Porzio U
By using a semi-quantitative reverse transcriptase-PCR assay (RT-PCR) we have analyzed dopamine transporter (DAT), tyrosine hydroxylase (TH) and synaptic vesicle monoamine transporter (VMAT2) gene expression in rat mesencephalic (MES) primary cultures. Consistent with previous data obtained during rat MES ontogeny, the onset of DAT transcription in vitro is delayed in embryonic day (E)13, but not in E16, MES neurons when compared to that of TH and VMAT2. In co-culture, the addition of target striatal cells (STR) to E13 MES selectively increases DAT mRNA level in DA neurons during the first 3 days in vitro; cortical cells are ineffective. On the contrary, DAT gene does not appear up-regulated in E16 MES co-cultured with target STR cells, indicating that MES DA neurons respond to STR stimulation only at defined developmental stages. Up-regulation of DAT mRNA level by STR in E13 MES seems to require direct cell interactions since target cells do not exert their effect on DAT transcription when are separated from MES cells by a porous barrier, which only allows diffusion of soluble molecules. Thus maturation of DA neurotransmission in vitro appears to follow a developmental program which can be specifically modulated by their target STR cells.
Selective drawing disorders after right subcortical stroke: a neuropsychological premorbid and follow-up case study.
Publication Date: 01/06/1996, on Italian journal of neurological sciences
by Grossi D, Calise G, Correra C, Trojano L
We report the case of a patient affected by a subcortical lesion of the right non-dominant hemisphere, and demonstrate that he had selective constructional disorders by comparing his post-stroke performances with those assessed 18 months before the stroke. A detailed analysis was made of the visuospatial, perceptual, representational and executive competences involved in drawing tasks at one, two and six months post-stroke. Neuropsychological follow-up revealed the progressive recovery of all visuospatial abilities. This study provides some interpretative elements for constructional disorders and, in particular, for the closing-in phenomenon observed only during the subacute phase.
Autoinduction of androgen receptor mRNA in primary cultures of hamster (Mesocricetus auratus) harderian gland cells.
Publication Date: 01/06/1996, on General and comparative endocrinology
by Varriale B
The Harderian gland (hg) is a gland which occupies a large portion of the orbital cavity. In many species, a sexual dimorphism occurs, which suggests a gonadal steroid control of the hg. The present study examines, in primary cultures of hamster hg cells, the regulation of the androgen receptor mRNA (AR mRNA) expression. In dose-response experiments measuring the expression of AR mRNA, testosterone (T) (10(-12) M) induced a 1-fold increase of AR mRNA compared with unexposed cells, and this effect reached its zenith (6.2-fold) when cells were exposed to 10(-8) M T. In other experiments, cells were exposed or not to different drugs [T, T + flutamide (F), F, T + cycloheximide (Cy), Cy] for different times (up to 96 hr). These experiments showed a time-dependent increase of AR mRNA in the cells exposed to T, while in the cells exposed to F, T + F, T + Cy, Cy, and control (unexposed), a time-dependent decrease of AR mRNA was observed. The size of the hamster AR mRNA in these in vitro experiments was similar to that observed in other mammals (9.5 kb). It is concluded that primary cultures of hamster hg cells are a valuable model for studying hg cell activity and that in this system T autoinduces its own receptor.
Occurrence of androgen and estrogen receptor mRNAs in the harderian gland: a comparative survey.
Publication Date: 01/06/1996, on Microscopy research and technique
by Varriale B
In Rana esculenta the presence of an androgen receptor in both the male and female Harderian gland (HG) has been demonstrated. Hybridization analysis has evidenced a high degree of homology between the rat androgen receptor cDNA and the frog androgen receptor mRNA (fARmRNA). Correspondingly the molecular size of fARmRNA is similar to those described in mammals (9.4 kb). In in vivo experiments testosterone (T) increases the levels of fARmRNA. The use of the antiandrogen alone or in combination with T prevents the increase of fARmRNA. In the control animals a loss of fARmRNA has been observed. In primary cultures of HG cells, the steady-state levels of fARmRNA increase in the cells exposed to T. These results suggest that T exerts an autoinduction on its own receptor, increasing the levels of fARmRNA. In Xenopus laevis the HG shows a sexual dimorphism of the protein pattern. The female shows two major proteins (210 and 180 kDa). Administration of estradiol to the male shifts the protein pattern into the female one. In this respect an estrogen receptor mRNA (ERmRNA) has been found in the female gland and can be induced in the male one. No ARmRNA has been detected in either sexes. A similar sex dimorphism has been found in Gallus domesticus. The female pattern is characterised by a protein fraction of about 210 kDa, the male one by a protein fraction of about 180 kDa. In 4-day-old chicks no sex differences have been found. An ERmRNA is expressed in the female, while no ARmRNA has been detected in both sexes. Neither AR nor ER mRNAs have been detected in the chick HG. Among mammals the HG or the hamster (Mesocricetus auratus) shows an androgen-dependent sex dimorphism. In in vitro experiments T 10(-12) M induces a onefold increase of ARm-RNA with respect to unexposed cells. This effect reaches its maximum (4.4-fold) when cells are exposed to T 10(-8) M. The size of the hamster ARmRNA is similar to that observed in other mammals (9.5 kb). The above results suggest that in the HG the phenomenon of autoinduction occurs and that there is a relationship between the androgen or estrogen dependence of the HG and the digamety of the species.
Early upregulation of medium neurofilament gene expression in developing spinal cord of the wobbler mouse mutant.
Publication Date: 01/06/1996, on Brain research. Molecular brain research
by Pernas-Alonso R, Schaffner AE, Perrone-Capano C, Orlando A, Morelli F, Hansen CT, Barker JL, Esposito B, Cacucci F, di Porzio U
Homozygous wobbler mouse mutants develop a progressive paralysis due to spinal motoneuron degeneration. To understand the molecular aspect underlying the genetic defect we have studied the embryonic (from E13) and postnatal expression of the three neurofilament and choline acetyltransferase genes in each member from several wild-type (wt) and wobbler (wr) progenies. There are no variations among wt littermates at all ages studied. In contrast, analyses of neurofilament mRNA reveals a 3-4-fold increase of medium neurofilament (NFM) mRNA in wobbler mice (wr/wr). The pattern of increased NFM mRNA during development, prior to the appearance of the wobbler phenotype, among littermates (from heterozygous carriers) conforms to a mendelian inheritance of a single gene defect 1:2:1 (wr/wr:wr/+:+/+). Light and heavy neurofilament mRNA levels are also increased later in development exclusively in those individuals with high NFM mRNA values indicating that increase of the latter is associated with increase of the light and heavy subunit expression. Also NF proteins are increased. Expression of choline acetyltransferase gene is instead always comparable to normal control. Our study provides novel insights into the nature of the wobbler defect, strengthening the hypothesis that neurofilament accumulation plays a pivotal role in the etiopathogenesis of motoneuron degeneration.
Myotonic dystrophy: antisense oligonucleotide inhibition of DMPK gene expression in vitro.
Publication Date: 25/04/1996, on Biochemical and biophysical research communications
by Galderisi U, Cipollaro M, Melone MA, Iacomino G, Di Bernardo G, Galano G, Contrufo R, Zappia V, Cascino A
Antisense phosphorothioate oligonucleotides, targeted against the first codon starting region of DMPK mRNA, were successfully used in K562 and HepG2 cells to decrease DMPK expression. The most effective antisense oligo, MIO1, when added to K562 cells, shows a 75% reduction of the DMPK gene expression 6 hours after addition. The same molecule, when encapsulated in liposomes, delays myotonin mRNA decrease at 24 hours after cell treatment. This considerable success with such inhibition in vitro could be utilised to generate a cell model to study myotonic dystrophy (DM) chemio-physiological alterations.
Implantation of an EEG telemetric transmitter in the rat.
Publication Date: 01/04/1996, on Italian journal of neurological sciences
by Cotugno M, Mandile P, D'Angiolillo D, Montagnese P, Giuditta A
We describe a method of implanting a telemetric transmitter of EEG signals in the laboratory rat. The transmitter is available commercially and may be implanted in a subcutaneous pocket prepared in the hindermost dorsal region of the animal. The two stainless steel electrodes connected to the transmitter are led to the cranium through a subcutaneous tunnel, and are fixed to the cranium bones. EEG signals are collected by a receiver placed under the cage; reception of the signals is improved by suitably placed antennae. The method allows recording of EEG data from a free-moving rat during the expression of behavioral tasks in a limited space.
The gene, protein and glycan structures of laccase from Pleurotus ostreatus.
Publication Date: 01/02/1996, on European journal of biochemistry
by Giardina P, Aurilia V, Cannio R, Marzullo L, Amoresano A, Siciliano R, Pucci P, Sannia G
A member of the laccase multigene family in Pleurotus ostreatus has been cloned and sequenced. The gene structure has been determined by comparison with the corresponding cDNA, synthesized by reverse transcription/PCR amplification. The gene encode a laccase isoenzyme of 533 amino acids which has already been purified and characterized [Palmieri, G., Giardina, P., Marzullo, L., Desiderio, B., Nitti, G., Cannio, R. & Sannia, G.(1993) Appl. Microbiol. Biotechnol. 39, 632-636]. More than 92% of the protein sequence, including the N and C termini, has been verified by fast-atom-bombardment mass spectrometry, thus confirming the correspondence between the gene and its protein product. The protein was N-glycosylated Asn444. Glycan analysis showed the presence of only a high-mannose structure containing varying numbers of mannose residues. The presence of O-linked oligosaccharides as well as other post-translational modification could be ruled out by the mass analysis.