Mallory stain may indicate differential rates of RNA synthesis: I. A seasonal cycle in the harderian gland of the green frog (Rana esculenta).
Publication Date: 01/01/1992, on European journal of histochemistry : EJH
by Chieffi Baccari G, Marmorino C, Minucci S, Di Matteo L, Varriale B, d'Istria M, Chieffi G
When Mallory's trichrome stain is used, acinar nuclei of the Harderian gland of Rana esculenta display different affinities for the dye. Some of the orangiophilic nuclei show affinity for aniline blue (blue nuclei). In the Harderian gland of Rana esculenta their number and the intensity of staining with aniline blue may vary during the year. The affinity for aniline blue disappears following digestion of paraffin sections with RNAase, but not with DNAase or trypsin. Furthermore, in vitro incubation with [5, 6-3H]-Uridine shows a selective incorporation by the majority of blue nuclei. Therefore, the affinity for aniline blue is likely due to increased RNA synthesis. The increment of nuclear RNA shown by these methods is supported by the quantitative determination of total RNAs during the resumption (October) and enhancement (May) of secretory activity, when the percentage of blue nuclei of the acinar cells is at its highest levels of the year. The affinity of RNA-rich nuclei for aniline blue, while others are strictly orangiophil, is discussed on the basis of molecular structure of the dyes used in the staining mixture. Mallory's trichrome stain appears to be an useful tool for detecting changes in cell nuclear status.
Short-term memory and verbal learning with auditory phonological coding defect: a neuropsychological case study.
Publication Date: 01/01/1992, on Brain and cognition
by Trojano L, Stanzione M, Grossi D
A patient is described with a rarely reported linguistic syndrome: he could repeat words but not nonwords. The patient produced semantic paraphasias in repetition and could read both words and nonwords flawlessly. His basic difficulties were localized in auditory phonological coding, identifying a clinical picture called "phonemic deafness." Short-term memory and verbal learning results suggested that a standard, selective short-term memory defect can be induced by auditory phonological coding deficits as well as by "pure" short-term memory capacity limitation and other phonological deficits. Findings also provided evidence that lexical-semantic code can allow normal verbal learning.
Solution structure of deltorphin I at 265 K: a quantitative NMR study.
Publication Date: 01/01/1992, on Peptide research
by Amodeo P, Motta A, Tancredi T, Salvadori S, Tomatis R, Picone D, Saviano G, Temussi PA
Deltorphin I, a delta-selective opioid peptide, has been studied in a DMSOd6/H2O cryoprotective mixture by two-dimensional (2D) NMR spectroscopy in the temperature range 260 K to 305 K. The high viscosity of the solvent at low temperature mimics a distinctive physico-chemical feature of cytoplasm and allows the measurement of a NOESY spectrum rich in intra- and inter-residue effects. Backbone NOEs at 265 K can be calculated with good accuracy in terms of only two limiting conformers: one folded, with a mole fraction of 0.30, and another extended with a mole fraction of 0.70. This calculation is still a rough approximation of the complex conformational equilibria existing in solution but, to the best of our knowledge, is the first one for a flexible peptide, and represents an encouraging starting point for a quantitative evaluation of NMR data of small, flexible peptides in solution. The folded conformer consistent with observed NOEs has a shape surprisingly similar to those of unrelated, rigid, delta-selective opiates.
The active site of Sulfolobus solfataricus aspartate aminotransferase.
Publication Date: 15/11/1991, on Biochimica et biophysica acta
by Birolo L, Arnone MI, Cubellis MV, Andreotti G, Nitti G, Marino G, Sannia G
Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus binds pyridoxal 5' phosphate, via an aldimine bond, with Lys-241. This residue has been identified by reducing the enzyme in the pyridoxal form with sodium cyanoboro[3H]hydride and sequencing the specifically labeled peptic peptides. The amino acid sequence centered around the coenzyme binding site is highly conserved between thermophilic aspartate aminotransferases and differs from that found in mesophilic isoenzymes. An alignment of aspartate aminotransferase from Sulfolobus solfataricus with mesophilic isoenzymes, attempted in spite of the low degree of similarity, was confirmed by the correspondence between pyridoxal 5' phosphate binding residues. Using this alignment it was possible to insert the archaebacterial aspartate aminotransferase into a subclass, subclass I, of pyridoxal 5' phosphate binding enzymes comprising mesophilic aspartate aminotransferases, tyrosine aminotransferases and histidinol phosphate aminotransferases. These enzymes share 12 invariant amino acids most of which interact with the coenzyme or with the substrates. Some enzymes of subclass I and in particular aspartate aminotransferase from Sulfolobus solfataricus, lack a positively charged residue, corresponding to Arg-292, which in pig cytosolic aspartate aminotransferase interacts with the distal carboxylate of the substrates (and determines the specificity towards dicarboxylic acids). It was confirmed that aspartate aminotransferase from Sulfolobus solfataricus does not possess any arginine residue exposed to chemical modifications responsible for the binding of omega-carboxylate of the substrates. Furthermore, it has been found that aspartate aminotransferase from Sulfolobus solfataricus is fairly active when alanine is used as substrate and that this activity is not affected by the presence of formate. The KM value of the thermophilic aspartate aminotransferase towards alanine is at least one order of magnitude lower than that of the mesophilic analogue enzymes.
Androgen receptor in the Harderian gland of Rana esculenta.
Publication Date: 01/05/1991, on The Journal of endocrinology
by d'Istria M, Chieffi-Baccari G, Di Matteo L, Minucci S, Varriale B, Chieffi G
An androgen receptor has been identified in the cytosolic and nuclear extracts of the Harderian gland of the frog, Rana esculenta. A single class of high-affinity binding sites was found: Kd = 1.9 +/- 1.3 (S.D.) nmol/l (n = 26) for the cytosolic extract and Kd = 0.9 +/- 0.8 nmol/l (n = 15) for the nuclear extract. The presence of binding activity in both nuclear and cytosolic extracts and the low rate of ligand-receptor dissociation are characteristics that distinguish this receptor from a steroid-binding protein. The Kd did not show any sex difference and did not exhibit any secretory activity-related change. Binding in both cytosolic and nuclear extracts was specific for androgens (testosterone = 5 alpha-dihydrotestosterone); oestradiol-17 beta showed a 30% cross-reaction; moreover, specific binding of [3H]oestradiol-17 beta was not detectable. The binding capacity of the Harderian gland increased progressively in both fractions from October to December, reaching a peak in May, and decreased suddenly during July to August. The lack of any morphological sex-related difference in the Harderian gland of the green frog might be accounted for by the high amount of circulating androgens as well as a similar concentration of androgen receptor in both sexes.
New insights on mu/delta selectivity of opioid peptides: conformational analysis of deltorphin analogues.
Publication Date: 01/05/1991, on Biopolymers
by Tancredi T, Temussi PA, Picone D, Amodeo P, Tomatis R, Salvadori S, Marastoni M, Santagada V, Balboni G
The message domain of dermorphin (Tyr-D-Ala-Phe), a natural mu-opioid heptapeptide, has long been considered the main cause of the high mu selectivity of this peptide and of its analogues. The recent discovery, in the skin of Phyllomedusa sauvagei (i.e., the same natural source of dermorphin) and of Phyllomedusa bicolor of deltorphins, challenges this belief. Deltorphins, in fact, are three heptapeptides characterized by a message domain typical of mu-selective peptides, but endowed of an extremely high delta selectivity, the highest of all natural opioid peptides. A conformational analysis of dermorphin and deltorphins, based on nmr studies in DMSO and cryoprotective mixtures and internal energy calculations, showed that the enormous differences in receptor selectivity can be interpreted on the basis of receptor models for mu and delta opioids that recognize the same beta-turn in the N-terminal part, but discriminate for the conformation and polarity of the C-terminal part. Here we present the synthesis, biological activity, and conformational analysis in solution of three deltorphin analogues with very similar constitution, but with different net charge, different location of negative residues, or even without negative residues, which confirm these hypotheses and show that His4 can play a specific structural role.
Cataract risk factors: blood level of antioxidative vitamins, reduced glutathione and malondialdehyde in cataractous patients.
Publication Date: 01/01/1991, on Metabolic, pediatric, and systemic ophthalmology (New York, N.Y. : 1985)
by Libondi T, Costagliola C, Della Corte M, Facchiano F, Menzione M, Savastano S, Simonelli F, Rinaldi E, Auricchio G
Since many years experimental evidences have suggested an association between nutrition and lens opacities. A dietary deficiency of antioxidants and reactive oxygen scavengers may be involved in the pathogenesis of the "idiopathic" human senile cataract, as it has been demonstrated in some experimental cataracts. We tested the levels of ascorbic acid (vit. C), alpha-tocopherol (vit. E), reduced glutathione (GSH) and malondialdehyde (MDA) in the plasma or in the red blood cells (RBC) of 42 patients who were affected by surgically significant cataract and of 40 age-matched controls. Plasma vit. C mean level was 4.46 gamma/ml in cataracts and 4.62 gamma/ml in controls, while vit. E level was 7.70 and 7.09 gamma/ml respectively. RBC GSH was found to be 342 gamma/ml in cataracts and 346 in controls, while the MDA content was 4.06 picoMol/ml and 4.08 picoMol/ml respectively. The level of each tested nutrient or metabolite was not found to be statistically different between cataractous patients and controls, nor any significant trend was found to be present when the nutrients and metabolites were correlated to each other. Our results do not support the hypothesis of a nutritional deficiency in human senile cataracts. However, a defect in the antioxidative metabolism pathways could be present either systemically or at lens level.
Mixed transcortical aphasia: clinical features and neuroanatomical correlates. A possible role of the right hemisphere.
Publication Date: 01/01/1991, on European neurology
by Grossi D, Trojano L, Chiacchio L, Soricelli A, Mansi L, Postiglione A, Salvatore M
This paper reports the clinical features of 2 patients affected by mixed transcortical aphasia. Both of them had extensive damage of the left hemisphere. Cerebral blood flow measurement showed a marked decrease in left-hemisphere flow. In particular, speech areas were found to be morphologically and functionally damaged. The present findings, together with a critical review of the literature, served as a starting point for a discussion of linguistic features, diagnostic criteria and classical neuroanatomical interpretation of mixed transcortical aphasia. The possible contribution of the right hemisphere in determining this aphasic syndrome is suggested.
Active polysomes in the axoplasm of the squid giant axon.
Publication Date: 01/01/1991, on Journal of neuroscience research
by Giuditta A, Menichini E, Perrone Capano C, Langella M, Martin R, Castigli E, Kaplan BB
Axons and axon terminals are widely believed to lack the capacity to synthesize proteins, relying instead on the delivery of proteins made in the perikaryon. In agreement with this view, axoplasmic proteins synthesized by the isolated giant axon of the squid are believed to derive entirely from periaxonal glial cells. However, squid axoplasm is known to contain the requisite components of an extra-mitochondrial protein synthetic system, including protein factors, tRNAs, rRNAs, and a heterogeneous family of mRNAs. Hence, the giant axon could, in principle, maintain an endogenous protein synthetic capacity. Here, we report that the squid giant axon also contains active polysomes and mRNA, which hybridizes to a riboprobe encoding murine neurofilament protein. Taken together, these findings provide direct evidence that proteins (including the putative neuron-specific neurofilament protein) are also synthesized de novo in the axonal compartment.
Blood-brain barrier permeability to micromolecules and edema formation in the early phase of incomplete continuous ischemia.
Publication Date: 01/01/1991, on Acta neuropathologica
by Sampaolo S, Nakagawa Y, Iannotti F, Cervos-Navarro J, Bonavita V
The distribution patterns of ionic Lanthanum (La3+; mol. wt. 139) were evaluated after 15, 30 and 60 min of middle cerebral artery occlusion in perfused-fixed rats. Blood-brain barrier (BBB) permeability to Evans blue (EB) and horseradish peroxidase (HRP; mol. wt. 40,000) in vivo was also evaluated. Brain tissue specific gravity was measured. An increase in brain water content was found as early as 30 min following occlusion. HRP and EB extravasation was not observed. La3+ crossed the interendothelial clefts of venules and capillaries at 30 and 60 min and was seen in both extracellular and intracellular brain compartments at 60 min. La3+ extravasation was seen in nonedematous areas bordering the regions of water accumulation. Our findings suggest that the early phase of incomplete continuous ischemia is accompanied by changes in BBB permeability and the interendothelial clefts of venules and capillaries seem to represent one of the early sites of ischemic damage.
Conformational preferences of [Leu5]enkephalin in biomimetic media. Investigation by 1H NMR.
Publication Date: 11/09/1990, on European journal of biochemistry
by Picone D, D'Ursi A, Motta A, Tancredi T, Temussi PA
The conformation of [Leu5]enkephalin has been studied by 1H-NMR spectroscopy in media more like the actual environment in which the agonist-receptor interaction takes place than water, i.e. in three cryoprotective mixtures (dimethylformamide/water, methanol/water and ethylene glycol/water), in aqueous SDS and in two neat solvents, dimethylformamide and acetonitrile, whose dielectric constants (36.7 and 37.5) are intermediate between that of water and that of the lipid phase. In all cases examined, contrary to the studies in water or dimethylsulfoxide, we were able to detect numerous nuclear Overhauser effects, indicating that the media employed favour well-defined structures and/or reduce the internal motions of the peptide. Data from both organic solvents and cryoprotective mixtures suggest a 4----1 beta turn as the most probable structure of [Leu5]enkephalin in solution, whereas in SDS/H2O micelles the structural picture appears completely different, suggesting the presence of a 5----2 beta turn. The existence of two different preferred conformations of enkephalins may possibly be related to their ability to be effective towards both mu and delta opioid receptors.
Regenerated EDL muscle of rats requires innervation to maintain AChE molecular forms.
Publication Date: 01/08/1990, on Muscle & nerve
by Melone MA, De Lucia D, Fratta M, Cotrufo R
Extensores digitorum longi of rats, infarcted and denervated by different surgical procedures, were used to analyze by biochemical and cytochemical methods the acetylcholinesterase (AChE) changes during muscle degeneration, regeneration, and early or delayed reinnervation. Biochemical tests showed that the regenerating muscle produces globular AChE forms (36% of controls) and small amounts of A12 (16S) asymmetric form (5% of controls); at the end of the regeneration, innervation and electromechanical function are required for the complete recovery of globular forms, and are absolutely critical to prevent A12 (16S) disappearance. Cytochemical observations showed that, unlike nicotinic receptor, AChE deposited at the neuromuscular junction before ischemic necrosis is protected from breakdown, as is the basal lamina of muscle fibers. Taken together, these observations contribute to the understanding of the factors that play a critical role in muscle repair and are, therefore, of clinical relevance.
Autoinduction of estrogen receptor is associated with FOSP-1 mRNA induction by estrogen in primary cultures of Xenopus oviduct cells.
Publication Date: 09/07/1990, on Molecular and cellular endocrinology
by Varriale B, Tata JR
The number of nuclear and cytosolic estrogen receptors (ER) per cell and the steady-state levels of the mRNA encoding a tissue-specific, estrogen-inducible protein (FOSP-1) were measured as a function of time following the addition of estradiol-17 beta (E2) to primary cultures of Xenopus oviduct cells. After a lag period of about 12 h, 10(-9) to 10(-7) M E2 caused a 10 to 15-fold increase in FOSP-1 mRNA by 60 h, whereas it was only 2-fold with 10(-7) M progesterone. Under the same conditions, E2 doubled its own total receptor content within the first 12 h, reaching a 4-fold increase in nuclear ER by 48 h. Cycloheximide treatment in the presence of 10(-7) M estradiol reduced the functional ER content by 75.90%. Treatment with the anti-estrogen ICI 164,384 of oviduct cells in which FOSP-1 mRNA was pre-induced to high levels with the hormones caused a drastic reduction in nuclear ER and a total loss of FOSP-1 mRNA in 72 h. The close correlation between the kinetics of autoinduction of ER and the induction of FOSP-1 mRNA, as was shown earlier for vitellogenin mRNA in hepatocytes (Perlman et al. (1984) Mol. Cell. Endocrinol. 38, 151-161), strongly suggests that Xenopus egg protein gene activation by estrogen requires the up-regulation of its own receptor by the hormone.
New features of the delta opioid receptor: conformational properties of deltorphin I analogues.
Publication Date: 15/06/1990, on Biochemical and biophysical research communications
by Balboni G, Marastoni M, Picone D, Salvadori S, Tancredi T, Temussi PA, Tomatis R
Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.
Dissociation and reconstitution of bovine seminal RNAase: construction of a hyperactive hybrid dimer.
Publication Date: 01/12/1989, on Journal of protein chemistry
by Tamburrini M, Piccoli R, Picone D, Di Donato A, D'Alessio G
The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we described the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.