[Recurrent acute myocardial infarction in a patient with nocturnal paroxysmal hemoglobinuria]. / Infarto miocardico acuto recidivato in un paziente con emoglobinuria parossistica notturna.

Paroxysmal nocturnal hemoglobinuria is a form of acquired hemolytic anemia with a high incidence of thrombotic complications, generally in the venous district; arterial thrombosis is rare, and exceptional in the coronary tree. We describe the case of a man who had two episodes of myocardial infarction, both during a hemoglobinuric crisis; this patient was free from cardiovascular risk factors and angiography revealed that there was no coronary stenosis. The clinical course was favorable and the patient's response to thrombolytic and dicumarol therapy was satisfactory. To our knowledge, this is the second case of coronary involvement in paroxysmal nocturnal hemoglobinuria described in the literature.

Sepsis is associated with increased ubiquitinconjugating enzyme E214k mRNA in skeletal muscle.

Previous studies provided evidence that sepsis is associated with increased ubiquitin-proteasome-dependent protein breakdown in skeletal muscle. The 14-kDa ubiquitin-conjugating enzyme (E214k) has been proposed to be a key regulator of the ubiquitin proteolytic pathway. We tested the hypothesis that E214k message and protein levels are increased in skeletal muscle during sepsis. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. E214k mRNA and protein levels were quantitated after Northern and Western blot analysis, respectively, 16 h after CLP or sham operation. Sepsis resulted in a 70% increase in the 1. 2-kb E214k transcript in the fast-twitch extensor digitorum longus muscle, whereas no changes were seen in the slow-twitch soleus muscle. E214k protein levels were not influenced by sepsis in any of the muscles studied. Although the changes in the expression of the E214k 1.2-kb transcript paralleled the differential effect of sepsis on protein breakdown in fast- and slow-twitch muscle, the potential role of E214k in the regulation of sepsis-induced muscle proteolysis needs to be interpreted with caution, because the results demonstrated that increased message levels were not associated with increased E214k protein levels.

Kinetic studies of the branched chain amino acid preferring peptidase activity of the 20S proteasome: development of a continuous assay and inhibition by tripeptide aldehydes and clasto-lactacystin beta-lactone.

We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This assay is based on the hydrolysis of the fluorogenic peptide, Abz-Gly-Pro-Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu-Ala bond by the 20S proteasome with a kc/Km value of 13 000 M-1 s-1. Hydrolysis of this peptide is accompanied by an increase in fluorescence intensity (lambda ex = 340 nm, lambda em = 415 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hydrolysis products, Abz-Gly-Pro-Ala-Leu and Ala-Nba. Using this assay, we examined inhibition of the BrAAP activity of the proteasome by a series of tripeptide aldehydes, Z-Leu-Leu-Xaa-H. When Xaa = Phe, (p-Cl)Phe, and Trp we observe biphasic or partial inhibition of the BrAAP activity. In contrast, when Xaa = Nva and Leu, simple inhibition kinetics are observed and allow us to calculate Ki values of 120 nM and 12 nM, respectively. The inhibitors that exhibit simple inhibition kinetics for BrAAP activity are also approximately equipotent for inhibition of the chymotrypsin-like (ChT-L) and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities, dissociation constants varying by less than 25-fold, whereas the inhibitors that exhibit biphasic inhibition kinetics for BrAAP activity are >300-fold more potent for inhibiting ChT-L activity than for PGPH activity. Inactivation of the BrAAP activity of the proteasome by clasto-lactacystin beta-lactone is also biphasic. beta-Lactone inactivates approximately 60% of the BrAAP activity rapidly, with kinetics indistinguishable from its inactivation of the chymotrypsin-like activity. The remaining 40% of the BrAAP activity is inactivated by beta-lactone at a 50-fold slower rate, with kinetics indistinguishable from its inactivation of the PGPH activity. These results suggest a mechanism in which hydrolysis of Abz-Gly-Pro-Ala-Leu-Ala-Nba (i.e., BrAAP activity) occurs at two different active sites in the 20S proteasome, and that these two active sites are the same ones that catalyze the previously described ChT-L and PGPH activities.

Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes.

Deubiquitinating enzymes constitute a family of cysteine hydrolases that specifically cleave ubiquitin-derived substrates of general structure Ub-X, where X can be any number of leaving groups ranging from small thiols and amines to Ub and other proteins (Ub, ubiquitin). We have developed a general assay for deubiquitinating enzymes based on the substrate ubiquitin C-terminal 7-amido-4-methylcoumarin (Ub-AMC). Ub-AMC is efficiently hydrolyzed with liberation of highly fluorescent AMC by two rabbit reticulocyte deubiquitinating enzymes: isopeptidase T (IPaseT), a member of the gene family of ubiquitin-specific processing enzymes, and UCH-L3, a member of the family of ubiquitin C-terminal hydrolases. We used this new assay to probe kinetic and mechanistic aspects of catalysis by IPaseT and UCH-L3. Results from four series of experiments are discussed: (1) For UCH-L3, we determined steady-state kinetic parameters that suggest a diffusion-limited reaction of UCH-L3 with Ub-AMC. To probe this, we determined the viscosity dependence of kc/Km, as well as kc. We found complex viscosity dependencies and interpreted these in the context of a model in which association and acylation are viscosity-dependent but deacylation is viscosity-independent. (2) The kinetics of inhibition of UCH-L3 by ubiquitin C-terminal aldehyde (Ub-H) were determined and reveal a Ki that is less than 10(-14) M. Several mechanisms are considered to account for the extreme inhibition. (3) The IPaseT-catalyzed hydrolysis of Ub-AMC is modulated by Ub with activation at low [Ub] and inhibition at high [Ub]. (4) Finally, we compare kc/Km values for deubiquitinating enzyme-catalyzed hydrolysis of Ub-AMC and Z-Leu-Arg-Gly-Gly-AMC. For IPaseT, the ratio of rate constants is 10(4), while for UCH-L3 this ratio is > 10(7). These results suggest the following: (i) Deubiquitinating enzymes are able to utilize the free energy that is released from remote interactions with Ub-containing substrates for stabilization of catalytic transition states, and (ii) UCHs are more efficient at utilizing the energy from these interactions, presumably because they do not possess a binding domain for a Ub "leaving group".

Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells.

The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.

Kinetic studies on the inhibition of isopeptidase T by ubiquitin aldehyde.

Isopeptidase T (IPaseT) can hydrolyze isopeptide bonds of polyubiquitin (polyUb) chains, simple C-terminal derivatives of Ub, and certain peptides. We recently reported that IPaseT is regulated by ubiquitin (Ub); while submicromolar Ub activates, higher concentrations inhibit this enzyme [Stein et al. (1995) Biochemistry 34, 12616]. To explain these observations, we proposed a model for IPaseT involving two binding sites for Ub. According to the model, the two sites are adjacent to one another and are the extended active site that binds two Ub moieties of a polyUb chain. The "activation site" binds the Ub that donates Lys to the isopeptide bond. The "inhibition site" is adjacent and binds the Ub that donates the C-terminal Gly to the isopeptide bond. We now report that the interaction of IPaseT with the C-terminal aldehyde of Ub (Ub-H) is also modulated by Ub. In the absence of Ub, Ub-H inhibits IPaseT with a Ki of 2.3 nM, while at 0.6 microM Ub, where the "activation site" is occupied, Ki is less than 0.1 nM. At high Ub concentrations, where both the "activation" and "inhibition" sites are occupied, IPaseT cannot bind Ub-H. We also determined the kinetics of inhibition of IPaseT by Ub-H. In the absence of Ub, a two-step mechanism is followed. In the first step, Ub-H slowly combines with IPaseT to form a relatively weak complex (K1 = 260 nM) that slowly isomerizes to the final, stable complex that accumulates in the steady-state (k2 = 2 x 10(-3) s-1; k-2 = 0.02 x 10(-3) s-1). In contrast, Ub-activated IPaseT is inhibited by Ub-H through a three-step process. In the first step, Ub-H rapidly combines with IPaseT to form a complex (K1 = 10 nM) that slowly isomerizes to a second, more stable complex (k2 = 18 x 10(-3) s-1; k-2 = 1.5 x 10(-3) s-1). In the third step, the second complex converts to the final complex (k3 = 1.5 x 10(-3) s-1; k-3 < 0.2 x 10(-3) s-1). To unify the results of this study with our previous results on catalysis, we propose that binding of Ub either to catalytic transition states or to tetrahedral inhibition intermediates liberates more free energy than binding of Ub to the reactant state of IPaseT and that IPaseT can utilize this binding energy to stabilize both of these tetrahedral species. The overall effect is a Ub-induced increase in catalytic efficiency or inhibitory potency.

Kinetic characterization of the chymotryptic activity of the 20S proteasome.

In this paper, we report kinetic studies for the chymotryptic activity of the 20S proteasome. Major observations include the following: (1) Reaction progress curves that are recorded at concentrations of Suc-Leu-Leu-Val-Tyr-AMC greater than about 40 microM are biphasic and characterized by initial velocities that decay by a first-order process to final, steady-state velocities. (2) Also at [Suc-Leu-Leu-Val-Tyr-AMC] > 40 microM, initial and steady-state velocities are smaller than predicted from simple, Michaelis-Menten kinetics. (3) The first-order rate constant for the approach to steady-state has a complex dependence on substrate concentration and decreases sigmoidally as substrate concentration increases. These results indicate that the 20S proteasome is a hysteretic enzyme and is subject to substrate inhibition. To explain these observations we propose a minimal kinetic model with two critical mechanistic features: (1) the 20S proteasome has two cooperative active sites for Suc-Leu-Leu-Val-Tyr-AMC and (2) there are two interconvertible conformers of active 20S proteasome. To probe this mechanism in greater detail, we explored the kinetic mechanism of inhibition of the 20S proteasome-catalyzed hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC by the peptide aldehyde, Ac-Leu-Leu-Nle-H. Our studies reveal a nonlinear dependence of reciprocal steady-state velocity on inhibitor concentration (i.e., parabolic inhibition) as well as a nonlinear dependence of the apparent inhibitor dissociation constant on substrate concentration. Both of these observations are explained by binding of inhibitor at multiple sites on the enzyme. Taken together, the results of this study indicate that the 20S proteasome is a conformationally flexible protein that can adjust to the binding of ligands and that has multiple and cooperative active sites. These results support a view of the proteasome's substrate specificity in which (1) substrates are recognized and hydrolyzed by more than one active site; (2) each active site can bind substrates that possess a variety of P1 residues; and (3) the P1 residue plays a relatively minor role as a specificity determinant. Finally, we interpret the results of this study to suggest that, in vivo, the 20S proteasome requires conformational plasticity for its interactions with regulatory complexes and, after it has combined with appropriate regulatory complexes, to catalyze hydrolysis of proteins.

Mechanistic studies on the inactivation of the proteasome by lactacystin: a central role for clasto-lactacystin beta-lactone.

Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces differentiation in a murine neuroblastoma cell line. The cellular target of lactacystin is the 20 S proteasome, also known as the multicatalytic proteinase complex, an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In aqueous solution at pH 8, lactacystin undergoes spontaneous hydrolysis to yield N-acetyl-L-cysteine and the inactive lactacystin analog, clasto-lactacystin dihydroxy acid. We have studied the mechanism of lactacystin hydrolysis under these conditions and found that it proceeds exclusively through the intermediacy of the active lactacystin analog, clasto-lactacystin beta-lactone. Conditions that stabilize lactacystin (and thus prevent the transient accumulation of the intermediate beta-lactone) negate the ability of lactacystin to inactivate the proteasome. Together these findings suggest that lactacystin acts as a precursor for clasto-lactacystin beta-lactone and that the latter is the sole species that interacts with the proteasome.

Kinetic studies of isopeptidase T: modulation of peptidase activity by ubiquitin.

We have investigated the specificity of isopeptidase T toward peptide-AMC substrates based on the C-termini of ubiquitin. The substrates investigated were Z-Gly-Gly-AMC, Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC and were hydrolyzed by isopeptidase T with kc/Km values of < 0.1, 1, 18, and 95 M-1 s-1, respectively. In the course of these experiments, we observed that the hydrolytic activity of isopeptidase T toward these substrates is modulated by ubiquitin in a biphasic fashion. While submicromolar concentrations of ubiquitin activate isopeptidase T, higher concentrations are inhibitory. In the activation phase, the extent of stimulation of kc/Km varies with substrate and is 8-, 50-, and 70-fold for Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC, respectively. Kd for ubiquitin in this phase is, of course, independent of substrate and equals 0.10 +/- 0.03 microM. At higher concentrations, ubiquitin is inhibitory and titrates kc/Km with an average Ki value of 3.0 +/- 1.3 microM for all three substrates. To explain these observations, we propose a structural model for isopeptidase T that involves two binding sites for ubiquitin. We propose that the two sites are adjacent to one another and are the extended active site that binds two ubiquitin moieties of a polyubiquitin chain for isopeptide bond hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway.

The transcription factor NF-kappa B is sequestered in the cytoplasm by the inhibitor protein I kappa B alpha. Extracellular inducers of NF-kappa B activate signal transduction pathways that result in the phosphorylation and subsequent degradation of I kappa B alpha. At present, the link between phosphorylation of I kappa B alpha and its degradation is not understood. In this report we provide evidence that phosphorylation of serine residues 32 and 36 of I kappa B alpha targets the protein to the ubiquitin-proteasome pathway. I kappa B alpha is ubiquitinated in vivo and in vitro following phosphorylation, and mutations that abolish phosphorylation and degradation of I kappa B alpha in vivo prevent ubiquitination in vitro. Ubiquitinated I kappa B alpha remains associated with NF-kappa B, and the bound I kappa B alpha is degraded by the 26S proteasome. Thus, ubiquitination provides a mechanistic link between phosphorylation and degradation of I kappa B alpha.

[Physiopathology of vasovagal syncope: review of the most endorsed theories and recent findings]. / Fisiopatologia della sincope vasovagale: revisione delle teorie più accreditate e recenti acquisizioni.

Vasovagal syncope is associated with an abnormal reflex and the physiopathological mechanisms of the phenomenon overall are only partially known. Experimental and clinical studies suggest that the main factor which triggers the syncope is the brusque interruption of the alpha-adrenergic tone with marked, sudden peripheral vasodilation. Although documented, vagal hypertony, with consequent bradycardia and asystolia, is only occasional and is almost always a secondary phenomenon. The most commonly suggested cause of vasovagal syncope is a Bezold-Jarish reflex starting from the cardiac receptors in the walls of the ventricle, mediated by the paradoxical activation of afferent vagal fibres. However, recent studies are suggesting that there may be other pathogenetic mechanisms such as the paradoxical activation of the venous-atrial baroceptors and other "extracardiac" vascular receptors. The neuro-endocrine aspect of the vasovagal reaction is very complex and in spite of the many studies carried out on the catecholamine, renal-angiotensive system, arginine-vasopressin, and b-endorphine trends, there are still many points awaiting clarification. The response of the autonomous nervous system linked to age also require further research.

Role of gizzard myosin light chains in calcium binding.

The contraction of molluscan and vertebrate smooth muscles is regulated by myosin. Although the myosin and its associated two subunits, the regulatory light chain and the essential light chain, constitute the Ca2+ regulatory system in both types of muscles, the mechanisms by which Ca2+ signal is transduced are quite different. In molluscan muscles, the direct binding of Ca2+ to the regulatory system triggers muscle contraction. In vertebrate smooth muscles, however, phosphorylation of the regulatory light chain is the major triggering mechanism. We measured Ca2+ binding in gizzard myosin and in hybrids of scallop myosin containing gizzard regulatory light chain or in hybrids of scallop regulatory domain containing gizzard essential light chain. Isolated chicken gizzard myosin did not bind Ca2+ in the range of pCa 8.0 to 5.0 in the presence of 2 mM MgCl2, supporting the lack of the specific Ca(2+)-binding site in gizzard myosin. Phosphorylation of the regulatory light chain did not generate a specific (Ca2+)-binding site. The hybrid scallop myosin containing gizzard regulatory light chain showed a similar Ca2+ binding as native scallop myosin with a one to one stoichiometry of Ca2+ to myosin head saturating at about pCa 6.0 at pH 7.6. In contrast, the hybrid scallop regulatory domain containing gizzard essential light chain did not bind Ca2+ either at pCa 6.0 or at pCa 8.0. Control preparations reconstituted with scallop essential light chains bound 0.69 mol per mol Ca2+ at pCa 6.0 with no binding at pCa 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of the regulatory domain of scallop myosin: role of the essential light chain in calcium binding.

The regulatory domain of scallop myosin, consisting of a regulatory light chain (R-LC), an essential light chain (E-LC), and a portion of heavy chain, occupies the neck region of myosin. This domain is directly involved in the regulation of molluscan muscle contraction, which is triggered by direct Ca2+ binding to myosin. We have isolated a soluble functional complex (regulatory complex) comprised of R-LC, E-LC, and a 10-kDa heavy chain fragment in a 1:1:1 stoichiometry by clostripain digestion of the myosin head (papain subfragment 1). N termini of the heavy chain fragments were either leucine-812 or valine-817. The isolated complex retained the specific Ca2(+)-binding site and bound Ca2+ with a similar affinity and selectivity as myosin. The individual components of the regulatory complex were isolated after complete denaturation with guanidine hydrochloride. The regulatory complex was reconstituted from isolated light chains and the heavy chain fragment. The renatured complex regained Ca2+ binding quantitatively. To elucidate the function of the E-LC in Ca2+ binding, we constructed hybrid regulatory complexes. The hybrid complexes reconstituted with molluscan E-LC and R-LC regained the specific Ca2(+)-binding site, whereas the hybrid complex formed with rabbit skeletal E-LC [alkali LC 2 (A2-LC)] and scallop R-LC did not. The results demonstrate that E-LCs from myosins regulated by direct Ca2+ binding are required for the specific Ca2+ binding in the molluscan muscle.

[Primary angiosarcoma of the right atrium invading the pericardial cavity. Description of a case diagnosed by exploratory thoracotomy and surgically treated with success]. / Angiosarcoma primitivo dell'atrio destro ad espansione in cavità pericardica. Descrizione di un caso diagnosticato con toracotomia esplorativa e trattato chirurgicamente con successo.

The AA. report the case of a 43-year-old woman with an angiosarcoma arising from the right atrial wall and growing into the pericardial cavity. The patient presented with recurrent pericardial effusion initially responsive to medical therapy. The diagnosis was made at the exploratory thoracotomy. Repeated 2D-Echocardiograms did not help for the diagnosis in this particular case. The patient underwent surgical resection of the tumor, chemo- and radiotherapy. After 30 months there are no signs of recurrence or metastasis. In our opinion the frequent recurrence of pericardial effusion in the same patient should be regarded with special suspicion.