Protease Activated Receptor-2 Contributes to Heart Failure

Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs), in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure.     

A severe autosomal recessive acromesomelic dysplasia,the Hunter-Thompson type,and comparison with the Grebe type

Summary Two siblings with a short-limb dwarfing condition which we call acromesomelic dysplasia, Hunter-Thompson type are reported. Abnormalities are limited to the limbs and limb joints in this severe form of dwarfism. The middle and distal segments of the limbs are most affected. The lower limbs are more affected than the upper. We are aware of one previously published case of this entity reported by A. G. W. Hunter and M. W. Thompson in 1976. Dislocations of the elbows and ankles were present in all three patients and dislocations of the hips and knees in two. One of the siblings who did not have hip and knee dislocations clinically resembled Grebe chondrodysplasia, another severe acromesomelic dwarfing condition. However, radiological analysis suggests that while acromesomelic dysplasia, Hunter-Thompson type and Grebe chondrodysplasia are related, they are not identical. Grebe chondrodysplasia has been established as an autosomal recessive trait. It appears probable that the entity we describe has the same mode of genetic transmission.     

In Silico Insights into Protein-Protein Interactions and Folding Dynamics of the Saposin-Like Domain of Solanum tuberosum Aspartic Protease

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.     

Arterial effects of aldosterone and antimineralocorticoid compounds mechanism of action

The aim of our work was to study the mechanism of action of aldosterone and antialdosterone compounds on Na+ and K+ fluxes in vascular smooth muscle. In the long term, regulation of salt metabolism depends on aldosterone effects on Na+, K+, H+ and H2O transport by the renal tubules. Furthermore, it has been shown that aldosterone modifies several epithelial transports, inducing a positive sodium balance. The chronic in vivo administration of aldosterone modifies transmembrane ionic fluxes in vascular smooth muscle. Garwitz and Jones suggested that aldosterone may enhance net Na+ transport through the stimulation of the sodium pump. The results obtained in our laboratory indicate that aldosterone has a direct stimulatory action on ouabain-dependent and on ouabain-independent Na efflux. Furthermore, the mineralocorticoid enhances passive K permeability, as well as the Na pump dependent K influx. Both effects are blocked by antimineralocorticoid compounds. Recent experiments have shown that vasopressin potentiates some of the in vivo effects of aldosterone.     

Regional brain GABA metabolism and release during hepatic coma produced in rats chronically treated with carbon tetrachloride

Hepatic coma was induced in rats chronically treated with CCl₄, by means of a single injection of ammonium acetate. The activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), as well as the synaptosomal uptake and release of [³H]GABA, were measured in the following brain areas of the comatose rats: cortex, striatum, hypothalamus, hippocampus, midbrain and cerebellum. Hepatic coma was associated with a general decrease of GAD activity, whereas GABA-T activity was diminished only in the hypothalamus, striatum and midbrain. During hepatic coma, the K⁺-stimulated [³H]GABA release was notably diminished in the striatum and cerebellum, whereas a significant increase was observed in the hippocampus. [³H]GABA uptake increased in most regions after CCl₄ treatment, independently of the presence of coma. The results indicate that GABAergic transmission seems to be decreased in most cerebral regions during hepatic coma.     

MHC nonrestricted cytotoxic T cell clones with selective specificity from patients with transitional cell carcinoma (TCC) of the urinary bladder

Summary Lymphocytes from patients with transitional cell carcinoma (TCC) of the urinary bladder are more cytotoxic to bladder tumor cells than to a variety of control cells. This disease-related cytotoxicity has previously been shown to involve several mechanisms and different types of effector cells. To analyze further the nature of the effector cells operative in this system, peripheral blood lymphocytes from eight TCC patients were stimulated in vitro with TCC extract and cultured in the presence of interleukin 2 and allogeneic feeder cells. When tested for cytotoxicity in vitro on a target cell panel including both adherent and nonadherent cell lines, the lymphocytes killed a broad spectrum of targets in a major histocompatibility complex (MHC)-unrestricted fashion. When cloned by limiting dilution, clones were obtained which displayed a more restricted pattern of target cell killing. Some of the clones were highly but not exclusively selective for TCC-derived target cells. Phenotypically, these cells resembled mature T cells of CTL-type (CD8⁺/CD4^–). They also expressed the CD3/5 T cell antigen receptor complex but target cell killing was not MHC-restricted. The results of various inhibition experiments suggested that the CD3/TCR complex was involved in the cytotoxicity exhibited by these effector cells. However, its precise role in target cell recognition and the identification of the tumor cell structures recognised by the effector cells require further studies.     

The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio

Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (wt^i–) and mutant phaseolin glycoforms (dgly ₁, dgly ₂ and dgly _1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly ₁ and dgly ₂ mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly _1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp base pair(s) - DAF days after flowering - GUS -glucuronidase - kb kilobase - kDa kilodalton     

EPR-detectable redox centers of the periplasmic hydrogenase from Desulfovibrio vulgaris

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) belongs to the category of [Fe] hydrogenase which contains only iron-sulfur clusters as its prosthetic groups. Amino acid analyses were performed on the purified D. vulgaris hydrogenase. The amino acid composition obtained compared very well with the result derived from the nucleotide sequence of the structural gene (Voordouw, G., Brenner, S. (1985) Eur. J. Biochem. 148, 515-520). Detailed EPR reductive titration studies on the D. vulgaris hydrogenase were performed to characterize the metal centers in this hydrogenase. In addition to the three previously observed EPR signals (namely, the "isotropic" 2.02 signal, the rhombic 2.10 signal, and the complex signal of the reduced enzyme), a rhombic signal with resonances at the g-values of 2.06, 1.96, and 1.89 (the rhombic 2.06 signal) was detected when the samples were poised at potentials between 0 and -250 mV (with respect to normal hydrogen electrode). The midpoint redox potentials for each of the four EPR-active species were determined, and the characteristics of each EPR signal are described. Both the rhombic 2.10 and 2.06 signals exhibit spectral properties that are distinct from a ferredoxin-type [4Fe-4S] cluster and are proposed to originate from the same H2-binding center but in two different conformations. The complex signal of the reduced hydrogenase has been shown to represent two spin-spin interacting ferredoxin-type [4Fe-4S]1+ clusters (Grande, H. J., Dunham, W. R., Averill, B., Van Dijk, C., and Sands, R. H. (1983) Eur. J. Biochem. 136, 201-207). The titration data indicated a strong cooperative effect between these two clusters during their reduction. In an effort to accurately estimate the number of iron atoms/molecule of hydrogenase, plasma emission and chemical methods were used to determine the iron contents in the samples; and four different methods, including amino acid analysis, were used for protein determination. The resulting iron stoichiometries were found to be method-dependent and vary over a wide range (+/- 20%). The uncertainties involved in the determination of iron stoichiometry are discussed.     

A new experimental model for myocutaneous flaps: latissimus dorsi of the rabbit--an anatomic study

A new experimental model, the latissimus dorsi flap of the rabbit, was studied. This was found to be a relatively inexpensive research model. Its use is advocated for composite tissue transfer as transposition, island, or free myocutaneous flaps.