Expression in insect cells of active mature cruzipain from Trypanosoma cruzi,containing its C-terminal domain

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzaindeltac) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.     

Measuring [Ca2+] in the endoplasmic reticulum with aequorin

The photoprotein aequorin was the first probe used to measure specifically the [Ca(2+)] inside the lumen of the endoplasmic reticulum ([Ca(2+)](ER)) of intact cells and it provides values for the steady-state [Ca(2+)](ER), around 500 microM, that closely match those obtained now by other procedures. Aequorin-based methods to measure [Ca(2+)](ER) offer several advantages: (i) targeting of the probe is extremely precise; (ii) the use of low Ca(2+)-affinity aequorin allows covering a large dynamic range of [Ca(2+)], from 10(-5) to 10(-3)M; (iii) aequorin is nearly insensitive to changes in Mg(2+) or pH, has a high signal-to-noise ratio and calibration of the results in [Ca(2+)] is made straightforward using a simple algorithm; and (iv) the equipment required for luminescence measurements in cell populations is simple and low-cost. On the negative side, this technique has also some disadvantages: (i) the relatively low amount of emitted light makes difficult performing single-cell imaging studies; (ii) reconstitution of aequorin with coelenterazine requires previous complete depletion of Ca(2+) of the ER for 1-2h, a maneuver that may result in deleterious effects in some cells; (iii) because of the high rate of aequorin consumption at steady-state [Ca(2+)](ER), only relatively brief experiments can be performed; and (iv) expression of ER-targeted aequorin requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones. Choosing aequorin or other techniques to measure [Ca(2+)](ER) will depend of the correct balance between these properties in a particular problem.     

Comparison of different ligand densities for the manufacture of CB Hep-1 immunosorbents

Different ligand densities of monoclonal antibody (Mab) CB.Hep-1 were studied during covalent coupling on Sepharose CL-4B for recombinant hepatitis B surface antigen (rHBsAg) immunoaffinity purification. Ligand densities of 2.2, 3.2, 4.2 and 5.2 mg Mab/ml immunosorbents, respectively, were assayed during five cycles of immunoaffinity chromatography (IAC). Adsorption capacities averaged either 3.2 mg/ml (0.57 mg rHBsAg/ml immunosorbent/5.42 mg of total purified protein) or 5.2 mg/ml (0.56 mg rHBsAg/ml immunosorbent/5.05 mg total purified protein). Immunosorbents showed ligand leakage levels below 3 ng Mab/microg rHBsAg. Antigen purity was higher than 95% in all cases. The results suggest that a ligand density (LD) of 3.2 mg Mab/ml immunosorbent should be used for immunoaffinity chromatography because no significant differences were found in the ligand densities studied (P-value=0.012), which saves 40% of CB.Hep-1 immunosorbent manufacturing cost in comparison with 5 mg Mab/ml immunosorbent, which is currently used in large-scale production.     

Wnt signaling involvement in beta-amyloid-dependent neurodegeneration

Alzheimer's disease (AD) is a progressive dementia paralleled by selective neuronal death, which is probably caused by the cytotoxic effects of the amyloid-beta peptide (Abeta). We have observed that Abeta-dependent neurotoxicity induces a loss of function of Wnt signaling components and that activation of this signaling cascade prevent such cytotoxic effects. Therefore we propose that compounds which mimic this signaling cascade may be candidates for therapeutic intervention in Alzheimer's patients.     

The role of mitochondrial nitric oxide synthase in inflammation and septic shock

Nitric oxide and cytokines constitute the molecular markers and the intercellular messengers of inflammation and septic shock. Septic shock occurs with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle appears as one of the main target organs in septic shock, showing an increased nitric oxide (NO) production, an early oxidative stress, and contractile failure. Mitochondria isolated from rat and human skeletal muscle in septic shock show a markedly increased NO generation and a decreased state 3 respiration, more marked with nicotinamide adenine dinucleotide (NAD)-linked substrates than with succinate, without uncoupling or impairment of phosphorylation. One of the current hypothesis for the molecular mechanisms of septic shock is that the enhanced NO production by mitochondrial nitric oxide synthase (mtNOS) leads to excessive peroxynitrite (ONOO(-)) production and protein nitration in the mitochondrial matrix, to mitochondrial dysfunction and to contractile failure. Surface chemiluminescence is a useful assay to assess inflammation and oxidative stress in in situ liver and skeletal muscle. Liver chemiluminescence in inflammatory processes and phagocyte chemiluminescence have been found spectrally different from spontaneous liver chemiluminescence with increased 440-600 nm emission, likely due to NO and ONOO(-) participation in the reactions leading to the formation of excited species.     

A chemical,genetic, and structural analysis of the nuclear bile acid receptor FXR

The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.     

Progress of National Multi-tissue Bank in Uruguay in the International Atomic Energy Agency (IAEA) Tissue Banking Programme

The transplant law of 1971 based on informed consent, allows people to register their willingness to be a donor upon death. Since 1978 the governmental Institution, the National Bank of Organs and Tissues (BNOT), have been regulated the organ and tissue donation. Important progress was implemented in the BNOT and specially in the National Multi-tissue Bank (NMTB). Since 2001 with the participation in the IAEA Tissue Banking Programme, Quality System Management has been implemented in the NMTB. New bio-production for radiosterilized tissues for the first time and improved procedures were carried out. As a result an increased production of high-quality tissues was obtained and distributed for clinical use.     

Purification and properties of a new psychrophilic metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum

To go further into the characterization of the proteolysis exocellular system of the salmonid pathogen Flavobacterium psychrophilum, the purification and characterization of a novel protease designated Fpp2 (F. psychrophilum protease 2) was undertaken. A protease (Fpp2) hydrolyzing azocasein was purified. The Fpp2 can be defined as a metalloprotease, it had an estimated molecular mass of 62 kDa with calcium playing an important role in the thermostability of the enzyme. Proteolytic activity was optimal at pH 6.0-7.0 and 24 degrees C and activation energy for the hydrolysis of azocasein was determined to be 5.4 kcal mol(-1), being inactive at temperatures above 42 degrees C. All these results are characteristic of 'cold adapted enzymes'. Fpp2 proved to be a broad range hydrolytic enzyme because in optimal conditions it was able to hydrolyze matrix and muscular proteins. It can be concluded that the Fpp1, a previously characterized 55 kDa metalloprotease, and the Fpp2 protease were produced under different physiological conditions and were immunologically as well as biochemically different.     

Daily changes of GABA and taurine concentrations in various hypothalamic areas are affected by chronic hyperprolactinemia

This study was designed to characterize, in anterior, mediobasal, and posterior hypothalamic and median eminence, the 24h changes of gamma aminobutyric acid (GABA) and taurine (TAU) contents in adult male rats and to analyze whether chronic hyperprolactinemia may affect these patterns. Rats were turned hyperprolactinemic by a pituitary graft. Plasma prolactin (PRL) levels increased after pituitary grafting at all time points examined. A disruption of the circadian rhythm was observed in pituitary-grafted rats, whereas GABA and TAU content followed daily rhythms in all areas studied in controls. In the mediobasal hypothalamus, two peaks for each amino acid were found at midnight and midday. In the anterior hypothalamus, GABA and TAU showed only one peak of concentration at midnight. In the posterior hypothalamus, the values of both GABA and TAU were higher during the light as compared to the dark phase of the photoperiod. In the median eminence GABA content peaked at 20:00h, the time when TAU exhibited the lowest values. Hyperprolactinemia abolished the 24h changes of GABA in the mediobasal hypothalamus and reduced its content as compared to controls. Hyperprolactinemia advanced the diurnal peak of TAU to 12:00h in the mediobasal hypothalamus and did not modify the 24:00h peak. In the anterior hypothalamus, hyperprolactinemia increased GABA and TAU contents during the light phase while it decreased them during the dark phase of the photoperiod. In the posterior hypothalamus hyperprolactinemia did not modify GABA or TAU patterns as compared to controls. In the median eminence hyperprolactinemia increased the 20:00h peak of GABA and shift advanced the decrease in TAU content at 20:00h and its maximum at 24:00h as compared to controls. These data show that GABA and TAU content exhibit specific daily patterns in each hypothalamic region studied. PRL differentially affects the daily pattern of these amino acids in each hypothalamic region analyzed.