Thermodynamic Modeling of Internal Equilibria Involved in the Activation of Trypsinogen

Abstract

The effect of activating dipeptides, sequentially homologous to the Ile 16-Val 17 N-terminus of bovine β-trypsin (β-trypsin), on equilibria involved in the binding of strong ligands (i.e., n-butylamine, the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI) and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen (trypsinogen) was investigated at pH 5.5 (I = 0.1 M) and T = 21.0 ± 0.5°C; under the same experimental conditions, thermodynamics for the binding of strong ligands to β-trypsin was also obtained. The equilibria involved in the binding of activating dipeptides and/or inhibitors to β-trypsin and to its zymogen are described according to an induced-fit formalism, taking into account ligand-linked interaction(s) between different functional and structural domains of the (pro)enzyme possibly involved in the trypsinogen-to-β-trypsin activation pathway. The analysis of data is focussed on parameters describing interactions between the so-called Ile-Val pocket (where the Ile16-Val17/V-terminus of β-trypsin or activating dipeptides bind) and the primary and/or secondary recognition subsite(s) (where strong ligands associate) present in the (pro)enzyme. Such an analysis allows to dissect the contributions due to the primary recognition subsite, where small mono-functional ligands (e.g., n-butylamine) bind, from those of the secondary subsite(s), which are additional recognition clefts for macromolecular inhibitors (e.g., BPTI and PSTI).     

Do changes in the mechanical properties of articular cartilage promote catabolic destruction of cartilage and osteoarthritis?

Osteoarthritis (OA) is a joint disease characterized by cartilage degeneration, a thickening of subchondral bone, and formation of marginal osteophytes. Previous mechanical characterization of cartilage in our laboratory suggests that energy storage and dissipation is reduced in osteoarthritis as the extent of fibrillation and fissure formation increases. It is not clear whether the loss of energy storage and dissipation characteristics is a result of biochemical and/or biophysical changes that occur to hyaline cartilage in joints. The purpose of this study is to present data, on the strain rate dependence of the elastic and viscous behaviors of cartilage, in order to further characterize changes that occur in the mechanical properties that are associated with OA. We have previously hypothesized that the changes seen in the mechanical properties of cartilage may be due to altered mechanochemical transduction by chondrocytes. Results of incremental tensile stress-strain tests at strain rates between 100%/min and 10,000%/min conducted on OA cartilage indicate that the slope of the elastic stress-strain curve increases with increasing strain rate, unlike the reported behavior of skin and self-assembled collagen fibers. It is suggested that the strain-rate dependence of the elastic stress-strain curve is due to the presence of large quantities of proteoglycans (PGs), which protect articular cartilage by increasing the apparent stiffness. The increased apparent stiffness of articular cartilage at high strain rates may limit the stresses borne and prolong the onset of OA. It is further hypothesized that increased compressive loading of chondrocytes in the intermediate zone of articular cartilage occurs as a result of normal wear to the superficial zone or from excessive impact loading. Once the superficial zone of articular cartilage is worn away, the tension is decreased throughout all cartilage zones leading to increased chondrocyte compressive loading and up-regulation of mechanochemical transduction processes that elaborate catabolic enzymes.     

Functional properties of hemoglobin leiden (α2Aβ26 or7 Glu deleted)

Hemoglobin Leiden is an abnormal human hemoglobin in which a glutamic acid residue has been deleted from the β-chain at position 6 or 7. The α-amino groups of the β-chain N-termini in tetrameric hemoglobin A are thought to be directly involved in the binding of simple anions and organic phosphates (1). The deletion of the 4th or 5th residue of the A helix in hemoglobin Leiden shortens the N-terminus of the β-chain, and the results reported here show that the anion binding site has been affected. Hemoglobin Leiden shows a decreased response to inorganic phosphate, chloride, 2,3-diphosphoglycerate, and inositol hexaphosphate, both in equilibria and kinetics of ligand binding. Although hemoglobin Leiden shows an altered response to anions, neither the cooperativity of ligand binding nor the Bohr effect are significantly altered by the deletion. The decreased effect of cofactors seems to be due to a decrease in the strength of anion binding which may be attributed to the altered geometry of the anion binding site.     

Role of the phosphorolysis of deoxyadenosine in the cytotoxic effect of the combination of deoxyadenosine and deoxycoformycin on a human colon carcinoma cell line (LoVo)

In LoVo cells, phosphorolytic activity acting on deoxyadenosine plays a major role in the resistance to the cytotoxic effect of the combination of deoxynucleoside with deoxycoformycin. In fact, the observed dependence of toxicity on cell density appears to be related to the metabolic conversion of deoxyadenosine into adenine. The phosphorylation of the deoxynucleoside, which represents the first step towards the formation of the cytotoxic agent dATP, proceeds at a significantly lower rate as compared to the phosphorolysis of deoxyadenosine. The analysis of the levels of deoxyadenosine and its derivatives in the incubation media reveals that the rates of disappearance of deoxyadenosine and of formation of adenine increase in concert with the reduction of the effect on cell survival. J. Cell. Biochem. 80:241–247, 2000. © 2000 Wiley‐Liss, Inc.     

Characterization of sulfonylurea receptors in isolated human pancreatic islets

Current information on pancreatic islet sulfonylurea receptors has been obtained with laboratory animal pancreatic β cells or stable β-cell lines. In the present study, we evaluated the properties of sulfonylurea receptors of human islets of Langherans, prepared by collagenase digestion and density-gradient purification. The binding characterisitics of labeled glibenclamide to pancreatic islet membrane preparations were analyzed, displacement studies with several oral hypoglycemic agents were performed, and these latter compounds were tested as for their insulinotropic action on intact human islets. [³H]glibenclamide saturable binding was shown to be linear at ≤0.25 mg/ml protein; it was both temperature and time dependent. Scatchard analysis of the equilibrium binding data at 25°C indicated the presence of a single class of saturable, high-affinity binding sites with a K_d value of 1.0 ± 0.07 nM and a Bmax value of 657 ± 48 fmol/mg of proteins. The displacement experiments showed the following rank order of potency of the oral hypoglycemic agents we tested: glibenclamide = glimepiride > tolbutamide > chlorpropamide ≫ metformin. This binding potency order was parallel with the insulinotropic potency of the evaluated compounds. J. Cell. Biochem. 71:182–188, 1998. © 1998 Wiley-Liss, Inc.     

Molecular basis of α-thalassemia in Sicily

To evaluate the allelic frequency and genetic diversity of α-thalassemia defects in Sicily, both epidemiological and patient-oriented studies were carried out. For the epidemiological study, phenotypic data were collected on more than 1000 Sicilian individuals. Among them, 427 were explored at the molecular level for nine α-thalassemic variants known to be common in the Mediterranean region. Our data reveal an allele frequency of 4.1% for α⁺-thalassemia matching that of β-thalassemia in this region. The presence of α°-thalassemia (––^MEDI and ––^CAL) was observed only in the group of referred patients. Newly acquired nucleotide sequence data on the deletional breakpoint of ––^CAL allowed us to design a simple PCR-based procedure for exploring this allele. The data also provide additional information concerning the genetic mechanisms involved in such large deletions. Received: 8 August 1996 / Revised: 16 October 1996     

The Peripheral-Type Benzodiazepine Receptor Ligands [3H]Ro 5-4864 and [3H]PK 11195 Bind to the Retina of Rabbit, but Not of Turtle

Abstract: The binding of [³H]flunitrazepam, [³H]RO 5-4864, and [³H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [³H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [³H]RO 5-4864 and [³H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (K_D values of 24 ± 2.3 and 2.2 ± 0.8 nM, and B_max values of 440 ± 35 and 1,482 ± 110 fmol/mg of protein, respectively). The binding was temperature - and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [³H]RO 5-4864 and [³H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.     

Macroalgae exhibit diverse responses to human disturbances on coral reefs

Scientists and managers rely on indicator taxa such as coral and macroalgal cover to evaluate the effects of human disturbance on coral reefs, often assuming a universally positive relationship between local human disturbance and macroalgae. Despite evidence that macroalgae respond to local stressors in diverse ways, there have been few efforts to evaluate relationships between specific macroalgae taxa and local human-driven disturbance. Using genus-level monitoring data from 1205 sites in the Indian and Pacific Oceans, we assess whether macroalgae percent cover correlates with local human disturbance while accounting for factors that could obscure or confound relationships. Assessing macroalgae at genus level revealed that no genera were positively correlated with all human disturbance metrics. Instead, we found relationships between the division or genera of algae and specific human disturbances that were not detectable when pooling taxa into a single functional category, which is common to many analyses. The convention to use percent cover of macroalgae as an indication of local human disturbance therefore likely obscures signatures of local anthropogenic threats to reefs. Our limited understanding of relationships between human disturbance, macroalgae taxa, and their responses to human disturbances impedes the ability to diagnose and respond appropriately to these threats.     

Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection

Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.Ebola virus (EBOV) is one of the most deadly human pathogens, causing a severe hemorrhagic fever syndrome in both humans and non-human primates with fatality rates ranging from 50 to 70%.¹ The recent outbreak of Ebola Virus Diseases (EVD) in West Africa highlights the pathogenic nature of this virus, the high mortality rates and pandemic potential. To date, there have been over 27 700 cases and >11 280 deaths.^{1, 2} Although EVD is usually an acute illness, increasing evidences exist of persistent infections and post infection syndromes,^{3, 4, 5, 6} highlighting the need to identify immune correlates of a protective immune response.Defining human immune responses to EBOV infection, pathogenesis and correlates of protection are important for designing effective therapeutic and vaccination interventions. A decrease in lymphocytes has been observed in studies in mice,⁷ non-human primates⁸ and humans,⁹ and is attributed to apoptotic mechanisms.^{7, 10} Persistent B and T-cell activation has been described in four survivors as long as one month after discharge from the hospital, suggesting recurrent antigenic stimulation.¹¹ While aberrant immune responses have been described after EBOV infection (reviewed in^{12, 13}), and different patterns of inflammatory mediators have been associated with different clinical outcomes,^{9, 10, 11, 14, 15, 16, 17} data on human immune responses to Ebola virus remain scanty, due to difficulties in obtaining sequential samples through the course of illness and to limitations imposed by biosafety requirements for laboratory analyses.We conducted a longitudinal study aimed to characterize the kinetics of T-cell phenotypes, activation/differentiation profile, autophagic/apoptotic markers and functionality in two EVD patients from soon after symptom onset through their hospitalization until recovery.