Isothermal titration calorimetry determination of individual rate constants of trypsin catalytic activity

Determination of individual rate constants for enzyme-catalyzed reactions is central to the understanding of their mechanism of action and is commonly obtained by stopped-flow kinetic experiments. However, most natural substrates either do not fluoresce/absorb or lack a significant change in their spectra while reacting and, therefore, are frequently chemically modified to render adequate molecules for their spectroscopic detection. Here, isothermal titration calorimetry (ITC) was used to obtain Michaelis–Menten plots for the trypsin-catalyzed hydrolysis of several substrates at different temperatures (278–318 K): four spectrophotometrically blind lysine and arginine N-free esters, one N-substituted arginine ester, and one amide. A global fitting of these data provided the individual rate constants and activation energies for the acylation and deacylation reactions, and the ratio of the formation and dissociation rates of the enzyme–substrate complex, leading also to the corresponding free energies of activation. The results indicate that for lysine and arginine N-free esters deacylation is the rate-limiting step, but for the N-substituted ester and the amide acylation is the slowest step. It is shown that ITC is able to produce quality kinetic data and is particularly well suited for those enzymatic reactions that cannot be measured by absorption or fluorescence spectroscopy.     

Biochemical changes in the transition from vitellogenesis to follicular atresia in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae)

In this work, we have explored the biochemical changes characterizing the transition from vitellogenesis to follicular atresia, employing the hematophagous insect vector Dipetalogaster maxima as a model. Standardized insect rearing conditions were established to induce a gradual follicular degeneration stage by depriving females of blood meal during post-vitellogenesis. For the studies, hemolymph and ovaries were sampled at representative days of pre-vitellogenesis, vitellogenesis and early and late follicular atresia. When examined by scanning electron microscopy, ovarioles at the initial stage of atresia were small but still showed some degree of asynchronism, a feature that was lost in an advanced degeneration state. At late follicular atresia, in vivo uptake assays of fluorescently labeled vitellogenin (Vg-FITC) showed loss of competitiveness of oocytes to uptake vitellogenin. Circulating vitellogenin levels in atresia were significantly higher than those registered at pre-vitellogenesis, most likely to maintain appropriate conditions for another gonotrophic cycle if a second blood meal is available. Follicular atresia was also characterized by partial proteolysis of vitellin, which was evidenced in ovarian homogenates by western blot. When the activity of ovarian peptidases upon hemoglobin (a non-specific substrate) was tested, higher activities were detected at early and late atresia whereas the lowest activity was found at vitellogenesis. The activity upon hemoglobin was significantly inhibited by pepstatin A (an aspartic peptidase inhibitor), and was not affected by E64 (a cysteine peptidase inhibitor) at any tested conditions. The use of specific fluorogenic substrates demonstrated that ovarian homogenates at early follicular atresia displayed high cathepsin D-like activity, whereas no activity of either, cathepsin B or L was detected. Mass spectrometry analysis of the digestion products of the substrate Abz-AIAFFSRQ-EDDnp further confirmed the presence of a cathepsin D-like peptidase in ovarian tissue. In the context of our findings, the early activation of cathepsin D-like peptidase could be relevant in promoting yolk protein recycling and/or enhancing follicle removal.     

The CB1 Receptor Antagonist SR141716A Reverses Adult Male Mice Overweight and Metabolic Alterations Induced by Early Stress

Perinatal stress may cause metabolic and hormonal disruptions during adulthood. The aim of this study was to evaluate the effects of early postnatal nociceptive stimulation (NS) on body weight and other metabolic parameters during adulthood and to determine whether CB₁ endocannabinoid receptors (CB₁Rs) may be involved in these effects. Male mice were subjected to NS during lactation with a daily subcutaneous injection of saline solution. Subsequently, both control and NS‐mice were treated from day 40 to 130, with an oral dose (1 µg/g body weight) of SR141716A, a specific CB₁R antagonist/inverse agonist. Mice body weight and food intake was periodically evaluated. Adult animals were then killed to evaluate epididymal fat pads and metabolic parameters. NS did not influence food intake in adult animals, but caused significant increases in body weight, epididymal fat pads, and circulating levels of leptin, corticosterone, and triglycerides (TGs). Chronic treatment with SR141716A normalized these parameters, with the exception of corticosterone levels. This treatment also reduced plasma levels of glucose, insulin, and total cholesterol in both adult control and NS‐mice. In addition, fatty acid (FA) amide hydrolase (FAAH) activity (the enzyme able to hydrolyze endocannabinoids) from liver and epididymal fat of adult NS‐mice was decreased by 40–50% in comparison to activities found in same tissues of control mice. Results suggest that overactive liver and epididymal fat CB₁R due to early NS may be involved in late metabolic alterations, which are sensitive to chronic treatment with SR141716A.     

Presence of Echinorhinus cookei off central Pacific Mexico

On 19 May 1999, the capture of a neonate male of the prickly shark Echinorhinus cookei (377 mm L _T) off Michoacán Coast (18°40'N: 103°47'W) was recorded. The prickly shark was found at a depth of 132 m; it was collected over muddy bottom using a trawl net. In Mexico, representatives of this species are scarce o. the Pacific Coast; this new record extends the known southern range 261 km from 21°01'N to 18°40'N for the continental coast. The organism was deposited in the ichthyological reference collection of the Centro de Investigación en Alimentación y Desarrollo, A.C, Unidad Mazatlán (catalogue number, CIAD 01–03 Mazatlán). Morphometric and meristic characteristics of the specimen are given.     

First report of an intraerythrocytic small piroplasm in wild Iberian lynx (Lynx pardinus)

A wild injured Iberian lynx (Lynx pardinus) was taken from the Sierra Morena population. During the health check small intraerythrocytic piroplasms, morphologically indistinguishable from other feline piroplasms, were observed in Wright-Giemsa-stained blood films. Amplification by polymerase chain reaction of a portion of the 18S nuclear small subunit (NSS) rRNA gene and sequencing revealed similarity of the unknown organism with sequences obtained from Pallas's cat from Mongolia and from a domestic cat in Spain. In a retrospective (1993-2003) study of 50 Iberian lynx tissue samples, no amplifications of the 18S NSS rRNA gene of the organism were obtained. This is the first report of a naturally occurring erythroparasitemia in the Iberian lynx and the first documented case of naturally occurring piroplasm infection in a free-ranging felid from Europe.     

Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.     

Reactive oxygen species and development in microbial eukaryotes

Reactive oxygen species (ROS) have been regarded as inevitable harmful by-products of aerobic metabolism. Growing evidence, however, suggests that ROS play important physiological roles. This raises questions about the pathways that different groups of organisms use to produce and sense ROS. In microbial eukaryotes, recent data show (i) increased ROS levels during cell differentiation, (ii) the existence of ROS-producing enzymes, such as NADPH oxidases (NOX), (iii) the involvement of NOX in developmental processes, and (iv) a conservation in the signal-transduction mechanisms used to detect ROS. This shows that manipulation of reactive species, as strategy to regulate cell differentiation, is ubiquitous in eukaryotes and suggests that such strategy was selected early in evolution.     

Characteristics of in vivo murine erythropoietic response to sodium orthovanadate

Current knowledge about the effects of vanadium compounds on erythropoiesis is still reduced and even contradictory. The aim of this work was to evaluate the in vivo effects of a single dose of sodium orthovanadate (OV, 33 mg/kg i.p.) on CF-1 mice in a time course study (0-8 days). Murine erythropoiesis was assessed through a combinatory of experimental approaches. Classical peripheral and bone marrow (BM) hematological parameters were determined. Erythroid maturation in blood stream and hemopoietic tissues (59Fe uptake assays), BM erythroid progenitor frequency (clonogenic assays) and erythroid crucial protein expressions for commitment and survival: GATA-1, erythropoietin receptor (Epo-R) and Bcl-xL (immunoblottings) were evaluated. Neither BM cellularities nor BM viabilities changed noticeably during the study. Peripheral reticulocytes showed a biphasic increment on days 2 and 8 post-OV. hematocrits enhanced transiently between days 2 and 4. 59Fe uptake percentages enhanced in peripheral blood nearly two-fold over control values between 4 and 8 days (p<0.01) without changes in BM and spleen. Additionally, mature erythroid BM compartments: polychromatophilic erythroblasts and orthochromatic normoblasts increased by the eighth day. BFU-E colonies remained near basal values during the whole experience, whilst CFU-E colonies raised 60% over control at 8 days post-OV (p<0.05). GATA-1 and Epo-R were significantly over-expressed from the third until the end of the experimental protocol (p<0.01). Surprisingly, Bcl-xL showed a constitutive expression pattern without changes during the experience. Experimental data let us suggest that OV does not to cause bone marrow cytotoxicity and that it accelerates maturation of BM committed erythroid precursors. Moreover, there are significant correlations among erythroid-related protein expressions: GATA-1 and Epo-R and the frequency of CFU-E. In addition, Bcl-xL expression invariance during the time course study would indicate that the stimulatory effect of OV treatment on erythropoiesis was mainly exerted on the maturation of red cell precursors rather than on the antiapoptosis of erythroid terminal progenitors.