The Influence of Catalysis on Mad2 Activation Dynamics

Mad2 is a key component of the spindle assembly checkpoint, a safety device ensuring faithful sister chromatid separation in mitosis. The target of Mad2 is Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mad2 binding to Cdc20 is a complex reaction that entails the conformational conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformer. Previously, it has been hypothesized that the conversion of O-Mad2 is accelerated by its conformational dimerization with C-Mad2. This hypothesis, known as the Mad2-template hypothesis, is based on the unproven assumption that the natural conversion of O-Mad2 required to bind Cdc20 is slow. Here, we provide evidence for this fundamental assumption and demonstrate that conformational dimerization of Mad2 accelerates the rate of Mad2 binding to Cdc20. On the basis of our measurements, we developed a set of rate equations that deliver excellent predictions of experimental binding curves under a variety of different conditions. Our results strongly suggest that the interaction of Mad2 with Cdc20 is rate limiting for activation of the spindle checkpoint. Conformational dimerization of Mad2 is essential to accelerate Cdc20 binding, but it does not modify the equilibrium of the Mad2:Cdc20 interaction, i.e., it is purely catalytic. These results surpass previously formulated objections to the Mad2-template model and predict that the release of Mad2 from Cdc20 is an energy-driven process.     

Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation -- a review

Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further.     

Serological diagnosis of paracoccidioidomycosis in HIV-coinfected patients

Paracoccidioidomycosis should be differentiated from other opportunistic diseases in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients who live in Latin America. Laboratory investigation can begin with serological tests, which are rapid and efficient. In the present study, double immunodiffusion (DID), counterimmunoelectrophoresis (CIEP) and an enzyme linked immunosorbent assay (ELISA) tests were assessed for the detection of anti-Paracoccidioides brasiliensis antibodies in 40 patients coinfected with HIV. The results were compared to those obtained for 75 non-HIV-infected patients with endemic paracoccidioidomycosis. Anti-P. brasiliensis antibodies were detected in 65% (DID), 79% (CIEP) and 95% (ELISA) of the patients with HIV/AIDS, significantly lower rates than those detected in cases of endemic paracoccidioidomycosis, which were 89%, 99% and 100%, respectively. The reactive sera of HIV-infected patients also showed lower anti-P. brasiliensis antibody titres than those of non-HIV-infected patients. Despite the lower intensity of the specific humoral response, serological tests are useful for the diagnosis of opportunistic paracoccidioidomycosis in the HIV/AIDS population. We suggest optimization of the laboratory diagnosis by combining the ELISA test with CIEP or DID.     

An investigation of the occurrence and properties of the mitochondrial intermediate-conductance Ca2+-activated K+ channel mtKCa3.1

The mitochondrial intermediate-conductance Ca²⁺-activated K⁺ channel mtK_Ca3.1 has recently been discovered in the HCT116 colon tumor-derived cell line, which expresses relatively high levels of this protein also in the plasma membrane. Electrophysiological recordings revealed that the channel can exhibit different conductance states and kinetic modes, which we tentatively ascribe to post-translational modifications. To verify whether the localization of this channel in mitochondria might be a peculiarity of these cells or a more widespread feature we have checked for the presence of mtK_Ca3.1 in a few other cell lines using biochemical and electrophysiological approaches. It turned out to be present at least in some of the cells investigated. Functional assays explored the possibility that mtK_Ca3.1 might be involved in cell proliferation or play a role similar to that of the Shaker-type K_V1.3 channel in lymphocytes, which interacts with outer mitochondrial membrane-inserted Bax thereby promoting apoptosis (Szabò, I. et al., Proc. Natl. Acad Sci. USA 105 (2008) 14861–14866). A specific K_Ca3.1 inhibitor however did not have any detectable effect on cell proliferation or death.     

Prophylactic Administration of Bacterially Derived Immunomodulators Improves the Outcome of Influenza Virus Infection in a Murine Model

Prophylactic or therapeutic immunomodulation is an antigen-independent strategy that induces nonspecific immune system activation, thereby enhancing host defense to disease. In this study, we investigated the effect of prophylactic immunomodulation on the outcome of influenza virus infection using three bacterially derived immune-enhancing agents known for promoting distinct immunological profiles. BALB/c mice were treated nasally with either cholera toxin (CT), a mutant form of the CT-related Escherichia coli heat-labile enterotoxin designated LT(R192G), or CpG oligodeoxynucleotide. Mice were subsequently challenged with a lethal dose of influenza A/PR/8/34 virus 24 h after the last immunomodulation treatment and either monitored for survival or sacrificed postchallenge for viral and immunological analysis. Treatment with the three immunomodulators prevented or delayed mortality and weight loss, but only CT and LT(R192G) significantly reduced initial lung viral loads as measured by plaque assay. Analysis performed 4 days postinfection indicated that prophylactic treatments with CT, LT(R192G), or CpG resulted in significantly increased numbers of CD4 T cells, B cells, and dendritic cells and altered costimulatory marker expression in the airways of infected mice, coinciding with reduced expression of pulmonary chemokines and the appearance of inducible bronchus-associated lymphoid tissue-like structures in the lungs. Collectively, these results suggest that, despite different immunomodulatory mechanisms, CT, LT(R192G), and CpG induce an initial inflammatory process and enhance the immune response to primary influenza virus challenge while preventing potentially damaging chemokine expression. These studies provide insight into the immunological parameters and immune modulation strategies that have the potential to enhance the nonspecific host response to influenza virus infection.Influenza viruses cause acute, contagious respiratory disease. Despite the availability of vaccines and antiviral therapies, influenza virus infections cause considerable morbidity and mortality each year. It is estimated that during seasonal epidemics 10% of the world population is infected, resulting in 2 to 3 million severe cases and up to 500,000 deaths (1). The failure of conventional methods to prevent illness and death from influenza is attributed to the continuous antigenic variability of the virus due to mutations (antigen drift) and reassortments (antigenic shift). The inadequacy of current anti-influenza virus treatments is particularly concerning in the case of influenza pandemics with new viral strains for which effective vaccines would not be initially available. Thus, an antigen-independent prophylactic treatment that could nonspecifically enhance immune responses to negate or inhibit the progression of influenza virus infection would provide invaluable benefits.Several recent studies have explored the use of immunomodulation strategies as prophylaxis or therapeutic treatments to modify the immune response to influenza virus infection, thereby preventing or decreasing viral burden, disease symptoms, and mortality. These strategies have one of two distinct immunologic goals: either to increase immune system activation and/or Th1 responses specific against influenza virus, or alternatively, decrease inflammation and immunopathology. The first strategy has been demonstrated in animal models by administering host proteins/glycoproteins that function in immune defense, such as the pattern recognition receptor (PRR) mindin (28), milk-derived glycoproteins (61), and virally delivered interferon (IFN) cytokines (27). Immunomodifiers of microbial origin have also been used to enhance host response to infection, including the binding subunit of cholera toxin (CT-B) (49), Th1-promoting Toll-like receptor (TLR) agonists CpG oligodeoxynucleotides (ODN) (15, 82), poly(I:C) (81), 3 M-011 (23), and synthetic lipid A analogs (11). Immunomodulators used in the second strategy, with the aim to prevent detrimental inflammation, have been associated with improved infection outcomes and include enterotoxin mutant LT(S63K) (80) and anti-inflammatory COX-2 inhibitors (84). However, immunomodulation does not always result in beneficial responses to infection. Administration of Δ⁹-THC, an immunosuppressive compound, decreased cellular infiltration and increased viral load when given prior to and during influenza virus infection (7). Similarly, sphingosine 1-phosphate (S1P) analog, an immunotherapeutic agent, was found to suppress induction of T-cell responses to influenza virus (46). Lastly, fish oil-fed mice demonstrated reduced lung inflammation, cellular infiltration, and cytokine secretion but increased mortality during influenza virus infection (60).These studies highlight the need for experiments that clarify the consequences of various immunomodulation strategies on influenza virus infection and the particular requirements for generating a protective response. Furthermore, very little attention has been given to the mechanisms by which different immunomodulators with unique effector functions modulate the host response when evaluated in the same infection model. To address these questions and increase our understanding of the consequences brought about by prophylactic immunomodulation in pulmonary disease, we chose to compare the effects of pulmonary delivery of three well-characterized vaccine adjuvants on the outcome of influenza virus infection in a murine model. The immunomodulators used in this study are CpG, a nontoxic protein designated LT(R192G) that was derived from the cholera-related heat-labile enterotoxin produced by Escherichia coli, and CT. These bacterially derived agents, known to promote distinct effector functions, are excellent immunomodulators, as they induce strong immune activation and have been previously evaluated as components of influenza vaccines (29, 42, 49, 53, 56, 58). CpG ODNs are synthetic unmethylated oligodeoxynucleotides containing CpG motifs that trigger a TLR9-dependent MyD88 signaling pathway. CpG treatment results in potent Th1 cytokine expression (IFNs and interleukin-12 [IL-12]), activation of dendritic cells (DCs), NK cells, and B cells, and induction of Th1 cells and a Th1 antibody profile (30, 35, 83). CpG has been extensively studied in animal models of systemic and pulmonary infectious diseases caused by influenza virus (15, 82) and other bacterial, fungal, and parasitic pathogens (3, 9, 15, 17, 25, 34, 51, 77).Bacterially derived ADP-ribosylating enterotoxins, including CT from Vibrio cholerae and LT from E. coli, are robust systemic and mucosal adjuvants. Both in vitro and in vivo studies have demonstrated that CT induces secretion of Th2 cytokines (IL-4, IL-5, IL-6, and IL-10) by immune system cells, maturation of DCs, generation of Th2 and T-regulatory cells, and active suppression of Th1 responses (2, 32, 38, 39, 47, 49, 53, 56). Studies in vivo have also shown that intranasal delivery of CT-B, the binding subunit of the enterotoxin, combined with minimal levels of CT holotoxin, induces protective effects in influenza virus-infected mice (49). In contrast to CpG and CT, LT and LT(R192G) induce a more balanced cytokine and antibody subclass profile indicative of a mixed Th1/Th2 immune response (16, 45, 73). LT(R192G) has yet to be evaluated as a prophylactic immunomodulator, but another LT mutant, LT(S63K), has demonstrated some protective effects against influenza virus, respiratory syncytial virus (RSV), and Cryptococcus neoformans infections (80). Although safety concerns limit the use of native enterotoxins for intranasal or intrapulmonary use in humans (54, 76), animal model studies are warranted because they enhance our understanding of the initial responses that can ultimately lead to protection of the host against infection. In addition, the use of these enterotoxins in laboratory research has the potential to be translated into clinical application by using mutated low-toxinogenic derivatives that retain their immunomodulatory properties.In this study we used a comprehensive approach to evaluate the effects of intrapulmonary delivery of three strong immunomodulators prior to influenza virus infection in a murine model. We hypothesized that the unique immunologic effects induced by prophylactic treatment with CT, LT(R192G), or CpG would differentially affect survival, viral loads, and immune responses of BALB/c mice to influenza A/PR/8/34 (H1N1) virus infection. The relevance of this study to influenza virus disease pathogenesis and infectious disease immunomodulation strategies is discussed.     

Epiprofin/Sp6: a new player in the regulation of tooth development

Odontogenesis is governed by a complex network of intercellular signaling events between the dental epithelium and mesenchyme. This network leads to the progressive determination of tooth shape, and to the differentiation of these tissues into enamel-producing ameloblasts and dentin-producing odontoblasts respectively. Among the main signaling pathways involved in the regulation of tooth development, Bone Morphogenetic Protein (BMP), Sonic hedgehog (Shh) and Wingless-type MMTV integration site (Wnt) pathways have been reported to play significant roles. Recently, the phenotype of mice deficient in Epiprofin/Sp6 (Epfn) has been found to present striking dental abnormalities, including a complete lack of differentiated ameloblasts and consequently no enamel, highly altered molar cusp patterns and the formation of multiple supernumerary teeth. In this article, we review the interaction of Epfn with the BMP, Shh and Wnt pathways in the regulation of tooth development, based on the data obtained from the study of several genetically modified mice.     

Histone hyperacetylation affects meiotic recombination and chromosome segregation in Arabidopsis

In this study, the meiotic role of MEIOTIC CONTROL OF CROSSOVERS1 (MCC1), a GCN5‐related histone N‐acetyltransferase, is described in Arabidopsis. Analysis of the over‐expression mutant obtained by enhancer activation tagging revealed that acetylation of histone H3 increased in male prophase I. MCC1 appeared to be required in meiosis for normal chiasma number and distribution and for chromosome segregation. Overall, elevated MCC1 did not affect crossover number per cell, but has a differential effect on individual chromosomes elevating COs for chromosome 4, in which there is also a shift in chiasma distribution, and reducing COs for chromosome 1 and 2. For the latter there is a loss of the obligate CO/chiasma in 8% of the male meiocytes. The meiotic defects led to abortion in about half of the male and female gametes in the mutant. In wild type, the treatment with trichostatin A, an inhibitor of histone deacetylases, phenocopies MCC1 over‐expression in meiosis. Our results provide evidence that histone hyperacetylation has a significant impact on the plant meiosis.     

Bovine lactoferrin interacts with cable pili of Burkholderia cenocepacia

In this study we evaluated the ability of lactoferrin, the most abundant antimicrobial protein in airway secretions, to bind the surface structures of a Burkholderia strain cystic fibrosis-isolated. Burkholderia cenocepacia is a gram-negative bacterium involved as respiratory pathogen in cystic fibrosis patient infections. This bacterium possesses filamentous structures, named cable pili that have been proposed as virulence factors because of their ability to bind to respiratory epithelia and mucin. Previously, we demonstrated that bovine lactoferrin was able to influence the efficiency of invasion of different iron-regulated morphological forms of B. cenocepacia. Bovine lactoferrin showed to efficiently inhibit invasion of alveolar epithelial cells by free-living bacteria or iron-induced aggregates or biofilm. Results of the present study demonstrate that bovine lactoferrin is also able to specifically bind to B. cenocepacia cells and show that cable pili are involved in this interaction. The attachment of bovine lactoferrin to pili led to a reduced binding of bacterial cells to mucin. Since cable pili are implicated in mediating the bacterial interactions with mucin and epithelial cells, lactoferrin binding to these structures could play an important role in neutralizing bacterial infection in cystic fibrosis patients.