SNAP-23 functions in docking/fusion of granules at low Ca2+

Ca(2+)-triggered exocytosis of secretory granules mediates the release of hormones from endocrine cells and neurons. The plasma membrane protein synaptosome-associated protein of 25 kDa (SNAP-25) is thought to be a key component of the membrane fusion apparatus that mediates exocytosis in neurons. Recently, homologues of SNAP-25 have been identified, including SNAP-23, which is expressed in many tissues, albeit at different levels. At present, little is known concerning functional differences among members of this family of proteins. Using an in vitro assay, we show here that SNAP-25 and SNAP-23 mediate the docking of secretory granules with the plasma membrane at high (1 microM) and low (100 nM) Ca(2+) levels, respectively, by interacting with different members of the synaptotagmin family. In intact endocrine cells, expression of exogenous SNAP-23 leads to high levels of hormone secretion under basal conditions. Thus, the relative expression levels of SNAP-25 and SNAP-23 might control the mode (regulated vs. basal) of granule release by forming docking complexes at different Ca(2+) thresholds.     

Redox potential of quinones in photosynthetic reaction centers from Rhodobacter sphaeroides: dependence on protonation of Glu-L212 and Asp-L213

The absolute values of the one-electron redox potentials of the two quinones (Q(A) and Q(B)) in bacterial photosynthetic reaction centers from Rhodobacter sphaeroides were calculated by evaluating the electrostatic energies from the solution of the linearized Poisson-Boltzmann equation at pH 7.0. The redox potential for Q(A) was calculated to be between -173 and -160 mV, which is close to the lowest measured values that are assumed to refer to nonequilibrated protonation patterns in the redox state Q(A)(-). The redox potential of quinone Q(B) is found to be about 160-220 mV larger for the light-exposed than for the dark-adapted structure. These values of the redox potentials are obtained if Asp-L213 is nearly protonated (probability 0.75-1.0) before and after electron transfer from Q(A) to Q(B), while Glu-L212 is partially protonated (probability 0.6) in the initial state Q(A)(-)Q(B)(0) and fully protonated in the final state Q(A)(0)Q(B)(-). Conversely, if the charge state of the quinones is varied from Q(A)(-)Q(B)(0) to Q(A)(0)Q(B)(-) corresponding to the electron transfer from Q(A) to Q(B), Asp-L213 remains protonated, while Glu-L212 changes its protonation state from 0.15 H(+) to fully protonated. In agreement with results from FTIR spectra, there is proton uptake at Glu-L212 going along with the electron transfer, whereas Asp-L213 does not change its protonation state. However, in our simulations Asp-L213 is considered to be protonated rather than ionized as deduced from FTIR spectra. The calculated redox potential of Q(A) shows little dependence on the charge state of Asp-L213, which is due to a strong coupling with the protonation state of Asp-M17 but increases by 50 mV if Glu-L212 changes from the ionized to the protonated charge state. Both are in agreement with fluorescence measurements observing the decay of SP(+)Q(A)(-) in a wide pH regime. The computed difference in redox potential of Q(B) in the light-exposed and dark-adapted structure was traced back to the hydrogen bond of Q(B) with His-L190 that is lost in the dark-adapted structure and the charge of the non-heme iron atom, which is closer to Q(B) in the light-exposed than in the dark-adapted structure.     

Secretory phospholipases A2 activate selective functions in human eosinophils

Secretory phospholipases A(2) (sPLA(2)s) are released in large amounts in the blood of patients with systemic inflammatory diseases and accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils or eosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA(2). We have examined the effects of two structurally different sPLA(2)s (group IA and group IIA) on several functions of eosinophils isolated from normal donors and patients with hypereosinophilia. Both group IA and IIA sPLA(2) induced a concentration-dependent release of beta-glucuronidase, IL-6, and IL-8. Release of the two cytokines was associated with the accumulation of their specific mRNA. In addition, sPLA(2)s induced the surface expression of CD44 and CD69, two major activation markers of eosinophils. In contrast, none of the sPLA(2)s examined induced the production of IL-5, the de novo synthesis of leukotriene C(4) and platelet-activating factor, or the generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of the sPLA(2)s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes' effects. In addition, inactivation of sPLA(2) enzymatic activity by bromophenacyl bromide did not influence the release of beta-glucuronidase or of cytokines. Stimulation of eosinophils by sPLA(2)s was associated with activation of extracellular signal-regulated kinases 1/2. These results indicate that sPLA(2)s selectively activate certain proinflammatory and immunoregulatory functions of human eosinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.     

Cytochrome c(551) as a model system for protein folding

Considerable progress was made over the last few years in understanding the mechanism of folding of cytochrome c₅₅₁, a small acidic hemeprotein from Pseudomonas aeruginosa. Comparison of our results with those obtained by others on horse heart cytochrome c allows to draw some general conclusions on the structural features that are common determinants in the folding of members of the cytochrome c family.     

The GLT-1 and GLAST glutamate transporters are expressed on morphologically distinct astrocytes and regulated by neuronal activity in primary hippocampal cocultures

The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamate-induced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic protein-positive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamate-induced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.     

CCR5-reactive antibodies in seronegative partners of HIV-seropositive individuals down-modulate surface CCR5 in vivo and neutralize the infectivity of R5 strains of HIV-1 In vitro

Exposure to HIV does not necessarily result in infection. Because primary HIV infection is associated with CCR5-tropic HIV variants (R5), CCR5-specific Abs in the sera of HIV-seronegative, HIV-exposed individuals (ESN) might be associated with protection against infection. We analyzed sera from ESN, their HIV-infected sexual partners (HIV+), and healthy controls (USN) searching for CCR5-specific Abs, studying whether incubation of PBMC with sera could prevent macrophage inflammatory protein 1 beta (Mip1 beta) (natural ligand of CCR5) binding to CCR5. Results showed that Mip1 beta binding to CCR5 was not modified by sera of either 40 HIV+ or 45 USN but was greatly reduced by sera of 6/48 ESN. Binding inhibition was due to Abs reactive with CCR5. The CCR5-specific Abs neutralized the infectivity of primary HIV isolates obtained from the corresponding HIV+ partners and of R5-primary HIV strains, but not that of CXCR4-tropic or amphitropic HIV strains. Immunoadsorption on CCR5-transfected, but not on CXCR4-transfected, cells removed CCR5-specific and virus-neutralizing Abs. Epitope mapping on purified CCR5-specific Abs showed that these Abs recognize a conformational epitope in the first cysteine loop of CCR5 (aa 89-102). Affinity-purified anti-CCR5-peptide neutralized the infectivity of R5 strains of HIV-1. Anti-CCR5 Abs inhibited Mip1beta-induced chemotaxis of PBMC from healthy donors. PBMC from two ESN (with anti-CCR5 Abs) were CCR5-negative and could not be stimulated by Mip1beta in chemotaxis assays. These results contribute to clarifying the phenomenon of immunologic resistance to HIV and may have implications for the development of a protective vaccine.     

Isolation of the mustard Napin protein Allergen Sin a 1 and characterisation of its antifungal activity

Proteins and peptides belonging to the plant immune system can possess natural antibacterial, antifungal and antiviral properties. Due to their broad range of activity and stability, they represent promising novel alternatives to commonly used antifungal agents to fight the emergence of resistant strains. An isolation protocol was optimised to target proteins found in plants’ defence system, and it was applied to white mustard (Brassica hirta) seeds. Firstly, a ～14 kDa protein with activity against S. cerevisiae was extracted and purified; secondly, the protein was identified as the mustard Napin protein named Allergen Sin a 1. Napin is the name given to seed storage (2S) albumin proteins belonging to the Brassicaceae family. While several Napins have been described for their antimicrobial potential, Sin a 1 has been mainly studied for its allergenic properties. The antimicrobial activity of Sin a 1 is described and characterised for the first time in this study; it possesses antifungal and antiyeast in vitro activity, but no antibacterial activity was recorded. The yeasts Zygosaccharomyces bailii Sa 1403 and Saccharomyces cerevisiae DSM 70449 along with the filamentous fungi Fusarium culmorum FST 4.05 were amongst the most senstitive strains to Sin a 1 (MICs range 3–6 μM). The antimicrobial mechanism of membrane permeabilisation was detected, and in general, the antifungal activity of Sin a 1 seemed to be expressed in a dose-dependent manner. Data collected confirmed Sin a 1 to be a stable and compact protein, as it displayed resistance to α-chymotrypsin digestion, heat denaturation and insensitivity to pH variations and the presence of salts. In addition, the protein did not show cytotoxicity towards mammalian cells.     

The role of the Cdk9/Cyclin T1 complex in T cell differentiation

The Cdk9/Cyclin T1 complex is very important in controlling specific differentiative pathways of several cell types, including muscle cells and neurons. We recently demonstrated the involvement of this complex in B cell activation/differentiation. To check whether the Cdk9/Cyclin T1 complex is also involved in the T cell activation/differentiation process, we isolated different T cell populations by magnetic separation, based on their surface antigens. We observed that the expression level of Cdk9/Cyclin T1 increases in effector T cells (CD27(+)), as well as in activated T cells (CD25(+)) and memory T cells (CD45RA(-)), thus suggesting a specific upregulation of the Cdk9/Cyclin T1 complex following antigen encounter. We have previously demonstrated that in B cells, Cdk9 interacts in vivo with the E2A gene products E12/E47 (members of the basic helix-loop-helix family) which are involved in differentiation. In this article, we show that this interaction also occurs in T cells. This suggests an active role for the Cdk9/Cyclin T1 complex during lymphoid differentiation, through physical binding with E12 and E47. These preliminary results suggest that the Cdk9/Cyclin T1 complex may be important in the activation and differentiation program of lymphoid cells and that its upregulation, which is due to still unknown mechanisms, may contribute to malignant transformation.