The mouth of a dense-core vesicle opens and closes in a concerted action regulated by calcium and amphiphysin

Secretion of hormones and peptides by neuroendocrine cells occurs through fast and slow modes of vesicle fusion but the mechanics of these processes are not understood. We used interference reflection microscopy to monitor deformations of the membrane surface and found that both modes of fusion involve the tightly coupled dilation and constriction of the vesicle. The rate of opening is calcium dependent and occurs rapidly at concentrations <5 μM. The fast mode of fusion is blocked selectively by a truncation mutant of amphiphysin. Vesicles do not collapse when fusion is triggered by strontium, rather they remain locked open and membrane scission is blocked. In contrast, constriction of the vesicle opening continues when endocytosis is blocked by inhibiting the function of dynamin. Thus, fast and slow modes of fusion involve similar membrane deformations and vesicle closure can be uncoupled from membrane scission. Regulation of these processes by calcium and amphiphysin may provide a mechanism for controlling the release of vesicle contents.     

Kinetic analyses of the magnesium chelatase provide insights into the mechanism, structure, and formation of the complex

The metabolic pathway known as (bacterio)chlorophyll biosynthesis is initiated by magnesium chelatase (BchI, BchD, BchH). This first step involves insertion of magnesium into protoporphyrin IX (proto), a process requiring ATP hydrolysis. Structural information shows that the BchI and BchD subunits form a double hexameric enzyme complex, whereas BchH binds proto and can be purified as BchH-proto. We utilized the Rhodobacter capsulatus magnesium chelatase subunits using continuous magnesium chelatase assays and treated the BchD subunit as the enzyme with both BchI and BchH-proto as substrates. Michaelis-Menten kinetics was observed with the BchI subunit, whereas the BchH subunit exhibited sigmoidal kinetics (Hill coefficient of 1.85). The BchI.BchD complex had intrinsic ATPase activity, and addition of BchH greatly increased ATPase activity. This was concentration-dependent and gave sigmoidal kinetics, indicating there is more than one binding site for the BchH subunit on the BchI.BchD complex. ATPase activity was approximately 40-fold higher than magnesium chelatase activity and continued despite cessation of magnesium chelation, implying one or more secondary roles for ATP hydrolysis and possibly an as yet unknown switch required to terminate ATPase activity. One of the secondary roles for BchH-stimulated ATP hydrolysis by a BchI.BchD complex is priming of BchH to facilitate correct binding of proto to BchH in a form capable of participating in magnesium chelation. This porphyrin binding is the rate-limiting step in catalysis. These data suggest that ATP hydrolysis by the BchI.BchD complex causes a series of conformational changes in BchH to effect substrate binding, magnesium chelation, and product release.     

Estimation of binding parameters for the protein–protein interaction using a site-directed spin labeling and EPR spectroscopy

Sensitivity of the electron paramagnetic resonance (CW EPR) to molecular tumbling provides potential means for studying processes of molecular association. It uses spin-labeled macromolecules, whose CW EPR spectra may change upon binding to other macromolecules. When a spin-labeled molecule is mixed with its liganding partner, the EPR spectrum constitutes a linear combination of spectra of the bound and unbound ligand (as seen in our example of spin-labeled cytochrome c ₂ interacting with cytochrome bc ₁ complex). In principle, the fraction of each state can be extracted by the numerical decomposition of the spectrum; however, the accuracy of such decomposition may often be compromised by the lack of the spectrum of the fully bound ligand, imposed by the equilibrium nature of molecular association. To understand how this may affect the final estimation of the binding parameters, such as stoichiometry and affinity of the binding, a series of virtual titration experiments was conducted. Our non-linear regression analysis considered a case in which only a single class of binding sites exists, and a case in which classes of both specific and non-specific binding sites co-exist. The results indicate that in both models, the error due to the unknown admixture of the unbound ligand component in the EPR spectrum causes an overestimation of the bound fraction leading to the bias in the dissociation constant. At the same time, the stoichiometry of the binding remains relatively unaffected, which overall makes the decomposition of the EPR spectrum an attractive method for studying protein–protein interactions in equilibrium. Our theoretical treatment appears to be valid for any spectroscopic techniques dealing with overlapping spectra of free and bound component.     

Effects of chlordiazepoxide on footshock- and corticotropin-releasing factor-induced increases in cortical and hypothalamic norepinephrine secretion in rats

Noradrenergic and corticotropin-releasing factor (CRF) neuronal systems within the brain have been implicated in stress and anxiety. Synaptic release of cerebral norepinephrine (NE) is increased during stress, and following intracerebral CRF administration. Benzodiazepines are commonly used anxiolytic drugs but information on their effects on the stress- and CRF-related release of NE is limited. We have used in vivo microdialysis to test the effects of the benzodiazepine, chlordiazepoxide (CDP) on the noradrenergic responses to footshock and intracerebroventricular CRF in the medial hypothalamus and the medial prefrontal cortex (PFM) of freely moving rats. Footshock (60 x 0.1-0.2 mA shocks in 20 min) significantly increased microdialysate concentrations of NE in the first sample collected after initiating the footshock. In the hypothalamus, microdialysate NE was augmented 64% above baseline. A second footshock session (100 min after the first footshock) increased microdialysate NE to 313% of the baseline. Thus the noradrenergic responses to footshock were enhanced by preceding footshocks. CRF (100 ng) administered into the locus coeruleus (LC) almost tripled microdialysate concentrations of NE in the PFM. CDP (5mg/kg, i.p.) had no statistically significant effects on the basal dialysate concentrations of NE, but it significantly attenuated both footshock- and CRF-induced increases in dialysate NE. CDP may exert a direct inhibitory effect on the noradrenergic neurons, alter the input to LC noradrenergic neurons, or alter the ability of CRF to activate the LC noradrenergic system.     

Toxoplasma gondii: chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis

Toxoplasma gondii is responsible for fetopathy in farm animals and humans and severe disease in immunocompromised individuals (i.e. AIDS patients). Effective vaccines, inducing protective and long-lasting immunity to this global parasite, are still desired. In the work, we evaluated the immunogenic and immunoprotective activity of Escherichia coli chimeric Dr fimbriae bearing selected antigenic epitopes of three T. gondii antigens (SAG1, GRA1 and MAG1), in comparison with conventional recombinant antigens obtained in E. coli expression system. Our data demonstrate a very high protective efficacy of recombinant antigens supplemented with Freund's adjuvants, whereas chimeric Dr fimbriae as a vaccine proved non-protective. The recombinant antigen vaccine induced a strong specific antibody response and prevented the brain cysts formation by 89%. The results are promising and should be confirmed in further study on farm animals by use of less aggressive than Freund's adjuvant preparations.     

Study of the interaction of the Ig2 module of the fibroblast growth factor receptor, FGFR Ig2, with the fibroblast growth factor 1, FGF1, by means of NMR spectroscopy

Fibroblast growth factor (FGF) receptor (FGFR) consists extracellularly of three immunoglobulin (Ig) modules (Ig1-3). Currently, there are two competing models (symmetric and asymmetric) of the FGF-FGFR-heparin complex based on crystal structures. Indirect evidence exists in support of both models. However, it is not clear which model is physiologically relevant. Our aim was to obtain direct, non-crystallographic evidence in support of them. We found by nuclear magnetic resonance that Ig2 could bind to FGF1 not only via the primary site (present in both models), but also via the secondary site (present only in the symmetric model). Thus, our data support the symmetric model.     

Conformational studies of hexapeptides containing two dehydroamino acid residues in positions 2 and 5 in peptide chain

Conformational preferences of a group of hexapeptides containing two dehydroamino acid residues in Positions 2 and 5 in peptide chain were investigated by means of spectroscopic methods (NMR and CD) and theoretical calculations. In the case of dimethylsulfoxide (DMSO) solution, only peptide with free N-termini adopted rigid 3(10)-helical conformation, for the rest of examined peptides extended and "zig-zag" conformers were predominant. CD measurements showed that only in chloroform solution the conformational freedom of investigated peptides was restricted.     

Synthesis and geometry of methyl (methyl 4-O-acetyl-3-azido-2,3-dideoxy-alpha/beta-D-arabino- and -alpha/beta-D-ribo-hexopyranosid)uronates

The synthesis of methyl (methyl 4-O-acetyl-3-azido-2,3-dideoxy-alpha/beta-D-arabino- and -alpha/beta-D-ribo-hexopyranosid)uronates is presented. High resolution (1)H and (13)C NMR spectral data for all diastereoisomers and single-crystal X-ray diffraction analysis for methyl (methyl 3-azido-2,3-dideoxy-beta-D-arabino-hexopyranosid)uronate are reported. The planarity of the 4-OAc and 5-COOMe groups as well as the orientations of the aglycone and azide groups in the crystal lattice is discussed. The influence of the 5-COOMe group on the pyranose ring conformation is considered.