Promotion and inhibition of vesicle fusion by polylysine

Polylysine induced rapid aggregation of large unilamellar vesicles composed of phosphatidylcholine-cardiolipin (1:1 molar ratio) but not their fusion. Application of the terbium-dipicolinic acid fusion assay showed that addition of polylysine at nanomolar concentrations enabled a significant lowering of the Ca2+ threshold concentration for vesicle fusion from 9 to 1 mM. Analysis of the kinetics of fusion with a mass-action kinetic model showed that polylysine enhanced significantly the rate of aggregation but affected only slightly the rate of fusion per se. Maximal enhancement of overall fusion rates occurred at a charge ratio (polylysine/cardiolipin) of about 0.5. At larger polylysine concentrations, e.g., at charge ratios greater than 3, polylysine inhibited vesicle fusion.     

Circadian variations in melatonin-binding sites in discrete areas of the male rat brain

The binding of 125I-melatonin to synaptosomes prepared from whole brains of male rats of the CD strain and from the brain, hypothalamus and striatum of male rats of the Sabra-Wistar strain was assessed throughout a 24 h period. The animals were maintained under a daily schedule of 14 h light (05:00-19:00 h) and 10 h darkness. In whole brain preparations the density of binding sites at 18:00 h was higher by about 70% than at 02:00 h with no variations in apparent affinity of the binding sites throughout the daily period. Specific binding of 125I-melatonin was found in both hypothalamus and striatum of the male rat with a distinct diurnal variation in binding site density in the hypothalamus only. The density of 125I-melatonin-binding sites in the hypothalamus was maximal between 10:00 and 18:00 h and dropped sharply after the lights went off. The apparent 125I-melatonin-binding affinities in these regions were constant and very similar to those in whole brain preparations. The daily variations in densities of 125I-melatonin-binding sites in discrete brain areas may represent a diurnal rhythmicity in the responsiveness of the neuroendocrine axis to melatonin.     

The interaction between Sendai virus and cell membranes: A quantitative analysis of 125I-Sendai virus particles' association with human red blood cells

Intact Sendai virus particles were radiolabeled by the use of chloramine-T and Na ¹²⁵I. The method described is reproducible, efficient and appropriate for the preparation of large quantities of biologically active virus with relatively high specific activity. Gel electrophoresis analysis of the radiolabeled virus revealed that approx. 50% of the total ¹²⁵I incorporated in the virus are associated with the two viral envelope glycoproteins, while the remaining 50% are evenly distributed throughout the other viral polypeptides. The ¹²⁵I-virus particles were used to study some of the kinetic parameters of the interaction between Sendai virus particles and human erythrocytes. Binding of virus particles at 4 °C is irreversible, non-cooperative and exhibits a characteristic saturation curve. A maximum of 1–2 × 10³ virus particles bound per cell was derived from the saturation curve. Non-radioactive native virus particles as well as isolated glycophorin molecules competitively inhibit binding of the ¹²⁵I-virus particles to human erythrocytes. Incubation at 37 °C of the virus-erythrocyte complex resulted in the release of about 33% of the bound virus to the surrounding medium.     

Specificity of Na+ binding to phosphatidylserine vesicles from a 23Na NMR relaxation rate study.

23Na NMR relaxation rate measurements show that Na+ binds specifically to phosphatidylserine vesicles and is displaced partially from the binding site by K+ and Ca2+ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na+ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na+-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na+) fall in the range 0.4--1.2 M-1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na+ at 0.1 M Na+ concentration.     

Silencing of OsCV (chloroplast vesiculation) maintained photorespiration and N assimilation in rice plants grown under elevated CO2

High CO₂ concentrations stimulate net photosynthesis by increasing CO₂ substrate availability for Rubisco, simultaneously suppressing photorespiration. Previously, we reported that silencing the chloroplast vesiculation (cv) gene in rice increased source fitness, through the maintenance of chloroplast stability and the expression of photorespiration-associated genes. Because high atmospheric CO₂ conditions diminished photorespiration, we tested whether CV silencing might be a viable strategy to improve the effects of high CO₂ on grain yield and N assimilation in rice. Under elevated CO₂, OsCV expression was induced, and OsCV was targeted to peroxisomes where it facilitated the removal of OsPEX11-1 from the peroxisome and delivered it to the vacuole for degradation. This process correlated well with the reduction in the number of peroxisomes, the decreased catalase activity and the increased H₂O₂ content in wild-type plants under elevated CO₂. At elevated CO₂, CV-silenced rice plants maintained peroxisome proliferation and photorespiration and displayed higher N assimilation than wild-type plants. This was supported by higher activity of enzymes involved in NO₃⁻ and NH₄⁺ assimilation and higher total and seed protein contents. Co-immunoprecipitation of OsCV-interacting proteins suggested that, similar to its role in chloroplast protein turnover, OsCV acted as a scaffold, binding peroxisomal proteins.     

White Matter Hyperintensities in Parkinson’s Disease: Do They Explain the Disparity between the Postural Instability Gait Difficulty and Tremor Dominant Subtypes?

Background

Brain white matter hyperintensities (WMHs) commonly observed on brain imaging of older adults are associated with balance and gait impairment and have also been linked to cognitive deficits. Parkinson’s disease (PD) is traditionally sub-classified into the postural instability gait difficulty (PIGD) sub-type, and the tremor dominant (TD) sub-type. Considering the known association between WMHs and axial symptoms like gait disturbances and postural instability, one can hypothesize that WMHs might contribute to the disparate clinical sub-types of patients with PD.

Methods

110 patients with PD underwent a clinical evaluation and a 3T MRI exam. Based on the Unified Parkinson Disease Rating Scale, the patients were classified into motor sub-types, i.e., TD or PIGD, and scores reflecting PIGD and TD symptoms were computed. We compared white matter burden using three previously validated methods: one using a semi-quantitative visual rating scale in specific brain regions and two automated methods.

Results

Overall, MRI data were obtained in 104 patients. The mean WMHs scores and the percent of subjects with lesions in specific brain regions were similar in the two subtypes, p = 0.678. The PIGD and the TD scores did not differ even when comparing patients with a relatively high burden of WMHs to patients with a relatively low burden. Across most of the brain regions, mild to moderate correlations between WMHs and age were found (r = 0.23 to 0.41; p<0.021). Conversely, no significant correlations were found between WMHs and the PIGD score or disease duration. In addition, depressive symptoms and cerebro-vascular risk factors were similar among the two subtypes.

Conclusions

In contrast to what has been reported previously among older adults, the present study could not demonstrate any association between WMHs and the PIGD or TD motor sub-types in patients with PD.     

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.