Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles

Sterol structure influences liquid ordered domains in membranes, and the dependence of biological functions on sterol structure can help identify processes dependent on ordered domains. In this study we compared the effect of sterol structure on ordered domain formation in symmetric vesicles composed of mixtures of sphingomyelin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, and in asymmetric vesicles in which sphingomyelin was introduced into the outer leaflet of vesicles composed of DOPC and cholesterol. In most cases, sterol behavior was similar in symmetric and asymmetric vesicles, with ordered domains most strongly stabilized by 7-dehydrocholesterol (7DHC) and cholesterol, stabilized to a moderate degree by lanosterol, epicholesterol and desmosterol, and very little if at all by 4-cholesten-3-one. However, in asymmetric vesicles desmosterol stabilized ordered domain almost as well as cholesterol, and to a much greater degree than epicholesterol, so that the ability to support ordered domains decreased in the order 7-DHC > cholesterol > desmosterol > lanosterol > epicholesterol > 4-cholesten-3-one. This contrasts with values for intermediate stabilizing sterols in symmetric vesicles in which the ranking was cholesterol > lanosterol ~ desmosterol ~ epicholesterol or prior studies in which the ranking was cholesterol ~ epicholesterol > lanosterol ~ desmosterol. The reasons for these differences are discussed. Based on these results, we re-evaluated our prior studies in cells and conclude that endocytosis levels and bacterial uptake are even more closely correlated with the ability of sterols to form ordered domains than previously thought, and do not necessarily require that a sterol have a 3β-OH group.     

Utilization of Sialylated Glycans as Coreceptors Enhances the Neurovirulence of Serotype 3 Reovirus

Mammalian reoviruses display serotype-specific patterns of tropism and disease in the murine central nervous system (CNS) attributable to polymorphisms in viral attachment protein σ1. While all reovirus serotypes use junctional adhesion molecule-A as a cellular receptor, they differ in their utilization of carbohydrate coreceptors. This observation raises the possibility that carbohydrate binding by σ1 influences reovirus pathology in the CNS. In this study, we sought to define the function of carbohydrate binding in reovirus neuropathogenesis. Newborn mice were inoculated intramuscularly with wild-type strain type 3 Dearing (T3D) and T3D-σ1R202W, a point mutant T3D derivative that does not bind sialic acid (SA). Infected mice were monitored for survival, and viral loads at the sites of primary and secondary replication were quantified. Fewer mice inoculated with the wild-type virus survived in comparison to those inoculated with the mutant virus. The wild-type virus also produced higher titers in the spinal cord and brain at late times postinoculation but lower titers in the liver in comparison to those produced by the mutant virus. In addition, the wild-type virus was more virulent and produced higher titers in the brain than the mutant following intracranial inoculation. These animal infectivity studies suggest that T3D-σ1R202W harbors a defect in neural growth. Concordantly, compared with the wild-type virus, the mutant virus displayed a decreased capacity to infect and replicate in primary cultures of cortical neurons, a property dependent on cell surface SA. These results suggest that SA binding enhances the kinetics of reovirus replication in neural tissues and highlight a functional role for sialylated glycans as reovirus coreceptors in the CNS.     

Predatory Bacteria as Natural Modulators of Vibrio parahaemolyticus and Vibrio vulnificus in Seawater and Oysters

This study shows that naturally occurring Vibrio predatory bacteria (VPB) exert a major role in controlling pathogenic vibrios in seawater and shellfish. The growth and persistence of Vibrio parahaemolyticus and Vibrio vulnificus were assessed in natural seawater and in the Eastern oyster, Crassostrea virginica. The pathogens examined were V. vulnificus strain VV1003, V. parahaemolyticus O1:KUT (KUT stands for K untypeable), and V. parahaemolyticus O3:K6 and corresponding O3:K6 mutants deficient in the toxRS virulence regulatory gene or the rpoS alternative stress response sigma factor gene. Vibrios were selected for streptomycin resistance, which facilitated their enumeration. In natural seawater, oysters bioconcentrated each Vibrio strain for 24 h at 22°C; however, counts rapidly declined to near negligible levels by 72 h. In natural seawater with or without oysters, vibrios decreased more than 3 log units to near negligible levels within 72 h. Neither toxRS nor rpoS had a significant effect on Vibrio levels. In autoclaved seawater, V. parahaemolyticus O3:K6 counts increased 1,000-fold over 72 h. Failure of the vibrios to persist in natural seawater and oysters led to screening of the water samples for VPB on lawns of V. parahaemolyticus O3:K6 host cells. Many VPB, including Bdellovibrio and like organisms (BALOs; Bdellovibrio bacteriovorus and Bacteriovorax stolpii) and Micavibrio aeruginosavorus-like predators, were detected by plaque assay and electron microscopic analysis of plaque-purified isolates from Atlantic, Gulf Coast, and Hawaiian seawater. When V. parahaemolyticus O3:K6 was added to natural seawater containing trace amounts of VPB, Vibrio counts diminished 3 log units to nondetectable levels, while VPB increased 3 log units within 48 h. We propose a new paradigm that VPB are important modulators of pathogenic vibrios in seawater and oysters.     

Associations of CFH Polymorphisms and CFHR1-CFHR3 Deletion with Blood Pressure and Hypertension in Chinese Population

Dysregulation of the complement system has been linked to pathogenesis of hypertension. However, whether genetic changes of complement factor H (CFH) and its related genes are associated with hypertension is unknown. We genotyped three SNPs in the CFH gene cluster that are closely linked to age-related macular degeneration, namely rs1061170 (Y402H), rs2274700 (A473A) and rs7542235 (CFHR1–3Δ), and tested for their associations with blood pressure and hypertension risk in a population-based cohort including 3,210 unrelated Chinese Hans (50–70 years of age) from Beijing and Shanghai. We found that rs2274700 (A473A) and rs7542235 (CFHR1–3Δ) were both significantly associated with diastolic blood pressure (DBP) (β = 0.632–1.431, P≤0.038) and systolic blood pressure (SBP) (β = 1.567–4.445, P≤0.008), and rs2274700 (A473A) was associated with hypertension risk (OR [95%CI]: 1.175 [1.005–1.373], P = 0.048). Notably, the associations of rs2274700 (A473A) with DBP (P = 2.1×10⁻³), SBP (P = 8×10⁻⁵) and hypertension risk (P = 7.9×10⁻³) were significant only in the individuals with low CRP levels (<2.0 mg/l), but not in those with CRP levels ≥2.0 mg/l (P≥0.0807) (P for interaction ≤0.0467). However, no significant association between rs1061170 (Y402H) and blood pressure or hypertension risk was observed (P≥0.259). In conclusion, our results suggest that genetic variations in CFH and its related genes may contribute to hypertension risk in Chinese Hans.     

A genetically tagged Psb27 protein allows purification of two consecutive photosystem II (PSII) assembly intermediates in Synechocystis 6803, a cyanobacterium

Photosystem II (PSII) is a large membrane bound molecular machine that catalyzes light-driven oxygen evolution from water. PSII constantly undergoes assembly and disassembly because of the unavoidable damage that results from its normal photochemistry. Thus, under physiological conditions, in addition to the active PSII complexes, there are always PSII subpopulations incompetent of oxygen evolution, but are in the process of undergoing elaborate biogenesis and repair. These transient complexes are difficult to characterize because of their low abundance, structural heterogeneity, and thermodynamic instability. In this study, we show that a genetically tagged Psb27 protein allows for the biochemical purification of two monomeric PSII assembly intermediates, one with an unprocessed form of D1 (His27ΔctpAPSII) and a second one with a mature form of D1 (His27PSII). Both forms were capable of light-induced charge separation, but unable to photooxidize water, largely because of the absence of a functional tetramanganese cluster. Unexpectedly, there was a significant amount of the extrinsic lumenal PsbO protein in the His27PSII, but not in the His27ΔctpAPSII complex. In contrast, two other lumenal proteins, PsbU and PsbV, were absent in both of these PSII intermediate complexes. Additionally, the only cytoplasmic extrinsic protein, Psb28 was detected in His27PSII complex. Based on these data, we have presented a refined model of PSII biogenesis, illustrating an important role of Psb27 as a gate-keeper during the complex assembly process of the oxygen-evolving centers in PSII.     

A comparison of trends in population size and life history features of Atlantic salmon (Salmo salar) and anadromous and non-anadromous Brown trout (Salmo trutta) in a single catchment over 116 years

Hydrobiologia - We use a long time series of catch abundance from a recreational fishery over 116&nbsp;years to look for population trends in Atlantic salmon, and anadromous (sea trout) and...     

Aberrant striatal dopamine transmitter dynamics in brain-derived neurotrophic factor-deficient mice

Brain-derived neurotrophic factor (BDNF) modulates the synaptic transmission of several monoaminergic neuronal systems, including forebrain dopamine-containing neurons. Recent evidence shows a strong correlation between neuropsychiatric disorders and BDNF hypofunction. The aim of the present study was to characterize the effect of low endogenous levels of BDNF on dopamine system function in the caudate-putamen using heterozygous BDNF (BDNF(+/-) ) mice. Apparent extracellular dopamine levels in the caudate-putamen, determined by quantitative microdialysis, were significantly elevated in BDNF(+/-) mice compared with wildtype controls (12 vs. 5 nM, respectively). BDNF(+/-) mice also had a potentiated increase in dopamine levels following potassium (120 mM)-stimulation (10-fold) relative to wildtype controls (6-fold). Slice fast-scan cyclic voltammetry revealed that BDNF(+/-) mice had reductions in both electrically evoked dopamine release and dopamine uptake rates in the caudate-putamen. Superfusion of BDNF led to partial recovery of the electrically stimulated dopamine release response in BDNF(+/-) mice. Conversely, tissue accumulation of L-3,4-dihydroxyphenylalanine, extracellular levels of dopamine metabolites, and spontaneous locomotor activity were unaltered. Together, this study indicates that endogenous BDNF influences dopamine system homeostasis by regulating the release and uptake dynamics of pre-synaptic dopamine transmission.     

Evidence that D1 processing is required for manganese binding and extrinsic protein assembly into photosystem II

Photosystem II (PSII) is a large membrane protein complex that catalyzes oxidation of water to molecular oxygen. During its normal function, PSII is damaged and frequently turned over. The maturation of the D1 protein, a key component in PSII, is a critical step in PSII biogenesis. The precursor form of D1 (pD1) contains a C-terminal extension, which is removed by the protease CtpA to yield PSII complexes with oxygen evolution activity. To determine the temporal position of D1 processing in the PSII assembly pathway, PSII complexes containing only pD1 were isolated from a CtpA-deficient strain of the cyanobacterium Synechocystis 6803. Although membranes from the mutant cell had nearly 50% manganese, no manganese was detected in isolated DeltactpAHT3 PSII, indicating a severely decreased manganese affinity. However, chlorophyll fluorescence decay kinetics after a single saturating flash suggested that the donor Y(Z) was accessible to exogenous Mn(2+) ions. Furthermore, the extrinsic proteins PsbO, PsbU, and PsbV were not present in PSII isolated from this mutant. However, PsbO and PsbV were present in mutant membranes, but the amount of PsbV protein was consistently less in the mutant membranes compared with the control membranes. We conclude that D1 processing precedes manganese binding and assembly of the extrinsic proteins into PSII. Interestingly, the Psb27 protein was found to be more abundant in DeltactpAHT3 PSII than in HT3 PSII, suggesting a possible role of Psb27 as an assembly factor during PSII biogenesis.     

In vitro cytotoxicity of unsaturated oligo[poly(ethylene glycol) fumarate] macromers and their cross-linked hydrogels

Currently, oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels are being investigated as an injectable and biodegradable system for tissue engineering applications. In this study, cytotoxicity of each component of the OPF hydrogel formulation and the resulting cross-linked network was examined. Specifically, OPF synthesized with poly(ethylene glycol) (PEG) of different molecular weights (MW), the cross-linking agent [PEG-diacrylate (PEG-DA)], and the redox initiator pair [ammonium persulfate (APS) and ascorbic acid (AA)] were evaluated for cytotoxicity at 2 and 24 h using marrow stromal cells (MSCs) as model cells. The effect of leachable byproducts of OPF hydrogels on cytotoxicity was also investigated. Upon exposure to various concentrations of OPF for 2 h, greater than 50% of the MSCs were viable, regardless of OPF molecular weight or concentration in the media. After 24 h, the MSCs maintained more than 75% viability except for OPF concentrations higher than 25% (w/v). When examining the cross-linking agent, PEG-DA of higher MW (3400) demonstrated significantly higher viability compared to PEG-DA with MW 575 at all concentrations tested. Considering initiators, when MSCs were exposed to AA and APS, as well as the combination of AA and APS, higher viability was observed at lower concentrations. Once cross-linked, the leachable products from the OPF hydrogels had minimal adverse effects on the viability of MSCs (percentage of live cells was higher than 90% regardless of hydrogel types). The results suggest that, after optimization of cross-linking parameters, OPF-based hydrogels hold promise as novel injectable scaffolds or cell carriers in tissue engineering.