Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Epidural analgesia and cesarean delivery in multiple sclerosis post-partum relapses: the Italian cohort study

Background

Few studies have systematically addressed the role of epidural analgesia and caesarean delivery in predicting the post-partum disease activity in women with Multiple Sclerosis (MS).The objective of this study was to assess the impact of epidural analgesia (EA) and caesarean delivery (CD) on the risk of post-partum relapses and disability in women with MS.

Methods

In the context of an Italian prospective study on the safety of immunomodulators in pregnancy, we included pregnancies occurred between 2002 and 2008 in women with MS regularly followed-up in 21 Italian MS centers. Data were gathered through a standardized, semi-structured interview, dealing with pregnancy outcomes, breastfeeding, type of delivery (vaginal or caesarean) and EA. The risk of post-partum relapses and disability progression (1 point on the Expanded Disability Status Sclae, EDSS, point, confirmed after six months) was assessed through a logistic multivariate regression analysis.

Results

We collected data on 423 pregnancies in 415 women. Among these, 349 pregnancies resulted in full term deliveries, with a post-partum follow-up of at least one year (mean follow-up period 5.5±3.1 years). One hundred and fifty-five patients (44.4%) underwent CD and 65 (18.5%) EA. In the multivariate analysis neither CD, nor EA were associated with a higher risk of post-partum relapses. Post-partum relapses were related to a higher EDSS score at conception (OR=1.42; 95% CI 1.11-1.82; p=0.005), a higher number of relapses in the year before pregnancy (OR=1.62; 95% CI 1.15-2.29; p=0.006) and during pregnancy (OR=3.07; 95% CI 1.40-6.72; p=0.005). Likewise, CD and EA were not associated with disability progression on the EDSS after delivery. The only significant predictor of disability progression was the occurrence of relapses in the year after delivery (disability progression in the year after delivery: OR= 4.00; 95% CI 2.0-8.2; p<0.001; disability progression over the whole follow-up period: OR= 2.0; 95% CI 1.2-3.3; p=0.005).

Conclusions

Our findings, show no correlation between EA, CD and postpartum relapses and disability. Therefore these procedures can safely be applied in MS patients. On the other hand, post-partum relapses are significantly associated with increased disability, which calls for the need of preventive therapies after delivery.

     

1‐Undecene from Pseudomonas aeruginosa is an olfactory signal for flight‐or‐fight response in Caenorhabditis elegans

Animals possess conserved mechanisms to detect pathogens and to improve survival in their presence by altering their own behavior and physiology. Here, we utilize Caenorhabditis elegans as a model host to ask whether bacterial volatiles constitute microbe‐associated molecular patterns. Using gas chromatography–mass spectrometry, we identify six prominent volatiles released by the bacterium Pseudomonas aeruginosa. We show that a specific volatile, 1‐undecene, activates nematode odor sensory neurons inducing both flight and fight responses in worms. Using behavioral assays, we show that worms are repelled by 1‐undecene and that this aversion response is driven by the detection of this volatile through AWB odor sensory neurons. Furthermore, we find that 1‐undecene odor can induce immune effectors specific to P. aeruginosa via AWB neurons and that brief pre‐exposure of worms to the odor enhances their survival upon subsequent bacterial infection. These results show that 1‐undecene derived from P. aeruginosa serves as a pathogen‐associated molecular pattern for the induction of protective responses in C. elegans.     

Mixed PGPR consortium: an effective modulator of antioxidant network for management of collar rot in cauliflower

Abstract

Sclerotinia sclerotiorum is a cosmopolitan fungal pathogen causing stem and collar watery soft rot of cauliflower. Previous management of this disease with chemical pesticides caused hazardous results which lead to use of more eco-friendly microbial approaches. In the present study, consortia of Trichoderma harzianum TNHU27 and Pseudomonas aeruginosa PJHU15 were assessed for their ability in controlling Sclerotinia rot in cauliflower. The seedlings of cauliflower were challenged singly and in consortium with these two compatible microbes. The plants were evaluated upon challenge inoculation with S. sclerotiorum for changes in total phenolic content, the activity of defense and antioxidative enzymes. The microbial consortium comprising of T. harzianum and P. aeruginosa significantly enhanced the defense responses of the plant in comparison to pathogen challenged and unchallenged control. The study elucidates that plant beneficial microbes in the consortium may provide superior protection by induction of faster and enhanced defense responses in comparison to unchallenged and single microbe challenged plants under pathogen challenged conditions.     

Evaluating whether velar lobe size indicates food limitation among larvae of the marine gastropod Crepidula fornicata

Disproportionately large feeding structures have been used to infer food limitation in some marine invertebrate larvae, but few studies have investigated whether other factors alter larval morphology in similar ways. In this study, larvae of Crepidula fornicata were reared either at five different food concentrations of Isochrysis galbana (clone T-ISO) at a single temperature (22 degrees C) (Experiments I and II); or on three different phytoplankton species (Isochrysis galbana, Dunaliella tertiolecta, and Pavlova lutheri) at both high and low concentrations at a single temperature (22 degrees C) (Experiment III); or at high and low concentrations of Isochrysis galbana at four different temperatures between 16 and 25 degrees C (Experiment IV). Shell lengths and velar lobe dimensions were determined for individual larvae at intervals to monitor relative rates of velar and shell growth. In addition (Experiment V), fast growing and slow growing larvae in Experiment I were examined separately to determine whether velar lobes developed at similar rates (relative to shell growth) for fast and slow growing larvae within individual cultures. In general, velar lobes grew significantly larger, relative to shell length, when larvae were reared at low food concentrations (P<0.0001); for larvae of similar shell length, the velar lobes of those fed 1x10(4) cells ml(-1) were on average 17.7% larger than those of larvae fed 18x10(4) cells ml(-1) of T-ISO. In contrast, larvae fed different phytoplankton species at equivalently high food concentrations did not differ in relative velum size (P=0.2666), even though shell growth rates differed significantly for larvae raised on the different diets, indicating substantial variation in food quality. We also found that relative rates of velum and shell growth differed among fast and slow growing individuals within treatments. Temperature had no significant effect on relative rates of velar and shell growth within the 16-25 degrees C range tested (P=0.121), but may have altered the relationship between food concentration and relative velar growth. These results indicate that dramatically reduced food concentration induces disproportionate growth in the velar lobes of C. fornicata, but that interpretation of data from field-collected individuals of this species will be made difficult by the potentially confounding effects of temperature, food quality, and differences in individual growth potential. Assessments of food limitation using morphological measurements for field-collected larvae will need to be supplemented with other indicators before convincing conclusions about the extent of food limitation in C. fornicata can be drawn.     

Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Fold-recognition and comparative modeling of human α2,3-sialyltransferases reveal their sequence and structural similarities to CstII from Campylobacter jejuni

Background  

The 3-D structure of none of the eukaryotic sialyltransferases (SiaTs) has been determined so far. Sequence alignment algorithms such as BLAST and PSI-BLAST could not detect a homolog of these enzymes from the protein databank. SiaTs, thus, belong to the hard/medium target category in the CASP experiments. The objective of the current work is to model the 3-D structures of human SiaTs which transfer the sialic acid in α2,3-linkage viz., ST3Gal I, II, III, IV, V, and VI, using fold-recognition and comparative modeling methods. The pair-wise sequence similarity among these six enzymes ranges from 41 to 63%.     

Equilibrium unfolding of <Emphasis Type="Italic">A. niger</Emphasis> RNase: pH dependence of chemical and thermal denaturation

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A_MG1) into another (I_MG2).     

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.