Malate and malate-channel antibodies inhibit electrogenic and ATP-dependent citrate transport across the tonoplast of citrus juice cells

Citrus juice cells accumulate high levels of citric acid in their vacuoles when compared to other organic ions including malate. Uptake of citrate into tonoplast vesicles from Citrus juice cells was investigated in the presence of malate, and after incubation with antibodies raised against the vacuolar malate-specific channel of Kalancho? diagremontiana leaves. Antibodies against the vacuolar malate channel immunoreacted with a protein of similar size in tonoplast extracts from three Citrus varieties differing in citric acid content. Malate channel antibodies inhibited both delta MicroH(+)-dependent and delta MicroH(+)-independent ATP-dependent citrate transport, indicating common domains in both transport systems and to the malate-specific channel of Kalancho? diagremontiana leaves. Malate strongly inhibited electrogenic citrate transport, whereas ATP-dependent citrate uptake was less affected. Kinetic analysis of citrate transport in the presence of malate confirmed the existence of two citrate transport mechanisms and indicated that both citrate and malate share a common transport channel across the tonoplast of Citrus juice cells.     

Pulsatile growth hormone secretion decreases S-adenosylmethionine synthetase in rat liver

S-adenosylmethionine synthetase (AdoMet synthetase) is responsible for the synthesis of the major methyl donor S-adenosylmethionine. The AdoMet synthetase gene was identified by subtractive suppressive hybridization as being expressed at higher levels in the liver of rats continuously exposed to growth hormone (GH) than in rats intermittently exposed to the hormone. Further studies on the regulation of AdoMet synthetase showed that the activity and mRNA levels were higher in female than in male rats. Hypophysectomy increased AdoMet synthetase mRNA in both male and female rats. Combined thyroxine and cortisol treatment of hypophysectomized rats had no effect on AdoMet synthetase mRNA levels. Two daily injections of GH for 7 days, mimicking the male secretory pattern of GH, decreased AdoMet synthetase activity and mRNA levels. A continuous infusion of GH, mimicking the female secretory pattern of GH, had small or no effects on AdoMet synthetase activity and decreased the mRNA levels to a lesser degree than two daily injections. It is concluded that the lower AdoMet synthetase activity in male rats is due to an inhibitory effect of the male characteristic pulsatile secretory pattern of GH on AdoMet synthetase mRNA expression.     

ATP-induced sucrose efflux from red-beet tonoplast vesicles

 Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H⁺-ATPase to bafilomycin-A and NO₃ ⁻. Inhibition of the H⁺-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H⁺-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism. Received: 12 October 1999 / Accepted: 18 November 1999     

Estimating Urban Traffic Patterns through Probabilistic Interconnectivity of Road Network Junctions

The emergence of large, fine-grained mobility datasets offers significant opportunities for the development and application of new methodologies for transportation analysis. In this paper, the link between routing behaviour and traffic patterns in urban areas is examined, introducing a method to derive estimates of traffic patterns from a large collection of fine-grained routing data. Using this dataset, the interconnectivity between road network junctions is extracted in the form of a Markov chain. This representation encodes the probability of the successive usage of adjacent road junctions, encoding routes as flows between decision points rather than flows along road segments. This network of functional interactions is then integrated within a modified Markov chain Monte Carlo (MCMC) framework, adapted for the estimation of urban traffic patterns. As part of this approach, the data-derived links between major junctions influence the movement of directed random walks executed across the network to model origin-destination journeys. The simulation process yields estimates of traffic distribution across the road network. The paper presents an implementation of the modified MCMC approach for London, United Kingdom, building an MCMC model based on a dataset of nearly 700000 minicab routes. Validation of the approach clarifies how each element of the MCMC framework contributes to junction prediction performance, and finds promising results in relation to the estimation of junction choice and minicab traffic distribution. The paper concludes by summarising the potential for the development and extension of this approach to the wider urban modelling domain.     

Pretransplant HRCT Characteristics Are Associated with Worse Outcome of Lung Transplantation for Cystic Fibrosis Patients

Objectives

Peri- and postoperative complications diminish the outcome of lung transplantation (LTx) in patients with cystic fibrosis (CF). We hypothesized that the degree of pathological findings on pre-LTx high resolution computed tomography (HRCT) is associated with higher morbidity and mortality in CF.

Methods

All our CF patients undergoing LTx between 2001 and 2011 were included. HRCT examinations were evaluated according to a scoring system for pulmonary disease in CF patients, the Severe Advanced Lung Disease (SALD) score and for pleural involvement.

Results

Fifty-three patients were included. Dominant infectious/inflammatory disease according to the SALD score was observed in 10 patients (19%). Five (50%) of those patients died within one week after LTx, compared to 2 (5%) patients without dominant infectious/inflammatory disease (p<0.001). This difference in survival percentage remained also significant in multivariate analysis. Patients with infectious/inflammatory disease received more packed red blood cells; 26 versus 8 in the first week (p<0.001). Pleural thickening was associated with higher requirement (10 units) for blood transfusion during LTx, compared to patients with normal pleura (4 units).

Conclusions

The analysis of HRCT in CF patients according to the SALD score showed that dominant infectious/inflammatory disease is associated with a higher mortality after LTx. If confirmed in other studies, HRCT might aid estimation of surgical risk in some adult CF patients.     

Determinants of Fluidlike Behavior and Effective Viscosity in Cross-Linked Actin Networks

The actin cortex has a well-documented ability to rapidly remodel and flow while maintaining long-range connectivity, but how this is achieved remains poorly understood. Here, we use computer simulations to explore how stress relaxation in cross-linked actin networks subjected to extensional stress depends on the interplay between network architecture and turnover. We characterize a regime in which a network response is nonaffine and stress relaxation is governed by the continuous dissipation of elastic energy via cyclic formation, elongation, and turnover of tension-bearing elements. Within this regime, for a wide range of network parameters, we observe a constant deformation (creep) rate that is linearly proportional to the rate of filament turnover, leading to a constant effective viscosity that is inversely proportional to turnover rate. Significantly, we observe a biphasic dependence of the creep rate on applied stress: below a critical stress threshold, the creep rate increases linearly with applied stress; above that threshold, the creep rate becomes independent of applied stress. We show that this biphasic stress dependence can be understood in terms of the nonlinear force-extension behavior of individual force-transmitting network elements. These results have important implications for understanding the origins and control of viscous flows both in the cortex of living cells and in other polymer networks.     

Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.     

First evidence for Cambrian glaciation provided by sections in Avalonian New Brunswick and Ireland: Additional data for Avalon–Gondwana separation by the earliest Palaeozoic

The first evidence for Cambrian glaciation is provided by two successions on the Avalon microcontinent. The middle lowest Cambrian (middle Terreneuvian Series and Fortunian Stage–Stage 2 boundary interval) has an incised sequence boundary overlain by a fluvial lowstand facies and higher, olive green, marine mudstone on Hanford Brook, southern New Brunswick. This succession in the lower Mystery Lake Member of the Chapel Island Formation may be related to melting of an ice sheet in Avalon. The evidence for this interpretation is a muddy diamictite with outsized (up to 10 cm in diameter), Proterozoic marble and basalt clasts that penetrated overlying laminae in the marine mudstone. That eustatic rise was associated with the mudstone deposition is suggested by an approximately coeval rise that deposited sediments with Watsonella crosbyi Zone fossils 650 km away in Avalonian eastern Newfoundland. A sea-level rise within the Watsonella crosbyi Chron, at ca. 535 Ma, may correspond to a unnamed negative ¹³C excursion younger than the basal Cambrian excursion (BACE) and the ZHUCE excursion in Stage 2 of the upper Terreneuvian Series. Cambrian dropstones are now also recognized on the northern (Gander) margin of Avalon in continental slope–rise sedimentary rocks in southeast Ireland. Although their age (Early–Middle Cambrian) is poorly constrained, dropstones in the Booley Bay Formation provide additional evidence for Cambrian glaciation on the Avalon microcontinent. Besides providing the first evidence of Cambrian glaciation, these dropstone deposits emphasize that Avalon was not part of or even latitudinally close to the terminal Ediacaran–Cambrian, tropical carbonate platform successions of West Gondwana.