Characterization of sterol uptake in leaf tissues of sugar beet

The uptake of cholesterol has been characterized in leaf discs from mature leaves of sugar beet (Beta vulgaris L.). This transport system exhibited a simple saturable phase with an apparent Michaelis constant ranging from 30 to 190 M depending on the sample. When present at 10 M excess, other sterols were able to inhibit cholesterol uptake. Moreover, binding assays demonstrated the presence of high-affinity binding sites for cholesterol in purified plasma membrane vesicles. In the range 1–60 M, cholesterol uptake showed an active component evidenced by action of the protonophore carbonyl cyanide m-chlorophenylhydrazone. Energy was required as shown by the inhibition of uptake induced by respiration inhibitors (NaN₃), darkness and photosynthesis inhibitors [3-(3,4-dichlorophenyl)-1,1-dimethylurea, methyl viologen]. Moreover, the process was strongly dependent on the experimental temperature. Uptake was optimal at acidic pH (4.0), sensitive to ATPase modulators, inhibited by thiol reagents (N-ethylmaleimide, p-chloromercuribenzenesulfonic acid, Mersalyl) and by the histidyl-group reagent diethyl pyrocarbonate. The addition of cholesterol did not modify H⁺ flux from tissues, indicating that H⁺-co-transport was unlikely to be involved. MgATP did not increase the uptake, arguing against involvement of an ABC cassette-type transporter. By contrast, cryptogein, a sterol carrier protein from the Oomycete Phytophtora cryptogea, greatly increased absorption. Taken together, the results reported in this work suggest that plant cells contain a specific plasma membrane transport system for sterols.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - PCMBS p-chloromercuribenzenesulfonic acid - PMV plasma membrane vesicle - TLC thin-layer chromatography     

Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family

Putative phosphate transporters have been identified in a barley (Hordeum vulgare L.) genomic library by their homology to known phosphate transporters from dicot species. The genes designated HORvu;Pht1;1 and HORvu;Pht1;6 encode proteins of 521 and 535 amino acids respectively with 12 predicted membrane-spanning domains and other motifs common to the Phtl family of phosphate transporters. HORvu;Pht1;1 is expressed exclusively in roots and is strongly induced by phosphate deprivation. HORvu;Pht1;6 is expressed in the aerial parts of the plant with strongest expression in old leaves and flag leaves. In situ hybridization showed that HORvu;Pht1;6 is expressed in the phloem of vascular bundles in leaves and ears. In order to study the biochemical properties of HORvu;Pht1;1 and HORvu;Pht1;6, the genes were expressed in transgenic rice (Oryza sativa L.) plants under the control of the rice actin promoter and suspension cell cultures were generated. Cells derived from transgenic plants were able to take up phosphate at a much higher rate than control cells, demonstrating that both genes encode functional phosphate transporters. The estimated Km for phosphate for cells expressing HORvu;Pht1;1 was 9.06 +/- 0.82 microM, which is characteristic of a high-affinity transporter. The rate of phosphate uptake decreased with increasing pH, suggesting that HORvu;Pht1;1 operates as a H+/H2PO4(-) symporter. In contrast, the estimated Km for phosphate for cells expressing HORvu;Pht1;6 was 385 +/- 61 microM, which is characteristic of a low-affinity transporter. Taken together, the results suggest that HORvu;Pht1;1 functions in uptake of phosphate at the root surface, while HORvu;Pht1;6 probably functions in remobilization of stored phosphate from leaves.     

The monotreme genome: a patchwork of reptile, mammal and unique features?

The first specimen of platypus (Ornithorhynchus anatinus) that reached Britain in the late 18th century was regarded a scientific hoax. Over decades the anatomical characteristics of these unique mammals, such as egg laying and the existence of mammary glands, were hotly debated before they were accepted. Within the last 40 years, more and more details of monotreme physiology, histology, reproduction and genetics have been revealed. Some show similarities with birds or reptiles, some with therian mammals, but many are very specific to monotremes. The genome is no exception to monotreme uniqueness. An early opinion was that the karyotype, composed of a few large chromosomes and many small ones, resembled bird and reptile macro- and micro-chromosomes. However, the platypus genome also features characteristics that are not present in other mammals, such as a complex translocation system. The sex chromosome system is still not resolved. Nothing is known about dosage compensation and, unlike in therian mammals, there seems to be no genomic imprinting. In this article we will recount the mysteries of the monotreme genome and describe how we are using recently developed technology to identify chromosomes in mitosis, meiosis and sperm, to map genes to chromosomes, to unravel the sex chromosome system and the translocation chain and investigate X inactivation and genomic imprinting in monotremes.     

Apolipoprotein A-I induced amyloidosis

Amyloidosis is characterized by extracellular deposits of protein fibrils with a high content of β-sheets in secondary structure. The protein forms together with proteoglycans amyloid fibrils causing organ damage and serious morbidity. Intact apolipoprotein A-I (apoA-I) is an important protein in lipid metabolism regulating the synthesis and catabolism of high density lipoproteins (HDL). Usually, apoA-I is not associated with amyloidosis. However, four naturally occuring mutant forms of apoA-I are known so far resulting in amyloidosis. The most important feature of all variants is the very similar formation of N-terminal fragments which were found in the amyloid deposits (residues 1–83 to 1–94). The new insights in the understanding of the association of apoA-I with HDL, its metabolism, and its hypothesized structural findings may explain a common mechanism for the genesis of apoA-I induced amyloidosis. Here we summarized the specific features of all known amyloidogenic variants of apoA-I and speculate about its metabolic pathway, which may have general implications for the metabolism of apoA-I.     

Fallback foraging as a way of life: Using dietary toughness to compare the fallback signal among capuchins and implications for interpreting morphological variation

The genus Cebus is one of the best extant models for examining the role of fallback foods in primate evolution. Cebus includes the tufted capuchins, which exhibit skeletal features for the exploitation of hard and tough foods. Paradoxically, these seemingly “specialized” taxa belong to the most ubiquitous group of closely related primates in South America, thriving in a range of different habitats. This appears to be a consequence of their ability to exploit obdurate fallback foods. Here we compare the toughness of foods exploited by two tufted capuchin species at two ecologically distinct sites; C. apella in a tropical rainforest, and C. libidinosus in a cerrado forest. We include dietary data for one untufted species (C. olivaceus) to assess the degree of difference between the tufted species. These data, along with information on skeletal morphology, are used to address whether or not a fallback foraging species exhibits a given suite of morphological and behavioral attributes, regardless of habitat. Both tufted species ingest and masticate a number of exceedingly tough plant tissues that appear to be used as fallback resources, however, C. libidinosus has the toughest diet both in terms of median and maximal values. Morphologically, C. libidinosus is intermediate in absolute symphyseal and mandibular measurements, and in measures of postcranial robusticity, but exhibits a higher intermembral index than C. apella. We propose that this incongruence between dietary toughness and skeletal morphology is the consequence of C. libidinosus' use of tools while on the ground for the exploitation of fallback foods. Am J Phys Anthropol 140:687–699, 2009. © 2009 Wiley-Liss, Inc.     

Broadband Onset Inhibition Can Suppress Spectral Splatter in the Auditory Brainstem

In vivo intracellular responses to auditory stimuli revealed that, in a particular population of cells of the ventral nucleus of the lateral lemniscus (VNLL) of rats, fast inhibition occurred before the first action potential. These experimental data were used to constrain a leaky integrate-and-fire (LIF) model of the neurons in this circuit. The post-synaptic potentials of the VNLL cell population were characterized using a method of triggered averaging. Analysis suggested that these inhibited VNLL cells produce action potentials in response to a particular magnitude of the rate of change of their membrane potential. The LIF model was modified to incorporate the VNLL cells’ distinctive action potential production mechanism. The model was used to explore the response of the population of VNLL cells to simple speech-like sounds. These sounds consisted of a simple tone modulated by a saw tooth with exponential decays, similar to glottal pulses that are the repeated impulses seen in vocalizations. It was found that the harmonic component of the sound was enhanced in the VNLL cell population when compared to a population of auditory nerve fibers. This was because the broadband onset noise, also termed spectral splatter, was suppressed by the fast onset inhibition. This mechanism has the potential to greatly improve the clarity of the representation of the harmonic content of certain kinds of natural sounds.     

Captive Reptile Mortality Rates in the Home and Implications for the Wildlife Trade

The trade in wildlife and keeping of exotic pets is subject to varying levels of national and international regulation and is a topic often attracting controversy. Reptiles are popular exotic pets and comprise a substantial component of the live animal trade. High mortality of traded animals raises welfare concerns, and also has implications for conservation if collection from the wild is required to meet demand. Mortality of reptiles can occur at any stage of the trade chain from collector to consumer. However, there is limited information on mortality rates of reptiles across trade chains, particularly amongst final consumers in the home. We investigated mortality rates of reptiles amongst consumers using a specialised technique for asking sensitive questions, additive Randomised Response Technique (aRRT), as well as direct questioning (DQ). Overall, 3.6% of snakes, chelonians and lizards died within one year of acquisition. Boas and pythons had the lowest reported mortality rates of 1.9% and chameleons had the highest at 28.2%. More than 97% of snakes, 87% of lizards and 69% of chelonians acquired by respondents over five years were reported to be captive bred and results suggest that mortality rates may be lowest for captive bred individuals. Estimates of mortality from aRRT and DQ did not differ significantly which is in line with our findings that respondents did not find questions about reptile mortality to be sensitive. This research suggests that captive reptile mortality in the home is rather low, and identifies those taxa where further effort could be made to reduce mortality rates.     

Prevalence of Pulmonary Hypertension in the General Population: The Rotterdam Study

Background

Pulmonary hypertension is characterized by increased pulmonary artery pressure and carries an increased mortality. Population-based studies into pulmonary hypertension are scarce and little is known about its prevalence in the general population. We aimed to describe the distribution of echocardiographically-assessed pulmonary artery systolic pressure (ePASP) in the general population, to estimate the prevalence of pulmonary hypertension, and to identify associated factors.

Methods

Participants (n = 3381, mean age 76.4 years, 59% women) from the Rotterdam Study, a population-based cohort, underwent echocardiography. Echocardiographic pulmonary hypertension was defined as ePASP>40 mmHg.

Results

Mean ePASP was 26.3 mmHg (SD 7.0). Prevalence of echocardiographic pulmonary hypertension was 2.6% (95%CI: 2.0; 3.2). Prevalence was higher in older participants compared to younger ones (8.3% in those over 85 years versus 0.8% in those between 65 and 70), and in those with underlying disorders versus those without (5.9% in subjects with COPD versus 2.3%; 9.2% in those with left ventricular systolic dysfunction versus 2.3%; 23.1% in stages 3 or 4 left ventricular diastolic dysfunction versus 1.9% in normal or stage 1). Factors independently associated with higher ePASP were older age, higher BMI, left ventricular diastolic dysfunction, COPD and systemic hypertension.

Conclusion

In this large population-based study, we show that pulmonary hypertension as measured by echocardiography has a low prevalence in the overall general population in the Netherlands, but estimates may be higher in specific subgroups, especially in those with underlying diseases. Increased pulmonary arterial pressure is likely to gain importance in the near future due to population aging and the accompanying prevalences of underlying disorders.     

How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

The Z deficiency in α1-antitrypsin (A1ATD) is an under-recognized condition. Alpha1-antitrypsin (A1AT) is the main protein in the α1-globulin fraction of serum protein electrophoresis (SPE); however, evaluation of the α1-globulin protein fraction has received very little attention. Serum Z-type A1AT manifests in polymeric forms, but their interference with quantitative immunoassays has not been reported. Here, 214 894 samples were evaluated by SPE at the G. Fracastoro Hospital of Verona, Italy. Patients with an A1AT level ≤ 0.92 g/L were recalled to complete A1ATD diagnosis. In parallel, to qualitatively and quantitatively characterize A1AT, sera samples from 10 PiZZ and 10 PiMM subjects obtained at the National Institute of Tuberculosis and Lung Diseases in Warsaw, Poland, were subjected to non-denaturing 7.5% PAGE and 7.5% SDS-PAGE followed by Western blot. Moreover, purified A1AT was heated at 60°C and analyzed by a non-denaturing PAGE and 4–15% gradient SDS-PAGE followed by Western blot as well as by isolelectrofocusing and nephelometry. A total of 966 samples manifested percentages ≤ 2.8 or a double band in the alpha1-zone. According to the nephelometry data, 23 samples were classified as severe (A1AT ≤ 0.49 g/L) and 462 as intermediate (A1AT >0.49≤ 1.0 g/L) A1ATD. Twenty subjects agreed to complete the diagnosis and an additional 21 subjects agreed to family screening. We detected 9 cases with severe and 26 with intermediate A1ATD. Parallel experiments revealed that polymerization of M-type A1AT, when measured by nephelometry or isolelectrofocusing, yields inaccurate results, leading to the erroneous impression that it was Z type and not M-type A1AT. We illustrate the need for confirmation of Z A1AT values by “state of the art” method. Clinicians should consider a more in-depth investigation of A1ATD in patients when they exhibit serum polymers and low α1-globulin protein levels by SPE.     

Serum Magnesium and Sudden Death in European Hemodialysis Patients

Despite suggestions that higher serum magnesium (Mg) levels are associated with improved outcome, the association with mortality in European hemodialysis (HD) patients has only scarcely been investigated. Furthermore, data on the association between serum Mg and sudden death in this patient group is limited. Therefore, we evaluated Mg in a post-hoc analysis using pooled data from the CONvective TRAnsport STudy (CONTRAST, NCT00205556), a randomized controlled trial (RCT) evaluating the survival risk in dialysis patients on hemodiafiltration (HDF) compared to HD with a mean follow-up of 3.1 years. Serum Mg was measured at baseline and 6, 12, 24 and 36 months thereafter. Cox proportional hazards models, adjusted for confounders using inverse probability weighting, were used to estimate hazard ratios (HRs) of baseline serum Mg on all-cause mortality, cardiovascular mortality, non-cardiovascular mortality and sudden death. A generalized linear mixed model was used to investigate Mg levels over time. Out of 714 randomized patients, a representative subset of 365 (51%) were analyzed in the present study. For every increase in baseline serum Mg of 0.1 mmol/L, the HR for all-cause mortality was 0.85 (95% CI 0.77–94), the HR for cardiovascular mortality 0.73 (95% CI 0.62–0.85) and for sudden death 0.76 (95% CI 0.62–0.93). These findings did not alter after extensive correction for potential confounders, including treatment modality. Importantly, no interaction was found between serum phosphate and serum Mg. Baseline serum Mg was not related to non-cardiovascular mortality. Mg decreased slightly but statistically significant over time (Δ -0.011 mmol/L/year, 95% CI -0.017 to -0.009, p = 0.03). In short, serum Mg has a strong, independent association with all-cause mortality, cardiovascular mortality and sudden death in European HD patients. Serum Mg levels decrease slightly over time.