Decreased ANT content in Zucker fatty rats: relevance for altered hepatic mitochondrial bioenergetics in steatosis

Mitochondria from Zucker fatty (ZF) rats (a model for fatty liver disease) showed a delay in the repolarization after a phosphorylative cycle and a decrease on state 3 respiration, suggesting alterations at the phosphorylative system level. The ATPase activity showed no differences between control and ZF rats, implying alterations in other components of the phosphorylative system. A pronounced depletion in the content of the adenine nucleotide translocator (ANT) was observed by Western blotting, while no alterations were found in the mitochondrial voltage-dependent anion channel content. These data suggest that hepatic accumulation of fat impairs mitochondrial function, reflecting the loss of oxidative phosphorylation capacity caused by a decrease in the ANT content.     

The physiology of membrane transport and endomembrane-based signalling

Some of the important open questions concerning the physiology of the secretory pathway relate to its homeostasis. Secretion involves a number of separate compartments for which their transport activities should be precisely cross-coordinated to avoid gross imbalances in the trafficking system. Moreover, the membrane fluxes across these compartments should be able to adapt to environmental 'requests' and to respond to extracellular signals. How is this regulation effected? Here, we consider evidence that endomembrane-based signalling cascades that are similar in organization to those used at the plasma membrane coordinate membrane traffic. If this is the case, this would also represent a model for a more general inter-organelle signalling network for functionally interconnecting different intracellular activities, a necessity for the maintenance of cellular homeostasis and to express harmonic global cellular responses.     

Overexpression of Flavodoxin in Bacteroids Induces Changes in Antioxidant Metabolism Leading to Delayed Senescence and Starch Accumulation in Alfalfa Root Nodules

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.Symbiotic nodules have a limited functional life that varies among different legume species. Nodule senescence is the sequence of structural, molecular, biochemical, and physiological events taking place in the process that a mature and functional nodule undergoes leading to the loss of the nitrogen-fixing activity and culminating in cell death of symbiotic tissue (Swaraj and Bishnoi, 1996; Puppo et al., 2005; Van de Velde et al., 2006).Various models have been proposed to explain the mechanisms that trigger the process of natural or stress-induced nodule senescence. However, it is generally accepted that a senescence-inducing signal from the plant causes a decrease in antioxidant levels and thus an increase in reactive oxygen species (ROS) up to a point of no return. Numerous studies have shown that ROS and antioxidant systems are involved in natural (Lucas et al., 1998; Evans et al., 1999; Hernández-Jiménez et al., 2002; Puppo et al., 2005) as well as induced (Dalton et al., 1993; Becana et al., 2000; Hernández-Jiménez et al., 2002; Matamoros et al., 2003) nodule senescence. Nitrogen fixation is very sensitive to ROS, and nitrogenase activity drastically decreases during nodule senescence (Dalton et al., 1986).Antioxidant systems that protect cells from oxidative damage have been described in symbiotic nodules (Dalton et al., 1986, 1993; Evans et al., 1999; Becana et al., 2000; Matamoros et al., 2003; Puppo et al., 2005). These include the enzymes superoxide dismutase (SOD), catalase, and peroxidase. Another enzymatic system associated with ROS detoxification is the ascorbate-glutathione pathway, which includes ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR; Dalton et al., 1986, 1992; Noctor and Foyer 1998; Becana et al., 2000). Ascorbate and reduced glutathione (GSH) in this pathway can also scavenge superoxide and hydrogen peroxide.During nodule senescence, several ultrastructural alterations in the nodule tissues and cells have been observed (Lucas et al., 1998; Hernández-Jiménez et al., 2002; Puppo et al., 2005, and refs. therein; Van de Velde et al., 2006). Cytosol becomes electron dense, altered vesicles proliferate, and eventually the cytosol undergoes lysis. The number of peroxisomes increases, mitochondria form complex elongated structures, and symbiosomes change in size and shape and fuse during natural and induced senescence of nodules (Hernández-Jiménez et al., 2002). Damage of the symbiosome membrane is also detected (Puppo et al., 2005; Van de Velde et al., 2006).A strategy of delayed nodule senescence could lead to increased nitrogen fixation and legume productivity. Delayed nodule senescence together with enhanced sustainability under field conditions are among the key aims of legume improvement programs (Puppo et al., 2005). An interesting approach proposed to achieve delayed senescence is to induce nodulation in legumes using rhizobial strains with modified redox capacity (Zahran, 2001).The protein flavodoxin contains a FMN group acting as a redox center transferring electrons at low potentials (Pueyo et al., 1991; Pueyo and Gómez-Moreno, 1991). The FMN cofactor of flavodoxin can exist in three different redox states: oxidized, one-electron-reduced semiquinone, and two-electron-reduced hydroquinone. This property confers high versatility to flavodoxins in electron transport systems (Simondsen and Tollin, 1980; McIver et al., 1998). To date, flavodoxin has not been described in plants, as flavodoxin-encoding genes were lost during the transition of algae to plants (Zurbriggen et al., 2007) and, consequently, no homologs have been identified in the sequenced genome of Arabidopsis (Arabidopsis thaliana; Arabidopsis Genome Initiative, 2000). Flavodoxin is present as a constitutive or inducible protein in different microorganisms (Klugkist et al., 1986). In the nitrogen-fixing cyanobacterium Anabaena variabilis PCC 7119, flavodoxin is expressed under conditions of limited iron availability, replacing ferredoxin in the photosynthetic electron transport from PSI to NADP⁺ and in nitrogenase reduction (Sandmann et al., 1990). Reversible electron transfer from flavodoxin to NADP⁺ is catalyzed by ferredoxin NADP⁺ reductase in different pathways of oxidative metabolism (Arakaki et al., 1997). In its reduced state, flavodoxin might be able to react with ROS and revert to its original redox state in the presence of an appropriate electron source. This could probably occur without the associated molecular damage that metallic complexes in catalases or SODs suffer (Keyer et al., 1995). The presence of flavodoxin has not been documented to date in the symbiotic bacterium Sinorhizobium meliloti. In Escherichia coli, however, flavodoxin induction is linked to the oxidative stress-responsive regulon soxRS (Zheng et al., 1999). It has been suggested that flavodoxin and ferredoxin (flavodoxin) NADP⁺ reductase might be induced and have a role in reestablishing the cell redox balance under oxidative stress conditions (Liochev et al., 1994). The properties of flavodoxin suggest that its presence in the cell may have a facilitating effect on ROS detoxification. In fact, an increase in the amount of flavodoxin has been observed in some bacterial species subjected to oxidative stress (Zheng et al., 1999; Yousef et al., 2003; Singh et al., 2004), and transgenic tobacco (Nicotiana tabacum) plants expressing flavodoxin in chloroplasts show enhanced tolerance to a broad range of stresses related to oxidative damage (Tognetti et al., 2006, 2007a, 2007b).In this work, Sinorhizobium meliloti was transformed with the A. variabilis flavodoxin gene and used to nodulate alfalfa (Medicago sativa) plants. The effects of flavodoxin expression on nodulation dynamics, on nodule development and senescence processes, and on nitrogen-fixing activity were analyzed. Mechanistic insights suggesting putative roles for flavodoxin in protection from ROS and the induced delay of nodule senescence are likewise discussed.     

Sub-lethal effects of fenbutatin oxide on prey location by the predatory mite <Emphasis Type="Italic">Iphiseiodes zuluagai</Emphasis> (Acari: Phytoseiidae)

We used a Y-tube olfactometer to assess the sub-lethal effects of the acaricide fenbutatin oxide on the olfactory response of the predatory mite Iphiseiodes zuluagai towards odours from: (1) air or undamaged coffee plants; (2) undamaged or red spider mite Oligonychus ilicis-infested coffee plants; (3) undamaged or false spider mite Brevipalpus phoenicis-infested coffee plants. Predatory mite adult females were exposed to residues of fenbutatin oxide or distilled water on leaf discs during a period of 72 h prior experiments. When exposed to distilled water (control treatments), predatory mites significantly preferred undamaged plants over air, O. ilicis-infested plants over undamaged plants, and they did not prefer B. phoenicis-infested plants over undamaged plants. However, predatory mites that had been exposed to residues of fenbutatin oxide were neither attracted towards undamaged plants nor to O. ilicis-infested plants. Thus, fenbutatin oxide affected negatively the olfactory response of I. zuluagai. We conclude that sub-lethal-effect studies should be considered in pesticide selectivity programs since the ability of predatory mites to locate their prey may be negatively affected by non-lethal concentrations of pesticides.     

From the laboratory to the field: contrasting effects of multi-trophic interactions and agroforestry management on coffee pest densities

Only few factors influencing pest populations can be studied in the laboratory, but many population‐driving factors interact in the field. Therefore, complementary laboratory and field approaches are required for reliable predictions of real‐world patterns and processes. Laboratory and field experiments with the red spider mite, Oligonychus ilicis McGregor (Acari: Tetranychidae), and the coffee leaf miner, Leucoptera coffeella Guérin‐Méneville (Lepidoptera: Lyonetiidae), on coffee plants, Coffea arabica L. (Rubiaceae), were combined to study the relative importance of biotic interactions, including resource preferences and natural‐enemy impact, and habitat factors, such as agroforestry type and management intensity, on coffee pest densities. In the laboratory, leaf discs cut from undamaged coffee plants were significantly preferred by red spider mites over those from plants infested with conspecific mites, leaf rust pathogens [Hemileia vastatrix Berkeley & Broome (Uredinales)], or coffee leaf miners, resulting in higher reproductive success. Similarly, undamaged plants were preferred by coffee leaf miners over red spider mite‐infested plants. However, in the field, red spider mite densities were positively correlated with coffee leaf miner and leaf rust densities, thereby contrasting with laboratory predictions. Hence, our study suggests that the importance of resource preferences and fitness expected based on laboratory experiments was suppressed by environmental conditions in the field, though other unassessed biotic interactions could also have played a role. Furthermore, intensified agroforestry was characterized by higher red spider mite densities, whereas densities of its major natural enemy, the predatory mite Amblyseius herbicolus Chant (Acari: Phytoseiidae), were not related to agroforestry management. Densities of coffee leaf miner and its main natural enemy, a eulophid parasitoid (Hymenoptera), were not affected by management practices. In conclusion, patterns found in the laboratory did not hold for the field, emphasizing the difficulties of extrapolating small‐scale experiments to larger spatial scales and the need to combine both approaches.     

Agroforestry management affects coffee pests contingent on season and developmental stage

1 Management of vegetational diversity in agroecosystems is a potentially regulating factor of pest population dynamics and may affect developmental stages in different ways.
2 We investigated the population dynamics of red spider mites, coffee leaf miners, and coffee berry borers in three management types of coffee agroforests: increasing plant diversity from a few shade tree species (simple-shade agroforests), intermediate-shade tree species (complex-shade agroforests) to high-shade tree species (abandoned coffee agroforests) in Ecuador. Furthermore, we studied how changes in agroforestry management affect population stage structure of each coffee pest.
3 Our results show that agroforestry management affected seasonal patterns of coffee pests in that higher densities of red spider mites were observed from August to December, coffee leaf miners from December to February, and coffee berry borers from May to July. Moreover, specific developmental stages of red spider mites, coffee leaf miners, and coffee berry borers differed in their responses to agroforestry management. During all stages, red spider mite reached higher densities in simple-shade agroforests compared with complex-shade and abandoned agroforests. Meanwhile, coffee leaf miner densities decreased from simple-shade to complex-shade and abandoned agroforests, but only for larvae, not pupae. Similarly, only coffee berry borer adults (but not eggs, larvae and pupae) demonstrated a response to agroforestry management. Environmental variables characterizing each agroforestry type proved to be important drivers of pest population densities in the field.
4 We emphasize the importance of considering seasonal differences and population structure while investigating arthropod responses to different habitat types because responses change with time and developmental stages.     

Transgenic chloroplasts are efficient sites for high‐yield production of the vaccinia virus envelope protein A27L in plant cells†

Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost‐effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein‐based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500‐fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast‐synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines.     

<Emphasis Type="Italic">Calycogonium bairdianum</Emphasis> (Melastomataceae: Miconieae), a new species from the Cordillera Central,Dominican Republic

Calycogonium bairdianum, a new species, is here described from the Cordillera Central of the Dominican Republic. It is compared to C. impressum, to which it is likely related. Although Calycogonium is not monophyletic, C. bairdianum and C. impressum may be related to other species in the genus that exhibit acarodomatia formed by hairs at the two major vein junctions on the leaf abaxial surface, a likely synapomorphy. Calycogonium bairdianum is distinguished from C. impressum by its relatively glabrous (vs. pubescent) and larger leaves (i.e., usually [2.9–]4.5–8.1 cm vs. 2.1–3.7 cm long) with veins that are plane to only slightly impressed adaxially (vs. more strongly impressed).     

Multifactorial approach and superior treatment efficacy in renal patients with the aid of nurse practitioners. Design of The MASTERPLAN Study [ISRCTN73187232]

Background

Patients with chronic kidney disease (CKD) are at a greatly increased risk of developing cardiovascular disease. Recently developed guidelines address multiple risk factors and life-style interventions. However, in current practice few patients reach their targets. A multifactorial approach with the aid of nurse practitioners was effective in achieving treatment goals and reducing vascular events in patients with diabetes mellitus and in patients with heart failure. We propose that this also holds for the CKD population.

Design

MASTERPLAN is a multicenter randomized controlled clinical trial designed to evaluate whether a multifactorial approach with the aid of nurse-practicioners reduces cardiovascular risk in patients with CKD. Approximately 800 patients with a creatinine clearance (estimated by Cockcroft-Gault) between 20 to 70 ml/min, will be included. To all patients the same set of guidelines will be applied and specific cardioprotective medication will be prescribed. In the intervention group the nurse practitioner will provide lifestyle advice and actively address treatment goals. Follow-up will be five years. Primary endpoint is the composite of myocardial infarction, stroke and cardiovascular mortality. Secondary endpoints are cardiovascular morbidity, overall mortality, decline of renal function, change in markers of vascular damage and change in quality of life. Enrollment has started in April 2004 and the study is on track with 700 patients included on October 15th, 2005. This article describes the design of the MASTERPLAN study.     

Guidelines for indications for coronary revascularization in The Netherlands

In 1996 the Minister of Public Health, Welfare and Sports in The Netherlands published a 'Planning Decree Special Interventions in the Heart'. She requested from the professional organizations guidelines for the indications for interventions in the heart. A working group was formed with representatives from the Dutch professional organizations for cardiology and thoracic surgery, to address this issue for patients with coronary artery disease. The working group confirmed the need to discuss all patients who are considered for either elective or emergency revascularization during a multidisciplinary consultation in (or with) one of the specialized Dutch hospitals. During this meeting of the 'heart team', at least one interventional cardiologist and one thoracic surgeon should be present. There are three possible outcomes of the heart team's consultations for each patient: drug therapy only ('conservative management'), coronary surgery or catheter intervention. For each case, the team should indicate the expected benefit, the risk of the intervention, the urgency and the estimated waiting time. The guidelines presented in this paper address these issues for three patient categories: stable angina pectoris, unstable angina pectoris and acute myocardial infarction.