Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

CO(2) Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays

Conditions for optimal CO₂ fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO₂ fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO₂ assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V_max for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C₃ plants.     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.     

Extra‐pair offspring are less heterozygous than within‐pair offspring in American redstarts Setophaga ruticilla

The vast majority of bird species are socially monogamous; however, extra‐pair paternity is nearly ubiquitous and a number of theories have been proposed to explain the prevalence of this mixed mating strategy. Here, we test the genetic compatibility hypothesis – the idea that females seek extra‐pair copulations with males whose genes are more compatible with her own. For this study, we examined eight years of paternity data (2004–2011) from a Nearctic‐Neotropical migratory bird, the American redstart Setophaga ruticilla, breeding in southeastern Ontario, Canada. We predicted that females paired with genetically similar males (higher relatedness) would be more likely to produce extra‐pair offspring and that extra‐pair offspring would have higher levels of heterozygosity than within‐pair offspring. Alternatively, because this population experiences high levels of immigration, females may produce extra‐pair offspring with more genetically similar males because of the potential for outbreeding depression. Using five highly variable microsatellite markers, we examined patterns of relatedness among social pairs as well as measures of offspring heterozygosity. In contrast to our predictions, we found no difference in relatedness between social pairs where the females produced extra‐pair offspring and social pairs where the females produced only within‐pair offspring. However, extra‐pair offspring were significantly less heterozygous than within‐pair offspring. Together, these findings suggest that females a) are not engaging in extra‐pair fertilizations based on relatedness to their social mate and b) appear to be mating with extra‐pair males that are more genetically similar to themselves. We suggest there may be benefits for females to mate with genetically similar extra‐pair males in highly outbred populations with high rates of immigration, such as for maintaining co‐adapted gene complexes or genes coding for local adaptations.     

Immuno-epidemiology of chronic bacterial and helminth co-infections: observations from the field and evidence from the laboratory

Co-infections can alter the host immune responses and modify the intensity and dynamics of concurrent parasitic species. The extent of this effect depends on the properties of the system and the mechanisms of host-parasite and parasite-parasite interactions. We examined the immuno-epidemiology of a chronic co-infection to reveal the immune mediated relationships between two parasites colonising independent organs, and the within-host molecular processes influencing the dynamics of infection at the host population level. The respiratory bacterium, Bordetella bronchiseptica, and the gastrointestinal helminth, Graphidium strigosum, were studied in the European rabbit (Oryctolagus cuniculus), using long-term field data and a laboratory experiment. We found that 65% of the rabbit population was co-infected with the two parasites; prevalence and intensity of co-infection increased with rabbit age and exhibited a strong seasonal pattern with the lowest values recorded during host breeding (from April to July) and the highest in the winter months. Laboratory infections showed no significant immune-mediated effects of the helminth on bacterial intensity in the lower respiratory tract but a higher abundance was observed in the nasal cavity during the chronic phase of the infection, compared with single bacterial infections. In contrast, B. bronchiseptica enhanced helminth intensity and this was consistent throughout the 4-month trial. These patterns were associated with changes in the immune profiles between singly and co-infected individuals for both parasites. This study confirmed the general observation that co-infections alter the host immune responses but also highlighted the often ignored role of bacterial infection in helminth dynamics. Additionally, we showed that G. strigosum had contrasting effects on B. bronchiseptica colonising different parts of the respiratory tract. At the host population level our findings suggest that B. bronchiseptica facilitates G. strigosum infection, and re-infection with G. strigosum assists in maintaining bacterial infection in the upper respiratory tract and thus long-term persistence.     

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.     

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.