Mechanism for increased leaf growth in elevated CO2

The effect of exposure to elevated CO₂ on the processes of leafcell production and leaf cell expansion was studied using primaryleaves of Phaseolus vulgaris L. Cell division and expansionwere separated temporally by exposing seedlings to dim red lightfor 10 d (when leaf cell division was completed) followed byexposure to bright white light for 14 d (when leaf growth wasentirely dependent on cell expansion). When plants were exposedto elevated CO₂ during the phase of cell expansion, epidermalcell size and leaf area development were stimulated. Three piecesof evidence suggest that this occurred as a result of increasedcell wall loosening and extensibility, (i) cell wall extensibility(WEx, measured as tensiometric extension using an Instron) wassignificantly increased, (ii) cell wall yield turgor (V, MPa)was reduced and (iii) xyloglucan endotransglycosylase (XET)enzyme activity was significantly increased. When plants wereexposed to elevated CO₂ during the phase of cell division, thenumber of epidermal cells was increased whilst final cell sizewas significantly reduced and this was associated with reducedfinal leaf area, WEx and XET activity. When plants were exposedto elevated CO₂ during both phases of cell division and expansion,leaf area development was not affected. For this treatment,however, the number of epidermal cells was increased, but cellexpansion was inhibited, despite exposure to elevated CO₂ duringthe expansion phase. Assessments were also made of the spatialpatterns of WEx across the expanding leaf lamina and the datasuggest that exposure to elevated CO₂ during the phase of leafexpansion may lead to enhanced extensibility particularly atbasal leaf margins which may result in altered leaf shape. The data show that both cell production and expansion were stimulatedby elevated CO₂, but that leaf growth was only enhanced by exposureto elevated CO₂ in the cell expansion phase of leaf development.Increased leaf cell expansion is, therefore, an important mechanismfor enhanced leaf growth in elevated CO₂, whilst the importanceof increased leaf cell production in elevated CO₂ remains tobe elucidated. Key words: Phaseolus vulgaris L., dwarf beans, elevated CO², biophysics of cell expansion, xyloglucan endotransglycosylase, XET, water relations     

Molecular phylogeny and phylogeography of ricefishes (Teleostei: Adrianichthyidae: Oryzias) in Sri Lanka

Ricefishes of the genus Oryzias occur commonly in the fresh and brackish waters in coastal lowlands ranging from India across Southeast Asia and on to Japan. Among the three species of Oryzias recorded from peninsular India, two widespread species, O. carnaticus and O. dancena, have previously been reported from Sri Lanka based on museum specimens derived from a few scattered localities. However, members of the genus are widespread in the coastal lowlands of Sri Lanka, a continental island separated from India by the shallow Palk Strait. Although recent molecular phylogenies of Adrianichthyidae represent near‐complete taxon representation, they lack samples from Sri Lanka. Here, based on sampling at 13 locations representative of the entire geographic and climatic regions of the island''s coastal lowlands, we investigate for the first time the molecular phylogenetic relationships and phylogeography of Sri Lankan Oryzias based on one nuclear and two mitochondrial markers. Sri Lankan Oryzias comprise two distinct non‐sister lineages within the javanicus species group. One of these is represented by samples exclusively from the northern parts of the island; it is recognized as O. dancena. This lineage is recovered as the sister group to the remaining species in the javanicus group. The second lineage represents a species that is widespread across the island''s coastal lowlands. It is recovered as the sister group of O. javanicus and is identified as O. cf. carnaticus. Ancestral‐range estimates suggest two independent colonizations of Indian subcontinent and Sri Lanka by widespread ancestral species of Oryzias during two discrete temporal windows: late Miocene and Plio‐Pleistocene. No phylogeographic structure is apparent in Sri Lankan Oryzias, suggesting that there are no strong barriers to gene flow and dispersal along the coastal floodplains, as is the case also for other generalist freshwater fishes in the island.     

Effect of Drought Stress on Certain Morphological and Physiological Characteristics of a Resistant and a Sensitive Canola Cultivar

Water stress is one of the main abiotic factors that reduces plant growth, mainly due to high evaporative demand and low water availability. In order to evaluate the effects of drought stress on certain morphological and physiological characteristics of two canola cultivars, we conducted a factorial experiment based on a completely randomized design. The findings show that drought stress exacerbations result in the plant's response to stress due to increased canola resistance caused by changes in plant pigments, proline, catalase, ascorbate peroxidase, peroxidase, superoxide dismutase and malondialdehyde, glucose, galactose, rhamnose and xylose. These in turn ultimately influence the morphological characteristics of canola. Drought stress reduces the concentration of carotenoids, chlorophyll a, chlorophyll b, total chlorophylls; however, glucose, galactose, rhamnose, xylose, proline, catalase, ascorbate peroxidase, peroxidase, superoxide dismutase, malondialdehyde (in leaves and roots) and the chlorophyll a and b ratios were increased. Reduction of plant height, stem height, root length, fresh and dry weight of canola treated with 300 g/l PEG compared to non‐treatment were 0.264, 0.236, 0.394, 0.183 and 0.395, respectively. From the two canola cultivars, the morphological characteristics of the NIMA increased compared to the Ks7 cultivar. Interaction effects of cultivar and drought stress showed that NIMA cultivar without treatment had the highest number of morphological characteristics such as carotenoid concentration, chlorophyll a, chlorophyll b, total chlorophylls a and b, whereas the cultivar with 300 g/l PEG (drought stress) had the highest amount of proline, malondialdehyde, soluble sugars and enzymes in leaves and roots. Increasing activity of oxidative enzymes and soluble sugars in canola under drought stress could be a sign of their relative tolerance to drought stress.     

A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.     

Caveolin-1 null (-/-) mice show dramatic reductions in life span

Caveolae are 50-100 nm flask-shaped invaginations of the plasma membrane found in most cell types. Caveolin-1 is the principal protein component of caveolae membranes in nonmuscle cells. The recent development of Cav-1-deficient mice has allowed investigators to study the in vivo functional role of caveolae in the context of a whole animal model, as these mice lack morphologically detectable caveolae membrane domains. Surprisingly, Cav-1 null mice are both viable and fertile. However, it remains unknown whether loss of caveolin-1 significantly affects the overall life span of these animals. To quantitatively determine whether loss of Cav-1 gene expression confers any survival disadvantages with increasing age, we generated a large cohort of mice (n = 180), consisting of Cav-1 wild-type (+/+) (n = 53), Cav-1 heterozygous (+/-) (n = 70), and Cav-1 knockout (-/-) (n = 57) animals, and monitored their long-term survival over a 2 year period. Here, we show that Cav-1 null (-/-) mice exhibit an approximately 50% reduction in life span, with major declines in viability occurring between 27 and 65 weeks of age. However, Cav-1 heterozygous (+/-) mice did not show any changes in long-term survival, indicating that loss of both Cav-1 alleles is required to mediate a reduction in life span. Mechanistically, these dramatic reductions in life span appear to be secondary to a combination of pulmonary fibrosis, pulmonary hypertension, and cardiac hypertrophy in Cav-1 null mice. Taken together, our results provide the first demonstration that loss of Cav-1 gene expression and caveolae organelles dramatically affects the long-term survival of an organism. In addition, aged Cav-1 null mice may provide a new animal model to study the pathogenesis and treatment of progressive hypertrophic cardiomyopathy and sudden cardiac death syndrome.     

Significance of inducible nitric oxide synthase in acute myocarditis caused by Trypanosoma cruzi (Tulahuen strain)

Chagas' disease, caused by Trypanosoma cruzi, is associated with myocarditis and expression of myocardial cytokines and inducible nitric oxide synthase (NOS2). To assess the functional significance of NOS2 in murine Chagas' disease, we infected NOS2 knockout (NOS2(-/-)) and C57BL/6x129sv (wild type) mice with the Tulahuen strain of T. cruzi. Serial transthoracic echocardiography was performed to assess the progression of left and right ventricular dysfunction in infected mice. Uninfected wild type and NOS2(-/-) mice served as controls. At day 10 post-infection (p.i.), infected wild type mice had larger left ventricular end-diastolic diameter (2.52+/-0.14-vs-2.11+/-0.06 mm, P<0.02) and right ventricle (0.6+/-0.2-vs-0 visual grade, P<0.02) as compared with uninfected wild type mice. At day 19 p.i., compared with uninfected controls, infected wild type mice had larger left ventricular end-diastolic diameter (3.30+/-0.29-vs-2.11+/-0.07 mm), left ventricular end-systolic diameter (1.86+/-0.29-vs-0.88+/-0.05 mm), right ventricle (1.6+/-0.2-vs-0 visual grade), lower heart rate (413+/-27-vs-557+/-25 beats per min), left ventricular relative wall thickness (0.44+/-0.05-vs-0.64+/-0.03) and fractional shortening (45+/-4-vs-57+/-2%) [P<0.05 for all]. In contrast, no differences in left ventricular end-diastolic diameter or fractional shortening were noted among infected and uninfected NOS2(-/-) mice at day 19 p.i. Compared with uninfected controls, infected NOS2(-/-) mice had significantly lower heart rate (272+/-23-vs-512+/-31 beats per min, P<0.01) and larger right ventricle (0.6+/-0.2-vs-0, P<0.05 visual grade). The magnitude of right ventricular dilation in NOS2(-/-) mice was less than that observed in infected wild type mice. At necropsy, the heart weight was greater (129+/-16-vs-109+/-7 mg, P=0.02) and myocardial inflammation more severe in infected wild type compared with infected NOS2(-/-) mice. Myocardial interleukin (IL)-1beta, IL-6, tumour necrosis factor-alpha, and interferon-gamma were induced in all infected mice. These data indicate that nitric oxide derived from NOS2 plays an important role in the development and progression of ventricular dilation and systolic dysfunction in acute murine chagasic myocarditis caused by infection with the Tulahuen strain.     

Cardioprotective effects of phosphoramidon on myocardial structure and function in murine Chagas' disease

Chagas' disease is an important cause of cardiomyopathy. Endothelin-1, a vasoactive peptide has been implicated in the pathogenesis of chagasic cardiomyopathy. C57BL/6 x 129sv and CD1 mice were thus, infected with trypomastigotes of Trypanosoma cruzi (Brazil strain) and these infected mice were compared with infected mice treated with phosphoramidon. This compound inhibits endothelin-converting enzyme and neutral endopeptidases and does not affect the growth of the parasite in culture. Phosphoramidon was given in a dose of 10mg/kg for the initial 15 days post-infection None of the C57Bl/6 x 129sv mice died as a result of infection. However, there was marked myocardial inflammation and fibrosis in infected, untreated mice. The hearts of the infected, phosphoramidon-treated mice showed significantly less pathology. Cardiac magnetic resonance imaging of infected mice revealed right ventricular dilation that was less severe in those treated with phosphoramidon. Phosphoramidon-treated CD1 mice survived the acute infection. Transthoracic echocardiography demonstrated left ventricular dilation and reduced percent fractional shortening and relative wall thickness. These alterations were also attenuated as a result of phosphoramidon treatment. These data suggest that endothelin-1 contributes to the pathogenesis of chagasic cardiomyopathy and interventions that inhibit the synthesis of endothelin-1 and/or neutral endopeptidase might have a protective effect on myocardial structure and function in murine Chagas' disease.     

Manganese administration induces the increased production of dopamine sulfate and depletion of dopamine in Sprague-Dawley rats

Sprague-Dawley rats were used as an experimental model for investigating the effects of manganese poisoning on the serum levels of unsulfated and sulfated forms of dopamine and its biosynthetic precursors, L-Dopa and L-p-tyrosine. Groups of rats were treated daily with Mn(2+) (20 mg or 40 mg; in the form of MnSO(4)) or Na(+) (20 mg; in the form of Na(2)SO(4)). High performance liquid chromatography (HPLC) analysis of the serum samples taken after a 50-day experimental period revealed that the serum level of dopamine sulfate increased by more than 10 times compared with untreated control rats or rats treated with sodium sulfate. In contrast, there was a dramatic decrease (by as much as 4.8 times) in the serum level of unsulfated dopamine in manganese-treated rats. The serum levels of L-Dopa sulfate and L-p-tyrosine sulfate were also markedly elevated, although not as much as those of dopamine sulfate. Meanwhile, the serum levels of unsulfated L-Dopa and L-p-tyrosine showed no dramatic changes. Atomic absorption spectrophotometric analysis revealed in general an accumulation of manganese in the four organ samples taken from manganese-treated rats. Compared with liver, heart, and kidney, the highest degree of manganese accumulation in manganese-treated rats appeared to be in brain. These results together suggested a role for manganese in stimulating the dopamine-sulfating sulfotransferases in brain, thereby leading to the depletion of dopamine in vivo.     

Developing and applying a benthic index of estuarine condition for the Virginian Biogeographic Province

A benthic index of estuarine condition was constructed for the Virginian Biogeographic Province (from Cape Cod, Massachusetts, to the mouth of Chesapeake Bay, Virginia) with data collected during summers of 1990 through 1993 by the US EPA’s Environmental Monitoring and Assessment Program (EMAP). Forty-eight metrics, based on attributes of the macrobenthos, were considered for the index, including measures of biodiversity, community condition, individual health, functional organization, and taxonomic composition. Salinity was correlated significantly with some of the metrics. Therefore, some metrics were normalized for salinity. The data used to develop the index (the calibration data) included equal numbers of reference and degraded sites, distributed equally across three salinity zones (<5, 5–18, >18‰). An independent set of data was used for validation. Linear discriminant analysis identified combinations of metrics that could best discriminate reference from degraded sites. The targets for correct classification were 90% of the sites for the calibration data and 80% for the validation data. Six combinations of metrics were identified. The final index was based on the ecological interpretation and relevance of the individual metrics and the ability to meet the calibration and validation targets. The final index consisted of three metrics: a positive contribution from salinity-normalized Gleason’s D (a biodiversity metric), and negative contributions from two taxonomic composition metrics, abundances of spionid polychaetes and of salinity-normalized tubificid oligochaetes. The index correctly classified 87% of reference and 90% of degraded sites in the calibration data and 88% of reference and 81% of degraded sites in the validation data. The index correctly classified sites over the full range of salinity (tidal-fresh to marine waters) and across grain sizes (silt–clay to sand).     

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.