Transgenic chickpea seeds expressing high levels of a bean α-amylase inhibitor

We describe a robust and reproducible Agrobacterium-mediated chickpea transformation method based on kanamycin selection, and its use to introduce the bean AI1 gene into a desi type of chickpea. Bean AI1 was specifically expressed in the seeds, accumulated up to 4.2% of seed protein and was processed to low molecular weight polypeptides as occurs in bean seeds. The transgenic protein was active as an inhibitor of porcine -amylase in vitro. Transgenic chickpeas containing -AI1 strongly inhibited the development of Callosobruchus maculatus and C. chinensis (Col. : Bruchidae) in insect bioassays.     

Are primed polymorphonuclear leukocytes contributors to the high heparanase levels in hemodialysis patients?

Patients on chronic hemodialysis (HD) are at high risk for developing atherosclerosis and cardiovascular complications. Heparanase, an endoglycosidase that cleaves heparan sulfate (HS) side chains of proteoglycans, is involved in extracellular matrix degradation and, as such, may be involved in the atherosclerotic lesion progression. We hypothesize that heparanase is elevated in HD patients, partly due to its release from primed circulating polymorphonuclear leukocytes (PMNLs), undergoing degranulation. Priming of PMNLs was assessed by levels of CD11b and the rate of superoxide release. Heparanase mRNA expression in PMNLs was determined by RT-PCR. PMNL and plasma levels of heparanase were determined by immunoblotting, immunofluorescence, and flow cytometry analyses. The levels of soluble HS in plasma were measured by a competition ELISA. This study shows that PMNLs isolated from HD patients have higher mRNA and protein levels of heparanase compared with normal control (NC) subjects and that heparanase levels correlate positively with PMNL priming. Plasma levels of heparanase were higher in HD patients than in NC subjects and were further elevated after the dialysis session. In addition, heparanase expression inversely correlates with plasma HS levels. A pronounced expression of heparanase was found in human atherosclerotic lesions. The increased heparanase activity in the blood of HD patients results at least in part from the degranulation of primed PMNLs and may contribute to the acceleration of the atherosclerotic process. Our findings highlight primed PMNLs as a possible source for the increased heparanase in HD patients, posing heparanase as a new risk factor for cardiovascular complications and atherosclerosis.     

An endo-1,4-β-glucanase expressed at high levels in rapidly expanding tissues

Plant developmental processes involving modifications to cell wall structure, such as cell expansion, organ abscission and fruit ripening, are accompanied by increased enzyme activity and mRNA abundance of endo-1,4--glucanases (EGases). An EGase cDNA clone, Ce14, isolated from tomato (Lycopersicon esculentum) has been shown to be identical to a tomato pistil-predominant EGase cDNA, TPP18. In addition to its previously reported expression during certain stages of early pistil development, Ce14 mRNA was also detected at high levels in the growing zones of etiolated hypocotyls (about 2.5-fold less than in pistils) and in young expanding leaves (about 3.5-fold less than in pistils). The abundance of Ce14 mRNA declined precipitously in older tissues as cells became fully expanded, and was barely detectable in mature vegetative tissues. Ce14 mRNA abundance was also low in abscission zones, and did not increase as abscission progressed. In fruit, Ce14 mRNA was present at low levels during fruit expansion, but was essentially absent during subsequent fruit development and ripening. Treatment of etiolated hypocotyls with ethylene or high concentrations of auxin sufficient to induce rapid lateral cell expansion and hypocotyl swelling also brought about an approximate doubling of Ce14 mRNA abundance, suggesting that Ce14 mRNA accumulation may be promoted directly or indirectly by ethylene. Thus, accumulation of Ce14 mRNA was found to be correlated with rapid cell expansion in pistils, hypocotyls and leaves.     

Construction of a flocculent Saccharomyces cerevisiae strain secreting high levels of Aspergillus niger β-galactosidase

A flocculent Saccharomyces cerevisiae strain secreting Aspergillus niger beta-galactosidase activity was constructed by transforming S. cerevisiae NCYC869-A3 strain with plasmid pVK1.1 harboring the A. niger beta-galactosidase gene, lacA, under the control of the ADH1 promoter and terminator. Compared to other recombinant S. cerevisiae strains, this recombinant yeast has higher levels of extracellular beta-galactosidase activity. In shake-flask cultures, the beta-galactosidase activity detected in the supernatant was 20 times higher than that obtained with previously constructed strains (Domingues et al. 2000a). In bioreactor culture, with cheese-whey permeate as substrate, a yield of 878.0 nkat/gsubstrate was obtained. The recombinant strain is an attractive alternative to other fungal beta-galactosidase production systems as the enzyme is produced in a rather pure form. Moreover, the use of flocculating yeast cells allows for enzyme production with high productivity in continuous fermentation systems with facilitated downstream processing.     

Low 8-oxo-7,8-dihydro-2′-deoxyguanosine levels and influence of genetic background in an Andean population exposed to high levels of arsenic

BackgroundArsenic (As) causes oxidative stress through generation of reactive oxygen species. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), a sensitive marker of oxidative DNA damage, has been associated with As exposure in some studies, but not in others, possibly due to population-specific genetic factors.ObjectivesTo evaluate the association between As and 8-oxodG in urine in a population with a low urinary monomethylated As (%MMA) and high dimethylated As (%DMA), as well as the genetic impact on (a) 8-oxodG concentrations and (b) the association between As and 8-oxodG.Materials and methodsWomen (N = 108) in the Argentinean Andes were interviewed and urine was analyzed for arsenic metabolites (ICPMS) and 8-oxodG (LC–MS/MS). Twenty-seven polymorphisms in genes related to oxidative stress and one in As(+III)methyltransferase (AS3MT) were studied.ResultsMedian concentration of 8-oxodG was 4.7 nmol/L (adjusted for specific weight; range 1.6–13, corresponding to 1.7 μg/g creatinine, range 0.57–4.8) and of total urinary As metabolites (U-As) 290 μg/L (range 94–720; 380 μg/g creatinine, range 140–1100). Concentrations of 8-oxodG were positively associated with %MMA (strongest association, p = 0.013), and weakly associated with U-As (positively) and %DMA (negatively). These associations were strengthened when taking ethnicity into account, possibly reflecting genetic differences in As metabolism and genes regulating oxidative stress and DNA maintenance. A genetic influence on 8-oxodG concentrations was seen for polymorphisms in apurinic/apyrimidinic endonuclease 1 (APEX1), DNA-methyltransferases 1 and 3b (DNMT1, DNMT3B), thioredoxin reductase 1 (TXNRD1) and 2 (TXNRD2) and glutaredoxin (GLRX).ConclusionDespite high As exposure, the concentrations of 8-oxodG in this population were low compared with other As-exposed populations studied. The strongest association was found for %MMA, stressing that some inconsistencies between As and 8-oxodG partly depend on population variations in As metabolism. We found evidence of genetic impact on 8-oxodG concentrations.     

Evaluation of HPFH and δβ-thalassemia mutations in a Brazilian group with high Hb F levels

Fetal hemoglobin (Hb F) is characteristic of the fetal development period. However, in some genetic conditions, such as hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia (δβ-thalassemia), Hb F continues to be produced in adulthood. We evaluated the frequency of two mutations of HPFH, HPFH-1 and HPFH-2 African, and two mutations in δβ-thalassemia, Sicilian and Spanish, in a Brazilian population. Peripheral blood samples were collected from adults from hospitals and blood centers in southeast and northeast Brazil. These individuals were healthy and without complaints of anemia, but had increased Hb F. Samples were submitted to electrophoretic and chromatographic analyses to quantify Hb F values and, subsequently, to molecular analyses to verify the mutations. In the molecular analysis, 16 of the 60 samples showed a heterozygous profile for the HPFH mutations, two for HPFH-1 and 14 for HPFH-2. In the same sample set, three were heterozygous for Spanish δβ-thalassemia and none were heterozygous for Sicilian δβ- thalassemia. The Hb F values in the HPFH-2 heterozygotes differed from those previously reported for this mutation. In this group, the HPFH mutations were more frequent than the δβ-thalassemia mutations. The finding of these mutations in this Brazilian population reflects the mixing process that occurred during its formation.     

Sparse sensillar array on Trioza apicalis (Homoptera, Triozidae) antennae-an adaptation to high stimulus levels?

To investigate the morphological basis for olfactory reception in the carrot psyllid (Trioza apicalis) we used scanning and transmission electron microscopy. Our study reveals a very sparse sensillar setup. We identify and describe several different types of single-walled sensilla likely to have an olfactory function, as well as mechanosensory hairs and intracuticular sensilla. A T. apicalis antenna is about 0.6 mm long and has 10 segments. Apically on the flagellum there are two conspicuous multi-porous single-walled bristles. There are six cuticular cavities on the flagellum; two smaller on the apical flagellomere, and four larger located on the lateral side of the antenna on flagellomeres 2, 4, 6 and 7. Each cavity contains two sensilla and there are three varieties of cavity sensilla. Mechano- and chemosensory hairs appear in low numbers on all segments but the third. Carrot psyllids most likely use olfactory cues to locate their rather strongly smelling host plants, and we argue that the low number of olfactory sensilla found in this insect may accommodate high concentrations of odour stimuli. There is no sexual dimorphism in the sensillar setup. In concordance with this, no sex pheromones have been described in the Psylloidea so far.     

Phenotypic divergence despite high levels of gene flow in Galápagos lava lizards (Microlophus albemarlensis)

The extent of evolutionary divergence of phenotypes between habitats is predominantly the result of the balance of differential natural selection and gene flow. Lava lizards (Microlophus albemarlensis) on the small island of Plaza Sur in the Galápagos archipelago inhabit contrasting habitats: dense vegetation on the western end of the island thins rapidly in a transitional area, before becoming absent on the eastern half. Associated with these habitats are phenotypic differences in traits linked to predator avoidance (increased wariness, sprint speed, and endurance in lizards from the sparsely vegetated habitat). This population provides an opportunity to test the hypothesis that reduced gene flow is necessary for phenotypic differentiation. There was no evidence of any differences among habitats in allele frequencies at six out of seven microsatellite loci examined, nor was there any indication of congruence between patterns of genetic variability and the change in vegetation regime. We infer that gene flow between the habitats on Plaza Sur must be sufficiently high to overcome genetic drift within habitats but that it does not preclude phenotypic differentiation.     

TGF-β1 and high levels of glucose do not increase insulin cell proportions in the avian embryonic pancreas

TGF-β1 is thought to decrease the proportion of embryonic pancreatic β-cells with respect to α-cells, whereas glucose is thought to enhance β-cell proportions in rats. However, chick pancreatic cells may respond in a dissimilar way to glucose. Thus, the effect of TGF-β1 on the proportion of β-cells in embryonic chick dorsal pancreatic buds (DPBs) in vitro was tested with short-term exposure to high levels of glucose. Five-day-old chick DPBs were cultured on growth factor-reduced Matrigel, which contains reduced levels of growth factors including TGF-β1, and a variety of culture media with and without high levels of glucose. TGF-β1 reduced the proportion of β-cells, as expected. A similar decrease in the proportion of β-cells occurred in the presence of high levels of glucose.     

Effect of high levels of dietary vitamin C on the blood responses of matrinxã (Brycon amazonicus)

Juvenile matrinx? (Brycon amazonicus) were fed with different dietary vitamin C (ascorbic acid or AA) concentrations for 2 months in order to evaluate its effect on the physiological condition of this species. Five treatments were tested: Reference diet-RD (350 mg AA kg(-1) and 45% crude protein commercial feed); C400, C500, C600 and C800 with supplementation of 400, 500, 600 and 800 mg AA kg(-1). Fish fed 800 mg AA kg(-1) supplemented diets presented greater weight gain and survival. Hematocrit, hemoglobin concentrations and erythrocytes showed significant increase (p<0.05) in C600 and C800 relatively to the others. Total leukocyte values were significantly higher in fish fed vitamin C supplemented diets relatively to those fed RD and leukocytes count of C600 and C800 were the highest among the treatments. Monocytes were the only cells of fish defense mechanism that presented variation, and C600 and C800 presented the lowest values (P<0.05). Glucose level was significantly different relatively to RD only in C400 and the plasmatic total protein values for fish fed vitamin C supplemented diets were higher than for fish fed the RD. Fish plasmatic ions (Na(+) and Cl(-)) were significantly lower for C500, C600 and C800 relatively to C400 and RD. The physiological profile and the growth performance of matrinx? suggest that diet supplemented with 800 mg AA kg(-1) can improve its performance and increase the survival rate.     

Is the onset of senescence in leaf cells of intact plants due to low or high sugar levels?

This review examines the hypotheses that developmental programmed cell death in leaves is mediated (i) by sugar starvation in the leaf cells or (ii) by sugar accumulation in these cells. Experimental evidence for both hypotheses is critically discussed and found to be lacking. For example, some papers show that sugars prevent senescence of cut leaves placed in darkness, and prevent low sugar levels in the leaves. In these tests, the sugars seem to replace photosynthesis, hence the results have little relevance to leaf senescence in intact plants in the light. Low nitrogen nutrition and high light results in earlier senescence than the low nitrogen treatment alone. This is accompanied by high sugar levels in the leaves. The results have led to the idea that accumulation of sugars is the cause of the additional effect, or more generally, that sugar accumulation is always the direct cause of leaf senescence. Results from over-expressing, or knocking out, hexokinase genes tend to support the high sugar hypothesis, but pleiotropic effects confound this conclusion. In addition, several experiments show the effects of treatments on senescence without the increase in leaf sugar levels. Nonetheless, sugar levels are usually measured in whole leaves. Such an overall level does not reflect the differences in the onset of senescence between tissues and cells, and can therefore not be used as an argument for or against either of the two hypotheses. It is argued that future work should determine the time line of the concentrations of various sugars in various cells and cellular compartments, in relation to senescence processes in the same cells. Taken together, the data are not decisive. It is possible that neither of the two hypotheses is correct.     

Breeding success and population trends in Adélie penguins in areas with low and high levels of human disturbance

The breeding performance and population trends of Adélie penguins (Pygoscelis adeliae) was studied at Esperanza/Hope Bay, Antarctic Peninsula, by comparing an area with low levels of human disturbance (LLD) and an area with high levels of human disturbance (HLD), close to an Argentine research station. From 1995/1996 to 2004/2005 (except for 1999/2000 and 2003/2004), the following population parameters were measured in both areas: (1) the number of breeding pairs, (2) the number of chicks in creches and (3) the number of chicks produced by breeding pairs. Counts were made for 26 breeding groups situated in the LLD area and 63 breeding groups located in the HLD area. The number of chicks per breeding pair was obtaobtained by following 100 marked nests in each area. All parameters were measured as described in the CCAMLR Monitoring Program protocols. The magnitude and direction (increasing or decreasing) of the changes in breeding population size and the number of chicks creched were similar in both areas. Overall, the number of breeding pairs decreased from 4,744 to 2,968 (37.4%) in the LLD area, and from 8,744 to 5,378 (38.6%) in the HLD area. The number of chicks fledged increased from 3,808 to 4,065 (6.7%) in the LLD area, and decreased from 6,991 to 6,712 (4%) in the HLD area. Breeding success (chicks fledged per marked nest) did not differ significantly between areas for most of the seasons compared. In 1996/1997, breeding success was significantly higher in the HLD area. Our data suggest that environmental influences currently exert greater effects than human disturbance on the penguin population at Esperanza Bay.     

Effects of high fat diet induced obesity on peripheral nerve regeneration and levels of GAP 43 and TGF-β in rats

The increasing frequency of obesity is important because of its accompanying related health problems. The effects of obesity on peripheral nerves have not been elucidated. We investigated the effects of obesity on sciatic nerve regeneration using electrophysiology, stereology, immunohistochemistry, histopathology and functional tests. We used control, obese, control injured and obese injured groups of rats. Electrophysiological results showed that nerve conduction velocity and EMG were same in the experimental groups, but the amplitude of the compound action potential of the control group was significantly higher than that of the obese group. Examination of the nerves showed that the control and obese groups had both larger axon diameters and thicker myelin sheaths. The number of myelinated axons was decreased in both of the injured groups. Axon diameters and myelin sheath thicknesses of the control injured group were significantly greater those of the obese injured group. There were no significant differences in functional tests among the groups. Although growth associated protein 43 immunostaining in the control injured group was significantly greater than that of the obese injured group, no significant difference was observed between the control and obese groups. There was no significant difference in immunohistochemical staining for transforming growth factor beta 3 between the control injured and obese injured groups. Our results suggest that obesity may affect peripheral nerve regeneration negatively after crush injury.     

Physiological response to heat stress of tomato ‘Micro-Tom’ plants expressing high and low levels of mitochondrial sHSP23.6 protein

Tomato ‘Micro-Tom’ plants were transformed for high or low expression of the mitochondrial small “heat shock” protein (HSP) (MT-sHSP23.6) to evaluate their response to high temperature. The plants were raised for 59 days under a controlled temperature, photoperiod and photon flow density and then subjected to heat stress for 24 h at 37 °C, followed by a recovery period under normal conditions (21 ± 2 °C). The cycle was repeated. The chlorophyll a fluorescence intensity was measured, and the parameters of the JIP-test were calculated. The gas exchange was also evaluated. The JIP-test showed significantly different responses of the genotypes to heat stress. The parameters of photosystem I activity and the net assimilation of CO₂ increased during the first stress cycle in genotypes with a high expression of MT-sHSP23.6 and in non-transformed plants; however, the net assimilation of CO₂ decreased in genotypes with a low expression of MT-sHSP23.6. The data suggest that MT-sHSP23.6 participates in the heat tolerance mechanism, considering that the suppression of this protein resulted in greater physiological damage during heat stress.     

Are saturating pulses indeed saturating? Evidence for considerable PSII yield underestimation in leaves adapted to high levels of natural light

The "saturating pulse" method of in vivo Chl fluorescence measurement has been widely used by physiologists and especially ecophysiologists, as it allows a simple, rapid and non-invasive assessment of PSII function and the allocation of absorbed energy into photochemical and non-photochemical processes. It is based on the accurate determination of the so-called Fm('), i.e. the fluorescence signal emitted when a "saturating" light pulse closes all PSII centers. In this methodological investigation, we examined whether the saturating pulse intensities required to obtain maximal fluorescence yields differ between leaves of various species receiving varying actinic light irradiances. It was shown that, in leaves adapted to comparatively high (yet realistic) levels of natural irradiances, the saturating pulses usually applied are not able to close all PSII reaction centers. As a result, there is a high risk of considerable Fm(') underestimation. Accordingly, the derived values of effective PSII yields and linear electron transport rates (ETR) are also underestimated, even at the highest saturation pulse levels afforded by commercial instruments. Since the extent of underestimation increases with actinic irradiance, the ETR versus light curves are considerably distorted. The possible reasons for the apparent inability of "saturating" pulses to close all PSII centers at high actinic light and the practical implications, especially in field work, are discussed.     

Extremely high circulating levels of 1α,25-dihydroxyvitamin D3 in the marmoset,a new world monkey

Compared to most mammals, the marmoset, a new world monkey, requires particularly large amounts of vitamin D to maintain normal growth. We compared serum concentrations of vitamin D metabolites in marmosets with rhesus monkeys and humans. The circulating levels of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] in marmosets were 4 to 10 times higher than those in rhesus monkeys and humans. But none of the marmosets exhibited hypercalcemia. In two marmosets which had suffered bone fractures, the 1α,25-(OH)₂D₃ levels were particularly elevated. These results suggest that the marmoset has an end-organ resistance to 1α,25(OH)₂D₃.     

Is hemiepiphytism an adaptation to high irradiance? Testing seedling responses to light levels and drought in hemiepiphytic and non‐hemiepiphytic Ficus

The epiphytic growth habit in many Ficus species during their juvenile stages has commonly been hypothesized to be an adaptation for avoiding deep shade in the forest understory, but this has never been tested experimentally. We examined growth and ecophysiology in seedlings of three hemiepiphytic (Hs) and three non‐hemiepiphytic (NHs) Ficus species grown under different irradiance levels. Both Hs and NHs exhibited characteristics of high light requiring species, such as high plasticity to growth irradiance and relatively high maximum photosynthetic assimilation rates. Diurnal measurements of leaf gas exchange showed that Hs have much shorter active photosynthetic periods than NHs; moreover, leaves of Hs have lower xylem hydraulic conductivity but stronger drought tolerance as indicated by much lower rates of leaf diebacks during the drought treatment. Seedlings of NHs had 3.3‐ and 13.3‐fold greater height and biomass than those of Hs species after growing in the nursery for 5 months, indicating a trade‐off between growth and drought tolerance due to the conflicting requirements for xylem conductivity and cavitation resistance. This study does not support the shade‐avoidance hypothesis; rather, it suggests that the canopy regeneration in Hs is an adaptation to avoid alternative terrestrial growth‐related risks imposed to tiny Ficus seedlings. The NHs with terrestrial regeneration reduce these risks by having an initial burst of growth to rapidly gain relatively large seedling sizes, while in Hs seedlings more conservative water use and greater drought tolerance for surviving the canopy environment are intrinsically associated with slow growth.