Effects of Desiccation on Phosphorus and Potassium Acquisition by a Desiccation-tolerant Moss and Lichen

The hypothesis that desiccation-tolerant mosses and lichensmay be more responsive to nutrient inputs accompanying intermittentdesiccation than mesophytic forest species was investigatedemploying species from semi-arid grassland in Hungary. Shootapices of the moss Syntrichia ruralis and marginal lobes ofthe lichen Cladonia convoluta were maintained for 7 weeks undercontrolled conditions. They were cultivated with or withouta weekly application of the major inorganic macronutrients,and either under constant hydration or with one or two 24 hperiods of desiccation each week. Growth of S. ruralis was stimulatedby nutrient additions, but lower weight increments were achievedwith increasing frequency of desiccation. All samples of thelichen showed positive growth, and no significant treatmenteffects were detected. A large net uptake of P occurred in nutrient-treatedmaterial of both species that was unaffected by the impositionof desiccation treatments. A smaller net uptake of K into theintracellular fraction was also observed when nutrients wereapplied, but in the moss this was against a baseline of decreasingK content. In contrast, more of the original K content was retainedin C. convoluta. In neither species was any clear evidence foundfor inhibition of nutrient uptake by the desiccation episodes.It is suggested that the lack of growth response in the lichenarises from an inability to bring together the additional nutrients,presumably mainly absorbed by the mycobiont, with photosynthateproduced by the photobiont. Copyright 2000 Annals of BotanyCompany Cladonia convoluta, Syntrichia ruralis, desiccation tolerance, mineral nutrition, phosphorus, potassium     

Conservation of the Caenorhabditis elegans timing gene clk-1 from yeast to human: a gene required for ubiquinone biosynthesis with potential implications for aging

Mutations in the Caenorhabditis elegans gene clk-1 have a major effect on slowing development and increasing life span. The Saccharomyces cerevisiae homolog COQ7 encodes a mitochondrial protein involved in ubiquinone biosynthesis and, hence, is required for respiration and gluconeogenesis. In this study, RT-PCR and 5′ RACE were used to isolate both human and mouse clk-1/COQ7 homologs. Human CLK-1 was mapped to Chr 16(p12–13.1) by Radiation Hybrid (RH) and fluorescence in situ hybridization (FISH) methods. The number and location of human CLK1 introns were determined, and the location of introns II and IV are the same as in C. elegans. Northern blot analysis showed that three different isoforms of CLK-1 mRNA are present in several tissues and that the isoforms differ in the amount of expression. The functional equivalence of human CLK-1 to the yeast COQ7 homolog was tested by introducing either a single or multicopy plasmid containing human CLK-1 cDNA into yeast coq7 deletion strains and assaying for growth on a nonfermentable carbon source. The human CLK-1 gene was able to functionally complement yeast coq7 deletion mutants. The protein similarities and the conservation of function of the CLK-1/clk-1/COQ7 gene products suggest a potential link between the production of ubiquinone and aging. Received: 15 March 1999 / Accepted 11 June 1999     

Kinetic Estimation of GFR Improves Prediction of Dialysis and Recovery after Kidney Transplantation

BackgroundThe early prediction of delayed graft function (DGF) would facilitate patient management after kidney transplantation.MethodsIn a single-centre retrospective analysis, we investigated kinetic estimated GFR under non-steady-state conditions, KeGFR, in prediction of DGF. KeGFR_sCr was calculated at 4h, 8h and 12h in 56 recipients of deceased donor kidneys from initial serum creatinine (sCr) concentrations, estimated creatinine production rate, volume of distribution, and the difference between consecutive sCr values. The utility of KeGFR_sCr for DGF prediction was compared with, sCr, plasma cystatin C (pCysC), and KeGFR_pCysC similarly derived from pCysC concentrations.ResultsAt 4h, the KeGFR_sCr area under the receiver operator characteristic curve (AUC) for DGF prediction was 0.69 (95% CI: 0.56–0.83), while sCr was not useful (AUC 0.56, (CI: 0.41–0.72). Integrated discrimination improvement analysis showed that the KeGFR_sCr improved a validated clinical prediction model at 4h, 8h, and 12h, increasing the AUC from 0.68 (0.52–0.83) to 0.88 (0.78–0.99) at 12h (p = 0.01). KeGFR_pCysC also improved DGF prediction. In contrast, sCr provided no improvement at any time point.ConclusionsCalculation of KeGFR from sCr facilitates early prediction of DGF within 4 hours of renal transplantation.     

Elevated Levels of Asymmetric Dimethylarginine (ADMA) in the Pericardial Fluid of Cardiac Patients Correlate with Cardiac Hypertrophy

Background

Pericardial fluid (PF) contains several biologically active substances, which may provide information regarding the cardiac conditions. Nitric oxide (NO) has been implicated in cardiac remodeling. We hypothesized that L-arginine (L-Arg) precursor of NO-synthase (NOS) and asymmetric dimethylarginine (ADMA), an inhibitor of NOS, are present in PF of cardiac patients and their altered levels may contribute to altered cardiac morphology.

Methods

L-Arg and ADMA concentrations in plasma and PF, and echocardiographic parameters of patients undergoing coronary artery bypass graft (CABG, n = 28) or valve replacement (VR, n = 25) were determined.

Results

We have found LV hypertrophy in 35.7% of CABG, and 80% of VR patients. In all groups, plasma and PF L-Arg levels were higher than that of ADMA. Plasma L-Arg level was higher in CABG than VR (75.7±4.6 μmol/L vs. 58.1±4.9 μmol/L, p = 0.011), whereas PF ADMA level was higher in VR than CABG (0.9±0.0 μmol/L vs. 0.7±0.0 μmol/L, p = 0.009). L-Arg/ADMA ratio was lower in the VR than CABG (VR_plasma: 76.1±6.6 vs. CABG_plasma: 125.4±10.7, p = 0.004; VR_PF: 81.7±4.8 vs. CABG_PF: 110.4±7.2, p = 0.009). There was a positive correlation between plasma L-Arg and ADMA in CABG (r = 0.539, p = 0.015); and plasma and PF L-Arg in CABG (r = 0.357, p = 0.031); and plasma and PF ADMA in VR (r = 0.529, p = 0.003); and PF L-Arg and ADMA in both CABG and VR (CABG: r = 0.468, p = 0.006; VR: r = 0.371, p = 0.034). The following echocardiographic parameters were higher in VR compared to CABG: interventricular septum (14.7±0.5 mm vs. 11.9±0.4 mm, p = 0.000); posterior wall thickness (12.6±0.3 mm vs. 11.5±0.2 mm, p = 0.000); left ventricular (LV) mass (318.6±23.5 g vs. 234.6±12.3 g, p = 0.007); right ventricular (RV) (33.9±0.9 cm² vs. 29.7±0.7 cm², p = 0.004); right atrial (18.6±1.0 cm² vs. 15.4±0.6 cm², p = 0.020); left atrial (19.8±1.0 cm² vs. 16.9±0.6 cm², p = 0.033) areas. There was a positive correlation between plasma ADMA and RV area (r = 0.453, p = 0.011); PF ADMA and end-diastolic (r = 0.434, p = 0.015) and systolic diameter of LV (r = 0.487, p = 0.007); and negative correlation between PF ADMA and LV ejection fraction (r = -0.445, p = 0.013) in VR.

Conclusion

We suggest that elevated levels of ADMA in the PF of patients indicate upregulated RAS and reduced bioavailability of NO, which can contribute to the development of cardiac hypertrophy and remodeling.     

Aging exacerbates hypertension‐induced cerebral microhemorrhages in mice: role of resveratrol treatment in vasoprotection

Recent studies demonstrate that aging exacerbates hypertension‐induced cognitive decline, but the specific age‐related mechanisms remain elusive. Cerebral microhemorrhages (CMHs) are associated with rupture of small intracerebral vessels and are thought to progressively impair neuronal function. To determine whether aging exacerbates hypertension‐induced CMHs young (3 months) and aged (24 months) mice were treated with angiotensin II plus L‐NAME. We found that the same level of hypertension leads to significantly earlier onset and increased incidence of CMHs in aged mice than in young mice, as shown by neurological examination, gait analysis, and histological assessment of CMHs in serial brain sections. Hypertension‐induced cerebrovascular oxidative stress and redox‐sensitive activation of matrix metalloproteinases (MMPs) were increased in aging. Treatment of aged mice with resveratrol significantly attenuated hypertension‐induced oxidative stress, inhibited vascular MMP activation, significantly delayed the onset, and reduced the incidence of CMHs. Collectively, aging promotes CMHs in mice likely by exacerbating hypertension‐induced oxidative stress and MMP activation. Therapeutic strategies that reduce microvascular oxidative stress and MMP activation may be useful for the prevention of CMHs, protecting neurocognitive function in high‐risk elderly patients.     

An Allosteric Signaling Pathway of Human 3-Phosphoglycerate Kinase from Force Distribution Analysis

3-Phosphogycerate kinase (PGK) is a two domain enzyme, which transfers a phosphate group between its two substrates, 1,3-bisphosphoglycerate bound to the N-domain and ADP bound to the C-domain. Indispensable for the phosphoryl transfer reaction is a large conformational change from an inactive open to an active closed conformation via a hinge motion that should bring substrates into close proximity. The allosteric pathway resulting in the active closed conformation has only been partially uncovered. Using Molecular Dynamics simulations combined with Force Distribution Analysis (FDA), we describe an allosteric pathway, which connects the substrate binding sites to the interdomain hinge region. Glu192 of alpha-helix 7 and Gly394 of loop L14 act as hinge points, at which these two secondary structure elements straighten, thereby moving the substrate-binding domains towards each other. The long-range allosteric pathway regulating hPGK catalytic activity, which is partially validated and can be further tested by mutagenesis, highlights the virtue of monitoring internal forces to reveal signal propagation, even if only minor conformational distortions, such as helix bending, initiate the large functional rearrangement of the macromolecule.     

A human memory T cell subset with stem cell-like properties

Immunological memory is thought to depend on a stem cell-like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and IL-7Rα(+) T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human stem cell-like memory T cell population is of direct relevance to the design of vaccines and T cell therapies.     

Physical activity attenuates the influence of FTO variants on obesity risk: a meta-analysis of 218,166 adults and 19,268 children

Background

The FTO gene harbors the strongest known susceptibility locus for obesity. While many individual studies have suggested that physical activity (PA) may attenuate the effect of FTO on obesity risk, other studies have not been able to confirm this interaction. To confirm or refute unambiguously whether PA attenuates the association of FTO with obesity risk, we meta-analyzed data from 45 studies of adults (n = 218,166) and nine studies of children and adolescents (n = 19,268).

Methods and Findings

All studies identified to have data on the FTO rs9939609 variant (or any proxy [r ²>0.8]) and PA were invited to participate, regardless of ethnicity or age of the participants. PA was standardized by categorizing it into a dichotomous variable (physically inactive versus active) in each study. Overall, 25% of adults and 13% of children were categorized as inactive. Interaction analyses were performed within each study by including the FTO×PA interaction term in an additive model, adjusting for age and sex. Subsequently, random effects meta-analysis was used to pool the interaction terms. In adults, the minor (A−) allele of rs9939609 increased the odds of obesity by 1.23-fold/allele (95% CI 1.20–1.26), but PA attenuated this effect (p _interaction  = 0.001). More specifically, the minor allele of rs9939609 increased the odds of obesity less in the physically active group (odds ratio  = 1.22/allele, 95% CI 1.19–1.25) than in the inactive group (odds ratio  = 1.30/allele, 95% CI 1.24–1.36). No such interaction was found in children and adolescents.

Conclusions

The association of the FTO risk allele with the odds of obesity is attenuated by 27% in physically active adults, highlighting the importance of PA in particular in those genetically predisposed to obesity. Please see later in the article for the Editors'' Summary     

Monoacylglycerol lipase inhibition-induced changes in plasma corticosterone levels,anxiety and locomotor activity in male CD1 mice

The hypothalamus–pituitary–adrenal-axis is strongly controlled by the endocannabinoid system. The specific impact of enhanced 2-arachidonoylglycerol signaling on corticosterone plasma levels, however, was not investigated so far. Here we studied the effects of the recently developed monoacylglycerol lipase inhibitor JZL184 on basal and stress-induced corticosterone levels in male CD1 mice, and found that this compound dramatically increased basal levels without affecting stress responses. Since acute changes in corticosterone levels can affect behavior, JZL184 was administered concurrently with the corticosterone synthesis inhibitor metyrapone, to investigate whether the previously shown behavioral effects of JZL184 are dependent on corticosterone. We found that in the elevated plus-maze, the effects of JZL184 on “classical” anxiety-related measures were abolished by corticosterone synthesis blockade. By contrast, effects on the “ethological” measures of anxiety (i.e. risk assessment) were not affected by metyrapone. In the open-field, the locomotion-enhancing effects of the compound were not changed either. These findings show that monoacylglycerol lipase inhibition dramatically increases basal levels of corticosterone. This endocrine effect partly affects the anxiolytic, but not the locomotion-enhancing effects of monoacylglycerol lipase blockade.     

Evaluation of exogenous siRNA addition as a metabolic engineering tool for modifying biopharmaceuticals

Traditional metabolic engineering approaches, including homologous recombination, zinc‐finger nucleases, and short hairpin RNA, have previously been used to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors. This approach has the following potential advantages: no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody (MAb) afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti‐CD20 MAb. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17‐fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an Antibody‐Dependent Cellular Cytoxicity (ADCC) assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 415–424, 2013