Enhanced hippocampus‐dependent memory and reduced anxiety in mice over‐expressing human catalase in mitochondria

Oxidative stress (OS) and reactive oxygen species (ROS) play a modulatory role in synaptic plasticity and signaling pathways. Mitochondria (MT), a major source of ROS because of their involvement in energy metabolism, are important for brain function. MT‐generated ROS are proposed to be responsible for a significant proportion of OS and are associated with developmental abnormalities and aspects of cellular aging. The role of ROS and MT function in cognition of healthy individuals is relatively understudied. In this study, we characterized behavioral and cognitive performance of 5‐ to 6‐month‐old mice over‐expressing mitochondrial catalase (MCAT). MCAT mice showed enhancements in hippocampus‐dependent spatial learning and memory in the water maze and contextual fear conditioning, and reduced measures of anxiety in the elevated zero maze. Catalase activity was elevated in MCAT mice in all brain regions examined. Measures of oxidative stress (glutathione, protein carbonyl content, lipid peroxidation, and 8‐hydroxyguanine) did not significantly differ between the groups. The lack of differences in these markers of oxidative stress suggests that the differences observed in this study may be due to altered redox signaling. Catalase over‐expression might be sufficient to enhance cognition and reduce measures of anxiety even in the absence of alteration in levels of OS.     

The ontogeny and population variability of human hepatic dihydronicotinamide riboside:quinone oxidoreductase (NQO2)

Dihydronicotinamide riboside:quinone oxidoreductase (NQO2) is an enzyme that performs reduction reactions involved in antioxidant defense. We hypothesized that NQO2 hepatic drug clearance would develop in children over time, similar to NQO1. Using human liver cytosol (n = 117), the effects of age, sex, ethnicity, and weight on NQO2 expression and activity were probed. No significant correlations were observed. Biochemical activity of NQO2 was as high at birth as in adults (0.23 ± 0.04 nmol/min/mg protein, mean ± SEM, range 0–1.83). In contrast, modeled hepatic clearance through the NQO2 pathway was up to 10% of adult levels at birth, reaching predicted adult levels (0.3 ± 0.03 L/h) at 14 years of age. Comparisons between NQO1 and NQO2 in the same livers showed that neither protein (P = 0.32) nor activity (P = 0.23) correlated, confirming both orthologs are independently regulated. Because hepatic clearance through NQO2 does not mature until teenage years, compounds detoxified by this enzyme may be more deleterious in children.     

Production of an active recombinant thrombomodulin derivative in transgenic tobacco plants and suspension cells

Thrombomodulin is a membrane-bound protein that plays an active role in the blood coagulation system by binding thrombin and initiating the protein C anticoagulant pathway. Solulin™ is a recombinant soluble derivative of human thrombomodulin. It is used for the treatment of thrombotic disorders. To evaluate the production of this pharmaceutical protein in plants, expression vectors were generated using four different N-terminal signal peptides. Immunoblot analysis of transiently transformed tobacco leaves showed that intact Solulin™ could be detected using three of these signal peptides. Furthermore transgenic tobacco plants and BY2 cells producing Solulin™ were generated. Immunoblot experiments showed that Solulin™ accumulated to maximum levels of 115 and 27 μg g⁻¹ plant material in tobacco plants and BY2 cells, respectively. Activity tests performed on the culture supernatant of transformed BY2 cells showed that the secreted Solulin™ was functional. In contrast, thrombomodulin activity was not detected in total soluble protein extracts from BY2 cells, probably due to inhibitory effects of substances in the cell extract. N-terminal sequencing was carried out on partially purified Solulin™ from the BY2 culture supernatant. The sequence was identical to that of Solulin™ produced in Chinese hamster ovary cells, confirming correct processing of the N-terminal signal peptide. We have demonstrated that plants and plant cell cultures can be used as alternative systems for the production of an active recombinant thrombomodulin derivative.     

High CO2 levels impair alveolar epithelial function independently of pH

Background

In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO₂) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO₂ levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO₂. The Na,K-ATPase consumes ∼40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO₂ on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function.

Principal Findings

We found that short-term increases in pCO₂ impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO₂, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCζ which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools.

Conclusions

Our data suggest that alveolar epithelial cells, through which CO₂ is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO₂ levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.     

Mycobacterium tuberculosis Bacteremia in a Cohort of HIV-Infected Patients Hospitalized with Severe Sepsis in Uganda–High Frequency,Low Clinical Sand Derivation of a Clinical Prediction Score

Background

When manifested as Mycobacterium tuberculosis (MTB) bacteremia, disseminated MTB infection clinically mimics other serious blood stream infections often hindering early diagnosis and initiation of potentially life-saving anti-tuberculosis therapy. In a cohort of hospitalized HIV-infected Ugandan patients with severe sepsis, we report the frequency, management and outcomes of patients with MTB bacteremia and propose a risk score based on clinical predictors of MTB bacteremia.

Methods

We prospectively enrolled adult patients with severe sepsis at two Ugandan hospitals and obtained blood cultures for MTB identification. Multivariable logistic regression modeling was used to determine predictors of MTB bacteremia and to inform the stratification of patients into MTB bacteremia risk categories based on relevant patient characteristics.

Results

Among 368 HIV-infected patients with a syndrome of severe sepsis, eighty-six (23%) had MTB bacteremia. Patients with MTB bacteremia had a significantly lower median CD4 count (17 vs 64 lymphocytes/mm³, p<0.001) and a higher 30-day mortality (53% vs 32%, p = 0.001) than patients without MTB bacteremia. A minority of patients with MTB bacteremia underwent standard MTB diagnostic testing (24%) or received empiric anti-tuberculosis therapy (15%). Independent factors associated with MTB bacteremia included male sex, increased heart rate, low CD4 count, absence of highly active anti-retroviral therapy, chief complaint of fever, low serum sodium and low hemoglobin. A risk score derived from a model containing these independent predictors had good predictive accuracy [area under the curve = 0.85, 95% CI 0.80–0.89].

Conclusions

Nearly 1 in 4 adult HIV-infected patients hospitalized with severe sepsis in 2 Ugandan hospitals had MTB bacteremia. Among patients in whom MTB was suspected, standard tests for diagnosing pulmonary MTB were inaccurate for correctly classifying patients with or without bloodstream MTB infection. A MTB bacteremia risk score can improve early diagnosis of MTB bacteremia particularly in settings with increased HIV and MTB co-infection.     

Cellulosomics, a gene-centric approach to investigating the intraspecific diversity and adaptation of Ruminococcus flavefaciens within the rumen

Background

The bovine rumen maintains a diverse microbial community that serves to break down indigestible plant substrates. However, those bacteria specifically adapted to degrade cellulose, the major structural component of plant biomass, represent a fraction of the rumen microbiome. Previously, we proposed scaC as a candidate for phylotyping Ruminococcus flavefaciens, one of three major cellulolytic bacterial species isolated from the rumen. In the present report we examine the dynamics and diversity of scaC-types both within and between cattle temporally, following a dietary switch from corn-silage to grass-legume hay. These results were placed in the context of the overall bacterial population dynamics measured using the 16S rRNA.

Principal Findings

As many as 117 scaC-types were estimated, although just nineteen were detected in each of three rumens tested, and these collectively accounted for the majority of all types present. Variation in scaC populations was observed between cattle, between planktonic and fiber-associated fractions and temporally over the six-week survey, and appeared related to scaC phylogeny. However, by the sixth week no significant separation of scaC populations was seen between animals, suggesting enrichment of a constrained set of scaC-types. Comparing the amino-acid translation of each scaC-type revealed sequence variation within part of the predicted dockerin module but strong conservation in the N-terminus, where the cohesin module is located.

Conclusions

The R. flavefaciens species comprises a multiplicity of scaC-types in-vivo. Enrichment of particular scaC-types temporally, following a dietary switch, and between fractions along with the phylogenetic congruence suggests that functional differences exist between types. Observed differences in dockerin modules suggest at least part of the functional heterogeneity may be conferred by scaC. The polymorphic nature of scaC enables the relative distribution of R. flavefaciens strains to be examined and represents a gene-centric approach to investigating the intraspecific adaptation of an important specialist population.