Na+/K+-ATPase α-subunit (nkaα) Isoforms and Their mRNA Expression Levels,Overall Nkaα Protein Abundance,and Kinetic Properties of Nka in the Skeletal Muscle and Three Electric Organs of the Electric Eel,Electrophorus electricus

This study aimed to obtain the coding cDNA sequences of Na⁺/K⁺-ATPase α (nkaα) isoforms from, and to quantify their mRNA expression in, the skeletal muscle (SM), the main electric organ (EO), the Hunter’s EO and the Sach’s EO of the electric eel, Electrophorus electricus. Four nkaα isoforms (nkaα1c1, nkaα1c2, nkaα2 and nkaα3) were obtained from the SM and the EOs of E. electricus. Based on mRNA expression levels, the major nkaα expressed in the SM and the three EOs of juvenile and adult E. electricus were nkaα1c1 and nkaα2, respectively. Molecular characterization of the deduced Nkaα1c1 and Nkaα2 sequences indicates that they probably have different affinities to Na⁺ and K⁺. Western blotting demonstrated that the protein abundance of Nkaα was barely detectable in the SM, but strongly detected in the main and Hunter’s EOs and weakly in the Sach’s EO of juvenile and adult E. electricus. These results corroborate the fact that the main EO and Hunter’s EO have high densities of Na⁺ channels and produce high voltage discharges while the Sach’s EO produces low voltage discharges. More importantly, there were significant differences in kinetic properties of Nka among the three EOs of juvenile E. electricus. The highest and lowest V _max of Nka were detected in the main EO and the Sach’s EO, respectively, with the Hunter’s EO having a V _max value intermediate between the two, indicating that the metabolic costs of EO discharge could be the highest in the main EO. Furthermore, the Nka from the main EO had the lowest K_m (or highest affinity) for Na⁺ and K⁺ among the three EOs, suggesting that the Nka of the main EO was more effective than those of the other two EOs in maintaining intracellular Na⁺ and K⁺ homeostasis and in clearing extracellular K⁺ after EO discharge.     

The Use of Anchored Agonists of Phagocytic Receptors for Cancer Immunotherapy: B16-F10 Murine Melanoma Model

The application of the phagocytic receptor agonists in cancer immunotherapy was studied. Agonists (laminarin, molecules with terminal mannose, N-Formyl-methioninyl-leucyl-phenylalanine) were firmly anchored to the tumor cell surface. When particular agonists of phagocytic receptors were used together with LPS (Toll-like receptor agonist), high synergy causing tumour shrinkage and a temporary or permanent disappearance was observed. Methods of anchoring phagocytic receptor agonists (charge interactions, anchoring based on hydrophobic chains, covalent bonds) and various regimes of phagocytic agonist/LPS mixture applications were tested to achieve maximum therapeutic effect. Combinations of mannan/LPS and f-MLF/LPS (hydrophobic anchors) in appropriate (pulse) regimes resulted in an 80% and 60% recovery for mice, respectively. We propose that substantial synergy between agonists of phagocytic and Toll-like receptors (TLR) is based on two events. The TLR ligand induces early and massive inflammatory infiltration of tumors. The effect of this cell infiltrate is directed towards tumor cells, bearing agonists of phagocytic receptors on their surface. The result of these processes was effective killing of tumor cells. This novel approach represents exploitation of innate immunity mechanisms for treating cancer.     

Cultured bacterial diversity and human impact on alpine glacier cryoconite

The anthropogenic effect on the microbial communities in alpine glacier cryoconites was investigated by cultivation and physiological characterization of bacteria from six cryoconite samples taken at sites with different amounts of human impact. Two hundred and forty seven bacterial isolates were included in Actinobacteria (9%, particularly Arthrobacter), Bacteroidetes (14%, particularly Olleya), Firmicutes (0.8%), Alphaproteobacteria (2%), Betaproteobacteria (16%, particularly Janthinobacterium), and Gammaproteobacteria (59%, particularly Pseudomonas). Among them, isolates of Arthrobacter were detected only in samples from sites with no human impact, while isolates affiliated with Enterobacteriaceae were detected only in samples from sites with strong human impact. Bacterial isolates included in Actinobacteria and Bacteroidetes were frequently isolated from pristine sites and showed low maximum growth temperature and enzyme secretion. Bacterial isolates included in Gammaproteobacteria were more frequently isolated from sites with stronger human impact and showed high maximum growth temperature and enzyme secretion. Ecotypic differences were not evident among isolates of Janthinobacterium lividum, Pseudomonas fluorescens, and Pseudomonas veronii, which were frequently isolated from sites with different degrees of anthropogenic effect.     

Effect of Toll-like receptor 2 and 4 of corneal fibroblasts on cytokine expression with co-cultured antigen presenting cells

Keratocytes are the first component to contact ocular pathogens when the epithelial barrier breaks down and the emerging evidences indicated keratocytes appeared to be one of the corneal cellular immune components. Little is known about the role of Toll-like receptors (TLRs) in keratocytes, although it has been well documented that keratocytes constitutively express various TLRs including TLR2 and TLR4. In this in vitro study, the authors focused on the role of keratocytes in corneal innate immune system and cross-talk of keratocytes with resident antigen presenting cells (APCs), especially through TLR2 and TLR4. Primary cultivated keratocytes (corneal fibroblasts) from C57BL/6 mice per se actively secreted pro-inflammatory cytokines, especially interleukin (IL)-6, with a dose-dependent manner in response to Pam3CSK4 or lipopolysaccharide (LPS) challenge. With co-culture of corneal fibroblasts with APCs per se, secretion of IL-6 and tumor necrosis factor (TNF)-α was markedly increased and it was counterbalanced by concurrent increase in IL-10 and tumor growth factor-β1. After Pam3CSK4 or LPS stimulation, this cytokine balance was completely broken down by overwhelming amplification of IL-6 and TNF-α secretion, especially in co-culture of corneal fibroblasts with macrophages, rather than with dendritic cells. Using corneal fibroblasts from TLR2 or TLR4 knockout mice, we could find the reversal of Pam3CSK4 or LPS-responsive dose-dependent increment in IL-6 and TNF-α. These results implied that corneal fibroblasts and their TLRs could be key components for the ocular homeostasis and pathogen-associated ocular innate immunity.     

Character displacement in the midst of background evolution in island populations of Anolis lizards: A spatiotemporal perspective

Negative interactions between species can generate divergent selection that causes character displacement. However, other processes cause similar divergence. We use spatial and temporal replication across island populations of Anolis lizards to assess the importance of negative interactions in driving trait shifts. Previous work showed that the establishment of Anolis sagrei on islands drove resident Anolis carolinensis to perch higher and evolve larger toepads. To further test the interaction's causality and predictability, we resurveyed a subset of islands nine years later. Anolis sagrei had established on one island between surveys. We found that A. carolinensis on this island now perch higher and have larger toepads. However, toepad morphology change on this island was not distinct from shifts on six other islands whose Anolis community composition had not changed. Thus, the presence of A. sagrei only partly explains A. carolinensis trait variation across space and time. We also found that A. carolinensis on islands with previously established A. sagrei now perch higher than a decade ago, and that current A. carolinensis perch height is correlated with A. sagrei density. Our results suggest that character displacement likely interacts with other evolutionary processes in this system, and that temporal data are key to detecting such interactions.     

Celastrol,an NF-κB Inhibitor,Improves Insulin Resistance and Attenuates Renal Injury in db/db Mice

The NF-κB pathway plays an important role in chronic inflammatory and autoimmune diseases. Recently, NF-κB has also been suggested as an important mechanism linking obesity, inflammation, and metabolic disorders. However, there is no current evidence regarding the mechanism of action of NF-κB inhibition in insulin resistance and diabetic nephropathy in type 2 diabetic animal models. We investigated the effects of the NF-κB inhibitor celastrol in db/db mice. The treatment with celastrol for 2 months significantly lowered fasting plasma glucose (FPG), HbA1C and homeostasis model assessment index (HOMA-IR) levels. Celastrol also exhibited significant decreases in body weight, kidney/body weight and adiposity. Celastrol reduced insulin resistance and lipid abnormalities and led to higher plasma adiponectin levels. Celastrol treatment also significantly mitigated lipid accumulation and oxidative stress in organs including the kidney, liver and adipose tissue. The treated group also exhibited significantly lower creatinine levels and urinary albumin excretion was markedly reduced. Celastrol treatment significantly lowered mesangial expansion and suppressed type IV collagen, PAI-1 and TGFβ1 expressions in renal tissues. Celastrol also improved abnormal lipid metabolism, oxidative stress and proinflammatory cytokine activity in the kidney. In cultured podocytes, celastrol treatment abolished saturated fatty acid-induced proinflammatory cytokine synthesis. Taken together, celastrol treatment not only improved insulin resistance, glycemic control and oxidative stress, but also improved renal functional and structural changes through both metabolic and anti-inflammatory effects in the kidney. These results suggest that targeted therapy for NF-κB may be a useful new therapeutic approach for the management of type II diabetes and diabetic nephropathy.     

Postprandial increases in nitrogenous excretion and urea synthesis in the Chinese soft-shelled turtle, <Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>

The objective of this study was to determine the effects of feeding on the excretory nitrogen (N) metabolism of the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis, with a special emphasis on the role of urea synthesis in ammonia detoxification. P. sinensis is ureogenic and possesses a full complement of ornithine-urea cycle enzymes in its liver. It is primarily ureotelic in water, and the estimated rate of urea synthesis in unfed animals was equivalent to only 1.5% of the maximal capacity of carbamoyl phosphate synthetase I (CPS I) in its liver. Approximately 72 h was required for P. sinensis to completely digest a meal of prawn meat. During this period, there were significant increases in ammonia contents in the stomach at hour 24 and in the intestine between hours 12 and 36, which could be a result of bacterial activities in the intestinal tract. However, ammonia contents in the liver, muscle, brain and plasma remained unchanged throughout the 72-h post-feeding. In contrast, at hour 24, urea contents in the stomach, intestine, liver, muscle, brain and plasma increased significantly by 2.9−, 3.5−, 2.6−, 2.9−, 3.4 and 3.0-fold, respectively. In addition, there was a 3.3- to 8.0−fold increase in the urea excretion rate between hours 0 and 36 post-feeding, which preceded the increase in ammonia excretion between hours 12 and 48. By hour 48, 68% of the assimilated N from the feed was excreted, 54% of which was excreted as urea-N. The rate of urea synthesis apparently increased sevenfold during the initial 24 h after feeding, which demanded only 10% of the maximal CPS I capacity in P. sinensis. The postprandial detoxification of ammonia to urea in P. sinensis effectively prevented postprandial surges in ammonia contents in the plasma and other tissues, as observed in other animals, during the 72-h period post-feeding. In addition, postprandial ammonia toxicity was ameliorated by increased transamination and synthesis of certain amino acids in the liver and muscle of P. sinensis. After feeding, a slight but significant increase in the glutamine content occurred in the brain at hour 24, indicating that the brain might experience a transient increase in ammonia and ammonia was detoxified to glutamine.     

Multisite Evaluation of Point of Care CD4 Testing in Papua New Guinea

Laboratory-based CD4 monitoring of HIV patients presents challenges in resource limited settings (RLS) including frequent machine breakdown, poor engineering support and limited cold chain and specimen transport logistics. This study assessed the performance of two CD4 tests designed for use in RLS; the Dynal assay and the Alere PIMA test (PIMA). Accuracy of Dynal and PIMA using venous blood was assessed in a centralised laboratory by comparison to BD FACSCount (BD FACS). Dynal had a mean bias of −50.35 cells/µl (r² = 0.973, p<0.0001, n = 101) and PIMA −22.43 cells/µl (r² = 0.964, p<0.0001, n = 139) compared to BD FACS. Similar results were observed for PIMA operated by clinicians in one urban (n = 117) and two rural clinics (n = 98). Using internal control beads, PIMA precision was 10.34% CV (low bead mean 214.24 cells/µl) and 8.29% (high bead mean 920.73 cells/µl) and similar %CV results were observed external quality assurance (EQA) and replicate patient samples. Dynal did not perform using EQA and no internal controls are supplied by the manufacturer, however duplicate testing of samples resulted in r² = 0.961, p<0.0001, mean bias = −1.44 cells/µl. Using the cut-off of 350 cells/µl compared to BD FACS, PIMA had a sensitivity of 88.85% and specificity of 98.71% and Dynal 88.61% and 100%. A total of 0.44% (2/452) of patient samples were misclassified as “no treat” and 7.30% (33/452) “treat” using PIMA whereas with Dynal 8.91% (9/101) as “treat” and 0% as “no treat”. In our setting PIMA was found to be accurate, precise and user-friendly in both laboratory and clinic settings. Dynal performed well in initial centralized laboratory evaluation, however lacks requisite quality control measures, and was technically more difficult to use, making it less suitable for use at lower tiered laboratories.     

Inactivation of ATM/ATR DNA Damage Checkpoint Promotes Androgen Induced Chromosomal Instability in Prostate Epithelial Cells

The ATM/ATR DNA damage checkpoint functions in the maintenance of genetic stability and some missense variants of the ATM gene have been shown to confer a moderate increased risk of prostate cancer. However, whether inactivation of this checkpoint contributes directly to prostate specific cancer predisposition is still unknown. Here, we show that exposure of non-malignant prostate epithelial cells (HPr-1AR) to androgen led to activation of the ATM/ATR DNA damage response and induction of cellular senescence. Notably, knockdown of the ATM gene expression in HPr-1AR cells can promote androgen-induced TMPRSS2: ERG rearrangement, a prostate-specific chromosome translocation frequently found in prostate cancer cells. Intriguingly, unlike the non-malignant prostate epithelial cells, the ATM/ATR DNA damage checkpoint appears to be defective in prostate cancer cells, since androgen treatment only induced a partial activation of the DNA damage response. This mechanism appears to preserve androgen induced autophosphorylation of ATM and phosphorylation of H2AX, lesion processing and repair pathway yet restrain ATM/CHK1/CHK2 and p53 signaling pathway. Our findings demonstrate that ATM/ATR inactivation is a crucial step in promoting androgen-induced genomic instability and prostate carcinogenesis.