Induction of ureogenesis in perfused liver of a freshwater teleost, Heteropneustes fossilis, infused with different concentrations of ammonium chloride

The induction pattern of urea cycle enzymes and the rate of urea-N excretion were studied with relation to ammonia load in the perfused liver of a freshwater ammoniotelic teleost, Heteropneustes fossilis, when infused with different concentrations of ammonium chloride for 60 min. Both urea-N excretion and uptake of ammonia by the perfused liver were found to be a saturable process. The V_max of urea-N excretion (0.45 μmol/g liver/min) was obtained at ammonium chloride addition of 1.18 μmol/g liver/min. The maximum induction of carbamyl phosphate synthetase (ammonia dependent), 200%, and of ornithine transcarbamylase, 120%, was seen by the addition of 0.58 μmol/g liver/min, and for argininosuccinate synthetase and argininosuccinate lyase of 150% and 115%, respectively, by the addition of 2.8 μmol/g liver/min of ammonium chloride. However, arginase activity did not alter in any of the concentrations of ammonium chloride added. An increase of ammonia load of 3–5 μmol/g wet wt from the physiological level in the perfused liver was sufficient to initiate and to cause maximum induction of most of the urea cycle enzymes activitty. These results further confirm the capacity of transition from ammoniotelism to ureotelism in this unique freshwater air-breathing teleost to tolerate a very high ambient ammonia.     

Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein

Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615–630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution.     

Defining the In Vivo Phenotype of Artemisinin-Resistant Falciparum Malaria: A Modelling Approach

BackgroundArtemisinin-resistant falciparum malaria has emerged in Southeast Asia, posing a major threat to malaria control. It is characterised by delayed asexual-stage parasite clearance, which is the reference comparator for the molecular marker ‘Kelch 13’ and in vitro sensitivity tests. However, current cut-off values denoting slow clearance based on the proportion of individuals remaining parasitaemic on the third day of treatment (''day-3''), or on peripheral blood parasite half-life, are not well supported. We here explore the parasite clearance distributions in an area of artemisinin resistance with the aim refining the in vivo phenotypic definitions.ConclusionsCharacterisation of overlapping distributions of parasite half-lives provides quantitative insight into the relationship between parasite clearance and artemisinin resistance, as well as the predictive value of the 10% cut-off in ''day-3'' parasitaemia. The findings are important for the interpretation of in vitro sensitivity tests and molecular markers for artemisinin resistance and for contextualising the ‘day 3’ threshold to account for initial parasitaemia and sample size.     

Does fish represent an intermediate stage in the evolution of ureotelic cytosolic arginase I?

Arginase catalyses the last step of the urea cycle. At least two isoenzymes of arginase are known; cytosolic ARG I and mitochondrial ARG II. ARG I is predominantly expressed in liver cytosol, as a part of urea cycle in ureotelic animals. The second isoform ARG II is primarily responsible for non-ureogenic functions, expressed in mitochondria of both hepatic and non-hepatic tissues in most vertebrates. Most micro-organisms and invertebrates are known to have only one type of arginase, whose function is unrelated to ornithine-urea cycle (OUC). However, in ureo-osmotic marine elasmobranchs arginase is localized in liver mitochondria as a part of OUC to synthesize urea for osmoregulation. An evolutionary transition occurred in arginase enzyme in terrestrial ureotelic vertebrates, with the evolution of ARG I from a pre-existing ancestral mitochondrial ARG II. This cytosolic ARG I activity is supposed to have first appeared in lung fishes, but the 40% and 60% distribution of arginase I and II activity in liver and kidney tissue of Heteropneustes fossilis indicates reconsideration of the above fact.     

Performance of the CareStart? G6PD Deficiency Screening Test,a Point-of-Care Diagnostic for Primaquine Therapy Screening

Development of reliable, easy-to-use, rapid diagnostic tests (RDTs) to detect glucose-6-phosphate dehydrogenase (G6PD) deficiency at point of care is essential to deploying primaquine therapies as part of malaria elimination strategies. We assessed a kit under research and development called CareStart™ G6PD deficiency screening test (Access Bio, New Jersey, USA) by comparing its performance to quantitative G6PD enzyme activity using a standardized spectrophotometric method (‘gold standard’). Blood samples (n = 903) were collected from Cambodian adults living in Pailin province, western Cambodia. G6PD enzyme activities ranged from 0 to 20.5 U/g Hb (median 12.0 U/g Hg). Based on a normal haemoglobin concentration and wild-type G6PD gene, the normal values of G6PD enzymatic activity for this population was 3.6 to 20.5 U/g Hg (95^th percentiles from 5.5 to 17.2 U/g Hg). Ninety-seven subjects (10.7%) had <3.6 U/g Hg and were classified as G6PD deficient. Prevalence of deficiency was 15.0% (64/425) among men and 6.9% (33/478) among women. Genotype was analyzed in 66 G6PD-deficient subjects and 63 of these exhibited findings consistent with Viangchang genotype. The sensitivity and specificity of the CareStart™ G6PD deficiency screening test was 0.68 and 1.0, respectively. Its detection threshold was <2.7 U/g Hg, well within the range of moderate and severe enzyme deficiencies. Thirteen subjects (1.4%, 12 males and 1 female) with G6PD enzyme activities <2 U/g Hg were falsely classified as “normal” by RDT. This experimental RDT test here evaluated outside of the laboratory for the first time shows real promise, but safe application of it will require lower rates of falsely “normal” results.     

Homogeneous Population of the Brown Alga Sargassum polycystum in Southeast Asia: Possible Role of Recent Expansion and Asexual Propagation

Southeast Asia has been known as one of the biodiversity hotspots in the world. Repeated glacial cycles during Pleistocene were believed to cause isolation of marine taxa in refugia, resulting in diversification among lineages. Recently, ocean current was also found to be another factor affecting gene flow by restricting larval dispersal in animals. Macroalgae are unique in having mode of reproduction that differs from that of animals. Our study on the phylogeographical pattern of the brown macroalga Sargassum polycystum using nuclear Internal Transcribed Spacer 2 (ITS2), plastidal RuBisCO spacer (Rub spacer) and mitochondrial cytochrome oxidase subunit-III (Cox3) as molecular markers revealed genetic homogeneity across 27 sites in Southeast Asia and western Pacific, in sharp contrast to that revealed from most animal studies. Our data suggested that S. polycystum persisted in single refugium during Pleistocene in a panmixia pattern. Expansion occurred more recently after the Last Glacial Maximum and recolonization of the newly flooded Sunda Shelf could have involved asexual propagation of the species. High dispersal ability through floating fronds carrying developing germlings may also contribute to the low genetic diversity of the species.     

CK2α is essential for embryonic morphogenesis

CK2 is a highly conserved serine-threonine kinase involved in biological processes such as embryonic development, circadian rhythms, inflammation, and cancer. Biochemical experiments have implicated CK2 in the control of several cellular processes and in the regulation of signal transduction pathways. Our laboratory is interested in characterizing the cellular, signaling, and molecular mechanisms regulated by CK2 during early embryonic development. For this purpose, animal models, including mice deficient in CK2 genes, are indispensable tools. Using CK2α gene-deficient mice, we have recently shown that CK2α is a critical regulator of mid-gestational morphogenetic processes, as CK2α deficiency results in defects in heart, brain, pharyngeal arch, tail bud, limb bud, and somite formation. Morphogenetic processes depend upon the precise coordination of essential cellular processes in which CK2 has been implicated, such as proliferation and survival. Here, we summarize the overall phenotype found in CK2α (-/- ) mice and describe our initial analysis aimed to identify the cellular processes affected in CK2α mutants.     

Molecular mechanisms underlying the inhibitory effects of bovine lactoferrin on osteosarcoma

Osteosarcoma (OS) is one the most common primary malignancies of the bone in children and young adults with high metastasis. The use of non-toxic naturally derived compounds is one of present strategies in OS therapy to reduce secondary effects and chemo-resistance. Lactoferrin (LF), a transferrin protein derived from milk, currently appears to be an anticancer agent. However, its suppressive effects on OS have not been fully investigated. Therefore, we aimed to examine the molecular mechanism underlying the inhibitory effects of bovine LF (bLF) on OS. OS cell lines (NOS1, U2OS, MG63, and 143B) and an osteoblastic (ST2) were treated with bLF. Effects of bLF on OS-cell proliferation and migration were examined by proliferation and wound-healing assays. Expression levels of low-density-lipoprotein-receptor-related protein 1 (LRP1) and cytokines including interleukin-1 beta (IL-1β), IL-6, and receptor-activator of nuclear factor kappa-Β ligand (RANKL) were measured using western blotting. Osteoclast formation was examined by co-culture of 143B, ST2, and bone marrow cells. We found that bLF down-regulated IL-1β, IL-6, and RANKL expression and suppressed phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 in 143B cells; bLF also drastically suppressed 143B-activated RANKL production in ST2 cells. This may have contributed to the reduction in the number of differentiated osteoclasts. Taken together, these data reveal that bLF down-regulates NF-κB to attenuate proliferation, migration, and bone resorption in OS and the OS-microenvironment. This study provides new findings and the precise underlying mechanisms of the inhibitory effects of bLF on OS. bLF can be a possible therapeutic agent for OS patients.     

Prevalence and Associated Risk Factors of Toxocara vitulorum Infections in Buffalo and Cattle Calves in Three Provinces of Central Cambodia

The prevalence and associated risk factors of Toxocara vitulorum infection in buffalo and cattle calves was studied in 3 provinces in central Cambodia. Fecal samples were collected from 517 calves between the age of 1-15 weeks and processed for nematode egg counts by a modified McMaster method. A total of 64 calves were found to excrete T. vitulorum eggs in their feces (12.4%; 95% exact CI: 9.7-15.5). The mean fecal egg count was 2,798 EPG (SD=16,351; range=0-224,400). A multivariable generalized linear mixed model showed higher odds of T. vitulorum infection for buffalo versus cattle, for animals aged 4-8 weeks versus younger and older ones, and for animals with strongyle infection. There was no association with fecal consistency. Farmers should be aware of the potential impact of T. vitulorum, and treat their calves at the age of 2-3 weeks with anthelmintics such as benzimidazoles or pyrantel.