Relationship between hydroxypyruvate and the production of oxalate in vitro

Chicken liver lactate dehydrogenase (l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to l-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD⁺/NADH ratio is greater than unity.Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the l-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, the l-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme.     

Tree dynamics and co‐existence in the montane–sub‐alpine ecotone: the role of different light‐induced strategies

Questions: Is light availability the main factor driving forest dynamics in Pyrenean sub‐alpine forests? Do pines and firs differ in growth, mortality and morphological response to low light availability? Can differences in shade tolerance affect predictions of future biome changes in Pyrenean sub‐alpine forests in the absence of thermal limitation? Location: Montane–sub‐alpine ecotones of the Eastern Pyrenees (NE Spain). Methods: We evaluated morphological plasticity, survival and growth response of saplings of Scots pine, mountain pine and silver fir to light availability in a mixed forest ecotone. For each species, we selected 100 living and 50 dead saplings and measured size, crown morphology and light availability. A wood disk at root collar was then removed for every sapling, and models relating growth and mortality to light were obtained. Results: Fir had the lowest mortality rate (<0.1) for any given light condition. Pines had comparable responses to light availability, although in deep shade Scots pine risked higher mortality (0.35) than mountain pine (0.19). Pines and fir developed opposing strategies to light deprivation: fir employed a conservative strategy based on sacrificing height growth, whereas pines enhanced height growth to escape from shade, but at the expense of higher mortality risk. Scots pine showed higher plasticity than mountain pine for all architectural and morphological traits analysed, having higher adaptive capacity to a changing environment. Conclusions: Our results support the prediction of future biome changes in Pyrenean sub‐alpine forests as silver fir and Scots pine may find appropriate conditions for colonizing mountain pine‐dominated stands due to land‐use change‐related forest densification and climate warming‐related temperature increases, respectively.     

Elucidating Emergence and Transmission of Multidrug-Resistant Tuberculosis in Treatment Experienced Patients by Whole Genome Sequencing

Background

Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years.

Methods and Findings

We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort.

Conclusions

Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.     

Induction of proliferation of neuroretina cells by long terminal repeat activation of the carboxy-terminal part of c-mil.

Expression of the P100gag-mil protein of avian retrovirus MH2 in cultured chicken embryo neuroretina cells was previously shown to result in the proliferation of normally quiescent cell populations. We show here that long terminal repeat activation of the carboxy terminus of the c-mil gene is sufficient to induce neuroretina cell proliferation.     

Regulation of L-Type Calcium Channels in GH4 Cells via A1 Adenosine Receptors

Abstract: Identification of A₁ adenosine receptors (A₁Rs) in a tumor cell line derived from rat pituitary (GH₄ cells) was performed by ligand binding and immunological experiments. Subsequently, the involvement of A₁Rs in the regulation of calcium conductance was studied in these cells. The agonist N ⁶-( R )-(2-phenylisopropyl)adenosine ( R -PIA) did not modify the intracellular calcium basal levels, whereas it inhibited the increase produced by 15 m M KCl depolarization. The antagonist 1,3-dipropyl-8-cyclopentylxanthine led to the opening of voltage-dependent cell surface calcium channels in the absence of exogenous KCl. The channels were of the L type because the effect was abolished by calciseptine and by verapamil. These results suggest that endogenous adenosine exerts a tonic inhibitory effect on calcium transport. This was confirmed by the high adenosine concentration found in cell supernatants (up to 1 µ M ) and by the calcium mobilization produced by exogenously added adenosine deaminase. In depolarizing conditions, the calcium peak in the presence of adenosine deaminase was reduced when cells were preincubated with R -PIA, thus suggesting that A₁R activation regulates the intensity of depolarization. These results demonstrate that adenosine is an important regulator of the physiological state of pituitary tumor cells by modulating, in an autocrine manner, the activity of L-type voltage-dependent calcium channels.     

Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.     

Population biology of the potato tuber moth, Phthorimaea operculella (Lepidoptera: Gelechiidae), in two potato cropping systems in Israel

The life cycle, within-field distribution, crop damage and impact of natural enemies of the potato tuber moth, Phthorimaea operculella (Zeller) were investigated in two potato cropping systems. The two systems differed in soil type (sandy vs. loess) which in turn affected the choice of cultivars, irrigation programme, insecticide application method (ground vs. aerial), and planting and harvest times. From mid-April to the end of May, almost twice as many moths were caught in pheromone traps in sandy than in loess fields. Highest infestation of tubers was found before harvest, and infestation was greater in loess than in sandy fields. Larval densities in foliage and tubers were significantly higher at the margins of the fields than in the centre. A significant positive correlation was found between adult catch and larval infestation on foliage in sandy fields but not in loess. Tuber infestation in sand was positively correlated with foliage infestation. No such correlation was detected in loess. Five parasitic wasps emerged from P. operculella larvae collected from commercial fields and volunteer plants: Diadegma pulchripes (Kokujev) and Temelucha decorata, (Gravenhorst) (Ichneumonidae) and Bracon gelechiae Ashmead and two other unidentified Braconidae. The most abundant predators at the field site were Coccinella septempunctata Linnaeus (Coccinellidae), Chrysoperla carnea Stephens (Chrysopidae), Orius albidipennis (Reuter) (Anthocoridae) and four ant species (Formicidae). Parasitism rate reached 40% and predation was estimated at 79%. Results are discussed with regard to the development of an integrated pest management programme for this important pest.