Purification and characterization of intracellular galactose oxidase from Dactylium dendroides

The intracellular galactose oxidase from Dactylium dendroides was purified to homogeneity with a 64% yield. The enzyme is a glycoprotein (7.7% neutral sugars, 1.7% aminosugars) with 72,000 Da of molecular mass. The enzyme showed nonlinear double reciprocal plots with O2 and D-galactose, suggesting cooperative binding for both substrates. The intracellular galactose oxidase catalyzes the oxidation of galactose derivatives and dihydroxyacetone but not of glycerol, glycolaldehyde, beta-hydroxipyruvate, and allyl alcohol which are substrates for the extracellular enzyme. Compared with the extracellular galactose oxidase, the intracellular enzyme showed higher carbohydrate content and sensitivity to diethyldithiocarbamate.     

Purification and properties of pyridine nucleotide-independent L-lactate dehydrogenase from Polyporus circinatus

Cell extracts of Polyporus circinatus grown on lactate catalyze the reduction of 2,6-dichlorophenolindophenol by l-lactate without the participation of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. The enzyme has been purified 78-fold and was homogenous by disc gel electrophoresis. The optimal pH was found to be 6.7. The Michaelis constant for l-lactate was 5.9 x 10(-4) M and the oxalate inhibition constant was 1.5 x 10(-4) M. The nature of the prosthetic group is discussed.     

Rhythmical timing and spatial scattering of foraging in a homer limpet {Patella rustica)

Homing to a more or less permanent scar after each foragingexcursion is a common movement pattern among intertidal gastropodsand chitons; however, details of the timing and spacing of foragingactivity have been investigated only in a few species. The presentstudy analyzes the short-term behavior of the limpet Patellarustica along the Tyrrhenian coast, Italy, using a motographictechnique to assess the fine organization of its foraging duringfavorable periods of sea roughness. P. rustica becomes activeonce the upper midlittoral is well splashed. It alternates foragingexcursions and resting at home with a periodicity slightly longerthan 12 h, suggesting a tidal-diel pattern. However, periodogramanalysis of the sea level oscillations during the study periodsrevealed no such rhythmicity because tidal oscillations werehidden by irregular variations caused by waves. As a resultof this time partitioning, limpets move, on average, less than50% of their potential activity time. Time partitioning maybe highly adaptive in reducing potential risks. Nevertheless,in the absence of clear external driving cues, the significanceof a very regular and apparently tidal pattern, fairly synchronousamong the different specimens, remains to be explained. Theactivity of P. rustica during each excursion is organized intothree parts: the outgoing journey during which grazing activityprogressively increases, a central part characterized by intensegrazing, and the return characterized by fast displacement anda more or less consistent trail following. Limpets head forrandom directions to reach foraging grounds in successive excursions,showing only a slight avoidance of the direction taken duringthe previous outward journey. This pattern produces a spatialscattering of grazing activity, allowing efficient exploitationof grazing areas distributed radially around home during subsequentexcursions.     

Metabolomics and lipid profile analysis of Coccomyxa melkonianii SCCA 048

Extremophiles - With an unsupervised GC–MS metabolomics approach, polar metabolite changes of the microalgae Coccomyxa melkonianii SCCA 048 grown under standard conditions for seven weeks...     

Head Movements Evoked in Alert Rhesus Monkey by Vestibular Prosthesis Stimulation: Implications for Postural and Gaze Stabilization

The vestibular system detects motion of the head in space and in turn generates reflexes that are vital for our daily activities. The eye movements produced by the vestibulo-ocular reflex (VOR) play an essential role in stabilizing the visual axis (gaze), while vestibulo-spinal reflexes ensure the maintenance of head and body posture. The neuronal pathways from the vestibular periphery to the cervical spinal cord potentially serve a dual role, since they function to stabilize the head relative to inertial space and could thus contribute to gaze (eye-in-head + head-in-space) and posture stabilization. To date, however, the functional significance of vestibular-neck pathways in alert primates remains a matter of debate. Here we used a vestibular prosthesis to 1) quantify vestibularly-driven head movements in primates, and 2) assess whether these evoked head movements make a significant contribution to gaze as well as postural stabilization. We stimulated electrodes implanted in the horizontal semicircular canal of alert rhesus monkeys, and measured the head and eye movements evoked during a 100ms time period for which the contribution of longer latency voluntary inputs to the neck would be minimal. Our results show that prosthetic stimulation evoked significant head movements with latencies consistent with known vestibulo-spinal pathways. Furthermore, while the evoked head movements were substantially smaller than the coincidently evoked eye movements, they made a significant contribution to gaze stabilization, complementing the VOR to ensure that the appropriate gaze response is achieved. We speculate that analogous compensatory head movements will be evoked when implanted prosthetic devices are transitioned to human patients.     

Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

Background

Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies. As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy.

Methodology/Principal Findings

Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections.

Conclusions/Significance

Multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and Giardia compared to microscopy, especially in samples exhibiting polyparasitism. The superior performance of multiplex PCR to detect polyparasitism and more accurately determine infection intensity suggests that it is a more appropriate technique for use in epidemiologic studies and for monitoring large-scale intervention trials.     

Behavior of the extremophile green alga Coccomyxa melkonianii SCCA 048 in terms of lipids production and morphology at different pH values

Extremophiles - The extremophile green alga Coccomyxa melkonianii SCCA 048 was investigated to evaluate its ability to grow in culture media with different pH. Specifically, Coccomyxa melkonianii...     

High-pass filtering of input signals by the Ih current in a non-spiking neuron, the retinal rod bipolar cell

Hyperpolarization-activated cyclic nucleotide-sensitive (HCN) channels mediate the I(f) current in heart and I(h) throughout the nervous system. In spiking neurons I(h) participates primarily in different forms of rhythmic activity. Little is known, however, about its role in neurons operating with graded potentials as in the retina, where all four channel isoforms are expressed. Intriguing evidence for an involvement of I(h) in early visual processing are the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors. Moreover, electroretinographic recordings indicate that these drugs affect temporal processing in the outer retina. Here we analyzed the functional role of HCN channels in rod bipolar cells (RBCs) of the mouse. Perforated-patch recordings in the dark-adapted slice found that RBCs exhibit I(h), and that this is sensitive to the specific blocker ZD7288. RBC input impedance, explored by sinusoidal frequency-modulated current stimuli (0.1-30 Hz), displays band-pass behavior in the range of I(h) activation. Theoretical modeling and pharmacological blockade demonstrate that high-pass filtering of input signals by I(h), in combination with low-pass filtering by passive properties, fully accounts for this frequency-tuning. Correcting for the depolarization introduced by shunting through the pipette-membrane seal, leads to predict that in darkness I(h) is tonically active in RBCs and quickens their responses to dim light stimuli. Immunohistochemistry targeting candidate subunit isoforms HCN1-2, in combination with markers of RBCs (PKC) and rod-RBC synaptic contacts (bassoon, mGluR6, Kv1.3), suggests that RBCs express HCN2 on the tip of their dendrites. The functional properties conferred by I(h) onto RBCs may contribute to shape the retina's light response and explain the visual side effects of HCN inhibitors.