Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.     

Small Mammals Ignore Common Rules: A Comparison of Vocal Repertoires and the Acoustics between Pup and Adult Piebald Shrews Diplomesodon pulchellum

A common rule for mammals vocalizing in the human audible frequency range (20 Hz–20 kHz) suggests that calls are higher in fundamental frequency (f0) in the young than in adults, because of the smaller sound‐producing structures of the young. Exclusions are rare, for example the pups of Asian house shrews (Suncus murinus) make some call types of the same or higher pitch than adults. In this study, calls from 62 piebald shrews (Diplomesodon pulchellum), 37 1 to 10‐d‐old pups from 10 litters and 25 adults were acoustically investigated in captivity. We found eight call types, all within the human audible frequency range: short and long low‐frequency squeaks with nearly flat contour, high‐frequency squeaks with modulated contour, high‐frequency squeaks with fractured contour, short and long screeches, clicks and whimpers. Seven call types were shared by pups and adults, suggesting that this vocal repertoire commences at birth. Against the common rule, the f0 of squeaks was the same in pups and adults, and the f0 of clicks and screeches was even higher in adults than in pups. These results suggest a non‐descending ontogenetic pathway that not follows the common physical relationship, of the lower f0 for the larger vocal folds.     

Effect of <Emphasis Type="Italic">m</Emphasis>-carbonyl cyanide 3-chlorophenylhydrazone on inorganic polyphosphates synthesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under different growth conditions

The effects of different concentrations of the protonophore uncoupler m-carbonyl cyanide 3-hchlorophenylhydrazone (CCCP) on the synthesis of inorganic polyphosphates (polyP) during the first 0.5 h of hypercompensation in the yeast Saccharomyces cerevisiae VKM Y-1173 growing on media with 2% glucose under low (hypoxia) or high aeration or with 1% (vol/vol) ethanol under high aeration were studied. It was shown that the yeast growth on ethanol was completely inhibited by 5 μM CCCP, while growth on glucose was inhibited by 25 μM CCCP, independently of aeration of the medium. The maximum rate of H2 absorption was shown at 2, 5, and 25 μM CCCP for the cells grown on ethanol, on glucose under high aeration, and on glucose under hypoxia, respectively. Against the decrease of total ATP level and total polyP, CCCP had a nonuniform effect on the synthesis of individual polyP fractions. CCCP maximally inhibited synthesis of the most actively formed fractions: polyPI during growth on glucose under hypoxia, polyPIII during growth on glucose under aeration, and polyPIII and polyPV during growth on ethanol. CCCP had no substantial effect on the synthesis of polyPII and polyPIV fractions, the formation of which seems to be less related to the electrochemical potential gradient of H⁺ ions.     

The ecology of the Calanus ponticus population in the deeper layer of its concentration in the Black Sea

The layer of daytime concentration of Calanus ponticus (VC andVI C) performing daily vertical migrations and the layer of‘winter stock’ aggregation are confined to the depthof maximal gradient of the main pycnocline under an unusuallysharp oxycline. The concentration layer thickness ranges from2 to 20–30 m and the Calanus concentration in it is >250ind. m^–3, sometimes being 3500 ind. m^–3 and evenmore. The population in the concentration layer is divided intotwo ecological groups: I, feeding and migrating specimens ofcopepodite stages V and VI, their body lipid contents being25–60 µg min.^–1; and II, non-feeding and non-migratingspecimens of copepodite stage V, their body lipid contents being100–150 µg ind.^–1. The relationship with oxygenconcentration was studied in both ecogroups. The experimentsshow that specimens of ecogroup II can exit at an oxygen concentrationof 0.06 ml 1^–1, but at such concentration falling intoanabiosis. They die at 0.04 ml O₂ 1^–1. Estimates of respirationof the group II specimens (‘winter stock’) showthat lipids they store are sufficient for 7 months' survival.Depth of Calanus concentration is determined by water densityrather than concentration of oxygen.     

Characteristic properties of metabolism of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> during the synthesis of α-ketoglutaric acid from ethanol

The study of free amino acid content in Yarrowia lipolytica cells grown on ethanol under thiamine deficiency showed that glutamate, alanine, and γ-aminobutyric acid (γ-ABA) occurred in the highest concentrations among the present 17 free amino acids. The culture liquid contained no amino acids. Analysis of the enzymes of oxidative metabolism in the yeast grown under these conditions showed that the cell-free homogenate contained substantial activity of glutamate decarboxylase, γ-ABA transaminase, and succinyl semialdehyde dehydrogenase. This result indicated the formation of succinate from glutamate in a reaction catalyzed by 4-aminobutyrate aminotransferase (γ-aminobutyrate bypass) under severe thiamine deficiency. These studies lead to the conclusion that cultivation of the yeast Y. lipolytica on ethanol under thiamine deficiency causes adaptive stress-induced metabolic changes. Increase of ammonium nitrogen consumption and excretion of α-ketoglutaric acid are indicative of physiological changes, the functioning of the γ-aminobutyrate bypass and high activity of malate dehydrogenase are manifestations of metabolic changes, and increased activities of the transamination reactions reflect the changes in nitrogen metabolism.     

Human biomarker discovery and predictive models for disease progression for idiopathic pneumonia syndrome following allogeneic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (SCT) is the only curative therapy for many malignant and nonmalignant conditions. Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication that limits successful outcomes. Preclinical models suggest that IPS represents an immune mediated attack on the lung involving elements of both the adaptive and the innate immune system. However, the etiology of IPS in humans is less well understood. To explore the disease pathway and uncover potential biomarkers of disease, we performed two separate label-free, proteomics experiments defining the plasma protein profiles of allogeneic SCT patients with IPS. Samples obtained from SCT recipients without complications served as controls. The initial discovery study, intended to explore the disease pathway in humans, identified a set of 81 IPS-associated proteins. These data revealed similarities between the known IPS pathways in mice and the condition in humans, in particular in the acute phase response. In addition, pattern recognition pathways were judged to be significant as a function of development of IPS, and from this pathway we chose the lipopolysaccaharide-binding protein (LBP) protein as a candidate molecular diagnostic for IPS, and verified its increase as a function of disease using an ELISA assay. In a separately designed study, we identified protein-based classifiers that could predict, at day 0 of SCT, patients who: 1) progress to IPS and 2) respond to cytokine neutralization therapy. Using cross-validation strategies, we built highly predictive classifier models of both disease progression and therapeutic response. In sum, data generated in this report confirm previous clinical and experimental findings, provide new insights into the pathophysiology of IPS, identify potential molecular classifiers of the condition, and uncover a set of markers potentially of interest for patient stratification as a basis for individualized therapy.     

Ultrasonic bouts of a blind climbing rodent (Typhlomys chapensis): acoustic analysis

ABSTRACT

The peculiar acoustic structure of ultrasonic bouts of blind climbing rodents Typhlomys might provide insight on their potential function. We examined 1481 bouts consisting of 1-6 pulses; 49.7% of them were single-pulse bouts. Bout duration and inter-bout interval depended on the number of pulses per bout, whereas period from start of a previous bout to start of the next bout was constant (80.0±2.9 ms). Ultrasonic pulses (540 pulses measured in a subset of 234 bouts) were short (0.68±0.15 ms) sweeps with fundamental frequency slopes from 127.3±6.3 kHz to 64.1±4.6 kHz and peak frequency at 93.3±7.4 kHz, emitted within bouts with inter-pulse periods of 13.03±3.01 ms. Single pulses and start pulses of multi-pulse bouts were lower in frequency than other pulses of the bouts. In contrast, pulse duration was independent on pulse position within bout. Pulses of Typhlomys were reminiscent of echolocation calls of Murina and Myotis bats, but higher in frequency, much shorter, fainter, displayed a convex contour of frequency modulation and only the fundamental frequency band without harmonics and were organized in bouts, that is not characteristic for bat echolocation. Most probably, Typhlomys uses their ultrasonic pulses for call-based orientation during locomotion, including climbing and jumping among bush branches.     

Oxidative Damage to DNA under the Action of an Alternating Magnetic Field

The content of damaged 8-hydroxy-2-deoxyguanosine nitrogenous bases in the blood DNA of healthy donors and patients with epidermolysis bullosa after exposure to an alternating magnetic field of 550 ± 30 A/m in the frequency range from 3 to 60 Hz in vitro was determined using an enzyme immunoassay. The degree of oxidative DNA damage in epidermolysis bullosa was almost twice as high as in healthy donors. It was shown that there was a significant increase in the level of 8-oxoguanine in the DNA of both groups after the magnetic field treatment, which depended in a complex way on the frequency. The resulting effect is explained by the generation of reactive oxygen species under the influence of a magnetic field and the disruption of DNA repair processes.

     

On criteria of optimal conditions at ballistic disintegration of microbial cells

Summary The present study examines the processes of ballistic disintegration carried out to release active biopolymers. The relationship between the activity of unit volume of homogenates and the time of disintegration has been obtained. Correctness of this relationship for any process of disintegration can serve as the criterion of optimal chemical composition of the medium in which this process occurs. Only two quantitative criteria have been found sufficient for comparison of any regimes of ballistic disintegration. The theoretical conclusions are proved by experiments in ballistic disintegrators of two types.Torulopsis candida cells were used to release the succinate oxidizing enzyme.     

Uronic acid content in polysaccharides: Estimation by decarboxylation involving use of gas chromatography

A quantitative method for determining uronic acid content in different polysaccharides has been described. The decarboxylation reaction was run with 57% hydriodic acid (HI) in a small flask attached directly to the gas circuit of a chromatograph. After the reaction was completed, the resultant carbon dioxide was supplied to the chromatographic column. A sample uronic acid (0.5–10.0 μmol) yielded quantitative results with a relative standard deviation of 2%. The contribution of nonuronic materials to the error did not exceed 0.6%. The total analysis time amounted to 50–60 min.