A STABLE ISOTOPE METHOD FOR MEASUREMENT OF THYMIDINE INCORPORATION INTO DNA

A method has been developed for the measurement of DNA synthesis in vivo using the incorporation of multilabeled, non-radioactive thymidine. Simultaneous intraperitoneal injection of hexalabeled thymidine and tritiated thymidine into a normal adult rat resulted in the incorporation of both labeled nucleosides into the DNA of cells undergoing replication. The DNA of several tissues and organs was analysed, including liver, thymus, spleen, bone marrow, and small intestine. Following extraction with hot trichloroacetic acid, acid hydrolysis, and thin-layer chromatography of the hydrolysates, the isotopic compositions of the thymine products were determined by field ionization mass spectrometry and by scintillation counting. The relative incorporation of radioactive and stable isotope-labeled thymidine was similar in all tissues, and corresponded to the ratio of the two labeled nucleosides in the injected material. These results indicate the feasibility of utilizing thymidine multilabeled with stable isotopes for measurement of cellular proliferation rates in conjunction with cancer therapy.     

Rate of non-adherent cell loss from long-term cultures of murine bone marrow: effect of medium conditioned by the adherent cell population

The rate of random spontaneous cell loss from the non-adherent cell population of long-term cultures of murine bone marrow was determined. The measured rate of non-adherent cell loss is affected by previous conditioning of the medium in which the non-adherent cells are suspended. Specifically, the measured rate of non-adherent cell loss is significantly slowed when the non-adherent cells are suspended in medium conditioned in long-term haematopoietic cultures instead of fresh medium. It appears that a subset of the adherent cell population is the source of the factor or factors within the medium which result in this slower measured rate of non-adherent cell loss. This effect may be due to the stimulation to division of haematopoietic progenitor cells, offsetting the lysis of other non-adherent cells.     

Determination of imipramine in plasma by high pressure liquid chromatography and field ionization mass spectrometry: increased sensitivity in comparison with gas chromatography mass spectrometry

A quantitative method is reported for the determination of imipramine in plasma samples in the low nanogram and subnanogram range. The sensitivity and precision of the technique, which involves high pressure liquid chromatography and direct probe field ionization mass spectrometry, are approximately an order of magnitude greater than are offered by gas chromatography mass spectrometry with selected ion monitoring using deuterated or other types of internal standards. [2H6]Imipramine, labeled in the ethylene bridge and in the aromatic rings, serves as the isotopic diluent. The method has been used for the determination of the comparative bioavailabilities of two different commercial preparations of imipramine. In these tests, subjects ingested a 25 mg tablet of one or the other drug preparation together with a solution containing an equivalent amount of imipramine deuterated in the ethylene bridge ([2H2]imipramine). The latter served as an internal check for intrasubject variability in absorption of the imipramine tablets. Typical results from one of the subjects are presented.     

Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis

The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997-1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.     

Revised classification/nomenclature of vitiligo and related issues: the Vitiligo Global Issues Consensus Conference

During the 2011 International Pigment Cell Conference (IPCC), the Vitiligo European Taskforce (VETF) convened a consensus conference on issues of global importance for vitiligo clinical research. As suggested by an international panel of experts, the conference focused on four topics: classification and nomenclature; definition of stable disease; definition of Koebner's phenomenon (KP); and 'autoimmune vitiligo'. These topics were discussed in seven working groups representing different geographical regions. A consensus emerged that segmental vitiligo be classified separately from all other forms of vitiligo and that the term 'vitiligo' be used as an umbrella term for all non-segmental forms of vitiligo, including 'mixed vitiligo' in which segmental and non-segmental vitiligo are combined and which is considered a subgroup of vitiligo. Further, the conference recommends that disease stability be best assessed based on the stability of individual lesions rather than the overall stability of the disease as the latter is difficult to define precisely and reliably. The conference also endorsed the classification of KP for vitiligo as proposed by the VETF (history based, clinical observation based, or experimentally induced). Lastly, the conference agreed that 'autoimmune vitiligo' should not be used as a separate classification as published evidence indicates that the pathophysiology of all forms of vitiligo likely involves autoimmune or inflammatory mechanisms.     

Solution structure of molybdic acid from Raman spectroscopy and DFT analysis

Protonation of produces the well-characterized polymolybdates, but at concentrations below 10⁻³ M the dominant species is monomeric molybdic acid, H₂MoO₄. It is likely to be the species adsorbed on manganese oxide, a process thought to control levels in the ocean, because of the strong proton dependence of adsorption. The molecular structure of H₂MoO₄ is elusive, since it occurs only in dilute solutions. Using 244 nm laser excitation, near resonance with O → Mo charge-transfer electronic transitions of H₂MoO₄, we have detected a 919 cm⁻¹ Raman band assignable to ν_sMoO. Using DFT, we have computed geometries and vibrational modes for the various structures consistent with the H₂MoO₄ formula. We tested the computations on a series of Mo(VI) oxo complexes with known vibrational frequencies, at several levels of theory. Best agreement with experimental values, at reasonable computational cost, was obtained with the B3LYP functional, employing a LANL2DZ ECP basis set for Mo and the 6-311+G(2df,p) basis set for O and H. Among the possible H₂MoO₄ structures only those based on the MoO₃ unit, with one, two or three coordinated water molecules, gave a scaled frequency for ν_sMoO that was within two standard deviations of 919 cm⁻¹. Best agreement was obtained for MoO₃(H₂O)₃. The MoO₂ and MoO structures gave frequencies that were too high. The Mo(OH)₆ structure could be excluded, because its vibrational frequencies shift down strongly upon H/D exchange, whereas the 919 cm⁻¹H₂MoO₄ band shifts up 1 cm⁻¹ in D₂O.     

Ultrastructure and osmoregulatory function of the kidney in larvae of the Persian sturgeon Acipenser persicus

The localization of Na(+) , K(+) -ATPase (NKA) and the ultrastructural features of kidney were examined in larvae of the Persian sturgeon Acipenser persicus (L 31-41 mm total length and 182·3-417·3 mg). Investigations were conducted through light and electron microscopy and through immunofluorescence for NKA detection. The kidney nephrons consisted of a large glomerulus and tubules (neck, proximal, distal and collecting), which connected to the ureters. Posteriorly, ureters extended and joined together into a thin-walled ureter terminal sac. Ultrastructurally, the glomerular cells (podocytes) possessed distinctive pedicels that extended to the basal membrane. The proximal tubule (PT) showed two different cells. The cells lining the anterior part of PT possessed apical tall microvilli (c. 2·7 μm), a sub-apical tubular system, a basal nucleus and dense granules. Posteriorly in the cells, the sub-apical tubular system and granules were absent and round mitochondria associated with basolateral infoldings were found; the apical microvilli were reduced. Distal and collecting tubular cells showed the typical features of osmoregulatory cells, i.e. well-developed basolateral infoldings associated with numerous mitochondria. No immunofluorescence of NKA was detected in the glomeruli. A weak immunostaining was observed at the basolateral side of the cells lining the neck and PT. A strong immunostaining of NKA was observed in the entire cells of the distal tubules, collecting tubules and in some isolated cells of the ureters. In all immunostained cells, the basolateral region showed a much higher fluorescence and nuclei were immunonegative. In conclusion, the epithelial cells of kidney tubules had morphological and enzymatic features of ionocytes, particularly in the distal and collecting tubules. Thus, the kidney of A. persicus larvae possesses active ion exchange capabilities and, beside its implication in excretion, participates in osmoregulation.     

Ordinary stoichiometry of extraordinary microorganisms

All life on Earth seems to be made of the same chemical elements in relatively conserved proportions (stoichiometry). Whether this stoichiometry is conserved in settings that differ radically in physicochemical conditions (extreme environments) from those commonly encountered elsewhere on the planet provides insight into possible stoichiometries for putative life beyond Earth. Here, we report measurements of elemental stoichiometry for extremophile microbes from hot springs of Yellowstone National Park (YNP). Phototrophic and chemotrophic microbes were collected in locations spanning large ranges of temperature (24 °C to boiling), pH (1.6–9.6), redox (0.1–7.2 mg L^?1 dissolved oxygen), and nutrient concentrations (0.01–0.25 mg L^?1 , 0.7–12.9 mg L^?1 , 0.01–42 mg L^?1 NH₄⁺, 0.003–1.1 mg L^?1 P mostly as phosphate). Despite these extreme conditions, the microbial cells sampled had a major and trace element stoichiometry within the ranges commonly encountered for microbes living in the more moderate environments of lakes and surface oceans. The cells did have somewhat high C:P and N:P ratios that are consistent with phosphorus (P) limitation. Furthermore, chemotrophs and phototrophs had similar compositions with the exception of Mo content, which was enriched in cells derived from chemotrophic sites. Thus, despite the extraordinary physicochemical and biological diversity of YNP environments, life in these settings, in a stoichiometric sense, remains much the same as we know it elsewhere.