Sphagnum divinum (sp. nov.) and S. medium Limpr. and their relationship to S. magellanicum Brid.

Sphagnum magellanicum has been viewed as being a predominantly circumpolar species in the northern hemisphere, but it occurs in the southern hemisphere and was originally described from the southern parts of Chile. It is an ecologically important species in mire ecosystems and has been extensively used as a model to study processes of growth, carbon sequestration and peat decomposition. Molecular and experimental studies have, however, revealed genetic structure within S. magellanicum, and morphological differences associated with these genetic groups. Here we describe Sphagnum divinum in Sphagnum subgenus Sphagnum (Sphagnaceae, Bryophyta) as a new species, based on molecular and morphological evidence. Sphagnum medium is reinstated as a distinct species and is epitypified. Consequently, a new species concept of S. magellanicum is presented including an epitypification. Important morphological characters to separate these three species in the field and under the microscope are presented. Ecology and distribution differ among the species; S. divinium has a wide habitat range including mire margin, forested peatlands and moist heaths, and a circumpolar distribution around the northern hemisphere. Sphagnum medium seems to be more restricted to ombrotrophic mire expanse habitats and shows an amphi-Atlantic distribution in the northern hemisphere. Sphagnum magellanicum has a very broad ecological niche in peatlands and is found in most mire habitats in Tierra del Fuego on the southern tip of South America.     

Decreased pulmonary diffusing capacity of divers over a 6-year period

Non-smoking, male, professional firemen divers (n = 15) underwent two pulmonary function tests (PFT) separated by 6 years. Measured data were compared to European Coal Steel Community recommended reference values to permit cross-sectional and then longitudinal study. Higher vital capacity (VC; P < 0.01) and forced expiratory volume in 1 s (FEV₁; P < 0.05), and lower maximal mid-expiratory flow (MMEF) coefficient with VC (MMEF/VC; P < 0.05) were observed in both PFT. Diver's pulmonary diffusing capacity (DL_CO) and the coefficient with alveolar volume (DL_CO/V _A) showed significantly (P < 0.001) different evolution profiles than those expected from predicted values. In divers, DL_CO and DL_CO/V _A decreased from 104.0% to 91.4% and from 106.4% to 91.5% of predicted values respectively. Changes in DL_CO and DL_CO/V _A correlated positively with the initial measurement of DL_CO (r = 0.67, P < 0.01) and DL_CO/V _A (r = 0.74, P < 0.01) respectively, whereas no correlation between changes in pulmonary gas transfer function and age or diving history parameters was found. Thus, it is suggested from our observations that hyperbaric atmosphere exposure increases the effects of aging on pulmonary diffusing capacity and that pulmonary gas transfer function should be regularly tested in professional and recreational divers. Accepted: 22 February 1997     

Biotinylation sites of tumor necrosis factor-alpha determined by liquid chromatography-mass spectrometry.

Tumor pretargeting with biotinylated antibody/avidin complexes improves the therapeutic index of systemically administered biotin-tumor necrosis factor (TNF) conjugates. Since the number of biotins in this conjugate is known to be critical for activity, we have characterized the structure of different biotin-TNF conjugates, prepared by reaction with d-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester and identified the biotinylation sites by trypsin digestion, reverse-phase chromatography, and electrospray mass spectrometry analyses. The results have shown that N-terminal valine is a preferential biotinylation site at pH 5.8, half of biotins being located on the alpha-amino group of this residue in a conjugate bearing one biotin/trimer (on average). Moreover, evidence has been obtained to suggest that the remaining part of biotins are linked to the epsilon-amino group of lysine 128, 112, and 65, while lysine 11, 90, and 98 were practically unmodified. No evidence of O-biotinylation of serine, threonine and tyrosine was obtained.     

X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: Detection of heterozygotes by selective medium

Skin fibroblasts from five unrelated males with X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency and from their families have been exposed to medium containing 6-thioguanine. This purine analogue selects against cells with normal hypoxanthine-guanine phosphoribosyl transferase activity and therefore permits detection of mutant cells in heterozygous populations. The results of these studies are compared to those obtained by autoradiography of single-cell clones of skin fibroblasts from the same subjects. In each case, the results of the selective method are similar to those obtained by clonal analysis. The use of selective medium therefore provides a sensitive means to detect heterozygosity at this locus and may provide a general method to select cells with X-linked markers from heterozygous populations.This work was supported by grants from the U.S.P.H.S. (#HD 00486), The Joseph P. Kennedy, Jr., Memorial Fluid Research Fund, and the National Foundation for Neuromuscular Diseases, Inc.     

Hybridization of somatic cells derived from mouse and Syrian hamster: Evolution of karyotype and enzyme studies

Somatic hybrids of drug-resistant mutant hamster and mouse cell lines have been isolated and propagated in long-term culture and have been studied in respect to karyotype and three enzymes. During the course of propagation the long-surviving hybrid clones show progressive loss of telocentric chromosomes associated in at least one case with loss of mouse enzyme. Hybrid clones showed hybrid molecules for malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6PGD) made up by recombination of parental subunits.This work was supported by National Institutes of Health Grant HD 00486.     

Central and peripheral mechanisms in the control of the diurnal rhythm of flashing in Luciola lusitanica (Charp.)

The mechanisms underlying the diurnal rhythm of flashing in L. lusitanica were investigated. It was shown that the diurnal rhythm of flashing was present also in animals kept in continuous darkness, showing that it was not directly determined by the periodic variations of the ambient light intensity which take place through the nicthemeron. During the hours in which the animals did not flash, photogenic volleys of normal size and contour could still be recorded, although at a lower frequency; however, they were not followed by light emission. Statistical controls ruled out the possibility that the absence of flashing during daytime was due to the reduced repetition rate of the photogenic volleys. The ablation of the gonads performed during day-time was followed by the reappearence of a flash in the wake of each photogenic volley. It is concluded that two mechanism control the diurnal rhythm of flashing, namely; a central mechanism, responsible for the decrease in volley frequency; and a peripheral mechanism, of gonadal origin, which is responsible for the inhibition of the photogenic organ.     

Increasing the Time Resolution of Single-Molecule Experiments with Bayesian Inference

Many time-resolved single-molecule biophysics experiments seek to characterize the kinetics of biomolecular systems exhibiting dynamics that challenge the time resolution of the given technique. Here, we present a general, computational approach to this problem that employs Bayesian inference to learn the underlying dynamics of such systems, even when they are much faster than the time resolution of the experimental technique being used. By accurately and precisely inferring rate constants, our Bayesian inference for the analysis of subtemporal resolution dynamics approach effectively enables the experimenter to super-resolve the poorly resolved dynamics that are present in their data.     

Role of lysine187 within the second extracellular loop of the type A cholecystokinin receptor in agonist-induced activation. Use of complementary charge-reversal mutagenesis to define a functionally important interdomain interaction

Activation of guanine nucleotide-binding protein (G protein)-coupled receptors is believed to involve conformational change that exposes a domain for G protein coupling at the cytosolic surface of the helical confluence, although the mechanisms for achieving this are not well understood. This conformational change can be achieved by docking a diverse variety of agonist ligands, known to occur by interacting with different regions of these receptors. In this study, we focus on the importance of a specific basic residue (Lys187) within the second extracellular loop of the receptor for the peptide hormone, cholecystokinin. Alanine-replacement and charge-reversal mutagenesis of this residue showed that it had no effect on the binding of natural peptide and nonpeptidyl ligands of this receptor but markedly interfered with agonist-stimulated signaling. It was demonstrated that this negative effect on biological activity could be eliminated with the truncation of the first 30 residues of the amino-terminal tail of this receptor. Complementary charge-reversal mutagenesis of each of the five conserved acidic residues within this region of the receptor in the presence of the charge-reversed Lys187 revealed that only the Asp5 mutant fully reversed the negative functional impact of the Lys187 charge reversal. Thus, we have demonstrated that a basic residue within the second extracellular loop of the cholecystokinin receptor interacts with a specific acidic residue within the amino terminus of this receptor. This residue-residue interaction is nicely accommodated within a new molecular model of the agonist-occupied cholecystokinin receptor.     

Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria

In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199-208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (Delta Psi), the compartment with a lower dication concentration positive. However, the Delta Psi values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at Delta Psi >100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian Delta Psi, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered Delta Psi. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria.