First evidence of the tooth eruption sequence of the upper jaw in Hyaenodon (Hyaenodontidae,Mammalia) and new information on the ontogenetic development of its dentition

Juvenile material with the main focus on the upper jaw of the fossil predator Hyaenodon was evaluated to study the tooth eruption sequence and to examine the ontogeny of its dentition in detail. The comparison in size of milk to permanent teeth indicates a growth rate of 12–16 % in Hyaenodon. The thin section of a deciduous canine of a North American taxon shows four dental rings. Based on the knowledge of recent carnivores, this implies an age of 3–4 years in the last stage of tooth eruption and thus a long juvenile phase. The mandibles ascertained the most recent established tooth eruption sequence for North American and European species. For the first time ever, juvenile material from Asia is documented and interpreted. This study likewise shows a difference in the sequence of the upper jaw: the first upper premolar erupts before the first upper molar in North American species, whereas the European taxa show an earlier eruption of the first upper molar. This fact further confirms the divergence between the Hyaenodon lineages from North America and Europe.     

State-of-the art data normalization methods improve NMR-based metabolomic analysis

Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples.

     

Time course of pump inhibition by ouabain in amphibian epithelia

Inhibition by ouabain of rheogenic Na⁺ transport across the basolateral membranes of frog skin is found to be manifest within 3–4 min. This rate of pump inhibition is not different from the rate of diffusion through extracellular tissue layers between the serosal bath and the actual site of action, i.e., the epithelial cell layers. It is concluded that the well-known slow time course of decrease in transepithelial current flow is due ionic redistribution and conductance changes of the epithelial membranes secondary to pump inhibition.     

Microelectrode studies of potential difference responses to changes in stromal K+ in bullfrog cornea

The effects of changing stromal K+ were studied using microelectrodes in an in vitro preparation of frog cornea. The intracellular potential (V0) responded in two opposite ways under short-circuit conditions: (1) depolarization (normal response) when stromal K+ was increased from 4 to 20 or to 79 mM, about 30 mV per 10-fold K+ concn. change; (2) a hyperpolarization (anomalous response) of 10 mV maximum when stromal K+ was increased from 0 to 4 mM. The increase from 4 to 20 or 79 mM decreased or even reversed the short-circuit current (Isc). The transepithelial conductance (gt) increased when K+ was increased to 79 mM but no change occurred in the apical membrane fractional resistance (fRo). Increase of stromal K+ from 0 to 4 mM increased Isc and minimally changed gt and fRo. Ouabain (10(-3) M) abolished the anomalous responses, that is, the increases in V0 and Isc when stromal K+ was increased from 0 to 4 mM. These results are interpreted in terms of two K+ conductive pathways in the basolateral membrane of the corneal epithelium, a Nernstian conductance and an electrogenic (Na+ + K+)-ATPase pump transporting more Na+ than K+ ions per cycle. The normal or anomalous potential difference responses to changes in stromal K+ appear to depend on the relative resistance of the two pathways at the time stromal K+ is changed.     

Na+ currents generated by the purified (Na+ + K+)-ATPase on planar lipid membranes

Purified (Na+ + K+)-ATPase from pig kidney was attached to black lipid membranes and ATP-induced electric currents were measured as described previously by Fendler et al. ((1985) EMBO J. 4, 3079-3085). An ATP concentration jump was produced by an ultraviolet-light flash converting non-hydrolysable caged ATP to ATP. In the presence of Na+ and Mg2+ this resulted in a transient current signal. The pump current was not only ATP dependent, but also was influenced by the ATP/caged ATP ratio. It was concluded that caged ATP binds to the enzyme (and hence inhibits the signal) with a Ki of approx. 30 microM, which was confirmed by enzymatic activity studies. An ATP affinity of approx. 2 microM was determined. The addition of the protonophore 1799 and the Me+/H+ exchanger monensin made the bilayer conductive leading to a stationary pump current. The stationary current was strongly increased by the addition of K+ with a K0.5 of 700 microM. Even in the absence of K+ a stationary current could be measured, which showed two Na+-affinities: a high-affinity (K0.5 less than or equal to 1 mM) and a low-affinity (K0.5 greater than or equal to 0.2 M). In order to explain the sustained electrogenic Na+ transport during the Na+-ATPase activity, it is proposed, that Na+ can replace K+ in dephosphorylating the enzyme, but binds about 1000-times weaker than K+. The ATP requirement of the Na+-ATPase was the same (K0.5 = 2 microM) with regard to the peak currents and the stationary currents. However, for the (Na+ + K+)-ATPase the stationary currents required more ATP. The results are discussed on the basis of the Albers-Post scheme.     

Effects of light and acetate on the liberation of zoospores by a mutant strain ofChlamydomonas reinhardtii

In light-dark-synchronized cultures of the unicellular green algaChlamydomonas reinhardtii, release of zoospores from the wall of the mother cell normally takes place during the second half of the dark period. The recently isolated mutant ls, however, needs light for the liberation of zoospores when grown photoautotrophically under a 12 h light-12 h dark regime. The light-induced release of zoospores was found to be prevented by addition of the photosystem-II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Furthermore, light dependence of this process was shown to be abolished when the mutant ls was grown either photoautotrophically under a 14 h light-10 h dark regime or in the presence of acetate. Our findings indicate that the light-dependency of zoospore liberation observed in cultures of this particular mutant during photoautotrophic growth under a 12 h light-12 h dark regime might be attributed to an altered energy metabolism. The light-induced release of zoospores was found to be prevented by addition of cycloheximide or chloramphenicol, antibiotics which inhibit protein biosynthesis by cytoplasmic and organellar ribosomes, respectively. Actinomycin D, an inhibitor of RNA synthesis, however, did not affect the light-induced liberation of zoospores.Sporangia accumulate in stationary cultures of the mutant ls. Release of zoospores was observed when these sporangia were collected by centrifugation and incubated in the light after resuspension in fresh culture medium. Since liberation of zoospores was not observed after dilution of the stationary cultures with fresh culture medium, we suppose that components which interfere with the action of the sporangial autolysin are accumulated in the culture medium of the mutant ls.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea