Studies on the catalytic mechanism of H2-forming methylenetetrahydromethanopterin dehydrogenase: para-ortho H2 conversion rates in H2O and D2O

H₂–forming N ⁵,N ¹⁰ –methylenetetrahydromethanopterin dehydrogenase is a novel type of hydrogenase that contains neither nickel nor iron-sulfur clusters. Evidence has been presented that the reaction mechanism catalyzed by the enzyme is very similar to that of the formation of carbocations and H₂ from alkanes under superacidic conditions. We present here further results in support of this mechanism. It was found that the purified enzyme per se did not catalyze the conversion of para H₂ to ortho H₂, a reaction catalyzed by all other hydrogenases known to date. However, it catalyzed the conversion in the presence of the substrate N ⁵,N ¹⁰ –methenyltetrahydromethanopterin (CH≡H₄MPT⁺), indicating that for heterolytic cleavage of H₂ the enzyme-CH≡H₄MPT⁺ complex is required. In D₂O, the formation of HD and D₂ from H₂ rather than a para–ortho H₂ conversion was observed, indicating that after heterolytic cleavage of H₂ the dissociation of the proton from the enzyme-substrate complex is fast relative to the re-formation of free H₂.     

A receptor for protein import into potato mitochondria

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.     

Identification of Ser-Pro and Thr-Pro Phosphorylation Sites in Chicken Neurofilament-M Tail Domain

Abstract: The tail domain of the midsize chicken neurofilament polypeptide (NF-M) contains several different types of Ser-Pro and Thr-Pro putative phosphorylation sites. We determined which of these sites are actually phosphorylated in vivo. Chick sensory neuron cultures were incubated in [³²P]phosphate, and the cytoskeletal fraction was mixed with a neurofilament fraction prepared from adult chicken brain. NF-M was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with chymotrypsin, and two large fragments were isolated. These were individually cleaved with trypsin, endoprotease Lys-C, or endoprotease Glu-C, and peptides separated by two-dimensional high-voltage electrophoresis and thin-layer chromatography. ³²P-labeled phosphopeptides were eluted from the cellulose plates and subjected to microsequencing and mass spectometry. We found that of 21 potential Ser-Pro and Thr-Pro phosphoacceptor sites, at least 20 are phosphorylated in vivo: all four Lys-Ser-Pro sites and at least 16 of the 17 Lys-Xaa-Xaa-Ser/Thr-Pro repeats. In addition, a novel Ser-Pro site in the extreme carboxy terminus is phosphorylated. This site, which has no proximal Lys residue, is also found in mammalian NF-M, but has not been reported to be phosphorylated. Together with three casein kinase I sites we have found recently in the acidic amino-terminal segment of the tail, a total of 24 or 25 Ser and Thr phosphoacceptor sites have now been located in the chicken NF-M tail.     

The stationary state of epithelia

A tissue is a geometrical, space-filling, random cellular network; it remains in this steady state while individual cells divide. Cell division is a local, elementary topological transformation which establishes statistical equilibrium of the structure. We describe the physical conditions to maintain stationary the epidermis (of mammals or of the cucumber), in spite of the fact that cells constantly divide and die. Specifically, we study the statistical equilibrium of the basal layer, a corrugated surface filled with cells, constituting a two-dimensional topological froth. Cells divide and detach from the basal layer, and these two topological transformations are responsible for the stationary state of the epidermis. The topological froth is capable of responding rapidly and locally to external constraints, and is a good illustration of the plasticity of random cellular networks.Statistical equilibrium is controlled by entropy, both as a measure of disorder and as information, and is characterized by observable relations between average cell shapes and sizes. The technique can be applied to any random cellular network in dynamical equilibrium. Mitosis as the dominating topological transformation and the fact that the distribution of cell shapes is very narrow are the only inputs specific to biology.

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Resume Un tissu est, à première vue, un pavage aléatoire d'une surface ou d'un volume par des polygones (polyèdres) topologiques, les cellules. Ce pavage reste dans un état stationnaire alors que les cellules se divisent constamment. Nous décrivons les conditions physiques nécessaires à l'état stationnaire de l'épiderme (des mammifères et du concombre), en dépit du fait que ses cellules se divisent et meurent. En particulier, nous étudions l'équilibre statistique de la couche basale, une surface couverte de cellules constituant une mousse topologique aléatoire. Les cellules se divisent et se détachent de la couche basale, et ces transformations topologiques sont responsable de l'état stationnaire de l'épiderme. Cette mousse topologique est capable de répondre rapidement et localement à des contraintes externes. C'est un exemple de plasticité de structures cellulaires aléatoires.L'équilibre statistique est contrôlé par l'entropie qui est ici à la fois une mesure du désordre et une quantité d'information. Il est caractérisé par des relations facilement observables entre les formes des cellules et leurs dimensions. Les seuls éléments spécifiques aux tissus biologiques sont la mitose comme transformation topologique dominante, et l'étroitesse de la distribution des formes de cellules.

     

Purification and properties of rabbit uterus preuteroglobin mRNA.

The mRNA for preuteroglobin, a precursor of the hormonally induced protein uteroglobin, has been partially purified from the endometrium of progesterone treated rabbits. The purification procedure starts with total endometrial polysomes and involves treatment with proteinase K and dodecylsulfate, chromatography on oligo(dT)-cellulose, sucrose density gradient centrifugation, electrophoresis in polyacrylamide gels containing dodecylsulfate, and a second absorption to oligo(dT)-cellulose. The final mRNA preparation codes exclusively for preuteroglobin in a wheat germ cell-free system and migrates as a single band in polyacrylamide gels containing 99% formanide. The average length of the poly(A) segment is 60 nucleotides and the translation of the preuteroglobin mRNA is inhibited by m7G(5')ppp(5')A, indicating that it contains a "capped" 5'-terminus. Comparison with known standards yields a molecular weight of 200,000 (600 nucleotides) for preuteroglobin mRNA, approximately twice as many nucleotides as required for encoding the 90 aminoacids of its cell-free product.     

Induction of structural chromosome aberrations and sister chromatid exchanges in human lymphocytes in vitro by aristolochic acid

The medicinal use of Aristolochia clematitis has been known for some time. The main active agent of this medicinal plant is aristolochic acid, a nitrophenanthrenecarbonic acid. Very recently, however, the Federal Health Office withdrew the licence for all drugs containing aristolochic acid, because of the well-founded suspicion that aristolochic acid may be a very potent carcinogen. We investigated the induction of structural chromosome aberrations and sister chromatid exchanges (SCEs) by aristolochic acid in human lymphocytes in vitro. Cells were treated with the agent tested throughout culture time and during the G0 phase of the cell cycle. We tested concentrations over a range of 1 to 20 micrograms/ml. Both treatment conditions resulted in an increased aberration frequency. The induction of gaps and breaks as well as the induction of SCEs showed a dose-dependent increase. The number of SCEs per metaphase was enhanced by a factor of 2 to 3. If conventional cytogenetic methods had been applied in time, one would have recognized the mutagenic risk of aristolochic acid earlier.     

Chronopharmacokinetics and Cardiovascular Effects of Nifedipine

Orcadian phase dependency in pharmacokinetics and hemodynamic effects on blood pressure and heart rate of different galenic formulations of nifedipine (immediate-release, sustained-release, and i.v. solution) were studied in healthy subjects or in hypertensive patients. Pharmacokinetics of immediate-release but not sustained-release and i.v. nifedipine were dependent on time of day: immediate-release nifedipine had higher C_max (peak concentration) and shorter t_max (time-to-peak concentration) after morning than evening application, and bioavailibility in the evening was reduced by about 40%. Orcadian rhythm in estimated hepatic blood flow as determined by indocyanine green kinetics may contribute to these chronokinetics. A circadian time dependency was also found in nifedipine-induced effects on blood pressure and heart rate as monitored by 24-h ambulatory blood pressure measurements. In conclusion, the dose response relationship of oral nifedipine is influenced by the circadian organization of the cardiovascular system as well as by the galenic drug formulation.     

Ungew?hnliches Verhalten von Zugv?geln in Gibraltar: St?rung durch Flugzeuge

Zusammenfassung Ein ungewöhnliches Verhalten von Singvögeln während des Frühjahrszuges wurde in Gibraltar beobachtet. Die tags auf dem Felsen von Gibraltar rastenden Vögel zogen in den Beobachtungsnächten nicht in die übliche Frühjahrszugrichtung, sondern entfernten sich nach allen Himmelsrichtungen vom Felsen. Das als Fluchtverhalten interpretierte Verhalten wird auf Manövervorbereitungen zurückgeführt, die zu einer außerordentlich erhöhten Flugfrequenz von Strahlflugzeugen um den Felsen herum führte.

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Unusual behaviour of passerine migrants in Gibraltar: disturbance by jet plane

Summary An unusual behaviour of passerine migrants could be observed during spring migration in Gibraltar. Birds having rested during daytime on the rock of Gibraltar, at dawn did not start to migrate towards northerly directions, but removed from the rock towards all directions. This was interpreted as a escape behaviour. It was attributed to the very intensified frequency of flights of jet planes around the rock, which occured in the course of preparations of military exercises.