Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Electrorotation--a new method for investigating membrane events during thrombocyte activation. Influence of drugs and osmotic pressure

The measurement of the spin of cells in rotating high-frequency electric fields ('electrorotation') make possible the investigation of dielectric membrane properties of single cells. This method was applied to membrane permeability changes accompanying thrombocyte activation and compared with light-scattering data. Describing the dielectric behavior of platelets by a single-shell model and assuming a sufficiently low membrane conductivity of 1 X 10(-7) S/m we found for nonactivated platelets a membrane capacity of 5.5 mF/m2 and the conductivity of the internal medium was estimated to be 0.12 S/m. Upon activation, the electrorotation decreased continuously, with half-times in the range of few minutes. This could be explained assuming a 500-fold increase in membrane conductivity. The application of both local anesthetics and virostatics inhibited the decrease of electrorotation, as did hypertonic osmotic pressure. In all cases this was accompanied by inhibition of platelet aggregation. Hypotonic solutions induced self-aggregation and spontaneous loss of electrorotation. It was concluded that the increase in permeability of the granule membrane is a crucial step in the release reaction and that the electrorotation method is able to detect the incorporation of the granule membranes into the plasma membrane during activation. The advantage of this electrorotation method is that it enables measurements on a single-cell level, thus avoiding interactions between platelets.     

Die Verbreitung von Vitamin K1 und Plastochinon in Pflanzen

Zusammenfassung Nach einem streng spezifischen Verfahren (Genauigkeit ±10%) wird die Verbreitung von Plastochinon 45 und Vitamin K₁ in Beziehung zu Chlorophyll a, -Carotin und, soweit ausgebildet, Blattfläche bestimmt. Geprüft wurden Vertreter der Cyano-, Chloro-, Charo-, Phaeo-, Rhodo- und Pteridophyta, Gymno- und Angiospermae.In allen Fällen wird bei Gegenwart von Chlorophyll auch Vitamin K₁ gefunden, begleitet von einem Überschuß an Plastochinon. Nur bei einem Teil der Rotalgen war kein oder nur sehr wenig PQ nachweisbar. Im allgemeinen liegen die Pigmentverhältnisse in den schranken von a:PQ:VK₁=100:10±5:1±0,5. Ausnahmen bilden, neben den Rotalgen, nur Aureablätter (a) und Sonnenblätter mancher Laubbäume (b). Bei (a) bleibt die Chlorophyllsynthese so stark zurück, daß trotz normaler Chinonausstattung pro Blattfläche und normaler Photosynthese extrem niedrige Quotienten a:PQ und a:VK₁ entstehen. Bei (b) folgt nach Abschluß der Chlorophyllsynthese und nach Erreichen maximaler Leistung ein starker Plastochinonanstieg, ohne daß sich der Vitamin K₁-Gehalt vergleichbar erhöht.Chromoplasten führendes Gewebe enthält die Plastidenchinone (PQ und VK₁) nur, wenn die Chromoplasten durch Metamorphose aus Chloroplasten hervorgegangen sind (Herbstlaub, Früchte), nicht aber, wenn sie überwiegend aus Proplastiden entstehen (Blumenblätter). Eine Chinonsynthese im Chromoplasten ist auszuschließen. Chloro- und Chromoplasten entbehrendes Gewebe führt auch keine Chinone (außer den Ubichinonen, die in Mitochondrien zu finden sind).Mit 4 Textabbildungen     

Synthetic lipid-anchored receptors based on the binding site of a monoclonal antibody.

Highly specific ligand receptor interactions generally characterize molecular recognition at cell surfaces and other biological systems. In this study we simulate a membrane receptor by fusing a monoclonal antibody fragment to a phospholipid. A sulfhydryl group in the hinge region of a monoclonal antibody fragment, was covalently linked to derivatives of phosphatidylethanolamines and phosphatidylserine via three different hydrophilic spacer arms. We investigated and characterized these lipid-anchored Fab-fragments which we have named 'Fab-lipids' in liposomal and monolayer systems. Methods for the monomolecular assembling of such films at the air/water interface and techniques used for their manipulation are outlined. We describe two possibilities for building a monomolecular receptor layer, consisting of two-dimensional pattern of oriented Fab-fragments with their artificial hydrophobic anchor embedded in a lipid matrix. In the first method a monomolecular film at the air/water interface was allowed to form from a vesicular suspension and driven into a phase separation, resulting in protein rich domains embedded in a protein depleted phase. This film was transferred onto a solid support in such a way that the established pattern was preserved. Alternatively, a recognition pattern was formed by directly cross-linking the Fab-fragments to preformed planar membranes composed of the reactive spacer-lipids and an inert matrix lipid. Specificity as well as contrast of the binding activity of the receptor layers were qualified using micro-fluorimetry.     

Paired sequence difference in ribosomal RNAs: evolutionary and phylogenetic implications

Ribosomal RNAs have secondary structures that are maintained by internal Watson-Crick pairing. Through analysis of chordate, arthropod, and plant 5S ribosomal RNA sequences, we show that Darwinian selection operates on these nucleotide sequences to maintain functionally important secondary structure. Insect phylogenies based on nucleotide positions involved in pairing and the production of secondary structure are incongruent with those constructed on the basis of positions that are not. Furthermore, phylogeny reconstruction using these nonpairing bases is concordant with other, morphological data.      

A Novel Cytotoxic Cerium Complex: Aquatrichloridobis(1,10‐phenanthroline)cerium(III) (KP776). Synthesis,Characterization, Behavior in H2O,Binding towards Biomolecules,and Antiproliferative Activity

The lanthanide complex aquatrichloridobis(1,10‐phenanthroline)cerium(III) [Ce(phen)₂(H₂O)Cl₃] (KP776) was fully characterized by elemental analysis, IR‐, and ¹H‐ and ¹³C‐NMR spectroscopy, as well as TG/DTA measurements, and its behavior in H₂O, important for the application as a chemotherapeutic, was studied. In addition, the binding of KP776 to nucleotides and single serum proteins was investigated by capillary electrophoresis, whereas binding to proteins in human plasma was observed by ICP‐MS. The compound shows promising anticancer properties in vitro: proliferation of human cancer cell lines is strongly inhibited with IC₅₀ values in the very low micromolar range.     

Effect of two β-alanine dosing protocols on muscle carnosine synthesis and washout

Carnosine (β-alanyl-L-histidine) is found in high concentrations in skeletal muscle and chronic β-alanine (BA) supplementation can increase carnosine content. This placebo-controlled, double-blind study compared two different 8-week BA dosing regimens on the time course of muscle carnosine loading and 8-week washout, leading to a BA dose-response study with serial muscle carnosine assessments throughout. Thirty-one young males were randomized into three BA dosing groups: (1) high-low: 3.2 g BA/day for 4 weeks, followed by 1.6 g BA/day for 4 weeks; (2) low-low: 1.6 g BA/day for 8 weeks; and (3) placebo. Muscle carnosine in tibialis-anterior (TA) and gastrocnemius (GA) muscles was measured by 1H-MRS at weeks 0, 2, 4, 8, 12 and 16. Flushing symptoms and blood clinical chemistry were trivial in all three groups and there were no muscle carnosine changes in the placebo group. During the first 4 weeks, the increase for high-low (TA 2.04 mmol/kgww, GA 1.75 mmol/kgww) was ~twofold greater than low-low (TA 1.12 mmol/kgww, GA 0.80 mmol/kgww). 1.6 g BA/day significantly increased muscle carnosine within 2 weeks and induced continual rises in already augmented muscle carnosine stores (week 4-8, high-low regime). The dose-response showed a carnosine increase of 2.01 mmol/kgww per 100 g of consumed BA, which was only dependent upon the total accumulated BA consumed (within a daily intake range of 1.6-3.2 g BA/day). Washout rates were gradual (0.18 mmol/kgww and 0.43 mmol/kgww/week; ~2%/week). In summary, the absolute increase in muscle carnosine is only dependent upon the total BA consumed and is not dependent upon baseline muscle carnosine, the muscle type, or the daily amount of supplemented BA.