Characteristics and regulation of active calcium transport in inside-out red cell membrane vesicles

Sealed, inside-out human red cell membrane vesicles, prepared by a modified method of Steck (Steck T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol 2, pp. 245–281, Plenum Press, New York), accomplish an ATP and Mg²⁺-dependent uphill calcium uptake with a reproducible maximum rate of 12–15 nmol/mg vesicle protein per min under physiological conditions. This maximum rate is increased by about 60–70% in the presence of a heatstable cytoplasmic activator protein (calmodulin) obtained from red cells. Calcium efflux from inside-out vesicles is smaller than 0.01 nmol/mg vesicle protein per min at intravesicular calcium concentrations between 0.1 and 20.0 mM.In the presence of Mg²⁺, active calcium uptake is supported by ATP, ITP, or UTP, but not by ADP, AMP, or p-nitrophenyl phosphate. The optimum pH for the process is 7.4–7.6, and the activation energy is 19–20 kcal/mol, irrespective of the presence or absence of calmodulin. Calcium uptake in inside-out vesicles is unaffected by ouabain or oligomycin, but blocked by low concentrations of lanthanum, ruthenium red, quercetin and phloretin. K⁺ and Na⁺, when compared to choline⁺ or Li⁺, significantly increase active calcium uptake. This stimulation by K⁺ and Na⁺ is independent of that by calmodulin.Concentrated red cell cytoplasm activates calcium uptake at low soluble protein:membrane protein ratios, while a ‘deactivation’ of the transport occurs at high cytoplasm: membrane protein ratios. A heat-labile cytoplasmic protein fraction antagonizing calmodulin activation, can be separated by DEAE-Sephadex chromatography. Based on these findings the regulation of active calcium transport in human red cells is discussed.     

Reconstruction of the proximal ends of retiolitid rhabdosomes (Graptolithina) from the Upper Wenlock and the Lower Ludlow

Detailed study of the ultrastructures on the cortical lists of retiolitids occurring after the lundgreni event has been undertaken. The extremely well preserved material from the Go?dap borehole of Poland (Baltica) enables classification of clathrial lists into two types—with single and double seams. Significantly different seam development is observed between two groups of retiolitids occurring before and after the lundgreni event. The study of seams and increments on the lists enables reconstruction of the membrane arrangement of the rhabdosomes. Reconstruction of the proximal ends, the arrangement of the ancora umbrella, and the layout of the proximal ventral and lateral orifices of five genera, Neogothograptus, Holoretiolites, Plectograptus, Spinograptus, and Quattuorgraptus n. gen., is presented. The new genus Quattuorgraptus is separated from Spinograptus because of the different development of the ancora umbrella and the width of the lateral walls of the rhabdosome.     

Changes in embryonic 8-cell nuclei transferred by means of cell fusion to mouse eggs.

Metaphase II and activated mouse oocytes were fused with 8-cell blastomeres, and morphological changes in the transferred nuclei were followed using light and electron microscopy. In metaphase II oocytes, blastomere nuclei underwent premature chromosome condensation (PCC) typical for S-phase nuclei: chromatin pulverization. Then an abortive spindle was formed without evident microtubule organizing centers. Blastomere chromosomes condensed to a lesser degree than meiotic chromosomes and lacked mature functional, trilaminar kinetochores. After parthenogenetic activation of these oocytes, blastomere chromosomes followed, in synchrony with oocyte chromatin, a similar route of changes (anaphase, telophase) and then reformed interphase nuclei of the pronuclear type. Remodeling of 8-cell nucleus thus occurred, but the integrity of the chromatin set was frequently disturbed by formation of micronuclei. If blastomere fusion with oocytes was done close to activation (either before or after parthenogenetic stimulation), the chances of remodeling of the nuclei decreased, because PCC was not regularly induced in all oocytes. In hybrids produced 60 min or later after oocyte activation, blastomere nuclei were maintained in interphase without any structural modifications. Multiple experiments in the mouse have shown that the nuclei from 8-cell stage transferred to enucleated oocytes and egg cells are not capable of substituting for pronuclear functions. Possible reasons for impaired functional reprogramming of 8-cell nucleus in the mouse are discussed in light of our present findings on the morphology of nuclei transferred before and after oocyte activation.     

Presence of covalently bound energy-rich phosphates in human tracheal smooth muscle myosin

In a preliminary report, the tracheal NaCl-myosin prepared from an old and a young subject was discussed. In the present paper, the total bound phosphate (P) content and its distribution is described in two parallel preparations of human muscle myosin. It was shown that a considerable amount of covalently bound P was present not only in NaCl, but in the fresh preparations of tracheal KCl-myosin. Analysing this phosphate fraction in the alkaline hydrolysate of RNA- and lipid-free preparations of myosin it was confirmed that phosphate was linked to the basic amino acid residues and their hydrolytic derivatives. As the phosphoryl binding sites are partly saturated, the phosphate concentration can be enhanced nearly three-fold compared to the fresh preparation. Phosphate incorporation is an autophosphorylation process depending on the ATP and Mg2+ concentration. Studying the actomyosin fraction in the presence of ATP it was found that its phosphate content can also be increased to a certain degree. It is supposed that the changes in phosphate content of myosin are associated with the formation of crossbridges between the actin and myosin filaments in the processes of muscle contraction and relaxation. The process can be influenced directly and indirectly by some natural factors and drugs resulting in the concentration or relaxation of bronchial muscles.     

Pharmacogenetically hazardous drugs: a proposed new scoring method.

Similar doses of a drug given to different individuals can result in widely disparate plasma concentrations and hence effects. Beside intraindividual differences also inter-ethnic differences of drug response must be taken into consideration. Both inter-individual and inter-ethnic variations of drug response are mostly related to genetic factors (polymorphism) involved in drug metabolism and kinetics. The farmacogenetic disorders involved clinically result in pharmacogenetic side effects. In order to avoid pharmacogenetic side effects, beside phenotyping of the patients, selection of drugs subjected to different pharmacogenetic disorders may be of great clinical importance. Therefore, a scoring method was carried out for the selection of pharmacogenetically hazardous drugs. With regard to both genetic and environmental factors influencing the drug response, 140 suspicious drugs were studied and classified with the method. Eighteen was the maximum point value for genetic and 12 for contributing factors involved, so 30 was the maximum point number in each drug studied. Out of 140 substances 50 drugs (qualified with 20 points or more) proved to be hazardous in different pharmacogenetic disorders, among them several widely used agents, e.g. Diazepam, Isoniazid, Phenytoin, Warfarin, Quinidine, Tolbutamide, etc. The article sums up the findings in a Table and comments them. This scoring method may be useful in drug safety and preventive medicine.     

Comparative investigation on the extractibility of acetylcholinesterase from normal and dystrophic muscles

Following combined administration of nonionic detergent and collagenase considerable differences were observed in extractibility of acetylcholinesterase activity from normal and from dystrophic rabbit muscles. The results obtained in the experiments carried out on vitamin-E deficient rats corroborated the authors' previous experiences obtained in vitamin-E deficient rabbits. However, as to the effect of the detergent, considerable differences were observed between the two species. If one compares the activities referred to 1 g muscle or to the total tissue mass it is clear that primarily the concentration of acetylcholinesterase was increased. The activity measured in the presence of butyrylthiocholine increased only to small extent in vitamin-E deficient dystrophy.     

Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization

We reinvestigated rearrangements occurring in region q13 of chromosome 11 aiming to: (i) describe heterogeneity of the observed structural alterations, (ii) estimate amplicon size and (iii) identify of oncogenes involved in laryngeal cancer progression as potential targets for therapy. The study included 17 cell lines derived from laryngeal cancers and 34 specimens from primary laryngeal tumors. The region 11q13 was analyzed by fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and gene expression microarray. Next, quantitative real time PCR was used for chosen genes to confirm results from aCGH and gene expression microarray. The observed pattern of aberrations allows to distinguish three ways, in which gain and amplification involving 11q13 region may occur: formation of a homogeneously staining region; breakpoints in/near 11q13, which lead to the three to sevenfold increase of the copy number of 11q13 region; the presence of additional copies of the whole chromosome 11. The minimal altered region of gain and/or amplification was limited to ~1.8 Mb (chr.11:69,395,184–71,209,568) and comprised mostly 11q13.3 band which contain 12 genes. Five, out of these genes (CCND1, ORAOV1, FADD, PPFIA1, CTTN) had higher expression levels in comparison to healthy controls. Apart from CCND1 gene, which has an established role in pathogenesis of head and neck cancers, CTTN, ORAOV1 and FADD genes appear to be oncogene-candidates in laryngeal cancers, while a function of PPFIA1 requires further studies.