Circadian Variations in the Affinities of Nad Kinase and Nadp Phosphatase for their Substrates, Nad+ and Nadp+ in Dividing and Nondividing Cells of the Achlorophyllous Zc Mutant of Euglena Gracilis Klebs (Strain Z)

-We have previously shown that NAD kinase and NADP phosphatase activities display circadian rhythms, in the soluble (SN) and membrane-bound (P) fractions of crude extracts of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis (which displays circadian rhythmicity of cell division). We determined if changes in the affinity of NADP phosphatase and NAD kinase for their substrates, NADP⁺ and NAD⁺, were occurring by calculating the ratios 100(velocity found in Km conditions/velocity found in saturating conditions). The rationale was that if the affinity remained unchanged according to circadian time (CI), these values should always equal 50, independently of any changes in enzyme quantity; values greater than 50 should indicate increases in enzyme affinity, and values less than 50 decreases in affinity. Our results indicated that these values calculated for NADP phosphatase exhibited a complex pattern of rhythmicity, while those for NAD kinase displayed circadian variations strongly correlated with the rhythms in enzyme activity. The curves showed troughs at CT 00-04 both in dividing and nondividing cells and peaks at CT 18-20 or at CT 08-14 in cells sampled, respectively, from a dividing or a stationary culture. Such variations are indicative of changes in the kinetic properties of the enzyme, which may reflect modifications in its affinity either for effectors (such as Ca2⁺-calmodulin) or for its substrate, NAD⁺. This may be due to (i) the expression of different isoenzymes at different CTs; (ii) different posttranslational modifications of the enzyme; or (iii) concentrations of effectors varying in a circadian manner.     

The proα2 (V) collagen gene (COL5A2) maps to 2q14→2q32, syntenic to the proα1 (III) collagen locus (COL3A1)

Summary A recombinant probe specific for the pro2 chain of human Type V collagen has been used for the localization of the corresponding gene (COL5A2) to chromosome 2. Regional mapping by in situ hybridization and analysis of DNA from humanxrodent cell lines indicated that COL5A2 is confined within the segment 2q142q32, thus syntenic to the pro1 (III) collagen gene (COL3A1).     

Abnormal electrophoretic mobility of spectrin tetramers in hereditary elliptocytosis

Summary Hereditary elliptocytosis (HE) is a genetically determined disorder of the red cell membrane. The main protein which composes the proteinaceous skeleton of the membrane is an elongated molecule named spectrin which is a heterodimer composed of two chains, and . In the membrane spectrin dimers are associated head-to-head to form tetrameric structures. We and other authors have reported that spectrin studied from many HE patients exhibited a dimer self-association defect (type I HE). A mutation in the head of the spectrin chain was mostly found in type I HE. We have previously described one of the three known spectrin pathological variants shown on mild tryptic digest pattern. This variant was characterized by the appearance of an abnormal 65,000-dalton peptide (Sp ^I/65). Using nondenaturating gel electrophoresis, we describe in this paper a triplicated pattern of the spectrin tetramer bands which is found in heterozygous HE cases displaying the 65,000-dalton variant. Study of a homozygous case allowed us to characterize the electrophoretic mobility of the abnormal symmetrical spectrin tetramer (₂ ^I/65-₂) and to study the correlation between the fraction of this abnormal symmetrical tetramer found in heterozygous patients and the amount of the 65,000-dalton peptide observed in spectrin tryptic digests.     

On the dynamics of the mass mortality of Diadema antillarum in Barbados

Widespread mortality of the black sea urchin Diadema antillarum occurred in the Caribbean in 1983; beginning in Panama in January, and having its major impact at Barbados in September. Mortality on ten reefs surveyed in Barbados was 93.2%, with the highest being 99.9% and the lowest 86.9%. Mortality on each reef was independent of the pre-mortality density on the reef. Urchins with test diameters between 20 and 40 mm were more severely affected than smaller or larger urchins. Populations on reefs exposed to incoming oceanic water suffered heavier mortality than those on protected reefs. Mean size of urchins was smallest on high density reefs. This may indicate a negative effect of density on urchin growth. At post-mortality densities, urchins may grow faster and reach sexual maturity sooner.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

Entrainment and phase shifting of the circadian rhythm of cell division by light in cultures of the achlorophyllous ZC mutant ofEuglena gracilis

The photosynthesis-deficient ZC mutant ofEuglena gracilis Klebs (strain Z) was cultured at 16°C on an aerated, magnetically stirred, mineral medium containing 0.1% ethanol (pH 7.0). Cell division could be entrained by a 12: 12 light: dark cycle (LD: 12, 12) or even by a one-pulse skeleton photoperiod (LD: 1,23) The rhythm free-ran in DD for at least 8 days with a circadian period (=25.5 h) in populations that had been previously entrained by LD. The freerunning rhythm could be phase-shifted by a single 1-h light pulse (3000 lx). The strong (Type 0) phase-response curve derived from the resetting effects of such signals given at different circadian times was similar to that for the photosynthetic wild-type strain. These results demonstrate that the presence of a functional chloroplast compartment is not necessary for the circadian clock to function inEuglena and suggest that phase resetting of the circadian clock by light occurs via a similar pathway in both photosynthetic and non-photosynthetic cell types.     

Effect of lysophosphatidylcholine on the NADH-ferricyanide reductase activity associated with the plasma membranes of corn roots

Plasma membranes from corn roots (Zea mays L.) were isolated by aqueous two-phase partitioning. A fraction enriched in a vanadate-sensitive ATPase showed characteristics of a plasma membrane ATPase. The sidedness of these vesicles was 89% right-side-out, as evaluated by the ATPase latency. A NADH-ferricyanide reductase was associated with these plasma membrane vesicles. The rate of ferricyanide reduction was 1.3 μmol · min⁻¹·mg⁻¹ protein and was strongly enhanced by the addition of lysophosphatidylcholine (LPC). The effect of this detergent on membrane solubilization and reductase activity was particularly studied. This type of detergent treatment revealed two pH optima (7.0 and 5.0) for the reductase activity, which exhibited biphasic kinetics in the absence or presence of the detergent. These data suggest that two or more reductases could be involved. In addition, membrane vesicle solubilization and determination of ATPase and reductase latency were simultanously studied. From these experiments, it is postulated that the reductase, which exhibits an optimum pH at 7.0 and is slightly stimulated by LPC, could be located on the external side of the plasmalemma. In contrast, the reductase at pH 5.0 strongly stimulated by the detergent treatment, is probably located on the internal side of the membrane, such as the catalytic site of ATPase. Finally, a possible direct action of LPC on the enzymes, is discussed.     

Involvement of the Dihydropyridine Receptor and Internal CaStores in Myoblast Fusion

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation–contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca²⁺associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca²⁺is mobilized via a machinery similar to that involved in excitation–contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 μM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca²⁺but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (α1 subunit of the L-type Ca²⁺channel) and the internal stores of Ca²⁺. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation–contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca²⁺is triggered by the type of mechanism already described in skeletal muscle.