[A method for predicting the onset of menarche]. / Eine Methode zur Vorausbestimmung des Menarcheeintritts.

For the period of 1981-1984 the author collected data on the menarche age of more than 32 thousand girls aged between 10-18.5 years by the so-called status-quo method. The evaluation was carried out by means of 19 factors. A model was developed on purpose to prognosticate the age of menarche.

Thioredoxins and the redox modulation of glucose-6-phosphate dehydrogenase in Anabaena sp. strain PCC 7120 vegetative cells and heterocysts.

Glucose-6-phosphate dehydrogenase (G6PDH) was isolated from heterocysts and vegetative cells of Anabaena sp. strain PCC 7120. Both enzyme preparations proved to be more active in their oxidized than in their reduced forms. At least one protein with thioredoxin activity was found in Anabaena sp. which, if reduced with dithiothreitol, deactivated the G6PDH preparations. The deactivated heterocyst G6PDH could be reactivated neither by O2 nor by oxidized thioredoxin. Reactivation of the enzyme was, however, achieved by oxidized glutathione or H2O2. The active form of Anabaena G6PDH was readily deactivated by heterologous thioredoxin(s). The Anabaena thioredoxin(s) modulated heterologous enzymes.

Modification of the fatty acid composition of phospholipids during the hypersensitive reaction in tobacco.

When Nicotiana tabacum cv. Xanthi nc. plants were inoculated with tobacco mosaic virus (TMV), grown at 33 degrees for 6 days, then transferred to 25 degrees C to induce necrotic reaction, a rapid saturation of microsomal phospholipid-bound fatty acids was observed. Lipoxygenase activity increased to four times that of the control activity prior to the observed changes in both the fatty acid saturation and the increase in permeability.

Interaction between hysteretic regulation and redox modulation of glucose-6-phosphate dehydrogenase from Anacystis nidulans.

The glucose-6-phosphate dehydrogenase (G6PDH) of cyanobacteria is a hysteretic enzyme which is also subject to redox modulation [FEBS Lett. 126 (1981) 85-88]. We have found that the hysteretic and redox properties of G6PDH exhibit specific interactions: (1) The hysteretic forms of G6PDH ('hypoactive' in equilibrium 'hyperactive'), obtained at pH 7.5 and 6.5, respectively, differ in their redox properties. The 'hypoactive' form is easily activated by oxidation whereas the 'hyperactive' form is easily deactivated by reduction. (2) At low G6P concentrations (greater than 1 mM) only the oxidized form of G6PDH has significant activity. An increase in G6P level diminishes the difference between the activity of oxidized and reduced G6PDH forms.

Regulatory properties of a fructose 1,6-bisphosphatase from the cyanobacterium Anacystis nidulans.

A fructose 1,6-bisphosphatase (EC 3.1.3.11) (FBPase) was purified over 100-fold from Anacystis nidulans. At variance with a previous report (R. H. Bishop, Arch. Biochem. Biophys. 196:295-300, 1979), the regulatory properties of the enzyme were found to be like those of chloroplast enzymes rather than intermediate between chloroplast (photosynthetic) and heterotrophic FBPases. The pH optimum of Anacystis FBPase was between 8.0 and 8.5 and shifted to lower values with increasing Mg2+ concentration. Under the experimental conditions used by Bishop, we found the saturation curve of the enzyme to be sigmoidal for Mg2+ ions and hyperbolic for fructose 1,6-bisphosphate. The half-maximal velocity of the Anacystis FBPase was reached at concentrations of 5 mM MgCl2 and 0.06 mM fructose 1,6-bisphosphate. AMP did not inhibit the enzyme. The activity of the FBPase was found to be under a delicate control of oxidizing and reducing conditions. Oxidants like O2, H2O2, oxidized glutathione, and dehydroascorbic acid decreased the enzyme activity, whereas reductants like dithiothreitol and reduced glutathione increased it. The oxido-reductive modulation of FBPase proved to be reversible. Reduced glutathione stimulated the enzyme activity at physiological concentrations (1 to 10 mM).l The reduced glutathione-induced activation was higher at pH 8.0 than at pH 7.0.

Isolation and properties of an isocitrate dehydrogenase from Anacystis nidulans.

A NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) was isolated and purified over 400-fold from Anacystis nidulans. The enzyme activity responded slowly to rapid changes in ligand (NADP+, isocitrate, Mg2+-ions) or enzyme concentration as well as to rapid changes in temperature. These are properties characteristic of the hysteretic enzymes. In addition, the enzyme activity was subject to product (alpha-ketoglutarate) inhibition. ATP, ADP and CDP also inhibited the enzyme. Unlike several other cyanobacterial enzymes, the isocitrate dehydrogenase of Anacystis is not under redox control.

Accumulation of guanosine tetraphosphate (ppGpp) under nitrogen starvation in Anacystis nidulans, a cyanobacterium.

The effect of nitrate deprivation on cell growth and nucleotide level was studied in Anacystis nidulans. A 10-fold reduction in nitrate level resulted in a drastic slowdown of growth. Upon addition of nitrate to the starving cultures, after a lag period, the cells resumed growth. Nutritional shift-down induced a transitory expansion of the guanosine tetraphosphate (ppGpp) pool, preceded by a transitory increase in GTP and ATP concentrations. After having reached peak values, the concentration of ppGpp, GTP and ATP dropped to the respective base levels. The expansion of the ppGpp pool was found to be due to an increase in ppGpp synthesis, rather than to a decrease in ppGpp breakdown. After nutritional shift-up, no decrease in the ppGpp level was found. In starving cells, a decrease in free amino acids was observed to occur concomitantly with the expansion of the ppGpp pool. The level of free amino acids started to increase simultaneously with the contraction of the ppGpp pool.

Redox modulation of a phosphatase from Anacystis nidulans.

Ascorbic acid (AA) increased the phosphatase activity (pH 6.8) in 10,000 g supernatants from Anacystis nidulans. The enzyme activated by AA was deactivated by dehydroascorbic acid (DHAA). The modulation by AA/DHAA of phosphatase activity in Anacystis appears to be specific; a number of other redox compounds, known to modulate other enzymes, had no effect on the Anacystis phosphatase. A purified phosphatase preparation from Anacystis was also deactivated by DHAA. In contrast, the purified enzyme was not activated by AA, suggesting that a factor mediating the effect of AA was lost during purification. Another factor was found to protect the purified phosphatase against deactivation by DHAA. The enzyme was characterized as a phosphatase with a broad substrate specificity, an apparent molecular weight of 19,000, and a pH optimum of 6.0-7.0. Dialysis of the enzyme preparation against EDTA abolished the phosphatase activity which could be restored by Zn(2+) ions and partially restored by Co(2+) ions. Crude extracts also contained a latent enzyme, the phosphatase activity of which could be detected in the presence of Co(2+) ions only. Zn(2+) ions did not activate this enzymatically inactive protein. The Co(2+)-dependent phosphatase had an apparent mol. wt. of 40,000, a broad substrate specificity, and an alkaline pH-optimum. Infection of Anacystis cultures by cyanophage AS-1 resulted in a decrease in phosphatase activity. The enzyme present in 10,000 g supernatants from infected cells could not be modulated by the AA/DHAA system.

Bacteriophage infection interferes with guanosine 3'-diphosphate-5'-diphosphate accumulation induced by energy and nitrogen starvation in the cyanobacterium Anacystis nidulans.

Anacystis nidulans accumulates large amounts of guanosine 3'-diphosphate-5'-diphosphate (ppGpp) upon nutritional or energy starvation induced by light-to-dark shift, treatment with carbonylcyanide-m-chlorophenylhydrazone (an uncoupler), or treatment with L-methionine-DL-sulfoximine (an inducer of nitrogen starvation). In contrast to healthy A. nidulans cells, those infected by AS-1 cyanophage do not respond with ppGpp accumulation when starved after about one-third of the complete infection cycle, except, to some extent, under extreme conditions when both nitrogen deprivation and energy deprivation are induced simultaneously (darkening plus L-methionine-DL-sulfoximine treatment). In contrast to cyanophage infection in Anacystis, infection with T4 phage of Escherichia coli CP 78 cells does not affect their accumulation of ppGpp under treatments identical with or similar to those applied in the experiments with Anacystis. This difference in response of phage-infected heterotrophic and photoautotrophic cells to starvation seems to reflect differences in control of nutritional or energy metabolism rather than differences in ability to synthesize ppGpp.

An iron-containing superoxide dismutase from Anacystis nidulans.

Superoxide dismutase (SOD) was isolated and purified from Anacystis nidulans to near electrophoretic homogeneity. The enzyme has a molecular weight of 37,500, as determined by gel filtration and SDS-gel electrophoresis. The enzyme molecule consists of two subunits of identical molecular weight. Proton-induced X-ray elemental analysis (PIXE) showed that the SOD of A. nidulans is an iron-containing enzyme; the Fe:enzyme mol ratio was found to be 1. The EPR spectra indicated that the active center contains high-spin ferric ion. Based on quantitative EPR data, we conclude that eseentially all iron ions were detected in the EPR experiments and were present in the Fe3+ active center. Effective g'-values were calculated from computer-simulated spectra and analysis of the g'-value anisotropy of the +/-3/2 Kramers doublet made the calculation of crystal field parameters possible. The symmetry of the Fe3+ ion in the SOD molecule was found to be close to rhombic (E/D=0.240).

Effect of light on the attachment of cyanophage AS-1 to Anacystis nidulans.

The effect of illumination on the extent and kinetics of the adsorption of cyanophage AS-1 to the blue-green alga (cyanobacterium) Anacystis nidulans was studied by using 32P-labeled phage. The initial rate of adsorption was not significantly affected by light. However, at Na+ levels used ordinarily to culture the alga ([Na+] = 11.7 mM), the total amount of phage adsorbed was doubled in the illuminated cultures, as compared with the dark-grown ones, over a wide range of multiplicities of infection (0.05 to 20). Upon a 10-fold increase in Na+ concentration in the medium ([Na+] = 0.11 M), the dark adsorption of the phage increased to the level of light adsorption found in low Na+ medium. The effects on phage adsorption of high Na+ concentration and light were not additive.

Polyribosomes in protoplasts isolated from tobacco leaves.

Polyribosomes (polysomes), active in an amino acid incorporation system in vitro, were isolated from tobacco leaf protoplasts. A comparison of polysome profiles indicated that the polysome/monosome ratio is greatly decreased in isolated protoplasts as compared to the intact leaf. In isolated protoplasts, a marked accumulation of ribosomal subunits was also found. The division of protoplasts, as investigated in the 8-cell and callus stages, was associated with a(n) (at least) partial regeneration of polysome profiles characteristic for leaves. Plasmolysis of leaves attached to the plant had no great effect on the polysome profile. However, leaf excision per se resulted in a dramatic loss of polysomes, even when the leaf tissue was floated on water. It is concluded that the isolation of the cell from its normal environment, and not the osmotic stress and associated increase in RNase activity, is the most important factor responsible for the loss of polysomes in isolated protoplasts.

Effect of "osmotic stress" on protein and nucleic acid synthesis in isolated tobacco protoplasts.

The incorporation of labeled precursors into RNAs and proteins of isolated tobacco (Nicotiana tabacum L.) leaf protoplasts decreases with increasing osmotic pressure in the incubation medium. The incorporation of precursors into RNA and proteins is linear for 15-18 h after the isolation of the protoplasts, irrespective of the osmolarity of the culture media. The uptake of precursors is also affected by the osmolarity of the medium. However, the osmotic stress-induced inhibition of incorporation of precursors into RNA and proteins is also apparent if the differences in uptake are taken into consideration in the calculation. Incorporation of (32)P into TMV-RNA is also inhibited by osmotic stress. As assayed by the double labeling ratio technique, osmotic stress has less unequivocal effect on TMV protein synthesis.

The postmaturational cleavage of 23 S ribosomal RNA in Anacystis nidulans is inhibited by infection with cyanophage AS-1.

The 23S (1.1 X 10(6) mol.wt.) rRNA of Anacystis nidulans undergoes postmaturational cleavage to produce 0.9 X 10(6) and 0.2 X 10(6) molecular species. We have provided evidence in double labelling experiments, in which we could distinguish between the fate of molecules synthesized before and after infection, respectively, that infection with cyanophage AS-1 abruptly and completely inhibits the cleavage of 23S rRNA in Anacystis cells.

Formation in the dark, of virus-induced deoxyribonuclease activity in Anacystis nidulans, an obligate photoautotroph.

In Anacystis nidulans, upon infection with cyanophage AS-1, after a lag period of 1 h the level of deoxyribonuclease (DNase) activity increaded rapidly up to 15- to 20-fold in 4 to 5 h in the light. In contrast, the ribonuclease and phosphomonoesterase activities increased significantly only 4 to 5 h after infection, i.e. as late as 1 h prior to lysis. In complete darkness, the nuclease levels remained unaltered. However, when the infected cells were exposed to light for 1 or 2 h after infection, the DNase level increased essentially to the same extent in the dark as in continuous light, although the complete replication cycle of the virus was impaired in the dark and cells lysed only in the continuously illuminated cultures. Inhibition of photosystem II with 3-(3,4-dichlorophenyl)-1-dimethylurea during the early illumination period strongly decreased the subsequent, infection-dependent increase in DNase activity in the dark. The virus-induced increase in DNase activity was also inhibited by chloramphenicol. The data suggest that, in spite of the obligate photoautotrophic nature of A. nidulans, dark metabolism is able to support fully the formation of some specific proteins if the triggering of their synthesis takes place in light.

Viral RNA synthesis is renewed in protoplasts isolated from TMV-infected Xanthi tobacco leaves in an advanced stage of virus infection.

TMV-RNA synthesis was shown to be renewed in protoplasts isolated from TMV-infected Xanthi tobacco leaves in which virus synthesis has already slowed down or stopped. This was demonstrated by the use of isotope techniques in three different disease situations: (a) in primarily infected leaves 8-10 days after infection, (b) in secondarily infected leaves 2 mo after infection, and (c) in stable green areas ("true dark green islands" according to the terminology of Atkinson and Matthews, 1970) of leaves exhibiting mosaic symptoms.