Nucleic acid synthesis and effect of glucose on its kinetics in cotyledons ofChenopodium rubrum l. during photoperiodic induction

In cotyledons ofChenopodium rubrum L. polydisperse RNA is synthesized in the region of the low molecular weight RNAs during photoperiodic induction. After short-time labelling the rate of 4s RNA synthesis was always higher in induced plants than in plants having obtained a light-break in the middle of the dark period. When glucose was added to the nutrient medium during the dark period of a single photoperiodic cycle the rate of nucleic acid (NA) synthesis was higher in non-induced plants than in induced ones at the termination of the dark period. In plants induced by two cycles in the absence of glucose the rate of NA synthesis at the termination of the second dark period was higher in induced than in non-induced plants. This difference is due to the differential kinetics of NA synthesis during darkness. In plants induced in the presence of glucose the peak of the rhythm in NA synthesis was advanced by 4 h relative to that found in plants induced in the absence of sugar. Thus, the termination of the dark period coincided with the negative slope of the oscillation in plants induced in the presence of glucose, while in plants having obtained a light-break NA synthesis decreased only slightly after having attained its peak. In plants induced in the absence of glucose the termination of the dark period coincided with the peak in the rhythm in NA synthesis. The rhythm in NA synthesis of the cotyledons during the dark period of an inductive cycle is out of phase with the rhythm in flower initiation.     

Variability in the amount of beta-globin mRNA in beta0 thalassemia

Globin mRNA isolated from a number of beta0 thalassemia patients of different ethnic origins was analyzed by RNA-cDNA hybridization and, in two cases, by fingerprint analysis of 125I-labeled mRNA. Quantitation of the relative amounts of alpha- and beta-mRNA by hybridization to purified alpha-and beta-cDNA revealed that in approximately half the cases, there was less than 1% as much beta-mRNA as alpha-mRNA. In the rest of the cases, low levels of beta-like mRNA were detected in amounts 4-12% as abundant as alpha-mRNA. There was variability in the yield of beta-like mRNA in patients of the same racial group, in the same patient at different times and in similarly affected siblings: beta-mRNA was virtually absent in some samples, whereas low but significant levels were found in other samples. In one patient, beta-like mRNA was not detected in peripheral blood RNA, but was present in the RNA of bone marrow cells. In one case, the thermal stability of the beta0 thalassemia mRNA-beta-cDNA hybrid was measured and found to be slightly lower than that of the authentic beta-mRNA-beta-cDNA hybrid. In none of the cases tested was there synthesis of beta-globin chains directed by beta0 thalassemia mRNA in a cell-free protein-synthesizing system, even when beta-like mRNA was detected in the sample by hybridization assays. mRNA from two patients was labeled in vitro with 125I, digested with T1 RNAase and fractionated in two dimensions. Analysis of the resulting fingerprints revealed the presence of prominent alpha chain-specific oligonucleotides without detectable beta chain-specific oligonucleotides, and thereby confirmed the results of hybridization assays showing absent or very low levels of beta-mRNA in the same RNA samples. Our results support the concept that beta0 thalassemia is heterogeneous in its molecular basis even within the same racial group: in some patients, it is associated with absent beta globin mRNA, whereas in other patients, it is associated with low but significant levels of nonfunctional beta or beta-like globin mRNA. The variable amounts of beta-like mRNA detected in different samples from the same patient, and in patients with the same genotype, indicate that as yet undefined factors can influence the yield of beta-like mRNA observed in beta0 thalassemia.     

Effect of different conditions of light on the nucleic acid fractions in cotyledons ofChenopodium rubrutn L.

The nucleic acid fractions in cotyledons of young Chenopodium rubrum plants exposed to continuous light, continuous darkness and short (8 h) day, respectively and labelled with³²P 24 h prior to harvesting were studied by means of chromatography on MAK columns. Some parameters of cotyledon growth (dry weight, cotyledon area, occurrence of mitoses) were also investigated. The changes in the nucleic acid fractions agreed with the dynamics of cotyledon growth. In continuous light the content of all fractions increased. The radioactivity of DNA and s-RNA did not undergo any great changes and only r-RNA increased. The specific activity of r-RNA increased slightly, that of soluble RNA and DNA was reduced. In continuous darkness the content of all the fractions did not undergo any great changes. The radioactivity as well as the specific activity of all fractions decreased. In short day the content of the nucleic acid fractions did not change conspicuously. Only the specific activity of s-RNA increased in a noticeable way while the radioactivity of r-RNA and the specific activity of DNA decreased and this of r-RNA did not change. The changes in nucleic acid metabolism were partially connected with changes in³²P uptake which depended upon light conditions but they were not merely a consequence of this fact. Obviously, there also exists a more direct relationship between nucleic acid synthesis and growth.     

INCORPORATION OF PHOSPHATE INTO RAT BRAIN DURING SLEEP AND WAKEFULNESS

Abstract— Labelled inorganic phosphate (³²P₁) was administered intraventricularly to unrestrained sleeping and waking adult rats. After about 20 min of sleep or a comparable period of wakefulness, as monitored by EEG and EMG, the animals were frozen in liquid nitrogen and the brains were analysed. One group of animals (A) was not previously acclimatized to the apparatus. A second group (B) was acclimatized. The specific radioactivity of a phosphoprotein fraction was elevated during sleep in group A but not in group B. The specific radioactivity of the phosphatides of group B was depressed in sleeping as compared with waking animals. This effect was not observed in group A. No significant difference was detected between the EEG patterns of sleeping animals in groups A and B, as evaluated by standard criteria. These observations suggest that the physiological conditions attributable to environmental, emotional or other determinants can influence shifts in brain metabolism during the sleep-wakefulness cycle.     

The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis

Canonical Wnt/beta-catenin signaling regulates the activation of the myogenic determination gene Myf5 at the onset of myogenesis, but the underlying molecular mechanism is unknown. Here, we report that the Wnt signal is transduced in muscle progenitor cells by at least two Frizzled (Fz) receptors (Fz1 and/or Fz6), through the canonical beta-catenin pathway, in the epaxial domain of newly formed somites. We show that Myf5 activation is dramatically reduced by blocking the Wnt/beta-catenin pathway in somite progenitor cells, whereas expression of activated beta-catenin is sufficient to activate Myf5 in somites but not in the presomitic mesoderm. In addition, we identified Tcf/Lef sequences immediately 5' to the Myf5 early epaxial enhancer. These sites determine the correct spatiotemporal expression of Myf5 in the epaxial domain of the somite, mediating the synergistic action of the Wnt/beta-catenin and the Shh/Gli pathways. Taken together, these results demonstrate that Myf5 is a direct target of Wnt/beta-catenin, and that its full activation requires a cooperative interaction between the canonical Wnt and the Shh/Gli pathways in muscle progenitor cells.     

The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots

Since copper (Cu) is essential in key physiological oxidation reactions, organisms have developed strategies for handling Cu while avoiding its potentially toxic effects. Among the tools that have evolved to cope with Cu is a network of Cu homeostasis factors such as Cu-transporting P-type ATPases that play a key role in transmembrane Cu transport. In this work we present the functional characterization of an Arabidopsis Cu-transporting P-type ATPase, denoted heavy metal ATPase 5 (HMA5), and its interaction with Arabidopsis metallochaperones. HMA5 is primarily expressed in roots, and is strongly and specifically induced by Cu in whole plants. We have identified and characterized plants carrying two independent T-DNA insertion alleles, hma5-1 and hma5-2. Both mutants are hypersensitive to Cu but not to other metals such as iron, zinc or cadmium. Interestingly, root tips from Cu-treated hma5 mutants exhibit a wave-like phenotype at early stages and later on main root growth completely arrests whereas lateral roots emerge near the crown. Accordingly, these lines accumulate Cu in roots to a greater extent than wild-type plants under Cu excess. Finally, yeast two-hybrid experiments demonstrate that the metal-binding domains of HMA5 interact with Arabidopsis ATX1-like Cu chaperones, and suggest a regulatory role for the plant-specific domain of the CCH Cu chaperone. Based on these findings, we propose a role for HMA5 in Cu compartmentalization and detoxification.     

CDK1 interacts with RARγ and plays an important role in treatment response of acute myeloid leukemia

Alterations in cell cycle pathways and retinoic acid signaling are implicated in leukemogenesis. However, little is known about the roles of cyclin-dependent kinases (CDKs) in treatment response of leukemia. In this study, we observed that CDK1 expression was significantly higher in bone marrow from 42 patients with acute myeloid leukemia (AML) at recurrence than that at first diagnosis (p = 0.04). AML patients had higher level of nuclear CDK1 in their leukemic blasts tended to have poorer clinical outcome compared with those with lower levels. We showed that CDK1 function is required for all-trans retinoic acid (ATRA) to achieve the optimal effect in U-937 human leukemic cells. CDK1 modulates the levels of P27^kip and AKT phosphorylation in response to ATRA treatment. Further, we show, for the first time, that RARγ in concert with ATRA regulates protein levels of CDK1 and its subcellular localization. The regulation of the subcellular content of CDK1 and RARγ by ATRA is an important process for achieving an effective response in treatment of leukemia. RARγ and CDK1 form a reciprocal regulatory circuit in the nucleus and influence the function and protein stability of each other and the level of P27^kip protein. In addition, expression of wee1 kinase and Cdc25A/C phosphatases also coincide with CDK1 expression and its subcellular localization in response to ATRA treatment. Our study reveals a novel mechanism by which CDK1 and RARγ coordinate with ATRA to influence cell cycle progression and cellular differentiation.     

The intracellular Arabidopsis COPT5 transport protein is required for photosynthetic electron transport under severe copper deficiency

Copper is an essential micronutrient that functions as a redox cofactor in multiple plant processes, including photosynthesis. Arabidopsis thaliana possesses a conserved family of CTR-like high-affinity copper transport proteins denoted as COPT1-5. COPT1, the only family member that is functionally characterized, participates in plant copper acquisition. However, little is known about the function of the other Arabidopsis COPT proteins in the transport and distribution of copper. Here, we show that a functional fusion of COPT5 to the green fluorescent protein localizes in Arabidopsis cells to the prevacuolar compartment. Plants defective in COPT5 do not exhibit any significant phenotype under copper-sufficient conditions, but their growth is compromised under copper limitation. Under extreme copper deficiency, two independent copt5 knockout mutant lines exhibit severe defects in vegetative growth and root elongation, low chlorophyll content, and impairment in the photosynthetic electron transfer. All these phenotypes are rescued when the wild-type copy of the COPT5 gene is retransformed into a copt5 knockout line or when copper, but not other metals, are added to the medium. COPT5 is expressed in vascular tissues, with elevated levels in roots. Taken together, these results suggest that COPT5 plays an important role in the plant response to environmental copper scarcity, probably by remobilizing copper from prevacuolar vesicles, which could act as internal stores or recycling vesicles to provide the metal cofactor to key copper-dependent processes such as photosynthesis.     

Involvement of propeptides in formation of catalytically active metalloproteinase from Thermoactinomyces sp

The metalloproteinase from Thermoactinomyces sp. 27a (Mpr) represents secretory thermolysin-like metalloproteinases of the M4 family. The Thermoactinomyces enzyme is synthesized as a precursor consisting of a signal peptide, N-terminal propeptide, mature region, and C-terminal propeptide. The functional molecule lacks the signal peptide, N-terminal propeptide, and C-terminal propeptide, which indicates the processing of these regions. Until now, it remained unclear if the N-terminal propeptide is involved in the formation and functioning of Mpr, and the role of the C-terminal propeptide was also unclear. In this work, a Bacillus subtilis AJ73 strain expressing Mpr without the C-terminal propeptide- encoding region being involved has been obtained. The absence of the Mpr C-terminal propeptide had no significant effect on the formation of the functional molecule and did not interfere with the protease secretion in B. subtilis AJ73 cells. Strains producing the N-terminal propeptide, mature region, and mature region covalently bound to the N-terminal propeptide were generated from Escherichia coli BL-21(DE3) cells. Functionally active Mpr forms could be produced only in the presence of the N-terminal propeptide, either covalently bound to the mature region (in cis) or as a separate molecule (in trans). Thus, the Mpr three-dimensional structure is formed according to the propeptide-assisted mechanism with no requirement of a covalent bond between the N-terminal propeptide and mature region. Moreover, Mpr variants generated in cis and in trans differed in their specificity for certain synthetic substrates.