Morphological Responses of Wheat to Changes in Phytochrome Photoequilibrium

Wheat plants (Triticum aestivum L.) were grown at the same photosynthetic photon flux (PPF), 200 micromoles per square meter per second, but with phytochrome photoequilibrium ([unk]) values of 0.81, 0.55, and 0.33. Plants grown at [unk] values of 0.55 and 0.33 tillered 43 and 56%, less compared with plants grown at [unk] of 0.81. Main culm development (Haun stage) was slightly more advanced at lower values of [unk], and leaf sheaths, but not leaf lamina, were longer at lower [unk]. Dry-mass accumulation was not affected by different levels of [unk]. Three levels of PPF (100, 200, and 400 micromoles per square meter per second) and two lamp types, metal halide and high pressure sodium, were also tested. Higher levels of PPF resulted in more dry mass, more tillering, and a more advanced Haun stage. There was no difference in plant dry mass or development under metal halide versus high pressure sodium lamps, except for total leaf length, which was greater under high pressure sodium lamps (49.5 versus 44.9 centimeters, P < 0.01).     

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163–171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Assignment of the gene coding for human cytochrome c oxidase subunit VIb to chromosome 19, band q13.1, by fluorescence in situ hybridisation

Summary A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-11-dUTP and detected by fluorescence. Subsequent R-banding indicated that the cytochrome c oxidase subunit VIb gene is localized in band 19q13.1, extending the evidence that the human nuclear genes of cytochrome c oxidase are not clustered.     

Protein phosphorylation in the facultative chemolithotrophThiobacillus novellus

The phosphorylation of at least five proteins with M_r of about 160,000; 93,000; 85,000; 45,000; and 29,000 respectively was demonstrated in crude extracts from the facultative chemolithotrophThiobacillus novellus. The incorporation of [-³²P]phosphate from ATP into these proteins was dependent on the presence of magnesium ion. The phosphorylation reactions were found to be reversible and required 12.5 mM NaF for maximal activity, indicating the action of phosphatases. In addition, 3,5-cAMP had little effect on protein kinase activity, whereas Ca²⁺ alone was weakly stimulatory. This activation was enhanced by the addition of 3,5-cAMP. Ca²⁺ with calmodulin had a strong stimulatory effect on phosphate incorporation into the proteins. A highly purified preparation containing only the 160, 93, and 85 kDa proteins phosphorylated histone, whereas the uptake of³²P by the three proteins was inhibited. Rabbit muscle phosphorylase b prevented incorporation of radiolabel only into the 160 and 93 kDa proteins.     

Accumulation of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA in embryonic rat hepatocytes. Evidence for translational control during the initial phases of hepatocyte-specific gene expression in vitro

The aim of this study was to establish whether the initial accumulation of hepatocyte-specific proteins after hormone induction is regulated at the pretranslational and/or the translational level. To this end, mRNA molar concentrations were determined and compared with rates of protein synthesis from previous studies [van Roon, M.A., Charles, R. & Lamers, W.H. (1987) Eur. J. Biochem. 165, 229-234]. In vivo, carbamoylphosphate-synthetase mRNA starts to accumulate at day 17 of pregnancy. Phosphoenolpyruvate-carboxykinase mRNA starts to accumulate only just prior to birth. Embryonic day 14 (i.e. 8 days before the expected day of birth), livers were chosen to study the regulation of the initiation of hepatocyte-specific mRNA accumulation in vitro. Accumulation of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA is regulated by the same hormones as accumulation of the respective proteins. The rate at which carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA molecules accumulate in cultured embryonic hepatocytes is relatively low, compared to that of postnatal hepatocytes. However, the increase of the rate of synthesis of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase protein is even 3-6-fold slower than that of mRNA. This shows that initially mRNAs accumulate intracellularly to a relatively high concentration without being efficiently translated or translatable. Only after the mRNA concentration reaches a plateau of 72 h and 48 h respectively, the cellular capacity to synthesize the respective proteins increases. Therefore, the translational efficiency is certainly one of the major rate-limiting factors of the initial phases of expression of the hepatocyte-specific genes for carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase.     

Developmental and hormonal regulation of carbamoyl-phosphate synthase gene expression in rat liver: evidence for control mechanisms at different levels in the perinatal period

Carbamoyl-phosphate synthase gene expression is found to be primarily regulated by conditions that enhance hepatic glucocorticosteroid levels (hormone injections) and cyclic AMP levels (induction of diabetes). After birth, changes in the level of carbamoyl-phosphate synthase protein follow changes in the level of carbamoylphosphate synthase mRNA, suggesting a pretranslational control mechanism. In fetal rats, carbamoyl-phosphate synthase gene expression is regulated by the same factors as in adults. However, both the level to which carbamoyl-phosphate synthase mRNA can accumulate and the extent to which mRNA can be translated appear to be limited, indicating control mechanisms at the pretranslational and translational level. Finally, in the immediate postnatal period, a transient but pronounced decrease in the rate of degradation of carbamoyl-phosphate synthase protein may play a role in the accumulation of the enzyme.     

Estimation of the stability of globular proteins

The interpretation of ΔG (the free energy change for the reaction, globular conformation ? randomly coiled conformation, in the absence of denaturant), in terms of the free energies of transfer of various parts of the protein molecule from water to denaturant solution, is unsatisfactory because the latter are assumed to be identical to the transfer-free energies of similar groups attached to smaller model compounds. We have made empirical adjustments to transfer-free energy theory that make possible linear extrapolation of the free energy of denaturation of a protein from transition region to zero denaturant concentration. The modified theory, used to analyze the denaturation of proteins by guanidine hydrochloride and urea, allowed us to calculate reasonable values for Δα, the average change in accessibility to solvent of the component groups of protein.