Regulation of Carbon Partitioning in Source and Sink Leaf Parts in Sweet Pepper (Capsicum annuum L.) Plants : Role of Fructose 2,6-Bisphosphate

Area expansion rate, partitioning of photosynthetically fixed carbon, and levels of fructose 2,6-bisphosphate (fru-2,6-P₂) were determined in individual parts of developing leaves of sweet pepper (Capsicum annuum L.). The base was rapidly expanding and allocated less carbon to sucrose synthesis in comparison to the leaf tip, where expansion had almost stopped. The change in leaf expansion rate and carbon partitioning happened gradually. During day time levels of fru-2,6-P₂ were consistently higher in the leaf base than in the leaf tip. Leaf expansion rate and carbon partitioning were closely related to day time levels of fru-2,6-P₂, suggesting that fru-2,6-P₂ is an important factor in adjustment of metabolism during sink-to-source transition of leaf tissue. The levels of fru-2,6-P₂ changed markedly after a dark-to-light transition in the leaf base, but not in the leaf tip, suggesting that regulatory systems based on fru-2,6-P₂ are different in sink and source leaf tissue. During the period upon dark-to-light transition the variations in level of fru-2,6-P₂ did not show a close correlation to changes in the carbon partitioning, until the metabolism had reached a steady state.     

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.     

Denitrification and oxygen respiration in biofilms studied with a microsensor for nitrous oxide and oxygen

Depth distributions of O₂ respiration and denitrification activity were studied in 1- to 2-mm thick biofilms from nutrient-rich Danish streams. Acetylene was added to block the reduction of N₂O, and micro-profiles of O₂ and N₂O in the biofilm were measured simultaneously with a polarographic microsensor. The specific activities of the two respiratory processes were calculated from the microprofiles using a one-dimensional diffusion-reaction model. Denitrification only occurred in layers where O₂ was absent or present at low concentrations (of a fewM). Introduction of O₂ into deeper layers inhibited denitrification, but the process started immediately after anoxic conditions were reestablished. Denitrification activity was present at greater depth in the biofilm when the NO₃ ^– concentration in the overlying water was elevated, and the deepest occurrence of denitrification was apparently determined by the depth penetration of NO₃ ^–. The denitrification rate within each specific layer was not affected by an increase in NO₃ ^– concentration, and the half-saturation concentration (K_m) for NO₃ ^– therefore considered to be low (<25M). Addition of 0.2% yeast extract stimulated denitrification only in the uppermost 0.2 mm of the denitrification zone indicating a very efficient utilization of the dissolved organic matter within the upper layers of the biofilm.     

Iron overload of the liver by trimethylhexanoylferrocene in rats.

Iron-deficient female Wistar rats were fed a diet, which contained 0.5% trimethylhexanoylferrocene, over a 56-week period. This dietary iron loading resulted in a progressive siderosis and enlargement of the liver with a maximum iron content of 947.0 +/- 148.0 mg (vs. 0.07 +/- 0.04 mg in iron deficiency) and a maximum organ weight of 39.4 +/- 6.6 g (vs. 6.9 +/- 1.4 g in iron-deficient control rats). Up to 43 weeks, whole liver iron rose by increase in iron concentration (max. 28.0 +/- 6.1 mg/g wet weight, w.w.) as well as by enlargement of the organ. Afterwards whole liver iron increased solely by ongoing hepatomegaly. At the commencement of iron loading, stainable iron was almost exclusively stored by hepatocytes equally throughout all areas of the liver lobule. Later, the distribution of iron-loaded hepatocytes became strikingly periportal, and, in addition, Kupffer cells as well as sinus-lining endothelia began to store iron. Animals with a liver iron concentration of more than 10.4 +/- 0.75 mg/g w.w. showed no further increase in ferritin and haemosiderin within hepatocytes. Iron-burdened Kupffer cells/macrophages, however, accumulated permanently, hereby forming intrasinusoidal and portal siderotic nodules and areas. First signs of liver damage such as necrosis of single hepatocytes and mild fibrosis began at a liver iron concentration of 14.7 +/- 1.4 mg/g w.w. With advancement of iron loading, focal necrosis of hepatocytes and iron-burdened macrophages took place, and significant perisinusoidal as well as portal fibrosis developed. Cirrhosis, however, the final stage of impairment in iron overload of the liver in humans, could not be induced in this animal model up to now.     

Mitochondrial malate dehydrogenase from watermelon: sequence of cDNA clones and primary structure of the higher-plant precursor protein

The isolation and sequence of a cDNA clone encoding the complete mitochondrial malate dehydrogenase (mMDH) of watermelon cotyledons is presented. Taking advantage of the polymerase chain reaction technology partial cDNA clones from the central part, the 3 part and the 5 part of the mRNA were obtained with oligonucleotides based on directly determined amino acid sequences. Subsequently, two complete cDNA clones for mMDH were synthesized with a sense primer corresponding to the nucleotide sequence of the amino terminal end of pre-mMDH and two antisense primers corresponding to the major alternative adenylation sites found in the mRNA.The amino acid residues for substrate and cofactor binding identified by X-ray crystallography for pig heart cytoplasmic MDH are conserved in the 320 amino acid long mature higher-plant mMDH. A presequence of 27 amino acids is present at the amino terminal end of the precursor protein.     

Oxygen transport in the blood of arctic mammals: adaptation to local heterothermia

Summary The oxygen binding of whole blood from humans and two arctic mammals, reindeer and muskox, has been studied as a function of carbon dioxide and temperature. All bloods display a marked Bohr effect with Bohr coefficients in the range –0.44––0.73. The Bohr effect is more pronounced at 20°C. The temperature sensitivity of reindeer and muskox blood expressed by the apparent heat of oxygenation, H, is almost three times lower than that of human HbA under the same experimental conditions. This thermodynamic difference gives special benefits to arctic mammals with large heterothermy by safeguarding oxygen unloading at very low ambient temperatures.     

Properties of plasma low density lipoproteins in patients with hypertriglyceridemia and ischemic heart disease

It has been shown that low-density plasma lipoproteins in patients with ischemic heart disease and hypertriglyceridemia are heavier in density, smaller in size, more negatively charged and more inclined to peroxide modification and aggregation than in healthy persons. The protein in the composition of such lipoproteins deviates towards the water phase, which may result in the masking of the domen, recognized by the BE-receptor and may lead to hyperlipidemia of a retaining character.     

The regulation of proenkephalin expression in a distinct population of glial cells.

The expression of opioid genes was examined in isolated populations of glial cells in primary culture. Northern blot analysis of purified type I astrocytes, oligodendrocytes and mixed oligodendrocyte-type-2-astrocyte lineage cells derived from cerebral cortex demonstrated robust expression of proenkephalin mRNA exclusively in type I astrocytes. The expression of proenkephalin mRNA was stimulated by the beta-adrenergic agonist isoproterenol, and 8-(4-chlorophenyl thio)adenosine 3'-5'-cyclic monophosphate (cpt-cAMP). Both of these compounds regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into type I astrocytes. HPLC and immunoassay of the cell culture media revealed significant levels of unprocessed proenkephalin secreted by the cell and this secretion was stimulated by isoproterenol and cpt-cAMP. The relatively high levels of proenkephalin expressed suggest that enhanced expression in astrocytes may be important during neural development, in trauma-induced gliosis and in neuroimmune interactions.     

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein.

Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-beta-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of approximately 200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched approximately 300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.     

Use of diethyl squarate for the coupling of oligosaccharide amines to carrier proteins and characterization of the resulting neoglycoproteins by MALDI-TOF mass spectrometry

The 8-methoxycarbonyloctyl glycosides of GlcNAc, Gal1-4Glc, Fuc1-2Fuc1-3GalNac and Fuc1-2Gal1-3[Fuc1-4]GlcNac were converted to primary amines by reaction with neat ethylenediamine and then coupled to bovine serum albumin (BSA) using diethyl squarate as the connector. The average degree of incorporation of the sugar onto the protein, as well as the molecular weight distribution, could be conveniently determined using matrix assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry thus avoiding cumbersome structure-dependent colour-tests or analysis of cleaved ligand. The present coupling method has the advantages of proceeding under very mild conditions, yielding controlled incorporation values and can reliably be used for the coupling of very small amounts (mg) of oligosaccharide.This paper is dedicated to Sen-itiroh Hakomori on the occasion of his 65th birthday